International J. of Healthcare & Biomedical Research, Volume: 1, Issue: 1, October 2012 P:6-12
The use of Serum & Urinary Biochemical Markers of Bone turnover in Post Menopausal
Women.
K.Satya Narayana1, Sravanthi Koora2, G.T Sivaraja sundari3, & I. Anand Shaker 4
…………………………………………………………………………………………………………
1
PhD Scholar - Dept of Medical Biochemistry, Bharath University, Chennai, Tamilnadu, India.
2
Lecturer - Dept of Pharmacology, Asan memorial Dental College & Hospital, Chengalpattu, Tamilnadu, India.
3
Reader - Dept of Pharmacology, Asan memorial Dental College Hospital, Chengalpattu, Tamilnadu, India.
4
PhD Guide - Dept of Medical Biochemistry, Bharath University, Chennai, Tamilnadu, India.
Corresponding author & e-mail address: -Satya Narayana K. satya79700@gmail.com
………………………………………………………………………………………………………….
Abstract:
Background: The period during which the female sexual cycle ceases and female sex hormones diminish rapidly to
almost none at all is called Menopause. Low bone mass and micro architectural deterioration of bone tissue, is most
common in postmenopausal women which leading to enhanced bone fragility and a consequent increase in fracture risk.
Menopause is the single most important cause of osteoporosis.
Materials & Methods: The study group 60 postmenopausal women in the age groups of46-66 years were taken as case
from our OP. As control, 60 healthy volunteers with age group of 25 to 45 years were recruited. The investigations
carried out were Serum alkaline phosphatase, calcium, phosphorus, Total protein, Serum 25-hydroxy vitamin D3 (25OHD), Serum estradiol. In 24 hours urine sample urinary calcium, Urinary phosphate & Urinary hydroxy proline.
Results: There were increased Serum alkaline phosphatase, Total protein, Urinary calcium, Urinary phosphate, Urinary
hydroxy proline & decreased serum calcium, 25-hydroxy vitamin D3 (25-OHD), estradiol levels in postmenopausal
women comparatively premenopausal women
Conclusion: estrogen deficiency induces bone resorption in cancellous bone leads to general bone loss and reduces bone
strength resulting in osteoporosis. Hence, estrogen replacement therapy may reduce the risk of osteoporosis. The 25hydroxy vitamin D3 (25-OHD) insufficiency was also been found with calcium imbalance in postmenopausal women
which is an important in follow up of therapy. The results also suggests that simple, easy, common biochemical markers
such as urinary hydroxyproline, total serum ALP, serum calcium, Urinary calcium and phosphate could be used as a tool
to assist health care professionals to predict bone turnover to minimize fracture due to osteoporotic changes
Keywords: 25-hydroxy vitamin D3 (25-OHD); osteoporosis; estrogen; Post menopause.
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Introduction:
The period during which the female sexual cycle ceases and female sex hormones diminish rapidly to almost
none at all is called Menopause. It occurs between 45-55 years of age. [1]. Due to menopause ovarian follicles
loss its function, which results in decreased production of estradiol and other hormones. Decreased levels of
estrogen leads to increased osteoclast formation and enhanced bone resorption, which intern leads to loss of
bone density and destruction of local architecture resulting in osteoporosis [3]. The prevalence of osteoporosis
increases with age, by WHO definition up to 70% of women over the age 80 years have osteoporosis [2]. In
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International J. of Healthcare & Biomedical Research, Volume: 1, Issue: 1, October 2012 P:6-12
India experts groups peg the number of osteoporosis to increase by 36 million by 2013 [ 3]. During the first
five years after menopause a women can lose as much as 25% of her bone mass. If left untreated this
condition can cause painful, debilitating fractures [3]. Osteoporosis is related to well-characterized secondary
causes (endocrine, renal, inflammatory, malignant diseases, etc.). Other causes such as genetic (parental
history), anthropometric (low body weight), reproductive (hypogonadism, premature menopause, lactation,
etc.), nutritional (low calcium intake, vitamin D insufficiency, high sodium or protein intake) or lifestyle
(smoking, sedentariness) conditions can also affect the acquisition of peak bone mass or lead to excessive
bone loss after peak bone mass was attained [4].
Approximately 30% of postmenopausal women sustain at least one osteoporotic fracture. However, the true
incidence of these fractures is difficult to assess because a large number of vertebral fractures remain
asymptomatic. Several studies reported Osteoporotic hip fractures occur in the third and fourth decades after
menopause; they are twice as common in women as in men. BY 90 Years of age, about 33% of women and at
least 17% of men sustain a hip fracture [6-9]. Life time losses may reach 30% to 40% of the peak bone mass
in women. The occurrence of Osteoporosis in postmenopausal women is very common problem especially in
India who is exposed to many of the risk factors like prolonged amenorrhea, low calcium diet, lack of
exercise, Vitamin D deficiency. But there are very few Indian studies regarding the prevalence of osteoporosis
in postmenopausal women and also regarding the biochemical markers which indicate bone turnover[10-13].
With the above background we aimed the assessment of biochemical markers of bone turnover which will
provide valuable information for the clinical utility of diagnosis and monitoring of metabolic bone disease to
assess the risk of osteoporoses in postmenopausal women.
Materials & Methods :
The study group 60 postmenopausal women in the age groups of 46 to 66 years were taken as case from
our OP. As control, 60 healthy volunteers with age group of 25-45 years were recruited. None of the subjects
were receiving any form of drugs. Subjects with the habit of smoking and taking alcohol were also excluded
from the study, pregnant women and patients on oral contraceptive pills, subjects with fractures in previous
one year and women on hormone replacement therapy or any other medications are excluded from this study
becouse that may affect serum and urinary profile. All the experimental procedures were approved by the
Institute Human Ethics Committee and informed consent was obtained from all the participants.
After an overnight fast of 12 to 14 hrs, blood is collected by Vein puncture in the morning. It is and
serum was separated by centrifugation and Serum alkaline phosphatase, calcium, phosphorus, Total protein,
Serum 25-hydroxy vitamin D3 (25-OHD) was estimated by optimized standard method, Serum estradiol was
estimated by immune enzymatic assay.
24 hour urine samples were collected in clean plastic containers. The pH of urine was adjusted to > 2 by
adding 2ml HCl and mixed thoroughly. Total volume measured and aliquotes were stored at -20°C until
analysis. Twenty four hour urinary calcium & Urinary phosphate was estimated by the spectrophotometric
method. Urinary hydroxy proline was estimated by modified neuman and logan method. Height and weight
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International J. of Healthcare & Biomedical Research, Volume: 1, Issue: 1, October 2012 P:6-12
was measured in meters and kilograms respectively and BMI was calculated by using the formula Wt/Ht 2
(weight in kilograms divided by height in (meters)2
The data obtained was analyzed, and the differences in the mean of various parameters were compared
using student’s t-test. Statistical analysis was performed using software SPSS windows.
Results:Table 1 Clinical characteristics variables in Study group and controls (mean±SD) Age, BMI,
Serum & Urinary biochemical markers
Parameter
Study group
Control group
p value
Age
56.09 ± 9.91
35.44 ± 9.66
<0.01
BMI
25.10 ± 2.12
S.Ca
2.10
2.32
mmol/l
(+/-) .12
(+/-) .11
S.Phos
1.17
1.18
mmol/l
(+/-) .18
(+/-) .20
S.ALP
161.4
129.9
IU/L
(+/-) 33.66
(+/-) 13.21
Estradiol
50.33
75.76
pg/ml
±11.42
±12.38
U.Ca
206.05
146.8
(mg/gm Creatinine)
(+/-)18.13
(+/-) 16.79
U.Phos
520.2
458.85
(mg/gm Creatinine)
(+/-)13.3
(+/-) 14.72
S. Protein
6.28 (+/-)
6.30(+/-)
gm/dl
2.2
2.9
U. Protein
128.04
110.4
mg/day
(+/-)12.31
(+/-) 29.96
U. Hydroxyproline
107.75
61.66
mg/day
(+/-)10.45
(+/-) 9.58
S. 25 OH D3
17.75
28.61
ng/ml
(+/-)10.45
(+/-) 10.58
25.38 ± 4.59
NS
<0.001
NS
<0.001
<0.01
<0.01
<0.01
NS
NS
<0.0001
<0.001
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International J. of Healthcare & Biomedical Research, Volume: 1, Issue: 1, October 2012 P:6-12
Table 1 shows the main demographic characteristics variables in study group and controls. Subjects and
controls have differed with respect to age. Body height, weight and BMI were showing a little variation even
though it was statistically not significant. There was a significant decrease in serum calcium, estradiol, 25 OH
D3 levels in study group comparatively with controls. Women with postmenopausal had significantly higher
serum alkaline phosphatase, urinary calcium, urinary phosphorus, urinary hydroxyproline when compared
with control group. There was a variation in serum phosphorus, serum total protein & urinary protein of the
study and control groups but it was not significant.
Discussion:
BMI was not significant in postmenopausal women, in comparison to premenopausal women.
Literature says that a low BMI is one of the risk factors for increased bone turnover( 14). However, we could
not find such a correlation in present study. We found a higher proportion(35 subjects) of women with low
BMI in post menopause comparative controls.
Bone is a connective tissue that provides mechanical support to the body vital organs and act as
reservoir of calcium and phosphate as 99% of calcium and 85% of phosphate are present in skeleton. Peak
bone mass is achieved during the third decade of life which gradually declines leading to osteopenia which
predisposes to osteoporosis.(15) Various risk factors are involved for the decline in Bone Mineral Density
(BMD) including dietary deficiency of calcium, phosphorus and vitamin D. We found the subjects with
postmenopausal had 25-hydroxy vitamin D3 (25-OHD) insufficiency which is not different than that found in
the control group and this also been reported in the general French adult urban population [20]. Some studies
[21] reported that bone resorption markers raises when serum 25-OHD levels falls down, it is unlikely that
vitamin D insufficiency was the main cause of osteoporosis in our population. The 25-hydroxy vitamin D3
(25-OHD) insufficiency been found with calcium imbalance in postmenopausal women. This might be
because of decrease in estrogen levels. Estrogen deficiency at menopause increases the rate of bone
remodeling which results in high turnover bone loss. In this study there is a decrease serum estradiol levels in
postmenopausal women when compared to premenopausal women. Hence present study supports the view
that estrogen deficiency induces bone resorption by which releases calcium into the extracellular space and
which in turn suppresses PTH secretion, calcitriol synthesis, and intestinal absorption of calcium in cancellous
bone leads to general bone loss and destruction of local architecture and reduced bone strength resulting in
osteoporosis.(16-19 )
Serum alkaline phosphatase (ALP) is the most commonly used marker of bone formation that plays an
important role in osteoid formation and mineralization. Normality of serum ALP levels is thus an important
diagnostic criterion in the diagnosis of osteoporosis (20). There is a significant increase in mean total serum
alkaline phosphatase activity when compared to controls. This shows that the bone mass continues to decline
with menopausal age. ALP is a ubiquitous enzyme that plays an important role in osteoid formation and
mineralization. It is observed that ALP levels were high in early postmenopausal women when compared to
those of premenopausal women as a result of the inhibitory effects of estrogen on bone turnover ate which is
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International J. of Healthcare & Biomedical Research, Volume: 1, Issue: 1, October 2012 P:6-12
dependent on age and body mass index.(22) The concentration of total serum protein increases in post
menopausal women non significantly as compared to controls.
There was a significant correlation of urinary calcium and phosphate were found in the
postmenopausal group compared to the premenopausal group. Similar results are reported in a study by
George BO (24) which has highlighted the fact that biochemical parameters can be used to monitor bone loss
in elderly persons. Baseline levels of bone markers is not only helpful in diagnosing osteoporosis in early
stage but also for predicting future bone loss in postmenopausal women. This also been reported by
Deutschmann et al (23) who has found association of hypercalciuria with severe osteoporosis. The significant
correlation suggests that the factors that increase the urinary excretion of calcium and phosphate would affect
the quality of bones both in males and females. Calcium salts in bone are embedded in collagen fibrils, 13% of
which is mainly hydroxyproline. During bone loss, collagen fibrils are broken down thus calcium and
hydroxyproline is excreted in the urine. Urinary hydroxyproline is thus considered as an index of bone
resorption and a major determinant of bone status (20). In the present study there was a significantly increased
urinary excretion of calcium & hydroxyproline in postmenopausal women when compared to control.
Sachdeva. et al, also reported same observations in their study (19). Similar observations were reported by a
number of other studies (20, 21). By taking into consideration of various criteria, methodology availability
and feasibility we have chosen these parameters. Our study also amply demonstrating the usefulness of
urinary profile and its significance in predict Osteoporosis in post menopausal women.
Conclusion: estrogen deficiency in postmenopausal women induces bone resorption in cancellous bone leads
to general bone loss and reduces bone strength resulting in osteoporosis. Hence, estrogen replacement therapy
may reduce the risk of osteoporosis. The 25-hydroxy vitamin D3 (25-OHD) insufficiency was also been found
with calcium imbalance in postmenopausal women, which is an important area in the follow up of therapy.
The results also suggests that simple, easy, common biochemical markers such as urinary hydroxyproline,
total serum ALP, serum calcium, urinary calcium and phosphate could be used as a tool to assist health care
professionals to predict bone turnover to minimize fracture due to postmenopausal changes.
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--------------------------------------------------------------------------------------------------------------------------------Manuscript submission: 26 September 2012
Peer review approval: 04 October 2012
Final Proof approval: 16 October 2012
Date of Publication: 16 October 2012
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