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Osteomalacia and Vitamin D Deficiency among Urban Saudi Adolescent Girls Abstract Objectives To determine the frequency of clinical osteomalacia caused by vitamin D deficiency among Saudi adolescent girls in Riyadh, Saudi Arabia. Methods Our study evaluated 25(OH)D in 2000 school girls living in Saudi Arabia. This crosssectional study recruited girls ages 12-18 (mean age 16.13±1.77 years)in Riyadh city, KSA from September 2011 to June 2014. Fasting blood samples were obtained measuring serum25(OH)D, parathyroid hormone (PTH ) in addition to biomarkers of bone remodeling. A clinical diagnosis of osteomalacia was considered in subjects with 25(OH)D less than 25 nmol/l in the presence of bone pain , muscle weakness with elevated alkaline phosphatase , and normal or low serum calcium and phosphorous. Results Vitamin D deficiency (Serum 25(OH)D <25nmol/L) associated with bone pains , muscle weakness , and with elevations in serum alkaline phosphatase consistent with a clinical diagnosis of osteomalacia was documented in 2.3% of the entire cohort.Vitamin D deficiency was found to be more prevalent in the summer than in the winter period . Serum PTH showed a significant inverse association with 25(OH)D at levels below 40nmol/L (r = -0.21;p<0.001). Conclusions Vitamin D deficiency is a common health problem among Saudi adolescent girls. In a good number of girls, this can progress to osteomalacia. To counteract this deficiency, a national program should be designed to increase intake of vitamin D rich foods, sun exposure and supplementation of food with vitamin D. Acknowledgments The authors express their appreciation and gratitude to King Abadulaziz City for Science and Technology (KACST) for funding this study (AR-28-91). Key Words:Osteomalacia, Parathyroid hormone, Bone turnover, 25 (OH)D. 1 Introduction Osteomalacia is defined as reduced or incomplete mineralization of normal osteoid tissue following closure of growth plates 1,2. It results from inadequate exposure to sunlight, poor intake or reduced absorption of vitamin D. Manifestations include impaired bone mineralization and in severe cases hypocalcemia and/or hypophosphatemia. Vitamin D deficiency has re-emerged as a major health concern in the 21st century in both developed and developing countries 3,4 and remains the most common cause of osteomalacia 5,6. Vitamin D deficiency may also have extra-skeletal manifestations, which are being further evaluated in randomized clinical trials. Osteomalacia secondary to vitamin D deficiency is a common clinical problem encountered in the Saudi population, including Saudi adolescent females 7,8. The condition is associated with nonspecific generalized muscle and bone pain as well as gait abnormalities and may be misdiagnosed as fibromyalgia or arthritis. The presentation of vitamin D deficiency may range from an asymptomatic state to clinical osteomalacia. This study was undertaken to determine the frequency of clinical osteomalacia defined as 25OH)D level of ˂ 25nmol/l in the presence of an elevated alkaline phosphatase and with clinical features of muscle and bone pain in adolescent girls recruited in intermediate and secondary schools in the entire city of Riyadh, Saudi Arabia . Methodology In this cross- sectional study, a total of 2000 school girls (aged 12-18 years) from intermediate and secondary schools were recruited during the period September 2011 to June 2014. The study was conducted in Riyadh city (latitude, 24.6º N). School girls from southern, northern, central and western parts of Riyadh City were enrolled using the stratified random technique. Proportional allocation method was used to determine the number of students recruited from each stratum. Within the stratum, one or more schools were selected randomly for recruitment. Informed consent from each student's parent or guardian was obtained through an official letter from the principal investigator and his team. A written assent was obtained from participants. All consenting subjects completed a generalized questionnaire, which included basic demographic data, medical history related to bone health, and lifestyle data. . Direct sun exposure to face and arms in minutes per day during weekdays and weekend in the last week was recorded. Dietary intake with emphasis on dietary vitamin D and calcium using the 7-day semi quantitative food frequency questionnaires were adapted for use from previous local studies. 9,10. The questionnaire consisted of Saudi food list, definition of portion sizes , and assignment of frequency of consumption with special emphasis on food items with calcium – in dairy or non-dairy products- or foods fortified with vitamin D . A specific section of the questionnaire was used to record vitamin D or calcium supplements intake. Subjects were asked if they had bone aches and pains. Pain was recorded as "present" or "absent" as perceived by the adolescent girl. Proximal muscle weakness was tested by abducting shoulders against resistance. Weakness was reported if muscle strength was 2 less than grade 4 using the medical research council (MRC) system 11. Waddling gait was reported if while walking on a straight line, there was waddling and exaggerated lumbar lordosis. Bowing of the legs was identified if there was (outward) bowing of the lower leg in relation to the thigh. None of the subjects were using calcium or vitamin D supplements. Anthropometrics were obtained by a general practitioner. Height in centimeters was obtained using a standard stadiometer and weight - without shoes – in kilograms was obtained. Definitions of normal weight, overweight, and obesity in this age group was according to expert committee recommendations regarding the prevention, assessment, and treatment of child and adolescent overweight and obesity 12. Fasting serum samples were obtained during winter months – December to February – and during summer months – June to end of August - . Blood samples were frozen at – 80 º C. Batches were analyzed within 3 days of receiving samples for the following: serum calcium, phosphorous, albumin, alkaline phosphatase, creatinine, 25(OH)D and PTH. Osteocalcin and carboxy- terminal telopeptides of crosslinks of type I collagen (CTX) were analyzed in 500 girls selected at random. Vitamin D inadequacy was defined based on the cut-off recommendations by the European Society for Clinical and Economic Aspects of Osteoporosis and Osteoarthritis (ESCEO)and the Institute of Medicine (IOM) of serum 25(OH)D below 50nmol/l. 13,14. Osteomalacia was clinically diagnosed in subjects with 25(OH)D deficiency of<25nmol/l) in the presence of bone pain and muscle weakness as well as the following biochemical features 15 :elevations in serum alkaline phosphatas,normal or low serum calcium corrected for albumin,andormal or low serum phosphorous level. Biochemical assays :The serum levels of calcium, phosphorous, and alkaline phosphatase were determined using Dimension Xpand Plus autoanalyzer 9Siemens Healthcare Diagnostics). Serum 25(OH)D was measured using the electrochemiluminescence binding assay using Roche (Cobas e 411 analyzer) .All measurements were performed in A RIQAS – Randox International Quality Assessment Scheme- participating laboratory of King Khalid University Hospital. The intra assay coefficient variation (cv) was 2.2-6.8 while the inter assay cv was 3.4-13 %. Serum PTH was measured using elecrochemiluminescence assay (Roche-modular E-170). Bone profile and creatinine were measured using the colorimetric method (Dade Boehring Dimension RxL Max). CTX and osteocalcin were determined using electrchemiluminescence immunoassay by the Roche automated system. Statistical analysis : SPSS version 21 (SPSS Inc., Chicago, IL, USA) was used for data analysis. Continuous data were presented as means, medians , and standard deviation while frequencies were presented as percentages (%). Independent T-test was used to compare differences of normally distributed variables and Mann-Whitney for nonGaussian variables. Chi square test was used to test association between categorical variables. Person coefficient correlation (R) was used to test correlation between two continuous variables. Ascatter graph using the linear model was used to display the correlation. Significance was set at p=0.05. Results Table 1 highlights the demographic characteristics of the subjects. Majority of the adolescents recruited were in secondary education (62.6%). Based on daily sun exposure, 3 face, and arms, more than half of the subjects (55.8%) acknowledged that they expose themselves to sunlight for less than 10 minutes daily, and only 29.5% expose themselves for more than 20 minutes. The overall prevalence of obesity (weight of 95 th percentile or greater) was 5%. Approximately 2 out of 5 subjects (43.5%) reported bone pain and almost 1 out of 5 subjects (17.8%) reported pain on walking. 40.8% of the girls had 25(OH)D levels ˂ 25 nmol/l in winter , while it was in 63% in summer. Clinical osteomalacia was identified in 2.3 % of the entire cohort based on the criteria outlined above. However , there were 69 girls (4.5 %) who had biochemical features of 25(OH)D ˂ 25 nmol/l with elevated alkaline phosphatise , but with no clinical symptoms or signs suggestive of osteomalacia such as bony pain or muscle weakness. Fresh milk intake was poor in the present cohort, with 55.5% consuming less than one cup daily. In contrast, 77.4% of the subjects consumed 1-6 cups of carbonated drinks on a daily basis (Table 1).Cream was consumed by 8.6% daily and 16.9 at least once weekly. More than one third of the girls did not use it (37.5%). White cheese consumption followed the same pattern where 18.1% used it daily and 16.3% used it at least once weekly. Full cream yoghurt was used daily by one eighth of the studied girls (12.9%), but only 5.2% used half cream yoghurt daily. Around one third of the sample (33.8%) did not consume full cream yoghurt and around one half (51.1%) did not consume half cream one. 31.7% of the girls ate canned fish rarely. One fifth of them (19.7%) ate canned fish 1 -3 times per week, 11.2% ate it daily and 14.9% ate it every once every two weeks. Eggs were consumed by 7.9% daily, 17.5% 1-3 times per week, and 8.8% once every two weeks. Around one fourth of the sample did not consume eggs at all. Daily calcium intake was 691.1 mg in the vitamin D deficient girls - 25(OH)D ˂ 25 nmol/l – while it was 1358.89 mg daily in girls with normal 25(OH)D levels ( p < 0.001). Table 2 shows the demographics of subjects with a mean age of 16.13±1.77 years; mean age of menarche was 12.76±1.2 years. Differences in mean biochemical parameters based on season were also presented in table 2. Circulating 25(OH)D levels were significantly higher during the winter season – December to end of February - (p<0.001) as well a significantly higher serum calcium level was observed in the winter months than in the summer months – June to end of August - (p<0.001) . Of note, serum ALP and PTH were also significantly lower in the winter group as opposed to the summer group (Table 2). The mean serum calcium level in winter was 2.29 ± 0.19 mmol/l while summer it was 2.06 ± 0.74 mmol/l. There was negative correlation between serum calcium and alkaline phosphatse (r=-0.139, p< 0.01). The correlation between 25(OH)D and alkaline phosphatase was) r=0.051),p >0.05. Table 3 shows the vitamin D status of subjects stratified and compared according to different risk factors. Vitamin D deficiency was most prevalent among subjects with no sun exposure (40.3%) and those with minimal intake of fresh milk (56.7%). Furthermore, there is a significant difference in the prevalence of vitamin D deficiency in relation to carbonated drinks consumption, with those consuming more having a higher percentage of vitamin D deficiency than those who consume less than once per day (p<0.001). It may be possible that this is due to lower milk intake in those with a high intake of carbonated drinks. With regards to the weight, vitamin D deficiency was more prevalent among those with normal weight (80.8%) than (5.7%) in obese subjects. The prevalence 4 of 25(OH)D deficiency was higher among those with normal circulating osteocalcin than those with elevated levels. The prevalence of 25(OH)D deficiency is significantly higher in the summer than winter season (p<0.001). Figure 1 shows that there is a negative correlation between low 25(OH)D level in general and PTH levels ( r=0.024) but the correlation was not significant (p=0.351). The figure shows that at levels of 40 nmol/l, the PTH starts to increase. At the level of 40 nmol/l the negative correlation was r=0.15 and it was statistically significant (p˂0.001). Discussion The present study documented a high prevalence of vitamin D deficiency among Saudi adolescent girls. This confirms many previous reports indicating a strikingly high prevalence of vitamin D deficiency in the different age groups and in both sexes of the Saudi population 16-18. The degree of sun exposure was generally minimal in the majority of the girls and it is difficult to know the time of day that the sun exposure occurred. Other investigators have previously suggested a certain duration of sun exposure (including arms and legs for a period of 5 to 30 minutes) considering differences in the intensity of the sunlight exposure as well as in skin pigmentation. [21]. The UVB necessary for cutaneous conversion of 7-dehadocholesterol to pre-vitamin D3 is 290-315 nm. The difference in the vitamin D levels between summer and winter was striking and is the opposite of what has been reported from countries at higher latitudes, which found higher vitamin D levels during summer months, when people are more likely to be outdoors and obtaining increased sunlight exposure 19-22. A similar observation to ours was made by Al-Daghri and his group 23 which suggests that although sun exposure in Middle Eastern and Gulf States may be minimal, an increase may occur in winter when people may be more outdoors, and this could raise vitamin D to modest levels. It is to be noted that in relation to sun exposure, the particular timing of exposure was found to be of importance. Alshahrani et al found that the optimum time for vitamin D skin formation in people living in the Riyadh area during summer is from 9:00Am to 10: 30Am and from 2:003:00pm. In winter, it was suggested that the optimal time for sun exposure is from 10:00am-2:00pm 24. With regards to the seasonal variation, the Canadian Health Measures Survey (CHMS) examined 25(OH) vitamin D levels in a representative sample of the Canadian population, and, in 456 girls aged 12-19 years, the mean 25(OH) vitamin D ranged from 60.8 nmol/l (winter, no supplement use) to 76.8 nmol/l (summer, no supplement use) 25.The IOM report suggests osteomalacia and rickets are most likely to be associated with 25(OH)D levels below 30 nmol/l [14]; and in CHMS, this was found in 3.8% of Caucasian boys and girls and 17.5% of “non-Caucasian” adolescents aged 12-19 years (non-Caucasian includes people of Chinese, South Asian, African, Filipino, Latin American, Southeast Asian, Arab, West Asian, Japanese, Korean, Aboriginal, and other racial backgrounds). An earlier study of 1753 Québec schoolchildren (871 girls) aged 9, 13, and 16 years, found a larger proportion had low vitamin D status, and in particular, 5 2%, 8%, and 10% of 9-, 13, and 16-year-old girls had frank vitamin D deficiency (defined as a serum 25(OH)D ≤ 27.5nmol/l) 26. The nutritional status of the girls, in relation to vitamin D deficiency and osteomalacia, has revealed that a small number of the girls consume sufficient milk or other dairy products. This appears to be cultural or related to the currently prevailing dietary habits of girls in this age group. This low dairy intake was coupled with a relatively high intake of soft drinks. Worthy to note is that milk in Saudi Arabia is fortified with vitamin D – 400 iu/liter of milk. This could further exacerbate a pre-existing vitamin D insufficient state possibly present from infancy. Our study revealed a significant proportion of subjects (2.3%) had vitamin D deficiency in the presence of elevations in alkaline phosphatase consistent with a clinical diagnosis of osteomalacia. It is evident that our cohort had variable degrees of vitamin D insufficiency, with evidence of secondary hyperparathyroidism observed in approximately10% of subjects. A significant percentage of these individuals may have had elevations in serum PTH within the normal reference range. 27. Ardawi et al found no threshold of serum 25(OH)D at which PTH levels plateaued in Saudi men 18. However, secondary hyperparathyroidism was evident in 18.5 % and 24.6 % in pre and postmenopausal women with 25(OH)D below 50 nmol/l 17. In our study, the negative correlation between serum vitamin D levels and PTH appeared at around 40nmol/l. This is lower than in some other studies. However similar observations have been documented before. In Chinese premenopausal women, a serum vitamin D level of 40nmol/l was found to be the optimum level to "suppress" PTH 28. In an older study by Lips, a level of 30 nmol/l was enough to suppress PTH level in elderly subjects 29. Interestingly, in Finnish adolescent girls, it was observed also that at levels of 25(OH)D of 40 nmol/l or above, PTH would start to be suppressed 30. This observation in our girls – in contrast to some other studies-may be a reflection of elevations in serum PTH within the normal reference range. It is also evident that many girls were having variable degrees of hypocalcemia in summer, which could contribute to the clinical observation of osteomalcia. It is also possible- although unlikely- that magnesium deficiency may be present in our study population , resulting in a blunted rise in PTH due to low intracellular magnesium31,32. Intracellular reductions in magnesium can result in a paradoxical block of PTH secretion 33,34 .We did not measure serum magnesium in our study population and this requires further evaluation. However, hypomagnesaemia is generally rare, except in severely malnourished elderly individuals or critically ill patients or those with poor gastrointestinal absorption. It is also possible that our young subjects displayed a different PTH response in comparison to the elderly population. This requires further study in well designed randomized controlled trials. Ethnic differences have been noted in the response of PTH and BTMs to vitamin D deficiency. Aloia et al found that in African American women, the PTH does not decline rapidly when serum concentrations of 25(OH)D are more than 40nmol/l 35. Dawson-Hughes et al emphasized the great debate in this subject,but suggested that the optimum vitamin D level that appears to suppress PTH levels ranges from 30-99 nmol/l with a cluster in the 75-80 nmol/l range36. A recent meta-analysis by Bjokrman suggested that responses of PTH to vitamin D supplementation are not only 6 determined by the baseline PTH levels and changes in vitamin D status, but also by age and mobility of the patients 37. In our population, and particularly in this age group, it would seem that levels of 40-50nmol/l may be considered sufficient to suppress PTH. Some studies failed to showa specific correlation between vitamin D and PTH 38,39.Investigators reporting this "lack of association” claimed that PTH secretion is primarily regulated by extracellular calcium concentrations. Individuals with a blunted PTH response have shown a lower serum calcium concentration, a reduction in bone turnover and protection of bone density in comparison with those who have hypovitaminosis D and secondary hyperparathyroidism. In a study by Barnes et al, there was no significant effect of vitamin Dsupplementation on bone turnover markers or PTH concentrations 40. In short, it would seem that this lack of consistency in the relationship between 25(OH)D levels and PTH could be related to important confounding factors including age – especially during puberty -,gender, pre and postmenopausal states, degree of mobility, status of calcium intake , and ethnic background. The authors acknowledge these limitations. Boys were not included , as the study concentrated on females who are at a higher risk for vitamin D deficiency. The diagnosis of osteomalacia was made clinically and was not confirmed by bone biopsy. Nevertheless, the present study is the first large scale observation to document the prevalence of vitamin D inadequacy and clinical osteomalacia amongst Arab adolescent girls in the region and raises the clinical importance of vitamin D deficiency in this under- studied population. In summary, a high prevalence of vitamin D deficiency amongst Saudi adolescent girls was noted , with a greater frequency of this condition in the summer months than in the winter months. This may be a reflection of the severe heat in the summer resulting in girls avoiding the outdoors as much as possible. Subjects with vitamin D deficiency had lower calcium intake, higher intakes of soft drinks , and had minimum sun exposure. Vitamin D deficiency was accompanied with subnormal calcium levels and elevated PTH levels. PTH appears to plateau around 25(OH)D levels of 40nmol/l. A biochemical diagnosis of osteomalacia was evident in a significant number of Saudi adolescent girls. Serious attempts should be undertaken to address this serious health care issue and its consequences in this population. Girls should be encouraged to obtain sun exposure in schools and homes, even for short time. Morefood fortification with vitamin D for different dairy products, cereals and flouris needed in Saudi Arabia. Screening for vitamin D deficiency is necessary in this age group in order to prevent rickets and osteomalacia. References 1. Shoback D, Marcus R, Bikle D . Metabolic bone disease in basic and clinical endocrinology. Greenspan FH, Gardner DG Eds. 7th edition. 2004; Lange Medical Publishing, New York. pp 295-361 7 2. Francis RM, Selby PL. Osteomalacia. Baillieres Clin Endocrinol Metab.1997;11: 145-160. 3. Plehwe WE. (Vitamin D deficiency in the 21st century: an unnecessary pandemic? Clin Endo .2003; 59 (1):22-4. 4. Ward LM; Vitamin D deficiency in the 21st century: a persistent problem among Canadian infants and mothers. CMAJ.2005;172(6):769-70. 5. Holick MF. 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Medical Research Council Aids to the examination of the peripheral nervous system, 1981; Memorandum NO. 45, Her Majesty's stationary office,London. 12. Barlow SE and the Expert Committee. Expert committee recommendations regarding the prevention, assessment, and treatment of child and adolescent overweight and obesity: summary report. Pediatrics 2007; 120 Supplement December 2007:S164— S192. 13. Rizzoli R, Boonen S, Brandi M L, Bruyere O.Vitamin D supplementation in elderly or postmenopausal women: a 2013 update of the 2008 recommendations from the European Society for clinical and economic aspects of osteoporosis and osteoarthritis (ESCEO). Curr Med Res Opin 2013;29(4):305-13 14. IOM (Institute of Medicine) dietary reference intakes for calcium and vitamin D. Washington DC: The National Academic Press.2011. 15. Thatcher TD, Clarke BL.Vitamin D insufficiency. Mayo Clin Proc.2011;86(1):50-6 8 16. 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Vitamin D insufficiency and the blunted PTH response in established osteoporosis: the role of magnesium deficiency.Osteoporos Int 2006;17(7):1013-21. 32. Steen O, Khan A. Role of magnesium in parathyroid physiology.2015 In Maria Luisa Bradi ML . Brown EM editors .Springer-Veralg Italia.2015; pp 61-68 33. Quittere U, Hoffmann M, Freichel, Lohse J.Paradoxical block of parathyroid is mediated by increased activity of Ga subunits. J Biol chem.2015; 276 (9) 6763-6739 34. Mori S, Harada S , Okazaki R , Inone D , Matumoto T , Oqata E. Hypomagnesemia with increased metabolism of parathyroid hormone and reduced responsiveness to calcitropic hormones. Intern med.1992; 31(6), 820-24 35. Aloia JF, Talwar SA, Pollack S, Feuerman M, Yeh JK .Optimal vitamin D status and serum parathyroid hormone concentrations in African American women. Am J. Clin Nutr 2006 ;84(3):602-9. 36. Dawson-Hughes B, Heaney RP, Holick MF, Lips P, Meunier PJ, Vieth R.Estimates of optimal vitamin D status.Osteoporos Int.2005;16(7):713-6. 37. Björkman M1, Sorva A, Tilvis R. Responses of parathyroid hormone to vitamin D supplementation: a systematic review of clinical trials. Arch Gerontol Geriatr.2009;48(2):160-6. 38. Reinehr T, Desousa G, Alexy U, Kresting M. Vitamin D status and parathyroid hormone in obese children before and after weight loss. Eur J Endocrinol.2007;157:225-32. 39. Singhellakis P, Malandinon FC, Parron CJ, Dannelli AM .Vitamin D deficiency in white, apparently healthy free-living adults in temperate region. Hormons.2011; ( Athens )10 (2 ) :131-143 40. Barnes MS, Robson PJ, Bonham MP, Strain JJ, Wallace JM. Effect of vitamin d supplementation on vitamin D status and bone turnover markers in young adults. Eur J.Clin Nutr 2006; 60 ( 6) : 727-33 10 Table 1. General Characteristics of Subjects included in the study Parameter N Education N (%) 2000 11 Intermediate Secondary Daily Sun Exposure <10minutes 11-20 minutes >20 minutes Physical Inactivity (%) Obesity (%) Bone Pain (%) Pain on walking (%) Osteomalacia Daily Fresh Full Cream Milk Intake <1 Cup 1-3 Cups 4-6 Cups Daily Intake of Carbonated Drinks <1 Cup 1-3 Cups 4-6 Cups Note: Data presented as frequency (%) 748 (37.4) 1252 (62.6) 1116 (55.8) 294 (14.7) 590 (29.5) 1124 (50.6) 75(5.0) 870 (43.5) 356 (17.8) 33(2.3%) 1110 (55.5) 568 (28.4) 322 (16.1) 452 (22.6) 876 (43.8) 672 (33.6) 12 Table 2. Demographics and Differences in Biochemical Profile of Subjects According to Season Parameter Mean ± SD Age (years) 16.13 ± 1.77 Age at menarche (years) 12.76 ± 1.2 N 25 (OH)D ( 50-125 nmol/l) Calcium mmol/l ( 2.1 -2.5 Winter Summer 1529 471 30.6 (3.5-155.0) 21.3 (7.5-131.5)** 2.29 ± 0.19 2.06 ± 0.74** 47.15 ± 10.69 48.16 ± 20.59 98.21±52.45 130.11±100.67** 5.06±5.57 5.98±5.55* 40.29 ± 21.34 38.53 ± 28.15 0.37 ± 0.24 0.37 ± 0.26 mmol/l) Creatinine µmol/l ( 50-110 µmol/l) ALP u/l (35-100 u /l ) PTH pmol/l (1.3-6.8pmol/l) Osteocalcin ng/ml (11.0-43.0 ng/ml) CTX µg/ml (0.10-5.94 ng/ml) Note: #denotes median (minimum-maximum); 13 *denotes p<0.05; **denotes p<0.01 Table 3 Prevalence (%) of Vitamin D Deficiency, Insufficiency and Sufficiency According to risk factors Deficiency Insufficiency Sufficiency Daily Sun Exposure 0 minutes 260 (40.3 ) 82 (15.0) 41 (9.6a 1-10minutes 199 (30.9) 240 (44.0) 81 (19.0) 11-20 minutes 87 (13.5) 107 (19.6) 44 (10.3) 21-30 minutes 60 (9.3) 65 (11.9) 238 (55.7) >30 minutes 39 (6.0) 52 (9.5) 23 (5.4) Daily Fresh Full Cream Milk Intake <1 Cup 251 (56.7) 265 (54.9) 76 (53.9b ) 1-3 Cups 113 (25.5) 138 (28.6) 52 (36.9) 4-6 Cups 79 (17.8) 80 (16.5) 13 (9.2) Daily Intake of Carbonated Drinks <1 Cup 85 (15.5) 164 (27.8) 48 (26.5c ) 1-3 Cups 250 (45.7) 257 (43.6) 70 (38.7) 4-6 Cups 212 (38.8) 168 (28.5) 63 (34.8) Weight Underweight 21(3.4) 23(3.4) 5(2.4 )ͩ Normal 497(80.8) 553(82.2) 176(85.4) Overweight 62(10.1) 62(9.2) 144(9.6) Obese 35(5.7) 35(5.2) 5(2.4) 270 (40.2) 324 (48.3) 77 (11.5f ) Presence of Bone Pain Osteocalcin Elevated 190 (33.5) 189 (28.5) 67 (31.9g ) Normal 377 (66.5) 473 (71.5) 68.1 Season Summer 317 (63.5) 139 (27.9) 43 (8.6h ) Winter 660 (40.8) 460 (28.4) 498 (30.8) a b c d e f g h p<0.001; p=0.038; p <0.001; p=0.6; p<0.001; p=0.05; p=0.16; p<0.001 14 Figure 1Inversecorrelation between 25(OH)D and PTH (R-0.024; p=0.351) 15