the prevalence of type 2 diabetes mellitus ( t2 dm ) is rising globally , especially in asian indians . decreased bone mineral density ( bmd ) is an established complication of type 1 diabetes mellitus and has been attributed to early age at diagnosis leading to decreased bone accrual , long duration , prolonged poor glycemic control and high insulin doses . several studies have shown that bmd at the lumbar spine and femoral neck were either similar , increased or rarely decreased in comparison with healthy controls . the exact pathophysiology behind the bmd changes in t2 dm has not been well elucidated . bone mineral density in diabetics is influenced by factors like calcium balance , osteoblast function , advanced glycation end products in bone collagen , inflammatory cytokines like leptin , peroxisome proliferator - activated receptors ( ppar ) isoform status and vitamin d status . high bmd in most of the recent studies with t2 dm is secondary to lower bone volume and functional hypoparathyroidism . there are no studies regarding bmd status in t2 dm without known complications in the asian indian population or of the various factors affecting it : viz sunlight , vitamin d status and lifestyle . hence , we studied the bmd profile and its relation if any , to clinical , hormonal and metabolic factors in asian indian t2 dm patients . it is a cross sectional prospective observational study comparing the bmd of t2 dm patients with that of sex matched healthy volunteers who attained peak bone mass ( pbm ) . diagnosis of t2 dm was based on american diabetic association ( ada ) criteria , 2008 . subjects with t2 dm were recruited from outpatient endocrinology department of tertiary care hospital in india . sex matched healthy control subjects who had crossed the age for pbm were recruited from community based health camps . the study was approved by the institutional ethics committee and a written informed consent was obtained from all participants . two hundred and fourteen t2 dm patients were screened for the study of which 21 refused to participate in the study . one hundred and ninety four ( 97 men and 97 women ) t2 dm patients and 571 healthy volunteers ( 262 men and 309 women ) aged between 30 to 50 years formed the study participants . postmenopausal status , smoking ( > 1 year ) , chronic alcoholism , serum creatinine > 1.4 mg / dl , pre - existing hypogonadism , hypopituitarism , thyrotoxicosis , past or present drug intake that may alter bone metabolism ( e.g. , vitamin d supplements , steroids , bisphosphonates , cytotoxics , anticonvulsants , thyroxine ) and evidence of diabetic end organ complications constituted the exclusion criteria . diabetes and other medical conditions were ruled out with appropriate investigations in controls and their baseline demographic data with bmd were used for this study . the diabetic study group underwent biochemical and hormonal evaluation along with bmd and study related procedures . a detailed clinical evaluation of the diabetes patients with respect to its treatment ( sulfonylureas , metformin , thiazolidinediones and statins ) and presence of complications was carried out . subjects with body mass index ( bmi ) > 25 kg / m were considered obese . sunlight exposure ( to exposed areas especially face and hands ) in terms of duration of outdoor activity , weekly schedule and outdoor attire the  rule of nine  was adapted to estimate the fraction of body surface area exposed to sunlight by each subject 's usual outdoor attire . physical activity was evaluated by global physical activity questionnaire developed by world health organization ( who ) . nutritional intake was calculated using a questionnaire with specific reference to dietary intake of energy , protein , calcium and phosphorous . biochemical investigations included serum calcium , phosphorus , alkaline phosphatase ( normal range 30 - 150 iu / l ) , albumin , creatinine , glycosylated hemoglobin ( hba1c ) , fasting lipid profile and urine calcium creatinine ratio ( ur.cr/ca ) . hormonal investigations included parathyroid hormone ( pth ) ( intra assay coefficient of variation ( cv ) : 5.5 - 6.3% , inter assay coefficient of variation ( ce ) : 7.9 - 8.6% and analytical sensitivity ( s ) : 3 pg / ml ) , serum 25-hydroxy vitamin d ( 25(oh ) d ) ( cv : 8.2 - 11% , ce : 9.6 - 12.5% , s : 1.5 ng / ml ) , follicular stimulating hormone ( fsh ) ( cv : 2.3 - 3.7% , ce : 5.4 - 6.7% , s : 0.1 iu / l ) , leutinising hormone ( lh ) ( cv : 4.8 - 6.5% , ce : 7.2 - 26% , s : 0.1 iu / l ) , estradiol ( e2 ) ( cv : 6.3 - 15% , ce : 6.4 - 16% , s : 15 pg / ml ) , testosterone ( t ) ( cv : 4.9 - 17% , ce : 6.0 - 12% and s : 15 ng / dl ) , sex hormone - binding globulin ( shbg ) ( cv : 4.1 - 7.7% , ce : 5.8 - 13% and s : 0.2 nmol / l ) , total tri - iodothyronine ( t3 ) ( cv : 5.4 - 13.2% , ce : 7.7 - 15.6% and s : 35 ng / dl ) , total tetra - iodothyronine ( t4 ) ( cv : 6.3 - 8.4% , ce : 6.7 - 9.8% and s : 0.4 g / dl ) , thyroid stimulating hormone ( tsh ) ( cv : 3.9 - 13.8% , ce : 8 - 17.5% and s : 0.004 iu / ml ) and urine free deoxypyridinoline ( ur.dpd ) ( cv : 8 - 15% , ce : 8 - 20% and s : 6 nm ) . we estimated calculated free testosterone ( cft ) from total testosterone and shbg , using the method of vermeulen et al . , by a computer program ( free and bioavailable testosterone calculator , developed at the hormonology department , university hospital , ghent , belgium and available at http://www.issam.ch/freetesto.htm ) . all hormonal investigations except 25(oh ) d were done by chemiluminescent immunometric assay with immulite 1000 system of diagnostic products corporation , los angeles , usa . serum 25(oh ) d levels of > 30 ng / ml , 20 - 30 ng / ml and < 20 ng / ml were taken as suggestive of vitamin d sufficiency , insufficiency and deficiency respectively . assessment of bmd was performed with the dual energy x - ray absorptiometry ( dxa ) ( hologic inc . measurements included dxa of the total hip , left femoral neck , intertrochanter , ward 's area and lumbar spine ( posterior anterior projection ) . all the above sites were used to divide the diabetes study group into groups i [ normal bmd ( z score > -2 ) ] and ii [ low bmd ( z score < -2 ) ] , but only the total hip and lumbar spine were used to compare bmd in the study group and the controls . bmi matched controls were selected with bmi criteria of 22 - 30 kg / m in men and 24 - 30 kg / m in women . the short term in vivo precision ( cv% ) of dxa unit were as follows : lumbar spine-1.09% , femoral neck-3.29% and total hip-1.26% , intertrochanter-3.09% , wards area-2.04% . bmd was reported in terms of g / cm and also in z - scores . z - scores were based on the caucasian normative database in the absence of normative data in asian indians . all results are expressed as mean  standard deviation of the mean ( sd ) and median . the statistical significance between means was calculated by student 's t - test , analysis of variance or mann - whitney u test when appropriate . multivariate linear regression analysis was used to determine independent associates of bmd in the diabetes group . assessment of bmd was performed with the dual energy x - ray absorptiometry ( dxa ) ( hologic inc . measurements included dxa of the total hip , left femoral neck , intertrochanter , ward 's area and lumbar spine ( posterior anterior projection ) . all the above sites were used to divide the diabetes study group into groups i [ normal bmd ( z score > -2 ) ] and ii [ low bmd ( z score < -2 ) ] , but only the total hip and lumbar spine were used to compare bmd in the study group and the controls . bmi matched controls were selected with bmi criteria of 22 - 30 kg / m in men and 24 - 30 kg / m in women . the short term in vivo precision ( cv% ) of dxa unit were as follows : lumbar spine-1.09% , femoral neck-3.29% and total hip-1.26% , intertrochanter-3.09% , wards area-2.04% . bmd was reported in terms of g / cm and also in z - scores . z - scores were based on the caucasian normative database in the absence of normative data in asian indians . all results are expressed as mean  standard deviation of the mean ( sd ) and median . the statistical significance between means was calculated by student 's t - test , analysis of variance or mann - whitney u test when appropriate . multivariate linear regression analysis was used to determine independent associates of bmd in the diabetes group . the diabetes study group consisted of 97 m and 97 f each , respectively ( age range : 30 - 50 years ) with t2 dm without micro or macrovascular complications ninety eight ( 58 : fand 40 : m ) patients ( 51% ) were obese . the general characteristics of the study group in the diabetes study group , 156 ( 76 : mand 80 : f ) had normal bmd , i.e. , group i. bmd greater than the mean of the age and sex matched caucasian controls ( z score > 0 ) was seen in 75 of 156 with normal bmd ( 48% ) . low bmd was present in 38 ( 21 m and17 f ) , i.e. , group ii . the control group was significantly younger and had a lower bmi than the study group . a comparison of baseline characteristics in t2 dm showed a significant increase in weight and bmi in group i ( m and f ) [ table 2 ] . the mean duration of t2 dm ( 41.1 vs 41.3 years ) was not significantly different in groups i and ii , respectively . glycemic control was similar in both groups i and ii ( mean hba1c : 7.26 vs 7.44 ) . bmd in t2 dm patients was significantly higher than that of the control group in all regions of interest except in total hip in men [ table 3 ] . in a subgroup of controls with matched bmi ( m-161 ; f-114 ) , the difference in bmd and z - scores between the diabetes group and the controls was no longer significant at both lumbar spine and total hip . comparison of different baseline characteristics in t2 dm patients based on bmd comparison of bmd of control and diabetes study cohorts patients on statin ( atorvastatin 10 - 40 mg , mean of 12.7 mg ) treatment for a minimum of 6 m were compared in group i ( n-109 ) and group ii ( n-23 ) . there was no significant difference in the use of statins between them in our study . patients exposed to thiazolidinedione ( rosiglitazone ( n-6,2 - 8 mg , mean : 4.7 mg ) ; pioglitazone ( n-16,15 - 45 mg , mean : 28.8 mg ) ) were evaluated for any correlation with bmd . there was no significant difference in use of thiazolidinediones between groups i and ii ( m and f ) . this could be due to the small subgroup size ( only 22 patients were on thiazolidinediones ) . the mean calorie intake was similar in groups i and ii ( m and f ) . in group i the calcium intake was 782 mg / d and 590 mg / d in males and females respectively , while in group ii it was 715 mg / d and 514 mg / d , respectively [ table 4 ] . the calcium / phosphorus ratio was 0.45 and 0.47 in group i males and females respectively , while in group ii it was 0.53 and 0.57 respectively . calcium / protein ratio ( mg / g ) was also low ( < 13 ) in groups i and ii ( m and f ) . comparison of dietary parameters in t2 dm patients based on bmd parathyroid hormone was normal in group i ( m and f ) but elevated in male patients in group ii [ table 5 ] . serum calcium , phosphorus , alkaline phosphatase , u.ca/cr and u.dpd/cr ratios were not significantly different among groups i and ii ( m and f ) . there was also no correlation in levels of e2 and cft with bmd ( m and f ) . comparison of biochemical parameters in patients with t2 dm based on bmd there was no significant statistical difference in 25(oh ) d levels between groups i and ii ( m and f ) [ table 6 ] . in the diabetes study group as a whole , 8.2% , 42.3% and 49.5% of men had normal , insufficient and deficient 25(oh ) d levels respectively . similarly , 2.1% , 34% and 63.9% of women had normal , insufficient and deficient 25(oh ) d levels respectively [ figure 1 ] . the mean 25(oh ) d level of our diabetes subjects was 20.3 ng / ml ( m ) and 17.1 ng / ml ( f ) . there was no correlation with the 25(oh ) d levels and bmd in our cohort . distribution of vitamin d status in t2 dm patients based on bmd distribution of vitamin d status in type 2 dm patients physical activity was found to be moderate in groups i and ii ( m and f ) and there was no significant difference between them . similarly , the sunlight exposure was comparable in both groups i and ii ( m and f ) , ( 143.54 vs 132.05 minutes per week in males and 108.19 vs 92.06 min per week in females respectively ) . a multiple linear regression analysis of affecting factors in the diabetes study group ( both men and women taken together and separately ) , showed bmi and weight to be independent predictors of bmd at both the spine and hip [ table 7 ] . sex was an independent predictor of bmd at the total hip in the whole study group . other variables like age , estradiol , free testosterone and calcium intake , when evaluated , were not independently associated with bmd . the mean calorie intake was similar in groups i and ii ( m and f ) . in group i the calcium intake was 782 mg / d and 590 mg / d in males and females respectively , while in group ii it was 715 mg / d and 514 mg / d , respectively [ table 4 ] . the calcium / phosphorus ratio was 0.45 and 0.47 in group i males and females respectively , while in group ii it was 0.53 and 0.57 respectively . calcium / protein ratio ( mg / g ) was also low ( < 13 ) in groups i and ii ( m and f ) . parathyroid hormone was normal in group i ( m and f ) but elevated in male patients in group ii [ table 5 ] . serum calcium , phosphorus , alkaline phosphatase , u.ca/cr and u.dpd/cr ratios were not significantly different among groups i and ii ( m and f ) . there was also no correlation in levels of e2 and cft with bmd ( m and f ) . there was no significant statistical difference in 25(oh ) d levels between groups i and ii ( m and f ) [ table 6 ] . in the diabetes study group as a whole , 8.2% , 42.3% and 49.5% of men had normal , insufficient and deficient 25(oh ) d levels respectively . similarly , 2.1% , 34% and 63.9% of women had normal , insufficient and deficient 25(oh ) d levels respectively [ figure 1 ] . the mean 25(oh ) d level of our diabetes subjects was 20.3 ng / ml ( m ) and 17.1 ng / ml ( f ) . there was no correlation with the 25(oh ) d levels and bmd in our cohort . distribution of vitamin d status in t2 dm patients based on bmd distribution of vitamin d status in type 2 dm patients physical activity was found to be moderate in groups i and ii ( m and f ) and there was no significant difference between them . similarly , the sunlight exposure was comparable in both groups i and ii ( m and f ) , ( 143.54 vs 132.05 minutes per week in males and 108.19 vs 92.06 min per week in females respectively ) . a multiple linear regression analysis of affecting factors in the diabetes study group ( both men and women taken together and separately ) , showed bmi and weight to be independent predictors of bmd at both the spine and hip [ table 7 ] . sex was an independent predictor of bmd at the total hip in the whole study group . other variables like age , estradiol , free testosterone and calcium intake , when evaluated , were not independently associated with bmd . bmd in t2 dm is determined by various pathogenetic factors and several acquired conditions pertaining to diabetes . circulating insulin / insulin growth factors and estrogen levels are the most important among the pathogenetic factors . a majority of studies on bmd in t2 dm have documented increased or normal bmd in different areas of the body using different modalities . fremantle diabetes study ( fds ) by rakic et al . documented an increase in bmd in t2 dm in both sexes . the bmd in the femoral neck were 0.851 and 0.808 gms / cm in males and females respectively , and in the total spine was 1.117 and 1.031 gms / cm and was greater than the age and sex matched non - diabetic controls . . this could be largely due to the fact that indians typically have bmd which is approximately 5% lower than the caucasian population . tuominen et al . , and sosa et al . , documented normal bmd in t2 dm . decreased bmd was documented by guven et al . , and al - maatouq et al . guven et al . , documented a femoral neck bmd of 0.769 and 0.716 gms / cm in men and women respectively and lumbar spine bmd of 0.912 and 0.845 gms / cm in the same cohort , respectively . both the studies , done in a predominantly muslim population had control groups with higher bmd . a majority of studies show no correlation of bmd to glucose control . however , the fremantle study of 194 t2 dm patients showed that hba1c was independently associated with bmd at the hip and femoral neck . the diabetes study group when compared to the non - diabetic control group had a higher bmi and this was statistically significant in both sexes in our study . studies done by strotmeyer et al . , have uniformly shown bmi to be an important variable affecting bmd . obesity itself is known to be associated with an increased bone mass in most studies . since t2 dm is preceded by a period of insulin resistance , it is postulated that hyperinsulinemia may confer a protective effect on bmd , either directly through elevated fasting insulin or indirectly through bmi . obesity could have the additional favorable effect of decreasing shbg levels and increasing the production of metabolically active free estrogen . it leads to increased osteoblastic differentiation and may also reduce osteoclastogenesis by altering the expression of rankl and osteoprotegerin in stromal cells . the aromatization of androgens to estrogens in adipose tissue represents an important source of estrogen , which is anabolic for bone . statins are reportedly associated with an increase in bone morphogenic protein-2 , a protein that plays an important role in osteoblast differentiation and bone formation . documented an increase in bmd in the femoral neck and total hip but not at the lumbar spine in patients on statin therapy . ppar- is essential for normal adipocyte differentiation and proliferation as well as fatty acid uptake and storage . schwartz et al . , documented increased bone loss at total body , trochanter and lumbar spine in women with the use of thiazolidinediones . it is in contrast however , to the study by harinarayan et al . from south india where the calcium intake was less than the indian council of medical research recommendations of 400 mg / d across socioeconomic status and vitamin d levels . there was a significant difference in pth levels in group ii in men as compared to group i. this can not be explained by vitamin d deficiency , as the vitamin d levels were comparable in groups i and ii ( m and f ) . the mean 25(oh ) d level of our study group was lower than that in fds which could be attributed to increased skin pigmentation , crowded housing with limited sunlight exposure , smog , and the angle of ultraviolet rays . duration of sunlight exposure is positively correlated with bone density and 25(oh ) d levels in various studies . there was no significant difference in sun exposure between groups i and ii ( m and f ) subjects with t2 dm . vitamin d deficiency and insufficiency was seen in 50% and 42% of male diabetes study subjects and 64% and 34% of female diabetes study subjects , respectively , despite adequate sunlight exposure . cross sectional studies have shown a positive correlation between bmd and exercise while interventional studies suggest that high impact exercises are better at increasing bmd . moderate physical activity has also been documented to lead to better bone health in studies in t2 dm in contrast to heavy physical activity . physical activity was found to be moderate in groups i and ii ( m and f ) in our study and there was no significant difference between them . most recent studies regarding bone mineral changes in t2 dm have shown increased density . in our study , more than 80% of subjects with diabetes had normal bmd . life style measures including adequate sunlight exposure , good calcium intake and moderate physical activity seen in our subjects could be significant factors contributing to normal bmd . however , the effect of age is likely to be minimal once it has crossed the age for peak bone mass . the bmd of t2 dm patients is moreover , greater than the controls , who recently attained peak bone mass . comparison with the caucasian database could have resulted in overestimation of low bmd and underestimation of normal bmd in our study . a comparative study of factors affecting bmd in the non - diabetic control group would have added strength to our study . bmd of t2 dm asian indian subjects following up in a tertiary care hospital in india was similar to that of the general population . weight and bmi had the most significant impact on bmd in both men and women .