Menopause

5.
MENOPAUSE
Improved health care services and socio-economic growth have led to an
increase in life expectancy at birth and the number of elderly persons in our
country. This has posed a new challenge to the health needs and care of elderly
men and women. Among women, menopause is an endocrine deficient state
whenin the ovaries cease to function and undergo a variety of physiological
changes that are at times unpleasant. Epidemiological studies have shown that
during this phase women are at increased risk for arterial diseases, prone to
urogenital problems and osteoporosis. Of these, osteoporosis is the most common
consequence of menopause and currently is considered a major public health
concern. Osteoporosis and its associated risk of fractures are preventable if
diagnosed in time. At the Institute, studies to develop modalities for prevention of
osteoporosis, assessment of its prevalence among Indian men and women,
development of assays for biochemical markers of bone turn over for diagnosis
and management have been initiated. Studies are being pursued to understand
molecular and cellular mechanisms regulating osteoporosis and the role of pro
inflammatory cytokines in development of osteoporosis. Osteoporosis is no more
considered as a disease of women and it is clear from various studies that
osteoporotic fractures are not uncommon in men. A project to study impact of
environmental, nutritional, life-style and genetic factors on the bone health of men
has been initiated. In addition to the molecular and clinical aspects, there is a need
to promote awareness about menopause/andropause related problems amongst
the community, programme managers and services providers. The Institute will
soon initiate programmes in this direction.
5.1
Determination of the Prevalence of Osteoporosis in Indian Women
by DEXA Technique: Therapeutic Intervention in High-risk Women
(Funded by Ministry of Health and Family Welfare, Government of
India)
Principal Investigator:
Rashmi Shah
Project Associates:
Lalita Savardekar, Shalini Baji,
Pramodini Phatak, Pratibha Sonawane,
Kavita Lad, D. Balaiah and U. Iddya
Collaborators:
Bhavin Jankharia, Anand Parihar, Radiologists
Vipla Puri, Consultant, Hinduja Hospital
Duru Shah, Consultant Gynecologist
Duration:
1999-2003
Osteoporosis is a problem that is increasing worldwide. In post- menopausal
women, an accelerated bone loss occurs which ranges from 1 to 5 per cent per year.
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Dual Energy X-Ray Absorptiometry (DEXA) is the “gold standard” for measuring
bone mineral density (BMD). A woman is considered osteopenic, if her bone
mineral density score is between 1 to 2.5 standard deviation (SD) below the mean
for young adults, and osteoporosis if it is more than 2.5 SD below the mean for
young adults. It is well established that there are interethnic variations in BMD
measurements.
This study has been initiated to develop norms and determine the prevalence
of osteoporosis in Indian women and to screen high risk women by the DEXA
technique, educate women about preventive measures for osteoporosis, counsel
them about the available non-hormonal and hormonal treatment regimens (diet,
exercise, calcium supplementation, HRT, bisphosphonates, etc.) and to evaluate
the efforts of HRT or alternate treatment on bone loss and other metabolic
functions. The study will also assess women’s acceptance towards treatment
taken.
The target for the study is 450 normal women in the age range of 25 to 75 years
(100 women in each 10 year period up to the age of 65 and 50 women in the 65–75
year age group). We have enrolled 423 women up to 31 March 2002. BMD was
measured at the spine (L to L ) and hip (femoral neck) using Hologic QDR 1000
1
4
DEXA machine and the foot (calcaneum) using Hologic Sahara 0058 1000
ultrasonography machine. Women whose medical history/findings suggest
factors affecting bone mass were excluded from the study.
Our preliminary data reveals that the mean BMD measurements at the foot,
spine and hip in Indian women are approximately 15 per cent lower than those of
age matched women in the USA (Hologic) (Fig. 62 A,B,C). The local reference
database will be useful for accurate interpretation of BMD measurements. The
detection rate of osteopenia and osteoporosis by USG is lower than that by DEXA.
Our study also shows that a reduction in bone mass appears earlier in the spine
than at the hip and that there is an age-related increase in osteoporosis (decrease in
BMD values).
A total of 60 women detected to have osteoporosis by DEXA were offered
treatment options namely HRT with calcium + vitamin D, bisphosphonates with
calcium + vitamin D and only calcium + vitamin D. Twenty women in Group 1
opted for HRT (combination of estrogen and progesterone) along with calcium +
vitamin D, Group 2 women took bisphosphonates along with calcium + vitamin D
and women in Group 3 received only calcium and vitamin D.
These subjects were monitored for bone turnover markers and DEXA. The
biochemical markers were estimated at baseline, 3, 6 and 12 months post
treatment, and DEXA at baseline and 12 months post treatment. The biochemical
marker panel included a bone resorption marker, Cross Laps (CTx)and a bone
formation marker, osteocalcin, along with intact PTH and vitamin D, which are
designated as associated markers.
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The mean baseline urinary CTx values are 1642 mg/mmol creatinine. The
mean serum osteocalcin levels are 8.3 ng/ml. The levels of serum vitamin D and
plasma intact PTH are 23.1 ng/ml and 31.1 pg/ml, respectively. Analysis of the
changes in the markers and DEXA findings at the end of 12 months of therapy is
ongoing.
BMD values of foot
2
BMD reading (gm/cm )
1.2
A
Clinic
Hologic
1
0.8
0.581
0.6
0.514
0.4
0.555
0.519
0.562
0.467
0.545
0.414
0.2
0.471
0.35
0
25-34
35-44
45-54
55-64
65-75
Age group
BMD values of spine
1.2
B
Clinic
2
BMD reading (gm/cm )
1.044
1.033
1
0.8
BMD values of hip
C
Hologic
0.983
1
0.892
0.907
Hologic
0.89
0.86
0.815
0.917
0.812
0.8
0.809
0.6
Clinic
1.2
0.749
0.774
0.71
0.662
0.6
0.4
0.4
0.2
0.2
0
0.79
0.683
0.721
0.661 0.609
0
25-34
35-44
45-54 55-64
Age group
65-75
25-34
35-44
45-54 55-64
Age group
65-75
Fig. 62: Comparison of BMD values at foot, spine and hip: Indian data vs American
database.
Women’s acceptance to treatment taken and side effects, if any, while on
therapy were also studied. Of the 60 women who took either HRT or
bisphosphonates along with calcium and vitamin D or only calcium and vitamin
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D, 54 women felt more energetic, fresh, their pain in their back and legs decreased
or disappeared and they were able to work more and felt less tired. The remaining
6 women (HRT: n = 3, bisphosphonates: n = 2, calcium therapy: n = 1) did not find
any difference in their health status. Some of the women experienced diarrhoea (2
cases on calcium therapy), heaviness of breast and vaginal dryness with vaginal
itching (HRT: n = 9) and generalized itching all over body with rashes (HRT: n = 6;
bisphosphonates: n = 7). In spite of these complaints the women continued
therapy except one in the calcium group. Three women were asked to discontinue
HRT (after 6 months, 7 months and 10 months of use respectively) as their followup mammography revealed changes that suggested that HRT should be stopped
and these women were re-evaluated and post-HRT mammography was done 3-6
months later which did not show any abnormalities.
Individual and group counseling services regarding osteoporosis are held
regularly at the “Elderly Women’s Clinic”. There is a need to increase awareness
among health care providers and women in general, about menopause and also
the therapeutic measures that can be taken so as to improve the quality of life of
the elderly women.
5.2
Assessment of Prevalence of Osteoporosis in Adult Population in
India (ICMR multicentre study)
Principal Investigator:
Rashmi Shah
Project Associates:
Lalita Savardekar, M.I. Khatkhatay,
Rajlakshmi Srinivasan, Suchita Utekar,
Reshma Sathe, Harvinder Kaur Sudan,
Madhu Singh and K. Chavan
Collaborator
Nilesh Shah (N.M. Medical Centre)
Duration:
2002 -2006
The objectives of the study are to: (i) establish peak bone mineral density
(BMD) reference values for Indian men and women; and (ii) assess the prevalence
of osteopenia and osteoporosis in Indian population.
To establish BMD reference values one hundred each, men and women in the
age range 20-30 years from the high socioeconomic group while for the prevalence
study, a total of 1500 subjects (750 men and 750 women) ranging from 30 to 70+
years and from three socio-economic groups will be studied. Biochemical tests
will be carried out in 20 per cent of the randomly selected subjects. Osteopenia
and osteoporosis will be identified using the values generated in the first part of
the study. The enrollment of volunteers is ongoing.
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5.3
Biochemical Markers for Early Diagnosis of Osteoporosis (Funded by
the Department of Science and Technology, Government of India)
Principal Investigator:
Meena Desai
Project Associates:
M.I. Khatkhatay, U.M. Donde,
A.H. Bandivdekar, K.V. Bhanu Prakash,
Rashmi S. Shah and Lalita Savardekar
Duration:
2001-2005
Osteoporosis is considered a major public health problem with an estimated
1.3 million fractures a year. Bone mineral density measurements by DEXA and
assays of biochemical markers of bone turn over are widely used parameters for
the assessment of bone status. The aim of the project is to: (i) develop simple
immunoassays for selective bone formation markers (osteocalcin and bone
specific alkaline phosphatase) and bone resorption markers [C-terminal collagen
type 1 (CTx) and pyridinium crosslinks (Pd and Dpd)]; and (ii) establish reference
values of the same in Indian women.
Purification of osteocalcin
Osteocalcin, predominantly synthesized by the osteoblasts, is considered as
a useful marker of bone formation. In an attempt to develop an ELISA for its
estimation calcin, was first extracted from bovine bones. Crushed bovine bones
were decalcified with 20 per cent formic acid, dialysed and purified further using
ion exchange chromatography. Approximately 600 µg of 98 per cent pure
osteocalcin was obtained from 90 g of bovine bones (Fig. 63) and used for
immunization to generate polyclonal antibodies. Monoclonal antibodies to
human osteocalcin are being procured in order to develop a sensitive
immunoassay.
Screening for bone metabolic and bone turnover markers
Two hundred and six normal healthy women in the age group of 20-60 years
(50 women in each 10 year period) and 60 menopausal osteoporotic women on
anti-resorptive therapy were screened for markers of bone metabolism (intact
PTH, calcium, phosphorous) and markers of the bone turnover osteocalcin and
CTx.
The data obtained from these women were categorized into 5 yearly age
bands. All these women had normal calcium and phosphorous levels ranging
between 8.5 and 11.5 mg/dl and 3.0 and 5.0 mg/dl, respectively (Fig. 64 A and B).
In the present study, the PTH levels were observed to increase with age, which
may lead to increase in bone turn over and consequently increase in bone loss with
age.It was observed that the mean PTH levels increased from 29 ng/ml seen in 21103
0.25
1
0.2
0.8
Abs (280 nm)
Abs (280 nm)
25 year age group to around 40 ng/ml in the 55-60 years age group. The levels of
calcium and phosphorous were within the normal range indicating that there was
no metabolic bone disorder in these women.
B
A
0.15
0.1
0.01
0.6
0.4
0.2
0
0
1
20
39
58 77
Tube No.
96
115
1 12 23 34 45 56 67 78 89
Tube No.
C
D
L1
L2
L3
L4 32 k
1
45 K
Abs (280 nm)
0.8
30 K
0.6
20.1 K
0.4
5.8 k
14.3 K
6.5 K
3.5 K
0.2
0
2.5 K
1 9 17 25 33 41 49 57 65 73 81 89 97
Tube No.
Fig. 63: (A) Sephadex G50 chromatography profile of 20 per cent formic acid extract
(Fraction Nos.48-77 active fration). (B) Ion-exchange chromatography
profile of the pooled active fractions obtained from Sephadex G50
chromatography (Fraction No.s 30-50 active fraction). (C) FPLC
chromatofocussing profile depicting the isoelectric point (4.0 + 0.5) of the
protein (Fraction Nos.69-73 active fraction). (D) The silver stained SDS
PAGE analysis of active fractions, Lane 1: Molecular weight markers,
Lane 2: Sephadex G50, Lane 3: Ion-exchange chromatography and
Lane 4: Chromatofocusing on 16.5 per cent Tricine gel electrophoresis.
Bone formation was higher during early twenties as reflected by the higher
levels of Osteocalcin in the age group 21-30 years. Once peak bone mass is
achieved around 30 years, bone formation slows down and bone resorption
exceeds bone formation with increase in the levels of CTx (Fig. 65 A, B, C).
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mg / dL
4.5
Phosphorous
A
4
3.5
g / dL
10
20-25
B
26-30
20-25
26-30
31-35
36-40
41-45
46-50
51-55
56-60
Calcium
9
8
31-35
36-40
41-45
46-50
51-55
56-60
Age in years
Fig. 64: Serum phosphorous (A) and calcium (B) levels in 206 women in the age
group of 20-60 years.
In the 60 menopausal osteoporotic women on anti-resorptive therapy, there
was a significant decrease in the bone turnover markers at 3 and 6 months of
therapy indicating retardation in bone loss (Fig. 66 A, B, C). CTx was observed to
be a sensitive marker for monitoring the therapy. Women receiving
bisphosphonate showed up to 30 per cent decline in the levels of CTx at 3 months
and up to 80 per cent at 6 months post therapy, compared to HRT therapy
indicating that the resorption was arrested more efficiently following supplement
with bisphosphonate.
Studies are ongoing and 300 normal healthy women in the age group (20-60
years) are being enrolled to establish reference values for the four markers of bone
turnover which will be correlated with DEXA findings.
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Intact
50
A
PTH
B
Oc
C
CTx
pg / mL
40
30
20
10
0
14
12
ng / mL
10
8
6
4
2
µMol/mol Creatinine
0
10
9
8
7
6
5
4
3
2
1
0
20-25
26-30
31-35
36-40
41-45
Age in years
46-50
51-55
56-60
Fig. 65: (A) Serum parathyroid hormone (PTH), (B) osteocalcin (Oc) and (C) urinary
C-terminal collagen type 1 (CTx) in 206 women in the age group of 20-60
years.
106
A
Calcium + Vit D
% change from baseline
3months
6months
0
-20
-40
-60
-80
Osteocalcin
C-terminal telopepeptide
-100
B
3months
Bisphosponates + Cal + Vit D
3months
6months
0
% change from baseline
% change from baseline
C
HRT + Cal + Vit D
-20
-40
-60
-80
-100
6months
0
-20
-40
-60
-80
-100
Fig. 66: Bone turnover markers osteocalcin (Oc) and C-terminal collagen type 1
(CTx) for monitoring anti-resorptive therapy in 60 women.
5.4
Relevance of Changes in Bone Related Proteins and their Utility for
Diagnosis of Osteoporosis in Indian Women (Funded by Ministry of
Health and Family Welfare, Government of India)
Principal Investigator:
M.I. Khatkhatay
Project Associates:
Meena Desai, U.M. Donde, Vrinda Khole,
Rashmi S. Shah and Lalita Savardekar
Duration:
2002-2005
The objectives of the study are to establish peripheral levels of cytokines in
Indian women and to correlate them with biochemical markers of bone turnover to
be used as an early predictor of osteoporosis.
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Non-competitive ‘Sandwich‘ ELISAs were optimized for interleukin (IL-1), IL6 and TNFα. In the pilot study conducted on 80 samples comprising of healthy
normal and regularly menstruating (n = 34), postmenopausal (n = 29) and
postmenopausal osteoporotic women (n = 17), the mean levels of IL-6 were
significantly elevated in the postmenopausal women (0.40 ± 0.05 IU/ml)
compared to the normal healthy women (0.29 ± 0.05 IU/ml). The levels were
further elevated in the osteoporotic menopausal women (0.60 ± 0.06 IU/ml)
indicating that IL-6 is a good marker for the diagnosis of osteoporosis. The study
will be further expanded.
5.5
Genetic Factors Contributing to Osteoporosis: Study of Gene
Polymorphism in Vitamin D Receptor Gene and Estrogen Receptor
Gene in Indian Population (Partly funded by WHO)
Principal Investigator:
M.I. Khatkhatay
Project Associates:
Meena Desai and Rashmi S. Shah
Duration:
2002-2004
The evidence that genetic factors contribute to osteoporosis originates from
family history, race, gender and twin studies. Based on familial studies, it has
been reported that individuals from families where the members have sultered
frequent fractures are at a greater risk of developing fractures. Caucasian and
Asian women are more likely to develop osteoporosis than African-Americans.
There is a strong evidence that peak bone mass is determined genetically. Genes
responsible for calcium uptake and regulation of osteoblasts and osteoclasts are of
prime importance. These include genes for vitamin D receptor (VDR), estrogen
receptor (ER), IL-1 and IL-6. Polymorphism in these genes are linked with low
bone mineral density. Polymorphisms reported in VDR are Fok I in exon II, Bsm
I, Apa I between VIII and IX and Taq I in exon IX. In ER gene, polymorphism is in
Puv II and Xba I in intron I.
PCR for all the genes have been standardized. The study will help in
identifying women who are at a greater risk of developing osteoporosis due to
their genetic make up.
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