ass skake that

NOTES
......ON ......
PHYSIOLC)GICAL
CHIGM:IS~[1RY,
BY
.
ALEX IUS McGLANNAN,
~
AI'lSOOTA'rE
PROl'-EBSOR
-,
<-F
.
PHYSIOI,OGICAL
lVL
n.
CHElV.IIS'I'RY,
COI...
LI<~GE 0)'<'----
PHYSICIANS
AND SUHGE()NS,
IHOO.
in the plant
r(~duetion processes and s:vntheses,conliving force into potential energy or chemical tension~
are the prevailin~
forces,we find in th0 animal body ~he rsversec I eav ag e arid ox t da t z ori pro~ess es ~wh i en c orivsr r chen;icg,lt ens ion
into livlng foro6.
While
verting
b;
This difference must not be o~errated,nor must a sharp line
considered to exist between plants and animals6 There are syn- .
thetic processes occuring in tho bodies of animals;and cleavage
procssse~ occur in some plants.ln
animal organiem the proc0Sses of oxidation and cleavage are j:;dominant~whi.le in plants,
those o r' reduction an d synthesis most often o ccur ,
CHRMICAL COMPOSITION
OF ~'HE ANTMAL BODY.
An elementary analysis of an 8nlmal body ,Shows the presan~e
of the e!em0nts,C,H;O,N~P,Cl,Fl,Si,Ca,Mg,Ne,K,andFe~With
the
oCC~sioDal presence of Al,Mn,Cuvand some other elements Which
Occur irregularly and cannot be looked upon as normal constituentse
The elem?nts C,N;Oiand H,oacur in the largest proportion.Or
the
metals Ca,Na,~nd K,are fuostaBbundant. The various SUbstances
reSUlting from the combination of the elements are classified
into the organic and inorganic constituents.
The organic class is sUbdivided into the nitrogenous and nonnitrogencil5 groups.
NT'I'POGENOUS
organic
bOLtiC:S
form the cn i e r part of the solid
animal tissues and are fMEru~.xfound to a consIderable extent in
the circulating fluids1the secretions and excre~ions. They belong
for the most part to a group of SUbstances known as the proteids
or are SUbstances closely related to the m8mbers of this group,
and for this reason are classed together as the protein SUbstances
The proteins are a most important ~roup of sqbstances oCcu~ing
in all liVing tissue ~and will be considered in detail later.
The nitrogenous waste products Urea,Uric Bcid,and the Xanthine
Bases,Creatinin
&~.Pigment8,ferments?and
other bodies belonging to
this class will be trijated of in connection with other SUbjects.
NON-'NI'l'ROGENOUS
Fats,and carbohydrates,will
be considered in detail later on.
Pormlc.aeetic?proprionio
and butyric acids,are present in sweat
and othe~t l uL:h:. Lac U. c a c 10. occur s in muscle. Phencl occurs in
various CJcbinations in the ur~ne and intestinal canal.Benzoic
<.
aci.rl!
the
ttl
ally
C)iJgUtat:::,
l1u!J}~~-:
']'.h&
~:r·.-i.~1·:3 i~l
~.ncrga.n.i!) constitL1cnts
pact."directlY
freel
food
.....,
most
s ys t en1 111-lal t ere' d ~;:;O.l.·IJe
r,":.s: f?
• -
80;1;e a r e -n6wlv;"ar",'p,;;'
'C''''
.a ,
n
-'
--.
..J ~-
-j.~.,.~
'.• 1 J, t.:
.~.
'Y
~~
\......
-",I
~.
t ncr g an ; c; C 1 a::; s i:=: :.0 e;:: t;;\ i
~~~liri (~c:}~~titue~t~u
';'he
G!lSE:S.~
gas60uil
L,'-"'A,-
~J
~J \, J \ .•
-~J..l.J
mattt:::rs
...... ...,. ....
or
i
I'
-:..~
~
the
1 (~e j
[.June!
.£..
J.~'
...
("....
c~ ,...r
...
,""\"'. ":
-
..
v ..~
··u
;¥".;
"r':)
e {.t. t:..; r
throug"i
1..
l,.
.... c
(l~::1.l1
p
art
t"''-...
,'.
';"-::1.
.':./-~
..L
~
v ~3'!...
......
(
lor
t he.
1;116
...
-'va:'.'l., &,t1<;
c~~f -r-f'c.
." ...
1""
Water.
'!: .h e
i. n L)
~ .~ 1. q U 1
g 2.:3 e G'0;:;
d;
b:J<~Y 8:"13 oX'ygen~
the
ill
~
,..,
1-'"
.
...
J.<yHr\·)e.ren"la~lG
hy(;r';';i:!Oi'~ :?,::" W(~J.I CJ,s
r'n'"
t;,
f...,....·,
d..i.
.f
pEUG
.... ~.:\
n;+~~'ger'
c~·~o·cu·~r"t·".
~""""L""'-'
....
""c'-'-,
l ........
~ ....
J
1
t..<...
~L.Jt ... wL:.,,1.!:Jl1.'.:..iE;
bon.iG a~~:i(L(rho fir::>t
two in tilE; b}.co~"'j;~;,d
~li'fd:r''''1Q~
>
/:;.x-r~
t....,~_d, .!oJ.L ......
of
, J' he
and
t-,·liYl'"'I.-..,
,-=. __•
$
..Li.
:J..rll":::;,-;._tJ-.;'tr!;('
c.. ...(
an d dri..c;k;?.L::e;:
o ~.~,·i·A
sr r-::·r j
(1 (~l·'.-lr) '.,'",I::
-
.in t h e C)v";)v
C2rbanate2~and
some
t
and
sUlphates
1\
~
r-.«...
1J0:(L,ivorE1,,'::,;](i
[",?r:;: I)j
~~.UY·~~:-:
.... 81..i.
(;&.1"'-
2n '-~ n i. U- Ci-! 8n s.n d C8.I b ~n',· 2'~ e,:l2,nd.
:::.,;.'-i. pl', ;~r i'" t',,:: Co i~lY L~r"c't:~en are
i:!th8
in:,-:;;:;tijJ3.1
t::~~~L'3.~,Ga,r~-.;-,:,i,., [:',jj.!~
~>o,
pi"ei~C:;;lt
In the
bleod
f~:1d ot.!.c~r rJ.u,i(~_,': .. and .1.'-.,' e·.V.i·'!'c-1,·:,."...•."! -"'J
',''"',,''C
("l~"'-""~~
',.
the
<-........
:i. l ..
l~ J
'.
lUt!gs,.a[l:::~ in YGr-y rn:il1ut2 "L~ )'-~d! (::,y tilc' ;c;l':i'r:;,
OX}gen
fcrIll,OC{~
~-
•
•
-"'~
-
~.-
-
-'-
.~
.~
••
t.;" ... ...: OJ'
v
thrLl L'No··th1rdE'
cr
C'l;:; w,-':i.ght
or
bodY"~~d
irnpart~~~e ~f wstc!· as a 0008cituSDt
J(
the
is sh~wn In a strickin~
mann~r
by irS :2mo~2:.1~
sny
h(~ate&
F~,uft101.p~-"r_lY
WA'l'~~E,rGrm~~mor(~
lJ'"
-.....
all
·~/~;--
-
....
.... ,f .. ,....
~.;",~,
"-~L.J,,.
~
-
...
-
t~J":"
.. 11\.-,
·;
...""Or-l·C).")
\.....r.{,~
.._J.
..
C.!.
:
charac.t::;risti:;
.l..t .....
---
In
j
'~J
1
--
\....
l
-
)":jy"T
1 ( ..._
pt1y.::;ic:c"J, pr'..Ji.JGrtle::
Dre
r~(,-fi~H:r,.eX50V§1{CJ52r~,"~p~$f~l\
and
flexible,becomes
J ~ ..:..
Cl ~
t·...... L
r (; (~0 g :ni z a b l e .. -
~ i
its
l,.
,.;"
\.,
... ~
~
...-;.
tht:'
wl'!.C:','3
animal
body
tis~ue,be
,..--.,f-"$
";·Cr-.
'.
~
[
'ti-, ....
-Ya\",
• ...:.
+e ....
.a.?
:::'28tl'oved,:e.n,,!
harJ"brittl(~
What
and
.)'~
all
the fluids
or the
body -. bla,jc"lYIDPh
etc'··\~'aterls
the
cool'lent,and
by lts
mc:a03 alone
cir'cula.tion
uf llutrif;nt
matter
is possible.It
is the medium alsc in WhICh all
aliments
are
dIssolved
bofore absorption.as
welles
thu moans by whicn all?
AX~(}pt ga~eous~Waste
producTS
are rerrloved~1'f1eprOCG~32es ot' :3ecre~
tion~transudationland
nut:ltion
depe~d
on itG pr0sence
for
theIr
Oi:rf'ormance,
~·:'lr·er8.1
'l'he gr\~ater
part
of t..'8 W:':llsr oresent
i1: as
such
frOIlJ v::d; uutlc,s
food and
cn~ result
of the
chomi0Bl
'."·ganisrn.
E1
Into
'1'he ::'o~:s of
,:~xGrGtion
the fl.limentary
SOLIDS,
Sodium
()f
the
~reat8r
In
water
tJ18
f:r~)rD
canal"
I;
t.hEtn
11"1
ohlcrides
are
0reSB!]t
~n n~arly
tJ 1 \) d \I the q l] ~~rl tit ~y () f ~;c d i 11m e h!. c~r
the
Ii').;.]."!". uf'
-in
the
'::...,].~_
t!~l(:- 'jr..nor
~;.~.0C(!
of
cf
that
functions
perts
·.~_S
i.:~(~J81.1J..)r.ts~
rut2;:=,'Sltlm
:s<':,J,t
.L"
i:::: !Jl'8sent~'-:bi)j>c;~~
rnaf-,Zcl33iurn
"'......
nd flutGoln
,Ie i '-ii.i} e Z l
so:.::rce
of their
the
211
.l .-1 e
<T8,j"c.'2rc
ind
teeth;
f';OL1S ~);":hr3~~-'f."11.liL~:3 ~
s.nd
;I
principal
sa . .l::"r~e
2,n;')~(:~t2
C31~1.urr..nota3s-;'um.,::odJ.l1.m.and
t"""
.'
,
nea:cly every tissue
tee r. i'! ; P 110 S P .h a -c 8 0 r
·the
!:')dy i:::; :J.Dtim::'T.~~l~.
:.;c;rmGred
].'lith
ktd~;e:y-~·.~ ~:.:i.1'.: t()-dJ<';~:;L:;: }.~,xtf)nt from
and pot8ss~ur
bod Y
oC·~lJ.r
furmance
L'Je bO(lY
is tal,erJ
...1. !1.L18,11amount
is
the
r'U!;1
111l:1.~.·,~8k.irl~,
the muse1.r3s
7lLe
uaDti.t.y
Cal{"::'um fluJ::-tde.!
~,n ;[;1;:),;·_3
traces
,jrink
Dh:::.;scl'ICl'l.6'::
_.
~lra found
in tht
Jones and
S t-c:
i n V' e r- y 1 ;J. [ e; c:. q iJ 8. n t :i. t Y, and 1s
IJ6.!"dness
':)j}
"r.h:;.ch t.he proper
per.'
EC,j"ie
depends.
casC:'Sj3.S
..
\';1
;.
'l\h~-" l):nOSp~~[:,~.e
org
ao
an
1.1,
g8:18ri~1
tho
org an i c
or
:::dl8.pr:'l
3 ....
"1 +c.~
.
rOtassiurn
and
be
ee~'j
o
s
the alkalini1:y' c)f
d
soft
i um
emov
d by
e
p
h
o
i ds
w.i t
rio
tne
with
ut
destr'tT-y-ing
i.t~
rri-
f}.exible~
7rrtn-;
a t es
a
aft eF-..UH: rf}movAJ~ce-r-
tOlJg119Hnd
s
p.ho
~nc6rporated
intimately
r
the \Qon(~l whi,:h
left
~.~~~ 118
is
~i=:~.clum
()f.
ITiiHrli,but
the
c
ar-bona
t es
i
ma i n
ta i.n
bloodo
th~
in bones an~ teeth,brit
in mUch
sn:~a.llt"r ouant rtv ;:!'12n ·::r.8. os p hates Lt iE found i.no tn er p ar t s ,
'l'he S;-()EtlJ. c onc r e troris of the lSixinterIl81
eer(Otoliths-f
CiT::,;) p ompo s
eJ ot~ 01-ystslline
~a2ciJm0carbonate,and
are the only inor~anic
. cry [3 t ::1 },. ~? 2 X t ~':;
t i r~g 8.. E.; S '1.1 G h l., nth
e ~=)0 dy' o
Po t as s i. ~lrn:- ;-~:'0 d t ur» an d, c 1:·:L_L
c i 'urn s 1..11
[J ria t (~S
a.r e f o un c in
srna,ll 8.n:-01J11 t·
In mOG~ o~· :i16 suli,Js aD~i flui.ds of the body·.PotasSit1ffi aci,i
S Lll ph ate
:i. s feu n dec· [j IS1, t; r:,:"j
iT 1 t h
ph c; no 1 s j, nth 9 e x c ret ion e •
Sili·;:cn
o c o ur s 0.11 n;_;,nu, cO ijU8Qt.lty
i.n the lJrine~8.nd
the blodd.
rl"t],(·f::S
oC it l·l£:t\·~e !·)Pf-)~~ f()t ..~:n ·;:i-l S()i -;.r: bones? hair and other
pa,rts.
Calcium
oarbonate
o~curs
«
'J
Ih'.()N~ ~\fl~t}::~fJ;;;t)eci[3~l
c,r the
maiter
withtl~8
Per~-oxide
tiOll
troll
~:.lct::,~e :f
bJ.()(')..i~')f
an
vrhlC1'l
:i.8 in
el~eount
the colorillg
be given
in connec:-
11{ar:noglobtn~i
will
b~l0Cd~
:~~:r0:C1is
qLlf.llltities
in the
ir! J..YTrpll and
C,h,y-let
c.l~)\.}:i-j~.~·n.~)()f' ~~eru;'!li' fj.bi"iIi
1.·.i.1G,;~1.~.i.,~~k·
~~n.d cfth.?:l"'~.
rluid~; ;6..rld a salt
of iroll prot!8,bly
a ptloS;Il:-i2.te~ezJ.s-:~8
j.n tb~ --·htlir,bla(~k
pigIIlent
alld
~l.stles
Df
or
,.oc>nes)r:nl"ls.c.les
found
D.nd
J.n V6r.y
sITJal1
t'iss,lJ6sljand
O'Lflb't"'
j
lead are prObably merely 13-ooiin minute quantities
qith
the fo
I\}\1::-Dl:~itc1,L;a:)ga!]PSf':1?e':)PF'er~8nd
dental
food"end
,
deuosited
beigB
Blo~ent3?whi0h
;')ot exr,',rE)ted
in some
neccs:::::::xy'
ps:::'t·"
depositorl
in ttlB
In
at
feces
are
absorbs
..i a.nd
tissue
or organ~of
whioh however
they form no
th ?
~:;Ctrr,e mar/it-er
A.r'ofmi9~being
absorbcd,may
be
the
wit.h
the
t
li~er' and
'j'td.<::; C1C1.:·:;E::l:ti(,~~tlon
a. gro~.:p~~~rlgof
of the bo dv"
taken
':')1'lCS
~78,rtolJ.~~
ott)er parts~
~~·rthe
(:(Iust::'luonts
H~at~~:.a,7c.s
,
or
c()r.)stitutin,g
-
1.1'1e t;ooy,is
tl1e
food
2.1so
nt~J.fr8
re~tly frol~ ~'pg6table IDatt0!'~r'erld6rs it 0b?ious the.! tile saUlS
elements
take part i~ the formB~i0n
of animal
food,withoUt
regard
to
tl,e
DL1,tlJrc
a{
t.he
Lt"(~:t~nc:1."
F,)CD~·~,,)re thc)''3e (~U():JCDi~ices?which
when
te.kcm
into
the
tJUdYt
have no ijljm~loU2
action C):l the org<:111is.m?wtnc:h 881""e as a sour-ce
of 8nergYji:1l-1~:
Gen replf:1,':"::
those
cOiJstituents
of theE body that
have b(;e"'l
CG;J~:'::rj(3d tr: :{':1,t.·,:.~,olisrn~{)r C8.11 preveni.. 01- diminish
the
C 0~·rl·~'~~UJ£r'ttic i; () f S l"J. C" t1 ;'~c·~Il.\~,"t 1. ·t. U e '0 t s
'l',tlf:' nitrt;'t{'3C:UU::
[!Y\'J
~:'s:r'\il:'"p p~~~j_ms.r·t.ly to
relJair
the
wasted
t-.i~.~~::ue~~.,).:,!:")ii
::.~~s·t.hn ·::ur:.·'r.l:~_~"_l"CF..~fIi·1CJ1.J~,: ill t,ile :ask of ~~ust~?1.iriii)bf
Co
animt"l haeat,and
voluntary
and involuntary
m<.Jt~on.
Tb e inorganic
food products
a1'8
usually
taken
Lnt o the
wlth urgBni~ material,or
dissolved
ln drinking
water.
prOxlmate
princi;les
are those foud products
WhICh
through
the animal body apparentlY
unaltered.
Ml.lk c on t a i ns all
the DG~e::;sary
o r g an i c and
inorganIc
body
,!!n
xe o
0BSS
o ons
t
it
«
uents
of human food.
Cows milk has alaimed
the aLtention
of investigators
to a
greE'ter extent t hen any o t ne r animal sUbstance'l"he rr e qu e n t ')~purtunity for of making analyses,Bnd
the plentJfUl
su}ply of material~have
contrihutad
to make the results of the study Vdr}
complete.A~cordinglY
a study of t~B composition
and cunstitllonts
of milk wlll lead TO B ~nowledge
of many of the important
r~ets
PhYSlolo~ical
chemleal
interBHt.
Cows milk like all
ot~ers
is an emulSl0n,~onsisting
of very
finely dlV~d8d fat,suspanderl
in a solution
of proteids,mllk-su~ar,
and s a Lt s
Lt
is an opaquej,vhite
or yellowish
White lHllJid,in
thin
1 a J e r s b 1 u e is 1;1 wh i t e , has ai' fa in t ()d0 r 8 n d H In 11 d , s 1 i is [1 t 1 y s \-/~~
et
t ast e vt'he sp.g.of
milk is ";etvreen lO'~H,and
lO:i5.
'r'ne color
of mi l.k is due to t he suspended
fat globules
and to
of
v
r
the
dissolved
casein.
Sxperiment
I.Rxamine a drop of milk under the mlcroscope.
Notlce
the varIOUS sized globules
~resent.-Com~are
the microscopIC
appearance
of whole rni Lk skim m il k and colostrum
milk.
The raa~tlon
of ~erfectlY
fresh milk l~ general~Y
amphoteric,due
to the presence
if mono and di calcium
phusuhate.When
exposed
to
the air,milk
gradually
becomes more and more acid,because
of the
conversion
of milk s,]~ar into laotic acid,ty micro-organIsms.
~xper.~!-rest
the reaction
of perfectly
fresh mIlk WIth sensitive
litmus papAr.-Also
some milk that has ex~osed
tothe alr for a day
or
t IVO.
Exp.3.-Boil
same fresh milk in a test tubeand note thatvfresh
emphoteric
milk will not coagulate
,but forms a s~um,whi~h
reforms
after removel.
Allow 30me milk to stand a few days,then
bOll,-a cuagulum
IS
formed.
If milk is sterilized
by heat,and
contact with bacteria
~revented
the formation
of lactic acid does not take place.
The addition
or various
antlseptics,salicylia
ac~d,borax,eta.will
prevent
the 'formation of acid for some t rrne.
F',Xp.l±-'l'O a r ew c v c v o t' milk add a m,ixture of equal
parts of
tincture
of guiacum and ozonized
oil of turpentine:-a
blue color
is formed.Repeat
with boiled milk
Plac~
400 c.o.of
milk in a lar~e
beaker
and dIlute with
&
liter
of water;add
~radually,starring
slo~ly,enou~h
a~atic acid,
to p r e c i.p i t a t e the
oa s e i n an d Le av e the
llCju:i(1 o e tve e n the
fLs k es
~ha flakes of ~asein carry the fat down with thAm. {an excess of
a c i d is to be av o i rte da lAl Low tr.e c o ag u Lum to fall
to the
bar torn of
tho v e.s s e Lr p o ur off the s up e rna t en t !,iquld.t'11ter
first throlJii,h
rnu
muslin
finally
thr0ugh
a t'old~d
(lIter
pe~er.
IPlltrate
A.) Pour
«n the ooag cLum rn the bt3aker,f)OO
(~.t;.0t'
Ni-lter,stJr
~antlY
an o
a110"'1 to s e t t Le sRernov e the
s up e r nat an t 1 )·41.:1(1 as c ornp Le t e Ly as
p os s t o j e by de"8nl8tion.!o
remo'/e
mos t «r the
,,'<31.";r r om tn e
eoae~ulurn,press
b~tween
f"{)lds
of (try Li.n on , t h e n tJlc(}s
(he (}(I.Oit!,Jlllrn
in a dry murtar and ~rlnd tt WIth fOOe~c.absolute
alcohol.~llow
to s t nn d half
an hOdr
In
»on t ao t w r.t n ~he 3,1IJvhol,then
flilf)!"
thro~dh
8 dry filtr ~aper
and collect
the fIltrate
in an Rvaporat i ng dish.'1'o
separate
t r.e c a s e r n anu the
fat
;!ress
t r e (H,'R,.~ulum
between folds of Ilnen and ~ork It In a dry ~00~.a.rla3k ~l!h [00
c s c •. jl:her.;~llov(
\0
s t an d 24h()~lrs
;ll~'ltatl.ng
oCl,.}l1s1onally;fJlt·:q'
t hr c ug n a dry
r i Lt o r p8.iJer,anrl
i'/8,sh w.i t h e rne r s Un it o t h a f:i:noru3.1
and a l c ob o Li c rt Lt r a.teaj e.l.Low the
ot h e r to evaporate
SpUnlan'c.'t}'lS1¥'
and remove t ne last
t r ac es of a Lc oh oI an d e t r.e r by eV8fJuratiD;j
over
a "later
t:ath. f~utter
faT.. )tJla,~e
t h e aas em
in :;; rno'r t e.r a.n d
rut
with a ~Astle
untIl
the ether
~1/Bporates
and B dry
powder
1S
j
obtElne,1.
Plao~
th
r r e e rlftm~
e e l e ar
t.o
rlair
filtrate
its
A, in
\I'oluM~.'I'r,n
a SaU(lAp~"!n
'"r>ms.lnu1g
~rH.i
boi
prc,r'O;ids
I
,WW2
0Y',~r
a
<cv·,:,~C;6~;U-
1a. u!d.
• (;olle,:1
on a filter.lvash
','(j,lt'
'....
a.tt~r flnd dr', With al(~ot",(jl 8ntl ,O"Tt'
er,as
dlrelHed
for ~asaln.·fhe
fllt.rate
lSDJl~ filrlher
evaporay~~d
until"the
deposition
of cf\l~ium !Jhas:Jhau~
r,~2i;ses t1um}-iinH,(,r
;1Dll1
the
volume is reduoe{1
to 75".c.,Allow
to ~ool,filrr?,r
and'VAsh
t.h""
calcium
~hosphate WIth w8ter,~va~orBt9
the filtrat.e
over
the w~ter
bath,to
rh~ consisten,}y
eT e d 2!fJ os t ted.
of
syrnp.On
coo IJ. 11;,i; (~rystals
ur
milksugar
GO MlIJ1ft:f
D".1,8 Li<V,fT\J.
a. XBntho~rotele
Reaction.-Heat
a small
quantIty
uf ~lDumin
with
concentrated
nltrlc
a<:id,-Che
alb'JlnHl
ur the sulution
turns
yellow.Allow
t~ aool and add an excess of ammonla,the
color
uhang~l
tv oran!~e.
b.~111ons
reaotion.~our
afe~ c.~.or w~t~r on a small Qllsntiry 0f
albumin.Add
t c.~.
Millona reagenr
and haat
to boiling-the
albumin turns red.
l(,Tl10n5 rea~ent is made by dlssol'leing
H10grams
of mercury
in
the same we1l!iht of lhp.nitrc
'.\.}lO,nnd
adding
to tt'le iJOQl s(Jlulion
two volump,s of watnr.
'8<.11 a small quantity
or 81bumln
in ~ v:tttnlb,-" '..tth ~bsolutd
aloohul.fI'ilter,~'!~:;h
vtith al~ohul
'.hen \\ilT.h ,Ht'h:r,t',ndus~1 t"e
meldl isl j'0r th~ following
tests_
a
small -quant'lty
of albmn'il1 by "ttotrtng
witn 1fi-a-c.i..al ..
aoetia.- Allow to aool and holding the test tube in an inolined
position
allow ~ c.c. of concentrated sulphuric acid to flow
down its side. A violet
or purple color deveLop es Where the two
liquids meet. ( Adamkiewiazs reaction.)
d.- To some dry albumin add concentrated h)drochlorlc
a~ld.-~he
albumin turns deep blue,-on standing the color fades. ( Llebarmans
.e,..... Df ss o Lv e
reaction ...
)
e.-Detecti.on of n i r r og en in albumin."", P1,,\)(: a small
Quantity
if dry~. albumin
in R test tUba with 8 or 10 times Its
weight of sOda-lirnc,snd h8at the mixture over a fres flame.
Ammon ia is iSiven off
and GAD be identlfied
o v its odor/Uts ac t Lon
on litmus paper ~n copper sulphate paper,and the fumes whioh it
forms with hydrochloric aaid.
F.-'J'lv, La.se ai gn e t es t s v-P'La ce 8 small quantity of albumin in a dry
test tUb8~ad~ a bright Uhip of SodluID,and heat carefully over a
free flame, Imser26 the end of the tUbe WhIle it is still hot in a
teaker contaIning
TO c.c. water. FIlter and to the filtrate aad a
rew drr-'I<:',)f
f6rr.ic
c hLo r i de and fsrrous s uj pna t e s Hea t nearly to
boil~n~ 2nd eftGr coolj.ng aCIdify wlth hydrochloric aOld.-A precipitate of Pruss ian Blue is formed.
g.-' DC::>:)0:::;.on of G'J,lpl'iur
in albu!1nn.-Mix a small quan t i t y o t
albumin wlth several p~rts
of oxidation mIxture (~otasslum nitrate
and sodium carbonate)
and heat In a dry test tUbeuntil all organic matter is burned away.Whlle
stlll hot immerso the tUbe in 10
XI c.o. of water contained in a small beaker. ACIdify with hydrochloric acid1filter and to the cle8r filtrate add a few, drops of
barium chloride,-a
precipitate is formed at on~e,or will a~~~ar on
....
'
standlng~
I
n.-.Detestion
of reduced
s ujpnur
s
»
'1'0 a
r ew
ide
add a drop or two of lead aaetate.Place
al bumrn in the sol ut ion and boil. "he mixture
the formation of lead sulphide.
2.
,,,.~.of s o dium h)drox
a small quantity
turns de,rtl b ecaus e of .
'f
~
GASRTN.
)
'1'0further free the casein from fat ,mix half of i twi th 125 c. ~
of water and place 1n a mortar. Add drop by drop (stirring withKK ~
a pestle)
a one per cent.solution
if caustic soda untilthe great~
er part of the casein is d is soLv e dst'he alkali
rr;ustbe added sloWl~
and the solution must not remain alkaline after thoroughly
stirring.Pres the soll~tion from fat andsus~ended matter by filtration
repeating the filtr8tion
until an ap~roximately
clear
filtrate is
obtained. Acidify very faintly wlth aaetic acid and wash the ~r8-·
cipitate
withwater by decantation.Flnally
filter and wash.After
drying the cBsein with alcohol and ether,repeat the react loriS
given tInder coagulated albumin.Reserve
eD*gh ansein for the fol-,
lowing testsv
•
a. IW.itate
w i th '1'1,.1[81" that has been rnade alkaline
with sor rum
carbonato.-The
casein
dlssolves.-Aoidify
the solution with acetic
acto"
bo
ed
Gri~~
.
~ 3rnall DITOunt
of
easeln
in a
mortar
with freshly
prepar
calCIUm
Hepeae
s
and aCIdIfy the filtrato
W:i.th J. 1:"'1" \"21 pr, inlJlscc:
Cif
carbonate.Fllyer
the
exp(-)rlrnent
c • - Mix
idation
a
qua TJ t 1. ty (j r ens e 1 n lV:1 t h S i:1V':; r " 1 ,J E' r t s 0 r o x _
fUse
as dir,c;,:t8d
Linder d,-~J:i:1:~:i(Jn of ::;Ulp,Fd'
In
S rna 11
mixture
WIth acetic
a~id
c aI ..
>i:-,ri: ,;ar.b.
and
albumin, IlSlng ni tr",
lnstep.d
of hydr0,;hlori"
aCl", tor- ""'''ttY,n"
the solution.
Poor a small qUADTIty
mt' thIs
SLIUtlon
Into
an 0X0ess
of ammonIum mc!ly"bdate
and ('/':,Ttj·).A ',';"'lln\,,.
~;J'-. ".·",·~l'.j,;,.L·,.·_.~.,"'.,
....
"."
.. ,'"f
h'S"
'-"''Y''
lYbdate
of ernr,10nium
3 BUT','!':P ?t.,'l'.
Pla{:o
::-J
C.c.of
o r'
i,~r'8ms
in:5
is
form~~d.
vessel
~r",
J.n
.. _
"
roOe•
8.
"._
G.
,
,<l.·L'
-
'"'
Ler
~_).!.'.'.)
_...
flCi:3K 1:1";;1
i ntc
t ....
f.' nl"·'>.l·t"-~,.·l·t'·I·~·l"-··"'\'
ii,
the
.~
pc;tssn
l:aUSCJe;
Wat8r,i'our
..
....
t'
il.vL ..Jl ....
;-
,11Sso1ve
"'1'
.ne
lU
i..i
.
r~;:iSi{,
b~;h
~i:jd
:~ont8.lnedtho
1'1')1 1>/1 th tD(~. ;~.,(::f e.l';ohUl
,,:r'<1
the WaShings
to the ~ontdmt3 or the flask.H at tha mixture on 3
water
bAth
un t ij a drop of 1{ is found
t o be~ '~omplet:;}
.sultit>ll
rrr
"~I
a t (0) r , i n d .i ,;a tit) g e ()mpIe t e sap en 1 fie 8. t ion. - 1;'] h ,Jr) t 1"1,:, 5':J,::;()': 1 f J ;; d t 1() n
is complete
pour the materIal
~nt() an evaoOratln0
Ul~h An,: ~V0~0ratE' the aleohol
over
the
Water baCh.When
~hO OdO'~:ot'nJ';vt',:.JI
1~
no
n
lorger
l"ppar';1 t,add
:jO r:.G.uf'
diLt2t~.3 SUliJh;:;rJ..e
Ct\;i'l.f;y
tl'J.s
crc!:tt}
mept
the
ss! ts or
the
fatty
p..::ids
21'H dn,,~
08(>.;
':'\I'd :JLli;.T,.J'::q.,: 1(1,
can bn re60~niz0d
by its 01or.
''i!'1tch
4.-CAlfGltYM
PffC)SPtfA1'R.
DiSsolve
aci d.
e.!-fI'O
nilri~
t'he
ex;~,,;::;s of
acid
solution
an
Make
a
8bout
and
wc;rrn~-2
eold
yello,\-'
..lU('.:
i30C;"~.dj
a sinall
'::JI('I;;
[..J01:T.iuTi of
'if
lJr')G.l,)itD'C:;
of
Ine
t,ile
a:.~etl';:
aW!liOOX8,1-
5 MII,KSU()AH.
Decant the motber llquor
S5 far as pussiblo
B~d dry the crystals
of mIlk sugar nlth;c~r on rtlter
pat~H:;r or on lJ()('OllS
pLji.'-;s.!-"or
i.J;:rlfic~tion,dlSSolve
the
sugar
In
about
TOO
C.C.
uf
Water
and
B.ld
a
11
U.
'l!'>
.•
,,'•.
'
:"
~
..
'tt1f- .1 \"
~ha""'()"'l t·I'.~~
h.a.at t'u" b"I'll'nJ
and
t-·'·ll~lr
"-""'11(~
_hJ-Gt.~;V-[),;..J-urr::.t.:)
on
the watflr
bAth
to ;>,;"j .::.c.G.nd 8.1101'1 to :::;(;:;.nd J.n a ,~oul til;:;c:e for
84
.'<J
,',
hours.Colleot
a.,-A small
quantit,Yheat""d
fIltratIon
and dry 00 filtAr
paper.
on a. {JlatlnurJj
fpil
Turns
b,l"OVofn,til" ....elp
as a caramel
odor,carbonizes
Bn1
the
aSh.
14'01' the
(T)
fOllOWing
2 grams
("2) T08.C.
erYstals
by
reA,~ion5,rnaI(,J
of mIlk sugar,dissolve(j
of (T) diluted WIth
fInally
two
90
burns,leBvlng
verj
little
,sol!.it.i.ons.
in TOO G.C.
Water.
\1!.'nr-3r.
C.c.
b.PF.HLT~Tns 'fRS'i'. 'j'O;;' C.(~. illixedf"ehlings
SOll)tior:
add:~
G.C.
of mater
and boil.
Alta 8 few drops of SOlUtIon
(T)
and ,~ont.lf:ue t()
boil
.The ;,rello\'1 prolnpitate
(Gl;OH1/'trst
rormi'~(l eiE,O:,::<;;s
un boili. i5
n
to red ~uprous oxide.
c.
of
first
a~
marked
MOOFiF.S'l'ES'J'.fJ~r cent.·:austic
yellow,
after
''-'0 :"i e.c.
potash
of
solut.ion
solution
and
(1)
2.<1<1,b.n
boj_l.'\!it~
il
SJ:>uOnJlhu
n.1tri.,::
9.;,~i(1 SUlUtIun
1:11kalln-:;; 'lJ.th
a,~I(j,,1nd add 8 tow' drUtlS
uf alFU.c;onll:m
of calcIum oxalate.
port<i;.iDn
nlfi,a.::idify t','j_th
ate.Apreci~ltata
If)
'3,mmonlum molYbdata
phosphomolYbJate.
b.-.
P,hlsphr,ru
Gal,~i.uF;
oqual 'Jo!u
me
~Oll1tluD
lurns
then brown,and
de"'(~lopes a carar.rF~l (ldor:;h:i,'.:h
the addition of SUlphuric
acid.
1S ITerii'
d~ pismuth test. Make 5 c.c.ur solution (I) strongly alkallne
wil'h sodium carbonate. Aad a small quantity of bismuth sUbnitrate
and boil.-A gray or~
black precipitate
is formed.
e.- ~o 5 G.C.solution
(1) add a few drops of sodium carbonate ad
Rnd a little freshly prepared solution of potassIum ferricyanide.On heating a colorless solution results,due to the conversIon of p
potassium ferrieyanide into potassium
ferrocyanide.
b.- '1'0 5 C.C.of silver nitrate add enough dilute ammonia to dis
solve the precipitate fIrst formed,and in addItion one drup of
caustic soda.To this solution add a faw drops of solution (1) and
heat.- Amirror of metallic SlIver will be depOSIted.
g.- Indigo test -To a portion of SolUtlon (2) add enough Indigo
carmine to produce a blue color,and make alkaline with sodium carb
onate9- On heating the blue solution turns successively red,yellow
and under favorable conditions becomes aolorlesseCool a portion of
the DolorlBss solution and pour it several tilles from one test XMR
·ube into another;-the colors are reproduced in reverse order •
._~iha following tests will dIstinguIsh milk sugar frum grape sugs.
·...Lch responds to all the
foregoing tests.
Shake 30 C.C.of solution (1) with a small piece uf yeast,flll
into a sma~l fermentation tUbe,and alloW ~o stand 1n a warm plaae
for 24 hoyrs.Make a similar test with a 2 per cent.solutlon of
grape sugar.The gas whieh collects in the upper part of rhe tUbe
is carbon dioxode,and is a measure of the extent of fermentation.
Milk sugar does not ferment with yeast.
i.- Put 5 grams of milk sugar in a 100 G.G.flask with 15 C.G.din
lute nitric acid and 5 C.C.concentrated nitric acid.Heat with a
free flame,under the hood,until a violent reaotion takes place.Remove the flame and alloW to stand 24 hours. Pour off the nitrIC
acid from the crystals of mucic ao rd wh i cn have been depos i t ed ,
Wash with water by deeantation,filter
and dry on filter paper.Dissolve a small qlilantityof mucic acid in an exsess of ammonia and
evaporate to dryness on a water bath. (Hood.) Heat a portion of the
reSidue in a dry test tUbe-pyrrol IS formed by the decomposition ~
of the ammonium mu ca t e and can be de t ee t ed by t1;)e
red color wn t cn
it imparts to a pine stick mOistened with concentrated hydrochloric acid and hel(1 In the mouth of the t ub e s Re pea t t r.e exp er arnent xi:
with grape sugar •
J. Weigh accurately
3 grams of pure mIlk sugar,dissolve In
water and make up the solution to too C.G.in a measuring flask.
After thoroughly miXing fill a 2 deCImeter polarimeter tUbe WIth
the solution taking a~re that no air bUbbles are introduced. Find
the zero of the instrument,insert
the tUbe and turn the analyzer
to the right until the position of extinction is reB~her.Read the
.B8B~angle and calCUlate th? §y~cific rotati()n from the formUla
-r-:
.A D.
=-A..Y
I. W
A D.represents
the angle of specIfic
rotation,
V
is the volume of the solution under exarnin Tlon,
;1'1
r ep r es erits the
'!f:df£ht
of material
in V,
L
represents
the length
of the p o La r rme t e r t uo e ,
a
represents
the obSBrved
angle.
Calculate
the weIght
of mBter~al
In the solution ooothe assumption
that the s p ee Lf ac rotation
01' rm Lk sugar
IS .);6.:):\
P PIece exaotly
50 C.C.of the mIlk sU~8r solutIon
In a Plorenue flask with abollt to C.C.d11ute
hydrochlor1c
aoid.Boll ~entlY
r or ~() mi.nu t es n ear-Ly n eu t r aLa ze the a ci d w i t r, !J(Jifdsred
s o da urn .car
bonet8,trBnsfer
to a 100 C.G.measuring
flask
and after coolln~ fl1
to the mark with water.PolarJ.ze
the solutIon
In a ~ dm.tub~,multiply the observed an~le by two and aompare w~th the rotationObserved before hydrolYsis.
j
~,O'rIO"J
ON MU,K.
Of" kf':NNIN
Prepar e a solution
of rennin
b y a i a so Lvorig .lgram' of t h e commercial
~reparatlon
in 100 G.G.of water, and filter th~ solutIon.
a. Pre~aration
of paracasein.
Plae8 100 C.G.of milk In a beakor,
add f~~~XMX
TO C.G.of the rennin sOlution.Mix
thoroughly
and alloW
to stand at 40 C.for a short tlme.A coagulum
cunsisting
of fat and
casein soon f'o rrns an d an a qu eous fluid (the whe,v,,'ihi'~h con t a rn s
prot ei d ,milk sugar and sal ts;) .1Sf.Jr~3SS ed out •'rh~s a c t i ori 13 s ugg es t rv e of lhe clotting'
of the blood.'j'he ce.se i n ob t a i.ne d b" c~oagulationwith
rennin is oalled pare-~asein
and differs from the matet
ial prAClpitat~d
from mIlk by aeetie aaid,
~llow the coagulated
milk to stand 24 huurssnJ
de~ant theXKke
Whey as cornp Let e Ly as possiblf:.Stlr
t h e co,<iI3ulum wr t n wat or In a I!l
mortar,filter
and wash. Press the p a r a ca s e an dryvith
linen, and
treat a small portion of the material
with absolute
aldohol and
ether as under casein Use thIs portion to make tests a and b ,~lva
en for cas e i n ,
b. Heat a por tron of the wh ey to bOlling,-n
voluminous
eo ag ul.um
IS formed.
a. WIth a portion
of the ~hey make the guiacum-turpentine
test.
d: Warm 2S C.G.of milk
to 40 C.add 2 C.G.of a Tper cent solution
of arnmon i um oxalate and th en 5G.G.rennin
s o Lu t Lonv S'tLr and allow
te stand.-No
alot IS formed.When
convln~ed
that ;oB~uIBtionvill
nc t occur 'Under these c i r cums t an c es ci v i o e the WIXLm e
into t wo
purtlons.To
the first add an excess of calcium chlorlde.Boil
the
s e eon d p or r i o n and add oal c i um ch Lo r t d e .- The iJresenae
of c a l c a um
salts is essential
for the pre~lpltat'on
of paracasein,but
not for
the action of renn~n.Cumpare
this reaetion
With
the clotting
of
blood.
~ Influence
of acids and alkalies on rennin coagUlation.
Tnto each or three t es t t ub es A_R,andG,placo
TO G.G.cnllk.'l'o
A
add JOdrops 0 f • r.per oen t , ny dr o ch Lo r Lc a c i d and to Ba fEF'1 dr op s of
sodium carbonate.Place lOC.a.rennin solut10n in each tUbe,warrn to
40 C.a,nd note the order i n w'hieh eo ag uLa t Lo n takes plal}e.
j
j
II
4
D~'
... ~
1: ~~T
J~~
(;
J. •• \.r
.1.
'.
.....
fI,"
1~
"J""
'.
T (~
"';
(j'~'
_.,.,'~
!~"
.....-••
1,\'1 I' C ,~ (',~R,"r ~\T Ci r.L ,ri"', 1\1 ~l
t ~ ~!t.),.;4~·~i1
}... "t" ' ,
~
....
.r ~
S 2, t u r 8 t ;.; ~() () r: r, c f !e:li Irt
(I () LT I" ~. ,...~c- \,
j"
'r.. a\, ..r- :i .\)..
1"'1
.~;;••";:'-~'
'-'-'..,.
'"
t
Cl
<"J~
~""'f
·'\.J·'f.J
;;::"I.,l .......
-
• -
,,:,,:tti'l
0
U
1.-
•.,
~
-',.
~
,
1'':11
D.'\r'r' ',.'\\!1' l'~F:. l""
,..,."
l ~V
'. 1"'.'1\1'..
"1
f i ':'":;.
y !) n·.'1\i,y~~(~(9
J' ".J..1 t ....•
':·1"
.'
;I,
•
r:
'1 '1' "j
,t' l•• ,!..}
I"l'"
,,,.'J,lf...
l' .",.':
";' lfCLu.J
r r·~l.:;
"I:;
(T:-......1t'~;,f.{••.l~'/J~
",J', "..,.
...
'" _
'"' ~(
:,n:1 ph
J'tJ<: ~~. F; (;~llI(\'j)
•t 'f":.1", ..\.J '1
,..,Po
,,", ••"
'c'
1.' , .. c'' 'L"
.~.
",-i
fJ."
t~
<' ('i "-
a: ('\-:.
~...,
t-'- .._j..
i. UTI] S 111 C'h~.t0; 'i'h') f; t l'l 'I L F;~t (j.( (: ,': (',n'::1 L! L [J c\ f (:2c;;,:; c:;:L .:"g' r:m
I.:~?' < 18.0~· eli; J. ob t,J. i:Li.' B 0 i:1 the
r j. J. t rat ~" ,~I.e,:j:l ;!>,:~bi.:!m :i.$ r:'OJ,g1.1'.l., a t 0i~.
F J.::w (' t h J n;c.<~~lT p)' e '2' ::'1":i 'I: E:'-',::;:; :~ {l (;;, J'J':~::'::' t a!' f..n d !~{!'
.i11:i
:Vt; '\'{,i!: n ;.;;0)
io;::,
fat
t
0
;!lS,s!l
(:iE
0
O.Oow'~:it0'J:'.A.tt~o'~~
s:'8.r:,d:i:-:,g
hours
2;1,
tion uf egsai~6~8n.~ont~inin~
~_-
&s
... ..;
M
idfq.,'rd
j,t
ac_,.{~ -.
~
(1t
-,
.
,,<.',13
.1
of"
e ;<.\1
~,. _.~
....,
-', P. 'y"'~i,15. ,t-'·.;Cc,.
;......; 't '" t ,....
I
Pr;j,ng.,·!-nJ<)'Cl'"or
pr Qijce-dLrig
the
m~t0ri~1
o,porti.on
j,l~ inil]~~'t'u
ti:.tratc
if, 2. sol~'~
in the aame condition
fil;;~H'li·
..Tho
this
~
the
f;iC1l-utic.n
[-~d(( e.0e~ic
,(,'10
j~r~I~J~,(~t)0:"
p(>l·~j.O:l
rd::'k
1;)xp0ri;:,::~rl'r.
l~~Pi~,G:t.~
on
clIO
co
1AC'l'TO
/C
';'0
~
".
,...
grt:t;';:1,;:.
Ok
";'",..".
AC1D
g:::'E,,;~l~';of
\:I~n::'
fI' .
';..;:-;
(",.3..r).,),).(l}~'"'J~.''J\'''
" ..-.......
,~l,""t:.
:'I
~
r,'g.r~M2~N·;·J\'tION,y
slliJ,tl'"
j".'~
.'"'
'"'"
1,)"
(';;;'!:H~ol".od in !jO(; G,Cill,if'
.,I/fi'tl;)l',r;l..J,110
CI.\.J.. .. S"''i,·1"'~ w",.,.~.,<:,IJ.t~i
'\r t...... r1 I:'
C!J
":1
~'"L
", t .,.j..,1, Y
'vYLl,.:.
. .J. /1~ UL"'.L,
'J
{..
"Y}
~ ...
o-j -/
oI~
<OJ
or old ohG~~0.Allcw th~ miXlurs to stand in s thormostat at kG O~
for 8 01' 7CHr~J:;~I})\t.
ti:H', :~:G:l of' t.hifol tl1i;';;: JH'm.t t.o bf.)j.:,1.ng ;<mrl filter
VlhiJ.f) 1.h'c, ~d(';L:ir." J.::;. !l..J·~" :;~tH~ fj'hp. fi,1r:r;,1te
ot'J ,'00.1.:\:(1/'-1' 'will
cl61H:e:.it
QrYsta,ls ~f ~irlc la0tat&o
H(;(;l',i",-::L311izB
cho c,alt
under the
the c~Y8tel~
the mut/lor liquor
.
of
frmr~
end dry ~AtW6en
or
[fi~;'I)'I'/6
by
[Il:;,lr
fJUs.:d.nlJ
tho
ha.g b~;en p:rEHdpitatede!"iIt0);,
until
aU. hY"d:t·ogt.Hl 1tulp.tlida
in e sepuratin~
funnel
add
and
filter
tho app8~'('Fml;!&
the ,rY8tQ~S
from
iliJtic~
paper.When
,
mo16.-:mlEHI
~at·c()""lfl';'1ti{.i
tlWGal
or
llcld,contains
,zinc.: la~tate
HS ge.s into
Wl:lt":H"
microsaopeeSepal'aT6
in the air Ule sb.l't contai.tls
:hzp..tian~WhiJ.f')
the
7.ine
3aIt
i~' WHH ,1ul'msntaUon laetic
~)·YJJtaLJ.:lzation"
Sf~}t
hl,)t
allowed to dry
01' crYfHal'"
we.'t(:;j):'
(If:
a~id
Whio!'1
two mOlecules
.1.11\'fE1,ri!l W~tcr'
ii'J
j.som(;.r-
or water
L'\nd '~Z:I'J(:irilpczo tiie
thewarw
sol"lt :i,r)t.l un t i}, t),11. triP; Z10"
und nea;t the: l':tltl'Sti:J
1;'0 ~t wa~t)'r' bath
hft~J
tl0en
tnt,
IH>lt:.tj,on
aoid and
extract
0xf,JolJ.C;,L .Pl 11IH3
B drop of sulphuric
1f01ume
of ctilOr.F'i.ltt}):'"
":4r;~ t~\,)n;lOn of' ettlEtl'i.t
·,(,rough a dry filtel·
parH:r irlt',j a dry t'la5;k.~va'porate
the ether O'lor a Wt1,t~r bEHh Whi~h has been pl"'I~'l/iou31y fl0attH! to
boiling.AVOID TH~ NEI0HBORHOOD O~ FLAMES AnD USE A.CONDENSINn ARtWi~6
'.'l.it.lI
e,1 solutioIl
t"P~ /'7I:.,FOta.:i..
RANGEMENT.
~ermen!8tion
lRot1e 80i4 obtaibed in this way ia 8 '01orlessliqvid
solUble in all proportion! in water,otnar and alGoho1~
Urr81manns test (or laati~ acid.To an aqueou$ Dolution or lactit actd add e. fAW dNipe of e~J(ld~ UCf;:trmannfll
reagent
i 10 G, c~or
a 2 perce~t gol~~ion
or
e~rboltc a~id in watar,CO ~11~h a f~w
drops or forrid dhlorid6 ha9~b0en
added)-AlmOb
yell~w oolDr.
OtJMn 1.'I'A'J'TVF: M'?Al/1S Tn Of,' MILK"
Determinetion of speeirid S~6v1ty. Obtairi 8 sUfti~1ent
Ao
quant1~
ty ci' milk for all
the ~lH:tl,yti~
work wlli6~) rol1ov"';:I~F'in&
UJfi/ SP.gd,
by meaDe
ot a la~tomBter abd use this number for oaleulating
the
we~ght
of ltd-lIt r't'o~n the ~iwwn Yolut(j~ 'ilhi~~h :1..z UtH'7& it.., t.b~ f01,10,,-
in~ a~rA~minBrioDa.
netermination of~ totalproteid by Kjeldahls melhod of determining
nitrogen- This method IS based on tho f~ct that nltrogenou~
sUbstances
on prolOnged heating
with sulphuric
acid are aompletely
oxidized,
the nitrogen
being converted into ammont um s u l pha t e r'h i,s
oxidatiun is spoken of as the di~estion.1he ammonium sulphate
is
R.
v
then
decomposed
bjo" distillation
with
an alkali,and
the
ammonia
distilling over 1$ oolleated in a known quantity
of a standerd
acid solutIon.By
titration
with a standard alkall,tha amount of ae
i<1 left un ejiang e d is determined,
the difference
between
t h as number
Bnd the amount of SCld taken represents
the number of C.C.neutralby armncn iaj rmaj t rp l v rng t n i s by the proper
co e rri c t en t the
cuan t a t y of nitro.gen
is obt'~ined.MultiplY the ·,it.Hght of n i t r og en
b.y t·,. :37 and the ,prodtlct
wil1be
t n e ''1eight
of p r o t e i d p r e s en t .
Ized
requited.-2
gJeldBhl
dlstil1in~
dahl digestion fl.asks,H
K.]eldahl :hstilling
for buret ttol.
ApparatUs
Pea-gents requir~d.
Pure hre'l:"eurl~
burettes,~ kJel
tUbe,i'l,con deris e r s t arid
flasks,2
j
o.xlde,pure oon c en t ra t e d sJlphur-
granulat&d zlnc,B
solution uf litmus,a 33 pereent
solutIon
or l"otaMllum hydr'oxide,B.
s·tshdard
s o Lu t a on of so d i um hydroxide,a
standard
solution or sulpbU~i~ Qoid,~ ~olution of phenol-~hthalein
in al~ohol.
~ha digestion.
{HOOd.) Pl&ce 5 C.O.~ilk in a dry KJolJahl dlges
t t on flaS'k;Add 20 n.C.ofconcentrated
sUlphuri~
811<1 and about
an
gram of mercuricoxide.?lace
the flask in an in~lined
posItion
and
heat g en tLy with a free
flarne until
frothing
ee aa es s Ln case frothing is eX'ct3ssive a sm$;11'p'leiJe or parrafine
may be added.Hradtta.lly
raise
th(~ temp@ral'Gre
tint';l t'hesulfJhuril~
a·;~ld boils briskly.No
fur ttl er a t tent lot'! is :rer~uired unt i 1 t he fig, t er ial na s cec ome co Lor'>l(!ss;ari"~sult
whien
is usita.[ly ob t at n e d in ~'>'O 01" ~~'Ormnu t.e s s Af t e r
coolihg
earefully dilute
witn about
T000.C.of
water,keepln~
the
rm xtur e oooi bY ag at a t i cn under a J(~t af(~old wa ter ,
The Distilla~.l0n_
Art·anga. the distilling
a.~i)aratus.ln
the r s c e r v
e r flladeexa~'ctl:V 50C.G.Qf:"deci-normal
Slllph!jri;~
8;}id or a t s ec:uiv a Lent tit stRndard'
auj;d' -brany
stren!5th.ifl"ansfer
the mat er ra.I
from the digestiorifl'flsk1'Co
the distilling
rlask,llsirlg
t3.bOiH 100
C.C.of d'1stilled
W:atfJf" for i"1i'lSlng.Adda
fev{ drol!S
of t->henol-phtha.
lein an(1 neutralizewlth
Strong KOtI. 3t'it1~ a srtall iJl&t;e of ZInc
into tne flaslt,:rrfake
alkaline
with
:I<OH, and immedIately
attach
the
flaS'k to 'the <hstilling
apparatus.Distill
off about 1.60 C.G.of
the
liquid
And titr.te
the excess of amId in the re~eiver
WIth sod~um
hjdhoxide.From
the result
of the titratIon
oaloulate
the weIght of
nltrogen,and
mUltIplY by t>.37 for the Wei~ht of the total
proteids
Supposing
26.:3 G.e.of
<1d(~l-normt-n alkell
were neC:'3ssary to neutralize
the exoess
of a'::ld In the reoeiver,
then 50minu3
~~;).j or;J.47
lCBCld
~.O.of acid w(~t'e n8utralized
by ammonia.As
on6 G.G.or (1eCl-nol·mal
ac:id Bullaion
1S equlvalent
tu .OOI7f:Jrams of nrnmonla or .0014 of
nltrogen,the
total nitrogen
present
is ~4_'7 x .OOL: (jl' .O~->,:±ot\gram
.O~.,4!)B x o.:~7 is .224:3 grams of protAid
in r. ~:.C.()r' milk,hbout
4.b percent.
5,~::,~ {;:klJ'
~v ~~
d,~·ld
]~L!
.~~-
.~.
I{()
!)
C;r,~C,) ,:.:.'~f
p r: ,) t (-;
:~~~~
U .:
~,";],.. /::~d
:',~i.t,":,,·~;,,~,~
:J<I.J
(>~."j ':..
,~'::,7}:~h.C)
:~'o:;<" -~cfV~"it:.h
j,"11IJilel s L~,dd·.
3\:';'PjJ.'!,··~)L j.'ng
E'i:
;r:f~'(l~'je'~j ~
a
f,~·'f:·'.)': :~'l.p~~~Ft; ,;{
J"~~i(JJ:{[;\l,d
t 'i c~ ;~,:~
t~
t~j, ~~)
::0
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C,:~·:~t};:ic~,i·l~~
~C·:::~,£1[X·~:··~~"~: :id
:r
~ r:.~():~,.i;t,:t'!)r~
()1'
:;li~ p i c r i ; r~I::~~,~:'l
E~.~~~,~J
fJ~(D.l(1
or
,:;!,) I":'~i"/'~J,~~t:;::l~ ,~",:':j. (:~ r (';;3,g :f) ~'::;::-~.;.;' ~t''~~~1u ~,'~)e (3) c' C, f~ me .~~:~1J.r~i It s f :i... :,i.:.):~ ) :l'J 5~) C ,".C D j"! G
~)'~1~~
'f; t t {;::hri r" E'.~.t c:: ~JtE:"r d ; .~\)/~; :~j~}~
e: t t .~\'?:. I [) (~t' C :,,'~ [it eJ.ll f' ~'lgiJ ~.:cpr st' f;i () 7... ~Jt Lon
;\"1
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'~~(J:hlj.T~'gr:3t,~:-~'ttrG~t~3 ~3GI':;Lttor;,.:a dj.l~'!1~'.',t'~,
[;~:)I.ut:~()ri 'Ji' ·~s.'~·.!(:i~~~,},~z~
C.l' P;';-::8,r::~Bil1
/
'J ;:~ c rl :.~"
j. 't .:;."f" r) ~.~.'~n1U IS
Li.,~n :":i: ;) l'~.-~~)).
:t d (:;':;1 ;-,~:):~"1,1-:: i ,:~:,_.! .c, ,i'i pet ;:;',CI~: i ~)·Y~~f 7::; ~;:" 'to .:' (~ :V'~~~r,
i ~-':,;:
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t e '( ["I ;~, ~~; G :~.,\)
fll"" ~~P B. ;'" ,~j,t J f) [I ':)f 1~
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~J.'~ ;;, '~';:~·l:" p 1 i:,1 (l~;3' ;; oX ~~~: t I y 2 t~ C., G.) !Ii i 1 ~
i1'1 a :?~5f) (;:\C~\}tl:.:'3D~3tjr"j,t')tr tJ.,s.Gk ~~!1'S~ c.:t.··~'Ut(i 'V~,th
,~:f.5Ci O'40()dj.stiJ.10~i
i~l
~ater0Add
i UIZl
ti0n
or
sod
r ~l
}
10 C~Cofell.~.in~s~(JpP0r ~O].uti0n~aIlci ~f'Le~ m:~"~in~a~j the
dr' c ,;~j. (,l r; S c::l. ~.~'~;i ~)~J 'iJo ~~ r (1W' dr- ()'p £3 ,~.. "t £.i, or: i tr,l~j t?$ t ~;,r:g t ,L1fj
t.hE; liq'!Jid. ftft.s!t
7;'~,,·";i'l ,~jJd,(~l.t:~J.\7):~l~.Vv~it!'l8€,r!(.:.::iti"\:,e
15;'tnl1.1S
1.1,jr
l~;~
.~~"".' •.~~.
r',.lJ,;~1..l
.1).':;
!.~
r.·t .."l'!
;. ..;; 'V""
~::,,,.·,t
v.;...!.., ... ,,,.lJ,
c.'
"F?.rJ
:"\'..."~,
t'" '~'
lJo,I~'~h.dt,: ...... JC;;:.,.t
ti~' t:~
", .../:1..:..:,,,
It "'~'~·l·i
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n('h'::... ,j",.
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),"\I... ~.·J
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e a. ~
;J8Jper
h~~
~ -t ':"'"
~ r ~,
.,
....... ;,;.(,.t .. ,.... L",';;)/.
,_.' • .1.
the flas~~ to tl)G m8t~k W~t~, aietill0d wste!'~~'ilA pre~if)it~~~e shol)ld
stll,side rap1dlY?].B~ivj,1:~ a pe~f~t,tJ,y cl,0~r ~clorle8a li,qtlj.duIt i~
imr.J Or' t H~~:!
t t:~i-i~~t,
t t
.1i q'~).'j
6 ~~:~~
·t:} Or ;.
1::' .1 ~.l\~' ':..~
c' \ '..".~~.,':.lJ~~).
j~(; ~,\t (.~
f2;
t tiA. t
,a.:n ::ft- ~<C;' ;)g ,f:~ () t E: 1}{ a 1. j« !~JJ,I~~;' t, :)<J.n r'l .~.~d ,~~
d ,~,
d ,:~,
ft; :?: C ~r...~,"j~n 1:3 {:i ~j, t· :.,~(,)t t 11~' r r' c e "l p:
t;D,'t~?::l E.1J.~.:;t1;~1.f: D, ~iJ,C?,f,:
\b!:;tDrd~1i fill~l."~lg
::rl'.1J flEtc,l·: ·t·~ t~1'Z: ~nSY'k~t:1d~~ a
lit1::e ffi0r·c~0~~~r 0u,~~,prlat3~al~~ ;~0r~ to ttJ~ ~J~\,·t~al f)oint R~ain
l~~
(~'!
:,:}.
'7,,~
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'~~J
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1 ~ ~;t' '~h.1'j~" () ~J;6,~".~
::t./ rot :':~i ~!
f:~ 1,),:t 1 tJ 't"t t c;.; ~~.
t t :L.i,.; l,~t~~!;j l:'::) 'L; r .:~
l~:,!t:/ ~ :~ (1 ~~I:1. ; .. i q tA Y.. d
t t"11,'" ';:';~.2 ~l
~o}
~~Jr d r-:\"
f' j;, ~L-:;<~;~" i,tl t ~,)a d ~.~
tV' f 11,,~,~:
t.~~h·:~
t~':;~;t ,~rri!:~,~~::L"C
i <;~';~ '·"·f· :~11 ~,. r;;., r):~i,Z~dr' Jl' b'i],:i<i~:,o~ t (~,~,rl~1I;) ~~,
i'iC pr ?~·F.t~t{~{~"
l'fJ i Ii.;: ';~':~lrl~~
~':';
r' i·.~ ~Jt :t (} (~I,. ri-~{~~J.!:1U.;,." $ r~c:"'~.
n rat f11. j!~ :~~ c ',.(: ~, F Eil~~li. ~·1~~;:'1 C:) P'l,": n't
solutio~~and 1~ (J .. (:~7Gh!~nist~1At~'atG aolutio~~in'to a sui'~ebl~re~'3 F- t 1":\ r' 1 ~:'I { d ~;.~l.~;t ~~;
Vi ;~~
t 1"1 ~':~)
G
!}:~:1t J3; r- b~rl·j t;, ~3I~:J~i;, t () j:" ('J j.l i 1::;:0' r: .:\ d~l ~j.x~;.j,; 'r" 1 y
20 r;"i\~,:".~·
~h<?i ;'cilk f3iJgFir' ~~t<i.utj.()n ~H:;i ~~!ea'~
t,.:~ bot:l.ir~g as r'fi,~Ji.'HY
a~ p0~~it)~~~~006tf.l)t)a
to t~o1,1 r~~ 8i~ mi~utfl~oAil')w ·tha ~rGoipitate
11
!;\
to sUb~i,d~ a:~~ if
D.;Y1Qti.r::
~ r~ I~
",~..:",j-.J:
tot
fJ
u g ~,i.~.
"'1'
1" ~''~.i.~t.:· ::;,
......·F~.;Jtti,
,;.~.
"
'% - '~' "1 J., ~'
:~.~~}.4. •.l"i",>~~
eaeh additinn~until
t (=~":,i"n:3,.~Jf{; •.r~:"'~~~:
h el't'
fe"f
~':'~Ol)r:f~l';~
•
.
1: ., •••
',
~ .• , ..
""fJ
:~3,'f:'
..
.;,
liquid
til~
jJC~ 11.1 ':
()
i {)
rJ
~'J8.[1,
t)a~ a blu~ ~010r S~ insuftioi~~t
~) ~;~r:.~rJ ~o~~.ij
dt,:;r J ~"'l:;"1
)'\r"i:"I'>
( .... ",.' (.....
··:l.I,~;'
,.~::
a t 1(3 h
v, ......
{~
i··~"
,-o,~.
(,'7>1
..
a POil1t i8 ~~~~l'f~&~t}er3
t :'.'.
€~
li'''~!'1'f{~
"
"':1
~~",/<
[~l
"
'!~1;P~;~
~"1G.t ~Jn
'rh
..
t')~"·""·.1h
\o.
J
~"'~.~::,
..
rzt "C'r~
,.':,;:'N
, ~f,l''i.ouJv!)LtJ,.L\4t
~:. ~ r( r., r.
j,t
'~'~'\l
""~:,,~.if,\v'
ito
::;
,:,r1t :~
r~tlt)
I-,.~
i~ imp0B~ible
t J i q u. i ~ ~.;.'.
b 1 ~),t2; ~)~'
,i';t':~i ....',~~
:r "i'O,,'
..:,"~':'~--'·t"-'"~.',
:.1,
' ~ .,1
"~<,
~~i
v·,-~
'"I'
~'~'J."
-;
••,
""1
':'_..\0
;J r:.1t ~; Ff j, I 1:. ~~Xf.
~l(;'·f·\f";~
,tZ'; ..I,.
t {)
to""1·
:v 0"1
to
BI
;!;e ...,€.~&;nii!f'i
,~."
"
"~"
l~~ t!1G tj,ltr2~b in ~ t0~t tU~G ~cnt~i.~liD~{0ns Cg{Jr·~~eti~
aeid~and
a t" ~,~'r dr ~}~.:fj c~?p ('}t f:~::1 ~ 1:~:'Y~it!F' T'T;' 0 (~ ,Y't:;~
ti i ':C~l r:(~
) {7 f ()t'J·t":,i':;', '~~ i..:')~-;,
-:)
~,~
~~
f;.!1, I) c:: (t J~~j, t ~
~j
d
pr e e Ip t t at e shows
e op per' is s t ill
that
co n t a Lne d i.n t n» sol,aian.-
Assuming that this result is obtained "lith :d4 G.C.stl,;,arsolution.
And tnat no copper is round when a determination is made ~1th 2~
C.e.sugar solution;tn~n
the quantity
necessary for exaot reduction
is between theso li!nlts.
more determinations are made us rng 20 G.G. of tm x e d ~'ehlings
s oLu t aon an d boiling ea cn t rrne e xae t Ly t) nu nut es v'rwo limits
wIll
tnus be Obtained whieh differ bY one fourth a G.C. The mean I
between these lImits is taken as the vulume of mllk su~ar solution
Which will".(,!xactly'
precipitate
the copper from 20 G.G.f<·ehlin~s
solution.By
expe r rmen t 134 rm Ll i grams of rm Lk sugar nave boerrro und
to preclpitate
eBactly 20 C.C.Fenlin~s
solution,and this w8i~ht is
con t a i ne c in the v o Lurne o rs ug ar solution
used.By a simple calculation the per~entage of sugar In the ffi11k 1S found.
SlJppOSe 24. T2:) e.G.were the mean above referred t o j trren the aalculation
wlllbe
as follo\ ...
s;Z'~.l;·>'j G.C.of a T - 10 d:&.lution,repl'esent
~.4}25 G.C.of ~ure m~lk.therafore
2.4125 G.C.uf ~ure milk oontain
.l~
milk sugar,snd
1:00 C.G.of
mllk contain
'.).::>5 grams,a~oordin~
to the proportion
2.4T25:
tOO::
.t:')lj,:
x
'rwo
j
-
x - 5.55.
,
Polarimetric
det~rm1nat1on
of milk sugar. Pla~e exaotly
50 G.C.ot
milk in a too C.G.~ee.suring flask,add about 2 C.C.~~ Millons roagen t an d (i1lto the mark ....
1th water,Allo'" the pre"if;ltste to SU.b'side,filter a portion
of the solution
t r.r
ough a dry filter
p ap er
j
and
polarize.
Determination of fat. The following method dSfisnding on the rapid
se~aration
of th~ rat from milk,bY reagents anJ the use of tho
a8ntriruge,gi~es results sufficiently aeourate
for all ~ractiaa1
i:i lJr'poses.
Apparatus
pipettes
Solution
PUEel
rAquirod.-~
centr1fuge,~lth
graduated
milk
bottles,t~o
5 and T 0.G.
t. Gonslsts •
oi1,37 GG.
wood aloohol,I3 cu.
A~id hydrochlorlc,~0
cc.
SolutIon
2.eonslst1ng of sUl~hurl~ aCId
The sample should be taken from the middle of the m i Lk ang or nul'S'
ing,as the fIrst milk is r1dher and the last poorer than the 8vsraye.~ co.of the s8mpl~ is introduced into tha milk bottle by maans
or the pipette;one cc.vf solution l.is added and shaken bY hand.
Then
by ~earis of a large pIpette
solution l.lS added little
by
11ttle w1th shakIng,until"
the buttle is filled.lt is then ?laced
in t n e e ent r t rug e and ;vhirled
for one or t wo mt nut es when the fat
ris~s to the top in a olear yellowish layer,and can be read off In
direct perc~ntages.lf the should be vary rich and When examinin~
cream dilute withwster
end mBke.pr0~er corr8~tions.
Yhe great mass of the organic constituents of th~ animal body
consists of amorphous,very
complrx sUbstanoes.of
high maleo lIar
weight,eorHaining
nitrogen.'lhes~ sUbst8nces"'hl~h
Fire classed toge
t
t her
In
ca I Le d rne PRO'n~lNS;are either true
t
ds
corlies closely l'~i8tlBf:lntarShe~.thegroup a.re called !Senerically
the protein sUbstances,although
In a few cases
the term 1S used to
designate in a s~ecial sense the proteids.
An elementary analysis of a protein WIll give the followin~
average result.
c. ~O to ~r. ~er~ent
og
e
Rgr-.Up
p
h.? to 7.2
N. T'J to lB.
o. 21 to t~
H.
r
o
e
a
j
or-
•
•
•
s •• [j to 2.5
• •
The quantity of nitrogen will most often be found very close to
16 percent.,so that a mUltipli~ation of the wei~ht of nltrogen
bj a factor
betwef)n b •• )'7 and 5• .s'),0suall,Y"
oLos e to t~.2:).Nlll!Sivf.1
the weIght of the protein under examination.
No mo le cuLa r formUla has b8(1D assigned to tho proloins.'i'helrnolec
ui er \'IEu-ght has been cal~ulate(t to be aboutU)OOO.
A eons ider'!.t
a on
of the possible .ariety of stru~tural relations in so large ~
molecule explains the d1ff1culty of rindin~ a JOnstltutlonal Corrou
ula for these bodies.
General propertiese "I'he,)' are as a rUle,arnorphous,non .1iffusable,
colorless,odorleas
f.lnd nearly tFi.st~less."rhey ar e variable
as to
solubility.They are all opti4ally a,t1ve t~rnin~ the ~18ne of
polarized light tQ the left.
When prot ~~ids are aoted on by su~erheEte(t
steam, r arments ,by -s ci d
or a.lkall,certain
decvmpos1.tion products are tormed, var)int'
''/lth
the ag en t ,and the eond r t rona under "7hi~h it aets.'l'hey all r es uj t
in the format ion of simpler eompounds ,creak a.ng downthe COmdlllj;l tad
protein rnolecule.the process being spoken of as cleavage or splltting.An example of a cleavage process
taking place in a sUbsyance
outside this group.is the alcoholic fermentatipQ uf Klu~ose.
~
..........
-
G H
0
~tj'..,.
.:.:;:-~n:: .... 0
,> (' O
_
M·'
.<..~
When the sfJlitting pro~e8s is accompanied
bY the de,:wmposition
of
water,and the taking up or its ~onstituents
it is known as hydrolytic cleavage;an example of this form Qt cieaYa~e is the
decotjpositiCJn of fats by steam,
") I :t.j
S ~'11
0
ltr3~P2 )3+:-1
H~O
OH
~r~( 011 ) 3.. ~:~
C
H
IH
. ..
0
:.1"
..d
•
.)IJ.
cleavage. are i~portant in metabolism,a.nd
are most frequent
11' met with
the process
of digestion.In
those chemical Changes
conver t ing complex sUbs t anees into 8J.m~ler ones, the at urns ent ar '3stat~ of sta~ler eqllibrium and satisfy stronger affinities,~un)
veTtIng chem1cal potential ener~i 1nto living anergy.
'!'hese
in
WheT'la proteid
is treated with an alkall;part
of the nitrogen
is split off ~s Rmmonia.
~fter cleavage with hydrochloric
aeid,
nitrogen
ean be obtained
in three forms,-as
an~unla,as
dl-amidO
compo~nds,precipitable
by phosphotungstIc
acid,anct 8S monoam~do
acidS,not
precipitable.
A part of the sulphur separates
on tOllIng with alkalies,as
sulphide,and
uan De deteeted by lead aoetate.What
remains
~an only
be detected after r us i on WIth OXIdation
rm x t ur e s ( See properties
and reactions
of the constltuentsof
milk.) In most ..r-o
t e i ds the
quantity of sulphur SpIlt off 1S about half of the amount of total
sulphur.The
proteid Doleaule
therefore
contalns at least two atoms
of sulphur.
When p r-ot e i ds undergo hydrolytic
cleavage, they r i r s t form
proteid bodies of lower molecular
weight,vBryin~
8~curding
to the
proteId aated upon,and
the nature of the decomposln~
agent. In the
~resenoe
of acid or alkali,bodles
InsolUble
in neutral solution
are formed,known
as a c i d or a Lk e.Lt al b urni ne f'he ~roteolytl~
enzyme
cause the formation
of albumoses
or ~ruteoses
and peptones,Purther
cleavage SllCh as reSUlts
from lOeB boiling wIthstron~
aaids,the
prolonged
R~tion of enzymes,Rr
oacterlB,Bives
rise to the amidoacids tyrosin,leucin
and aspartis
a~id.On far rea~hing
decomposItion the hiaton bases,lysin
histidin,and
ar8aninare
Obtained.
v
GLA,55 lPTGA'iTO~J
OF 'fHF
PPO'n~TNS.
A classifioation
of the protelns,based
on true sClantific
prin
ciples,is
impOSSIble in the ~r8sent state of our knOWledge
of the
s uo s t an ces sPor convena ene e an s t uo y the," are d i v rue d zn t o three
groups.Simple proteids,Gompound
protiects,and Albuminoias.~ach
group is SUbdivided.
s lMPL~
'rne
PRO'I'i~:
IDS
0t(~.1H D;.;TNOUS BODl RS.
Lca I reactions
of the p ro t e i ds are d1vided
Ln t o two
classes,the
precIpitation
and the color reactions.
A. Precipitation
reaotions.
1.-Coagulationtest.
On heating a proteid solution,lt
is ~hanged
the temperature
necessary
dependM1ng
on the proteid present,and
with favorable
external
conditions18s,the
presenoe of neutral
salts,and the proper reaction of the solut10n,most proteidS se~arate in the solid state as ~oagulated proteids.
anaj.yt
An alkaline proteid solution will not coagulate on boiling,a
neutral solution only incompletely,so
the reaction must be acid fD
for coagulation!The
neutral
liquid is boiled and dilute Bcetic
acid added carefullY,boiling
before adlting each drop.Tf dilute
n i t r t c aCi.d i~ used,ad<1.after
bo i Li ng abou t one drop of acid fur
j
each cc.of solution used. if too little nitric BOld be,add~Jt~
soluble comb i.nat aon of the a,oid and the proteid is formed, which 1,s
precipitated
by more acid.A proteid
solution aonta1ning ~ ~m~11
quantity of salts,will
require the addition of a rew drops or
saturated solution of Ha Cl,befor~ boi11ng,or
thO heatin~ test may
fail,especially
with acetic aeid,whon
the protB1a ~olution is
dilute.
2. HaIlers test.Place in a test tUbe Hbout IOao,pure nltr1a aoid,
allow the proteid solution to flow 1o\1n suoh a manner that the
liquids do not mix. A White opaque rtng of Ijoal6ulated protold
forms
at the line of junotion.
3. Precipitation
by metallic salts. Plaoe ) co.uf proteid solution
in each or three test tUbBs;a,b,and o~~c B,edd about Zor. ,oppBr
SUlphate scf ut i onvr o b,alid eas i e or naut re I l",aa a ce t a t.ej t or
c,add merouric chloridew~'heau reaetlone
explain tho UDe or vroteid
solutions as antidotes
In eaSBS or poiaonlng
oy these mBt~l~,
4. To 5 co. proteid solution add a few drops of aaltla 8ald and
som~ potassium
ferrooyanide,a
voluminoua
preelpitate
1~ formed.
6. Saturate a solution of protoid with ammonium qulpriMttf!
-lit pra,,!p
itate is formBd,exeept
in a solutiun of popton8A
Picrfo,trichlorao'tio Ph08photun~itia)~ho~phomolYba1c
Gnd tlnnl0
aoids,pot.e,ssium mer eur i e 1od1de,~nd al'H).hol)~ll t)r~"lplt.,ti:)
~roteid soiutions,under
s~edial ~und1tionB.
Coagulation
is dist1nguish~d
from pr~oipitation,ln
b#lnij th0
result or R ohange or alteration
or the proteid 1n its JhemldBl
an d physioal prop"rties,usually
man1rt'8t~d rn iti "tHftn~{, or
Soa,llbi11ty.
s, Color re8~tions.
Xantho-prota1e
reaotion. See proporties anrl r0BattDn~ of the ~gnstituents or milk.
Wlllons react1on;aa above,
Adamkiewioz reaotion-
N
H
L1~bermanns
reaction
H
Biuret reaation.-To
5 OGJof proteid solution add an ~qu~lvolume
or sodium hydrox1de,and
after mixlnM ndd ono or two dru~~ or ~
dilute solution or oopp0r sulvnet9.-A
v101~t to pLnk color 1~ obtained aocording to tn~ amount of ~opp~r ~olution,~nd
th~ nature
or the proteid used.It mBy be n~e~.~ary to h~Bt the lolut1on,to
Obtain a 41stln~t rhaotionq
Any of these color react1on~ 04n b~ obt~ine4 w~tn oth~r .ub.t~no ••
but the proteids alone ~111 r~upond to Bll rlv~.
Millons and the xantno-prot~lc reft~t1on ~r~ ~1v~n by ~ub~tane ••
Of the ..,henol group and indicate th" pra80nce or i1n itrOm~tLo
nucleus 1n the proteid moleeule.A~amk1~w1Gzs
r~~~tton l~ con.ider-
ed
a furfurol reaotion,brOU~ht about by a ~arbonYdrAt~ ~roup 8.
~ell as an aromatic group in the prot~id.Ll~bermftnn" i~ 3 furfJrol
reaction. THe b1ur~t r~a~tion 1. ~lv~n by ~3v~ral nun-prot~ld Mllbstances and seetns to indicate the r~re."nf1Bor fJ.t let.l~t tWQ fJONH ..
,.
2
8S
groups,united
to a .tn~le C or N atom,~r
on& ur mor~
CQnk ~rouV.
united in open chain.
GI!ASS
11" 1 CAT r 0\1 0;<' PFO'j'F:IDS.
Those proteids ocauring formsd in the animal body and whi~h ~an
be isolated from it without losing thelr origInal properti~s,are
call ad
?"TA'fl V f'~ PPO'I' E 1 DS.
MOdiflcationsobtained
by thB.action of heat,chemIcal
reagents,and
proteolytic enzymes on t h e na trv a proteIds,are ;~alled DFf'~IV';:L)
PFO'ff?TDS.
The dIfferent SOlUbility of proteids,&nd the effect of certain
neutral salts on their solutions
is the baSiS of the following
clasSlfication.
1. Protelds siluble in al~ohol.
2e1m and
Gliadin,re~resent
class,and are of scientific interest only.
2.
Proteids
solUble
in
divided into two grou~s.
water, insolUble
in
alCOhol.
this
't'h i s c Las s IS
a. Albumins
These sUbstances ara solUble In water,Rnd era nut
precipitated by weak aCIds or alkalles,or ty s8tur&ting thelT solu
tion with
MgS04 or NaCl.'fhey
are preljlpitated by large quantities
of~min,~ral !'t{nds or m.~tallic
salrS,Rnd by satl1ratlon
of t ne i r
SOlUtIon
:vl!th
ammonillm
sulpnate."hej
ate
b.} I1da"t,ln
;;oei5ul&too
nH1
neutral salts.
b. Peptones,are s!Jbstane~ls rl:3sultintS
from the dig\1stlon of proteId
'fhey are not preoipitated
bj( saturation1· ... ith
ammonium S'.lliJhat':l,and
are not coagulated by heat.
presence
of
8. Proteids solUble in dilute neutral
alCOhol and in W&ter.
This class inoludes
salt
solution;insolubla
the globulins,vitelltns,and
In
the hetero)pro)
Important glObulins are 1,acto-globulin
of milk,serom
globulin of blood serum,myo-globUlin,mY~sin,muSdRlin,mYOSinogan
of
muscle,fibrlnogen
of blood.1he vitellins Inalude the crY~tallin
of the lens,and vlt~llin of th~ egg YOlk.Hatero-proteoses
ara
teos.s.'J'he"
formed
during digestion of ~ruteiJ.
The glObUlins and vitellins are easily distin~uished from hetero
proteoses,by being ~oagulated by heat.
GlObulins ~re pre~ipitated
When theIr solution is saturated ~tth
arnmoni.um SUlphate,magnestum
sU1fJhata or sodium "hloride;thdY :1.1"'"
also
precipItated
When the solution
is half
saturatt!d
with aminon i.
urn SUlphate.
Vitellins
differ from glObulins in not being salted out
The hetero-proteoses
will be considered in oonnection
gos t ion.
'fhe
With
var ious globul ins a.nd v 1 t ell ins are cons ider ei.t
tissues
of WhiCh
th(~.y
f()!")IJ
iJart.
fhe
In
NaCI.
~ith di-
by
c onne
c t ion
4. Proteids soluble in dilute acids or alkalies,insoluble
in
aleohol,water,and
salt solutions.
Alkali albuminate,is a variety of deri~ed proteid,obtained
when
native proteid is 83ted upon by an alkali,1'he ahange takes placo
slowly in dilute solutions,at the ordinary temperature,but more
rapidty on heating.
Acid albuminate,is obtained when a nati~e prote~d is digested with
dilute acid.
Alkali and a~id albuminates have certain propertias in ~ommon.'(hey
are insoluble in water and dilute'sRIt solut10n,but eas1ly solUble
1n water in the presence of' a small quantity of 8cio or alkali.
~his solution does not coagulate on boiling,but 1S ~recipitated on
neutralizing With acid or alkal1.A solut10n in dilute aJid as ~omf
is completoly preclpitated bY saturation ~ith ffia~neslum sulphate,
ammonium sulphate,or sodium ¢hloride,but a solution in dilut~
alkali is not precipitated
Although agreeing 1n these reaations.ao1d and alkali albuminates
arc essentiallY differen,.In the modification of a proteid by an
alkali the chemical ¢hange is essentially different from that
oeouring When the proteid is acted on bY an acid,and the products
are of differant natures."he alkali albumlnates ha~e de~ided acid
properties;they disso~~e in water,on the addltion of calciw~ carbonate,with the e1imination of (~arbon di-oxicte;a ~roperty the a3id
albuminates do not possess. Dilute solutions of alkalles act mor.
enor~eti~allY on proteids,than do a1ids of corresponding dilution.
Whep treated with alkaliespart of the nitrogen,and often part of
the sulphur,is split off from the proteid;'for this reason we can
~onvert acid albuminate into alkali albuminate,but oannot reverse
th~ process.When a solution of a~ld albuminate is made in wateT,bY
the ald or a small amount of alkali,no alkali albuminate is formed
but a solUble ~ompound of the a~id albuminate and the alkali. When
alkeli albumin is dissol~ed with aoid a eompound of alkali albumln
and acid,but no acid albuminate forms.
Anti-albumid,is an acid albuminate obtained during a rapid digestion with proteolytic enzymes,or by boillng proteid with dilute.
sulphuric acid.snd is characterized by its resistanoe to dl~esti~e
fluids,and by its ne~ati~e reaction with ~illons Teagent
by
DYs-proteose is a modification of hetero-proteos6,obtained
leaving this material in contact 'vith water for a lon~ tlme.
6
Proteids InsolUble without decomposition.
Coagulated proteids,may be obtained from native proteids by the
action of heat,aeids.alcohol
&c.rhe nature of the prouess of ~oa~uletion is unknown.Ooagulated
proteids are insolUble witheut
decomposition.
BlOOd fibrin,is a coagulated prot81d ObtaIned by the con~ersion of
rt~rinogen of the blood into fibrin by an enzyme.
CO~POUND
PR01F.lDS.
When a simple proteid is boiled with a dilute acid or alkeli,we
obtain by the rirst hydrolytie oleayage.a sUbstance of simpler
stru~ture,but still proteid in its nature.By repeating the aleavage we will e.entu8l1y obtain a non-prote1d.When a oompound
proteid is treatad in u similar manner,we have formed by the first
splitting,two sUbstances,ene
proteid,snd the other non-proteid in
its nature.During
the splittin~ of the compound proteids,the
simple proteid formed ia immediately con~erted into acid or alkali
albuminate,depending
on the agent usec.1in the hydrolysis. All a"id
or alknll albuminates bein~ id~ntical in their properties,a
.
classification
of compound proteids must depend on the non-proteid
results of hydrolyais.
Haemo~lobins,are
compound protieds yielding a molecule oontainin~
iron.'j'heyare all solUble in water,and ooagulated bY heat with
decomposition.~hey
are beat studied in connection with the blood.
GIy~~proteids,ar~
those compound proteids yielding a proteid,and a
sUbstance ~apable of redu~ing oupr1c oxide.
Muoins,are secreted bY the larger muouous ~lands of the bodY,and
will be considered in conneotion with saliva,which a true mu~in.
~ucoids or muoinoid. resemble muoin. in all their propert~es
exeept in being preei~ltated bY acetic aoid.
Amyloid ocours pathologically
in the splean,liver and kidneys as
infiltration,an4
in .erou. membranes as granules with concentric
1~yer8.Amyloid
y1e1,. QD boIling with dilute hydroohloric acid,
sulphuric acid and a reducing sUbstanoe.lt
is split by the action
or an alkali 1nt8 prot_i. and chon4roitin-sulyhUri~ aci4,an etherial sulphurio 80i4 or the oompQsttion
C 1~2("}'J°l:4HS05
~ueleo-prot.ids.are
compound prote,ids ¥ie.ldi~g on hydrolysis,
nucleins and a simple proteir."'I'henueleins: are especiallY resistant to the action or pepsin hydroohlorie ecla.ano are usually
Obtained as an insolUble ~es1due after tn'e 4igest ion of nucleo-pro
teids With t'hl. r:eagent.
'I'henuc~eo"'prot.'icl
.. can be loo,~ed upon as a combination of
compound and s~ple prot.id.~he nu~leins bein~ eomp!ex molecules
re,:;embling 4ompoundproteids.,'ne
nue Lerns are (1i~ided into two
clesses.
Cell nU~leins,Whioh
yie14 on hydrolysis,a proteid,ortho-phospnorio
acid,and Xanthin bases;occuT ohiefly in the nuclei of the cell,but
are found in the protoplaam,and may •• pas~ into the animal fluids
on the destruotion or the cell.
Pseudo-nucleins
yis1d on clea,a~proteid
and ortho-phosphoric
acid,
but no xanthin base •• ~hey are considered to be deoomposition
products or the true or cell nucleins.Casein
and the nuclein of
eFK iolk belon-s to thu. gloaa.'l'f\u lel.t~~r~ubiltance contains
ar on
In additi~n to the usual constituents
of the group.
Nucleo-histon,is a variety of nueleo-proteid,yielrling on hydrolY
sis with .8 percent hydrochloric acid,nuclein and a proteid SUbstance soluble in hydroohlori~ 8<'id an d wh i ch differs from other
proteids in being insoluble in an excess of ammonia. This proteid
sUbstance is known as HtS~ON.
Phospho-carnic
aoid,is a complex substance
combining
the aharac
teristi~s of the nucleo-proteids with soma of those of the glycoproteids.It yields on hydrolysis,in addition to other sUbstances~
phosphoric acid,a reducing carbohydrate,and a peculiar proteid,
carnic a~id.
~LBUMTN01DS.
'i'hes e
are protein bodies, whose general prop er t aea sugg es t them
to be anorna.Loussimple protei •• 'l'he,occur ch'ierl y as cons tit uents
of the animal skeleton and or the skin and its appenda~es.They
exist as a rule in an insolUble condition in the organism,and shOW
marked resistance to the ordinary proteid solvents,and to reagents
in general.'
Keratins,oocur as the chief oonstituents of the horney structure
of the skin an d its appenda6es.l\eratins o-re characterized by containing proportionately large quantiti.es of sulphur,part of wh:i.ch
is in loose oombination.On dae~ eleaYa~e,the keratins give the
s~ne products as the protei4s.1t is probable that the keratins are
derived from proteids bY SUbstituting proteid oxygen,bY sulphur.
Keratins dissolve s10"'IY in caustic: alkalies,especially
on heating
'i'hey ar e not disso}1red bY pepsin b¥droohlor a e so id, or by t r yp s in
solut ion.'i'heygive the xantho-proteic Millons and the bluret r ea e
tion.
Elastinloccurs
in connecstive
tissue.'l'heligamentum nuchae contains
mUch elastin.
On deep ~leavaKe elastin yields the same products as the prot
teids,except that glyeocoll repl~ces aspartic ~cid.The molecule
contains a very small J:)ercentage of SUlphur.
Elastin is insolUble in strong alkalies at the ordinaey tempera'
ture,and only slowly..on boiling.lt is very slowly s oLub Le in co Ld
sulphuric acid,but
easily dissolves in Warm nitric acid. Gold
hydro~hloric acid easily dissolves elastin derived from the aorta,
While elastin from the ligamentt~ nuchae is disso&ved w~th diffi
cUlty.F.lastin gives tne xantho-protei4,Millons
and the biuret
reactions.
Collagen,is the chief oonstituent or fibrous tissue,and of the
organic portion of bones.noll&~en of bones is called ossein.
Mixed with other materials collegen occurs in cartila~e as chon
drigen.
On boiling with water,or more redi~y in the presenee of acid,
.ollagen" is oon.erted into gelatine;which when geated to 130 c. is
transformed into collagen. Collagen can therefore be considered as
the anhydride of gelatine.
Gelatine on hydrolysis,resembles
the proteids in most of its
products,but differs in yielding large quantities of ~lycoooll,and
in not producing any tyrosin.~his last difference is important,and
is accentuated by the products of the putrefaction of gelatine,
which are free from indol and ska t oI s t'ne properties of gelatine
will be further studied in connection with bone.
Reticulin,oceuring
in the reticular tissue,and Skelatins forming
the skeletal tissues of invertibrates are members of this group.
The cleavage products of the protein sUbstances ,will( be taken
up,in connection with digestion.
F'A't'S.
Pats are widely distributed constituents of the animal organism
occuring in every organ and part of the body.~he bone marrow is
the tissue richest in Fats,and the intermuscular connective tIssue
the fatty tissue in the abdomen,and sUbcutaneous connective tissue
are the most important deposits of fat.ln plants the fat is found
in the seed,~ruit and oocasionally in the roots.
The fat of the animal body consists partly of fat absorbed from
the food,and deposited in the tissues,and. partly
of . fat formed
,
.
'
'.
in the organism,from proteids and oarbohydrates.When
food contains
an excess, of fat the exoess is stored up in the fat ty t issues and
when the food becomes deri.ient1n
rat
this
store is drawn on .In
.
.
starvation no tissue deoreases as much as the fatty.'l't:'le
fatty
tissue of the animal' body is a depot Where,durirtg proper alimEmtation,nutritive material of great importanoe,is stored,to be
given off as it may be n8eded.
The animal fats consist chiefly of the compound ethers,stearin,
palmatin and olein.The proportion of each present,varies in
fiirrerent fats,the fat of children is relativelY richer in stearin
than adult fat.ln solid fats stearin and palmetin havethe preponderance,and the quantity of olein is ::sreaterin t.hemore liqUid
ones.rhe solid stearin and palmetio have loW melting points.
These ethers are triglycerides,formed
by the replacement of the
hydroxyl hydro~en of the tribasic alcohol glycerin,by the fatty
acirl radicles.tn certain rats,particularly
in milk fat,gly~erides
of butyric caproia,capryllic and capric acids occur,with the first
mentioned three.In addition to the the stearin,palmatin
and olein,
the animal Cats also contain the gly~erides of lauric,myristic and
arachidic acids. While glycerine is the alcohol ehieflyoccuring
in
the animal fat,certain higher alcohols have been shovm to exist in
~.;
the
oo dy ,
The fats
hydrolytic
are oharacterized
by the acia and alcohol
formed by
cleavage
of their
molecule.'fhis
cleavage,whieh
may be
produ~ed by f9~ments.super-heated
steam or caustic
alkalies
is
called saponification.The
term is used in general
to denote the
hydol y';::~; s[.'1.:i. t t ing of a compound ether.
The Gynthesis
of fats
from their
component
alcohols
and aClds
with removal of water has nerer
been made artificlallY,and
requires
tile 58iective
action of the liVing cell
for its perform)
ance.
When exposed
to the air for a long tilllfJifats
are ,t£composG,Lj into
fatty acids and alcohols.The
fatty
a.ids arc oxidized,producing
v oLat i16 c ompoun ds of unpleasant
odol"'.l'ats
so de e.ompos s d are s a Ld
to
,
be
.
rEi.·"[lGj.Qc
Free fatty
a c i ds occur in v ar Lous proportions
in t h e rae of th~
body" and (i. e formed
dur- ing the digest ion of ne u t r a I fats, Uncombir,
ed alechols are very rare,the
only oCCuring io sUfficiont
amount
to be of interest
is cholesterin
ChcJ.esterin,is
an unsaturated
aleonol
of
the
composition
C')'7H/,
"-!'
_9H,
.",0
req~ir'ing
the addition
of ten atoms of hydrogen
to saturate
its
moI e cuj o , It is a constituent
of all ~el113 and is (cunei in C:,J!'t1binaileD wlth stearic
and palmitic
acids
in the bloOd. skin and b~ain
Chclesterin
fats arB very resistant
to the action
or putrefa.etive
b a o t or ia; and the oc cur ance of rnes fa t s i'l combina t j~.on wa t h the
keratinf3~pl'e;.·ents
the deeomposition
of t r.e skin sur ree e of the
livin15 bodY,bY the bacteria
whi'Ch are eone t ant t y pree~,l1t on it.
Chole::iterin
o c cur s in large
quantity
in the bile and i6 the c h i ef'
constituent
of biliary
calculi.
Its properties
and reactions
will
be given in connection
with bile.
The simplest.fd~m
in ~ombination
with
in sPSi"JJ8.cetti;which
alcol1C:.t
of fat is one consisting
of a monacid al~ohol
a monobasic acid.Such
a fat is eetin
oddUring
yields
on hydrolysis
palmitic
a c a d and cetyl
0
,-;
x>
,.0 C
i'
H
II;;' ~3'L 2 16 33
catin
W A~or ~U~h SUbstances
ILL,
acoid
m'l·tl""b'''''l'·
~.,
a~ •..
c,
...
oS
C
H
COOH ~ C
15 31
Palmitic
acid
H
CH OH
15 31 2
cetyl
aleohol.
only one form of combination
d
It'
,
an mu I-aCId a.lcohols
are
is possible.
conside . .ed, the
of combinations
are multiplied
in prorr~ion
to
1
bl'
,
.
,·'--,!.l,-,,,
~.
ep aeea
e groups an the acid or aJeoho'- .'I'he com•.
LILD~"1'ci()I1
of mono·-basic acid and glyeerin
allows
three :V£tTiatons
as one., t VIC' O~~ three
of the hydrOXYl groups ~\~l;,the
"v
glY,,:c:~in are replaced.The
combination
in WhiCh allthree
Y'epL;.eod
forn'ing a saturated
compound is the one oc~uring
j.n th'eOiC
f)fJ,'::"sible
t h Co
"''''~''·
..
variety
c.r nf r
~re'
bOdy,
"
--'7;;.;;;.;;
a tri-a,~id
base is combined in molecular
proPoi'tion
with a
:,c aci
a neutral
sal t is ordinarily
formed. Under certain
10
"~Lj(.n
t
08, wi th
or ganl.~ ula t er ial ,the 1" esul t will be the formation
01
,a
compolled
haVing
at the same time~ the propertiE:s
of salt
acid
~11n. 'baseo
~ \:?l!en
l
:: ~_l'~~,~,~
\
Glycerin phosphoric acid is a compound of this nature obtained
by the combonation of glyoerin and ortho-phosphoric
acid,with the
elimination of water.~he reaction is represented in this equation
c
H (OR) ...PO (OH) ~ 0 H (OH)
3 5
3
3
3 5
eo (OH) 2'0
2'
..r H
O.
2
1he hydroxyl left in combination with the glycerin radicla can be
replaced by acid ra4iclas,while
the phosphoric acid hydrohyl.is
replaceable by basic or alcohol radicles.
Lecithins are phosphorized fats occuring in all organs and
tissues of the body,particularly
in the central nervous system.
fhey are formed by replacement of the ~lycerin
hydroxyl of glycerin phosphoric acid,bY fatty acids.and the hydrogen of one of the
phosphoric acid hydroxyls by the residue of the complex alcohol
cholln.Distearyllecithin
is represented by the formUla
03H5(OT6H3702)2°cl~-002H4NOH(OH3)3.
'l'Hesynthesis has never been accomplished, but the s t rue t ure
of the moleoule is tnovm from the resUlts of its decomposition.
Distearyllecithin
yielding on hydrolysis cholin,stearic acid and
glycerin phosphoric acid.
Cholin is a complex aloohol.belonging
to the group called ~ydramines.tt can be looked on as ammonium hydroxide,in which three
htdrogenatoms
have been replaced bY methyl, and a fourth hydrogen
bY the combination with the di-acid alcohol of ethene.
Closely related to oholin are neurin and muscarin,substances
rei
SUIting from the putrefaction of animal matter,and belonging to a
class of SUbstances known as the animal or cadaveric alkaloids,or
ptomaines,on account of their similarity in Chemical composition.
and physiological action,with the vegatable alkaloids.
Neurin,can be Obtained by remoVing the components of water from
cholin.Poisoning with this sUbstance,gives
ri~e to symptoms elmost
identical witn those of Tleus.and suggest the possibility of
decomposition in the intestine,in this condition,resulting
:
in the formation of neurin;the absorption of which is the daUse of
the symptoms. (Auto-intoxication.)
Muscarin.more poisonous than neurin,can be obtained by adding a
atom of oxygen to its moleCUle.
F.pinepherin is one of the three alkaltjds.oocuring
normally in
the animal body.It is obtained from the suprarenal capsule.and has
a marked action in raising blood pressure.
EXAMINA110N
OF
ADIPOSE
Separation
of fats and connGc~~V~ tiSEUS
P:~ace ab ou t 10 grams of a d i poa e t iss ue :fir efr.rab::" Y - r ;:":1I t:l C
hog} t hc t hac been cut into small p i.ec es an d sUbsequent.:':' 61';:;\:.~-:.r:
as finely
as possibl& in a mortar,in a flask;pour upon it bO oc.of
absolute
alcoho,l;and
heat to b,oiling over' a water na t h The greater
part of the, fat dissolves.
in t h e: alcohol,leaving
the o onn ec t Lve
tissue
e t e .. undissolv:ed,.Fi.lter;bring
the residue
on the filter
p ap er r and' wash r rr-s.t with alcohol
then- with ether.Allow
the ether
t.o evapor at.e from the residue
and: test
ror proteid
bY mearia o.r the
xan.tho-prot-elc.
rea~,tion. or b,y means of' I,til.lons.- reag en t.. Evapora t.a
the. filtrate
&f.1'd' \"18s.hings over
the water bat.h;on c;o.oling the, f'at
i
c
slowly
solidifie.s,
I~ Rub a small
piece
Rea~tions
of Fats.
of f~t on a piece
of glazed
paper~-the
paper
becomes semi-t r enspar en t •
2. '1'0 a few c.c.. o,f alcohol t;add 1 or 2 drop.s of ve-ry dil ut e caus.t t e
soda and then sufficient
rosolic
acid'
t opr oduoe an int.ense
red
color.rC-o. this- mi,xtu.re add an ,.~.j,. etheria.l
solution
of r a t tone
drop of fa.t d:isscolve'd' in a fe'''''! "-. of ether.) -'}!he color of the
solution
is not ehanged,the
{..-\os react
neutral ..
3. Mix a drop, o£ r-a'~ with a small: quan.t it y of pot as s ium b is ulphate
place this mixt.ure in a dry test
t ube j an d heat-the
characteristic
penetrating
ddor of
acrolein
is no~ieeable.
4. Heat a small portion
of the fat in a test
tube with sodium
earbonate
solution-nhe
fat is not saponified.Caustic
soda acts in
the same way at room temperature.
Ss>p.<>mlr1C&t
:ion of r c t s •
To 100 ce.of alcohol
add, gradually
!O grams of flnely
pOWdered
caus tic potas.h.lf
the. mixt utre: a.t any t rme becomes, too hot t. cool in
3. stream
o.f water.When th'e, a,loo:.nalwill not d'issolve
any moDe of
t.he potash a1,10\'I to' s-tand\;:and toenP;O\1l~J" o'f"f the clear sup er aa t.ant,
1 iqui.d from the: F'e"s:idU:e-.
Melt 50 grams. Qif' }.a·p·d
a su:itabaetre!S$,e-l
wi th a lit tl&.
alcohol
br ing i·nto a 5@6 ClC-fl-a'$.tt:p,o:\W ~on, it t.he alcohO,licsolUtion of caustic
p.otas·h;-and. heat over t,he water bath until. a dro.p
of the mixture
is completely
dissolve4,
in water. Wh.en saponifiea·
t ion is complete pour the o.ontents of the flask
into an evaporat
ing dish or a beaker containing
500ec.of
water;heat
over the water
bath until
the aleohol
has evaporated;and
then add dllute
sulphuric acid until
the solution
is distinctlY
acid. Allow to remain on
.('1
the water bath until
the fatty
acids form a layer on the surface
of the liquid,and
then allow to cool. The layer of fatty
acids
contains
palmitic,stearic
and oleic
acids.~'he
liquid
contains
glYc
erine.
Decant the liqUid upon a filter
paper (the filtrate
to be
used in the preparation
of glycerin.
).Break the fatty
acids into
s~all
pieces with a glass
rod bring upon a fliter
pa?er~and wash
flrst
With tap wate!" until
the wash water does not ~'eact acid,then
with a little
distilled
water.
Reactions
of a mixture
of fatty acids ..
Repeat exo er tment e 1,2 an d 3 't204ll-er 1!.eactions of fats.
4. To a half saturated solution of sodium carbonate;add
some of
the ratty acid mixture.The
fatty acids dissolve with the liberation of carbon di-oxide;and sodium soap e are formedu Cool the
mixture-it
solidifies to a jelly.
5. ":<ri.ng 2 grams of the fatty acids in a beAker and pour on them
100 cc~of water;haat and dissolve the fatty aoids by adding a
slight excess of caustic soda-soap solutiDil.'l'.!'l;·-e
f<>llowing reactions are to be made wii. th small portions of this eolution',vhile it
is still warm"
a. Add to a small portion one cc.hydrochloric
acid-the fatty acids
are liberated.
b. Add to a small portion a solution ~r calcium chloride-the
mixture does not foam on shaking.
e , Add to a third portion lead acetate -wnr t e precipitate.whioh
on warming becomes stickey lead plastero
d. Add to a few cc.of the soap solution one or two drops of sweet
oil~and shake the mixture,-homogeneous
milk like mixture (~mulsion
rhe soaps have the property of emUlsifying fats~Repeat tho exper~
ment but instead of using soap solution use a few drop2 of sodium
carbonat/;; solutionoEmulsion
takes p-lace if the oil contains a
little free fatty , acid;absolutely
neutral fats free from 80ia are
not emUlsified.
6. Place in each or two dry test tubes equal portions or fatty:
acids and fat.Place both tUbes in a beakar which 1s pa.rtly filled
with water;and heat carefully with a small flame,st1rrin~
the
water to maintaan an even temperature-the
meltin~ point or the
fats is lower than that of the fatty aeids.
Palmitic and stearic acids can be separated from oleic acid,bY
erYstallization
from an alooholio solution. Oleic aoid oan be
obtainsd in a pure state fpom the mother liquor ,bY oonvertin~ it
into lead
iOleate~decomposing
this salt with hydrouhlorio
acid in etherial solution,purifying
by repeated
extraotion of the
etherial solutian with water,in 'a separating funnel,and evaporating"
Separation
of Glyoerin.
The filtrate from the fatty acids contains gly¢erin~potaBsium
SUlphate and free sulphuric acid.Pilter tho solution;neutralize
With sodium earbonate;evaporate
first over a free flame later oyer
the water bath ~M; much as Posslble;and
then ext r-aet the residue
With 50 eCoof alcohol.After
standing a short time,filter:evaporate
as completely as possible over the water bath;and extract again
With 25 CCQof absolute alcohol.Remove
from the water bath and ~-....
after COoling
pour into a small flask;and add 25 e~~or ether.
Shake thoroughly;
and allow to stand 24 hour s ,Most or the salt.
are
removed by thi~ process.Pilter;end
allow t06yaporate atfirst,1n a
warm pla~etand then warm gently over the water bath. Glycerin
remains behind as a light yellow syrup with an intensely sweet
tast e.
Reactions of Glycerin.
1. Make the acrolein test with a few drops
of glycerin.
2. Mix a little borax on a wathch glass with a drop of glycerin;,
and heat a platinum wire brought in contact With the mixture in a
colorless flame. The flame is colored green. Di~801ve the remainder of the glycerin in water;add caustio soda;thena drop or two of
~ehlings solution.On heating there is no reduction of copper-
OARBOHYDRA'f1'~S•
The carbohydrates form the ohief portion of the solids of plant
structure. They occur in relatively small quantity In the animal
bodY,either free or as a constituent of compound ~rote1ds.CBrbohydrates are ext,raordinarily important as food,serving as fuel to
yiel4 heat and muscular po_ar.Any excess of carbohydrate material
absorbed is deposited in the liver as glyeogen,or is converted
into fat and as such stored up.
~he carbohydrates include the various augars,starohes,gums,
cellulose &e. and several s ubs ta.noes obtained s yn t be t i"al1y. 'rh e y
are distinguish~d by the sUffix ose,added to a root signifying
their origin ,or some other chara.cteristic.AII carbohydrates are
diVided into three groups:the mono,di and polysa.ccharides.
MMONOSACCHARIDES,are
divided into trioses,tetroses,pentoses,nexoseS,heptoses &c. according to the number of oxy~en atoms presant in
the molecule.Of these groups the pentoses and hexoses are of
Physiological importance,the others are,however,of the ~reatest
scientific interest.AII monosaccharides are either aldehydes or
ketones of polyhydric alcohols,and are known acoordingly as aldos
es or ketoses.'rhe hexoses are derived from an a Lcono Ljwnos e moleCUle is made up of two primary alcohol groups in combinat1on with
four secondary alcohol groups.Wnen this alcohol is oxidized an
aldose is formed it the primary group is acted upon;while a'ketosa
reSUlts from the oxidation of the secondary.
CH20H f CHOH) §HOHCH~JOH-t 0
HeXYl alcohol
CH2·0H(CHOH),,:>CHOHCH20H-~O
-,
:::. CHZOH (GHOH) §HOHCOH1 H 0.
2
Aldose
=:GH_OH(CHOH)
2
Ket as e.
3
GOCH OH.
~..
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..ff,],;; mir)t'ii~:;M !;;;·;·.·U.'
.•l~;
t hf'; t ,6 ~j t ('l;ft h.,~~~:i.l.;r; ~~{~{7r~· [,:~tl.
,~;"t1.1
t'l t ~~
{! (}~i' t:l){-;"t t irJ~(;)r.
j"~
c;t'!~ered7
:;;-,~.:';~~~~"~~/;
..:.f·.·-gi::~:~~~
. :i:'~:~~~
;: f:r~:~
~:~:~;
::i::;~o~~~N:~own
~'i
6$ M
n
'~..
\~,)~,j)'fU"),fr'1H;~:,-: (}H~OH(CH(}"H)4C:JlH41·!~\i~0·'~;·f';;;/;~
Cll:,::l-t(GHOH)4
=
CHNNHC H ;6 S
H NNHC6H5
Z
6nS -;. HZv -:-
CHZOH (CHOH) 3CCHNNHCtiH5NNHC
...~·1
~li. ..
osazone.
'~·l'F:.
llyc.TcgenL3 not evolved but acts on another rnoLe cuLe of
anil in and a.rIlIIOIli~t
r<:~:"tlYlhydrazine, forming
H NNHC
2
H
1" 2
65
H
=
H N4J ...
285
NH
3
•
rhe osazones are yellow crystalline sUbstanoes;which
dirfer in
melting po i n t ao Lub i La t y and other properties
,in each sugar.'I'hey
are very useful means of separating sugar. from other materials in
sa:utlon,and from each other.fhey are of the utmost importance in
the sy~thesis of sugars.
On cleavage with gentle heat and strong hydrodhlorio acid,osa
zones yield phenYlhydrazinA'hydroehlori~eBnd
keto-aldehyde compounds,called osones;
CHZOH (CHOH )3COH (NNHCSH5'2
+ 2 H20 + 2HCl =.
j
2CoHsNHNHZHCi'"
CH20H(CHOH)~COCOH.
080ne.
By reducing the osone with nascent hydro"eQ,from
zinc
8cid,a ketose is obtained;
GH20H (CHOH) 3COOOH
to
2H::
and
a ce t
ac
P":
GN20r. (CHON) ~-iCOCH
"Iha ketose is obtained in this way from an isomeriC aldose,-as
in the oonversion of glucose into levulose.
The monosaccharides are colorless odorless,sweet,substances
neutral in reaction,radilY soluble in water,
sparinglY inalcohul
insoluble in ether.They are optically active,some laevo,others dex
tro-rotatory:the optically inactive modifi~ations are formed bY
the union of two opticallY oppoaed componants.Soma are fermentable
with yeast;some undergo lactic,butyric
and mue f Lag anona
fermentation.
Dextrose,levulose and ~alacto.e of the hexoses,anJ aribinose
and xylose of the pentoeea,8re the only monosaocharldes of reAl
interest in physiological chemistry.
DF.X"ROSF:,
(glucose,e:rape-s\Jgar),o4curs
il\ large q uan ta t y in :3
nUmber of sweet rruits,Qften aeoompanie4 by levulose,Tt occurs in
the intstinal
t rae t n during ditrestion,also
in sma Lf quan t Lt y in the
blood and lymph,and in trades in the urine,and other arnimal t1ssue
and fluids.ln diabats the quantity of aex t rose in the urino~may
re8ch 5 percent or m~re.
Dextrose can be Obtained artifidially by boiling staroh with
dilute mineral acid,dextrin being formed intermediately. It can be
Obtained by the hydrolytic ~leavage of other compound sU~Brs,and
~lUeosides.ln the body it can be formed from proteids.
Dextrose usually ~cdurs
as a thick syrup,but
can
b9
arystallizeG.
,It
is easily soluble in water,the solution is dextrogyrate,and
:shows birotation;that
is the angle or rotation graduallY diminish~
.es,when the solution is allowed to stand.~he opposite action is
:called hemi-rotation.The
specific rotation is 52.ti in a one to
.fifteen percent solution.
· Dextrose reduces metallic oxides,and is easily and completely
ifermentable with yeast.On oxidation it yields at first gluconic
acid;th8il saccharic acid.With phenylhydrazine
it forms an osazone
.soluble :n ooiling alcohol,1nsoluble
in water,and melting at 204 205 C. Pive grams of dextrose,will reduce 1000 cc.Fehlings solute
LEVYLOSE,is a ketose and crystallizes with difficulty. Its
·watery solution is Laevogvr-a t ev r t ferments with yeast,and g i ves
the same reduction tests aad osazone as glucose.Levulose
can be
detected by warming a few drops of the suspected liquid with a
solution of resorcin in diluted hydrochloric acid{I-~),when if
·levulose be present,the liquid will become red,and a precipitate
·settle,which forms a red solution in alcohol.5.43 grams of levulose reduce IOOOcc~ Fehlings solution.
Levulose,occurs mixed with glucose in fruits,and is formed by the
hydrolytic clea\age of cane sugar and some other carbohydrates.In
Extraordinar~ cases levulose is found in the urine of diabeties.
This sugar is of special dietetic importance in diabetes because
of itq being redily assimilated.
GALACTOSE,is an aldose,obtained on the hydrolytic cleavage of
milk sugar,and some other carbohydrates.It
is alsoobtained from
eerebrin,by heating with dilute acids.
Gelactose crystallizes in needles,and is someWhat less solUb16
than dextrose in water. Its solution is dextrogyrate and snows b1rotation.It ferments with yeast,but is acted on very slowly.Its
osazone is slightlY soluble in water,easilY soluble in hot aleohol
and melts at 193 C. Asolution of galaotose 1n glacial acetic acid
is optically
inactive.On
oxidation it yields galactonic
and then mucic acid.5sIl grams of galactose reduce 1000 cc.
Fehlings solution.
ARIBINOSF.,with XYLOSE,is formed
and found in a large number of
plants,and is of omportanee in building up other constituentse
The herbivora
absorb and utilize the pentoses as food.In men
some pentose seems to be absorbed and used.'l'heypass redily into
the uTine,seldom however, in SUfficient quantity to interfere with
reauction tests for dextrose.Pentoses have been found in the uriny
or persons addicted to the use of morphine.
Ar"
.
1b~nose 1S an aldose,crystalline
sweet soluble in waterqthe
solut·
.
,
,
~on 1s dextrogyrate,sp.rotation
I04.ti It forms an osazone
m~lting at 157-158 C.,does not ferment with yeast,and on warming
W~th hydrochloric acid containing phloroglucin forms a red solut10n.4.3 grams of aribinose reduce 1000 ~e.Fehlings solution.
•
Xylose resembles aribinose in all of its properties,excert
in sp.
rotation,whieh. is only 18.:l,the melting point of the osazone,I59160 ~. ;and in reducing power.4.4
4 grams of xylose reduce 1000cc.
Fehlings
solution.
DISACOHAR ID~~S
'fhe important d Ls ac cbar Ldes , are cane sugar and milk sugar
occuring ready formed in nature,and maltose and asomaltose,produced by the hydrolytic cleava~G of more complex cerbohydrates"Maltose has been obtained from dextrose by the action of the enzyme
maltaae,the process being one of building upa complicated ~arbohydrate from a monosaccharide;called
reversion.
The disaccharides on hydrolysis Yield two molecules of hexoses,
they are therefore looked on as anhydrides derived from two mono'saccharides with the re~oval of ine molecule of water.~he dlsaccharides are divided into two groups;those Which do reduce oxid~s
of metals.~and t r.ose which do not.
CANE SUGAR or daccharose,occurs
in the stalk of sugar cane,in the
root of the suga~ beet,the trunk of the maple,and in other plants.
it is easily crystallized, and very soluble in water,Its solution
is dextrorotatorY,with
a specific rotation of 66.5
On hydrolysis cane sugar yields dextrose and levulose.
CIZHZZOII
r H20 -;:. OHZOH (CHOH) 4 COR ... CHZOH (CHOH) SCOCHZOH.
cane sugar
dextrose
levulose.
The levulose turns the plane of polarization more to the left,than
the dextrose does to the ri~ht,and the mixture obtained after the
cleavage has an opposite rotation to the cane sugar itself.For
this reason the mixture is called invert sugar,and the change
inversion. The term inversion is used to distinguish the cleavage
of a compound sugar into monosaccharides.
Cane sugar does not respond to the reduction tests.It is not
direotly fermentable with yeast~but is eaSIly so ,atter inversion.
Inversio~ can be brought about by an enzyme,invertin,oceuring
in
the yeast;by boiling with dilute hydrochloric acid;and by the
inverting enzyme occuring in the intestinal canal.On oxidation
cane sugar yields a variety of products,depending
on the oxidiZIng
agent,and the intensity of the process.Boiled with concentrated
nitric acid,it first splits into dextrose and levulose.wl1ic~ are
further changed by prolonging the action,yielding among oth&r products,saccharic and oxalic aGids.
MALTCSE,is forMed by the action of diastase of malt,saliva~or
pancreatic jUice on starch,~nd also transitorilY,bY the a~tion of
SU~Phuric acid on starch.It is crystalline,redily
solUble in w~tet
SlIghtly in alcohol,insoluble
in ether. Its solution is dextrorota
tory and shows bi-rotation;sp.rotation
137. It ferments with yeast
and reduces metallic oxides like dextrose.7.78 grams of maltose
reduce 1000 cc.Fehlings solution.lts osazone malts at 206 C.,and
is more soluble than glucosazone.
By maltase ~nd other hydrolytic agents,maltose is inverted forming
two molecules
of dextrose.
ISOMALTOSE, is produced
with maltose during the hydrolysis of
starch.It is easily soluble in water, the solution 1S dextrorotatory,sp.rotation very close tothat of maltose.Jt ~ives the ordinary
reduction testsli~
not fermentable,and forms an osazone melting
between 140 .. :53 C.,soluble in hot water,and hot absolute alcohol
tsomaltose
has heen produced
hydrochloric
bY the reversion
of ~lucose
by fuming
acid.
Lactos6;milk sugar~ occurs in the milk,and is occasionally found
in urine ~uring pregnancY,and after the ingestion of R large
quantity of this sugar.lt is arystallizable,faintlY
sweet,hatder
and less swoet aDd less soluble than cane sugar.tts solution is
dext~orotatory,sp.rotation
52.5
Lactose is not fermentable with yeast,but is easily made to undergo lactic qcid fermentation.It peduces metallic oxides,and forms
an osazone melting near 200 O.On hydrolysis it yields ~lucose and
galaetose.Laetose
differs from cane sugar in reducing metallic
oxides,and from dextrose and maltose bY not fermenting with yeast
and bY yielding mucic acid on oxidation with nitric acid.
POLYSACCHARIDES.
'l'hisgroup includes a number of complex carbohydrates, occur in~
in all vegetable mat t er ,'l'heyare all amorphous and e rr r er from the
members of the other groups in haveing no sweet taste.Some are
solUble in water others swellup in water,and some are apparently
unchanged by it,Repeated hydrolysis converts them for the most
part into monosacahBrides.
~he polysaocharides a~e ordinarily
divided
into the;starches,
gums and celluloses.
SrI'AROHES.
THE starches include starch,inulin and glycogen.
They are SUbstances of the cornposition9(Ctflo05)x'
STARCH,amYlum~occurs
ext~nsivelY in plants,especiallY
in the
seeds,roots and trunk, where it is stored as the reserve food.It is
a white~odorless and tasteless substance,consisting
of smallgrains
Which vary in size and shzpe in different plants.These grains
prObably eonsist of two different sUbstanees,granulose
and cellUlose~of wtlioh tho first only goes into solution on treatment with
di~~tat_ic enzymes. _
Starch
is insoluble
in cold water,alcohol
BDd ether.On
heating
with water,the
starch grains
sW~ll up and burst,forming a pasts.On
boiling
the paste
,soluble
s t a r cn {AMYI,ODEX'fRINOF AMIDUI,IN),is
formed.So
uble st~roh
is also formed e.s an interm~diate
step or
the conversion
or starch into suger,bY ~nzyrnes or dilute a"ids.
Starch
does not reduoe metallic
oXides,nor
ferment
wi~h yeast.
With iodine
it gives
a charaoteristic
blue aolor.On
hydrolysis,
starch
is converted
into ~lucose,several
interme~iate
com~ounds
being formed.
TNULIN,is soluble
in warm w'ater wa t nout forming a pastG.'J'he
sOQltion is laevogyrate,snd is colored red or yellow with Iodine.
It forms Levu l os e on boiling
with dilute
s u Ipn ur Lc s;Jld.DiastHtic
enzymes have very slight effect on inUlin.
Glycogen,stands
Intermediate between the starches and Jextrlns.
It WIll be ~onsidered
in connectIon with the l~ver~
GUtd~ •
'Ihe gums are
dextrins.
'fhe vegetable
divided
mucila~es
into
are
the
vegitable
un ampcr t arrt
muc i Lag ea and t n e
in [Jhysiological eh erm s e
rs t r y ,
DI"X'I'RINSare o108ely
rala ted to the s t ar ene s ,b;~ lng 'formed dur Ing
1heir
diges t ionR.nd ;liel din~h 1 ike them. dext rose on comp1at e h y drol~
8 is. 'I'he muoilages
;:field not only dextrose
but var t o us o t ne r hex oseS,and an abundance
Qf pentoses.AlcQhol
pre~ipltates
the dextrins
and mueilages.Rasi~
lead acetate
p r e c rp i t at ea the muoa Lag es but
not the daxtrins.
DgX')'RIN (Rritish Gum ),is
p r o.duce d by heating starch
to ;dOOor
210 C. ,or after
moistening
'f"ith nitric
8did
to 100-110 G. ,also
by
boil ing starch
with dt.lute
acid, and by the act ion of eriz ymes on
stercsh.Tn
thp.se processes,several
steps have been noticed.Soluble
starch is first
~roduced,~lving
a blue color with iOJ±ne;from
this
a sugar.and
ERY'I'HRODEX1'RIN (colored red with a o d i ne I are 8~lit
by
hYdrolysis.Srythrodextrin
is furthor spllt,formingACHROODEX~HIN
(not colored
by iodu~e),m-rtltodextrin,and maLt-os e s Pr-om aohr oouex trin,more malto-dextrin,and
maltose
is split,and finally
the hYdrolysis
r es m t s in
the formation
of dextrcse,i'lith SOUle malto-dextrin,whioh
rafuses to split
further.
Commercial'dextrin
is a mixt~e
of these
~arious
dextrins ~ith
some sugar.
The dextrins
are sG1uble in water,the
solution
is dnxtr06yrate.
Whether dextrins
reduce
Fehlin~s solution
.when pure is 38 yet undecided; the commercial
er t icl e does, and it 1S iirobable t riat pur e
dextrins
are reducing
agents.'J'hey
ar u not iHrectly re r men t ab Le.
Gf!:LIJULOSES.
CellUlose
forms the en i e r cons t r t uen t of the wal t s of p l.an t
cells, \"{hile" t h ey are young. In the older
cells
the ce l l u l os e 1S
lncrusted ~lth lignin.
Cellulose is enar-aet er ized by its insolubi Ii t y.Ammoniac!:!.l
solution of eupric oxide {Schweitzers rea~ent),is the only speclfic
solvent for cellulose.
8y the action of concentrated sulphuric·acid,cellulose
is aonyerted into
YLOID,a substance givin~ a blue color with iodine.On
bQiling with dilute acids amyloid yields a monosaccharide.
Strong nitric acid conyerts cellulose into nito-celluloses,nhi4h
are yery explosiye.
HEMMICF.LLULOS~,differs from cellulose in dissolving on coiling
with dil~te mineral aClds,with a splitting of the molecule forming
monosaccharides.
In the hum~an intestinal tract,celluloses undergo partial decom
position, forming soluble products whose nature is not definitelY
known.
~:NZYM.ES
•
Hydrolytic cleavage,as has been shown,can be brought about outside the body;bY heat,with or without the aut ion of acids or alkalies,and by the action of certain sUbstances called ferments. In
the body~wher.e these cleavages ere so important,the high temperature or the chemical reagents necessary for the splitting,would
cause immediate death;consequentlY
in animals,the change must be
brought about by the ferments.
Ferments are substances that de~ompose others,\vithout being
ohanged themselves or permanentlY ut il ized. 'rhe act lV 1t Y of all
ferments is impeded by the accumilation of the products of the
fermentation. their activity
is oontrolled by the temperature,and
character of the solution in whi~h they act.Every ferment has a
certain temperature at which it is most active,the optimum temperature. A higher temperature first impairs end than destroys its
actiVity. All ferments are permanentlY destroyed by heating to
100 C.with water.Coo11ng impairs their actiyitY,but eyen after
freezing they regain their power,when carefully brought to the
proper temperature.Some act best in acid,others in alkaline,and t
others in neutral solution of certain concentration for eacn
ferment.
During activitY,some
ferments
galn in power,while
oth~rs nelther
gain nor loose,.
ORGANIZF.D F~:RMEN'rS,make up the first class-.'fheyare .1iYin~ .
unicellular
organisms. Fermentation resulting from theIr actIvlty
is closely related to the life of the cell,and the formation of
new cells. Salicylic,boric and arsenous acids,chloroform,and
thymol,phenol and all disinfectants,will
in certain concentratIon
kill these bodies.
ENZYMES or unorganized ferments,constitute
the seeond group.
They are sUbstances resulting from the a~tivity of special eells.
generally of the hi~her animals.1t is probable that the a~tivity
of organized ferments is due to their secreting an enzyme;and that
the increase 0
ower observe in organized ferments during aotiv
ity,is due to the increased amount of enzyme secreted by the constantly increasing number of cells~nzymic action is not noticeably impaired by disinfectants.This
diYference is probably d~e to a killing or crippling of the cells
of the organized ferments,praventin~
a $6cretion of enzyme.
The chemical composition of the enzymes is not knownj As yet no
enzyme has been prepa.red in the pure s t a t ev'rnev may be extracted
from the cells by means of water ~nd glycerin.The solution in
glycerin is very stable. When in solution they are easily Obtained
by precipitationof
some other sUbstance from the same solutioR;the
enzyme being ca.rried down with the preoipitate.The activity of
their solution is usually destroyed by heating to eO C.
EnzymIC action is
catalytic,and is a physical action,
resemb lang the act ion of so Lut ions of colloid metals. '1'heanalogy
is shown by a solution of a colloid metal,causlng the liberation
of oxygen from hydrogen di-oxide,the inversIon of cane sugar,the
decomposition of calcium formate with liberation of hydro~en and
the formation 'of calcium carbonate and carbon di-oxide;anJ by the
loss of activity on boilingCatalytio action has been explained on the hypothesis th3t
oxygen is capable of eXisting in both the bivalent and tetravalent
state;ohangin~ from the tetravalent to the bivalent in the presence of the
catalytic agent.
The important enzymes are classed as.AMYLOLYTIC or diastatio,
PRO'fEOlIYTI
C, S'l'F.A1'OL Y'I'YO or fat spl itt tng, INVI:t~R'rlNG, an d COMlULA'l'
INn. They are found in the human body In the dIgestive Juioss,and
In the blood.The glucoside and urea splitting enz~rnes are found
tn plants.
DIGF.SrflON.
The pro~Ass 0
igestion is both me~han1~al and chemi~al.~he
meehanlcal action causes the disintegration of the food materials,
preparin~ t~em for the ~hemicBl action of the dlgestivs agents;
further the food mass is mechanically propeled along the entire
length of the digestive tra-ct,being brought i.n t o successive contBct with the various digestive secretions.
Some nutritive sUbstances are soluble in water,and pass dIrectly into the s~:{stem;but the greater part of food material r3quires
o
a chemical change,usuallY
an hydrolysis,before
bAing ubsorbed
This cleavage is brought abuut bY the fluids secreted oy the
:?
digestive glands.
'I'hestudy of the process of digestion,begins
witt)
the stUdy of
the chemical composition and Bction of the digestive flULds.
Although the fluids are ~istinct in their aelioh and oomposition,
and the expressions oral,gastrie and intestinal digestion are
used to con~enientlY designate the Chief alterations of the foods
in these portions of the tract;it must be remembered that these
changes are interdependant ,and 18ny disturbance of one process,will
effect the rest.
ORAL D1Gj<~srl'T0N
I
SALIVA AND SAL1VAPY
DIGRSTION.
1n the ~outh the coarser portions of the food are ground and
torn by the teeth,an4 with the softer materi al,thoroughlY disinta
grated and m~xed with the saliva.
SALIVA is the mixed secretions of the ~arotid,submaxillary,sublingual and buccal ~lands,in men.Tt is albumInous and mucous in
its nature.
Saliva is a watery solution of abuut 5 percent of SOllds.Tna
important solids are ptyalln,an amylolytic enzyme,proteid,mu~in
and saLt s v't'h e saLts are alkaline and earthy phosphates,ca,rbonates
and chlorides;and potkssium sUlphocyaniJe.Saliva
is normally alkaline in reaction,but may be neutral or aeid.Pathologically
saliva
may contain sugar,in diabetes;melanin
in Addisons dosease;or bile
pigment in i.;terus.Leucine and urea have been found in saliva an
uraemia.
The amount and the quality of saliva secreted,is governed bY
certain nerve influences.Stimulation
of the glosso-phar~n~eal
nerve,produces a watery secretion from the parptid gland,containig
lng litlle proteid,diastatic
enzyme or salts. Irritation of the
sYmpathetic inhibits secretion;but stimulation of both nerves,
Causes an abundant floW of parotid saliva,which'contains
much
~anic
matter but little salts.
The sUbmaxillary gland,on stimulation of the chorda tympani7will
produce an incr.asedamount
of salivB,called chorda saliva;whioh
is poorer in soc1lidconstituents,than
the vis~id,s?mpathetic
saliva
produced by the gland on stimUlation of the sympathetic. Section of
both nerves
ives rise to a thin watery secretiun-the paralytic
saliva.
ihe alkalinity of the saliva aids its diastatic function,but is
more illportant beeaus e of the...
s t rrnulating effect or the weak bas t e
solution on the floW of gastric Juice.Acidity of the saliva haa
been observed in many diseases and even in health,so that its
diagnostic significance is not grea t st'h
e formation of lactic acid
by termentation in the mouth;is a common cause of a~id saliva.
The quan tity of sal iva secret ed in a day is about
100 c <J. The
floW is easily excited by reflex stimulation;as by the smell or
sight of food;or bY chewing of some insoluble sUbstance. Tn salivation t her e 1S a great rnc eeas e of the r t ow of s aLi va an d thp.
resulting emaciation,is probably due to the draIn of water from
the system ~nrl the dilution anti neu t ra Lt za t i on of the f~astric
Juice,bY' the alkaline fluui.Diminution occurs but se Luom. an d IS
always assocIated with ~eneral rlisturbance of secretion.
Prya Lan t he d i a s t at t e en zyme occurs in t rie saliva of all an una.Ls
except the pure carnivora. 1t is characterized by its action In
eonv er t i r»: starch into sugar s Th e enzyme acts best 8 t 40 C. ;in a
neutral
"~solution,although
it is very actIve in a weak alkaline
soLu t i on and is active in acid solution up to .2 percent un ner-a
I
acidistronger acid stops Its action,but doe~ not kill thH enzym3
unless the ae i d be concen t ra t edvr ne en zyrne t her e r or-a retains its
activity in the stomach,as long as tne gastric Juice does not
exceed this acidity_
The activity of ptyalin,varies w i t r, different s t a.rch es a ccor d
ang to. the amount of cellulose they contain an d also Whether theb
are bOlleJotraw.lLs
activIty is Im~aired by teaJon account of
the tannin it containstless so by coffee and ~oca,lts actIvity,
also is modified by such drugs as atropine,which inhibit secretion
'rhe activity rs increased by common s a.Lt hor se ra{jish,~inger,rnustard &~.and by SUbstances stimUlating the secretory nerves.The
ptyalin occurs in greatest proportlon in the first portion of
saliva seereted,beooming
less ~n each successive portion as saliva
ti on is prolonged.Amixed
saliva of half an hours s ecr e t Lon under
stimulation,will
contain
an average proportion of ptyalin ,and is
.
always used for- the estimation of this enzyme'S a ct Lv i t y ,
As it is not possible to colleet and weigh the ptyalin,its
quantity in saliva is measured by its activitY,bY means of the
method of daae t aaLme t r y , (See rea e t aons of saliva.)
Salts, Theso consist chiefly of alkaline chlorides,mixeJ with
earthy phosphates,and calcium bi-carbonate.Sulphocyanate
of potassium is a variable oonstituent.
Yuein of saliva is a true glycoproteid,and
is easily isolated
from saliva and recognized.
Glucase,is a second enzyme occuring in saliva,it converts
maltose into dextrosei
i
i
j
c
j
'fhe i ('11<t~s 8:nd some other
S81i~ary
~lands.1his
~ower
ab8or~tiQn.
dl·ugs.arahs.bit1l811y
IS used in rleasurin~
secreted
OJ the
the rapIdity
of
(l. neral
P98Jt ions
of rni xe d Saliva ..'
St.imuIBte
the r OIV of sa1i'la b'y p18cin~ in tl"l":t mouth ~. Sl'lall"Jlass
rod rounded at both ends. Gclll"~t
r n a sui t a e l e vessel
a.n d f 11 t.e r ,
a. 'fest t rie reaction
'of fresh
salivai
....
ilh s ens i t rv e I r r.nus :,d:lper.
b. Allow
Observed
a rew oe.of sa11va
due to
to
the preo1pitation
stand
of
8
Jay ur two -n ~louJlness
~alcium
oarbonate.
ft1d acetic acid to a few ee.of s81i'l~-a praoipitate
idsoluble in an exce~s of rh~ acid- Muoin~
d.
ld
IS
formed,
d. ~o a fp,w co.of saliva add nitric acid - a preoi~itate is rorme~
soluble on heating with R yello~
Qolor;u~on the ad~ition of an
excess
of allr.al
i the color
enang e s to oran~~e.o;anthojJr~t ~10 r":;I=i<;.
e. Add Mill-ana raaMsnt
to a t e« O~ .. or saliva
and t;o,il,,-el trier
pr e e ap r r a t ed proteid
e r t ne soll.~tion t ur-ns rad.
f.
or
1£1
t
h€
A...
td eaus t ac p ot aan to a small port ron of saliva
an d t r. en a Jrop.
t wo of
dilute
oopper SUlphate solution
-8 blutst'!
Violet
'~olor
fJroduceJ
eSf)eciallY on4~~rmin5. Biuret rea c t ron ,
he~ognit ion of Potassium sul{jho-::yar.1.de
f' Y,t;NS ~,i,. the saliva·-:ro
a '1e. no. o~ the sall~a
a~J one drop ~f ~Ji",arg~c.J.~~
a"ld end 6..
f &w... ,lrops
of v er Y t.lll ut t~ ~err 1c chlor Ide sol ut 10n •.~ red co Lor a t lor
i]Ue to the ror-ma t acn of iron s u lp noe yen Lde as p r o du c e dEeco~nltion
of Vt100n in the Sali,.va.-lnto-,:lOOcc.of
;"bsalute a Lco n-al
~our 20 - 30 ceo of 8811'18 tnot
filtered)
and Shake th~rQUHhlY.
Place in an ~rlenm~er
flask,lay thQ flask on its diJe.ana
alloW
i,"1'~ p r e e t p t t et e t o subslde.Pour
orf the flnld
and n1f»~at the operation ~ tlmes;than filter.cQllA~ting
the preCIpitate
~n a sr~ll
filter,wash
with absolute aleohol,then with ettt~r 804 ~laGe In a
dessieator
for ~ hours.
1. 'I'he c1ry Sllustance
is insoluble
1n water. Upon t~e c.t1dition
of
one dr op of ~8US t i IS sode .• it passes
,Jradually
into sol u t ion. 'fhis
Solution ~ive8 with a trace of ~o~per sulphate th& biurot
re~dtim
2. Roil the remaind(~r of the dry m\l...:in for 8 rew minutes with
a
small quantity or dilute hydro~hloric
8oid;allo\Q
to cool ar}\.t make
a~kaline with caustic soda. Add a few drops of mixed PehlinJs solution and boil.~~ithe'}r a yellow or a red fire~ipi tate
is produc:"d.
a. Praparation of starch past~o~Powder finely one grem of i~areh
in a mortar and add 10 cecof water rUbbing with the pestle until a
homogeneous mixture is obtained~Pour lnto a beaker containing '90
,ceo
of boiling water.and stir until a smooth paste is formed.
On a clean wh1te surface (a ~orcelain ~late) arrange a s~ries
of drops 0 dilute iodine solution.Brlng the starch paste to a
temperatur
of 40 O. Place Bxactly 10 ~e.of the paste 1n a te.t
t~be and add to it one ac.oe saliva shaking thoroughly.KEEP the
mixture, at 40 C.and at the end of one minute remove a drop Oflt
,and add to the first drop of the iodine series;repeat this every
minute until the color of the mixed drops is extinguished,indicating the conversion of all star~h into simpler sugars.With normal
~aliva the color w111 disappear in six minutes.
Recognition of the products of salivary digestiono
'(0 TO cc.of starch paste add one ce.of saliva,and
digest at
~O elafer5t~eRtpfmthetd~gested
mixture in a test tUbe and add a
small quantity of Pehlings solution,on boiling the oopper is
reduced.Repeat the experiment with boiled salivao
b. Pill a fermentation tube with the digested material and add a
small piece of yeast;allow to stand in R warm olqce for 12 hours.
Influence of ~oids and alkalies on salivary digestion. Into eaoh of
four test tUbes a,b,o,and
d,place 10 cc~ of starch solution.To a
add one cc.of a 0.25 percent solution of hydrochloric acid,to b
one cc.of 0.5 per~ent acetic acid solution,anJ to
o,one co.or a
0.5 percent solution or sodium carbonate.Add one cc.of saliva to
each of the four tubes and shake thoroughly.Arrange
on a por~elain
surface four rows of iodine drops lettering the rows to correspond
to the test tUbe~ ..
Place the test tUbes in a water bath and heat to
40 C;At the end of one minute withdraw a drop from each tUbe by
means of a cllass rod, bringing the drop in contact with the first
drop of iodine in its corresponding row;repeat this process every
minute as directed under diastasimetry ;for 8 minutes.
GASTRIC
DTGEsrION.
-
'rhe food,after trituration and miXing with saliva,lon the mout n,
passes through the oesophagus into th e stomach. Here the food mass
is SUbjected to a kneading process and is acted on by the gastric
jUice,which with the assistanoe of the swallowed fluids,reduces
the mass to a liquid state.Part of the food is now absorbed
through the stomach wa.ll,but most of it passes on through the
pylorus into the duodenum as ehyme.
Our present knOWledge of the composition and'mode of secretion
o~ gastric jUioe is due in great measure to the use of gastrie
flstulae;made on animals and occuring accidentally in men.
Th~ aeeretion or the gastric jUiee is not continuous,and
ia
brought about by ch6m1eal irritation or the gastric mucuous memb~ane or by paychic influence. A passionate desire for food,~111
e~us~ a flow or th~ jUine,but
always after a pause of about rour
minut~s.Chemical
irritation of the gastrio mucosa,as by the ak
alkaline IDeas of food and saIiTa,causes
a slower but mora continuous 110w.~he pr~senee of free acid in gastric JUioe cannot be
demen!trate4,until
about 20 minutes after the - - swallowing or
food.'!his is uue to the power of pr o t e ads to form neutral compound
~ith r.ydro~hloric acid.Durin~ this time the ptyalin of sali98 is
aotive in the hydrolysis of starch. There is a ~onstant formation
of lactic a~id during this early stage of gastric digestion,Which
however c8asea,when the hydrochloric a~id acid reaches .07 percent
~hf quant1ty of jUi~e secreted durin~ digestion varies With the
quantity and quality of the food.'l"hlS action of various foods,meat
bread and milk
1S arranf$ed in tJro~ressi'le series as folloi'8 ,
.bY Hammerstein.
A.eidity
Digestive Acti"ity
Duration of activity.
Meat
SF fi:AD
Bread
Milk
~eat
keat .•
B~ead
Milk
MILk.
Composition of Gastric JU1ce.
'I'ht~astr 1c. Jui"e, can hardly be Qbtained pure and free from foo4
residues,or from mucuous and saliva.In man it ia a nearly oolor1esa fluid or an acsid taste and a distinct acid reaction.
"'he aoid re•• t1on dependa on the presenc~ or free aCId, chiefly
hydrochlorio.Yhe
quantity or hydrochlo~i. 80i4 in normal JUice is
from .02 to .03 pe~oent.Arter partaking or food,pat1cularly
aftor
a meal "rich 1n carbohydrates,lactio
aoid oocurs abund~ntly,an4
sometimes acetic and butyric aoids."he persiatant occurenceor
lactic acid with a diminution or abscence of hydrochloric acid in
the ~aatric JUioe is indicative of dancer of the stoma~h.
Perfectly fresh ~astric Juice Gontains a little nucleoproteid
and on standing for some time contains albuDMises. "he remainder of
the organio matter consists of a small amount of mucin and two
enzymes.pepsin and rennin.'j'he inorganic material is a mixture of
the ohlorides or sodium,calcium,potassium
and ammonium;Wlth
the
earthy and iron phosphates.
'lhe free hydrochloric acid and the pepsin and rennin,are the
physiologically
important constituents of the ~astric JuiGe.
Pepsin is ~haracterized-bY
its action on proteids.lt is active
Only in acid soIUtion;rormin~
peptones •
.tn the conversion or slm~le proteids into peptones,by gastric
JUlce;ACID
ALBUMINA~·F. is first formed.The acid albuminate is split
forming compounds poorer in carbon. called primary proteoseS.'l'hese
aTe or two kinds,one solUble in water,called PRO~O-PRO~EOSE;th.
other insolUble in water,but solUble in dilute salt s'lutions,
called H~T~RO-PROTEOSg.The
proteoses differ,according
to the
protein from which they are derived.Proteoses from ~elatirie.are
g eLat ca es rrom fibrin fibrinoses,from'albumip
eIbumos ea ~diiBy
further sot ion, T hft!l!e pri"'1F!.ry
prot eos es form dent er6-prutao,S.(j., -. '.
DEUTERO-PROTEOSE
is finally split forming P~P~ON~.
. .~
The prote,oses are precipitated When their solution is saturated with ammonium suj.pha t e l p ep t onas ar e not .Primary pro t eo sea
~re preoipitated from neutral solutions by saturating with sodIum
ehlor1de~deutero-proteoses
are not preoipitated from neutral 801ution,bllt are,after the solution !las been acidified by aceti~ acid.Copper sUlphate aomplet~lY preaipit8tes the primary proteoses,but
does not erfeet deutero-proteose.Peptone
is the end produ¢t of
gastrio digestion,is diffusible;and is not chancled by the further
aetion of pepsin.
~he change of proteids into peptone is a pro~ressive reaotion,
all the intermediate products eXist at the same time in a ~astrio
digestion;their relative quantity depending on the time the process has progressed. A complete change of all 'the proteid into
pe~tone,is prevented by the accumilation of th~ products of peptic
activity.
Compound proteids are split by gastric JUice,after Which the
digestion of the simple proteid portion proceeds as above.Th~
nucleoproteids yield a peptone free from phosphorous;tn6 nuclein
split off being unchanged. The AalbuminoiJs,are
characterized by
not yielding ~ny hetero-proteose,showing
an absence of this sidechain of the proteid
molBcule.Co~lagen
is first changed ihto
gelatine Which then forms sucoessively an acid albumin,protoproteose,deuteroproteose
and gelatine
peptone,Elastinis
not
changed bond
the formation of protoprot60s~.Keratin.
are not
decomposed by
Igastric juice •
j
. Rennin is a milk curdling enzyme.lt is present! phJ.iolo~icallY
in all human gastric Juice.Pathologically
It is absent in oarein
oma ,atrophy anc ohronic oatai-rhof the slomach.ThelJresenoe
or
rennin in gaatri. JUice is shown by its aotion on milk. (See the
direotions for the examination of milk.)
Hydrochloric acid of gastric JUice is all derived from the
sodium chloride of the food,bY the pO~6r of selective absorption
po •••• ed by the oells ,of the ~8strio muoosa.The oell. split the
aQdium ahlor1de,by hydrolysis,sending
the alkali in,to the blood
and the a.id into the lumen of the gland.
'l'h~chief runction of the hydrochloric aoid 18 to aid the enztm1c action ;as a d1sinfectant it has sli~ht power in,,~o weak a
solution.
The quantity of a~id in the gas trio Juice is constantly lowar
than normal in diseases Where the gastric mucosa is affected with
catarrhal inflamation,cirrhesis,or
fatty or amaloid degeneration.
It 1s usually diminished in anaemia and neurasthenia.In carcinoma
it is frequently entirelY absent.
An exoessive
secretion
of hydrochloric
acid oocurs in ulcer or
the stomaoh .An inorease of the acidity is present ~n cases of
hyperaciditY,depend3nt
on direct irritation
of the mucuous membrane by the food;~orupon
abnormal secretion
,the result of
"disease of the nervous system.
EXAMINA1'ION OF GASTRIC
JUICE.
Clinically
the examination
of 'gastric Ju~ce is an important
aid to diagnosis
in diseases of tho stomaeh.The
activity of this
secretion
depending
to a great extent on the quantity and quality
of the food;it is necessary
in order to obtain results' capable of
comparison,to
administer
to thB empty stomach a mebll'lhose composition is constant
,and to evacuate
the stomach at a definite
interval
from the ingestion.For
this purpose several test meala
are recommended
of which [~\\7alds,given below~is
the roost convenient
as digestion
if! at its height at the f)l},j of an ho ur an.d the s t omaen contents
are then comparitively
clean and easily removed.
Rwalds test breakfast,consists
of 70 grams of white bread and
300 cCQof weak tea.~he stomach is emptied after the lapse of an
~our.
j
'l'he digested rnat er t a j is removed
from the stomach by means of a
soft rUbber tUbe;and
the miwture
is filtered and tested by means
of the following
reagents.
Recognition
free hydrochloric
acid.
In the absence of a SUfficient
supply of gastric jUice,prepare
the
following
solutions.
or
P.- Dilute
one c c s o f hydroshloric
acid with 89 c c s o r water.'l'his
solution
contains about .25 percent of acid.
B.- Dilute
10 cd.of A with 40 CC.of water.
c. Dossolve 0.8 grams or lactic acid in ICO c~~of water.
D.- Dissolve 2 grams of e l bumos e in 100 c e s o r watf31"'cl"ilter
..
Reactions
of these solutions
towards the following
reagents.
1. 'l'ropaeolin. 0.0.
au
To a few cc.or A. add a few drops of the tropaeolin
Solution.Cherry red colorQRepeat
the experiment
with solution Do
b.-Dilute
a portion of A with equal volumes of water and of D;add
a few drops or tropaeolin
solu~ion
and compare with the colors
previouslY
obtair.ed.
!5
c. ~o about
ce~of C ad~ a few d~ops of tropae~li~ solution,and
dl v i de the mi x t ur e in to t riree n ear 1 y equal par t s •1-.2 all <: 30 'fo lade!
an equal volume of water!to
2 an equal volume
sodium Chloride
solution{3
percent) ;to 3 an equal volume of e on c en t r at au sodium
~hlor1do solution_The
solutiona
1 and 2 are not ehanged,3
changes
Its oolor to rose~
oi
d. -Bring a few drops of eaoh of the solutions
A,e, and D into a
porcelain
dish add a few drops of tropaeolin
solution and evanorate over the water bath to dryness
(avoid strong heating)oAn
.
intense blue color of the residue is obtained. Albumose
in small
quantity does not interfere
with this reaction.
2.
G{Jl\jZBURG~)
REAGEN'!'.
To a few drops of solution A in a porcelaih d~shadd
a !e~ dro
drops of' tIle re ag en t and evapor at e over the wat ar bath (a,void
e xeen
sive h6~tingJ-purple
red rasidueo
bo Repeat
t~e experiment
with Band
C separate]y;also
with'a mixt ur e of' j:'; end Do Albumose does not Lnt er rere w i t h ""1"" ,:,,,,.~,,,,'·7~~ .!._~
un:Lcss f)1'8Sen"t in great excess;laetia
acid
does Dot give the test
Ba
•
Repeat
the tests Dade
mi x t ur c ~Ls ::1GI" est
.~
..."
_
\0
reagent.
under Gunzburgs
less e xp enrsLv e
G,bl e ana
_
th2TI
Iw
J
\i,r
'i'h e
.......,,::
...'\.,;
>_
.....
J
r es cr c rn
Gunzbur~s.enJ
~ ,
is
said to be equally se~si[iveo
Prepare
ferri.c
UCfelmsns
~3111-o1"i,de to
reagent by the addition of a few drops of
10 e e s o r a 2 percent solution of oarboli0 amid
a~ To a few c~Gof solution
A add a few drops of Uffelmans
reagent~
decoI or:t za t :f~0'1-1c
b. Repeat
experiment a;but use solution C -lemon yellow colo:o
Co
Mix equal volumes of A and C and add a few drops of tIle reagent
. ~on
ye.now color"Hyal'ochlorie
acid in this strength
d oe s 'Jot
. p r-even t
ecogn i.t i.on
lactic
d4 To about
15 cc&of C add Uffelmans reagent and divide the
solution into three portions (I) (2) and (~).To (I) add an equal
volume of w~terlto (2) an equal volume of 3 perGent sodium 0hlorida solution?to
(3) an equal volume of sa t ur-at ad sodium ehler-ide'
the
r
of
acid"
soluti0n;-(3)
is dacolorizedo In general the presence
chloride
doss not interfere with the reaction.
of sodium
e a Mt~ 26 ee , of A'i of C~.and of D an d. shake thon:HJ.gh].y xn a separ-:
ating f~nD91 with an equal vol~me of 6th8r~S0?Brato ~J~0 sth0rial
extrac~:( i:1,::1Jt n.:tt8r
t hr ough f" dr y filter<7A:U.ow
the ()th,~;l~ t o 0'1'8.[<"
orate vdissolve the residue in a little
water and to ~ p~rtioD add
Ufre':"L~r.\3!lS ",r·0Fc1:-2"6I.lt;:.ltl-l.is me t ho d is u-sed \~"henG"
..r e r t11D SO.LU~1.011 ~Jr
lactio B0id is colorod,Bs in the DBse of stomach contents;it also
-':
s ep
ar
f:"",
at
('Ii:;
J.clGt:i.e
ReGogGitioD
clf
The pras0Dca
8,C iii
r r-om
;0;
ubs
t
an
o
o
s
which
g:t VEl a
~
imi18.l'
co
Lo
r
,
PEPSINg
of pepsin
in
a solution
is shown by the POW6? of
the :30~t.l.lti0i.1 to dj.t'Sost
albumino'l'he
quantity of p ap s Ln p:'osent
measured ''0y th e amount of c.oaguj a.ted albumin di.ges t e d b:l"the
j,s
801u''~
.
tierl in a definite
time
Frepare
h solution of 005 grams of pepsin in 500 co-of a a25
percent solutioil of hydrochloric
aeid"
When estimating the proteolytic
aotivity
of gastric JUice~add
·t~ 25 0coof jUiC0 enough hydrochloric
aoid to make the
ad.dity
about 0,,02 percent.
In Gach of fivG test tUbes place 6ne gram of fibrinoTo test
t ub e CA) add one c c -c r gastric juice or the pepsin hy dr oc h Lor'
ic acid sclutioD.~o tube (n) ad~ two cc~of the di~astive rluid~TO
I:
..
(C) add 3 cc. to (D) add 4 cc. and to
(E) 5 cc.-Plaee
the tUbes
in a water bath
at 40 G. for an no ur s At the end of r n rs time the
change In~tha bulk of the proteid in each tUbe
will give an idea
the quantity of pepsin
present.With
normal gastric
JUice,sll
the fIbrin
in tUbes (D) and (E) ,will
be dissolved.
or
Estimation
or the quantity of hydrochlori~
aaid.
rhe aeictity of gastric Juice as manifested
by lts react~on
wlth
litmus or phenol-phthalein,lS
dUG nut only to free
~dld ,but alsQ
to acid salts. A quantitative
estlmation
of the acidity
of ~astrlc
..1uiee,wii.hout·reiSard to its
source is termed trie estimation
of
total
acidity
and 18 usually
expressed
as hydrOChlorIc
acid.
The estimation.
To TO c o o f t he filtere('
gastric
JUice ad d a few drops of
phenol-phthalein,place
the im x t ur e in :3 beaker and a dd about :65c\}.
of distille.! water.From
a burette add slowlY,dith
constant agitat ton deai-normal
solution
of sodium or potassium
h.vdroxid(~,u!'til
the liqUid in the beaker hRS a sli~ht
reddlsh color,whiah does
not disappear
on stirring.
Ft'rom the number of c e s o f a Lke.Li used ca Leu Lat e the quantity of
v
a e i d pr e s e n t
,
Congo red is not ch arig e d by 20111 salts
or
organic
acids,a
titration made .with this sUbstanoe as an indicator
will show the
amount of FREr~ HYDROCHLORtC
ACTD.
.
r
Artificial
I. Preparatl.Qn
a"~le
,
I
pepsin
Gastri~
Ji6Bstion.
digestive solution- Tlisti()l~ei grams
in a litre of 0.4 percent
hydTochloric acid.
of
the
2. lilatar tal for .ti~ as t ion.
A. ~resh bloo~ fibrin or coe~ulated
fibrin
~r6served
of
in chloroform
\'Ister.
B. 'I'ne meat
residue
from the preparation
or er ea t an e f~c. (see
muscle)
c. 1'he Whi t e s of Ti:) eggs t nat ha.e been c:oagulated by boiling,
and
then finely divide~.
~T~h matertal may be a4~e4 d.1rectly to the ji~estive sol~tion,but
·(Joe.gola,-edmaterial
shaUl d ne treated
as follows ,before adJi t ion.
Plaoe the co aguj a t ed materi.al
In a saucepan,s<M one liter of ~ater
and one ee , of h'ydro~hlorie
acid; and heat
ov er the 1ft!ter bath
until
the material
gelatlniz6s.....
1Io. tCl 0001.
Bring the p r o tea d Into the nottle
c:onta1oiog the digest ive fluid
and place in the th~rmostet
at 40 C. for ten daysAt the ent of this
time filter
t ne dif5eaniye pro4ucts r rr st
through linen, t hen thro~h
a la.rge folded filter.
Pla..:e the fil
trate
in a saucepan
and Narming to 50 c. o.er the water bath
neutralize with sodium earbonate.T( 8 precipitate 1. formed,filter
and evaporate
the filtrate first
o.,er a free flame,later
over th&
•
w~ter bath,to about 800 COe If
~n th~ course of e~aporation a
co~gulum is thrown out,filter before the solution becomes too oin~
~entrated.Tt is necessary during the Avaporation to kaep the
solution neutral.The evaporated residue is a solution in water and
s o-t tum .-;hlo'"'iri0.
of the ~lbumose an d peptone.
Separation
of Albumose::.and Peptones
by
weans of ammonium sulphate
TOO grams of finely pow-rer ed ammonium sulphate in a'
pour upon it the solution of a Lbumoa es and p ep t one j anu r ue
with a pe~tle in order to obtain a saturated solution_
The albLmoses are precipitated at first as a sticky scum,which
b) stirring may be brought together
into a v~saous mass \~ich
adheres to the ~estleeD~cant the solution (to be used in the preparation of peptones ) from the precipitated
albumoses;wash the
lEtter by decantation with a saturated solution of ammonium suIphats?place in a sauce pan and dissolve ln a little water, Add
er-o ugh "water to make the solution measure
one liter,boil over a
free flame (replacing with hot water that lost by eva~oration) and
add powcer-e d oar i um carbonate in sma LL por t aons until ammonia'
ceases to be ~iven off:and a filtered portion after ac~difYlng
with b y crocn.lor f c acid no longer gives a p recap i.t a t e l"ith barium
ch Lo r Lde ..Wpen the ammonium sulphate is completely de comp os ed ,
filter and add to the filtrate first ammonia
and then ammonium
oarbonate solution as long as a precipitate is formed;~armtallow
to stand a short time and filter.F.vaporate th9 filtrate to a small
volume over the water bath (best in a porcelain dish' ),allON to
cool and ~recipitate with an excess of 85 percent alcohol.Allow
In6
albumoses to remain in ~ontact Nith the alcohol for 2 hours
then decant the liquid and bring the albumoses in contact with '
absolute al<?Ohol for half an hour.'rhe a Lbumoe es wn i cn a t first are
S?ft and s t acky are hardened by this treatment and may be rUbbed
w~th a pestle to & fine powder.Collect
on a smooth filter vaah
wlt~ ether and press on (lIter paper.
\~he albumoses obtained in this manner are a m~xture of
prqto- and hetero-albumos~.
dysPlaee
mor t ar j
\
reactions of albumose.
Dissolve
5 grams of albumose in 100 ce.of water
~arming~Filter the solution
and
by gently
fA)
B .
use CQr the rea~tionB.
,
011 a fe~ cc~ of the SQlut10n;it
remains
Efter the addition of aceti~ acid.
unChanged even
(B) AcidUlate a portion of the solution -ith acetic acid and add
~~ equal Volume of saturated solution of sodi
..'
t i on becomes e.Lou dy cLear-a ' 'IV
.
~
chlol".l4e.'lhe solaclOUdy again lfheo c~Ol.
ng hen heat 1S applJ.e.lf
an~ b-e~oming
(0)
Add a little SOdium Chlorid.e sO'1ut"on
to
a Ib umos e solution;and then one or ~.
two drs ~ a few c~. or the
---- --'-,-' .~_,~
__~ _ -; __
.~.
~ __or n i'tl" t c aci<Ct. Ei ther
~
a cloudiness or a precipitate is produeed;both disappear on th~
a~dition or more acid.The solution on standing or on heating turns
yellow;the yelloW eolor changes to orange wh~ caustie alkali is
added.
(D). A~idulate a portion of the solution with a f~w drops of
acetic adid and add potassium ferroayanide solution.A cloudiness
iff p rodu ced ,which disappears on n ea t t ng (often not e omp Le t eLy ) ,
(F).
A.dd to a ,Jortion of tne solution half its volume of caustic
s~da solution and then one or two drops of a dilute solution of
copper sUlphate. (Biuret reaction).
~,\odifi8dBiuret r ea et ron- Ad d to a portion of the dilute solu~ion
half its volume of caustic soja s olu t aon s HoLd i.ng the test t uee
inclined,pour a dilute solution of oopper sUlphate upon the mixt ure in the
test
t ub e In such a manner as to a v o ad mixlng.'fhe
characterIstic aolor developes At the line of contact.
(p). Dilute 5 0C. of the solution with 45 ec.of water.Aad to small
portions of the solution,mercuric chloride,tannin.and phosphotun~
stic aeid.ln A8Ch-ease a preci~ltate is formed.
(B)~ A~d Millons reagent to a small portion of tha solution and
bo11-a red precipitate or a red solution is fo~rned.
Preparation
~
of peptones.
'fhs satura.ted solution of ammonium slllphate.whieh was deoanted
from tM' pr.ecipi.
t a t ed a Ibumca ee ,eont aana peptones ari d a small quantity of deutero-a.lhumo,s~.·rofree from traces of albumoses,ctissolve
in this solution
by heating 20 g rams .or ammonium sulphate;allow
to
cool E\Iid f 11 t ar ,B~rore o on t Lnua ng the s epara t i:on "take out a small
portion of the fl1trate.and add an excess of strong CBusti~ ~otash
solution and a few drops of dilute ~opper solution.If an intdnsely
eolored solution results,peptones are present in
sufficient
quantity to warra.nt t ho t r isolation.
free the aoLu t ion from the gr.ater part of the emmon tum
su!phate,evaporate over the water bath to a small 71olume,allow to
cool end filter or de4ant.
Bring the filtrate into ~ sau~e pan,add one liter of water and
treat with barium oarbonate~o.
exae t Ly as in t n e preparation of
a,lbumoses.Peptones prepared in this manner usually eontaln oonsid_arable inorganio salts.
Reactions of peptones-
"0
Pept~nes respond to all the reaotions
excep t on,' C) , (D)•
TNtEsTINAL
as given un~er a1bumoses
DtGF.S~tON.
Normally the digestive seeretions of the stomach and those that
operate in the small intestine,are maintained in a s t a t e I
of
physiolo~ical equilibrium, the entrance of the chyme into the dUo~
denum provoking a reflex secretion of the ~ancreatic and b~liary"
fluids to an extent sUfficent to ne~tralize the acid contents of
the stomach,and to establish the de~ree of alkallnltY,most suitable for the action of the ferments ~hich complete the action or
digestion. A moderate increase in the acidity of the ~astric
contents is followed by an increasod secretion of baLe and paner e-at t e JU10f}; an rj is iJrobably the eaua e of t ne oecur-ance of hyp er-acaa
ity of the gastric jUice 1n certain functional diseases of the
liver and p~ncrens,th~ increased acidity bein~ a stirnulus,~rovlded
by natur~ to aause B more active secretion.
Prom these facts it is seen that the digestion and sUbsequent"
absorption of food depends not only upon the integrity of tha
liver and pancreas, but also upon the due performance of th& fun~tions of the stomach.
The secret ions of the pancreas are t \'/0 in number; t he external
or pancreatic
jui~e;and an internal secretion ,having glyoolytic
.
power,lfhich is ,teli'lered .i rree t j v into the bLoo d c whe r-e it changes
glycogen into suga.r.'l'.he
occur anc e of sugar in the urine in disease
Qf the pancreas is probably due to interference with this function
'l'hepancreatic JUice 1S an aLk aI ine fluid con t arnrng sod tum
cerbonate in excess of any oth~r salt,and five ferments;trYRsin,a
proteolytic ,amylopsln,a diastatic,steapsin,s
fat splittin~,an
emUlsifying and a milk curdling enzyme. rhe secretion of this
juice is intermittent,and its character depends on the kin~ of
food ~aken end the sta~eof
,tigestion.'l'he
secretion increases
rapidly
after eating,and rea.ches its maXimum in the course
of the first three nour-ss Pr-om this time tht~ secretion diminishes
butm..-=.y
again in areas e from the 5th. -7t1'lo
hour ,when larl'l6 quant ities/of fooa,genp.rally pass from the stomach ~nto the intestine.
Prom this time on it gradually decreases until it finally stops at
the 15th.-Idth.hour.
'}'hequality of the food has ,al1 unmistakeable influence on the
composition of the
Juice an~ the quantity of tne different
enavmes ,'j'h3 jUice is always riches t in diastat io anzyme after a
bread ~iet,and riehest in steclpsin after a fat meal.
.
.
AMYLOPSIN,~s closely related to ptyalin,and converts stareh,raw
or boiled,into erythrodextrin,achroode~trin,and
finally into maltose and dextrose.'}'hedextrose is prObably formed bY the invertin
of the intestinal juice.
Steapsin,splits neutral fats into fatty acids and glycerin.'tne
ratty acids,the alkali and the emUlsifying enzyme act on another
portion of the fat and form an emUlsion.
TRYPsIN.oonver. s pro
ds into peptone in alkaline
or neutral
solution,acting
most energeti~allY in a weak alkaline mixture.
The proteid is first con~erted into alkali albuminate,~hich is
then changed into deutero-proteose;no
primary proteoses being obtained.The deuteroproteose is hydrolizad formin~ peptone,similar
to the peptone of gastric di~estion.This peptone (A~PHO-PEPTONE)
is split bY the further aetion of trypsin -formlng a pe~tone of
lower molecular weight,called AN'n-P£t~P'l'ONF.;and
amidO-~,cl.ds.
AMIDO AC1DS,rormed
aspartic
bY triptic
acid and tryptophane.
'l'YROSlNand L"~UGlN,are described
duets of pancreatic diKestion~
Asparti1 acid is amido-succinic
#
dl.~estion are tyrosin,leuein,
w'ith the reactions
acid
of thO'
produes
CZH3NHZ(COOH)2
fryptophan is a chromogen rorm~n~ a viol~t color with bromi~e •
The quantity of acids formed from tne proteid 1S about equal to
the quantity of anti-peptone formed,indicatin~ a splitting of the
proteid mole~ule into halves,the HEYl and AN~I ~roups.
't'hehemi group is most prominent in hetero-proteose,and
if this
proteose be Obtained pure and a~ted upon by trypsin,th8 hyJrolysis
~ill result in the formation of-more amjdo-acids then anti-peptone
The tript10 digestion of protoproteose g~ves e result the
reverse o~ this,showing the predomin6nc~ of the anti group in the
molecule.~he digestion of sU~h SUbstances as ~elatine and antialbumid,which do not form he\erO-proteose,will
form anti-pe~tone,
and no amido-acids-
Artifi~iallY the anti-peptone can be hydrolyzed
osses,and
some other undefined ~ompoun4s.
forming h1ston
'rHF. HlS'l'ONB ~SES or PRO'l'Al41NES,
arg inin, h is t t o m and lY6 in, are
for~ed Whenever a proteid is pushed to the limit or trypti~ digestion.~hey are looked on ,as bein~ the essential nu~leus of the
proteid moleoule,the amido-aciJ,and other ~roupings bein~ joined
on as side ehains •.
The hlston bases oocur mixed in 41fferent proportions in the
s~ermatozoa of salmon,stur~eon and some other fiSh,and are easily·
separated from another by hy~rolysis.
LY~lN,the first base Obtained from a proteid,is di-amiJo caproio
aela. C5Hg(NHZ)ZGOOH.
ARG1NIN,so called from the beautlfil compounr it forms~ith
silver
nItrate
1.
a .con~ugatlon
.
.
.
\ ~s
of urea wlth
ornithin, (di-amido ~aleric
_ Le Lds on hydrolysis
.
•"he COmposltlon
of arglo1n
18
aCl.d),
'1t'.l"h 1 t y
. .
.,.
NH2CNH-~4H?(NH2)2GOOH.
~
f;
Histidiri,has not been assigned
formula is CoBgN3 Z.
0
a structural
. 'J'heseprotamines \'lill respond
tlons.~articularly
the biuret,in
formula;lts
empirical
to se~eral or the proteid'reacWhich they ~ive a pink color.
.
• t
When a concentrated
solution of protamines
bodies are precipitated.
hol,these
is
poured
into
aloo-
Tn a natural
digestion
the absorption
of peptoDes,~EI;kes
place
b~fore th~ trypsin ean form protamines.
p
t a JUi~e
ompuon d
t a
t
on
tr.(~ r esut t in~~ compounds. '!he s i mpLe flroteld
is cb ang e d as above.
tn the aBse of nucleoprote1ds,
the nucleins,true
and pseuJo,
are dissolved
and hy,ir olyzed.Coll~gen
IS not directly
aigested
by
trypsin
but only after
oonversion
into gelatine,which
is easily
oon ver-t e d into e:{elatine p ep t on e s Fd as t Ln is cria.nge d as by the.
~astr1e
JUioe.i'eratins
art:' not decomposed.
The
an
c
r
e
a
c
splits
APTIPfCIAL
Preparation
of the
the
P~NCREArlC
products
Plaoe 250 grams of fibrin
c
of
p
r
o
e
ds
and
a
c
s
DIGESTION.
trypsin
proteolysis.
in a "2 liter bottle;pour
upon it
~ ceo of dhloroform and add 5 ec.of
one
liter
or water
~ontaining
&atur at ej s o or um c ar eona t e solution
and either
~~;or :-1 grams of fJan- ,
or e as po\vder or pancreas
pUlp.Shake
thoroughly'
cork,anl1
!Jh'"ce in
e thermostat
~'J.t 40 C. for
t en \1ays.rrr-'8
mixture
must remain alk3.line,and
oa c t e r La j criang e be prevented
by the a da i t Lor, of t nyrnoI
or 0hloroform ,
if ne"'l~ssary. t,t t he
en a of t hs f' r r s t :4"k hours,
fi1 t er a few ~C. of t he digest ive mi xl ure an.i to t h e r i It r at G ad d
hromine water.-~
violat
color
lndloates that digestion
is progress
ing. (8romine or tryptophan
~eaation.
)
.1t the end of ten days pour
the .son t en t a of t h e bottle
rn t o a
s auc epan ,aei,tulate
fA.i.ntly ~1.tn acetic
a.~i<itheat to boiling
anu
Llt0r.F;',apQre.te
the filtrate to about I5{)-200 co.and a I Low to
S~Etnd. in a oo()l ~~ace. Within
a ff!I¥ no ur-s a large
quan t Lt y of tyros an W~ll be c1epOS1t-e.d,usually·
in the fum of very fina needles.
Decant the mot ne r itflucr
(to be use.' in th~ i-'l'epa.ratioo of Leuc t n )
from 1h~ t yr os In thro ugh 1 inen t ';ash hast ily bydeeantat
ion ,'lith a
small quantity
of eol,t w'ater,brinJ
into a small flask,andiissolve
In about 100 co.of hot water to Which a little ammonia has been
added.,f"ilter
tne aoluti.on
In an eVBporatin~
-hSh,heat
over the
tVaterbath
until
'"mmoni..a has blteD dri"en
off and allol'l to cool.
~:xamine a. few or the ~ryatals
<if tyroOBill UI1.i-ar the mloroscope;
'~oll':lct the remai.n<l~ran
a fIlter
~ash 'Ni-ttl ~ter
and dry.
Ty'rosin 18 r,groxyprlenyl
ami,1o pro~rio.ni.e
a-.:~d.
.
'"'H ~. OH
v
I,!}
4~.... C .. H (NH
~~~j'
2.
) eOOH
Tt IS sraringl,y
801UblH in 001,1 water,more
SolUble.in
boilin,J
VI
\rater,~llTJost
Insoluble
in str~>niS al~ohol
an,: quite insolubll/' in a
ether •.. t IS rf>dl1"l;' solu"le
In a 'I' .•
i
lk l'
.
',.s,an: a "8 1es,hn'!
In solutions
Of alkaline salts. oJ
.
•
J
U
-
(j
,t>
f
ea",tlons of tyrosine
Place a few erystala of tyrosin on a slide and warm gently-th6Y do
not melt.~at ~ryste18 found in pus resemble tyrosin in ~en8ral
appearan.eebut
melt "hen warmed,and are insoluble in hydroohloric
act.1.
To a ~ery small quantity of tyroain add water and a fe~ dro~.
turns rose-rad and upon
or J4illons reagent. On boil ing thetmixture
dert..tondin~ a aeepe~ red color deyel,pes.All phenols and their
et1v8S ~ive this reaction.
Perias reaetion.-Place
a small uantity
of tyrosin upon a \Vatch
and h ~a.t for
ti!lass,adJ & feW' drops or concentrated
sulp.huric
aeld
an
d pour into
hale
an hour over 8. boiling water bath.Allow to ~ool
,.
1f -ce. of water contained in a porcelain evaporating dish. 'farm
"neutralize
with powderod barium carbonate,filter
while hot,e~aporate the filtrate to a few au. and a~d a Yery dilute ferric ohloride
solution-a violet color.
f'reparat ion of I.tF:UCIN.
t'~vap(lrat
e t ne mother liquor decant ell frOID t he tyros in er ys t aa.ls,
,
over the water bath to a thin syrup and examine microscopi~allY
the seum forme~ on the surface.Tn
this impure form leucin usuallY
orystalli~e.s
in r01Jl14.4 balls
"hi·~h s now radiatlng linet.hBri.ng
th8'
mixtur6 int~ a tlask,pour u~on it se.erel times its volume of ~O
p er-e en t alcohol,
heat on the ivat ar bSU'1. alloW to eoo I and r i.1tar.
Pl'he alcohol1e
extract
contains moat or the leucine F,ve.porate the
al~ahol on the water bath,dlssolve
the residue in water and boil
~ith freshly
prelJared lea,;1hydroxilie.Allow
to cool,filter,rree
the
(1ltrate from lea4 ~ith sulphurette4
hydro~en anJ evaporate to a
small volume.rha leu~in ~hich 1s pre~1pit8ted on standin~,~~ best
separated
from the mother liquor by transferrin5 to a porous clay
plate.
Leucin (ami,1o-isobutyl
a ee t ae acid.)
G4H9GH(NH:3}COOH.
is a ~onstant produot or the rtecomposition of proteins enzymes or
ott'Hr hydrolyt 10 at:ents. As usually O-btained, in animpure form it
cryst.a.lli&ea 1a r.Qun~1ed lumps \,hi en orten shO'" rad-iatiug IS t r iat ion
When pure it forms White ):Sl.i.tteriD~
flat crystals • .It 1$ easily
soluble in hot weter~le •• alQ 1!'1 ~C)ld \'ater.s()l~ble
in aloonol,insolu~le ia eth~r.
Reaotion, or leucin.
in a .;11")' test t ube a ver·i 8ID811 t:'0rtionor leue1n, it
SUblimes 1n the form of woolly flakes. te nea t e c ab""'e its meltln~
point (170 C.) it decomposes into ~arbon Jioxide an~ amylamine.tne
latter sUbstance ha~in~ a onaracterist1~ odor.
Heat slowly
Place a little leu~in in 8 4ry test tUbe. add a piece ot causti~
loda(stiek form) ,one cmJlong an~ 8 drop or two of water.Heat until
lhe caustic me •. l'ts;ammonia is given orr. Allow to cool,dissOl.~
111
l 1it t Le water and ac1~ulate with 11il ut e sulphUr ic sc·t a-the odor
.r valerisn10 acid ~s notaiceable.Leucine
bY this treatment take.
ip oxygen and rtecomposes into valerid acid ammonia and carbon di)xide.
01sso1ve a portion of the leuein in water,d~colorize
if necesskry With animal chareoal,filter,make
alkaline w'ith caustic s-Oda
and add 1-2 drops of copper SUIPhaie solution.1ne copper hydrate
({hiah is precipitated at first diSS~lves on Shakinl3 giving a blue
solution Whi~h is not reduced on heatin~ •
•S~herors reaction~(OnlY applicable to Yery pure leucine )Eyaporate carefully to dryness on a platinum foil with nitric acia 8
small portion of leucin;the residue will be almost transparent and
turn yellow or brown on the addition of caustic alkali.Ir this be
again very carefully ~oncentrated with the alkali an oil drop is
obtained,I'fhich runs oyer the platinum in a sj?heroidal state.
DTASTA,'IC
ACTION Off'PANCRRA'fIN.
Prepara some stareh paste (A) as l.1irectedun<1er salivary digestion.Dissolve otte gram of paner-eatan in 500 cc.of water(B) and
after s t an dang at 40 C.for two hours,filtertha solution.i'ix in a
test tube equal volumes of (A) and (8) and heat at ~!40 C.'l'he
material becomes transparent,reduces
Pehlin5s solution anJ is not
colored blue upon the addition of iodine solution.Repeat the test
using a boiled solution or pancreatin.
The ~ancreatin solution itself Should be tested ~ith Pehlin~s
solution as the commercial article is (re~upntlyadulterated
~ith
sugar.
Recognition
of a fat-splitting
Ferment
(SrF.APSI~)
These experiments must be made with the fresh panoreas.
Shake a gram or two of butter,
fat with a few ee -cr luke
warm water to which one drop of caustic soda has been added,and
after eooling skake with an equal yolume of ether.P~ur oCf a
portion of the etherial extract on a watch ~lass and allow the
ether to evaporate(neutral butter fat)
Obtain a pl~ce of fresh pancreas the size of a pea and mix it
intimatelY by rUbbing with the butter fat on the watoh glass.CoY~
with an inverted watch glass or t h s same size and place in a
thermostat at 40 C. In a few minutes the odor of butyric
acid may be recognized.
nrind part of a pancreas with a little water to a thin paste,and
divide into two portions; (A) and (B)~Boil t c destroJ the ferment
th~ portio~ (A).Shake a gram of butter fat with luke warm water,
add a few drops of rosolic acid solution &nd enough dilute caustic
soda solutio~ to turn the indicator.Mix equal quantities or the
Cat mixture'~ ~iA)' and (B)Ir the resulting mixtures are not plainly red add drop by drop dilut e s o d i urn carbona.t e sol ut ion. Ke3p th'tt
mixture at 40 CQ for 12 hours.The mixture in (A) does not ~hange
its color; the mixture in (8) turns y-elloW'due to the acid libera
ted frolI!the butter fat.
In a similar manner the d1astatic and fat-splitting properties
of cystic flUids,l'{hichare thought to originate in the pancreas
maybe
t asted .,
'rH~~L1VER.
The functions of the liver are digestive, excretory and assimilative.Tts
digestive and excretory functions are carried out
largelY by its'secretion-the bile.tne digestive action of bil~ is
very slight,being ~eaklY dlastatic,anJ a.iding in the emulsifying
~r fats ..
As an excretory organ the liver is very important.It removes
thO effete haemoglobun,which
it converts into bile pigment,and the
waste products 9f protein metaoolism,som€ of~hi~h
are formed in
the liver, itself,oth'21rs are der aved from theblood.'l'ne liver also
has the valuable function of modifying or destroying eertain drugs
and food products,dangerous
to the organism.This fun~tion is of
the utmos t irnport an ce in prot eet ing the bo"iy agains t a ser ies of
more or less poisonous products formed during digestion.
Tn respect of nutrition its functions are most varied and complex.,'i'n
e liver elaborates and mOttifi.esalmost every product of
digestion brought to it by the blood through the portal vein;
changing not only the peptone and sugar,but also tr.ansforming the
products of deeper cleavage,the amido-acids,histon
bases and ammonistand carbon Jioxide?forming urea and bUilding up more complex'
bodies.
This activity is evidenced by the formation of glyco~en,the
excretion of bile acids,and the forma.tion of urea. Urea and uric
acid are found in larger quantity than in the blood,th& xanthin
bases,lactlc acid ani cystin are found in considerable quantity.
GLYCOGEN.
Gly;~ogen is a poj ys ac cnar Ldej hav fng properties intermediate
between starches and dextrins.'l'heliver builds up 5lycogen Cromthe
•
simple sugar absorbed
form the carbohycrate
from the d1~e.ted material ..... a rule. but oan
\fhen the ..
te~ is free trom this materia,l_
It o.csur. most abundantly after
a '!l~ftl
ri~t't in carbohVdrat •• ,
rea.hing
it. maximum 14 or ttl hours after
eating. 'l'he an'ora~e quantity or gl)'cogen 15 from 1 to 4 percent
of th.•·liver v/eil!Sht.Aamall
quantity of glycogen occurs in the mus~les .and its presence can
be dete~ted in almost e~ery tisaue ot the body.
<Hyeogen is an anwrphous \vhite t88t61e88 and o.lorless
k>0\fq.r.lt
ia solUble in water,the solut1on
is dextrogyratG.5p.r.I~J.;~.lt
does not reduce Fehlinge solution,and
i8 not decomposea bY boiling
with caustic alkali.By
\Uastatio
enz;}tmes it is eonvt~rted inlo
maltose.and
dextrose,end forms dextrose when boiled with dilute
acid.
Preparation of glyco~en. Mince 50 grams or tresh liver,and add
to it ~500 cc.of boiling
water conta1Din~ a small quantity
of
aeet1c 8~id.Strain the~paleScent
solution
through muslin. The
solution
contains besides
gly~ogen,some prot6in.~0 free from the
'letter,conoentrate the solution by ev.poration
to 8 small ~olume,
and 844 a1ternatelY A. few Ilrops or hyc1ro~hloric acid and of potassium mer~uri~ iOdide as lon~ a preoipitate
is forme4.Pilter a portion or the liquid,and
t es t with 8oci4 and iodide
i .tQIDai(e sure
that the protein
ia completely removed. When this is tne oase.filter,rirst through mualin then tnroufSh paper.alld add to the
filtrate
2 volumes or allJoho~tirring thQrQu~hl)i.Pour orf the
aupernatant liQui j an d 1t8.sh the lireoipi tated glycogen wi th ;;5 per
cent aloohol by d~cantat1on.and
cov.r ~itn abMolute al~ono~or
an
hour or more.Colleot
tbe ~lyco~en on • f~lter an~ dry between
rol~
pa~er.
or filter
Reactions
or
~l Y"4ogerh
Pissol •., 8<1•• tllyoo~.n in \f8ter bY ~.ntl.
.solUtioD-rea.blahoe
to solUble starch.
Tv a
portion
of
tfl
fJ
heat. Ano~e.le.c:ent
8Qlut ron a4 a few drop.
or
iodine.
A re44ish
brown eolor for ... -Resemblance to erytbrodextrin.
'Hotl. Gother portion or the sol ut ion with Fehling. solut ton, -no
re4uot1on ooeura.
'1'0 some gl,.40tfOl1 solution
a4 a rew 4ropa of dilute hydrochloric
"e.ei~ an~ bo~l;"n~ol Mel neutrallae.'fo
erie portion ad iodine solutio
to another a4d 'ohl1nga solution and boil·.'rhe glycogen is s"lit
by
this treatllMitnt.
To 80m. glyoogen solution a4 Oft$ co. Qf _al1v. and keep the mixture at 40 c.ror ten minutes.At the end Qf this time test a portion or the solution with io~1ine;another portion with Fehlinge.
r
'rrie bile
is arm x t ur o r)f the s ec re t Loris or the liver
cells an4
t~e muous secr(tert by the tlanJs
of the biliary passages
anj by
the mucosa of the gall
blc,!inr.
\on;n:;l
bile is a t.;olJor! yellow to'
olive
greeD colored liquid,uith
a faIntly alkaline
reaction,and
a
sp.g ..ofabout
TOIO.'f'he avorStse daily
quan t t t y ts about 760 cc s con-s
tainlng about 2 percent
of solids.
Its chief constitu8nts
are :_
Bile pigments~bilirubin
ana blliverdin.Bile
salts,glycocholate'and
taur~Cholat8
of sOJium MucuoUS,Which
ISB
complex muaoid nucleo~rot0iJ.~r01~sterin.
Fats inaluctin~ leclthins,and
soaps~Inorganio
salts;aLout
~8 peroent,consistin~
of sodium chloride
and phosphate
with smFll
El:1Ount
of SOodi.lln c ar boriat e Lr-on pl'losphate,c81-cium
salts
and occas ~n.El1y
coppel".
The'secretion
of bile
is con t t nuo uaj eue a t s d i s etiar-ge into the
duo4<en~ is intermittent;the bile b~ing stQred
in the gall-bladder
unt il us.e a, '['he rate
of flow' u; irr'6gular. and influenced
chiefly
by
taking food,increasiny
4W"i.ng 4ig~stion
and fal1iJ:1~5 wh en food is
withheld.The
dri!l1kin~'Qr
large quantity
O!,'<"Fter
;~auses ::tII;
6
j
iner~esed
flQ" of 'bile,and
intestine
is a po.~rfUl
the
stimulus
ab ao r'p t Lon 'Of f)i.le salts
to t ne s"tt~l"'oeti'O:n.
from
the
'l'HE BILE S~I;'rS AND ACIDh.-" are the SQ,,1iua ae.Lt s (If ;;lYl:o.;ooli.c
an" taurocholio aaitts.On fl;V'1r'Glysi.s glY':Q4hGli~ ri.:.iJI yie.l4s oholalioac
id and glyco~o11 {ami.1o-aeet s e HcirlJ" T.l'n':r-vo~ol.i.c a-ctd yiel dB
In •• ~.i t j,(:ITJ t c cho La I ic ac 1d t aur- :i.ne (ami JO-'lt'hYl sulphonic
a~ul).
Ch'OLAL t o ;J;'tlJ o c cur-s in t ne ur in e Ln i:;{~rU3 ~its
compos it ion is
C Iif
CHOF{~N
~~,~ti
(~InOOOH
2:3
..
'l'he t"'st.~s :"0"" ttl,,, r<t..~~)gniti'O~
Qf
th~:Me at:i.J::; c.rC given
,rith
the
,aostl;y'
the.
4irect1~ns fGr the e~~~liDntio~ af bile.
BtLE PIG~'''~S ..The bile
pigments ar{:# Dl1m-'::1"QUS b'lt
".ult of post mOl't(~~ ~han5e,or GeoOt1t' in ti:'-e$I(!l!CS
e.a11.¥ Th3 important
,-..Ii, ';';".'.18nt-iil P;l'r,
'i~l
"r">"L'n
,-;;-,"'1
h;!
.fC' ~
....... "...... ,_ s.
""~.....
_...... .., .....
'-J,
fiIleIRUBi:N, is a reddish
it Gloaely l'€S81T:lbies,.lt
reaet i.«1s} •
yell~w
tdi'l1es
',"
..
oJ• .J-
c;,r'~
..
?'hY~'Siologi
...
"iV"'l';'.'...
,,'w
."
'.
"
.~.
p.i.g~nt ~ deri.ve~ (:'011<!k1.e.t:~etinlllfh.i.oGtl
nur:Jle:t""ou..sGOhH~"S w~en
~){i.~.!.7thi
..
BILIV'RHDIN is form~l4! ~eQ ~tn ~.lkal ine 'Solut Ion 'Of bil iru-'in
expOsed to the air,or otherwise mil~lv oXiJized.
!Sdo;'
1a
r;.aner2.1
\iiHh a
(A).Th~
(8).
'./
)
",.l..
-e
'J
t:J ....
.. J
bile
<>
car
'.J
tn e follo;~Lli~
of bile,v~rifY
portion
reaction
of
Mo i/8;ipitate
1\1 'or'o"l
;}lV':OS
i"1
(.
tests.
Sl:i811
either
_8
neutral
is pro(lUced by
prer'i(litate-Lluc;in
sli~htlY
nl~all~e,
boilHlg.
nucl..::-al(1)rnin,·J(!lorl.~.s
.
.rct- ..
1
t'"'"
~.I
or
st[",tCJji;'l~ll;=O;
t',3r~bile
8P..lt3
<.~f!'
(D). ~lkBl~as
~h&nie the color but ~o not produce
(E) .e ~CL'Sih\3n
8d<.;811. slowly
i)reclf;ltatl~
c,t fIrst
a fJreC:~jJita'>::"
b.;lG
IDucin:tn;::';:1
I
gaT i \lID ~ h lor i ,1e ;
( H'),
I cholie
.,' e c o g n l't'1'
(}n of
'10 200
c cs o f
en
ad
Ij e
(1
t0
a precipltate.
aciO:i1ves
(G). the salts
of
h
l'/h
the
- bile
salts,<.1.nd
the
ox bile
metals
heavy
In
an
an alkaline
ti.Le
·.>,V8,poratin~
a o r os
e
dish,aJd
:jO,J;lDS
Gh~l.r;~081::"liX t nor ougn Lv an1'~vaporat9
to .rr yne s s
,'''',t''''''.
bat'
h P'~"rlO<'C>
th'"
..n v j":'><.!ldUpc"D'\
\10w,19r
as finely
~:J. -;'j
v \J
• ,'--'
u.,;
......
~.,
.~ 1 ~. -'
t"'
.
1.
j
.. 1 •. ~,
....
,1
_ij~nir15
in general
D'FJ.l
\
,;Uuf
sulutiun
ovo,:
8:::;
~....
or
t
an i:
no
,j[.:.,'.r"j,i:l',~
-
mortar1place
in a dry fla.:;k;an<.t
extract with 8bSo~ute
al,:;~)h(jl
~eatin~
o~er a water
bathaAlloW
to cool;f11ter ·into a dry fl~sk~
ad~ 8n~U~h ether
to produce
a permanent
cloudinass,0ork1anJ
allow
to stand in a cool
plate fur 24 hours.~he
blla salts crystallize
In the form ~f White silky ne3Jles(Plattners
crystallized
bil~")
Co Ll o c t t h a e r ys t a l s on a filter
anJ dry,Use a erie per cen t sOl: ci,,:,
10
8.
l
t ion tOl'
t n e fe,llo\"rinr!
t es t ,
Pettencoffers
test, '10 e r ew c c s of
:) drops of a ten percent
solution
tUbe inulineJtallo~
three or four
the solution
in a test
tUi.,e:u-1(i
r
of cane sugar,t 1en
hclJtn,s,
th'~
oe~of eonoentrated
sU1J~uri~
to flo,v do"m the side
so ttU'l.t the acid
forms
a 1::<:"<31' Cit H,')
bottom."·]h"lre
th-3 liquids
meet a purple
violet
col,)y dGv:Jlci-,e0~
Holdinf~ J:rl'3 tU(J8 un(h~r a stream
of cold '.vater
,,::r,?,Ju811\
mJ.x ::'h'3
contents-a
dap purple
solution(avoid
much hsat).lnto
82Gh of two
test tutes (A)
c~ntatning
a few ccoor ~12cial
acetic acid,anJ
(B)
;;onta.:Lnin"J
a fe'~'l ,~;.;,or
alcof10ltJ.Jonr
Fl sma,].l l.;llentity
of tfl.'::: lJurjJle
"oi,j
sol ut ion.
The solution
in
,~)
s~aatro50o?ically;~iv8s
~hOWS a ~reBn
fluorescence;and
when eXEmined
a bnnd in ~rBen.The
solution
in (8)s~ows
imffl8.:1iately
after
m:i'iHn6
th';; same ban:1;iJjJon
8taPdin!~
the solo.ti<c~n
turns
br'own;nn<:t>";ive8 two bands-one
in io5reen;the
ottll-:)r in clllBo
Pet tenkoffol's
test
1S 15iven
by thebil'3
direct ?RDLl
due to the presence
of cholalic
acid.
nl:-COC]lOIic
aci,j;{''Ln
be si3tJarated
from taurocholic
ing to a watery ~olution
ether,snd
enouL5h sulphuric
is
3~id
a;..;parentlY
bY:idd-
of the
bile
orystals.a
few drDps
of
acid
tocause
a ~louJiness.On
;3tHnd.in,~
such a mixture
ja~oslts
fine needles
of glYCOChOlic
acid.
When bile
is boiled
for several
hours
with
hydroChloric
acid,
for
Seversl
hours I a dar~: resinous
body is thrown
out of tn~ solut.ion
..
'I'his black
bOuY~\"hich
becomes
brittle
on coolin,-~,ls
dyslysin1T.ile
e.nhydri ,~e 0 f"~(..) 1 f' 11, C 8 C i ,1" 'l't- '''! ." ],;(",0' r:~)l i'~ ',r, d t ,?u:" () r;:'::..l i. l:: f' C i,18
are hydrolyzed by tH.l~ l,·'~~il. :c~to':n::;
e;l.~(;();;oll8.n:J
t.aur ar.
formed
are left in the solution.'fo separate them the solution is oon een-e
tratec a.nd poured into 15 volumes of alcohol,W'hen the taurin is
precipitated.
t
-
EXAMINA' JON OP BTL1ARY O~LCULt.
Boil the freshly powdl3red stones with water in order to r emov e
tra~es of bile that are usually present;fllter and allow the residue to dry"F.xtraet the dry residue exhaustively with a mixture of'
ether and aleohol(equal parts by volume) filter each extract
through e dry filter into a dry flask;and evaporate the united
extracts to a small volume.On standIng or possiblY during the
~vaporation,cholesterin
crystals are deposited. (Examine under the
m i c r o s c op e l
0
free the cholesterin
from fat, dissolve in uO fH~reent aloohol
(25 ce. )add a small piece of caustic potash,heat in a flask
over
the water bath;transfer
the contents of the flask to an e~aporating .1ish an d evaporate
to dr yness .1'ake up the res idue in water; and
shake in a separating funnel with ether.Separatethe
~therial from
the alkaline fluid;and allow the former to evaporate.
1'0
on a slide;cvver with a cover
51ip;ard
brinle(enough sulphuric
acid in e on t ae t with the edge of
the slip to fill the space between the two glasse~.Then brlng a
drop or two of iodine solution in contact with the ed~e of thQ
51 ip-tt-8 c rys t aLs ar e colore d graduall;'y' er own then v ~olet light
blue ..gr aen ,86.'rhese colors do not al'l{aysoccur in the order gi'fen
(A)
Place a few drops of cholesterin
often p rec ee ded or followed bY others.
Place a few c rys t.a'l
s of cholesterin a n a small porcelain dish,
with a few drops of hydrOChloric acid,and ferric chloridecEvaporate over th~ wat~r bath. (Blue color.).
(0). Dissolve a little cholesterin in a few ce.of chloroform;add
3D equal
volume of concentrated sulphuric a~id~and shake thorough1.y.'l'he
sUlphuri~ ac i d solution un der' the chloroform exhibits a
~reen fluorescence;tne
ohloroform solution turns first red, then
violet.Tf a little of the Chloroform solution is poured into a wet
test
tUbe;the solution is decolorized;if concentrated sulphurio
Ecid is then added to the contents of the tUbe. the original color
and
are
(8).
brought ba~k.
Tn & very dilute solution of cholesterin the reaction is someWhat different-yellow at first,then
red coloration of the chloroform~yel1ow coloration of the acid with green fluoreseence.
iD}.TJiebermans
reactiono·-Dissolve
a IHtle
cholesterin
bY heating
in acetic anhydride;and after cooling add concentrated sulphuric
aciJ.The mixture gives the eolors;~ink,red,blue,and
fina111 green.
IS
are hydrolyzed by this treatment.The glycocolland tBurin
formed
are left in the soLu t i on vt'o separate them the solution is concentrated and poured into 15 volumes of aLc onc Lj wnen the taurin i~
pr iC! C 1 pit ~'ted
,
I
0
EXAJ.'. 'N.A'l'ION
OF
81 IJl ARY
CAl.CULl.
Boilthe freshly powdered stones wa t n water in order to remove
traces of bile that are usually present:fllter
and allow the residue to dryeRxtraet
the dry residue exhaustively With a mixture of'
eth~r and alcohol(equal parts by volume) filter each extract
through a dry filter into a dry flask;and
evaporate the united
extracts to a small VOluffi8.0nstanding or possiblY Juring the
avaporation,cholesterin
crystals are deposited. (Examine under the
m.i e r o s c op e L,
'
r r ee the crioLe s t er f n from fat 1 dissolve in bO percent alcohol
(25 co. )add a small piace of caustic potash,heat
in a flask over ths water batn;transfer
the contents of the flask to an evaporating dish an d evaporate to dryness. Take up the res idue in wa tar; aftd
shake jn a separating
fuunel with ether.Separate
the etherial from
the alkaline fluid;and allow the former to evaporate1'0
(A) Place a few drops of cholesterin
on a slide;cover
with a cover
sljp;and brincl enou~h SUlphuric
acid in contaet with the edcle of
the slip to fill tha space between the two ~lasseH.rhen
bring a
drop or two of iodine solution in contact with the edge of th~
s I i p-r t ne c rys t a I.s ar e colored graduallj' or own then violet 1 19h t
bl u e ~gr e en , gB. Thes e colors
do not a Lways occur in the order g 1v en
and are often preceeded or followed by others.
(B).Place a few crystals of cholesterin in a small porcelain aish,
with a few drops of h~drochloric acid:anJ ferric chloride.Evaporate 0'1 er th,' Ws; t er bat h ~ (B I u e co rcr l(C). Diss()lv8 a little cholesterin
in a few cc.of chloroform;add
an equal volume of concentrated
sUlphuri~
aciJ)anJ shake thoroughly.The suipnur tc a c i d soll1tj.cn':nder t rie cn Lor of cr'm exhibits a
~reen fJuorescence;the
ctlorcform
solution turfis first red~then
violot.Tf a little of the Chloroform solution is ~oured intD a w~t
test t.uee j t n e s o tu t ron is decolorizea;if
concentrated
su Lphur Le
Ecid is then sdded to the contents of the tUbe, the original color
v
~;rought ba ck .
In e very dilute solution of cholesterin
the reaetlon is someWhet different-yelloW
at first,then red coloration of the ch1oroform~yellow coloration of the acid with green fluoreseence.
(D).Liebermans
r8actioo.-Dissolve
a little ~holesterin by heating
in acetic anhydrid8;and
after cooling add concentrated
sulphuric
aciJ"The mixture ~iges the eo1ors;~ink,red,blue,and
fina1l' gr~en.
is
· ~he residue of the stonRtinso~Uble in ether and alCOhQl,aonsists of the inor~enic salts,anJ ~ile pigment.
W&sh the insoluble r~siJue ~ith ether,cut off the upper part of
the filter papRr on whi~h th~rA is usually somA cholesterin;and
wash ~
residue with about 20 e e s of' ,Hlute hydrochlorio aci..",
pourin~ the filtrate repeatidlY upon the residue.
The filtrate contains ~alcium salts,recognmzed bY addingammonia
aeetie at;}idand ammonium oxalate;possiblY traces of copper,reeognized bY addin~ a few ~rops of potass1um ferroeyanido to t~a hY-'
drochloric acid solution-~uprie ferrocyaniJe.
Wash the r':3sidUerernaitlin~on the filter paper 'nth wat er until
t r.e 8si.j is remove:i;~lf'.eethe
filter a nd residue in aNurm place,
illth
unt i I dry;eut into pieces;place a n a cr y ~:rlenmyer flask,'ofarm
B I1ttle chloroform, and filter through a dry riltero~he
filtrate
dontains bilirubin. pour a small quantity upon a watch glass$sllow
the chloroform to evaporate spontaoeoUsly;anJ
examine the residue
mieroscopically-ill
d,efined rhombic platas,or prisms-
GMELTNS R~ACTION
!
This is extremely
delicate and may
bd
~pplied in various ways.
fA) .. /:J.ft3W c~.of the chloroform solution are placed in a test t uo e
and an equal amount o t' yello" nitric acid added bY pouring it down
the side of th~ inclined tUbe in such a manner that the liquids do
not mix.At the line of Junction colored rings appe8r,bain~ yelloW
nearest th9 Bcid;and progressivelY red tViolet,blUe,greBn,passin~
upwards-
(8). 6 few ~rops
or
the solution
are placed on 8 porcelain slab;
and a (lrOp of yelloW fuming nitriC aeid is brought into contact
with it. A play of colors is observed at the Junction of th3
flui(1s.
(0) _ :::;hake
:Jart of the ehloroform sol',aion,'1ith a c aLu t e so lium
carbonate solution-the coloring matter is taken up by the alkaline
sCiJ!utionthe chloroform losing its "olor more or less eornpletelY·
~his action of its ehloroform solution~jistin~uish0S
bilirubin
v
from oth&r pigments found in the animel'bod ,
(D).
Exp ose the 8,1:<',,'1ine
solution of bilirLl'~ir:
t o t h e [11",'
::,D
open vessel-it turns green due to t ne fUrL tic, 'J U;" 0i~i',
r .i n ,
BI.,OOD.
'}'heblo()'Jcan e e e ons t uer ed 8.S a fluid tissue.'fhB cell C;LIi:;
suspended in the transparent liquid intercellular sUbstanc8,-the
plasma.
~he blood is alkaline.The alkalinity ,oelaulate4 as sodium carbonate IS about .5 ~ereent.The alkalinity is due to tne presence'
of alkaline carbonates and phos~hates.
�rhe specit'ic
gra-tity'
as
Tr.Hw..'ht bY &.dding A. drop of
zol; ir -t"t sinks to the bot
floats
on t~~ surface,add
liqu1d.~hen
determine the
'ltt.'" oonstituents
of tne
'1esaels anti organs,markEhtlY
blood oonta1ns 22 par~ant
ide, bY volume: venous bloo.t
betwe~n I~o
and 107';),8.n«1 can be deter
blood to a mixture
of bhl(#TofQrm and ben
tom A.\tet mora ohlorotorm,lf
trt& clfop
benzol;unt1l it floata m1d~ay 1n t~.
ap.g.of
the mixture.
bloo,1 ,vary,in tt'H~ blood of di.ffereDt
in t ne veins anu arteries."he
art'3rial
Qf oxy~en,and ~O percent of uarb~n Jioxcontains
Y.(u 5 fJercent 0, an.l "'b peroent
CO2• "'he ~a.e80CI1Ur in loose
oomb1na t ion nod t'ree in the plasma.
when the clood of ma~1mals 1~ remo.ed from the vessela it is
converted"more or less rapidlY ,into a Jelly li~e n~s.,bY cosgulation.~he ohan~e is the result
of the aotivity of a ferment,
thrombin,existin~
in the blood in an un4 •• elQpe~ form,on f1br1nogen.formiDg fibrin.The fibrin eneaOK1 •• tne o~rpua4le. in 1t~
rn8.~es.an4 tl'\sre ia '~r8duall, pr ••••• out. ol.ar Y~11ow rlui~,tha
blood serum.
1he coagul.tlGD or ~lgQ4 •• n ~. pt•••nt.4 in •• rlou.
ways. After
th13"iraJeotion Qr a1b .... e intg a •• 1n or a tkI.s.t.h"bloolo1 do\}f.inot
coagulate on le •• 1D~ the ~od1.Jr t~. bloo4 be ~aWD 41re4tly
into
a satur.ted 80lut1on Or _.ntl.twa auljtllate.inth., proport1un or
a ·tot.or1nto a .olutton ot jiU, .....
v.ala'e.80 that the mixture
<Jontai". tlt l ••• t. t iJere..ftt or o.al.,e.no'.g-..culat t-., tale. pl ••••
The ~l...a obt.1b~d rrom auth ~Ioo•• i. &no~ .a pepton.,.alt
an~
oxa1a'te plasma. Tf th~ b1CXld ia De~t.n ,*Ul"ln~ ooafjult..
tu,ft, t ne
fH.rinatJparatss
tn thr<ta4. or
th~ 11'i\1141~ ,1Hfibrinate4 ~l00d.fhG ~r6.8nt1on or oo.~ul.tlon
of ~luud by .alt_,is ,tue
to the pre.ipt tat iQn or 4t8141um.
-.
The b1004 oell. arll r~4. whlt~ .Ul0 Uut ~lQv,t plaqu" ••
'fhe ro4 oOell_ !)ontain 1'l.U.~~lODla.
a oom~O\ln(ipr.ot.ll1, 4vn\a.1Il'.l'n~
_irGn~a,n4 h •• lhg U'I& prop~rt)" of rOl"11l1Dat a lQQ. t! OCiHDblnatJ.vn wl t: h
Qxygen.~~e dlrrerenoe tn oolor or .enOU4 an. a!"tarla1
01QQd 14
(ftl\".ea,an,1
lar~(,ly
ttue t~' the
dlrr.rentn.&4IOIVglotl.i.n ••
~Qntaln .lar~. ~unt or nU~1.o~rot~14,~~~
The 1~u4ooyt8.
.,lyoog$n an4l!1 tl".48 of myosin.
rl'h,~ pl&81l8 oonta.tn.
3 ~rotlJli1.tOn.
'
~lfJ.ll1ts\JrwrA
albUIIln,ani
two
~10bul~na,fibtlno~.n
and •• ~UQ~glooUllh •
...Amon~ OU\~r.bOCh~"
fllllt "'lth, 1" tne • ..,rum.-,te '1!"&6,uric acid,
3r"et~,4arb_&',par-al
•• tl0,.~'h' ~lptlU"~d e.O"".,wrnol1is. and xanthin
base •• Ut!xtroae ani 40(". ~re
.~le"
.I.ltora
..a goOOUr lIlormally'.T.n
~~4~tlun to the fibrtn rerme~t,blood tontalns a ~ltaolyt1~ and an
lnvert.i.n~ vnzyme.
(A) Defibrinated
blood.
General reactions.
(A). Litmus paper-Moisten
a pie~e of litmus paper with a concan
trated solution of sodium chloride. Puncture the end of the finger
'lith a sterilized 'needle,placI:3 tM~ drop of'
blood obtained on
the Ii t rnus paper t er a row secon ds and t hen was n the blood fro,ll
the paper by means of the salirie solution~As a ruld the alkaline
'reaotion aan be clearly seen.
(8). Ilitmus 801\1.t1on.
Pour a La t t Le red litmus solution on a piece
of porous plate,obtain
a Jrop of blood as abovB,place it on the
red' surface fOI!'" Ft r ew seconds and then wash off wi t h running water
a b(reenish b Lue spot is not Lc eab Le Where t rie -Ir op touched
Gui,acum and turpentine oil. '1'0 10 oc s of' wa t er Elt:d a f'~\" urop s
blood,then a f,,;'4 c e or t i riot ur-e of iSuiaaum an o wix,~"inally add
a faW' i,~c.of ozonized
turpentine oil and: shake t ho roughLy -tne
mixture is ~olQred blue.
(D). Hydrogen dioxide. Add to une cc.of blood two or three cu.ot
hydrogen dioxide-intense
frothing dUe to the esoape of oxygen. The
oxyhaemogj o o rn is d(~!jomposeJ. (Catalytic a c t Lonv },
IG}.
of
v
Spectroscopic
examination-
dbser ipt ion t ns v iQlet
ri~ht.
assumEod to be on th{1 observers
Tn t h('! fol1ow'ing
ana of the spectrum, is
(A) Oxyhaemoglobin.
Dilute 10 cc.of bIooa ~ith 90 of water and
filter~~laQe ~art of the solution in a test iUbe and examIne With
t h e ap ec t roe cop e s v'h
en the solution is so concentrated the spectrum
will prObably be entirely shut off as far as the yellow or orange
but on ~radually dilut1ng wlth water
a spectrum is finally seen
Which ShOWS two absorption bands to the ri~ht od the D line.The
right hand band is broader faInter and less sharply Jefinnd than
the other and the color of the l~ght Which emerges from the left
limb of the Ler t hand is yellow.'llhen the solution is tur t n er
(jia:utet~
the ban-rs disappear s amu l t eneo us j y.
/
(8). Reduced haemoglObin. When a solution of oxyheemoglobin is tra
treate~ with a reducing agent the colorin~ matter loses oxy~en an~
is changed into haemoglobin. Stokes flUi(1 is the most sui tabla
reagent for reJucing;and is prepared as follows. Dissolve 3 ~r8ms
of sa~cted
orrstals of ferrous sulphate in eold water and add a
cold aqueous solution of two grams of tarta.ric or f.Jitrica )d.
Make up with ~ater to 8 ~olume of 100 ~e.a.nd immediately before
using add amnorlia until the precipitate \Vhich forms at first is
redissol~ed.The solution should not be exposed to the air and ~s
unfit for use 24 hours after it is ~repared.
Pre-pare e solution of oxyhaemot51obin \'1hichIvill snow the cha.raot er rs t re absort>tion bands.Allow 8. few cr-ops of Stokes solution to'
floW into the solution in sueh a manner that air bUb~les ere not
carried down with hte liquid.The colQr of the solutlon ~hanges to
a purple or violet and the s~edtrUm shoWs a s~ngle broad ,diffuse
and. p oor Ly define4 bBnd as though two oxyhaemot5lobin bands naI
gone together and had been displaced to the left. When the solution
is agitated with air its color Changes to bri~ht red ana the dpectrum is that of oxynaemog Lob m(C).Mnthaernoglobin. To a dllute solution of blood add a drop or
tw'o of a fre~hiy prepared TO p ereen t solution of potassium ferricy
anido.'l'he~olor of the solution becomes brown and the spectrum
s hows an absorption band in the red and in addition two uan-ts of
more or less uneertain character \'1hichoccupy the pos i t aoo s of the
bands of oxyhemoglobin. When the solution is t reat ed with Stokes
fluid the methaemoglobin ehanges to haemoglobin,Oxyhaemoglobin
bein~ formed intermediatelY and on B.gitating with air the haemoglobin is ~hanged to oxYhaemoglObin.These
changes ~aneasilY
be'
followed with the spectros~ope.
(D). Aaid neematin ~o a few drops of undiluted blood add a drop or
two or aee t t c a 0 iii. '}'hehnamoglobin is broken up into a his ton call
ad globin Bnd a non-proteid substanee ealled haemin.rne solution
whioh reSUlts is aln~st black but on diluting with water is seen
to be red.Tha spectrum has a band in tn0 red almost. but not quite
eoan 0 idt,:,nt
\l7i th the corresponding
band of methaemo~lobin. 'l'he- .
, his ton under these conditions is of course further decomposed.
(F.) •. Alkaline na ema t m 'fa a porti.on of the acid haematin sofutaon
b.dd sodium hy,jroxh1e un t il the preoipitate wnich forms has redi.ssolved.The solution will be alkaline and if properly Jiluted will
shoW a poorly defined band to the left of the n line.It is
usUt'.llyobser'l1edhowe'l1erthat the entire speotrum is absorbed
except the red.
(F}.Reduced haematin (Haemoehromogen)Reduce a portion of alkaline
haemetin solution ..
<lithStokes fluid ana after f/roperly ~1iluting
examine with the spectroscope. Two very dark end Sharply defined
bands are seen betWeen D and ~ which seem to be coincident ~ith
the )raud5:
p r ...
·,d"LL~Gd by o;~yhaern(Jg!.J)btD.It will
be noted however, that
emerges on the left
of the left
band is plainly
greei1oBY·iilutin~~
t he cvlutici.!
with
TJlai:s.:.· the b3.r!~; on t ho right
may be madE t~ disappoar,while
th0 ather ~and is still
very dark.
HARMfTOPORPHY2TN, Place a few oeD of nODc8ntratsct suIDhuric
acld
the
lightvilJi(;.'1.
if In
. a t es
•
th •
estt J:ut.,e s t i..r r.n t o t118 a c z. ef a drop or~ b I 001.~ an d1- exemano
sol~tion.Two ba~~s are seen on opposite
sides
of the
D line,
The
one
on
t he left.
darke~ on ;he
:.~
right
La: ....:;:·Ovi t ho
s~de
0113
on
the
rig11t
IS
broad
and muen
than on the leCto
CARB0NMONCXTDE HAC~On1GBIN.
Pass
aSLGw
C;J~'r':mt
of illlj~nii1ating
gal::' ~wh~Ch conta:'.ns
about 7
pdr~ent
8f c~rbonmonoX~de~thrcugh
50 eeeo: blOOd until the color
of [he bloo'i ia brig!~t r0~~
Spectre,s
cop i c t es t e .·-'Exe.min~ a p or t ton of
'l'he banJ!Cj occupy nearly
the s a.ne ~Ics:'..tif)n
og!obin.rreat
bands
pei·s"5..2
t
the so:utjon
with reduci~g
t ne carbonmonoxi de blood
as the bands of ')xyhaemAgents (Stokes fluid)?th&'
v
Ohem:1:;a1. tests"
'l'wo ~olutimTs
are
requ:i.re~·-a
solution
of oxyhaemoglobin
.b Loo d (A);a
solution
o r carbon
monoxide
bloOd{B).
IQ '1'0 a 'fev'! cocof
;A) add one halt
its
Volume of st!'ong caustic
potash solution.-Fcpeat
th& ~xperiment
with (8), (A)deposits a
brownish
r e d 01" reddish
grt38il fJrecipitate,
is) usually- deposits
brilliant red.-Tn
dilute
sollltion (A)1.18!-iOSlts
a brownish g r ey ,
preclpitat~71 {Aj i n dilute
s(jlution}un"~i~r
t h e s e circumstances
becomes murky
I"'tllc:ren
tun1S light
reJ,'lhe
liquid
,1eposits
red
flakes on sten~ing~
2.
'l'o TO Ccoo!'
LA)
ferrocye.n:idc;and
add 15 (;~.·of a ~~O percent
2 t":·"o~' ac e t t o ~1;,;id",-·E8pQat
a
solutif)(\
I)f" potassium
tile e;~peime!1t
with
(8) and a!ter allo~ln~ to stand some time compare the colors; (A)i§
greyish bro~n or green; (B)giv&5 a bright red colorb
30 nix a small qu~-mti~y
of (A)vrith <1 times its volume ofl'/ater.'l'o
a p or t ion
one percent
Gf
tnl::::
diJ.ute
solution
add three times its volume
of a
the experiment
with
(B)
scl:1tioTI c1' tanninQRepeat
Haemin test <Bl00~ stain
tcst,)Evaporate aarefully
a small drop of
dilute
sodium chloride
solution on a slide~?lace a few particles
of the suspeoted
m~terial or a drop or blood on the thin layer
of
s~lt crystalsa00ver
~ith a cover glass and bring enough glacial
aeet:i.c
acid on the sliJe
and in oontact with the edge of the slip
to fill
the space between the glasses~Heat
until bUbbles of gas
begin to form ~As ~vaporation
pro~eeds adJ glacial
aceti~
acid
drop by dro.p fr(m~ the edge of t h:e Cover glass, whGn a reddish
brown
tinge appears
rBmo;e
further [rom the flame and allow the
acid
to evaporat€:< iEJ-'~DmirJ€r1lIDiCroscopjcalIY"
~,
I
'l'he blood
as wej.~. arc e ar ouc fluids
arid c.not ner ~.nsol'.1blee'l'he
serum
c on t e f ns on e proteid
form.::1' is serum ~lbumin
solUble in water
the latter serl~n ~lobulin {held in solution by the salts of the
blood~'1'he two pr o t e s ds mar bo pr-e c Lpi t a t s d t og e t ne r or s e p ar a t e Ly
by using c9rtain saltso
To 25 oe,,()f blood serum in a mortar a<.!d 20 grams or ammonium
sUlphate and rub with a pestle until the fluid is saturated
with
the salt~8oth
p r o t e a d..s ar e pre~i~:'.'tated
e ompLe t e Ly -PdLt er' through
a dry filter
paper,aoidulate
the :iltrate with aoetic
acid and
b011-no changeD
I
'1'0 25 ee ..o f serum ad~ 25 e c s o f a s a t ur a t e d s o j u t i cn of ammonium
sUlph8t~;rilte~
and wash with a Eatura~ed solution
of thB salt.
Serum globulin is precipitated
an d serum albumin is in soj ut aon ,
Hea t the filtrate to boiling·~serum
aLbumdn is coagu],ateaoPlace
a
little
of the globulin in a test tUbe and pour water on it -the
proteid d1ssolves by virtue
of the Emall qua~tity of salt
adhering
to it. Heat \he solut1on
to boiling-eoagulation~
Saturate 25 co.of serum wi th roagnes Lum sUlphate<>-~Serum glObUlin
is preeipitated,serum
Serum albumin
and
albumin
glObulin
remains
give the
in soj ut f.on ,
~ommon proteid
reactions.
,
,
In the muscle we find a flui6,the
muscle plasma~resl:tmblin!S
the
blood p08sma;\'(h.ich
coagulates
spontaneously
~separatillg
a proteid,
myosin and yielding
a s er um-j'he plasma is rapidlY
coagulated
after
death and is an important
fac:tor 1i"l the c aus e t i on of rigor
mortis.
Dead mus oLe con t a Lns S l)\~era.lprot e ids, SOUle of Which ar e sol u-ble in water and ~alt
sclution~a~d
some insoluble4Myoalbumin,
myosin .muscul1n,and
myoglobulin
belong to the first
grouP7and the
stroma auba t sn ce b eLong s to the La t t er '"
•
Mus<;le contains
a complex Hlyco'~nucleo
proteiu~MYO-·PHOSPHOCARNIO
ACID, \Y'hi~h t s beleived
to
be the s our ee of en er g y in the muscle"
The extract ive bodies of the muscle aloe ampor t ant ,and ar e both
nitl·ogenous
and non-nitrogenous"Creatintthe
xanthin
bases,urea and
uric acid are found"They seem to be the result of ~rote1d wetabolism'iin the museleoln,osit,glycogen,suge.:;;> end lactic
acid are non-« ,
nitrogenQus
extraetives~onstantly·presentc>
Inosit
is prObably an aro~ati~
alcohol,but
1S often classed as
a carbohydrate.Glycogen
may be absent from dead muscle~being
transformed. into dextrin and sugar post-mortemoLa~tic ~cid of th&.
In~sclB id'sarcolaetic
acid,darived
from
several
sources-
The XAN'I'HINR ~S~:S'so far isolated from the animal cell 81,~ TI in
nUml:Hr.'I'hey are all
rourid in the muscLe an d are •roris t an t p .. o duo t s
of the
cleavage
of nualeins, (cell nueleins),They
can be obtained
by synthetic processes
from uric acid,and an in~reased
secratio~
0f urIc acid follows
their
ingestion as food.Yhey
are all descr~bed as s ucs t Ltut aon pro-mets
of plJFDJ,the
mo LecuLar' structure of
v hich .is
c nar eo t er as t t c of the gronp,and is known aa the pur an
Due 1e us or r int5. 'l'he t' crmu la is given b elow :the nurno ers of t he
members being a dte Ii for o onv en i en c e in des i~nfcl
t inH t he subs tit u-·
j
/
t
ion
,
eompoun d s ,
,
/
4C~~
I ':J
~
2
q
~:: N
y'S:11hin
is known
trioxypurin(2-,:-E),lS
8S
2-nHoxypurin,an\.)
ShoVID
co
I
the
q
ij
C
-
its
formulae;
HN -
00
GO
i
,..
H
,..,
I
:0
N -....
, .
n .
2'.f
./
8
C
relation
to
ur r c ac a d
\
cII - ~n'T_
..
C -
by
b
N/
f.1i~ -
Xanthin
Pecogn{t10n
pr ot eras of mus cIc ,
~'!ix 100 grams or finely
..Uvided
I ean beef ,iith :300 tP'ams uf water
8~d allow
to I
stand
at room temperature
for J hours. Pour the
mixture
on a linAn f~lter and prees the residue with the hand,snd
of
some of the
filter the solution~
Test the reaction
of the filtrate toward sensitive litmus paper,
8,r. d t owar d Lacmo r dv'r es t the reae t t on of a solution
made from meat
ttat has stood some time.rhe presence
of mono and di calcIum ~hGS.
pta t e anti the formation
of sa r co La e t tc a c i d ,explains
the p e cu I ia.r
reaction of the solutions.
Fractional Coa~ulation. Pour TO eG.of the filtrate
into a test-·
tUbe and place in a small beaker halt fUll of water.Heat
the wate}
i~ the beaker slowly ,at the same time holding
a thermometer
in
t h e liquid
in the test tUbe.Be.oreen 50 -51) Gea C02q',5ulation occurs
Pilter and heat the filtrate to d5 C.-a sBcond ooa~ulation.Fllter
aDd heat the Cilirateto
75 C. a third ~oag~lation
occurs.
~xtrao,;
the meat residue with a Ifi percent amrnon rum Chloride
solutioD,adding enough of the latter to form a moderately thick
peste. Coyer the vessel allow to stand 24 hours and filter.
Pour a quantity or the filtrate into a. la:Li::l
excess
of water.
e
Myosin is precipitated.
The precipItate
is ,tissoJ.vea
v/ith great
by extremely
dilute hyarochloric acid formind
aCId albumin.On
n)itutralizin~
the acid solut~on
with SOdium hydroxide the adid albumin is precipitated and passes Into solution
When an excess of'
alkali
has
been
added.
,
' .
Plaoe a fe~ drops of the filtrate U~ion a pieoe of rock salt.A
film of myosin is deposited as the solution
becom6s saturated \Yith
salt.
r~w
Boil a
filtrate
from
oxalat·".),
co.of
the flltrate.The myosin is ~oa~UI8tod and the
the Coagulum contaIns
ea Le rum, ('feat
'-'nth 8.ll1mon~um
!
"he constituents of b~ne are or~ani.,L:aloium
,and iron phOSPhate)
And' organic (Qssl3in),1'he iTon ia yrobabl:v'
derived In part,from
the
b Loou of b·'One.-Oasein
.is a Collagen.anl1
on boilin~ iVith dilute
said Jields gelatln~.
rhe mineral matter can be removed,~ilhout
alterincl the appearance of th3 bone;t:y allow',ing the non e to stand in con t ae t with
dilUte hydrochloric acld for 2' hours.8y
this
treatment the Don.
b,eeooes i-I iabl.e.
of Gala till •
or i~alatin 25 eC.of <l1stille,j 'Vatdr and alIa,,"
the end of this time t n e tSelatin
is a.'Iol1en
Eeact
r
Pour upon a I5 am
t,o stand 24 hours.At
dissolvet1.Decant
10n$
8.(lJ 8 ~c.Of
distilled
"(star;
the iSelatin
IHssolveS;Fwa
than Cool
th~, gd~tinous
mass t nus Obtained add about 50 cc s o r 1Vater;and
heat a~ain 9Ver the Wat~r bath.1his solution
of ~elatln after
it
but
neat
"0
no t
over
I.he
water
the
'Vater
bat./'! until
cooled,IS to be use4 for th~ follOWing
tests.
"011 a portion of the aolution after a1.ing to it a re_ drops of
aeetic a~id-no preeipitats I~ formed.
has
Add to anotner ~ortlon
aaatio aoid and potassi~
rerrooyanlde-no
it from albumin
and alt;umoses.).
T~ stew c~.a~d mer~uri~ chloride solution-no preaipitate.
Addition or tannin ,or hYdro.nlorl. aaid ona phoSPhotungsti.
aoid give a voluminous preol~itate.
l-:r'9~lpitate(Distj.nguishes
90il a portion ·,lh oao third lt8 vol~.
of nitric aoiO-8 faani
yellow ~olor t~ pr0JUced,ShO~ing
the presenco of an aromatic
/
ra,1itJal.
<'laustie soda &'1,1 a little
copp er sUlphnte:Ji'Te
e bll.~~ violet
e oj.or without
a t r a e e or red. (.Dist.in~uishes
a t rr orn a Lcumos ea tl,nd
p~~on~s. )By heatin~ to boilincl tif little copper sulphate 1$ aJded...,.th~ ,,,alor turns
s omewhet )"c·,d;i.t' more I~OP?er ;sulpha.te
is us e a ,
boilln!~ does not ;~hi~nefe the color.
Bo11in~ with ~illons
rea~ent produdes a faint pink or rad Jolor~
'l'his reaction
dis t in,>{11t sn es gelatin
from tJTvt e-i.1::;ln
benElral; ;0<1
sho~s the abs en c e of t he t yr os a n il,rOup.
'fhe addition
or cr omm e wa t er p"O'.lUr-,:t'"S a heavy yellow precipi
tat9,possesaln~ tou~h a~h~siY8 propertia&.
n~~l~tine pos s e s s es the rlro~ert~·
ot'hol<ting
many tJrecipl.tates
in
s usp ens rcn
in a r i ne Lt dlvided
conJltivn,so
that
U'J.1 pass thr~ugh
filter
pe.~er;o:r of p,.~"an In~ rne (ormation
or preJlpitates.
'l'HF: URTNF~.
'l'he urine
is
th·r~
most
amp or t e.nt
an rma I oxeretion.it.
is
t
ne tn3e,ns
of eli~in8ting
tne nitrogenous
waste material,as well as most of
thewatHr
and solUble
nu ner aI sUbstflntH~8 •.~. stUd}' of thH ~omposit t on or tho. urine
wlll g-i",e important
imformation re~arJi"ng me t ao-«
ol1sm,the naturA of the ¢h9rni~al pro~es3a8 takin~ place in thu
body~and th~ condition of the urinary
organs.
In health
and on a mt xe a ditH, the quan t i t y (if ur an e seer ete<t in
i. acout
THOO ceo t,~0nt8ininH
50-tj() ~;,ra.z.nsof soliJs
tof
/!:~j grams
IS urea.
"'hn r ea e.t Lon is D.cid or ampno t er a e ,
and the sp.g.about
T020.
:\lmost all the ni trogen
of urine
is searete.~
as ure~~.·l'he othf.lr
nitro~enous ftonstituents are uria Rcidtxanthin bases,and
araatinin
tfhe constituents
of urine
are cla5GififH~ as follows.
!.- Produ-::ts
of norma.l IJetaboliS{J).
:2. ?ro\1U(~ts of foo(! impurities.
3. SUhstances due to impurities
in the fOOd.oot chan~eJ in the
organism.
~. Products elabor~ted
by the body as ~roteotlYes
a~ainst drugs
and fOOd impurities.
5. Produe ts seor6t ed bY' c,?r tain
indi.viduals.
(1 diosjrn ~ras y. ) •
~).
1\:8. ter,ials
no t connee ted "lii th metabol iSInt but the rasul t of
:6,4 lJours
;vhi~/"; :3:; to
deo.~raposition 10 the intestine.
7. Pro~~~ts of disturbed
metabolism.
B. SUbstanoes
ooeuring pathologically_
rhe followin~ table Sh0WS the constituents
acaording
as they result from the metabolism
of urin8,8rranw~J
of rood element.
r"OOdS.
Ij At:F.lON.
Proteid.
Nl'rRO(j~~N.
?HOR PHORUS.
sUI,PHTJR.
Proteid.
Nuoleo-prot.
Phosphates.
Prot ai J.
Phosphoric
Slulphates.
l?ci.1 iier i-v-
Preformed
a t rves .
Oonjui:Sate•
Carbohydrate.
fi'a r,
ff:xeretion.
Carbon rli-oxide.
!
UrEl8
Uri~ acid
Hippuri~
aeid
Creatinine
Aromoni&.
Xanthine
bases.
TNOROANTC
II:
UnoxidizeJ
Sulphur.
BAS~S.
SOIJi urn, pot ass Lum, magries rum and c aLe ium oxides.
The inorganic bases forme~ in metabolism,oceur
in insufficient
.quantity to saturate the acids ~re5ent.The
excess of acid is taken
up by ammonia prepared
b.y the organism
for this
purpose. Wnene"ler
'the bOdy forms a protective sUbstance,it always ma.kes an excess of
the material.The
eXCHSS of aromonra r orme o in this case 1S como rneu
WItt the carbon di-oxide formIng carbamic acid,(amido-carboni,
801e;); I~hi~h corne rnes wi th another molecule of a.mmonia forming
anmon rum e ar eama to. This sal t is depr ived of one moLe cuj e of «at er
in the liver,and converted into urea.
Uric acid is formed in the liver ~robably bY the oonJugation of
lactic
a~id
a.nd ammon La iw i t n urea.
Hippuric acid occurs abundantly in the urine of herbivora,and in
human urine after the ingestion or benzoic aCld·or any of its precursors.
Creatinin is derived from creatin,of whi~h it 1$ the anhYdri~e.
Creatin is present
in the muscles ~t all times,and the excretion
or ~ree t inin is d1reetl y aap en dan t on the amoun t - of muscle (meat) in
the diet.'l'ha
daily quantity
of oreatinin is small on a mixed diet
an o is no measure of rnus cuj ar- metabolism.
~he preformed
Gulphates are alkaline salts of sulphuric acid.
Conjugate sulphates are etherial
combinations of the acid and eroma.tio alcohols.
Class 2.
Class 2.
~he ~entoses
found
in
the
are generally the result of food impuritY,but
urine after lar~e doses of morphine.
are
Glynuronin a~i1 ocnurs ounJu~ated;uauallY
after taking chloral
or camphor.
'i'h,;:, acid occupies a position int.ermediate bet'·'/een
the IDOno and
di-basic acids obtained trom tn9 sugars.It uan be looked on as a
dibasic acid one or whose carboxyl groups has been reduced to the
aldehyde state.'l'he
composition of glycuronic acid is
'COH(CROH)4COOH
80th the atove
reduae dopper solutiofi and may be ~onfused with
dextrose. Distinguishing tests will be ~iven later.
(,
I
'/,
II.
Class 5..
GYS'l'T.N .is am t r-ogsn, .,
_ some cases In large qua~:
lus.
Alkaptoni~ a~ids,re4uoe
kno~n,-homogentisie
a~id
C6H3{OH)Z02H30HCOOR.
Por tests to distinguish
of su~arI in urine.
aompoun d eon t a m ang sulphur. It
tty and may ba Jeposited,forming
o eeure in
a calcu-
r
solution ..
l'wo ofthe,se
aei-1s are
CtiH3 (OHJ;JCOOH.
and ur-o Leu czna e acid,
Penlings
these acids tram dextrose,sse
detaction
\\.
Class 6..
Nueleo-albumin,o,,~urs in the urine normallY,it is easily distin~uished from serum albumin.
-As a result or putrafaction in the intestine there are excreted
in th~ urina,inJlcal
,skatol,phenol,pyrocateanin,sulphuretted
hydrogen,para-~resol.
Gr ,3501, WhiCh is usually ex cr-et e c ,1ike the other aroma t i¢ al·JOhol
in combination vrith potassium acid sUlphate,i~ methyl paroxypnenyl
G'itl;OHGH3.
lndol
nitrQgenouM phenol deri9atives.rhey are formed by
the decomposition of proteids.
Indol is
./ CH~'
in
tne liver this if; oxidized into
C6H4
ca.
inr1Qxyl,
/" COR ~
~
NH /'
C6H3"
»: CH.
---
'i'he indoxyl
is conjugated
doxyl potassium sUlphate~
with potassium
NH
./
acid sulphate, forming
10-
Patholo~ically any of the normal constituents mny be increased,
or 1iminished in quantity. Almost any constituent of the body can
occur in the urine durinJ disease ~rocesses.Those
of interest as
aids to diae:nosis
or o t her-wtss elinieally.are <1eserib,e<1 in the
direetion~ for the exsminatiQn 6r 'urine.
1. General
Reactions.
'Pest t h e r-eac t i on of fj"eSh ur-Lna also the r-eac tI on of urLne that has stood
for some time- the former
is usua I Ly acid and clear,
the later
alkaline
and
cloudy.
With small quantities
of filtered
ur i ne make the followine;
tests,
2. Add hydrochloric
acid and heat- dark coloration
of the urine,
on standing possibly
a deposition
of uric acid.
3. Add caustic
soda solutionprecipitation
of phosphate,
which on heating forms, into flakes
and on s t and i ng eradua11:" settles
t.o the bottom of
the vesselThe precipitate
rna:' or )'la:r not be colored.
Cr~'stals
of ammonde pos it ad whe n the so Lut ion is allowed
Lum. magna
Ul:'1 phosphate
are usually
to st and
Boil a portion
of the urineif the r-e ac ti on is acid,
the urine
remains
clear;
if neutral
or alkaline,
a precipitate
of pho spha't e s usually
occurs.
Addition
of a few drops of acetic
acid dissolves
this precipitate.
4. Add bar rum chloride
solutiona white pr-e c Lpi t a t e cons Ls t Lng of bar-Lura
sulphate,
and bar1111'1pho sp hat e ; add i tLon of hydrochloric
acid dissolves
a
part of the precipitate.
50 Add silver
nitfate
solutiona white precipitate
of silver
chloride
and
silver
phosphate.
The latter
is dissolved
by nitric
acid.
6 Add basic
lead acetate
solutionThA voluMinous precipitate
contains
lead chloride,
lead phosphate,
lead sulphate,
and the creater
part of the
colorinc
mattRr of the urine.
Filter;
the filtrate
is noarly
co1orlosso
Basic lead acetate
is used in manv cases to de c o Lo r i ze ur-Lrie ,
81
.-1
0
u. Preparat ion of Urea.
Evaporate
SOO ci c , of urine
(fir'st
over a f r-eo f Larne later
over the wat e r-«
bath) to the consiste~cy
of a thill svrup,
cool with ice water,
and add about
twice its volume of nitric
ac I d ( 7>~~;;t) that has been be I Led unt i 1 c olorle ss
and then cooled down to zer o , A'lLo» to stand a short tLme an d collect
the
crystals
of urea nitrate,
u s i rig a suction
pUlllr perforated
po r-ce Lai.n plate
etc,
and wash w i t h a little
cold 'colorless
n l trLc acid.
Dissolve
in as
little
hot vate r as possible
and as tnt, so Luti on e;ently boils,
add smaLL
successivE:' portions
of potassium
l'f:r;'lcl.l1sanate until
t he solution
of urea
nitrate
is coLor-Les s ,
Al.Lo» so Luti on to cool and ciraw the :'jother
liquor
from the crystals
7lith the filter
purrp , Press dry 0;1 a filter
paper and
weigh ap}!l~oxir;w,tel~'.
Dissolve. t.he urea n i t r a t e in a small quan ti t y of hot
water and add SODe what more than the 8Guivale~t
quantity
of powdered barhun carbonate.
Exaporate
to dryness
on t ne wat er-eba'th , ex t rac t the powdered residue
with a suall
cuanti t v of bc i Li.ng abso Lut e alcohol
and allow
the ail.coho Li c solution
to e(~o1.. R~cl"rstallize
n·,.t': ria t e r-La I from hot alcChol
Wind the Doltine
point of the"product.
Urea is the no s t i npo r-t ant n i t.r-ogeriou s c ons t i t ue nt of the urine
representinG under nornal
conditiollS,
a~out 85~ of the total
nitrogen
elioinated
by t he ki dne y s , Cherliea11y
cons icier-ed it is t 1'16 arnide
of carbonic
ae id and
~s represented
b:' the formula
(C 0 IH 1-", ).
It crystallizes
from alcohol
l.n large quadratic
prisms,
which P1eit
132°C is very s()ll1ble in \"later,
less soluble
in alcohol
and insoluble
in ether.
g
ltt?
RBACTIONS
01;1 UREA ..
1. Heat carefully a small portion of urea in a dry test-tube the urea
melts and a' sublimate of ammonittm carbonate is ft~xx
formed (strong odor
of ammonia).
'Continue the heating until the melted mass begins to solidify. Af'tercoolinc
dissolve the residue in' wat er (addine; a little caustic
soda) and make the ~:~~~x
biuret test ..
The following reaction takes place when urea is heated as above.
(Biuret)
(,
2
cot
,
N H 2'2
=
(C
° '2
N H (~ H2 )2 ~ N H3
a portion of Urea in a dr~T test-tube until the whole mass becomes
solido
After/cooling
dissolve in water (adding a little caustic soda) and
then acidulate with hydrochloric
acid" - a precipitate of cyanuric acJ.d..
20 Heat
3 C 0
(N H B) 2'::;' C~
H3°3
N3
+- 3
N H:3
(cyanuric acid)
/
30 fo a small portion of a solution of urea in water add mercuric
nitrate- a white precipitate
is f'o rme d the ccmpo sIt t on of which depe~-~~
the concentration
and relative quant Lt Le s of the two solutions used.
When
/Jhe,solutions
are neutral and dilute;,> a salt is formed havine a composi-- tion represented by the formula ( IT H2 )2e 0
He (N 03 )2 • 3 BeO•
1'2,
This is the salt formed in the reaction on which Liabie's volumetric method
for the determination
of urea is based"
4. Dissolve a few crystals of urea on a wa.tch glass in a few drops of
water and add a concentrated
solution (If oxalic acid~ precipitate
of urea
cxa.Lat.e , / (C 0 Hz H4 )2 CzHz04
H:P /.
+
Examine
5 .. Repeat
(4) but use nitric acid instead of oxalic acid:. Urea nit-rate
Examine mic:coscopically 0
is l'0 rme d ( CON 2 H4 o' If N 03 )
6 ..
Heat a small portion
of' urea
p
C o 'H2
.,
·'4'
+2
w i.t h
r.r
·'2
c au st Lc soda solution ..
0 e
C 0 2 ~'~"
2'" l'T H 3"T,L
N2
°
•
7. To a small drop of norcury add nitric acid warm the acid and add a
little urea- a mixture of Co! and No is eiven off~
...
..
8
0
To a little bromine
ro>,
..;Z 0
'R'
water Ln a test ..-t ube add caustic
( NaO··,Iir)
To t h I s solution
Sodium hypobromite
is for58d
of ur-e a ,
.,I,?, T\T
l. 2
. .~ .
C 0 N2 H4 4 3 Na 0 3r ~ o r:
oJ
0
j,
1
r)
H
.." ...~
0
,.l
..,...
0'
soda solut'i:-onadd a solution
3 Ua
Br.
Prepara.tion of Urj.G~,·Acid..,
To IOoCfCoco
of urine add IOcr':'.:; Co of hydr-o ch l.or Lc acid and allow to stand
in a cool place for 24 hours
Collect the tSJlt}rt'tk"kx crystals of highly
COlored uric acid and wash with water-· (Examine nicroscopically).
Ill"
0
0
Suspend the crystals
in a sma'l L quarrt Lt y of wat e r , heat,
add enough c aus't I c
soda to dissolve
the uric
acid and then decolorize
by boiling
"Kith animal
charcoal.
Filter,
make acid with hydrochloric
acid,
heat to boiling
and
allow to 0001- Uric acid ~
obtained
in this
way is a colorless
crystalline
powdor- Collect
on filter
paper,
wash'with
water and allow to dry.
Uric acid is very insoluble
in cold water
(1: 15000), insoluble
in aID cohol and ether,
solublH
in caustic
alkalies;
in ammonia it is scarcely
soluble.
•
REACTIONS OF URIC-ACID.
1.
Hurexide
testSee "Exaru nat.Lcn ' of Urinar:r
Calculi".
and
2 •. Schiff's
reac t ion Dis solve a 1 it t Le ur-Lc ac id in s od Ium carbonate
Place a drop of thin
snlution
on Filter
paper previously
moistened
with
nitrate
of Silve 1'",. The s i Lve r nitrate
is reduced
to silver
and a stain
is
formed.'
,
3. Boil n solution
of urI o ac id in Call s t ic soda w i th a small amount of .
Fehlings
fluidreduction
wit& production
of~a frey reddish
precipitate.
If the cor/per salt
is in -exc e s s , t he Latt e r is ohtatnc d ,
It is obvious
from this
reacti~ll
that
some care must be exercised
in ~akine reduction
tests
for suear in the urine.
4. To a s od i um c ar bonate
so Lu" ion of u r I c acid add mage n s La mixture
(Mg
S04 or ITg
c12"
N H4 Cl ~ N Eo ... H20),
and then silver
nitrate
so ...
7
lution.
Uric acid is precipi.tated.
as a celatinous
maenesiosilver
salt.
Reaction
(1) is ir.:J.portant for the recognition
of uric
acid reaction
(4)
for its
isolation.
Uric acid is cot a Lways deposited
on adding hydrochloric
acid to the ur Lne : in such a case it is ne ce s sar y to use reaction
(4) in order to show the presence
of ur i c acid.
This test
is also used
for the quantitative
deterMination
of uric acid.
IV. Preparation
of Hippuric
Acid.
To 300 c. c. of horse s urine
a dd a sl iGllt e xc e s s of mi lk of lime, warm ,
filter,
evaporate
the filtrate
to a snall
bulk and treat
when cold,
with
hYdrochloric
acid in slieht
excess,
The hydr ochf.o r t c acid decomposes the
hippurate
of calcium
Lt ber at Lng hi ppu r Le acid which separates
at once in
the for.m of needles
(usua:U.:i hiGhl:' colored).
Collect
at the crystals
on
a perforated
porcelain
plate
(usinC the suction
PUMp.) diSSOlve in hot
water
(to whi ch a lit tIe ammcn i a has be e n added) and deo oLor i ze wi th
animal «.ant charcoal.
:"i1 tel' while hot , evaporate,
the filtrate
to a small
volume and whe'n cold add hydr-o ch'Lor i.c ac id- Collect
the c r-y s t a Ls wash with
a Ii ttLe wat er and r-ecr yat.a.I Li ze fr-o m hot wat er
(Examine microsaopioeJ.ly).
Hippuric
acid,
(C 11 C0 C H !i S COOE.) is nonobasic
and forms salts
whi(;J::I.
2
6 5
«
are
CGld
for the most part
soluble
in water,
It is soluble
with difficulty
in
wat.e r readily
in hot vat.e r , Lns oLubLe in ether.
Helt.ing point
100°C.
REACTIONS
OF ~IPPURIC ACI~.
1. Heat gently
a sma.l.'Lportion
of t h e ac id in a clay in a dry test-tubethe acid melts,
and solidifies
aeain OD cooline.
Heat atain
nore strong~
the melted acid turns
red,
Give.s a 8ublinate
of ben7.oic acid and an odor
simi.l.ar to that
of oil of bitter
a Lmond s , du e probably
to herrzo Lon i.t r LLe
and hydrocyanic
acid.
The red ooLor is d118 to the decomposition
of ~'
o.o.e8l.l ( C H (N H 2)
C (\ o H) The sublimate
of benzoic
acid has a ..
2
very characteristic
microscopic
appearance,
is roluble
i11 sodium carbonate
..and. is reprecipitated
from this
solution
by hydrochloric
acid.
v.
Recognition
of Oxalic Acid.
'To 500 e ,c , of urine add a few Co 0 e of calcium chloride
solution,make
alkaline
with ammoni a , acidulate
faintly
w i t h acetic
acid and a.llow: to
stand 24 hours.
(Add a few CoCo
of an alcoholic
solution
of th~nnol) •.
At the end of this
time filter
t.nr ough a smaI L filter,
wash with a little
water,
pLae e the filter
paper in dilute
hydrochloric
acid,
warm gently
and filter.
Evaporate
the filtrate
to a small volmne, make alkaline
while still
warm with N H3 acidify
with acetic
acid and allow to stand for
a few hours in a warm placeD
Examine the precipitate
microscopically.
VI. Recognition
of Creatinine"
Make 200-300 c ,c . of urine faintly
aLkaLi.ne with milk of lime, .and add
calcium chlor:i.de solution
unt i I all the phosphates
are precipitated-·
Filter,
trash w'ith wat.er and evaporate
the fj,J. trate
... wash water
(3fter
faintly
acidifyine
with acetic
acid) to the consistency
of a syrup.
To
the warm syrup add XXX 50 coco of
alcohol,
transfer
to a small'beak'eT,
cover' with a elass
plate
and aJ.10','1to s tand in R cold place at least
eight
hours.
Filter
through
aSI,mll
fi Iter,
wash w i th alcohol,
evaporate
the
f U"traie
to 50-60 c "c allow to cooL, Add 1/2 c ..c.' of alcoholic
zn 012
cove r t.ho hreaker
\v1th a class
sclut ion (81' g. 1.20),
st t r thorouchl~r,
plate
and allow to stand. ~2-3 days in a cool placeCollect
the crystals
of creatinine
zinc chloride
and wash them with a little
alcohol.
1. Weylis ReactionPowder a portion
of the salt,
boil with a little
water,
allo'N to cool and filterTo the filtrate
add a few drops of sodium
nitropmissiate
a~1a-:ke-alkaline
w i t h caus t Le soda- deep red colo~tion
which quLck Ly fades to a s t raw co Io r , Act d if'v with acetic
acid and boilPrussian
blu~ or berliY' Greeno This test
can~ also be made directly
with the
urine,
but since' acetone
gives a similar
reaction,
it is necessary
to boil
the urine
and cool before
app Ly i.ng the test.
Jaffis
Reactiollo
To a portion
of u.r i ne add aqneol1s solution
of picric
acid and make alkali~e
with caustic
soda- a deep red color •. Acetone
yields
no such red color with this reagent.
961
0
VII Phenol(a)
Sulphate
Phenol,
of Potassiun ..
(e H
6 5
Reactions
of Phenol.
Prepare
a 2~ solution
and a 0.2;; so Inti on of pheno L and make the follCll'ing
tests
with each solution.
1. Add ferric
chloridea clecjp amsth:rst color 'which is dischare;ed
by···.
strong
ac:i,.cis- Hany phenol derivatives
(salicylicacid
- orthohy~C
acid)
give a z:m:kkxxsimilar
reaction ..
2. Add a t ew drops of TIillon's
reacent. and heat to boiline;
-intense
dark
red solution
or red precipitate
.. This reaction
will.show
1 part of
phenol in 60,000 parts
of wat er ; the color however in such dilution
is
pink.
This reaction
is also e;iYen by all rhenols.
3. Add bromine water- A precipitate
of 1'10nob;r'OYlD
phenol and .diGrOmphenol
is formed, which is characterized
by its pen8tratine;
odor.
Further
addition
of bromine produces
a yellowish
~hite
crystalline
precipitate
of
tribromphenol
(C6H;2Br'7pJ-I) e
Reactions
(2) and(3) are the most delicate
and the ones generallY
used- Reaction
(3) is also used for t he quantitative
determination
of·" -.. ,........
phenol but certain
precipitations
are necessary
to avoid the formation
of
bromoxytribrombenzene
I C H Dr 0 Br,Z: tetrabromphenol)
After the us~of'
..phenol, this
substance
m§.~T2be 3fOlmd as such in the urine.
Hor se ur me
and pathological
urine usually
contain
para-cresol
in place of phenol.
m-n
/
/
~
The reactions
of para-cresol
are similar to those of phena1, though
Les s pronounced..
The add it ion of ferric
chloride gives a dirt.~7-grey ...
co10r- (b) Phenol sulphate of potassium (C H 0 S03K).
6 5
Reco nition of Phenol in Urine ..
Phenol and cresol are foun
inurine In te
form of etherial
sUlphateso
To prove the presence of phenol it is necessary to first
decompose
by
heating with h~rdrochloric acid.
Malee 500 c .c , of urine alkaline
with Ua2 Co3and evaporate almost to
dryness on the water-bath..
Add
100 c.c. of sulphuric
acid and distil.
Makes tests for Phe~
with the distillateo
"yIII"
Pyrocate~nC
This substance occurs in human urine as a conjugat e sulphate and
caus~s alkaline
urines to turn dark on exposure to the air..
It readily
reduces solutions
of metallic
salts,
a fact to be taken into
consideration
when dealine with the presence or absence of suear in the
ur t ne , Prepare. a 0.2;( aqneous so Lut I on and make the following tests,"
10 Add dilute ferric
chloride
solutiongreen color.
The additidn
of
a little
tartaric
acid and then a.smorua changes the creen color to
cioleto
Acidulating
with acetic acid produces the green color again.
2 Add a Ii t t Le armon i a and then a fev! drop'S of silver nitrate
solution-immediate
reduction
to l1etallic
s i Lver
3 Addition of caustic
soda colors solution
e;:reen, br-own and black.
4. Addi tion of lead asetate
prec ipi tat es p~rl'ocatechin as a lead comPOund. The filtrate
from this precipitate
does not eive any of the
re~ctions
aboveo
'
0
;»
0
IX ~eni
tion of Judican_.
1. Jaffes methode To 5 c ;c , of urine add an equa.l volume of concen ...
l'rated
I-1" ce and ru x thorollghl~'"
Add drop by drop a n
soluti on of
bleaching pov!der un t il the maximumof color is at tained and s riake
gently with 1 CC of chloroform.
The chloroform is colored blue", It
is difficult
to e s t i.mat.s the quant Lt y of hypochlorit'e
ne ce s sa r-y to pro~.
duce the most intnnse blue and an excess of' the reagent oxidizes
indigobluea
The reaction
ix in any case takes place slOWly and does' not
always ShOT,r"
the presence of indican unl e s.s great care is taken"
Hlgtlky colored t'cd;;rwc:rx urine should be decolorized
w i t h basic lead
acetate
before making the test and any albumin present should be also
r emcve d ,
0
Gbermayers r';Iethodo
l'ii"Orin excess) and filter
4Q
To a port ion of urine
t hr ough a dr? f' d Lt.e r-,
add lead
acetate
To a portion of the filtrate
add an e-qua'l,colume of' concentrated
H cl ;
which contains' O~2-0.41 of ferric
chloride.
- Shake the so Lut ion. with
chloroform." ,.Ferrj,c chloride
does not oxidize indiGo blue ..
3. Mac I:unn t s !!ethod.
Boil equal volumes of urine and' concentrated
hydrochloric
acid that contains a trace of nitric
acid.
Cool and
agitate
with chloroform.
The chloroform is generally
colored v i oLet ,
and shows two absorption
bands near D- one dtle to indigo- blue, the
other to indigo red~
X Recognition of Unoxidized Sulphur.
Pour upon a small piece of zinc a little
hydrochloric
acid and allow
the acid to act a short time.
Pour the acid off and wash the zin~
with water.
Place the piece of zinc in a flask with 50 ocxfrcCo of
urine and add enough hydrochloric
acid to cause a gentle evolution ofhydrogen.
Suspend in or over the neck of the flask a piece of filter
paper noistened with lead acetate
solution.
The paper turns blacko
Only the so called neutral
s:tqi{ sulphur
of the urine eives 428 with
nassenthydrogen.
XI. R~cognition of Pepsin.
Agitate 200 co. of nrine with a shred of fibrin
b:T drawing air
through the urine .~fter
an hour or two' r-emove the fibrin
and wash
with water.
Pl~e t.he fibrin
in a test-tube
with a little
h;Trdochloric
acid (0.2~) and alloW to stand at 400·tmtil the fibrin
is decomposed~
Wi th the soLut ion make the biuret
test.
Repe at the experiment with
boiled urine.
XII. Inorganic Constituents.
1. Chlorides
_ Acidulate a few co of t be ur t ne with nitric
ac Ld and
Add silver nitrate.
A white precipitate
of silver chlorideo
2. Sulphates- Acidulate a portion of the urine with hydrochloric
acid
and add Ba cL. - a cloud of barium sulphate.
"
3. Etherial
sftlphates
Nix 20 co. of urine with 20 cc of' an alkaline
2
solution of barium chloride and filter~Boil the filtrate
with 1/
its volume of concentrated
h:rdrochlori o acid cloudiness
due to the
precipi tat,ion of barium sulphate.
(norniallY very little)
(Do not use
too much Hel).
4. Phosphates
_ l':ake 50 cc , of urine a'Lkal.Lne w l t h anmond a , and filte":~
Dissolve the predipitate
in nitric
acid and test t h- solution for
phosphoric acid w i th moLy bdLc solution,
make the filtrate
acid -wi t h
nitric
acid and test for phoaphor Lc acid with mol:rbdic sol'lJt.ion ..
,The phosphoric acid in the filtrate
represents
the excess of phos~
pnor t c acid over that required for the calcium and flagnesj,um
0
Q
XII Albumin.
The followine tests
should be nade with normal ur Lne and with several
sp02cimens of vathological
urine.
All material
should be. perfectly
cleaT
and if this cannot be effect »d by filtering
t nro ugh paper,
f il tel' .,
through asbe stos or ag i tate
the ur tne with magnes.ta (1<'15 0) and filter
1. The Boilin£.? Test.
Heat a portion of the urine to boilins
in a
test-tube
and whether a precipitate
has been formed or not , add concentrated
acid (nitric)
drop at a time until the volume of nitric
acid
used is about 1/20-1/10 the volume of t he urine.
In case a flocculen.t
precipitate
is produced the presence of alhumin may be considered
pro-ven',
Not. 1.
As a positive
test for albumin this reaction
has great
value, but a negative r-esu.l.t. is no indication
that albumin is about: ..Note 2.
Faintly alkaline
or amphotern urines may yield on boiling
a precipit~te
of phosphates which is indistinguishable
in its gener~l
apvearance from a precipitate
of coaeulated albumin; and an al~aline
uJ"1ne may rama in clear after boiling even t hough it contains Ej.lb'l.1min.
0
No.7.
The subsequent
addition
of nitric
phosphates
aftd precipitate
proteid
acid. will
dissolve
which remains
in
'boiling.
No.3.
If an acid urine
yeilds
acid,
albumin is absent
and there
no precipitate
is no necessity
.
precipitated
llt
solution
after
on boiling
and remains
for the additon
of
nitric'
Note 4. acid. When the quarrt I t y of albumin is snaIl,
the addition
of a
drop of nitric
acid to the boiled
urine produces
a precipitate
which
disappears
on shaking (xx the fluid.
When the urine
is rich enough
in salts
or when sufficient
acid has been added to render
the medium
J
incapable
of holding
acid albunin
in solution,
a permanent
preeipitate
is produced.
upon this
llrinciple
is based the following
very sensitive
te st.
Treat the urine with enol1gh acetic
ao id to p r-oduc e a marked ac id
reaction.
(2-3drops
of acid for 2OCc. of urine),
add 1/6 of its volume
of a saturated
s oLut ion Of/odium
chloride
and boil.
Under these
conditions
no precipitaj;/i;on
of phosphates
can. occur,
and e"lren a trace
of albumin \/ill
be c.<mVerted into ac id albtunin and sal~ed out by sodium
chlorides.
Note
5.
When this
test. is properly
executed
there
is no danger of
confusinc
albumin with uncles
albumin or albmI1O$e, for both- of these
substances
remain in the IllX nitric
solntion
wh i Le it is hot.
The same
also holds true for ur t c ac id and ur at e s , but in case of concentrated
urine
it is we Ll., to dilnte
a port ion of the ur t ne before
making the
te st e spec irilly
if the prec ipi tate for-ned b;' the concentrated
urine
is
grant1lar.
A pr-e c ill it ate produced
by the pr i gmerrt of Le teric
urine will
consist
of biliverdin
and will diesolve
in alcohol.
!Tote 6.
Aftor the L,.t e r na.L or external
admini fit ration
of snch drtl[;S
em
as 'tl'upentine,
toblJ-balsaM,cubcbo,
storax,
oil of sandal wood petrol
etc.
the urine may contain
resino
which conduct
themselves
like
albumin
as far as this
test
is concerned.
These resins,
howver, easily
pass
into solution
uron the addition
of alcohol.
Note 7
In case the test
y i eLds a sufficient
quanitty
of precipitate
to rilter
and wash, the material
should be subMitted
to the biuret
0
r-e ac t Lon ,
2 Heller's
Test.
Place ahout 25 cc. of clear
urine
in a tes~-tube
0
(better
a conical
slass)
and introduce
10 cc. of concentrated
nitric
ac id b:r means of a pil'otte
who se point
reaches
the bot tom of the vessel
The ni tiic
should be allov/ed to flow ve r y slowly so that the lizuids
do not mrx , but form a sharp
zone of contact.
Under these
conditions
albumin wi 11 be chanced to acid albumin whic h be ing ,.insoluble
in ni tri"
ac Ld ap pe ar s as a wh i te r ins in the 7.0n& of contact;
on at anding the
ring of acid albumin will Benerally
becomes wider ext.ending
upward int;o
the urine.
Note
1.
While this
reac~ion
is extremely
sensitive
it has not cethe
disadvantage
e ornmo
n to so Dan:, albmnin tests
of show ing the' prese.n
of
traces
of pr-ot e id "''lhich are nor-ma'l Ly present
in avery urine.
Note. 2.
In oa se s where the urine
is concentrated
or where there
is e xc e s s I V(~ quan t i t~T of uric ac id a e Loudy zone may appear on standing
This zone noweve r is always considerably
hieher
than the zone of con-'
tact
of the two liquids,
and will
dissolve
when the urine
in which
it is formed is carei'ull;'
removed from the nitric
acid and warmed.
No.. 80
Urines which contain as r~tch as 45 grains or urea' to the liter
may
deposit a crystalline
precipitate
of urea nitrateo
So concentrated
a
::artm:x urine shou.ld be diluted
before the albumin test is applied ..
Note 30
As in the boiling test a cloud for~ed by nitric
acid may
'6e referable
to unclaoalbumin,
albumose or urin acids..
The exclusive
of these substances can be accomplished by the methods giveno
Note 4
The' colored ~x
rings which are produced have nothing to
ao wIt~bumino
A ring which varies from rose to brick red is due
to the normal pigments of t he urine, a mahogany ring .Ind.Lc at e s Lnob i Ld.n;
while an excess of urinary indican produaew a blue ring which may be
so intense as to color the entire solution
after the liquids have been
mixed ..
3
The Potassium ferro e anide te st.
00 idify' a small port ion of urine
and add a fev{ drops of p t as s Ium. ferroc~ranide.
A clouding shows the
pr-e senc e of albumos of al imdn provided that the precipitate
is not
soluble on heating' and that addition of acetic acid alone did not produce a precipitateo
4
Trichloracetic
acid Test:
By means of a pipette
place 1 or 2
CC::-of an aqueous soltl,tion of the reagent (5p g - lQ147) in the bottom
of av t e st ...tube co ntaining
a few c c. of filtered
urine as in Heller! s
testo A white ring will be seen at the zone of contact between'the
two
fluids
its intensity
uarying with the amount of albumin present ..
The colored rings whd c h are formed when nitric
acid is 'Used are rae-ely
o bse rved ,
Serum albumin ser-um g Lo bu'l In and albumoses are precipitated,
the presence of the latter
being recognized by the fact' that the precipitate
dissappears
on boiling and reappear on cooling..
This test is
undoub t e d.ty one of the most sensitive,
and b:r its use it is possible
to
demonstrate albumin in urines with which the more COmMon
tests yield
negative resultso
0
0
0'
XIV...,
Recognition
of A'Lbumo se s ,
~esFthe 11ri1J.8-foral bumt n ~ -;"i"""'f--="i-::-t--:"i-s
pre sent ac idify
st r-ong l y with
acetic acid~ add an equal amount of saturated
sodium chloride
solution,
beD. and filter = On cooling a.Ibumo se s will produce a cloud wh i ch
disappears
on he at Lng and r-e appear on c ooLtng , make the filtrate
al·kaJ.ine with caustic
soda 'and add copper su Lphat e, (Biuret reaction) 0
.Use 0" 3 grains of albumose to 100 c c. of ur-Lne ,
XVQ DextroS9G,
following tests
should be made with each of four solutions
t2;zzz
(a) A 2f solution
of crape sug ar in wate r , (b) A 02% so Lut ion
of grape sugar in water ... (c) a 2~ solution
of grape sugar in normal
ur-Lns
{d} a 02% solut ion of crap'e sugar in normal urine
A number of
patb.o:Logical ur i ne s should then be examined, but must be free from
pT'oteid.s
before the tests are applied ..
10 Reduction Testso
These tests all depend upon the power possessed
bY-grape sugar to reduce various substances wh i ch eive evi.dence of this
reduction
by the formation of precipitates
or by changes in color"
As normal urines and moines which are sevreted after the administration
Of' certain
drugs o onat In sub s t anoe s which are reducing agents"
It is
clear these reduction
tests
are not to be re~arded as conclusive
of the
prasaQ~e of sugaro
------The
0
�A) Trommers Test. The urine is made strongly alkaline with sodium
~Ydroxlde and a 5% so ution of copper sulphate is added at a time until
the precipitated coppe hydrate no longer dissolves on shaking. The
liquid is then heated to he boiling point but should not be allowed to
boil. If sugar is present
yellow cloud of cuprous hydroxide will
be found which may gradually change to red cuprous oxide. When the
precipitate first Dk~kes its appearance the heating should be discontinued as the reduction will proceed rapidly and the reducing action
of other substances is eliminated by avoiding the boiling temperature.
This test has the disadvantage that an alkaline copper solution is
reduced by many substances which «Ia~XA occur in normal or pathological urine or which may appe~r in t.he same urine after the ingestion
of various drugs. f)uch substances are uric acid, creatine, creatinine,
allantoin, mucin, pyrocatehin, hydroquinone, me e;lynronicacid com--pounds, the alcaptonic aeids (homogentisic acid, urolanci~c acid),
urobilin gall pigments, moreover, after the inCAstion of certain drugs
anyone of a large number of these reduoing substances may appear in the
urine (b) With Fehling~s solution- When Trommer's Test is carelessly
executed an excess of copper over that required for a small amount of
sugar will be (\'onvertedinto black cupric oxide which may mask a small
quantity of cuprous oxide. This may be avoided by using mixed Fehling'
flUid, when any excess of copper over that which is required for the
reducing substances will remain in solution. All of the substances
which interfere with Trommer's Test must be noted also here.
(c) Bottger's Test
A port.ion of urine is made alkaline with caustic
soda, a little bismuth nitrate is added and the solution is boiled for
several minutes. The presence! of sugar' is shown by the appearance of
a black precipitate of bismuth or m bismuth suboxide, or by the
darkening of the undissolved biSMuth salt. This test has the advantage that bLsmubh salts are not reduced by many substances which
wasil~r reduce an alkaline copper solution- uric acid, pyrocatechin,
hydro quinone , creatinine and alcaptonic acids. '
(d) Hylander's Test
To ten volumes of urine add'one volume of
MYlander's reagent and boil the mixture for [-)
ninutes. The presence
of sugar- is shown by the eppearance of a black precipitate. Hylander's
solut ion is a pr-epar-at ion contatm ng bismuth, which is extremely sensitive to the reducing aetion of sugar. The test has shown the
pr-e sence of reduc Lng substances in norma l. urine.
2 Phenylhydrazin Test.
To 100 cc. of a urine add 2.5 grains of
phen~71hydrazin hydrochloride and 5 grams of sodium acetate: heat
3/4 hours over the water bath and filter while hot- On coolting
yellow needles of phenYeelucoOazone are depo s t ted. When t his substance
is pure it melts at ~~~x
2040 - 2050
3 The Fermentation Test. Shake a portion of the suspected urine with
some compressed yeast and fill into a fermentation tube. Allow tv tuand
in a warm place, preferably in a thermostat which is kept at 400anclnote
the collection of eas bubbles in the upper part of t"oo tube. A check
test should also be made with a one per cent solution of dextrose which
has been sh~ken ffitha portion of the same specimen of yeast. It is also
weL'lto make a second check test with water that has been shaken with
the yeast. The fermentation in each ease should proceed for twelve
hours.
4 Polariscopic Test.
Before applying this test it is nece saar-ytQ,
renove all proteide from the urine and to free from a greater part of
the coloring by prec1pitatine with a little neutral lead acetate. Read
the zer s of the tnstrument , insert the t ube which has been previously
filled with prepared urine and turn the polarizer unt LL extinction is
obtained.
The sensitiveness of the test depends entirely upon the
construction of the instrument and as r10st instruments are not capable
of showing a trace of sugar it is generally useless to try the test
when no positive reaction has been obtained w~th reduction tests.
0
0
10.
THE FOL:LOWING TABLE WIIJ' .. SHOW THE TESTS NECESSARY FOR DISTINGUISHING
SUGAR, FROU OTHER REDUCING AGEN'l'S,OCCURRING
•
••
Substance
•
•
•
"
••••••••
•
..
•
•
•
•
•
•
•
"
•••.••••••••
•
•
•
Fehling's Test
..
•
•
•
•
•
•
..
•
•
•
•
•
•
•
•
•
•
..
41
IN THE URDTE •
••••••••••
-.
••••••••
t.
Bismuth
.
Fermenting
tl
Phenylhydr-az i ng
Dextrose
Reduction
Reduction Ferments
Osazone
Dextrorotatory
Pentoses
Reduction
Reduction Negative
Osazone
Dextrorotatory
Glycm"ollic
Acid
Reduction
Reduotion Negative
Osazone
aevo-roratory in
urine
'1'
Alkapton!
Acids
Rednction
Negative Negative
Negative
Inactive
Lactose
Reduction
Reduction Negative
Osazone
Dextrorotatory
Uric Acid
Reduction
-Negati va Negat ive
Negative
Inactive
Creatinin
Reduction
Negative Negat ive
Negative
Inactive
Pyrocatephin
Reduction
Negat ive Negat ive
Negat i ve
Inactive.
. 1:
11.
xs ,
Reccgn LtLon of Aceto Acetic
The test
must bs made with fresh
AC1CL
l~,3H C O. C H2 COOR}
urine.
1. Tc a few e ;e , of ur-Ln« add ferric
e hl.or-Lde solution.
Should a
precipitation
of phosII: ate occur this Xci: is f Utered off and mor-e of
the. iron solution
is added to the filtrate.
A Bordeaux red color
indicat .:e s the presence of acete acetic acid.
•
2. Acidulate
a port ion of ur t ne (10-50 c o, ) wit.h dI Lut e sulphuric ac id
and extract with an equal colmne of' ether.
separate the atherial
extract
and shake it w i t h a little
Vt1ry di Lut,e fl~rric chloride solutionthe
presence of aceto aeetic acid colors the aqneous solution red.
X"ITI
Recognition
of AcetonQ/(rH3 ) ~ C
(l/
Diabet io Urine)
,-
Aci tone is a colorless
liquid havtng an agreeable odor.
It is sol- 0
uble in alcohol, water, and ether in all proportions.
BoilinG po~nt 58
To 250 of normal urine add 5 drops of acetone and an equal anount of
hydrochloric
acid; submit the material to distillation
and uS'S the first
portions of the dlstillate
for the followine tests:Iodoform Test.A few c c. of the distillate
are t:r-eated .r i th s evera
arops 0 a (llllte
s()lut ion of iodo-potassic
iodide and caustic
soda- prec ipi t at ion of iodoform ( eEl
) on warru ng which may be
readily r-ecognLzed by it s odor.
2. R€~rnold' S Test
Treat a few cc , of the di.stillate
with a snaL'L
amount of fresh!:' p r-e c ipitated
:rel10\, oxide of mercury.
This is prepared by p.cv.EKcd~'c{::::o= prec ip i tat Lng a soIut ton of mar-cur Lc chloride
(poisonous) wt t.h an alcoholic
soLurLcn of caustic soda.
If acetone
be pnesent a black color will result
in tho clear filtrate
upon the
addi ~i on of a few drops of ammcn
i um su Lphd de ,
XVIII Recognition of Bile Piements (Ioteric
Urine)
1'. Gmelinr s Test (See Biliar~r calculi).
2
Gmelin's Test
(";odified by Rosenbach- Filter
a Cltlantity
of urine
through filter
paper; r.hen br Ing a drop of ~re1low nitric
acid in con....
tact with the appr-oxi.ma t e Ly dry paper.
3 .. Make a portion
of the urine alkaline with sodium carbonate solution (1-2 drops) then add calcium chloride solution until the fluid
over the precipitate
formed after ahak Lng is colorless
or has the color
of normal urine.
Pilter
wash the precipdtate,
place in a test-tube
wi th alcohol and br me rnt o soLut ion by the arlo,it,ion of hydrochloric
ac ide Boil t h e clear soLut ion- e;:ceen and. blue c olol's.
Allow' to cool
and add nitrie
acid- the sreen soLutt on turns hltiEJ,violet. and red ..
This test is often positive
when (1) is negative and is recommended when
the urine contains a lare;e ar~;)'l!1t of indican.
4. Acidulate a por-tLon of t hs ur:tne with acetic acid and shake with
chloroform.
The lat tel' is colored yellow.
0
~~II
-
Recoenition of Blood Piements.
A
xy aemo 0 1n- Tie
aemoe;lobin.
1. 'Heller's Test.
".a e a por lon OJ t ..
l.'l urine
alkaline
with caustic
soda, and boil. A precipitate
o~ hasic phosphates is formed which, if
blood coloring matter is present is colored l'ed- at t ilnes when the urine contains a laree aMotmt of colol'inb nattar
(bile pie;ment- etc.) ..
It may be difficult
to appreciate
ths color of the precipitate.
In this
case filter
off and dissolve in acetic acid, when if clood pi~ent
be
present the solnt ion becon-us red, and the color vanishes gradually upon
exposure to the air.
No. 12.
2. 8uaiacum
Test.A mixture of tincture
of' enaiaet.un and ozonized oil
of turpentine
is allowed to floVl carefully
down the ?ide of a test
tube
upon the urine in such a manner as to form a distinct
layer
above the
lTrine- a ';{hit~ ring which grad1.lally turns
to blue 'ilill be seen to form
at the surface
of contact.
I: 3 To about 100 c c , of sus ec t ad urine add a small quantity
of a
solution
of eee' a Lbumfn in vat e r , Heat to boiling and, filter
the c oneulated
a Lbumf.n,
This coagulum will
contain haema t In , Grind the
precipitate
in a mortar with about 200c. of absolute
alcohol
and a few
drops of concfJntrated
sui\phuric
ac id transfer
to a flask, heat to
boiling
and filter.
After
t h e filtrate
has cooled make alkaline
with
canst ic soda.
Raduc tl w'i th ammonfnum sulphide,
and e xamf.ne spec tros-
copical1
haemochromogen.
examination.
(B) HAE;TATOPORPHYRPT.
A direct
speetroescopic
examillatio11
the urine for t h t s pigment is
generally
Hnsatisfactor~r
for t he reason that r.he field
is USUally
obscured by the presence
of the nther colored
snbstances
in. the urine.
It is therefore
advisable
to isolate
the p i gmen t b:r some pro-etlss of'
preci~itation
of extraction.
1. !lethod 01' (Tarrod.
Treat
100cc.
of suspected
urine
with alkaline
bariun chloride
so lut; ion un t i 1 the r eage nt no J.onger fo~ms a pl>ecipi tate
Haematoporph~Trin
under t he se conditions
is p:cecil'itated
L:. SC~6 f'o rm
of chemical combination with the phosphates
of the alkaline
earths.
Filter
throlle;1tl a sma Ll. filter
paper,
was the pr-e c I p t t at.s once w t t h water
and O:1.C8wit h absolute
alcohol.
P1ac e a wat ch c r-y.s t a I over the funnel
and a Ll ow t he alcohol
to drain
off as c ompLe t e Ly as possible.
Bring
the mo s t precj.pHate.
Lito
a s:1<:'J.J.l mor t ar-, ack a bou t ten drops of h~'droch1oric
ac t d- ~E1Cl E:~!011Gh alcohol
to form a t h i.e k paste.
,liter
crinding the paste,
tra:. sf'e i- to an Es Lenmeye i- f'La sk , UP ing a sT1al1 amount,
of absolute
alcohol
for "lashing,
a m warm the rnaterial
on a lIater-bath
A110'."to become perfectl:'
cool a nd filter.
The filtrate
is now examined
spectroscopica1).y.
If haemat opo r-phyr-Ln t s present
the vent c h '"racteristic ab s or-p t ion spectrum
of the suhs t.anc e in acid so Lut ion ":lill be seen •.
4. Direct
for
spectroscopic
or
E.iCAI'TINATr ON OT' URINARY
CALCULI,.
He.at a snaIl quantity
of t h8 f inol~T liOYldered stone of a platinum foil:
if the powder burns cor1111\~te1y 1t~avinc but little
ash,
it !.'1a:' contain
uric ac id, ffi1lnOn inm.'1 In'atl'l, e:ist in, 01' xanthine:
if i~, burns
incomPletely
1ea~Tinc cC:lsiclerab1e
ash, it 218.:' cO;'1trd.n 111'ic acid,
urates,
calcium, phosl~hate', macnesim'1 phOf~phatf!, HlnCnintlr1 nae;nesim:1 phospahte,
or ea1ciuD oxalate.
The i/lethocl to be
IJl1Y'Sllecl
in 1:he a:'tal:rsis depends
ul,on this dif'f'erence.
1. The POI/dar burns complete1:,
or 1eaves a mere trace of residue.
Digest a ~'ort ion of th:e-'lIY\i-IClei~-::'li th di1nt8 h?di~ochloric
ae id ',varming
gently.
Ca) It diSSOlves
cOJ11pldtel:r
-c:'stin
oX' xanthine.
To tflst
for
cyst in heat
u. ]. ].1't1El of t:h,.::; l,o',,'der 'dth
8.l'lrrlOnia and f'il ter- pour
the filtrate
On a watch-Blass
and allow to evaporate.
Exanine microSCOpicallyCystin crystallizes
in sioc-aided
plates.
Cystin stones
are genera11:'81'1a11, havfj H ~Tol1oH colo)' and SMooth surface.
No" 13
0
To t8st
for xanthinfl
make the' xanthin
test
with nitric
acid and caustic
sada(see chapter
on muscle).
Filter
and wash the residue
with water.
(b) It dissolves
~ncoMp18tely~
Residue,
Uric acid.
To t e s t for uric acid make the murexide testPlace a smaf.L quantity
of the re s rdue Ln a po r-ce Lai n dish,
pour upon it a f'ew drops of strong
nitric
acid and evaporate
over the 'vater-bath
to dr yna s a- a yelle"Nish
or reddish
residue
resultsAllow to cool and moisten
with a little
ammonI a- a reddish
pnr p Le color
due to t he f'o rrnaf ion of pmrexideAdd a drop or t wo of caustic
soda, a deep blue. ooLor , moi s t e n with
water and evaporate
to drynessthe color disappearsHurexide
is destroyed
by this
treatment
and. the test
dist·ineuishes
uric
acid from
xauthin
and guan i n ,
Uric acid stones' var-y Ln size,
are quite
hard
and cenerall:'
colored
ye Tl ow or brown.
2" Fil tr6.te"Ia:' c ont a rn armon i um chlorideTo t e s t for nlTlOnia, heat the solution
with sodium carbonate:
anmonf a
is recogni~ed
by its odor, alkaline
reaction,
etc.
II
The Povder
but
becomes
lea1Te sac
black
ollsiderable
owing to
the
ore;anic
substances
present,
residue.
Digest
a little
of tIE finAly powdered stone in dilute
hydrochloric
ac iel warmi ng gentl:r:
effervesence
shows the presence
of o ar-bonat e s ,
(a) Complete solutionuric ac id is absent ..
(b) Incomplete
solution
t he residue
may contain
uric acid proteid
substances,
epithelial
cells,
etc.
The' Gen8ral appearance
or a microscopic
exara i n at ion VIilJ. dec ide this.
l lake the murexide
testI~ any case the solution
is to be invGstincted
further.
To a small
pert ion of the s oLut ion add sod Iurc c ar-bonat e ; boil an d test
for a-mon i a
Dilute
th e remainder
of the so Lut ion with water,
f i1t~r,
make fain""ly
a.LkaL'ine Ylith armcn i a and acidulate
with acetic
acid.
Either
an appr-ox»
u.!latel:r clear
or a c Loudy solution
r e su.l.t s ,
The latter
deposits
Senerally
a whi te powdery prec LpLt nt e ,
The ~rellowish "Nhite flakes
Y(1ich
are s oroet trne s seen in. the liqllid
consist
of iron phosphate.
Filter,
dissolve
in h:rdrochloric
acid and add potassium
f'e r-r-oc yan Lde-« bLue
color
(Prussian
blue)
The white precipitate
consists
or- calcium
.
oxalate"
Filter,
(if the quantity
is not too sna Ll) vash w'it.h water,
dry and 510';' on the platinum
foil.
The c a Lc Lun oxalate
burns to calc i um car bonat e and calc iun oxide.
To the re s Ldue add a drop of hot
wat e r and test
the reactiona LkaLi ne , ac i d If'y w i th hydrochloric
acidef'f'e r-ve se nce ,
The filtrate
frOM iron phosphate
or calcium oxalate,
may
contain
calcium or Magnesium phosphate.
1.. To a sr.ial L quan t.Lt y of the filtrate
add ur any'L n i t r-at e /U02 (N03)2/
a yellouish
whf t e pr e e Ip t t at e of' u:nml~' phosphate
shows the presence
of phosphoric
acid ..
2 To the remai:-lder of' the filtrate
add amL"1oninumoxalate;
white pre .....
cipitF_te
shors calciumWarm, filjer
and make alkaline
'Nith ammonia
crystalline
precipitate
of ar''.moninUL''1
maGnesium Ilhosphate
s11o"-{sthe
presenna
of maGnesium"
0
QUAN1.ITATIVE fu~!uJYSIS OP URINE.
In all
quantitative
estimations,
it is absolutely
necessa.r~r to use
well mixed 24 hours
(night
and day) urine..
The vessel
used as a
receptacle
should be k.::-pt closed
as much as po s s LbLe during
this
t tme
and if necessary,
thymolor
some other disinfectant
should be added to
prevent
putrefaction"
}!leasure as acctlratel~r
as possible
the quantity
of urine
vo ided du ring the 24. hours,
us i ng a dr~T craduated
cylinder
0
DETERHINATlon
O:? sPECI3i'IC
GAVITY ..
If tho urine
is clear
and wi hout a deposit,
the specific
gravity
may be de t e rm i.ne d at once; ot he rw i se it will be necessar~r
to clarify ...
if urates
have been deposited,
warm sentl~r until
solution'
folloivs
and
a'llo'll to cool~ if suspended
particles
are present,
filter
..
1,_, By mean s of the Hydrometer
(urinometer)
The u r i ne whose specific
gTavi ty-:[stobeQetermined
is pl?-cecr in a wide cylinder
and to a void
foaming the liquid
should be allowed to run down the side of the
vessel"
The hydrometer
is introduced
into t.he liquid
and the specific
o
gravity
is read on the steM of the instrument
DETER!UNATION
OF rOTAL
SOLIDS ..
The amount of solids
can tle caluclated
1vith a stlfficient
degree of
accuracy
for clinical
pur-po se s by I'1ultiplyinC
the last
two figures
of
the specific
grqvity
by 2.33 (for adults)
or by 1.66 (for children)
the nunbe r obtained
indi cat I ng the amount of so lids
m every 1000 cubic
centimeters
of urine"
Example Spa gr" .; 1.03030 x 2.33 ;.:;69 9 grams ·
in 1000 subic centimeters.
DE'T'F.mU:'~ATI ON OT' CHI_OEInES.
R~srnt(s:xx
Heagent sand
apparatns
nece s s ar'y
(Reagents
are' prepared)
Cal A so tuti on of silver nitrate
of S11Chstrnngth
that
one c ,e , corresponds
to ten ~illiBrams
of soditun' chloride"
. (b) A solution
of potassium
chromate.
(1:20).
(c) oxidation
mt xt ur'e (3 })artslX.ctf. of dry potassi1.un nitrate
- one
r;art of dry sodium carbonat e , )
\d) Powdered calcium
carbonateo
>e) 011.eburette
complete
wi th float
and stand.
\f) one pipet te 110ee ) e
(g) Two porcelain
crucibles
with covers.
Place
in crucible
about 4 grams of oxidation
mixture
and measure 10
c c , of urine
into each. Evaporate
over the water-bath
to dryness
, and
he at very carefully
over a free ~rame, be 6innine
at the top and working
gr-adua l Ly t o var d ·the bottom of the crucible,until
the material
has
become whiteAllow to cool,
add a little
vater,
heat on the waterbath and transfer
the material
to beakersAcidif~r faintl~T with nitric
acid and warm Geiltly to expell
the carbon dioxide..
Neutralize
the
slight
excess of ncitric
acid wi t h a small quantity
of calcium
carbonate,
add a few drops of 11ota~sium chromate and titrate
to a faint
red color with silver
nitrateo
Q
Read off the number of cubic centimeters
of silver
I.titrate
used and
mu.ltiply
'by QI~X 0 01"
This gives the amount of chlorine
as sodium
chloride
contained
in 10 cc of urine»
From this
number can. be reck ....
one d t h e amount of sodium chloride
in the total
quantity
of ur me
vo Ide d ,
AJ.b.umi:;~~
and sugar,
.if present,
shou.Ld be removed before
the urine
is
add9d to the oxidation
'mixture,
so as to obviate
losses
from frothing
Which wou.Ld c t her-w I se oc cur"
0
0
DETERHINATION
-----.-·-·~_
.. ~
0;;' SULPHATEs"
.. .,.,..~"'""O*_,..-'-'- .........
,-..--·". ...... ·"Il"',.£4'C·,"'"_I...
Re.age n t s and apparatus
_
necessary
(Re ag en t s are prepared):
~
la.) Al.ka Li ne bar-I um chloride
mixture
(2 vo Lumes of a saturated
solut ion of ba.rium hydr at e and 1 volume of a saturated
solution
of
bar-Ium chloride)
"
{b) Saturated
solution
of barium chloride"
~c) 'I'"lVQ a sh'Le s s filter
pape rs ,
d~.Two p.orDelain
crucibles
with cOVRrso
e) A measuring
flask
100 cco
f) a pipestem
triabgle.
10 S:ctal Sulphates.··
To 100 c c , of clear
ur ine (in case it is c.onH
i--,"'~
~
c erit r a t e d % GOcc to which GOcc of water is added) contained
in a beake r ,
add 10ce or hydrochloric
acid and heat over a free f' Lame to gentle
boiling ~ Aft er b o i 1 I ng a bout ten rri nu t 8$, add a p inch or' two of
pOWdered. potassinm
pe r'manganat e and continue
to bo I L,
I:f after
this
addition
and subsequent
boiling
a light
amber colored
fluid
does not
repultJ
add ano t he r pinch of pe rrnang ana t e and cont Inue tohoil
(u sua.LLy
1/2 gram of permanganate
is sufficient
to brine about this result)
Continue
to boil until
the solution
is perfectly
cleara
Remove the
flame and add about lO·~l5> cc .. of barium chloflide
solution
that has
be en prt:~vir)usly heated
to boiling"
Stir
the liquid
with 2: class
red,
and &.ll()v/ to stand
on a bo i Li ng wat e r--bat h for ten rnf.nut e s ,
At the end
of t.h.i s time the b ar Lum su Lphat.e has for the most part fallen
to the
be t tom of' the breaker
and t he supernatant
liquid
has bec orne c Le ar '"
}l':it an as h i e s s filter
Lnt o a suitable
funnel;
moisten
it "lith a
very dilute
so Luti on of hydrochloric
acid and transfer
the barium
su.Lphat e t o the fi .Lt e r (1'he fi 1 tr at e should be perfe c t ly c Lear ) Wash
with hot water until
the filtrate
no loneer
gives a cloudiness
with
s.t Lvor n i tr-at e-. Wash once with alcohol
and then with ether~
Allow
the ether
to evap,)rate
and transfer
the filter
paper and. barium sulphate
to a weighed crucible
Place the crucible
en a pepestem tri-.
angle and hoat carefull:r
unt.il the filter
is' carbonized
then i~cline
the
cruGi"ble ar..d 810';1 l!.1:~i I the re sidue is whi ts ... Allow the erGC i ble to
cool ir.:. ,a de'sicatc.\J' and weigh"
The ·:mi.ght of the barium sulphate
multipl:l.ed
b:T 0,4206 gives the al:'1(Hmtof 81.11phuric acid and by 0,,34335,
the quan.ti ty cf 8n1.plmr trioxide
in the amount of urine
used for the
a.nalysisr,
2,:,. Con,jiJgate
f1ulphates·~
lUx 100cc of clear
urine with an equal
colu~iie-·c~J"---2-1l~-2]j{EL'T:rli8-soiution
of barium chloride.,
Stir
the mixture
thol~out:;hly
an.c1 after
allowi;:lg to stand a few Plimltes;
filter'
through
a
dr·y f'ilt:~:cs collecting
lOOeR" of t he filtrate
in a d:sy IOOcc ~ measuring
flasJ'~c
T'ransfer'
the lOOcc" of ciltrate
to a beaker,
acidjf~T with lOcco
01' hyd.:-c(;chl·:)rjc 8r:id boil
ten l'1inutes and heat over a boiling
vrater ....
~
bath UTJ.t:Ll. sup2rnatant
liflUid
is clear c
1'he precj.pitated
barium sulphate'
is removed to an ashless
fUter,
wash'ed,
dried.,
i!)cin.erati3d;
weighed~ etc.
exactly
as X under "TOrl'AL SULPHATES"o
I
-
~" -
"-,..
~
".:.>.
-':!'
"">c.-" .""'~ __.' _._-~_._.-
...
y
No. 160
DETERMINATION OF URIC ACID.
FOlin's; modification
of Hopkin's Method.
Apparatus and reagents required.
A burette graduated in tenths.
Amr;oniumsulphate.
10/,S'; Solution of ammoniumsulphate.
n 20 potassium permanganate solution.
1. In normal urine without deposit.
to 100 c c , 'of the urine in a beaker, add 10 gr-ams ammon tum sulphate;
stir until COMpletely dissolved,
and add anmond.a water, until the
react ion is ver-y faintly
alkaline.
Allow the mixture to stand 2
hour s , Filter
and wash the precipitate
with IO}:; solution
of ammonium
sulphate,
unt il the waahfng s are free' :rrom chlorides,
Rinse the precipitate
i!lto a 200 c.c. beaker, and dilute the mixture to 100 c.c. then
add 15 c.c. concentrated
sulphuric
acid,
and b.:itrate while hot. One
e oC. n/20 potassium parmangauat s so Lut ion is equivalent
to .00375 Uric
acid,.OOl must be added, as a correction
to the result
obtained.
In acid urine with -de po sLt ,
If the deposit be'onlYl1ric
acid or urates,
there is but little
clancer in neglecting ,it. Add the ammon tum chloride
to bring down
the uric acid that rema ins in solution,
and treat the entire precipitate
as directed
above.
The deposit most likely to lead to error
wou l d be on of 'e yst in- If this be suspe cted a few drops of ammon i a
s hou Ld be added and the urine warmed.
The chloride
of ammcnf um should
be added while the sample is still
warm.
3. In Alkaline Urines.
If
the---dElrJo-sIT-of-'-l)hosphatesbe very Lar-ge , it may be well to filter
it off, and wash with hot water: otherwise it May be neglected.
The
anmo n t un urate comes down on saturation
with ammonf.um chloride
nuc h __
more rapidly
in alkaline
urine.
The only objection
to adding a few
a!''l(lonia in all cases is the precipitation
of the phosphates and the
fact that the urate precipitate
appear-s to absorb
mor-e pLgmen t from the
alkaline
urine.,
Continue the process as in 1.
Ll" In Highly P!ernel?-ted Drines.
Sc\metimes though very rarely the uric acid crystals
':lay be so much
pigmented as to suCgest that an error in titration
might result.
In
such vc a se s the original
urate precipitate
may be deaoznpose d in the
presence of alcohol.
After washinc the precipitate
off the filter,
95% alcohol equal in ~lount to the water present is-added,
and after
acidifying
, the liquid is cautiously
heated to boiling
in the usual
ways ' and then, wit h a watch crystal
placed over the beaker, it is
digested for sone time over the water-bath
- The separated crystals
should be well washedIn the urines
that contain bile,
the biliary
pigments may come down in considerable
quantity,but,
in spite of this.,
the Ultimate errorar1pears to be small.
2.
DETER:,IINATION OF UR.8A.
If albuy~in is present
it m11stfirst
be removed hy boiling
(after
acidifyine with acetic acid if necessary)
and :rilterine
off ,the flakes of
c08clulated
proteid.
No , 17
0
/
A.pparatus and reagents required.
Doremus Uremeter.
Alkaline
Qll
solution of sodium h~Tporbromite (10 gr-ams sodium hydroxide
25 c c
water, to which 2.5 c.c. Brownine and 10 c.c.
¥
water have
een added.
1% Solution
of urea.
he solution should be freshly made.
Titltine the meometer, pour in enough hypobromite solution
to completely fill the long arm, and pass the bend.
Draw the urine into the pipette to the gradnation. Pass the
into the me ome t e r- as f'a r- as the bend,
and
c ompr-ess the
The nitrogen liberated, will colle ct.
of
the tube.
Each division indicates .001 gram,of urea, in
c. ur in.e.
IiIake
in the upper
,
a check de t erminat ion, wi th the
connect the r~sult accordingly.
1/1 urea
solution,
and