CORRELATIVE ANALYSIS OF VITAMIN D STATUS IN PEOPLE SUFFERING FROM TYPE-2 DIABETES MELLITUS IN EASTERN REGION OF NEPAL Affiliation

Introduc on Vitamin D deficiency exists as a common problem among popula on worldwide. The deficient vitamin D level leads to direct impact on various normal func oning of human body systems. It has been observed in few studies that decreased circula ng concentra on of 25-hydroxyvitamin D is associated with type-2 diatetes mellitus. The possible reason behind such occurrence is due to the direct effect of vitamin D on glucose metabolism. Vitamin D exerts influences on ac vity of pancrea c β-cell func on from where insulin is secreted. In addi on, the pa ents suffering from low vitamin D status show insulin resistance. These affected individuals with low vitamin D status exhibits impaired markers of glucose metabolism such as glycosylated hemoglobin. So, researchers have found vitamin D deficiency to correlate with type-2 diabetes and 80 % of obese adults suffering from vitamin D insufficiency state. In spite of such crucial significance, the correla ve studies related to vitamin D status and type-2 diabetes s ll remains obscure in eastern region of Nepal.


Objec ve
We aimed to inves gate status of vitamin D among type-2 diabe cs and analyze its possible correla on

Methodology
In the present study which was hospital-based and crosssec onal one, antecubital venous blood samples were collected from pa ents (n=100) in plain vials with informed wri en consent.Blood samples were allowed to clot and centrifuged for separa on of serum.The separated sera were further processed for determina on of glucose (fas ng as well as post-prandial) by spectrophotometry and es ma on of vitamin D with use of microwell Enzyme-linked immunosorbant assay (ELISA) technique.The data was interpreted by using SPSS so ware version 16.

Results
We found that 75 % type-2 diabe cs had suffered from "state of vitamin D deficiency".Among them, females, in contrast, were predomina ng which was interes ng in an analy cal point of view.

Conclusion
Our study reviews evidence on disorders related to type-2 diabetes mellitus and vitamin D status, especially in older people.The biological processes that lead to synthesis of vitamin D in human body ssues become less efficient when an individual's age advances with in addi onal occurrences of type-2 diabetes mellitus.We should therefore maintain the reple on of vitamin D in healthy older people via supplementary intakes and sensible sun-exposure.

INTRODUCTION
The vitamin D is accessible in natural foods meaning that 1 humans should consume them on regular basis.In addi on to their availability in natural foods, human body ssues 2 produce vitamin D in response to exposure of sunlight.Vitamin D deficiency, however, exist as common problem in 3 popula on. .The deficient level of vitamin D exhibits direct impact on normal func oning of human body systems.Bones and tendons require sufficient amount of vitamin D 4 as normal growth requirement.The reduced level of 25hydroxyvitamin D is associated with type-2 diatetes mellitus.The already known fact behind this occurrence is due to the direct effect of vitamin D on glucose metabolism.Vitamin D exerts influences on ac vity of pancrea c β-cell func on from where insulin is secreted.These affected individuals with low vitamin D status exhibits impaired HbA C. The inadequacy of vitamin D, further, retards 1 physical growth with a risk in fracture of hip bones among 5 elderly people.Maintenance of normal levels of vitamin D, therefore, is essen al for appropriate regula on of systemic processes.Thus, vitamin D deficiency is emerging as a serious health problem.Hence, es ma on of its plasma levels is essen al for determining the grade of an individual who may stand either under-deficiency or over-nutri on state.As it transforms metabolic processes of glucose and deficiency is worldwide being prevalent in stages that range from degree of newborn to infancy and adulthood, 6 determina on of vitamin D is crucial.
Moreover, numerous studies have reported for existence of higher incidences of vitamin D deficiency among type-2 7 diabe cs.So, con nua on of vitamin D deficiency may affect type-2 diabetes control.Vitamin D deficiency typically associates with worsening glycemic index and 8 increased insulin requirements.As per the data put forwarded by World Health Organiza on, a pandemic disease called diabetes mellitus affects 170 million pa ents with having an effect on more than double to 370 million pa ents by year 2030.Researchers, in contrast, have found vitamin D deficiency to correlate with type-2 diabetes and 80 % of obese adults suffering from vitamin D insufficiency 9 state.Scien sts have shown low vitamin D status to associate with development of type-2 diabetes as well as metabolic syndrome so that these consequences reflect as 10 incidences of vital clinical importance.In spite of suchcrucial significance, the correla ve studies related to vitamin D status and type-2 diabetes s ll remains obscure among type-2 diabe cs s in eastern Nepal.

METHODOLOGY Study design and enrolment criteria:
This was a hospital-based, cross-sec onal correla ve study carried out in the Department of Internal Medicine with Diagnos c Laboratory sec on at Birat Medical College and Teaching Hospital (BMCTH), Tankisinuwari, Biratnagar, Morang, Nepal.A total of 100 type-2 diabe c pa ents with age group ranging from 20 and 60 years-old were registered to meet the target popula on.Exclusion criteria were: pa ents using medica ons such as an convulsions, an tubercular drugs, calcitonin, gabapen n, cor costeroids and pa ents diagnosed with diseases like osteoporosis, osteomalacia, rheumatoid arthri s.In addi on to es ma on of their glucose levels, these pa ents were inves gated for vitamin D status in collected blood samples.The informed wri en consent was taken from all pa ents and study was approved by Ins tu onal Ethical Commi ee.All the par cipants had undergone detailed history, clinical examina on and laboratory inves ga ons using Performa designed for this study.
All pa ents were subjected to following inves ga ons: determina on of fas ng blood sugar (FBS) and post-prandial blood sugar (PPBS) as well as es ma on of vitamin D.

Sample collec on and serum prepara on:
Venipuncture was performed to collect blood samples under universal a en veness as described in manufacturer's 11 protocol.Antecubital venous blood was collected from pa ents in plain vials with informed wri en consent, strictly as per the norms recommended by Ins tu onal Ethical Commi ee.Blood samples were allowed to clot for five min and centrifuged at 3000 rpm for 10 min in order to separa on of serum.Sera were stored at-20 °C un l assessment.All steps were carried out under sterile condi ons and precau ons were taken to prevent blood samples from 12-13 hemolysis, as described earlier.

Determina on of glucose in serum:
The determina on of glucose levels in serum was done using spectrophotometer and glucose oxidase-peroxidase 14 assay kit by Tinder's method.The unhemolysed serum samples and normal as well as abnormal controls were used while es ma on, according to the instruc ons provided by manufacturers.

Es ma on of vitamin D:
Compe ve immunoassay was performed for es ma on of serum levels of vitamin D using direct chemiluminescent technology with ADVIA Centaur vitamin D assay kit.The ADVIA Centaur XP system was u lized while performing assay.The system was prepared a er loading reagent packs containing an -vitamin D-an body.Instrument automa cally performed certain ac ons that include: dispensing 100 μl serum into cuve e, adding 115 μl dithiothreitol, 200 μl solid-phase reagent followed by incuba on at 37 °C for 5 min and administering 200 μl lite reagent with incuba on at 37°C for 2.5 min.Then machine had separated, aspirated and washed the cuve es with reagent water.A erwards instrument had dispensed 300 μl each of acid and base reagents to ini ate chemiluminescent reac on.The pa ents were declared deficient if they demonstrated serum levels of vitamin D < 10, insufficient when concentra ons resided between 10-30, sufficient a er values > 30 and toxicity if > 100 ng/ml.Results were obtained according to selec on op on in system.Based on se ng up of the assay, system had reported serum levels of vitamin D in terms of ng/ml.

Data interpreta on:
The validity and reliability of test results were determined using control sera (from BIO-RAD).Data were analyzed under So ware Package for Social Science version 16 (SPSS 16).

DISCUSSION
In present study, we observed that majority ( 75 %) of type-2diabe cs who a ended the hospital in eastern part of Nepal suffered from "state of vitamin D deficiency" (Figure 2).Based on few studies carried out previously in rela ve perspec ve, vitamin D deficiency was shown to be more [16][17][18] common among type-2 diabe cs.
Our observa on stands in accordance with these reports.By contrast, in eastern part of Nepal, frequency of vitamin D status remained unknown.Thus we screened type-2 diabe c individuals to inves gate existence of associated status of vitamin D and observed preponderant significance of vitamin D deficiency in this area (Figure 2).In age group 51-60years females had higher prevalence of Vitamin D deficiency as compared to male.This has been the striking prevalence among old-aged type-2 diabe cs.In this analy cal point of view to discuss, Hanan A.H. had also obtained the similar data and so had reported the fact that 23 vitamin D levels were significantly higher than females.These findings, in the study carried out by Hanan A.H., had been sugges ve to the fact that vitamin D levels were affected by age, na onality, gender and health statues.In beneficial point of view to the pa ents having vitamin D deficiency, sensible sun exposure cause health benefits and 24 maintains the strong bones.

CONCLUSION
Our study reviews evidence on disorders related to type-2 diabetes mellitus and vitamin D status, especially in older people.Vitamin D is as essen al for bone health in adults and in children to prevent osteomalacia and muscle weakness.Skin provided vitamin D synthesis followed by UVB irradia on from summer sunshine and to lesser extent via absorp on from food.However, these processes become less efficient when an individual's age advances with in addi onal occurrences of type-2 diabetes mellitus.We should therefore maintain the reple on of vitamin D in healthy older people via supplementary intakes and sensible sun exposure.

RECOMMENDATIONS
Based on our observa on in the present study, we strongly recommend to supplement vitamin D in both diabe c and non-diabe c older people.

LIMITATIONS OF THE STUDY
As the present study has been carried out in an urban city Biratnagar, it would be more applicable if the rural areas would have been included.

Figure 4 :
Figure 4: Bar diagram showing variance of vitamin D level and Pie chart to demonstrate gender-based distribu on of 51-60 years old local diabe cs.21-30 years-old 31-40 years-old 41-50 years-old 51-60 years-old Vitamin D deficiency exists as a common clinical problem these days.According to the survey carried out by Mar na R., more than half of the adult people in Germany had 19 vitamin D levels below common threshold.Since then there had been a lot of a en on for vitamin D in scien fic community.Other study carried out by Daga et al in North India, 91.1 % of type-2 diabe c pa ents had vitamin D deficiency.In this study, vitamin D concentra ons were 7.88±1.2and 16.64±13.6ng/ml in diabe c and non-20 diabe c pa ents, respec vely.In addi on, Yousef A.S. had reported about prevalence of vitamin D deficiency in the 21 Kingdom of Saudi Arabia in every age group.According to Colorado prevalence study, Alejandro S. had shown commonness of vitamin D inadequacy in Portuguese older adults.In such study, na onwide cluster sample of 1500 Portuguese subjects > 65 years of age suffered from vitamin 22 D deficiency.In the present study, we observed that 75 % people who had suffered from type-2 diabetes mellitus stood in the state of vitamin D deficiency.When we divided such 75 % type-2 diabe cs into four sets designated as 'A', 'B', 'C' and 'D' consis ng of individuals as described above, our data laid us to note that 50 % (n=37) were pa ents s of th vitamin D deficiency (4 bar, Figure-3).Our findings were therefore sugges ve of predominant existence of vitamin D deficiency.According to study previously carried out by Barbara J.B., hypovitaminosis D exist as common problem worldwide, but it is more common and severe in older 10 people as we observed in our study.