Fulltext - Jultika

ACID-BASE BALANCE,
DENTINOGENESIS AND
DENTAL CARIES
TUULA
B ÄC K MA N
Institute of Dentistry
Experimental studies in rats
1999
TUULA BÄCKMAN
ACID-BASE BALANCE,
DENTINOGENESIS AND DENTAL
CARIES
Experimental studies in rats
Academic Dissertation to be presented with the assent
of the Faculty of Medicine, University of Oulu, for public
discussion in Auditorium 1 of the Institute of Dentistry
(Aapistie 3), on September 24th, 1999, at 12 noon.
O U L U N Y L I O P I S TO , O U L U 1 9 9 9
Copyright © 1999
Oulu University Library, 1999
Manuscript received 1.9.1999
Accepted 3.9.1999
Communicated by
Professor (emer.) Heikki Luoma
Professor Gary M. Whitford
ISBN 951-42-5362-0
(URL: http://herkules.oulu.fi/isbn9514253620/)
ALSO AVAILABLE IN PRINTED FORMAT
ISBN 951-42-5361-2
ISSN 0355-3221
OULU UNIVERSITY LIBRARY
OULU 1999
“We can lift ourselves out of ignorance,
we can find ourselves as creatures of excellence
and intelligence and skill.
We can be free! We can learn to fly!"
Richard Bach
Bäckman, Tuula, Acid-base balance, dentinogenesis and dental caries. Experimental studies in rats
Institute of Dentistry, University of Oulu, FIN-90401 Oulu, Finland
1999
Oulu, Finland
(Manuscript received 1.9.1999)
Abstract
High-sucrose diet and metabolic acidosis have some similar effects on bone and they both reduce
the formation of dentine. This series of experiments was conducted in order to get information
about the effects of acidosis and alkalosis on dentine during primary dentinogenesis and also to
ascertain if high-sucrose diet affects dentine formation via acidosis. Chronic metabolic acidosis
(0.25 mol/L of NH4Cl in drinking water), chronic metabolic alkalosis (0.25 mol/L of NaHCO3 in
drinking water) and chronic respiratory alkalosis (atmospheric pressure equivalent to an altitude of
3000 m) were induced in the rats immediately after weaning for 6 and 7 weeks. One subgroup from
each of the main groups was fed a high-sucrose (43%) diet and one a standard maintenance diet,
each ad libitum. The control groups had the same diets, but normal drinking water and atmospheric
pressure. All the rats were injected with tetracycline (to mark the onset of the experiment in dentine) and inoculated orally with Streptococcus sobrinus. The acid-base status was verified by blood
gas analysis at the end of the experiments. After sacrifice, fissure caries was scored with Schiff reagent and the areas of dentinal lesions and tetracycline-marked new dentine were measured from
sagittally sectioned mandibular molars. The mineral elements (Ca, Mg, F, Na, P and total mineral
contents) of the dentine formed before and during the experiment were measured with an electron
probe microanalyzer.
With the high-sucrose diet, respiratory alkalosis and metabolic acidosis promoted the initiation
and progression of caries while metabolic alkalosis slightly retarded it. With the standard diet, all
the experimental conditions slowed the rate of dentine formation and metabolic acidosis had the
most pronounced effect. The mineral analysis revealed a totally different pattern of mineralization
when the rats with metabolic acidosis (increased calcium and total mineral content) were compared
to the previously reported rats with a high-sucrose diet (decreased calcium and total mineral content). Besides this, metabolic alkalosis did not correct the effects of the dietary sucrose on dentine
formation and blood gas analysis showed no acid-base disturbances in the sucrose diet group.
Therefore, a high amount of sucrose in the diet slows the rate of dentine formation and reduces the
ability of teeth to resist caries attack by mechanisms different from those of metabolic acidosis.
Nevertheless, metabolic acidosis was found to be the most harmful state of disturbance in acid-base
balance for the teeth of young rats, especially with a diet containing a high amount of sucrose.
Keywords: acidosis, alkalosis, cariogenic diet, tooth
Acknowledgements
The work for this thesis was carried out in 1989-1998 in the Department of Pedodontics,
Cariology and Endodontics at the Institute of Dentistry, University of Oulu, Oulu, Finland.
I wish to express my warmest gratitude to my mentor and supervisor, Professor
Markku Larmas, D.D.S., Ph.D, who gave me the opportunity to enter the world of scientific research. His visions and ideas and his encouragement and support have helped me
to proceed on my scientific career ever since.
I am also grateful to the referees of this work, Professor Emeritus Heikki Luoma,
D.D.S., Ph.D. and Professor Gary M. Whitford, D.M.D., Ph.D., for their critical appraisal
and valuable comments on the content and also the language of my manuscript.
I thank all the former and present faculty and staff at the Institute of Dentistry, who
helped me to carry out this work, especially Sinikka Kortelainen, D.D.S., Ph.D., and Ulla
Pajari, D.D.S., Ph.D., who first introduced me to the methods involved, Sisko Huumonen,
D.D.S., Ph.D., who has helped and encouraged me throughout these years, Leo Tjäderhane, D.D.S., Ph.D., and Eeva-Liisa Hietala, D.D.S., Ph.D., for their valuable contributions to this thesis and all the staff of the Department of Pedodontics, Cariology and Endodontics for their understanding attitude towards me and my research.
I owe very special thanks to Päivi Laukkanen, M.Sc. for her help and guidance with
the biostatistics. I am also grateful for the help I was given by the staff of the Laboratory
of the Institute of Dentistry (especially Mrs. Eeva-Maija Kiljander), the Laboratory Animal Centre (especially Mrs. Päivi Moilanen), the Technical Department of the Institute of
Dentistry (especially Mr. Reijo Kettunen, Mr. Pasi Moilanen and Mr. Eino Kemppainen),
the Photography Laboratory (especially Mrs. Liisa Kärki), the Institute of Electron Optics
and the University Library.
I warmly thank my father Väinö Bäckman, who taught me to appreciate a good education and encouraged me to use my intelligence, and I cherish the memory of my mother,
Hilkka Bäckman, who taught me to conquer my limitations and fears. Finally, I wish to
thank my fellow skydivers, especially my team and my dear friends Hellevi Peltoketo and
Raimo Kemppainen for sharing so many moments of freedom and joy that gave me the
strength to accomplish this work.
8
This work was supported in part by a research grant from the Finnish Dental Society, by
the Medical Research Council of the Academy of Finland and by the Research and Science Foundation of Farmos.
Oulu, September 1999
Tuula Bäckman
Abbreviations
A
caries lesion in enamel (Schiff reaction)
ad lib.
ad libitum
ATCC
American type culture collection
B
advanced dentinal caries lesion (Schiff reaction)
B.E.
base excess
C
cavitation (Schiff reaction)
COMPO
back-scattered electron image
EPMA
electron probe microanalyzer
H
+
hydrogen ion
-
HCO3
bicarbonate
m-acid-stan
metabolic acidosis and standard diet
m-acid-suc
metabolic acidosis and sucrose diet
m-alk-stan
metabolic alkalosis and standard diet
m-alk-suc
metabolic alkalosis and sucrose diet
N
no caries lesion (Schiff reaction)
n
number of observations in groups
norm-suc
control group with sucrose diet
norm-stan
control group with standard diet
pCO2
carbon dioxide partial pressure
pO2
oxygen partial pressure
r-alk-stan
respiratory alkalosis and standard diet
r-alk-suc
respiratory alkalosis and sucrose diet
T
initial dentinal caries lesion (Schiff reaction)
10
Contents
Abstract
Acknowledgements
Abbreviations
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Review of the literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Acid-base balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Causes of disturbances in acid-base balance . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.1. Causes of metabolic acidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.2. Causes of metabolic alkalosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.3. Causes of respiratory acidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.4. Causes of respiratory alkalosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Maintaining of acid-base balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.1. Role of lungs in maintaining acid-base balance . . . . . . . . . . . . . . . . . .
2.3.2. Role of buffers in maintaining acid-base balance . . . . . . . . . . . . . . . . .
2.3.3. Role of kidneys in maintaining acid-base balance . . . . . . . . . . . . . . . . .
2.4. Acid-base balance and bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4.1. Metabolic acidosis and bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4.2. Respiratory acidosis and bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4.3. Metabolic alkalosis and bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5. Acid-base balance and teeth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.6. High-sucrose diet and dentine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.7. Dentinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Working hypothesis and aims of the study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Maintenance of the rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Diets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Conduct of the experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.1. Induction of metabolic acidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.2. Induction of metabolic alkalosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.3. Induction of respiratory alkalosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.4. Control rats (normalosis) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4. Anesthesia and blood samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5. Preparation and analyses of the tooth samples . . . . . . . . . . . . . . . . . . . . . . . . . .
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4.5.1. Quantification of dentine apposition . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5.2. Mineral analysis (EPMA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5.3. Caries scoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.6. Pilot studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.7. Statistical analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.7.1. Statistics in blood gas analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.7.2. Statistics in measuring dentine formation . . . . . . . . . . . . . . . . . . . . . . .
4.7.3. Statistics in mineral analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.7.4. Statistics in measuring caries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. Pilot studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. General health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. Blood properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.1. Metabolic acidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.2. Metabolic alkalosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.3. Respiratory alkalosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4. Dentine formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5. Dentine minerals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.6. Caries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.6.1. Areas of dentinal caries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.6.2. Caries scoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1. General health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2. Acid-base balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3. Dentine formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4. Mineral analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.5. Caries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
An attempt to distinguish the initiation of caries from its later progression in dentine led
to a development of a method in which the size of the carious lesion was calculated and
compared to the amount of dentine formed during the experiment (ie. during the cariogenic challenge) (Larmas & Kortelainen 1989). From the beginning, the working hypothesis was that because the secretion of dentine is the only known form of host response in
this tissue, its rate should reflect the magnitude of the response. Thus, the rate of secretion of dentine was supposed to be modulated by the rate of caries progression. There
were also reasons to assume, that the reactions of the odontoblast cells are different in
young compared to old teeth and also under active compared to arrested (or slowly progressing) caries lesions (Massler 1967).
In addition to its well-known caries inducing effect, a high-sucrose diet has been
observed to cause a reduction in the growth of dentine in the molar teeth of young rats
(Kortelainen & Larmas 1990, Tjäderhane et al. 1994, Bäckman & Larmas 1997, etc.) in a
dose-dependent manner (Huumonen et al. 1997). This was a surprising finding in the
sense that caries has previously been reported to accelerate the formation of dentine (e.g.
Massler 1967, Silverstone & Mjör 1988).
A further question arose, of what causes the reducing effect of a high-sucrose diet on
dentine formation. This effect is powerful enough to overwhelm the (theoretical?) promoting effect of caries on dentine formation. According to the literature, the way the
high-sucrose diet affects dentine systemically is completely unknown. On the other hand,
some information has been reported on the effects of a high-sucrose diet on another mineralized tissue, i.e. bone. It has been reported to cause loss of calcium from bone,
decrease in net renal tubular reabsorption of calcium (Lemann et al. 1970, Lennon &
Piering 1970) and even osteoporosis (Saffar et al. 1981, Tamura et al. 1983, de Tessieres
& Saffar 1992). All the same effects have also been reported in chronic metabolic acidosis (Barzel 1995, Green & Kleeman 1991). Collagen fibril formation and cross-linking are
inhibited by glucose in vitro (Lien et al. 1984, 1992). Metabolic acidosis slows down collagen synthesis (Krieger et al. 1992, Whiting & Draper 1981). Some of our pilot studies
showed slightly acidotic blood gas values in the rat groups fed on a high-sucrose diet
(Bäckman et al. 1996), which would suggest that one of the mediators of the reduction of
14
dentine formation might be the altered acid-base balance of the tissue. According to the
literature, it still remains unknown whether or not there are any common mechanisms
shared by these two states.
Bone and dentine have many similarities in composition and formation (Linde 1989,
Linde & Goldberg 1993). Therefore, a question was set whether a high-sucrose diet
causes any reduction in the dentine formation via an acidotic state. Based on the knowledge concerning bone, acid-base balance was thought to be one of the possible modulating factors both in the dentine formation and dentinal caries progression. An experiment
was planned to determine if acidosis would cause similar changes in dentine growth and
structure as a high-sucrose diet does. Chronic metabolic acidosis was induced with drinking water containing ammonium chloride.
In addition, the effects of chronic metabolic alkalosis were tested. As the opposite state
to acidosis, it was hypothesized that alkalosis would correct the effects of a high-sucrose
diet on dentine growth and structure if the effects of a high-sucrose diet were mediated
via acidosis.
Also, the overall effects of the altered acid-base balance on the composition and health
of the teeth were of interest. Acid-base imbalance is a common systemic disorder in many
diseases, in using certain drugs, in special diets, in high altitudes etc. Especially, metabolic acidosis in a mild form is quite common, it is detectable for example when a person
is losing weight or consuming protein-rich diet. (Brewer 1990)
Assuming that the changes in acid-base balance during the primary dentinogenesis
would cause changes in the calcium metabolism and collagen formation of odontoblasts,
and thus in the growth (and probably the structure) of the dentine, of a kind that resemble
those in bone, we set out to analyze the effects of chronic metabolic acidosis and alkalosis
and chronic respiratory alkalosis on dentine formation and mineral contents of dentine in
the molar teeth of young rats and whether these conditions have any effect on the progression of dentinal caries.
Chronic respiratory acidosis was not taken into this series of experiments because it
has little effect on bone (Lau et al. 1987, Bushinsky 1988, Bushinsky 1989, Chabala et al.
1991). Furthermore, it was impossible to maintain large enough groups of rats in the
hyperbaric chamber available. A pilot study with a small group was made, however, in
the hyperbaric chamber (1.5 bar) containing 27% O2 and 0.03% CO2 in N2. The results
suggest that respiratory acidosis may stimulate dentine formation in the mandibular
molars of young rats.
2. Review of the literature
2.1. Acid-base balance
A substance that can release or donate hydrogen ions (H+) is called an acid and a substance that combines with or accepts hydrogen ions is called a base. When an acid dissociates in a solution, it yields a free H+ and its conjugate base (with a negative charge).
The acid dissociation constant tells the strength of an acid: the higher the dissociation
constant, the more an acid is ionized and the greater is its strength. The concentration of
H+ in a solution is usually given as pH, which is a negative logarithm of the H+ concentration when expressed as moles/L. (Rhoades & Tanner 1995)
Acids in the mammalian body fall into two groups: carbonic acid (H2CO3) and all
other acids (so-called nonvolatile acids). All can be products of the metabolism. Carbonic acid is in equilibrium with the volatile gas CO2, which leaves the body via lungs,
whereas the other acids in the body are not directly affected by breathing. The nonvolatile acids are buffered in the body and excreted by the kidneys. (Rhoades & Tanner 1995)
Mammalian cells are very sensitive to intracellular changes in the H+ concentration
and also to that of the extracellular fluid, because the intracellular pH depends in part on
the extracellular pH. Nevertheless, they are not identical. The normal pH of the human
arterial blood is 7.40. In the venous blood it is slightly lower because of the higher concentration of the carbon dioxide. Intracellular pH values are lower than those of the extracellular fluid and range from 6.8 to 7.3 depending on the tissue and its metabolic rate.
When arterial pH is below 7.40, the state is called acidosis and when above it, it is called
alkalosis. (Ganong 1981)
Acidosis and alkalosis are both classified as either metabolic or respiratory, depending
on whether it is bicarbonate (HCO3-) (metabolic) or carbon dioxide (pCO2) (respiratory)
that primarily deviates from the normal range in blood (Martin et al. 1981). In metabolic
acidosis HCO3- and thus plasma pH (hydrogen concentration) fall, in alkalosis they rise.
In respiratory alkalosis pCO2 value and thus the carbonic acid concentration falls and pH
rises (Martin et al. 1981) and vice versa in respiratory acidosis.
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2.2. Causes of disturbances in acid-base balance
Various acute and chronic diseases cause severe changes in the acid-base balance of the
body. Reasons for small changes in the balance are still largely unknown, as well as all
their consequences, but they are known to be common. Acid-base disorders are especially common in sick children (Brewer 1990).
2.2.1. Causes of metabolic acidosis
The most common imbalance in the acid-base balance in the industrialized countries is
mild chronic metabolic acidosis caused by the diet rich in the animal protein. Proteins are
metabolized to organic acids. The typical American diet produces after metabolism
approximately 100 meq of acid every day (Barzel 1995). This kind of a diet has been
proved to cause aciduria and calciuria as a consequence of acidosis and thus a loss of
total calcium of the body (Breslau et al. 1988, Schuette et al. 1980, Licata et al. 1981).
Cola drinks that contain phosphoric acid are another acidosis-inducing ingredient of diet,
especially among young people (Barzel 1995).
In addition to the protein-rich diet, mild acute or chronic metabolic acidosis has been
reported in connection with diarrhoea (loss of bicarbonate), fasting (ketoacids) and heavy
exercise. Strenous exercise, like all states causing tissue hypoxia, elevates the amount of
the lactic acid in the extracellular fluids. (Brewer 1990)
The metabolism of carbohydrates and fats produce lower amounts of organic acids in
blood than the metabolism of proteins. All these acids are normally rapidly buffered and
excreted, but sometimes they accumulate in the body (Ganong 1981). When the kidneys
are not functioning normally they may fail to excrete the normal acid loads or reabsorb
the bicarbonate thus causing chronic metabolic acidosis. Renal failure and renal tubular
disorders, such as renal tubular acidosis, are known causes of metabolic acidosis (Brewer
1990). Kidney's ability to excrete acids also deteriorates with aging (Adler et al. 1968).
The symptoms of type I (impaired hydrogen ion excretion) and type II (impaired bicarbonate reabsorption) renal tubular acidosis are tiredness, loss of appetite, and growth
retardation in children. These diseases are usually a result of nephritis caused by infections or drugs. Also toxins and some congenital metabolic diseases may impair the renal
function. (Jalanko & Holmberg 1998)
Examples of life-threatening diseases that cause chronic metabolic acidosis are uncontrolled diabetes mellitus (diabetic ketosis) (International Classification of Diseases: ICD10 code E14.1), starvation (ketoacidosis) (E12.1) and hypoaldosteronism (E27.4) (Brewer
1990, Bichara et al. 1990). Ingestion of acidifying salts (for instance NH4Cl or CaCl2),
methanol or ethylene glycol (toxic metabolites formic acid and glycolic acid) or salicylate
overdose are sometimes found to be a cause of a serious state of acute metabolic acidosis
(Brewer 1990).
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2.2.2. Causes of metabolic alkalosis
Metabolic alkalosis may also be caused by some serious medical problems, for example
congenital chloride diarrhoea (E87.8), prolonged vomiting, hyperparathyroidism (E21)
and various neoplasms (Brewer 1990, Bichara et al. 1990). Also some diuretics, mineralocorticoid excess and ingestion of exogenous alkali are mentioned in the literature as the
possible causes for metabolic alkalosis (Brewer 1990).
Some renal diseases may also cause metabolic alkalosis. One example of this is Bartter
syndrome (E26.8), which is usually caused by mutations in the co-transport genes. It is
also reported to induce hypercalcinuria. (Jalanko & Holmberg 1998)
Other causes of mild chronic metabolic alkalosis in industrialized countries include
bulimia (F50.2) (loss of gastric acids) (Mitchell et al. 1987), calcitonin administration for
the treatment of osteoporosis (Escanero et al. 1991), a vitamin D excess or a vegetarian
diet (alkaline metabolites) (Bichara et al. 1990).
2.2.3. Causes of respiratory acidosis
Carbon dioxide formed by the tissue metabolism is in large part hydrated to H2CO3
increasing the total hydrogen ion load until CO2 is excreted in the lungs. Thus, impaired
(or suppressed) ventilation is the major cause of acute and chronic respiratory acidosis.
Possible reasons for the impared ventilation are lung diseases (for example asthma [J45],
cystic fibrosis [E84], pneumonia or pulmonary edema), impared lung motion, or certain
neuromuscular disorders (for example muscular dystrophy [G71], myasthenia gravis
[G70], or drugs that depress central nervous system) (Brewer 1990).
2.2.4. Causes of respiratory alkalosis
Respiratory alkalosis is probably the most common acid-base disorder among seriously ill
patients (Brewer 1990). Acute or chronic respiratory alkalosis is brought about by hyperventilation caused for example by hysterical hyperventilation syndrome (F45.3), brain
injury (Martin et al. 1981), anxiety, stress (Magarian 1982), hyperthyroidism (E05), liver
failure, fever (Brewer 1990), heart failure, anemia, pregnancy or a residence at high altitude (Krapf et al. 1991).
Human and other mammals hyperventilate at high altitudes, both at rest and during the
physical activity. The hyperventilation partially compensates for the lower tension of oxygen in the inhaled air. This leads to the lowered concentration of carbon dioxide (and carbonic acid) in the blood resulting in higher pH and respiratory alkalosis. (Hurtado 1971)
18
2.3. Maintaining of acid-base balance
Normally pH remains relatively constant both outside and inside the cells. Alterations in
the acid-base balance are resisted by extracellular and intracellular chemical buffers and
by respiratory and renal regulation. In the first place, kidneys and blood buffers attempt to
correct metabolic disorders and lungs attempt to correct respiratory disorders. (Brewer
1990)
Buffering in blood and extracellular fluid occurs in minutes. Acid or base added to the
body enter cells and bone slowly, over hours (Rhoades & Tanner 1995). In human body,
respiratory compensation for a metabolic disorder begins within minutes and is complete
in 12-24 hours. Metabolic compensation for respiratory disorder (increase of bicarbonate
in respiratory acidosis and decrease of bicarbonate in respiratory alkalosis) occurs more
slowly: it begins in hours and requires 2-5 days for completion (Brewer 1990). After the
compensations, the state of acid-base disturbance can be considered as chronic.
The change in pH in blood (produced when acid or base is added) is minimized by
chemical buffers, but they do not entirely prevent the pH change (Rhoades & Tanner
1995). In fact, in a disturbance of the acid-base balance, neither buffers nor the respiratory
or renal systems are completely successful in correcting pH until the underlying reason
for the disorder has been removed (Brewer 1990).
2.3.1. Role of lungs in maintaining acid-base balance
A normal adult produces about 300 liters of CO2 daily from the metabolism of foodstuffs. In the blood, CO2 reacts with water to form carbonic acid, which dissociates to H+
and HCO3-. In the lung capillaries they are converted back to CO2 and water and the CO2
is expired. (Rhoades & Tanner 1995).
As a secondary respiratory compensation, the lungs react to metabolic acidosis and
alkalosis. Metabolic acidosis stimulates breathing causing hyperventilation while metabolic alkalosis suppresses it. These are attempts to correct pH by changing the concentration of carbon dioxide and carbonic acid in the blood. (Rhoades & Tanner 1995)
2.3.2. Role of buffers in maintaining acid-base balance
Oxidation of proteins and amino acids produces strong acids, like sulfuric, hydrochloric,
and phosphoric acids, in the normal metabolism. These and other non-carbonic (non-volatile) acids are buffered in the body and must then be excreted by the kidneys (Rhoades &
Tanner 1995).
The most important extracellular buffer is bicarbonate, which usually buffers these
non-volatile acids. The kidneys regenerate the bicarbonate used in buffering by excreting
hydrogen ions in the urine as ammonium and titratable acids (Brewer 1990). Other major
19
chemical pH buffers in the body are inorganic phosphate and plasma proteins in the extracellular fluid, cell proteins, organic phosphates and bicarbonate in the intracellular fluid,
and mineral phosphates and mineral carbonates in bone (Rhoades & Tanner 1995).
2.3.3. Role of kidneys in maintaining acid-base balance
The kidneys have two important roles in the maintaining of the acid-base balance: to
reabsorb bicarbonate from and to excrete hydrogen ions into urine. 4500 mmol of bicarbonate are filtered into the primary filtrate of urine daily, but only 2 mmol of it are finally excreted. 70-80% of bicarbonate is reabsorbed in the first part of proximal tubule, 1020% in the loop of Henle and 5-10% in the distal tubule and collecting ducts. (Jalanko &
Holmberg 1998)
Carbonic anhydrase plays an important role in the reabsorption in the proximal tubule.
Disturbance in the reabsorption of bicarbonate in the proximal tubule leads to metabolic
acidosis, hyperchloremia and alkalotic urine. This disease is named as "type II renal tubular acidosis" (N25.8). (Jalanko & Holmberg 1998)
Renal tubules actively secrete hydrogen ions. Most of this takes place in the distal part
of the nephron, but active transport of hydrogen ions occurs in the proximal tubule, too.
The H-ATPase of the apical cell membrane secretes hydrogen ions into urine. For each
hydrogen ion secreted, one bicarbonate molecule is transported to the interstitial fluid,
from there it diffuses into the bloodstream. Fifty mmol of hydrogen ions are normally
excreted daily. (Jalanko & Holmberg 1998)
If the hydrogen ions are not properly secreted into the collecting ducts, the result is
metabolic acidosis, hypokalemia, hypocalcemia, nephrocalcinosis and an alkalotic urine.
This disease is called "type I renal tubular acidosis" (N25.8). (Jalanko & Holmberg 1998)
The maximal hydrogen ion gradient, against which the transport mechanism can
secrete H+ ions, corresponds to a urine pH of 4.5 in humans. However, three important
molecules remove free hydrogen ions from the tubular fluid permitting more acid to be
secreted: H+ is bound to ammonia, phosphate and bicarbonate to form NH4+, H2PO4-,
CO2 and H2O. (Ganong 1991)
The source of the hydrogen ions secreted by the tubular cells is not completely certain. It is probably produced by dissociation of H2CO3. The acid-secreting cells contain
carbonic anhydrase, which facilitates the rapid formation of H2CO3 from CO2 and water.
The renal acid secretion is mainly regulated by the changes in the intracellular pCO2,
potassium concentration, carbonic anhydrase activity and adrenocortical hormone concentration. (Ganong 1991)
2.4. Acid-base balance and bone
The main constituents of bone are type I collagen in the organic matrix and hydroxyapatite in the inorganic matrix. The mineral in the skeleton is being turned over throughout
life. Calcium in bone turns over at a rate of 100% per year in infants and 18% per year in
adults. Osteoblasts produce bone by secreting collagen that forms the matrix which then
20
calcifies. Osteoclasts are responsible of resorption: they erode and phagocytose bone. One
turnover cycle, in which one cavity is resorbed and filled again, is relatively slow:
approximately eight months. (Green & Kleeman 1991)
Inactive osteoblasts flatten out over the bone surfaces (Fig. 1). They make a partial
membrane, which separates the so-called bone fluid (which is in contact with the
hydroxyapatites) from the extracellular fluid of the adjacent tissues (Green & Kleeman
1991). The fast regulation of serum calcium occurs across this quiescent surface area
(Parfitt 1987). Inside the bone canaliculi, osteocytes are involved in this process (Talmage
& Grubb 1977). The tiny hydroxyapatite crystals present an enormous surface area in the
bone (100-200 square meters per gram of bone). Also, the bone is relatively well vascularized. This structure allows a rapid mobilization of the bone calcium. (Green & Kleeman 1991, Ganong 1991)
Acid-base balance has an effect on bone turnover, especially on the rates of bone
resorption and calcium mobilization. Bone mineral participates in the defense against
acid-base disturbances, especially against metabolic acidosis (Lemann et al. 1966, Green
& Kleeman 1991). The role of the bone mineral is important in the acid-base disorders, as
no appreciable change in the intestinal calcium absorption occurs (Bichara et al. 1990).
In the mammalian body, mainly three hormones regulate the calcium metabolism and
the bone turnover. 1,25-dihydroxycholecalciferol (vitamin D derivative) increases calcium absorption from the intestine and, indirectly, from bone. Parathyroid hormone mobilizes calcium from the bone and increases the urinary phosphate excretion. Calcitonin
inhibits bone resorption (Ganong 1981). Used as drugs, these hormones are also capable
of inducing acid-base disorders. Calcitonin administration (Escanero et al. 1991) and vitamin D excess (Bichara et al. 1990) have been reported to cause metabolic alkalosis.
2.4.1. Metabolic acidosis and bone
In mammals, the endogenous metabolism produces acids, mostly originating from the
proteins in the diet. The extracellular fluid bicarbonate buffers in part these acids, causing a decrease of bicarbonate in blood and thus a fall in systemic pH. Fall in pH is buffered by other buffers in the body, including the mineral phases of bone (Bushinsky 1995).
Bone contains large buffer stores, specifically salts of phosphate and carbonate (Rhoades
& Tanner 1995). In the process of skeletal buffering, calcium is released from the bone
mineral (Bushinsky et al. 1983).
If the acids are produced in great amounts or their excretion is impaired, the result is
the loss of bone. The kidneys react to the additional calcium in plasma by increasing calcium excretion (hypercalcinuria). As there is no change in the intestinal calcium absorption, the net result is a decrease in the amount of calcium in the body (Breslau et al.
1988).
The change in the total mineral content of the bone is marked in severe acidosis
(Lemann Jr et al. 1966, Green & Kleeman 1991). The effect of acidosis on the bone is
much greater in young mammals than in adults (first described by Jaffe et al. 1932). In
adult subjects, there is less bone buffering due to the lower proportion of bone water and
21
exchangable mineral surface (Burton 1992). However, even a mild acid loading may lead
over the years to osteoporosis (Sebastian et al. 1994). This kind of a mild acid loading
may be caused by, for example, the high-protein diet.
In the bone the cell-mediated calcium release is the most important and sensitive
mechanism of response to metabolic acidosis (Bushinsky 1989, Goldhaber & Rabadjija
1987). To a lesser extent, low pH also promotes physicochemical mineral solubility,
which does not depend on the cells (Bushinsky & Lechleider 1987). In addition to stimulating osteoclastic function, metabolic acidosis also inhibits osteoblastic bone formation
by slowing down collagen synthesis (Krieger et al. 1992, Whiting & Draper 1981).
In vitro, a decrease in the bone collagen synthesis and diminished alkaline phosphatase
activity occur in calvariae in metabolic acidosis, both indicating a suppression of osteoblastic function (Krieger et al. 1992). The genes critical to osteoblastic function are
altered by pH. In a group of the immediate early response genes (c-fos, egr-1, junB, c-jun,
junD), metabolic acidosis (pH 6.8) leads to a reduction in egr-1 stimulation, while metabolic alkalosis (pH 7.6) stimulates it. RNA for type 1 collagen reacts in the same way to
both acidosis and alkalosis. Increasing or decreasing external pH by 0.2 units causes a
significant change in the egr-1 stimulation. Thus, small changes in systemic pH may have
a significant effect on the expression of certain genes important for the osteoblastic function. (Frick et al. 1997)
The activity of osteoclastic enzymes in cultured calvariae is enhanced in metabolic acidosis (Krieger et al. 1992). Stimulation of the osteoclastic beta-glucuronidase release has
been reported (Bushinsky & Nilsson 1995).
Parathyroid hormone has similar effects as acidosis on the bone. It also induces the
cell-mediated bone resorption, suppresses the osteoblastic collagen synthesis and stimulates the osteoclastic beta-glucuronidase release. In vitro, additive effects of metabolic
acidosis and hyperparatyroidism on the net calcium efflux and the bone cell function have
been reported (Bushinsky & Nilsson 1995)
2.4.2. Respiratory acidosis and bone
Respiratory acidosis seems to cause mainly similar, but not as profound changes in the
calcium metabolism as metabolic acidosis. Alterations in the surface ion composition in
the cultured bone in metabolic, but not in respiratory acidosis, have been reported (Chabala et al. 1991). There is proton influx into the bone during metabolic acidosis, but not
during respiratory acidosis (Bushinsky 1988), and calcium efflux from the bone during
metabolic acidosis is greater than during respiratory acidosis in vitro (Bushinsky 1989).
In the cultured bone, the alterations in the ion composition in respiratory acidosis are
much less severe than in metabolic acidosis (Chabala et al. 1991). In vivo, respiratory acidosis does not appreciably increase the urine calcium excretion, although there is an
increase in the serum calcium concentration (Lau et al. 1987).
22
2.4.3. Metabolic alkalosis and bone
Metabolic alkalosis causes an influx of calcium into the bone, but the effect is not as
strong as the opposite effect of metabolic acidosis (Bushinsky et al. 1983). Also, metabolic alkalosis results in hypocalcinuria and thus a retention of calcium, while there is no
change in the intestinal calcium absorption (Bichara et al. 1990).
Neutralization of the daily metabolic acid load with base decreases calcium excretion
(Bushinsky 1996). In clinical studies, patients with a negative calcium balance have been
treated successfully with sodium bicarbonate (Lutz 1984) and potassium bicarbonate
(Sebastian et al. 1994).
In vitro, both mild (pH 7.5) and severe (pH 7.6) metabolic alkalosis cause a progressive decrease in the calcium efflux from the bone. The calcium efflux is inversely correlated with medium pH: the higher the medium bicarbonate, the less calcium efflux from
the bone (Bushinsky 1996). Also in several clinical studies metabolic alkalosis has
decreased bone resorption and even increased bone formation (Breslau et al. 1988, Licata
et al. 1981, Schuette et al. 1980).
Metabolic alkalosis decreases bone calcium efflux by stimulating the osteoblasts and
suppressing the osteoclasts (Bushinsky 1996). Alkalosis may alter the function of both the
osteoblasts and the osteoclasts to a similar degree or it may modify the function of one
cell type which then alters the function of the other. These mechanisms are not yet clear.
Alkalosis causes a decrease in the release of osteoclastic enzyme beta-glucuronidase,
which has an important role in the bone resorption (Bushinsky 1996). Also, the osteoblastic collagen synthesis is induced. The genes important for the osteoblastic function have
been found to react in metabolic alkalosis. In vitro, the osteoblastic early response gene
egr-1 and RNA for the type 1 collagen are stimulated resulting in induction of the odontoblast collagen synthesis (Frick et al. 1997). There is an inverse correlation between the
effects of metabolic alkalosis on osteoclastic enzyme release and osteoblastic collagen
synthesis (Bushinsky 1996).
In the process of resorption, the osteoclasts secrete protons between themselves and
the bone mineral. To prevent intracellular alkalinity, the osteoclasts must excrete the
bicarbonate generated for every hydrogen ion secreted. In metabolic alkalosis, the
increased concentration of the bicarbonate in the extracellular fluid may suppress the
osteoclastic hydrogen ion secretion. (Bushinsky 1996)
If the osteoclastic activity is inhibited by calcitonin, the influx and efflux of calcium
are still, although in lesser extent, correlated with the concentration of bicarbonate. This
indicates that the alterations in the bicarbonate concentration have also a non-osteoclastmediated effect on the bone. It remains unknown whether metabolic alkalosis also affects
the physicochemical mineral dissolution in addition to its effects on the cell-mediated calcium flux. (Bushinsky 1996)
Data relating to the alkali loads and the respiratory changes are scarce (Green and Kleeman 1991). No reports concerning respiratory alkalosis and the bone seem to have been
published.
23
2.5. Acid-base balance and teeth
There are numerous similarities between the osteoblasts and the odontoblasts. The principal difference between the osteogenesis and the odontogenesis lies in the fact that no
remodelling nor osteoclast-like cells exist in the dentine (Fig. 1). However, microorganisms seem to be capable of destroying enamel and dentine structure by a direct action
resembling osteoclasts dissolving bone (Brännström et al. 1980, Luoma et al.1984).
Fig. 1. Schematic drawing of the dentine (left) and the bone (right). Gray area = mineralized
tissue. Striped gray area = unmineralized new dentine (predentine) / bone (osteoid). Left:
carious dentine and cariogenic bacteriae up, odontoblast cells (pulp) down. Odontoblast
processes in tubules. Right: Active (round) and passive (flat) osteoblasts surround the bone.
Osteocytes are located inside the bone. They are communicating via the bone canaliculi with
each other and with the osteoblasts. In left upper corner, multinucleated osteoclast resorbs the
bone.
The odontoblasts are partly under the same metabolic regulation as the osteoblasts
(Linde & Goldberg 1993), and therefore the formation of the bone and the dentine are
probably regulated by similar factors. Thus there is a reason to assume that the changes in
acid-base balance have effects on dentine metabolism as they do on the bone. Indeed, in
previous studies we have found that chronic metabolic acidosis slowed down the rate of
dentine formation and the general body growth in the young rats (Bäckman et al. 1996).
In humans, several congenital chronic diseases, causing acid-base disturbances, result
into changes in dental health and development and the structure of the teeth. Congenital
persistent proximal type renal tubular acidosis causes enamel defects of the permanent
teeth (Winsnes et al. 1979). Also, missing and peg-shaped teeth, enamel hypoplasias and
excessive caries in carbonic anhydrase II deficiency syndrome with renal tubular acidosis
have been reported (Ohlsson et al. 1986).
24
Severe symmetrically distributed enamel hypoplasia in the permanent teeth was found
in a patient with chronic metabolic acidosis (congenital persistent renal tubular acidosis of
proximal type, capillary blood pH 7.07-7.15) (Koppang et al. 1984). Delayed shedding
and eruption, agenesia of a few permanent teeth and retarded tooth development were
also reported. The primary teeth were normal except for an extremely thin enamel. From
a 10-year old boy, several teeth had been extracted due to caries. His skeletal age was 3
years and dental age 2 years delayed.
Both chronic metabolic alkalosis (children with congenital chloride diarrhoea [E87.8],
Myllärniemi & Holmberg 1975) and chronic respiratory alkalosis (children with acyanotic congenital atrial septal defect in heart [Q21.1], Bäckman et al. 1990) have been
observed to increase caries resistance. Myllärniemi & Holmberg (1975) also reported
enamel defects and hypoplasias of varying severity in both the deciduous and the permanent teeth. The timing of the deciduous and permanent teeth formation and eruption was
normal.
Acid-base balance also affects the fluoride metabolism. The absorption rate of fluoride from the stomach is dependent on the pH of the gastric contents (Whitford & Pashley
1984). Plasma clearance of fluoride by the kidneys is related to urinary pH: acidosis
induces reduction in the renal clearance of fluoride (Whitford et al. 1976). High concentrations of fluoride and magnesium are found in the bone and the enamel associated with
the acidotic state (Angmar-Månsson & Whitford 1995).
Mineralization defects in the enamel of rats and dogs, resembling fluorosis, have also
been found in acidosis without an exposure to fluoride (Angmar-Månsson & Whitford
1990). Both chronic metabolic acidosis (exposure to NH4Cl) and chronic respiratory acidosis (exposure to 10% CO2) result in major disturbances in the rat incisor enamel (Whitford & Angmar-Månsson 1995).
In an experiment with young pups, chronic metabolic acidosis was induced with
NH4Cl (Angmar-Månsson & Whitford 1986, Angmar-Månsson & Whitford 1990). This
resulted in an increase in the amount of fluoride in teeth with no change in the phosphorus
concentration. Also in NH4Cl -induced acidosis with no fluoride supplementation, the
mineralization of enamel was severely disturbed with alternating layers of hyper- and
hypomineralization, having in some cases even cystic appearance in the microradiographic analyses. Chronic metabolic alkalosis (induced with NaHCO3) caused only minor
changes in the mineralization pattern. The ratios of Ca/P or Ca/Na did not differ between
these groups or compared to the controls.
With similar experimental setting, Driessens et al. (1987) found no differences in the
Ca/P or Ca/Na ratios in the molar dentine between acidotic, alkalotic and control pups.
Also, there was not a clear trend in these ratios as a function of the distance from the front
of the mineralization.
Metabolic alkalosis enhances the excretion rate of fluoride by the kidneys, which is
reflected in reduced fluoride levels in both soft and hard tissues. The disturbance in the
enamel mineralization associated with alkalosis is additive to that produced by fluoride.
In acidosis, the defective mineralization is attenuated by a supplementation with fluoride.
(Angmar-Månsson & Whitford 1990)
Calcium phosphate supplementation of the diet did not mitigate the defects in the
enamel mineralization associated with the chronic acid-base disturbances. Instead, it
worsened them, especially in chronic acidosis. (Angmar-Månsson & Whitford 1990)
25
Chronic respiratory alkalosis, caused by living in low pressure corresponding 5490
meters above sea level, also results in disturbances in the mineralization of the rat incisor
enamel. The enamel is severely damaged both macroscopically and microradiographically and uniformly bleached to the color of chalk. The incisor dentine contains numerous
small lacunae. Like in metabolic acidosis, hard and soft tissues have a higher concentration of fluoride. (Angmar-Månsson et al. 1984, Angmar-Månsson & Whitford 1990)
2.6. High-sucrose diet and dentine
Reduction in the growth of dentine in the molar teeth of young rats caused by a highsucrose diet has been observed in numerous studies (Larmas et al. 1992, Tjäderhane et al.
1995, Huumonen et al. 1997, etc.). This effect is independent on the other dietary constituents as well as on the severity of caries (Tjäderhane et al. 1994).
The results also indicate that the reduction of dentine apposition is not caused by the
caries, but by the systemic effects of a high-sucrose diet, because dentinogenesis was
slowed down even under the intact fissures (Huumonen et al. 1997). And vice versa:
slower dentinogenesis has not been found to increase (or decrease) the caries initiation or
progression. This result has been obtained by reducing the dentine formation by means
other than sucrose during the primary dentinogenesis (Huumonen et al. 1996).
Concerning molar fissures, it is difficult to confirm if microbial effects on dentine are
totally avoided or not. The bacteria seem to be able to invade the enamel through surface
microdefects. A cross-section reveals only one plane of enamel and the defects may
remain unobserved (Seppä 1984, Seppä et al. 1989). Also, dentine may be infected
through an incipient enamel lesion, even when no cavitation has occurred on the enamel
surface. This has been reported both in rats (Luoma A-R. et al. 1984, Luoma H. et al.
1987, Seppä et al. 1989) and in humans (Brännström et al.1980, Seppä 1984, Seppä et al.
1985).
During secondary dentinogenesis, the rate of the dentine formation and the caries progression both are less than 1/10 of that during the the primary dentinogenesis. This suggests that there is a connection between the rate of caries progression and the deposition
of the dentine. (Hietala & Larmas 1992, Kortelainen & Larmas 1994)
The concentration of sucrose in the diet must be high to reduce the dentine formation
in the rats. When young rats were fed on a diet containing 15%, 30% or 43% of sucrose,
significant reduction in the dentinogenesis was only seen in the animals with 43% of
sucrose in the diet. The rats were not inoculated with cariogenic bacteria. The critical
amount of sucrose seemed to be between 30 and 40 g/ 100 g. (Huumonen et al. 1997)
All kinds of high-sucrose diets seem to have the same effect on dentine formation in
the young rats. Autio et al. (1997) reported only a slightly stronger reduction in rats fed
on the modified Stephan-Harris diet (43% of sucrose, Table 1) than in those fed on the
R36 diet (special diet for growing rats and mice, Brood Stock Feed for Rats and Mice
R36, Finnewos Aqua Oy, Turku, Finland), in which most of the barley and wheat flour
were replaced by sucrose (41%) and casein was added to compensate the loss of protein.
26
Both of the sucrose diets mentioned above slightly increased the width of predentin
compared with the control diet (R36) (Autio et al. 1997). In this respect, the effects of
both sucrose diets were equal. The same effect, but more pronounced, has also been
reported in the study of Hietala et al. (1997), in which the modified Stephan-Harris diet
was used. The increased width of predentin indicates disturbed mineralization in the rats
fed on the high-sucrose diets (Butler 1995).
Also quantitative changes in the amounts of mineral elements of dentine of the young
rats' molars has been observed in connection with the high-sucrose diet (the modified
Stephan-Harris diet) compared to the standard diet (Ewos R3, Table 1) and to the modified Stephan-Harris diet in which sucrose has been replaced with potato flour (starch)
(Tjäderhane 1996). Calcium, phosphorus, fluoride, sodium, magnesium, zinc and the total
content of minerals in dentine were determined with SEM equipped with an electron
probe microanalyzer (EPMA). Reduction in all the elements measured, except F and Zn,
was found in the sucrose group.
In the study referred to above (Tjäderhane 1996), the dentinal Ca/P ratios did not differ
before or during the experiment or between the groups. A Stephan-Harris diet in which
sucrose had been replaced with complex carbohydrate (starch) gave identical results to
the Ewos R3 standard diet, which suggests that nutritional deficiences were not the cause
of the changes in the mineral contents in the dentine of the rats fed on the sucrose diet.
The high-sucrose diet has also been reported to suppress the rate of fluid movement in
the molar dentine of young rats (Steinman & Leonora 1971). The rate of the fluid movement was inversely related to the incidence of dental caries. The authors assume that the
products of metabolism (lactic acid) accumulate and the nutrient uptake decreases in the
dentine as a consequence of the suppressed fluid movement. Unlike the bone, the dentine
is avascular and thus more dependent on the fluid transport system.
The dentinal fluid movement has been found to be regulated by parotid hormone
(”parotin”) in rat (Leonora et al. 1992) and in pig (Tieche et al. 1994). Parotidectomized
rats had a suppressed fluid movement in the dentine regardless of the quality of the diet.
A high sucrose-diet reduces the secretion of the parotid hormone and thereby suppresses
the rate of the dentinal fluid movement in both the rats and the pigs (Leonora et al. 1992,
Tieche et al. 1994).
2.7. Dentinogenesis
The odontoblasts lining the pulp chamber produce the dentine. The dentine is a highly
permeable tissue, because densely packed dentinal tubules radiate from the pulp throughout all the layers. 15000 tubules/mm2 are present in the outer dentine and 55000 tubules/
mm2 near the pulp. The dentine contains more minerals than the bone: 70% of weight
consists of the minerals. Type I collagen is predominate in the organic and hydroxyapatite in the inorganic portion. (Linde & Goldberg 1993)
The dentine may be divided into intertubular dentine and peritubular dentine. The
former is the main product of the odontoblasts constituting the largest volume of the dentine. The intertubular dentine consists of a fibrous network of collagen with deposited
mineral crystals. The peritubular dentine forms a highly mineralized sheath around the
27
dentinal tubule (0.5-1 micrometers thick in humans). The peritubular dentine gradually
(partly or completely) fills up the dentinal tubules at some distance away from the pulp
chamber. (Linde & Goldberg 1993)
The first stage of the dentinogenesis forms mantle dentine on the dentine-enamel junction during the early stages of the tooth development. In human, the mantle dentine is 530 micrometers thick (Linde & Goldberg 1993). It is rich in proteoglycans and more
irregular and less mineralized than the following layers. (Jenkins 1978, Linde & Goldberg
1993).
The second stage, forming the next layer and consisting the most of the tooth structure,
is called primary dentinogenesis. Some confusion exists in the literature concerning the
ending time of this stage (Cox et al. 1992). The primary dentinogenesis is considered to
be finished and secondary dentinogenesis started at different phases in different publications, such as when the crown is fully formed, when the tooth erupts (Cox et al. 1992),
when the tooth becomes functional (Linde & Goldberg 1993) or when the root is fully
formed (Torneck 1994). The latter seems most reasonable, because (in humans) the tooth
metabolism becomes slower after the root apex is formed.
In rat molars, the dentine formation slows down gradually during both the primary and
the secondary dentinogenesis with no apparent transition from the former to the latter
(Johannessen 1961, Hietala & Larmas 1992, Kortelainen & Larmas 1994). The structure
of the secondary dentine is supposed to be slightly more irregular than that of the primary
dentine (Torneck 1994).
The next stage, tertiary dentinogenesis, occurs as a tooth response to irritations. Tooth
preparation made by a dentist, dentinal caries, attrition, abrasion and/or erosion are the
most common irritating factors (Cox et al. 1992). The tertiary dentine may also be named
according to the quality of the irritation: The dentine formed as a response to attrition,
abrasion or erosion is called "reactional dentine" to separate it from caries and preparation
induced "reparative dentine". The tertiary dentinogenesis may be absent even in a fully
matured tooth. The quality of the tertiary dentine seems to be dependent on the speed of
its formation: the faster it is formed, the more irregular it appears (Linde & Goldberg
1993, Torneck 1994).
Predentine is the innermost layer of the dentine, right next to the odontoblasts and the
pulp. It is a thin layer of unmineralized organic matrix, mostly collagen. This layer is
present also in an old tooth, in which the dentinogenesis is slowed down. (Linde & Goldberg 1993)
During all the stages of the dentinogenesis, permanent layers of dentine are formed.
Thus, a disturbance in any stage leaves persisting marks in the structure. This is different
from the bone, in which constant turnover exists.
Because of the confusing terminology concerning the different stages of the dentinogenesis and the lack of apparent zones of transition from one stage to another, the names
of the stages are partly ignored in this study. Considering the age of the rats during the
experiments (3-10 weeks), however, the dentine formed was mostly primary.
3. Working hypothesis and aims of the study
Among dentists, dental caries is thought to merely imply the dissolution of the tooth minerals by bacterial functions. In fact, this is principally the case in the enamel, and undeniably also in dentine, although perhaps less exclusively.
In the dentine, the vital processes are principally regulated by the cells of the pulp/dentine complex in a way that makes dentinal caries a process notably resembling the bone
resorption. Thus, the function of the cariogenic bacteria in the dentine may be comparable
to the function of the osteoclasts in the bone.
The rate of the destruction in dentinal caries seems to be associated with the rate of the
dentinogenesis. Caries proceed faster in the teeth of the young animals with rapid growth
of the dentine than in the teeth of the older animals (Hietala & Larmas 1992, Kortelainen
& Larmas 1994). On the other hand, a high-sucrose diet and metabolic acidosis slow
down the rate of the dentinogenesis and induce caries (Kortelainen & Larmas 1990,
Tjäderhane et al. 1994, Bäckman et al. 1996).
The aim of this work was to find out whether the high-sucrose diet and the acid-base
imbalance of the body have the same kind of systemic effects on caries (on cariogenic
bacteria) and on the odontoblasts in the dentine as they have on the osteoclasts, osteoblasts and osteocytes in the bone and whether these processes are connected. The slower
rate of formation, as such, of the dentine is thought to be analogous to bone resorption, as
no resorption by tissue cells occurs in the normal dentine.
The tested central hypotheses were:
1. Sucrose affects odontoblasts causing reduction in dentine formation and acceleration
in caries progression at least partly via metabolic acidosis.
2. Metabolic alkalosis eliminates most of the effects of sucrose, if the hypothesis number one is correct.
4. Materials and methods
4.1. Maintenance of the rats
All the experimental animals were Wistar rats (Wistar Hannover), 96 animals altogether,
and they were bred in the Department of Laboratory Animals, Institute of Dentistry, University of Oulu. Their maintainance and care and all the experimental procedures were
performed by persons licenced to that work. The animals did not suffer from pain or diseases of any kind during the experiments. The experimental protocols were accepted by
the Experiment Animal Committee of the Medical Faculty, University of Oulu.
The animals were housed 2 or 3 to a cage (Macrolon III) on a bed of European aspen
shavings at a temperature of 21°C and humidity of 40%-60%, and subjected to the same
lighting regimen (12 hours light and 12 hours dark) and the same frequency of human
handling. The rats were weighed once a week. The food and water consumption per cage
were recorded at intervals of three days throughout the experiment. These were rough
estimations, because no metabolic cages or feeding machines were used (individual cages
are not found to be suitable for caries experiments in rats, Baker et al. 1979).
4.2. Diets
A modified Stephan-Harris diet was mixed in our laboratory and used as the high-sucrose
diet and a commercial Ewos R3 diet (Brood Stock Feed for Rats and Mice R3, Ewos AB,
Södertälje, Sweden) as the standard one. The compositions of the diets are presented in
Table 1 and the nutritional values in Table 2. The diets are nutritionally acceptable for
growing rats according to the recommendation of National Research Council (1972).
Both diets were supplied in a powdered form and provided ad lib.
30
Table 1. Compositions of the diets.
Diet
Ingredient
Modified Stephan-Harris diet
Sucrose
43
(sucrose diet)
Wheat flour
22
Skimmed milk powder
32
Ewos R3 diet
(standard diet)
Amount (wt%)
Liver powder
2
Vegetable oil
1
Oat flour
28
Wheat products
50
Soya meal
7
Fish powder
7
Fodder yeast
3
Minerals
3
Animal and vegetable fat
1
Vitamins and trace elements
<1
Abbreviations: wt% = weight (g) per 100 g.
Table 2. The nutritional values of the diets.
Component
Unit / kg diet
Energy, kJ
Protein, g
Fat, g
Linoleic acid, g
Calcium, g
Phosphate, g
Sodium chloride, g
Magnesium, g
Potassium, g
Ferric, mg
Copper, mg
Retinol, mg
dl - α - tocopherol acetate, mg
Thiamin hydrochloride, mg
Riboflavin, mg
Pyridoxine hydrochloride, mg
Vitamin B12, µg
Calcium pantothenate, mg
Free fluoride, ppm
Modified Stephan-Harris diet
(sucrose diet)
15560
143
21.6
0.8
5.9
3.9
3.8
0.47
6
18
5.3
0.85
7.6
2.5
7.1
1.8
7.6
17.2
0.00
Ewos R3 diet
(standard diet)
12600
210
50
1.5
9.9
6.5
7
2
8
190
30
0.36
63
3.3
12
4
20
10
0.05
Abbreviations: RDA = recommended daily allowance, National Research Council (1972).
RDA
18420
133
55
2.4
5.6
4.4
6
0.4
20
38.9
5.6
0.67
35
1.5
2.8
7.8
5.6
8.9
0.00
31
4.3. Conduct of the experiment
The rats were 3 weeks old at the beginning of the experiments. Groups 1-4, groups 5-8
and groups 9-11 were run at the same time. Inside the groups 1-4, 5-8 and 9-11, the rats
were taken from the same litters. The same number of rats from each litter was placed to
each of the groups. Each rat was randomly chosen for its group. The rats were weighed,
marked and given an intraperitoneal injection of oxytetracycline hydrochloride (30 mg/kg
Terramycin®, Pfizer Corp., Brussels, Belgium) to mark the onset of dentine apposition as
a line visible in UV-light (Larmas & Kortelainen 1989, Hietala et al. 1993).
In order to induce dental caries, the mouths of the animals were inoculated with a fresh
suspension of Streptococcus sobrinus (ATCC 27531 K 1 Fitzgerald) on days 2 and 3 of
the experiment and then weekly. The repeated inoculation of all the rats with the same
bacteria also ensured the relative equality of the oral microbial flora in all the groups
throughout the experiment. In order to avoid large dentinal carious lesions (and the disturbances in odontoblastic metabolism induced by deep caries), the experiments were limited to six weeks' duration in the groups with the high-sucrose diet. Nevertheless, some
bacterial invasion may have been progressing deep in the dentine (Luoma H. et al. 1987,
Seppä et al. 1989) also disturbing odontoblast function. The duration of the experiments
was based on pilot studies. The cariogenic challenge was adjusted to be sufficiently long
for both the initial and advanced carious lesions to be present at the end of the experiments.
4.3.1. Induction of metabolic acidosis
The rats, referred to as acidotic, were divided into three groups and they all received ad
lib. distilled drinking water supplemented with 0.25 mol/L ammonium chloride (pH 5.06;
PHM62 Standard pH meter, Radiometer, Denmark; Angmar-Månsson & Whitford 1986).
The sucrose diet was given to one acidotic group (6 week experiment, group 1), while the
other two groups (6 and 7 week experiments, groups 2 and 9) were fed on the standard
diet (Table 3).
Distilled water was used (instead of the tap water) to ensure the similarity of the drinking water in every group throughout the experiments. The quality of the drinking water
was not an essential matter in these experiments, since all the groups consumed the same
kind of water (part of them supplemented with ammonium chloride or sodium bicarbonate).
32
4.3.2. Induction of metabolic alkalosis
Three alkalotic groups received ad lib. distilled water supplemented with 0.25 mol/L
sodium bicarbonate (pH 8.3; PHM62 Standard pH meter, Radiometer, Denmark; Angmar-Månsson & Whitford 1986). One group was fed on the sucrose diet for six weeks
(group 3), while two groups received the standard diet, one for six (group 4) and the other for seven weeks (group 10) (Table 3).
4.3.3. Induction of respiratory alkalosis
Two groups were kept in a hypobaric chamber with an atmospheric pressure equivalent to
an altitude of 3000 m (69,7 kPa) for 6 weeks. One group (group 5) was fed on the sucrose
diet and the other (group 6) on the standard diet (Table 3). They all received distilled
drinking water (pH 6.10; PHM62 Standard pH meter, Radiometer, Denmark).
The people taking care of the animals entered and passed the hypobaric chamber via
airlock, so that the pressure remained constant in the chamber throughout the experimental period. Also, the blood samples from the tails of the rats at the end of the experiment
were taken inside the hypobaric chamber to ensure the correct blood gas values.
4.3.4. Control rats (normalosis)
The remaining three groups received distilled drinking water (pH 6.10; PHM62 Standard
pH meter, Radiometer, Denmark) and were kept under normal atmospheric conditions.
One group was fed on the sucrose diet for 6 weeks (group 7) and the other two on the
standard diet for 6 and 7 weeks (groups 8 and 11) (Table 3).
Table 3. Grouping and treatment of the rats in the experiments.
Group
6 weeks
1
2
3
4
5
6
7
8
7weeks
9
10
11
Male
n
Female
n
Diet
Acid-base status
Group abbreviation
4
5
3
3
3
3
3
3
2
1
6
6
8
7
6
6
sucrose
standard
sucrose
standard
sucrose
standard
sucrose
standard
metabolic acidosis
metabolic acidosis
metabolic alkalosis
metabolic alkalosis
respiratory alkalosis
respiratory alkalosis
normalosis
normalosis
m-acid-suc 6wk
m-acid-stan 6wk
m-alk-suc 6wk
m-alk-stan 6wk
r-alk-suc 6wk
r-alk-stan 6wk
norm-suc 6wk
norm-stan 6 wk
3
4
5
5
6
4
standard
standard
standard
metabolic acidosis
metabolic alkalosis
normalosis
m-acid-stan 7 wk
m-alk-stan 7 wk
norm-stan 7wk
33
4.4. Anesthesia and blood samples
At the end of the experiment the rats were anaesthetized by a minimum respiratory suppression using a mixture of midazolam (Dormicum®; Roche, Basel, Switzerland), fluanisone-fentanyl (Hypnorm®; Janssen Pharmaceutica, Brussels, Belgium) and sterile
water 1:1:2 0.2 mL/100 g of rat weight, given intraperitoneally. While the rats were
unconscious, blood samples were taken from the cut tips of their tails into capillary tubes
containing heparin and an "iron flea" for stirring (review: Beetham, 1982).
The blood samples were used for the measurement of pH, bicarbonate (HCO3-), base
excess (B.E.), and oxygen and carbon dioxide partial pressures (pCO2 and pO2), with a
blood gas analyzer (Corning 168 pH/Blood Gas Analyzer, Corning Medical, U.S.A.).
After this, when still unconsious, the animals were killed in a carbon dioxide chamber.
4.5. Preparation and analyses of the tooth samples
The mandibular molars were prepared for the analysis of dentine formation, dentinal caries and mineral analysis using a method described by Keyes (1958). The mandibles were
dissected, defleshed and sectioned sagittally by using a 0.1 mm thick diamond disk saw
and water cooling, in an oblique parasagittal plane (Fig. 2).
34
Fig. 2. Sectioning of the rat mandible.
4.5.1. Quantification of dentine apposition
From the lingual halves of the jaws and teeth, the areas of dentine under the middle transverse fissure of the first molar, the mesial one of the second molar and that of the third
molar (Fig. 3) were photographed on Kodak Ektachrome daylight film, 400 ASA, in
ultraviolet light (460 nm, C2 200W/4 mercury vapor lamp, Philips, Belgium). Photographing was done under an Orthoplan Ploemopak microscope with 16x magnification
(Leitz, Wetzlar, Germany; subsidiary, Midland, ON, Canada).
35
The areas of dentine surrounded by a tetracycline-line and the pulp (Fig. 3) were measured planimetrically from video images by circumscribing them as they appeared on the
monitor (Salora 445 A RGB, Salo, Finland; camera: Hitachi VKM 96 E, Tokyo, Japan)
with a serial "mouse" connected to a PCVision Frame Grabber (Imaging Technology, Inc.,
Woburn, MA., U.S.A.) (Larmas & Kortelainen 1989).
1st
2nd
3rd
Fig. 3. Schematic drawing of sagittally sectioned mandibular molars of rat. Wide black line =
enamel. Grey area = dentine apposition measured. Dotted line = tetracycline-marked onset of
dentine formation during the experiment.
4.5.2. Mineral analysis (EPMA)
One of the two lingual halfs of the each sectioned mandible was embedded in epoxy resin as a bulk sample to eliminate the influence of the sample thickness, then polished and
coated with carbon. For analyses of dentine mineral element contents from the samples,
an electron microscope equipped with an electron probe microanalyzer (EPMA) (JEOL
JSM-35 Scanning Microscope with JEOL JCXA-733 Super Probe electron probe
microanalyzer with a ZAF-correction program; JEOL Ltd., Tokyo, Japan) was used.
Examination spot diameter was 10 micrometers, beam current 15 nA.
With EPMA, the amounts of calcium (Ca), phosphorus (P), fluoride (F), sodium (Na),
magnesium (Mg) and total mineral contents were determined. These mineral elements
were given in oxides (CaO, P2O5, Na2O, MgO and ZnO) with the exception of fluoride.
The actual weight percentages were calculated.
The analyses were made for the groups 2, 4, 6 and 8 (6 week's groups with the standard diet) because they did not include dentinal caries lesions. The measurements were
performed in two areas under the second (main) fissure of the first molars and the first
(main) fissure of the second molars: the first three spots between the dentino-enamel
junction and the tetracycline line, the second three spots between the tetracycline line and
the pulp (Fig. 4). The third molar was excluded because of the small amount of dentine
formed before the start of the experiment.
36
Fig. 4. Back-scattered electron image (COMPO) of rat's first and second mandibular molar
showing the spots from where the mineral elements were measured with EPMA.
4.5.3. Caries scoring
The areas of dentinal caries were measured from the same photographs under the same
fissures with the same method as the dentine apposition (Fig. 5). Method is described in
detail in Larmas & Kortelainen (1989) and Hietala et al. (1993).
37
Fig. 5. Photomicrograph of the crown of the third molar. The dentinal caries lesion is seen as a
fluorescent area (surrounded by dotted line). Fluorescent tetracycline line shows the onset of
dentine apposition during the experiment.
All the sectioned molars and fissures were also stained with Schiff reagent to reveal
the caries lesions in the dentine and enamel. The dye reacts with the aldehyde groups
originating from proteolyses (König et al. 1958). The lesions in each fissure (three fissures in the first, two in the second and one in the third molars) were examined and classified into one of the following grades: N = no lesion, A = enamel lesion, T = initial dentine
lesion, B = advanced dentine lesion or C = cavitation (scoring system of König et al.
1958). (Hietala et al. 1993).
4.6. Pilot studies
Before running the experiments described above, six pilot groups were made with six
corresponding control groups. These animals were Long Evans rats and they were three
weeks old at the beginning of the experiments. All these groups were fed on the highsucrose diet. Metabolic acidosis was induced for three and five weeks, metabolic alkalosis for seven and nine weeks, respiratory alkalosis for seven and respiratory acidosis for
six weeks. The rats with respiratory acidosis were kept in a hyperbaric chamber (1.5 bar)
containing 27% of oxygen and 0.03% of carbon dioxide in nitrogen. The other disturbances in acid-base balance were created the same way as above.
38
The dentine formation was measured basically the same way as mentioned before, but
older equipments were used. Because of that, and also because of the different rat strain,
the results can not be compared to those of the experimental groups. Thus, percentages of
the increase or decrease in weight gain, dentine formation and dentinal caries compared to
the corresponding control groups were calculated. Dentinal caries was explored by using
Schiff reagent. No statistical analyses were performed for the pilot groups.
4.7. Statistical analyses
Inter-examiner variations in determining the areas of dentine formed during the experiment (Larmas & Kortelainen 1989) and carious lesions (Hietala et al. 1993) by the above
methods were insignificant. Nevertheless, minimum number of persons were involved in
handling of the material. All blood samples were taken and all the sectionings of the mandibles were made by the author. The areas of dentinal caries (the author) and the areas of
dentine apposition (another researcher trained by the author) were measured by one person. Schiff staining and analyses were made by the author. One laboratory technician performed the EPMA analyses. The statistical analyses were made by the author, with help
and guidance of the biostatistician. The analyses were performed by using SPSS-program (releases 6.1.3 and 7.5).
4.7.1. Statistics in blood gas analysis
According to the Shapiro-Wilks normality tests, the observations of the blood gas analysis were not normally distributed and therefore non-parametric tests were used. KruskalWallis -test was utilized to declare the need of comparisons of each two independent
groups. The Kruskal-Wallis statistics is a direct generalization of the Mann-Whitney U test for more than two independent groups. It is an analogous procedure to the one-way
analysis of variance, but does not require making assumptions of observations to come
from normally distributed populations. Because the groups were small, p<0.1 was accepted as a significant difference. (Glantz 1989)
When values of some groups were different according to the Kruskal-Wallis -test, the
Mann-Whitney U -test was used to determine which differences of each two groups were
significant. The Mann-Whitney U -test tests the hypothesis that a treatment had no effect
when observations are in two independent groups (Glantz 1989). Groups were compared
in multiple pairs: Each group with metabolic acid-base disturbance was compared with
the corresponding group with the normal acid-base balance and the same diet (1 vs. 7,
2+9 vs. 8+11, 3 vs. 7, 4+10 vs. 8+11, 5 vs. 7 and 6 vs. 8+11). Also, the groups with the
same treatment, but different dietary compositions were compared (1 vs. 2+9, 3 vs. 4+10,
5 vs. 6 and 7 vs. 8+11). Because the multiple comparisons are ignored by the Mann-Whitney U -tests, the two tailed p-values of p=0.02 or less were considered as significant difference (instead of p=0.05).
39
The logarithmic scale in the pH values did not affect the statistical analyses, because
non-parametric tests were used. In the non-parametric tests, ranks are given to each of the
numerical values and the test is made by using these ranks, not the original values.
Because the data were not normally distributed, median (referenced by md) and variation
of measures (mainly presented by range from minimum to maximum) are given instead of
mean and standard deviation.
As metabolic and respiratory alkalosis and acidosis obtain the chronic stage in a few
days after the beginning of an experiment (Brewer 1990), the groups that had the similar
treatments for 6 or 7 weeks were combined (groups 2 and 9, 4 and 10, and 8 and 11). The
only values that differed significantly between the combined groups, were pH (p=0.002)
and pCO2 (p=0.002) in groups 8 and 11 (Mann-Whitney U -test). In group 8 the md of pH
was 7.34 (range 7.25 to 7.42) and pCO2 7.33 kPa (5.69 to 9.28). In group 11 the md of pH
was 7.41 (7.35 to 7.45) and pCO2 5.76 kPa (5.03 to 6.49).
4.7.2. Statistics in measuring dentine formation
In these experiments, the molars in the right and left side were regarded as independent
samples when the dentine growth was measured. Because of the genetic similarities of
the rats in this study, the molars in the right and left side of one rat can be considered as
equally dependent (or independent) on each other than the molars of different rats. The
genetics of the rats which are from the same laboratory rat strain, bred in the same place
and originate from the same litters are very similar.
Another reason for the combination of the right and left side was the small number of
the samples in each group. This was partly caused by the small number of the rats, partly
for discarding of the samples judged unacceptable for the analyses (the tetracycline line
was not visible in 93 teeth and sectioning was not of acceptable accuracy in 17 teeth).
The dentine formation during the experiment was measured from each molar separately. Thus, 33 individual groups were formed of the results. The measured areas of the
dentine were normally distributed in 31 groups out of 33, when p>0.09 was regarded as
normally distributed in Shapiro-Wilks -test and skewness and kurtosis were also considered. (Shapiro-Wilks normality test is appropriate when the groups are small.) The groups
not meeting the criteria for normality were as follows: group 11, first molar (p=0.01,
skewness -1.16 and kurtosis 0.79) and group 11, second molar (p=0.05, 1.22 and 3.48).
Considering the small number of samples in each group, these two groups did not prevent the use of the parametric tests.
Global testing between all the groups (F-test: one-way-ANOVA) was not needed,
because all possible pairwise comparisons were not appropriate. Comparison between the
groups was made the same way as in the blood gases, except that groups with the seven
week's experimental period (groups 9, 10 and 11) were kept separated and compared only
to each other (9 vs. 11 and 10 vs. 11). As obvious, less dentine was formed during the six
than the seven week's experiments preventing the combining of the six and seven week's
groups.
40
Comparisons of the two groups at a time was performed with independent samples ttest. Equality of variances were determined by using Levene's test (when p<0.09, the variances were unequal) and an appropriate version of the t-test was used. In the t-tests,
p=0.01 (or less) was considered as a significant difference, because of multiple use of the
groups in comparisons.
4.7.3. Statistics in mineral analysis
Because of the great variability, which is typical for EPMA measurements in the dentine,
median of the three measurements in each spot was chosen to represent that point. With
this method, the unacceptable values (the very high and very low values) are automaticly
excluded. Limits for acceptable values are not given which makes the method more reliable compared to the use of mean values. The chosen values were not normally distributed in any of the groups. Thus, non-parametric tests were used in comparisons.
Global test (Kruskal-Wallis) was used to show the differences (no differences were
found) between the groups in the mineral elements of dentine formed before the experiment. Wilcoxon signed-rank test (Wilcoxon matched pairs test) was used in comparing
the dentine formed before to that formed after the start of the experiment (the pre-chosen
significance level was 0.05). The Wilcoxon signed-rank test is analogous to the paired ttest, testing that the treatment had no effect when observations are from the same subjects (like before and after treatment) (Glantz 1989).
Each group was compared to the control group (group 8) and also to the groups 2 and
4 were compared to each other (Mann-Whitney U -test). In these tests, each value was the
calculated difference between the measurement before and after the start of the experiment. The tendencies of both molars in the results of the comparing of the groups were
analyzed and found quite similar, except that in fluoride and phosphorus, there was a difference between the groups 6 and 8 in the first molar but not in the second one. Also, in
sodium, there was a difference between the groups 4 and 8 in the first but not in the second molar. This was thought to be caused by the small number of the samples or by the
slightly different stage of development in the first and second molar.
Based on the results above, the genetic similarities of the rats in this study and the
nearly same developmental stage of the first and second molar, the mineral measurements of the first and second molars were combined. Thus, the first and second molars of
the same rat were considered as equally dependent (or independent) on each other than
the molars of different rats. Then, the groups were compared by using the Mann-Whitney
U -test. p=0.03 (or less) was considered as a significant difference, because of the multiple use of the groups in comparisons. Here, the number of the groups is smaller than in
comparing blood-gas values, thus the significance level is p=0.03 instead of p=0.02.
41
4.7.4. Statistics in measuring caries
In calculating the areas of dentinal caries lesions, based on the fluorescence of the carious dentine observed under the fluorescent light, such a large number of intact fissures
were present, that rational statistical analysis was not appropriate. As such, the results
supplement the other findings concerning caries. Groups with standard diet were excluded from this data because of the very small number of carious lesions.
The results of the Schiff staining were compared by using a method described by
König et al. 1958. In this method, intact fissures (N) are scored as 0 and enamel lesions
(A) as 1. Initial (T) and advanced (B) dentinal lesions and cavitations (C) are all scored as
7 to emphasize the difference between enamel and dentinal lesions. (This scoring system
has been previously used for example in comparing dysfunction indexes.) In every tooth,
the scores of each fissure were summarized (3 fissures from left side and 3 from the rigt
side in the first, 2 from both sides in the second and 1 from both sides in the third rat
molar). These sum scores were used in compairing the groups by using Mann-Whitney Utest (non-parametric data). Pre-chosen significance level was set at p<0.05.
5. Results
5.1. Pilot studies
The results of the pilot studies are given in percentages compared to the control groups in
Table 4. Two experimental groups, which had the same treatments (group 1 and group 3)
are included. All the pilot groups were fed on the high-sucrose diet. One control group
with normalosis and high-sucrose diet was run for each of the pilot groups (data not
shown). Caries was scored with Schiff reagent and T, B and C lesions were regarded as
dentinal lesions.
The numbers of the rats in pilot groups with respiratory alkalosis and respiratory acidosis were small and standard deviations were high. The results were not as reliable as
those presented in Table 4 and therefore they are not presented in it. In respiratory alkalosis the weight gain was higher in the males, dentine formation was decreased and also
dentinal caries was slightly decreased (the control group had plenty of caries). In respiratory acidosis, weight gain was slightly and dentine formation apparently increased. Dentinal caries were increased in the first and decreased in the third molars.
Table 4. Results of the pilot studies on the effects of metabolic acidosis and alkalosis.
Increase (+) or decrease (-) of the weight gain of the rats, and dentine formation and
dentinal caries in the 1st, 2nd and 3rd molars given in percentages compared to the
controls. All the groups were fed the high-sucrose diet (see Table 1).
Group
n
m-acid-suc
pilot 3 wk
pilot 5wk
exp. 6 wk
m-alk-suc
exp. 6 wk
pilot 7 wk
pilot 9 wk
Weight
Dentine formation
Dentinal caries
Male
Female
1st
2nd
3 rd
1st
2nd
3 rd
10
6
6
-27
-23
-11
-24
-28
-9
-25
-15
-9
-35
-29
-13
-4
-9
-3
+18
+32
+47
+29
+50
+33
+6
+39
+56
9
8
8
+4
+28
-5
+32
+4
+20
+16
-1
+8
+23
+4
+8
-3
-11
-13
-8
-42
-10
-23
-6
+1
-48
Abbreviations: exp. = experimental group, n = number of the rats.
43
5.2. General health
Two rats died during the ordinary experiment: one rat from the group 1 (m-acid-suc 6 wk)
and one from the group 9 (m-acid-stan 7 wk). Thus, the mortality of the animals was
within the usual limits, even though both of the rats that died were from the acidotic
groups.
No significant differences in general appearance and behaviour or food and water consumption were seen between the groups. The weight gain of the acidotic animals with
both diets (groups 1, 2 and 9), was lower than that of the others (Table 5). Statistical analyses concerning the weight differences were not appropriate because of the very small
number of rats in the groups, when the groups were divided into males and females. The
male rats with the high-sucrose diet seemed to gain slightly less weight than the males
with the standard diet with the exception of the rats with metabolic acidosis and standard
diet (group 2). In females, the order was the reverse (except in the group 2) and the differences between the groups were smaller. As expected, the female rats gained less weight
than the males.
Table 5. Mean weight gain (in grams) during the periods of the ordinary experimental
studies. The groups are arranged according to the weight of the males.
Group
Weight gain
Male
Female
144
99
6 weeks
2 (m-acid-stan)
1 (m-acid-suc)
166
118
5 (r-alk-suc)
178
117
7 (norm-suc)
186
130
3 (m-alk-suc)
193
123
8 (norm-stan)
201
118
6 (r-alk-stan)
205
111
4 (m-alk-stan)
208
134
7 weeks
9 (m-acid-stan)
191
110
10 (m-alk-stan)
239
138
11 (norm-stan)
250
136
5.3. Blood properties
Blood pH, base excess, bicarbonate, carbon dioxide and oxygen values of the rat groups
at the end of the experiment are given in Figures 6-10. No significant differences in these
values were found between the male and the female rats in any of the groups. Therefore
the values of the sexes were combined. Also, the groups that had the same experimental
44
settings for 6 or 7 weeks were combined, because the one week's difference in the duration does not make any difference in the blood gas values since the chronic stage has been
obtained in a couple of days after the onset of the experiments (Brewer 1990).
1: m-acid-suc (n=6)
2+9: m-acid-stan (n=13)
3: m-alk-suc (n=9)
4+10: m-alk-stan (n=16)
5: r-alk-suc (n=9)
6: r-alk-stan (n=8)
7: norm-suc (n=9)
8+11: norm-stan (n=18)
6,9
7,1
7,3
7,5
Fig. 6. Blood pH in the experimental groups. Each 7-week group is included in the
corresponding 6-week group: m-acid-stan (groups 2 and 9), m-alk-stan (4 and 10) and normstan groups (8 and 11) are combined. In box plots the box presents 1st and 3rd (upper and
lower) quartiles with the median value inbetween. The whiskers give the lowest and highest
values. Extreme values are marked as circles. n = number of blood samples.
1: m-acid-suc (n=6)
2+9: m-acid-stan (n=13)
3: m-alk-suc (n=9)
4+10: m-alk-stan (n=16)
5: r-alk-suc (n=9)
6: r-alk-stan (n=8)
7: norm-suc (n=9)
8+11: norm-stan (n=18)
-20
-10
0
10
20
Fig. 7. Blood base excess (B.E.) in each group given in mEq/L. For groups and box plot
presentation: see Fig. 6.
45
1: m-acid-suc (n=6)
2+9: m-acid-stan (n=13)
3: m-alk-suc (n=9)
4+10: m-alk-stan (n=16)
5: r-alk-suc (n=9)
6: r-alk-stan (n=8)
7: norm-suc (n=9)
8+11: norm-stan (n=18)
10
30
20
40
50
Fig. 8. Blood bicarbonate (HCO3-) given in mmol/L. For groups and box plot presentation: see
Fig. 6.
1: m-acid-suc (n=6)
2+9: m-acid-stan (n=13)
3: m-alk-suc (n=9)
4+10: m-alk-stan (n=17)
5: r-alk-suc (n=9)
6: r-alk-stan (n=8)
7: norm-suc (n=9)
8+11: norm-stan (n=18)
2
4
6
8
10
12
Fig. 9. Blood carbon dioxide partial pressure (pCO2) given in kPa. For groups and box plot
presentation: see Fig. 6.
46
1: m-acid-suc (n=6)
2+9: m-acid-stan (n=13)
3: m-alk-suc (n=9)
4+10: m-alk-stan (n=17)
5: r-alk-suc (n=9)
6: r-alk-stan (n=8)
7: norm-suc (n=9)
8+11: norm-stan (n=18)
4
8
12
16
Fig. 10. Blood oxygen partial pressure (pO2) given in kPa. For groups and box plot
presentation: see Fig. 6.
The groups with the same drinking water and pressure, but different diet, were compared (group 1 vs. group 2+9, 3 vs. 4+10, 5 vs. 6 and 7 vs. 8+11). No significant differences were found except between the groups with respiratory alkalosis (groups 5 and 6).
In group 5 (r-alk-suc) the concentration of bicarbonate (HCO3-) (p=0.01) and the partial
pressure of carbon dioxide (pCO2) (p=0.004) were lower than in group 6 (r-alk-stan) (Fig.
8 and 9) (Mann-Whitney U-test). In the following are presented the results, which were
obtained when each group with metabolic acid-base disturbance was compared with the
corresponding control group (norm-suc or norm-stan) (group 1 vs. group 7, 2+9 vs. 8, 3
vs. 7, 4+10 vs. 8, 5 vs. 7 and 6 vs. 8).
5.3.1. Metabolic acidosis
When blood pH, B.E. and HCO3- are all considered, the animals were acidotic in the
groups (m-acid-suc and m-acid-stan, groups 1 and 2+9) receiving drinking water supplemented with NH4Cl (Fig. 6, 7 and 8). pH was lowered in both diet groups, significantly
so with the standard diet (group 2+9, p=0.001) (Mann-Whitney U-test). In chronically
altered acid-base balance pH is buffered and thus closer to the normal value than in acute
stage, thus B.E. and HCO3- reveal the disturbances more clearly. B.E. (p=0.002 with the
high-sucrose diet in group 1 and p=0.0001 with the standard diet in groups 2+9) and
HCO3- (p=0.001 and p=0.009 respectively) were significantly lowered compared to their
controls with normalosis and high-sucrose diet (group 7) or normalosis and standard diet
(group .8+11).
47
5.3.2. Metabolic alkalosis
pH, base excess and bicarbonate values were elevated in the groups with alkalotic drinking water (m-alk-suc and m-alk-stan, groups 3 and 4+10) (Fig. 6, 7 and 8), which confirmed the alkalotic effect of the sodium bicarbonate containing water. With the highsucrose diet (group 3, Fig. 6), the elevation of pH was significant (p=0.008) compared to
the group 7 with normalosis and high-sucrose diet (Mann-Whitney U-test). B.E. (Fig. 7)
was higher with both diets (p=0.0003 with the high-sucrose diet in group 3 vs. group 7
and p=0.005 with the standard diet in groups 4+10 vs. group 8+11). Also HCO3- was significantly elevated by metabolic alkalosis (p=0.0004 group 3 vs. 7 and p=0.0004 group
4+10 vs. 8+11, respectively).
5.3.3. Respiratory alkalosis
The measured blood values (Fig. 6, 7, 8, 9 and 10) were typical to chronic respiratory
alkalosis in the groups 5 and 6 (r-alk-suc and r-alk-stan). Significantly alkalotic pH was
found only when the high-sucrose diet (group 5, p=0.0007, Mann-Whitney U-test) and
the standard diet groups (group 6 vs, p=0.01) were compared to the original group 8
(norm-stan 6 wk).
Here respiratory alkalosis is caused by the lack of oxygen leading to hyperventilation,
which lowers the concentration of carbon dioxide (visible with both diets: p=0.0003 with
the high-sucrose diet in group 5 vs. group 7 and p=0.002 with the standard diet in group 6
vs. group 8+11). Hyperventilation did not fully correct the lack of oxygen induced by
hypobaric conditions. With the standard diet in group 6, pO2 was significantly lowered
(p=0.0003) compared to the control group 8+11.
Lower concentration of carbon dioxide (and thus of carbonic acid), is compensated in
a chronic stage by reducing the amount of bases in blood. This way a mammalian body
tends to return pH towards normal. Thus B.E. was lower in the groups 5 and 6 (significantly so in the group 6 with the standard diet vs. group 8+11, p=0.0008). HCO3- was
reduced significantly with both diets (p=0.0003 with the high-sucrose diet in group 5 vs.
group 7 and p=0.0001 with the standard diet in group 6 vs. group 8+11).
5.4. Dentine formation
The amounts of the dentine formed during the experimental periods and the results of statistical comparison (independent samples t-test) are given in Table 6. For easier comparison, dentine formation in the second molars (as an example) is also presented in Fig. 11
and 12.
48
Table 6. Mean dentine formation in three molars in square micrometers x103 during the
experimental period.
1st Molar
Group
n
Mean
2nd Molar
SD
n
Mean
3rd Molar
SD
n
Mean
SD
6 weeks
1 (m-acid-suc)
10
180
25
10
148
32
8
341
34
2 (m-acid-stan)
10
209*
42
11
181*
33
12
349*
22
3 (m-alk-suc)
13
206#
28
14
168#
19
13
368
36
4 (m-alk-stan)
16
260
34
15
207*
23
16
391
34
5 (r-alk-suc)
14
176#
34
14
153#
25
13
342
25
6 (r-alk-stan)
18
243
21
18
204*
22
16
357*
34
7 (norm-suc)
16
197#
44
16
170#
34
15
353#
24
8 (norm-stan)
13
262
24
13
242
27
12
398
27
9 (m-acid-stan)
14
210*
27
11
163*
21
12
322*
45
10 (m-alk-stan)
18
249*
21
18
200*
20
155
380
42
11 (norm-stan)
17
292
20
17
238
22
168
387
36
7 weeks
Abbreviations: n = number, SD = standard deviation, * = significant difference (connected by ]) between the
experimental and corresponding control group (groups 1, 3 and 5 versus group 7, groups 2, 4 and 6 versus 8 and
groups 9 and 10 versus 11, each molar separately). # = significant difference (connected by <) between highsucrose and standard diet group (group 1 versus 2, 3 versus 4, 5 versus 6 and 7 versus 8, each molar separately)
(p = 0.01 or less in independent samples t-test).
1: m-acid-suc (n=10)
2: m-acid-stan (n=11)
3: m-alk-suc (n=14)
4: m-alk-stan (n=15)
5: r-alk-suc (n=17)
6: r-alk-stan (n=18)
7: norm-suc (n=16)
8: norm-stan (n=13)
100 000
150 000
200 000
250 000
300 000
Fig. 11. Dentine formation in square micrometers in the second molars in the six week's
experiments. The box presents the 1st and 3rd (upper and lower) quartiles with the median
value inbetween. The whiskers give the lowest and highest values. n = number of the teeth.
49
9:m-acid-stan (n=11)
10:m-alk-stan (n=18)
11:norm-stan (n=17)
100 000
150 000
200 000
250 000
300 000
Fig. 12. Dentine formation in square micrometers in the second molars in the seven weeks's
experiments. For box plot presentation: see Fig. 11.
According to Table 6, the reducing effect of the high-sucrose diet, as compared to the
standard diet on dentine apposition, was visible in all the high-sucrose groups (groups 1,
3, 5 and 7). In the first and second molars it was more apparent (p<0.001 in group 3 vs.
group 4, 5 vs. 6, and 7 vs. 8 in both first and second molars, independent samples t-test)
than in the third molars. In the third molars, the difference was significant between the
groups 7 and 8 (p<0.001).
When the rat groups with an acid-base disturbance and the high-sucrose diet were
compared to the controls with the high-sucrose diet (group 1 vs. 7, 3 vs. 7 and 5 vs. 7,
each molar separately), no significant differences were found, although the results followed the same patterns as in the groups with the standard diet. The effect of the highsucrose diet seemed to be independent of the acid-base disturbance.
In the groups with metabolic acidosis and the standard diet (groups 2 and 10), the
amount of dentine apposition was reduced in all the molars when compared to those in
the control groups (groups 8 and 11) (6 weeks: 1st molar p=0.003, 2nd p<0.001 and 3rd
p<0.001, 7 weeks: p<0.001 in all the molars.)
In metabolic alkalosis and the standard diet (groups 4 and 11), a difference (reduction)
was found in the second molar (p=0.001) in the group with 6-week's experimental period
(group 4), when compared to the controls with the standard diet (group 8). In the 7-week's
experiment (group 10), the amount of dentine was reduced in the first (p<0.001) and second (p<0.001) molar compared to the controls (group 11).
Statistically significant reduction in the dentine formation was also observed in respiratory alkalosis (group 6) in the second (p<0.001) and third (p=0.002) molars in the rats
fed the standard diet for 6 weeks, as compared to the group 8.
50
5.5. Dentine minerals
Only groups fed on the standard diet (the 6 week's experiments) were included in the
mineral element analyses in order to avoid caries induced demineralization zones. Ca/P
ratio was the same (1.8) in all the groups in dentine formed both before and after the
onset of the experimental period. Also, no differences were found between the groups in
any of the measured mineral elements of dentine formed before the experiment.
The amount of calcium in dentine formed before the experiment was higher than that
formed during the experiment in all the groups, except in the group 2 (metabolic acidosis), in which it was lower (Table 7). In addition to this, in group 4 (metabolic alkalosis)
the amount of fluoride increased and the amount of phosphorus and total mineral contents
decreased during the experiment. In the group 6 (respiratory alkalosis) the amount of
magnesium decreased and in the group 8 (controls) sodium, phosphorus and total minerals decreased during the experiment.
When the change in mineral values (dentine formed during the experiment - dentine
formed before the experiment) of the groups 2, 4 and 6 were compared (Table 7) to those
of the control group 8, the group 2 (metabolic acidosis) differed most: the values of Ca,
Mg, Na and total minerals were higher in the group 2. The amount of fluoride was lower,
but not significantly. When the "opposite" groups 2 (metabolic acidosis) and 4 (metabolic
alkalosis) were compared, the amounts of Ca (p=0.008, Mann-Whitney U -test), Mg
(p=0.03), P (p=0.005) and total minerals (p=0.007) were higher in group 2, but the
amount of fluoride was lower (p=0.027).
51
Table 7. Median (md) dentine mineral contents in dentine formed before (a) and during (b)
the experimental period, given in weight percentages. Minimum, median and maximum of
the difference between mineral contents in dentine formed during and before the
experiment. P-values for comparisons between b versus a and between the experimental
groups (2, 4, 6) versus the control group (8).
Mineral
Group
Calcium
2 (m-acid-stan)
4 (m-alk-stan)
6 (r-alk-stan)
8 (norm-stan)
Magnesium
2 (m-acid-stan)
4 (m-alk-stan)
6 (r-alk-stan)
8 (norm-stan)
Fluoride
2 (m-acid-stan)
4 (m-alk-stan)
6 (r-alk-stan)
8 (norm-stan)
Sodium
2 (m-acid-stan)
4 (m-alk-stan)
6 (r-alk-stan)
8 (norm-stan)
Phosphorus
2 (m-acid-stan)
4 (m-alk-stan)
6 (r-alk-stan)
8 (norm-stan)
Total
2 (m-acid-stan)
4 (m-alk-stan)
6 (r-alk-stan)
8 (norm-stan)
Before
md. a
During
md. b
Min.
Difference
Median
Max.
Diff. b-a
p-value
Comp. to control
p-value
30.89
32.35
31.79
31.69
32.05
30.52
30.24
30.69
-0.06
-7.92
-5.77
-8.14
0.60
-1.70
-1.20
-0.83
2.05
5.98
2.87
1.78
0.021*
0.034*
0.036*
0.036*
0.008#
0.607
0.983
0.37
0.28
0.46
0.35
0.35
0.27
0.31
0.33
-0.10
-0.18
-0.23
-0.22
0.04
-0.01
-0.07
-0.03
0.11
0.10
0.06
0.05
0.203
0.121
0.015*
0.078
0.017#
0.843
0.351
0.58
0.38
0.47
0.47
0.49
0.55
0.41
0.50
-0.33
-0.25
-0.43
-0.24
-0.05
0.16
-0.04
0.07
0.40
0.86
0.54
0.44
0.114
0.030*
0.363
0.061
0.031
0.304
0.078
0.55
0.48
0.55
0.60
0.54
0.46
0.43
0.52
-0.06
-0.18
-0.15
-0.32
-0.02
-0.03
-0.02
-0.09
0.05
0.29
0.11
0.09
0.203
0.171
0.256
0.006*
0.023#
0.105
0.036
17.48
17.30
16.38
17.89
18.50
16.89
17.01
17.45
-1.00
-4.70
-1.80
-7.83
0.36
-0.58
0.12
-0.41
2.02
0.45
2.83
1.74
0.241
0.003*
0.496
0.041*
0.031
0.937
0.071
84.24
83.48
82.78
87.28
89.62
81.96
84.86
84.02
-10.55
-14.48
-11.01
-32.25
2.82
-3.35
-0.86
-3.67
7.43
2.00
6.74
3.89
0.203
0.008*
0.363
0.031*
0.017#
0.968
0.330
* = significant difference between b versus a values inside the groups (p = 0.05 or less, Wilcoxon Signed Ranks
Test). # = significant difference between b-a values in the experimental versus control groups (p = 0.03 or less,
Mann-Whitney U -test).
52
5.6. Caries
5.6.1. Areas of dentinal caries
In Table 8, the areas of caries lesions were calculated only from the fissures, which have
dentinal caries. Only the groups with the high-sucrose diet were included, because there
were no dentinal caries lesions in the groups with the standard diet. The right and left side
did not differ markedly and they are combined. Also there was no difference between the
male and the female rats, thus their data are combined, too.
Table 8. Areas of dentinal caries lesions. Total numbers of the fissures examined (n) and
percentages of the fissures with dentinal caries lesions. Minimum, maximum and mean of
the lesion area in each group, given in square micrometers (calculated only from the
fissures with lesion). The experimental groups are arranged according to the number and
the mean size of the lesion.
Molar
first
second
third
Group
Fissures
Lesions
Area
n
%
Min
Max
Mean
21
81
800
13330
4810
1 (m-acid-suc)
12
67
440
9240
3980
3 (m-alk-suc)
18
11
490
5380
2940
7 (norm-suc)
18
44
40
7080
3420
5 (r-alk-suc)
22
91
640
9460
4580
5 (r-alk-suc)
1 (m-acid-suc)
12
50
680
8330
4630
3 (m-alk-suc)
18
22
210
1730
1057
7 (norm-suc)
18
56
930
12260
4470
5 (r-alk-suc)
19
100
5510
39350
16900
1 (m-acid-suc)
9
100
3140
25060
14760
3 (m-alk-suc)
18
78
1040
16830
7190
7 (norm-suc)
15
87
2140
16560
6500
In the first molars, the carious lesions were smallest in groups 3 (metabolic alkalosis)
and 7 (controls). In both of those groups, there were also the greatest number of fissures
with no dentinal caries. In the groups 1 (metabolic acidosis) and 5 (respiratory alkalosis)
the lesions were the largest and more numerous.
In the second molars, there were the least caries and fewest lesions in group 3 (metabolic alkalosis). Lesions were equally large in the groups 1, 5 and 7, and the most numerous in the group 5. For easier comparison, a box plot presentation is given in Fig. 13. In
the figure, the right and left side are also combined and the fissures with no caries are
included.
53
1:m-acid-suc (n=12)
3:m-alk-suc (n=18)
5:r-alk-suc (n=22)
7:norm-suc (n=18)
-2000
2000
6000
10000
14000
Fig. 13. Areas of dentinal caries in the second molars of the rats in the groups 1, 3, 5 and 7.
Extreme values are marked as circles and outline values as asterisks. For box plot presentation:
see Fig. 11.
In the third molars, lesions were generally larger and more numerous than in the first
and second molars. Lesions were smallest in the groups 3 and 7 and largest in the group 5
(not much difference between the groups 1 and 5). There was only a few teeth with no
dentinal caries, most of them existed in the group 3 (metabolic alkalosis).
5.6.2. Caries scoring
Schiff reagent revealed same amounts of caries in the groups with the standard diet
(groups 2, 4, 6, 8, 9, 10, 11 were compared to each other in pairs) (data not shown). In all
those groups, less than half of the fissures were affected by enamel lesions only, the rest
were intact.
The results of Schiff staining are given in Table 9. According to the dye penetration,
each fissure was classified as healthy (N), enamel lesion only (A), initial dentinal lesion
(T), more advanced dentinal lesion (B) or cavitation (C). Percentages of each of these categories are given separately.
54
Table 9. Results of Schiff staining of the caries lesions. Percentages are given of the
fissures with no caries (N) and lesions of different depths (A, T, B, C). The groups are
arranged according to Table 8.
Molar
first
second
third
Group
n
N
A
T
B
C
5 (r-alk-suc)
66
1,5
25,8
15,2
40,8
16,7
1 (m-acid-suc)
36
2,8
16,6
27,8
27,8
25,0
3 (m-alk-suc)
54
13,0
64,7
16,7
5,6
0,0
7 (norm-suc)
54
3,7
63,0
5,6
25,9
1,8
5 (r-alk-suc)
44
0,0
2,3
20,4
63,7
13,6
1 (m-acid-suc)
24
0,0
16,7
16,7
50,0
16,6
3 (m-alk-suc)
36
25,0
66,6
5,6
2,8
0,0
7 (norm-suc)
36
5,6
44,4
25,0
22,2
2,8
5 (r-alk-suc)
22
0,0
22,7
18,2
45,5
13,6
1 (m-acid-suc)
12
0,0
16,7
25,0
50,0
8,3
3 (m-alk-suc)
18
5,6
72,1
16,7
5,6
0,0
7 (norm-suc)
18
5,6
66,6
5,6
22,2
0,0
Abbreviations: n = number of fissures, N = no lesion, A = enamel lesion, T = initial dentinal lesion, B = advanced
dentinal lesion, C = cavitation.
Lesions were scored and the dentinal lesions (T, B and C) were combined for comparison of the groups. The results of this scoring (see: 4.7.4. Statistics) are presented in Table
10. The sides were combined.
Table 10. Minimum, median and maximum of the sum scores of the caries lesions in each
group (Schiff staining). Healthy fissures (N) were scored as 0, enamel lesions (A) as 1 and
dentinal lesions (T, B, C) as 7 to emphasize the difference between enamel and dentinal
lesions. The groups arranged according to Tables 8 and 9.
Molar
first
Group
5 (r-alk-suc)
1 (m-acid-suc)
3 (m-alk-suc)
7 (norm-suc)
second
5 (r-alk-suc)
1 (m-acid-suc)
3 (m-alk-suc)
7 (norm-suc)
third
5 (r-alk-suc)
1 (m-acid-suc)
3 (m-alk-suc)
7 (norm-suc)
Abbreviations: n = number of fissures, N = no lesion, A = enamel
advanced dentinal lesion, C = cavitation.
n
Min
Md
Max
66
8
15
21
36
8
21
21
54
1
3
21
54
3
9
15
44
8
14
14
24
8
14
14
36
1
2
8
36
1
8
14
22
1
7
7
12
1
7
7
18
0
1
7
18
0
1
7
lesion, T = initial dentinal lesion, B =
6. Discussion
6.1. General health
The growth of the animals, with the exception of metabolic acidosis, during the experiment indicated that the animals were growing normally and that the experimental conditions did not endanger their health. The animals with metabolic acidosis, however,
appeared healthy. Thus the effects of the experimental conditions evidently were not
reflections of severe problems in the general health.
The rats fed on the high-sucrose diet gained slightly less weight than those fed on the
standard diet. The modified Stephan-Harris and Ewos R3 diets differ in many aspects, but
their effect on the general health of rats has been shown previously to be equal (Larmas et
al. 1992, Tjäderhane et al. 1994, Autio et al. 1997). The diets are slightly under the recommended energy level (Table 2). Rats are known to regulate food intake to meet energy
need (National Research Council 1972) and food was freely available, thus no deprivation
of energy resulted.
Both diets are nutritionally acceptable for growing rats, although according to National
Research Council (1972), the amount of fat is too low in high-sucrose diet. Also some
minor deficiences in vitamins and minerals exist in both diets, but they do not endanger
the health of the animals (Huumonen et al. 1997). Control groups of both diets (group 7
fed on modified Stephan-Harris diet and groups 8 and 11 on Ewos R3 diet) were established to allow a comparison of each of the experimental groups with an acid-base imbalance to the corresponding control group with the same diet.
The reduced weight gain in the acidotic animals has been found to be a consequence of
disturbances in the bone calcium and muscle protein metabolism, causing retarded growth
(May et al. 1986). The same mechanisms that affect the bone are also likely to affect the
dentine, and our results do indeed indicate reduced dentine formation in the acidotic animals.
Metabolic alkalosis has been thought to cause corresponding anabolic changes in the
bone and muscle growth (Bushinsky et al. 1989). We found only a hint of the anabolic
effects: the rats gained a little more weight in the group with metabolic alkalosis and the
standard diet. Also one pilot group supported this finding (no weights were available from
the other pilot group).
56
6.2. Acid-base balance
Rats have slightly more alkaline blood than humans. Koppang et al. (1984) reported normal range in capillary blood of humans to be pH 7.38-7.44. The technique of taking the
blood sample from the cut tails resembles the measuring of blood gases from a capillary
blood sample in humans (Beetham 1982). In the study of Whitford & Pashley (1979),
7.46 was the blood pH of the control rats. However, lower pH values for the normalotic
rats have also been given (pH 7.41-7.45, Whitford & Reynolds 1979).
Whitford & Pashley (1979) considered blood pH 7.41 to be an evidence of a mild
chronic metabolic acidosis in rat. In their experiment, acidosis was induced by adding
NH4Cl (0.25M) to the drinking water. Mild chronic metabolic alkalosis was induced by
0.15M NaHCO3, respectively, and the blood pH was 7.53. Blood pH apparently depends
on the site from which the sample is taken, the device for measuring pH, the handling of
the blood samples and the condition of the animals (stress, anesthesia etc.) (Upton &
Morgan 1975). Thus, pH values are not entirely comparable between different experiments.
Our results concerning the blood-gas values in the normalotic groups followed closely
those taken from rats with cannula implanted in the aortic arch under anesthesia with pentobarbital or inactine (Brun-Pascaud et al. 1982). The acid-base balance was what has
been considered normal in the control groups without metabolic or respiratory disturbances (groups 7, 8 and 11), except that pCO2 -value was higher in several cases (Kaczmarczyk & Reinhardt 1975). Anesthesia tends to cause ventilatory depression and thus
raise the pCO2 values (Bar-Ilan & Marder 1980, Brun-Pascaud et al. 1982). Probably
because of that, pCO2 values in our experiments were above normal and the variation was
high within the groups. pH and HCO3- parameters are regulated by slower metabolic systems and can thus be considered more reliable when measuring the acid-base status.
The metabolic acidosis induced here was moderate relative to the previously reported
blood gas values for rats (Kaczmarczyk & Reinhardt 1975) and humans (Pesce & Kaplan
1987). The aim was to keep the changes in the acid-base balance moderate in order to
maintain the good health of the animals and to obtain a physiological stage (i.e. resembling diet-induced acid-base disturbance in humans). The B.E. and HCO3- values
revealed the differences better than did the pH, indicating the involvement of buffers for
correction of the pH values. This confirmed the chronic state (Pesce & Kaplan 1987).
In these series of experiments (unlike in one of the pilot groups, not presented here),
the high-sucrose diet itself was not seen to cause acidosis or any other distubances in the
acid-base balance. Blood pH is very well buffered, while the changes in the intracellular
pH are yet possible. The acid-base conditions and buffer mechanisms inside the odontoblast cells are extremely difficult (if not impossible) to measure.
The use of ammonium chloride for inducing acidosis has been under review because
some investigators have suspected that its effects on the bone may be attributable more to
the chloride ion than to the hydrogen ion and acidosis as such. This has been refuted by
testing the effect of various concentrations of nitric acid on the bone in vitro, whereupon
the results approximated those obtained with an addition of HCl when equivalent numbers of protons had been added (Goldhaber & Rabadjija 1987).
57
The results obtained in the blood gas analysis of the groups given sodium bicarbonate
in drinking water (groups 3, 4 and 10) indicated that the chronic metabolic alkalosis was
mild (Brun-Pascaud et al. 1982, Kaczmarczyk & Reinhardt 1975, Pesce & Kaplan 1987).
There has been discussion about whether orally administered sodium bicarbonate is an
appropriate tool for creating metabolic alkalosis in the rat because it causes a difference
of less than 3 meq in serum bicarbonate values (Atkins & Burg 1985, Knepper et al.
1985, Galla et al. 1991). In our experiments the alkalosis was intentionally kept at a mild
level in order to keep the animals healthy. Alkalosis is less tolerated than acidosis by a
mammalian body.
Metabolic compensation was clearly visible in the groups with respiratory alkalosis:
the amounts of bases and especially that of bicarbonate were lowered in order to buffer
pH closer to normalosis. In theory, metabolic alkalosis and acidosis are also compensated
by respiratory changes. However, this was not visible in our results, probably because of
the mildness of the metabolic acid-base disturbances or of the relatively small amount of
the rats in each group.
6.3. Dentine formation
The high-sucrose diet reduced the rate of dentine formation significantly in young rats,
which has also been found previously (Larmas & Tjäderhane 1992, Hietala & Larmas
1994, Autio et al. 1997, etc.). A reduced bone growth with a high-sucrose diet has been
reported in adult hamsters (Saffer et al. 1981) and rats (Hietala 1993). This lends additional support to the idea of the similarities between the odontoblasts and the osteoblasts.
Microbial invasion in dentine may occur even in incipient caries and possibly modulate the dentine development. Early invasion of oral microbes into white spot of human
enamel (Brännström et al. 1980, Seppä 1984, Seppä et al.1985) and invasion of two
strains of Streptococcus mutans into enamel of rat and further into dentine, while the
enamel surface apparently had not yet collapsed (Luoma A-R. et al. 1984, Luoma H. et
al. 1987, Seppä et al. 1989) suggest their early involvement in dentinal caries process.
This may be one of the reasons for the reduced dentine formation especially in the groups
with the high-sucrose diet.
One of the hypotheses tested in this work was that sucrose affects odontoblasts at least
partly via metabolic acidosis. Since the high-sucrose diet as such did not shift the acidbase status towards the more acidic end of the scale (as it seemed to do in one of the pilot
studies), the mechanism by which the sucrose effect on dentine is mediated is apparently
not via the acidotic state. Other evidence against this hypothesis is visible in the groups
with metabolic alkalosis: Although the alkalotic drinking water changed the pH status of
the blood, it did not correct the reducing effect of the high-sucrose diet on the dentine
apposition.
No differences in dentine growth were found between the male and female rats,
although their body weights differed significantly. Thus, (primary) dentine formation was
not connected to body growth and weight gain, confirming previous findings in young
58
rats (Kortelainen & Larmas 1990, Tjäderhane et al. 1995, Bäckman & Larmas 1997).
Therefore, it is unlikely that the lower weight gain per se affected the dentine formation in
the groups with metabolic acidosis, as the animals otherwise appeared healthy.
The main new finding here was the strong reduction in the dentine formation in young
rats associated with chronic metabolic acidosis. Both the pilot groups supported this finding. Acidotic animals and humans have disturbances in calcium and protein metabolism,
causing retarded growth and even protein catabolism (May et al. 1986). In our study,
chronic metabolic acidosis was indeed accompanied by reduced body weight and diminished dentine apposition.
Mild chronic metabolic acidosis is common among people with modern diet containing plenty of animal protein and cola drinks (Barzel 1995). The diet of young people also
contains a lot of sugar. If acidosis and high amounts of sugar potentiate each others
effects in young humans, like they seem to do in young rats, the development of the teeth
of numerous children may be affected.
When the primary dentinogenesis is still going on in the weanling rats (Baker et al.
1980) and the teeth are producing dentine at maximum efficiency, any disturbance can
probably cause a reduction in the dentine formation. This would explain the slightly
slower dentine apposition in metabolic and respiratory alkalosis.
6.4. Mineral analysis
The amount of mineral elements (except fluoride) seemed to be lower in the dentine
formed during the experiment than in the dentine formed before the onset of the experimental period (Table 7). The same pattern of mineralization has been found by Tjäderhane (1996) and by Huumonen & Larmas (in press). This is presumably due to a continuous mineralization inside the dentine, not only at the border of the pulp. Mineralization is
probably in a more advanced state in the dentine formed earlier. This kind of maturation
process may be typical for young teeth, in which the primary dentinogenesis is still proceeding.
The mineralization in the group with metabolic alkalosis followed closely to this "normal" trend. Differences between dentine formed before and during the experiment were
not as apparent in respiratory alkalosis and the "normal" trend disappared totally in metabolic acidosis. The weight percentage of calcium was even higher in metabolic acidosis in
the dentine formed during the experiment.
When the percentages of calcium in dentine formed during the experiment were compared between the groups, the result remained the same: calcium content was significantly higher in the group with metabolic acidosis compared to the control group (p=0.04,
Mann-Whitney U -test) and to the group with metabolic alkalosis (p=0.01, Mann-Whitney U -test). Surprisingly, the bone has been reported to lose calcium in metabolic acidosis (Green & Kleeman 1991, Bushinsky 1995).
One explanation for increased amounts of calcium in the dentine of acidotic animals
may be the absence of resorption by the osteoclasts or corresponding cells in the dentine.
Metabolic acidosis probably decreases the collagen synthesis by the odontoblasts in the
same way it decreases it in the osteoblasts. This would result in slower apposition of den-
59
tine. But, as resorption does not occur in the dentine, the mineralization proceeds normally. Two possible explanation are suggested to explain the higher than normal percentage of calcium: the increased amount of calcium available in the blood due to the
increased bone resorption, or more thorough mineralization of the dentine (more dense
structure) due to the slower rate of formation.
Tjäderhane (1996) found that the sucrose diet (modified Stephan-Harris diet) resulted
in half the amounts of the total mineral contents in the dentine formed before and during
the experiment, compared to the controls. (The experiment was started with 3-week-old
rats and lasted for five weeks.) Thus, compared to metabolic acidosis, the effect of the
high-sucrose diet was completely different. This gives more evidence (against our working hypothesis) that the reducing effect of metabolic acidosis and a high-sucrose diet on
the growth of dentine are mediated through different mechanisms.
In the study of Tjäderhane (1996) the amounts of calcium and phosphorus were also
lower, but the Ca/P ratio remained unaffected in the dentine formed both before and during the experiment in the high-sucrose and the control groups. In our study, the ratio was
1.8 in all the groups, which has been reported as a normal ratio in the dentine (Jenkins,
1978). The acid-base status did not affect this ratio in our study in rats nor in the study of
Driessens et al. (1987) in dogs. This may suggest that the mineralization processes are not
easily disturbed in the dentine.
6.5. Caries
In the groups with the sucrose diet, there was the smallest quantity of dentinal caries in
metabolic alkalosis. There was also markedly less caries in the control group with normalosis and high-sucrose diet than in metabolic acidosis and respiratory alkalosis with
the same diet. The group with respiratory alkalosis was slightly more affected by caries
than the group with metabolic acidosis. These results were confirmed with Schiff staining. (The pilot studies supported the findings in the groups with metabolic acidosis and
alkalosis.)
The occurrence and severity of dentinal caries were quite similar in the first and second molars, but different in the third ones. The first and second molars erupt almost at the
same time, the first one 1-2 days before the second. Their eruption time is approximately
the 16-18th day after birth (3-5 days before the beginning of the experiments) (Baker et
al. 1980).
The caries lesions were most advanced and most numerous in the third molars in all
the groups, even though the eruption of the 3rd molar occurs about 16 days later than that
of the 1st and 2nd molars. The eruption time of the third molar is the 32-34th day after
birth (11-13th day of the experiments) (Baker et al. 1980). The developmental stage of the
third molars and, perhaps, the elevated amount of cariogenic micro-organisms in plaque
at the time of eruption due to prolonged sucrose ingestion may make them more susceptible to caries.
60
Maturation of the enamel is incomplete at the time of eruption in all teeth, and this also
increases the amount of caries in the molars in young rats, especially in the 3rd molar.
Furthermore, the progression of dentinal caries has been found to be much slower in the
teeth of adult rats than those of young ones (Kortelainen & Larmas 1994).
Metabolic alkalosis or acidosis were induced by using the alkalized or acidified drinking water. Thus, some of the effects of those waters on caries were probably mediated via
the pH of the oral fluid locally. Bicarbonate-phosphate combinations (pH 7.4) added to
sucrose has been demonstrated to reinforce oral natural buffers and lessen plaque fermentative pH fall in vitro (Luoma & Luoma 1967, 1968, Luoma et al. 1970) and in vivo
(Luoma & Luoma 1968). With this method, caries prevention was observed in rats
(Luoma et al. 1968). Although blood was not studied, the additive effect probably also
reinforced the blood buffering.
In our study, respiratory alkalosis surprisingly had the strongest activating effect on
caries, in spite of the fact that no local effect on mouth was present. Respiratory alkalosis
is compensated in mammalian body by creating mild metabolic acidosis (and vice versa),
although pH is never fully corrected (Brewer 1990). Metabolic compensation for respiratory alkalosis might be strong enough to enhance caries, but more likely there are also
other, yet unknown effects, which accelerate caries progression in respiratory alkalosis.
The changes in the dentinal growth and mineral contents were different between the
groups with respiratory alkalosis and metabolic acidosis, which suggests that different
mechanisms were involved in these states. These aspects need more experiments to be
solved.
Previous research has demonstrated that chronic metabolic alkalosis increases the caries resistance of children's teeth (Myllärniemi & Holmberg 1975), a finding which gains
support from the present experiment with young rats' molar teeth. In spite of the small
reduction in the dentine formation in the groups with metabolic alkalosis, the odontoblasts may build dentine of better quality, which, together with a possible rise in salivary
pH, might increase the caries resistance.
Our results pointed to the harmful effect of the opposite state, chronic metabolic acidosis, on teeth. The results give reason to believe that, (in addition to the probable effects on
saliva, which were not studied here), metabolic acidosis has a systemic impairing effect
on the ability of pulp/dentine complex to resist caries attack, probably via the retarded
growth, the altered structure and/or some other changes in the growth and development of
dentine.
These factors are worth remembering in the treatment and prophylaxis of the teeth of
chronically ill patients, especially of such children. Also, if no other reason is found for
an exceptionally bad caries status, it seems to be worth while to check the acid-base status
of the patient. This may sometimes help in finding an undiagnosed general disease, as has
happened in our clinic (Bäckman et al. 1990). Furthermore, an interesting question arises:
what are the effects of acidosis inducing high-protein diet and cola-drinks, in connection
with candies and other sweet food, on the development and caries status of the yougsters'
teeth?
7. Conclusions
In the blood gas analysis, the experimental methods were proved adequate: moderate
chronic metabolic acidosis and mild metabolic alkalosis and respiratory alkalosis were
confirmed.
As expected, chronic metabolic acidosis resulted in reduced weight gain. Also, the
dentine formation in the molar teeth was slowed down significantly. The primary dentinogenesis is probably easily disturbed: a slight reduction in the growth of dentine was seen
in most of the experimental groups.
The high-sucrose diet also markedly reduced the growth of dentine, but apparently by
a different mechanism than metabolic acidosis. Firstly, the high-sucrose diet did not
induce acidosis in the blood gas analysis. Secondly, alkalosis did not eliminate the reducing effect of the high-sucrose diet on dentine formation. Thirdly, the effects of metabolic
acidosis on the mineralization of the dentine in molars were completely different from the
previously reported effects of the high-sucrose diet.
Chronic metabolic acidosis increased the weight percentage of calcium and total mineral content in the dentine formed during the experiment. This is different from the results
reported in bone and probably due to the lack of resorption and remodelling in dentine.
Metabolic acidosis and respiratory alkalosis promoted the initiation and progression of
the dentinal caries. This was only seen in the rats with the high-sucrose diet. The third
molars were most affected, which implies higher caries susceptibility of erupting teeth
with ongoing maturation process, when compared to the erupted ones with more
advanced stage of dentinal development, as found earlier. Metabolic alkalosis slightly
protected the teeth from caries.
Metabolic acidosis and the high-sucrose diet potentiated each others effects in promoting caries and reducing the growth of dentine in the teeth of young animals. Respiratory
alkalosis seemed to be harmful mostly concerning caries and metabolic alkalosis proved
to be even slightly beneficial to the teeth.
Answers to the tested hypotheses are:
1. Sucrose affects odontoblasts causing reduction in dentine formation and acceleration
in caries progression via processes other than metabolic acidosis.
2. Metabolic alkalosis did not eliminate the effects of sucrose on dentine formation,
which supports the finding mentioned above.
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