1. Dr Aysha Habib Khan Assistant Professor & Consultant Chemical Pathologist Department of Pathology & Microbiology and Medicine Aga Khan University, Karachi Vitamin D Deficiency: Insight from Local Experience aysha.habib@aku.edu

2. Introduction: Vitamin D Fat soluble hormone Major forms are Vitamin D2 (ergocalciferol) and D3 (cholecalciferol) Receptors have been identified for >30 tissues Function of the immune, reproductive, muscular, skeletal and integumentary system VDR best characterized on intestine, kidney and bone Critical for maintenance of bone health

3. Metabolism of Vitamin D

4. Functions of Vitamin D Calcitropic Functions/Classic target tissues INTESTINE Absorption of Ca, Mg and P BONE Direct effectIndirect effect I ncrease osteoclast activity, remove Ca & P from the bone KIDNEY Enhance the actions of PTH on Ca & P transport Mineralization of skeleton

5. Synthesis of 1,25(OH) 2 D in various cells (local production) via 1-hydroxylase enzyme that acts on the VDR Non Calcitropic Functions/Non-classical target tissues Functions of Vitamin D MUSCLE Increase cathelicidin Act locally on activated T & B lymphocytes Regulation of genes that controls proliferation, inhibit angiogenesis, induces differentiation & apoptosis Increased insulin ↑ protein synthesis via VDR, ↑ no. and size of type 2 muscle fibers IMMUNE FUNCTION CANCER CELLS ISLET CELLCVS DISEASE Decreased renin

6. Health Effects of Vitamin D & Calcium Intake: Institute of Medicine; Report Brief November 2010 Information about the health benefits beyond bone, were from studies that provided often mixed and inconclusive results and could not be considered reliable Evidence supports importance of Vitamin D in promoting bone health www.iom.edu/vitamind Health Outcomes Cancer CVD Hypertension Diabetes Metabolic syndrome Falls Immune response Neuropsychological functioning Physical performance Preeclampsia Reproductive outcome

7. What should be the optimal levels of VD? Potential Disease-Specific Biomarkers of Vitamin D Sufficiency Parathyroid Hormone (PTH) Calcium absorption Bone Mineral Density (BMD) Definition of Vitamin D Status At 20 ng/ml: PTH sub-normal Calcium absorption 65% less At 30 ng/ml PTH starts to rise Ca absorption is 65% greater then at 20 ng/ml BMD values are high

8. Serum Levels of 25 hydroxy vitamin D 10 20 30 40 50 “deficiency” “Insufficiency” “ Normal” ng/ml Hollick MF. NEJM. 2007; 266-280. Boonen S et al. Osteoporosis Int. 2004;15:511-519. Lips P. Endocr Rev. 2001;22:477-501. Heaney RP. Osteoporosi Int. 2000;11:553-555. Heaney RP. Am J Clin Nutr. 2004; 80 (suppl):1706S-1709S. Thomas MK. NEJM. 1998;388:777-781.

9. Vitamin D deficiency (25OHD) Increase Parathyroid Hormone Decreased 1,25(OH) 2 D Decreased absorption of intestinal calcium Increase 1-hydroxylase - increase1,25(OH) 2 D Increase mobilization of bone calcium Increase excretion of phosphates - hypophosphatemia Decreases bioavailability of calcium

10. Vitamin D deficiency osteomalacia Osteomalacia means “soft bones” Failure of mineralization of remodeled bone Bone pains in arms, legs, spine, and pelvis, with actual tenderness of the bones Progressive weakness Muscle weakness Waddling gait & muscle cramps High incidence of fracture than expected for age

11. Causes of vitamin D deficiency Primary 25OHD Deficiency: Inadequate sunlight exposure Low dietary intake Secondary 25OHD Deficiency Fat malabsorption Liver diseases Kidney disease Inherited conditions Type 1: Abnormal or absent 1-hydroxylase enzyme Type II: End-organ resistance

12. Main determinants of Vitamin D deficiency Poor intake Lack of sunlight exposure

13. Food International Units (IU) per serving Percent DV* Cod liver oil, 1 Tablespoon1,360340 Salmon, cooked, 3½ ounces36090 Mackerel, cooked, 3½ ounces34590 Tuna fish, canned in oil, 3 ounces20050 Sardines, canned in oil, drained, 1¾ ounces25070 Milk, nonfat, reduced fat, and whole, vitamin D fortified, 1 cup9825 Margarine, fortified, 1 Tablespoon6015 Pudding, prepared from mix and made with vitamin D fortified milk, ½ cup 5010 Ready-to-eat cereals fortified with 10% of the DV for vitamin D, ¾ cup to 1 cup servings (servings vary according to the brand) 4010 Egg, 1 whole (vitamin D is found in egg yolk)206 Liver, beef, cooked, 3½ ounces154 Cheese, Swiss, 1 ounce124 Food Sources of Vitamin D

14. Vitamin D Intake: A Global Perspective of Current Status Current food supply, dietary patterns & supplementation practices inadequate In high risk groups, supplementation Food fortification Mona S C et al: Jn. Nutrition March 2007

15. Entire need for vitamin D can be met by adequate exposure to sunlight The body does not overproduce vitamin D as prolonged exposure produces inactive metabolites In the absence of adequate sun exposure the body depends on dietary supply for vitamin D Daily need of Vitamin D

16. Recommendations for Sunlight Exposure Exposure of arms & legs for 5-30 min (depending on time of day, season, latitude & skin pigmentation) between 10am -3pm twice a week is often adequate With longer exposure to UVB rays, an equilibrium is achieved in the skin, and the vitamin simply degrades as fast as it is generated.

17. Factors affecting Vitamin D production on skin Factors affecting the transmission of solar UVB radiation to the earth’s surface Season Geographic latitude Time of day Cloud /fog Window glass Factors affecting the penetration of UVB radiation into the skin Individuals with higher skin melanin content Sun screen Ageing skin Excess skin cover Indoor life style

18. Risk Factors Dark skin, Children and those aged over 65 years Pregnancy Routine covering of face and body, e.g. wearing a veil Infant who has prolonged breastfeeding without vitamin D supplementation, especially if the mother is vitamin D deficient Housebound or institutionalised Living in countries at high latitude Chronic disease, e.g. malabsorption, renal or liver disease

19. Variability in individual 25OHD responses to supplementation Retested after 3 months of supplementation to confirm if target levels are achieved. Estimated average vitamin D requirement to reach level of 30ng/ml is 800 to 1,000 IU A lower intake may be adequate for individuals with regular effective sun exposure With risk factors: like Obese, osteoporotic, etc.: 2,000 IU/d – measuring 25OHD is recommended IOF position statement: vitamin D recommendations for adults (2010)

20. Vitamin D Deficiency: Insight from Local Experience Issues & Challenges (2002 – 2005) VDD manifesting in its extreme shape Management issues Lack of solid epidemiological data International recommendations & guidelines regarding desirable doses and levels may not readily apply to populations from the region Lack of funding

21. Research Questions Is it Assay? What is the status in our population What are the determinants of VDD? What is the optimal level for our population?

22. Is It Assay? Methodology reviews and validation studies Samples to external lab Participation in external quality control

23. High Prevalence of VDD in Out-Patients at AKUH 25OHD data May 2002 – December 2004 95% had VDD Low serum Ca and elevated AP were reflective of severe deficiency Elevated iPTH correlated with mild to moderate deficiency Serum Ca, P and AP are poor markers of moderate to mild deficiency, and cannot be relied upon as a screening tool Serum 25OHD and iPTH are better markers. Lubna M Z, Aysha HK et al: Vitamin D Deficiency in Ambulatory Patients: JPMA, 2008

24. Healthy Volunteers (n=123, 43% females; 57% males) January 2006-December 2007 URC funded Thirty eight participants (30.89%), have raised PTH (mean 107 ±18.04 pg/ml). Negative correlation between serum iPTH and Vitamin D levels (P=<0.001, r=0.3). Shireen M, et al. Prevalence and significance of vitamin D deficiency in apparently healthy adult volunteers in Karachi Pakistan. Clinical Biochemistry 2010

25. Healthy Premenopausal Female Volunteers (n= 174) (2007 – 2010, URC funded) Farhan Dar, Khan AH et al 92.8% of the females were identified as D deficient, 6.1% had insufficient levels 1.1% had optimal levels. High NTx 36.8% sHPTH 25.9% volunteers

26. Two main determinants: nutrient intake sunlight exposure Issues: Lack of tools for assessment of nutrient intake and sunlight exposure Determinants of VDD

27. Development and validation of a food frequency questionnaire for assessing macronutrient and calcium intake in women residing in Karachi, Pakistan (2006 – 2010) Romaina I, Khan AH et al 24 h dietary recall data at phlebotomy centers 4 dietary recalls List of food items Development of food composition table (dietary intake could be converted into nutrient estimates). Validated against NTx

28. VariablesFFQMean of 4 24h recalls MeanSDMeanSD Calcium610.7306.4462.1175.7 Mean daily Calcium intake estimated by FFQ and 24 h recalls

29. Development and Validation of Sunlight Exposure Measurement Questionnaire (SEM-Q) for use in adult population residing in Pakistan ( December 2009 to April 2010) Quratulain Humayun, Romaina Iqbal, Khan AH et al

30. Biochemical ParametersStatusFrequency (Percentage)Mean Ca (mg/dl) Low Normal High 18 (5.9 %) 285 (93.4 %) 2 (0.7 %) 7.8 9.06 10.4 Vitamin D (ng/ml) Deficient Insufficient Sufficient 276 (90.5 %) 16 (5.2 %) 13 (4.3 %) 6.36 25.05 38.73 iPTH (pg/ml) Low Normal High 2 (0.7 %) 173 (56.7 %) 130 (42.6 %) 10.26 58.45 143.39 NTx (nMBCE/L) Low Normal High 11 (3.6 %) 223 (73.1 %) 71 (23.3 %) 4.07 13.23 24.20 Bone Health Status in Healthy Premenopausal Community Dwelling Females in Karachi (n=300) 25 th July 2008 – December 2011, PSF Funded) IOF Young Investigator Award at Dubai

31. Comparison of Bone Health Status in Healthy Premenopausal Community Dwelling Females in 3 Towns of Karachi (n=300) 31 AH. Khan, G. Naureen, F. Dar, R. Iqbal

32. Nutrient Intake Per Day Total Calcium (mg) 686.2 ± 271.4 Sunlight Exposure < 30 Minutes 30 - 60 Minutes >60 Minutes 53.1 % 32.1 % 14.8 % Calcium Intake and SE of Community Dwelling Premenopausal Females In Karachi

33. Association of Housing Structure with Biochemical Parameters in Community Dwelling Premenopausal Females Residing in Karachi

34. Baseline serum 25OHD, Sr, Ca, P and iPTH were determined in 53 fasting volunteers, Followed by administrating (PO) 4.8 mg SrCl 2 /kg and Collecting blood at 0.5, 1and 4h to determine the absorption (AUC 0→t ) of Sr. Following the initial absorption test, volunteers received a single IM injection of 600,000 IU vitamin D 3. Two months later, the fasting serum and the Sr absorption test were repeated, as described above. Assessment of Calcium Absorption in Adult Pakistani Population before and after Vitamin D Administration Using Strontium as Surrogate Khan AH, Rohra D, Saghir S, Udani S, Wood R, Jabbar A

35. Average serum strontium (Sr) concentration-time curve and area under the time-course Sr concentration curve (AUC 0→4h ) in healthy Pakistani population following PO dosing of strontium chloride before and after the IM injection of Cholecalciferol (600,000IU) showing no impact of high serum 250HD level on the absorption of Sr (a surrogate of Ca absorption) from the GI tract Aysha HK, Dileep R, Sakil S, et al

36. Effects of intramuscular vitamin D supplementaion on biochemical parameter (mean  sd) of the study participants before and after the intramuscular injection of Cholecalciferol (600,000 IU) Variable Before (n = 53) After (n = 32) p-value Calcium (mg/dl)9.2±0.59.1±0.40.828 Phosphorus (mg/dl)3.9±0.7 0.358 25OHD (ng/ml)17.2±7.826.5±7.5<0.0001 iPTH (pg/ml)65.7±51.5 53.0  31.0 0.110 AUC 0→t (µmol h L -1 )206±193185±1890.308

37. Conclusion: A single vitamin D 3 injection of 600,000 IU significantly increase mean 25OHD concentration and tended to lower iPTH concentrations in subjects with initially low 25OHD status, suggesting to utilize this simple form of treatment to improve vitamin D status and to have a possible biological effect on Ca homeostasis. However, we found no obvious effect on Sr absorption, suggesting the possibility that maximal vitamin D-dependent Ca absorption had already been achieved in these subjects at a lower vitamin D status. Limitations: Ca intake was not assessed 1,25(OH) 2 D was not measured

38. Response of 25OHD (nmol/l) and iPTH levels (pg/ml) to 600,000 IU of injection cholecalciferol in healthy volunteers in Karachi, Pakistan (n=20) After 8 weeks of injection vitamin D, 25 OHD levels became optimal in 6 (35%) volunteers (mean 92.9±16.6 nmol/l). It remained low in 5 (25%) volunteers (mean 41.6±9.6) while insufficient levels were seen in 9 (40%) volunteers (mean 63.3±5.8). Aysha HK, Dileep R, Sakil S, et al

39. Recommendation Life style Changes Sunlight exposure Diet Physical activity Supplementation: 800-1000IU of VD 1000 -1200 mg of calcium/day

40. Why is D deficiency so extensive? 1. Genetics 2. What is the optimum level for our population? 3. Are we unable to manufacture D efficiently from sunlight? 4. Are we breaking down active D more rapidly? 5. Is it a difference in expression of Vitamin D receptor and its signaling pathways?

42. To me this picture highlights the popular believe that relaxation is derived from nature and beauty. As u can see there is a side view mirror of a car in the picture which to me means that a family has come to this spot from an urban city (car = urbanisation) and is trying to escape the fast city life and chilling in a lonely area with only natural beauty to enjoywhich to In the midst of the life we have grown used to living in, chasing money and dreams this picture shows that huge buildings busy roads crazy crowd are not sufficient for humans and nature, Gods creations are as much an integral part of human life and are infact a part of their needs (even if they dont know about it)