Serum Vitamin-D and related parameters in healthy women of reproductive age group

 

 

Abstract               Background: Vitamin D deficient or insufficient in India, being a tropical country, it was believed that Vitamin D deficiency is seldom seen in our country But, to our surprise, opposite scenario prevails. Aims: To determine serum vitamin D levels and Other parameters such as serum calcium, phosphorus and alkaline phosphatase levels were also evaluated. Materials and methods: 300 healthy women of reproductive age group from 15-49 years Women belong to suburban region. Vitamin-D, calcium, phosphorus, Alkaline phosphatase and exposure to sunlight in minutes were noted. Results: The mean serum 25-OHD level was 19.2± 8.3 ng/mL. Of the included patients, 85.2% (of 350) were vitamin D deficient, and only 15.4% (54 of 350) had normal serum levels. Among patients with vitamin D deficiency, 131 cases had severe deficiency, 87 had moderate, and 82 had mild deficiency. 114 cases (38%) were currently pregnant, among whom 83 were vitamin D deficient. 16.4% suffered from sever deficient, 16% with moderate deficient and 5.3% with mid deficiency. Mean sun light exposure daily and calcium has positive correlation with vitamin-D and has significance in the study population Conclusions: In the presence of lack of naturally occurring Vitamin D rich food fortification/supplementation with Vitamin D and adequate sunshine exposure should be given a higher priority among these women.

Key Words: Vitamin-D, Calcium, Phosphorus, Reproductive women.

 

INTRODUCTION

Calcium is an important macromineral required for vital functions. Calcium is important for growth and development of bones and teeth, enzyme action, mediation of hormonal responses, blood coagulation, muscle contractility and neuromuscular function. Calcium in blood exists in three distinct fractions: protein-bound(40%), free(48%), and inorganic anions bound calcium(12%). The equilibrium between free and bound calcium fractions is dependent on several variables. Free calcium is physiologically active but is not routinely determined because of technique-related limitations and higher costs. Therefore total serum calcium is routinely measured.^1,2 The major source of Vitamin D is sunshine exposure, even though a large percentage of the Indian population (>80%) is suffering from Vitamin D deficiency. Women from reproductive age group are exposed to greater risk of developing bone abnormalities and other associated disorders due to low Vitamin D levels. There is a lack of scientific literature on the status of Vitamin D and calcium in women of reproductive age from India. Hence, the present cross-sectional study was conducted to study the status of Vitamin D and calcium levels in women of reproductive age in Delhi, India.³ Bone health is determined by 18years as 90% of peak bone mass is attained. Decreased calcium levels may occur due to poor calcium intake over a long period of time (childhood), hormonal changes (elderly women) and low levels of vitamin D, which makes it harder to absorb calcium. Calcium stores will be utilized by body to perform different functions which lead to slow deterioration of bones and teeth with advancing age. Reduced calcium levels lead to osteopenia which reduces bone mineral density leading to osteoporosis. A long term deficiency leads to dental changes, cataracts, alterations in the brain and osteoporosis. A calcium deficiency may not have early symptoms. So to avoid future complications related to women and child health, serum calcium levels should be checked at an early stage and measures should be taken to improve calcium levels. The recommended daily allowances for calcium will be followed according to the National Institutes of Health(NIH). Assessment of vitamin D status of an individual is reflected by measurement of circulating vitamin D metabolites. Only two metabolites, namely, 25-hydroxy vitamin D and 1,25-dihydroxy vitamin D have received the greatest attention in biochemical estimation of vitamin D.⁴ Calcium and vitamin D are important to the reproductive female. Deficiency leads to serious skeletal consequences, yet optimum levels or sufficiency is widely debated. 

AIM
To determine serum vitamin D levels and Other parameters such as serum calcium, phosphorus and alkaline phosphatase levels were evaluated in healthy women of suburban region so that preventive and educative measures and screening programs can be encouraged to improve the calcium levels at an younger age in women.

MATERIALS AND METHODS
The present study is a single-centered prospective study which includes 300 healthy women of reproductive age group from suburban region. The study will be done on women from surrounding areas who will come for general check up to GVP Hospital. Only individuals who will be consented to participate in our protocol program will be eligible for inclusion.
Inclusion criteria: 15-49 years Women belong to suburban region. 

Exclusion criteria: Diagnosed cases of osteoporosis, osteopenia, Women suffering from hormonal disorders like hypoparathyroidism and hyperparathyroidism,
taking calcium supplementation, having renal disease and using any drugs which affect calcium level. Informed consent will be taken from all the women in our study in written and explained form in their own language. Approval from the ethical committee will be taken. 3 ml of venous blood sample will be collected in a plain tube. Sample will be collected after the patients had been seated for 10 min. Care will be taken to avoid muscular contraction in the limb during sample collection. Samples will be stored for <2 hours at 5°C until centrifugation if necessary. Serum will be separated and calcium levels will be analysed in semi autoanalyzer by OCPC End point method. The analysis will be done by using commercially available meril kit. Daily Internal quality control will be performed with assayed normal and assayed abnormal to assure the accuracy of result. Monthly External quality control assessment will be done to compare the performance with a standard reference laboratory. The maintenance of quality control ensures our results to be more accurate and supports our study on large number of subjects. 25(OH) D levels in serum were measured as a standard procedure at the department of biochemistry. 25-hydroxyvitamin D Assay done by using chemiluminescent immunoassay technology. The lower limit of Quantitation of the assay was 4.0 ng/mL. Specific antibody to Vitamin D is used for coating magnetic particles. Subsequently, the starter reagents were added, and a flash chemiluminescent reaction was initiated. Internal and external quality control was maintained by running a sample of known concentration of 25(OH)D along with the samples for analysis. Vitamin D deficiency was defined as serum 25(OH) D levels ≤20 ng/ml9. The levels were further categorized under mild deficiency (12–20 ng/ml), moderate deficiency (6 < 12 ng/ml) and severe deficiency (<6 ng/ml).Estimation of Serum Calcium done by using modified O-cresolphthalein Complexone method using kit. Optical density of 10μl of test serum was read against blank and Standard at a wavelength of 578nm using UV spectro- photometer. Normal values of calcium using this kit will be 8.4- 10.4mg/dl.
Estimation of Serum Phosphorus done by molybdate UV method using Liquimax Phosphorus Optical density of 10 μl of test serum was read against blank and standard at a wavelength of 340nm using UV spectrophotometer. In this method, normal values of serum Phosphorus is in a range of 2.4-5.0mg/dl.
Estimation of serum alkaline phosphatase by quantitatively by DEA buffer/DGKC method using Liquimax alkaline phosphatase. Change in the optical density of 20μl of test serum was read against distilled water at 405nm. Normal range of alkaline phosphatase ranges from 80-315. The data obtained will be expressed as Mean ± Standard Deviation (mean ± SD). The data will be entered in MS-Excel sheet and analysed by comparing with normal calcium levels.

RESULTS

The mean serum 25-OHD level was 19.2± 8.3 ng/mL. Of the included patients, 85.2% ( of 350) were vitamin D deficient, and only 15.4% (54 of 350) had normal serum levels. Among patients with vitamin D deficiency, 131 cases had severe deficiency, 87 had moderate, and 82 had mild deficiency.

 

Table 1: Vitamin D Deficiency According to Different Age Groups, BMI Groups and Seasons

total of 300 patients were included in this study with the mean age of 32 ± 15.2 years.

 

Table 2: The Severity of Vitamin D Deficiency in Non-Pregnant and Pregnant Women Based on Different Trimesters

114 cases (38%) were currently pregnant, among whom 83 were vitamin D deficient. 16.4% suffered from sever deficient, 16% with moderate deficient and 5.3% with mid deficiency.

 

Table 3: Descriptive statistics and laboratory biochemical parameters in correlation with vitamin -D

*Significant p-Value <0.05

Mean sun light exposure daily was found to be 25±32 (minutes) in the study population. Calcium, phosphorus and alkaline phosphatase levels were 9.0±2.1, 4.70±0.9 and 127±66 respectively. Mean sun light exposure daily and calcium has positive correlation with vitamin-D and has significance in the study population

DISCUSSION

A unique property of Vitamin D is that it can be produced endogenously in the skin following sufficient sunlight exposure; specifically exposure to ultraviolet B (UVB) radiation (280–315 nm). It is regularly reported that more than 80% of Vitamin D intake is from sun exposure.⁵ Other than its role in maintaining calcium and phosphorus homeostasis, promoting healthy bone mineralization, induction of cell differentiation, inhibition of cell growth, and regulation of apoptosis, Vitamin D is involved in regulating the functions of the female reproductive system. Hormones regulated by the Vitamin D system include estradiol, progesterone, human chorionic gonadotropin, and human placental lactogen, all of which are critical in maintaining the regulation of reproductive health.⁶ Maternal Vitamin D deficiency or insufficiency during pregnancy has been related to preeclampsia, gestational diabetes. ^7,8,9 conditions in bone disorder greater risk of cesarean delivery and preterm birth. ^{10, 11}It has also been associated with polycystic ovarian syndrome (PCOS), endometriosis, uterine leiomyomas, and invitro fertilization outcome.¹² India is a country with ample sunshine, despite the fact, Vitamin D deficiency continues to be a growing public health concern. Other than geographic factors and ambient UVB radiation there are individual-specific variables that affect endogenous production of Vitamin D, such as limited access to sunlight, air pollution, skin condition and pigmentation (skin type), time spent outdoors, type of clothing, and sun protection practices.¹³The mean serum 25-OHD level was 19.2± 8.3 ng/mL. Of the included patients, 85.2% ( of 350) were vitamin D deficient, and only 15.4% (54 of 350) had normal serum levels. Among patients with vitamin D deficiency, 131 cases had severe deficiency, 87 had moderate, and 82 had mild deficiency. The earlier studies conducted across the country have documented similar results of (96%), (76%), (58.5%), and (83%) among women of reproductive age.^14,15,16,17 A high prevalence of Vitamin D deficiency and poor dietary calcium intake is likely to worsen during pregnancy and may cause significant adverse health consequences in the newborn, including rickets and tetany. A recent meta-analysis concluded that Vitamin D concentrations were lower in women with PCOS compared with those without PCOS, independently of body mass index.¹⁸ The present study reveals a high prevalence of Vitamin D deficiency among Indian women of reproductive age.114 cases (38%) were currently pregnant, among whom 83 were vitamin D deficient. 16.4% suffered from sever deficient , 16% with moderate deficient and 5.3% with mid deficiency.In a study by Hovsepian on an Iranian sample of pregnant mothers in 2011 in the city of Isfahan, the trend of severe (26.9%), moderate (23.9%) and mild (19.6%) vitamin D deficiency seemed to be similar to that of our study, which highlights the significance of paying attention to serum levels of vitamin D in pregnant mothers, and women of reproductive age.¹⁹In another study by Hashemipour (2004) in Tehran reported prevalence of severe, moderate and mild vitamin D deficiencies was 9.5%, 57.6%, and 14.2% respectively. serum levels of vitamin D in young and middle aged women were significantly lower than the older group (14), which is in line with the results of this study. This difference can be justified by parenteral use of vitamin D plus calcium supplements that is commonly prescribed to older women in Tehran for prevention or treatment of osteoporosis.²⁰Serum calcium was positively correlated with serum Vitamin D in our study indicating that Vitamin D is involved in the absorption of calcium from the gut.²¹ Serum phosphorus and alkaline phosphatase levels were within the normal limits but not correlated with Vitamin D status in our study, was similar to the study conducted in Karachi .²² Vitamin D increases the efficiency of intestinal absorption of calcium to 30-40% and phosphorus absorption approximately by 80%.²³ Vitamin D deficiency state leads to secondary hyperparathyroidism which results in loss of phosphorus in the urine and decreases intestinal absorption of phosphorus. This cause low or low normal phosphorus concentration. Low normal calcium and low normal phosphorus both will cause insufficient calcium phosphorus product which is important for bone mineralization process. Defective mineralization causes rickets in children and osteomalacia in adults .²³ During deficiency of Vitamin D, there should be low levels of calcium and phosphorus but alkaline phosphatase levels should raise. But in our study, calcium was reduced but phosphorus and alkaline phosphatase levels were within normal limits indicating that bone mineralization was not yet affected. We found positive correlation between sun light exposure and Vitamin D status. It is already well known fact that major source of Vitamin D is the sun light. Our study was supported by a study conducted in Saudi where exposure to sun light resulted in 21⁄2 fold increase in 25-OH Vitamin D3 levels .²⁴ The need for improvement in vitamin status of the Indian population is both important and urgent. The Indian government needs to take substantive measures in this direction. Revision of RDA for calcium and vitamin D is required. Better facilities and technologies should be made available countrywide to enable timely diagnosis of clinical manifestations of vitamin D deficiency in individuals who need attention by the clinicians. Population-based programs at the national level must be developed to increase awareness of the problem at hand, to provide affordable vitamin D supplements and also to provide vitamin D fortified foods to the Indian populace at large. Research in this field needs continued support to provide a comprehensive picture of the ongoing vitamin D problem and also to study and monitor the effects.

 

CONCLUSIONS

Vitamin D deficiency is highly prevalent in Indian women of reproductive age. These women may possibly have a higher risk for development of osteoporosis and pregnancy-related complications in future life. In the presence of lack of naturally occurring Vitamin D rich foods in the country, food fortification/supplementation with Vitamin D and adequate sunshine exposure should be given a higher priority among these women. Even though sun light is adequate in this part of country, sun light exposure is not sufficient to synthesize Vitamin D in the body. Food products should be fortified to ensure sufficient Vitamin D. It is advisable to take oral intake of 800IU-1000IU Vitamin D daily. Sun light exposure daily for 10 minutes on face and arms is very much needed to raise the sun shine vitamin. Indian government should lay proper guidelines for attaining adequate Vitamin D levels in this subcontinent .

 

REFERENCES