Effect of Intramuscular Injection of Vitamin D on 25-Hydroxyvitamin D Levels, Glycaemic Control, and Liver Enzymes in Libyan Patients with Type 2 Diabetes Mellitus

Abstract

Background Vitamin D is a fat-soluble hormone that plays an important role in glycaemic control. In addition, it has a positive effect on improving liver enzyme function.

Aim This study was performed to examine the effect of intramuscular injection of vitamin D on serum 25-hydroxyvitamin D [25(OH)D] levels, glycemic control, and liver enzymes in Libyan patients suffering from type 2 diabetes mellitus (T2DM) with vitamin D deficiency.

Methods and Materials This cross-sectional study enrolled 100 T2DM (50 males and 50 females). Their serum 25(OH)D, fasting blood glucose (FBG), and liver enzymes were measured at the baseline and 12 weeks after treatment with vitamin D (200,000 IU) injection monthly for 3 months. Data analysis involved the estimation of mean ± standard error (SE) and comparison of means between pre and post-treatment values using paired t-test. Independent t-test was used to compare the means between males and females. The level of significance was set at p < 0.05.

Results Females had a lower 25(OH)D blood levels than males at baseline (7.03 ± 0.25 ng/mL versus 7.86 ± 0.26 ng/mL, respectively p < 0.02). 25(OH)D levels in both sexes was increased significantly from 7.45 ± 0.18 ng/mL to 26.69 ± 0.24 ng/mL after 12 weeks of vitamin D injections (p < 0.001), with no significant differences between male and females. FBG levels in both sexes was significantly decreased from 144.68 ± 1.84 mg/dL to 85.96 ± 0.34 mg/dL post treatment (p < 0.001). Alanine aminotransferase (ALT) was increased from 10.24 ± 0.17 U/L at baseline to 20.34 ± 1.15 U/L post treatment (p < 0.001). Similarly, aspartate aminotransferase (AST) was increased from 11.23 ± 0.21 to 20.57 ± 0.22 U/L (p < 0.001), and alkaline phosphatase (ALP) was decreased from 124.95 ± 1.15 U/L to 111.17 ± 1.27 U/L (p < 0.001). There were no significant differences between male and female liver enzymes either pre- or post-vitamin D injections

Conclusion Treatment with vitamin D injection showed a significant increase in 25(OH)D accompanied by decreased FBG and ALP levels and increased ALT and AST levels. Vitamin D levels should be monitored and adjusted in diabetic patients.

Keywords

25(OH)D - ALP - FBG - AST - ALT - vitamin D - Libya

Key Messages

Intramuscular injection of vitamin D for 3 months significantly increases serum 25(OH)D levels, approaching the sufficiency levels. Serum vitamin D levels are negatively related to FBG concentrations and ALP activity, and positively related to ALT and AST levels.

Introduction

Vitamin D is a fat-soluble multifunctional hormone that has a vast number of functions on bones and non-skeletal cells. Many studies demonstrated the expression of vitamin D receptor (VDR) in several cell types including pancreatic β cells.[1] [2] Vitamin D response element was detected in insulin genes[3] and it enhances the transcription of insulin receptor genes.[4] Moreover, evidence showed that the glycemic status is affected by the receptors of vitamin D located in the human liver and kidney.[5]

Previous cross-sectional and longitudinal observational studies reported the association between vitamin D status and risk of T2DM, and highlighted the role of vitamin D in modifying the risk of T2DM.[1] It is suggested that the positive effect of vitamin D on glucose adjustment occurs by the alteration of intracellular calcium levels, which leads to an increase in insulin sensitivity and insulin secretion.[5]

In addition, recent studies found an association between vitamin D deficiency and non-alcoholic fatty liver disease (NAFLD)[6] [7] [8]. NAFLD is considered as the most common cause of liver disease.[9] Sixty to seventy percent of patients with type 2 diabetes mellitus and greater than 34% of the population suffer from NAFLD.[10] [11]

Considering the high prevalence of vitamin D deficiency worldwide and its association with an increased risk of T2DM and NAFLD, and the lack of studies related to vitamin D deficiency in Libyan diabetics, the current study was performed to evaluate the effect of the intramuscular (IM) vitamin D supplements on glycemic control and liver enzymes in Libyan patients with T2DM and vitamin D deficiency.

Materials and Methods

The present study is a cross-sectional study performed from September to December 2019, at public health care centers in Tripoli, Ghadames, and Al-Zawiya in the western region of Libya. In total, 100 male and female patients, who came for regular checks, with a history of taking anti-diabetic medications and suffered from vitamin D deficiency, participated in the study. Each participant was informed about the study's nature, gave consent for participation and completed a self-reported questionnaire before the study was commenced. The study was approved by the Bioethics Committee of Libyan Center for Biotechnology Research, Tripoli, Libya, and conducted in accordance with the Helsinki Declaration.

Individuals' body weight and height was measured to calculate the body mass index (BMI), height was measured without shoes and while wearing light clothing in a standing position. BMI was calculated using the formula of weight (kg)/height (m)[2]. Other health-related factors including education level, smoking habit, physical activity, and place of residence were among the questions as well.

Study Design

At the beginning, baseline (pre-treatment) plasma concentrations for 25(OH)D, FBG and liver enzymes (ALT, AST, and ALP) of the participants were measured. Then, the participants under supervision of a medical doctor, received an IM dose of 200,000 IU/month of vitamin D (Cholecalciferol Pharma Developpement Chemin de Marcel, France) for 3 months, followed by the same measurements 1 month after the last dose of vitamin D injection. The inclusion criteria was as follows: (1) participants aged 25 to 62 years; (2) healthy individuals suffer only from T2DM for over 1 year; (3) unchanged anti-diabetic drugs. The exclusion criteria were as follows: (1) subjects with chronic diseases other than T2DM; (2) subjects with severe infections prior to the study; (3) patients using vitamin D or multi-mineral supplements over the past 3 months prior to the study; and (4) pregnancy or lactation.

Biochemical Parameters Measurement

Five mL of venous blood were collected by venepuncture from each subject after eight hours fasting, transferred into a white tube, incubated in a water bath at 37°C for 15 minute and centrifuged for 10 minutes at 3000 rpm at room temperature, then sera were separated and used to measure FBG, 25(OH)D levels, and liver enzymes including AST, ALT, and ALP. FBG was performed according to the manufacturer's instructions using a semi-automatic biochemistry analyzer Kenza Max BioChemistry, Biolabo Diagnostics, Kenza Biochemistry, France. Measurement of vitamin D was performed using ichroma II Boditech Med Incorporated, Gangwon-do, Korea. Ichroma vitamin D is a fluorescence immunoassay (FIA) for the quantitative determination of total 25(OH) D2/D3 levels in human serum/plasma. The activity of liver enzymes were measured using Biolabo SAS, Maizy kit, France.[12]

Statistical Analysis

All statistical analyses were performed using SPSS Version 22 statistical software package for Windows (SPSS Inc. Chicago, Illinois, USA). Descriptive analyses including number (N), percentage (%), mean ± standard error (SE), were performed to describe the general characteristics of the study population. Paired t-test was used to compare means between pre and post-treatment values. In addition, independent t-test was used to compare the means between males and females. P < 0.05 was considered statistically significant.

Results

Baseline Characteristics of Study Participants

The mean age of all participants was 45.6 ± 8.5 years with no significantly difference in age between males and females (p = 0.88). [Table 1] illustrates that the majority of the patients (82%) were overweight, and there was no significant difference between males and females in this regard (p = 0.67). The majority of the patients were married (79%), non-smokers (63%), and all smokers were males. Most of the participants do not practice any physical activity of any kind (84%). In addition, most of the patients had university education (87%), and 58% of them reside in Tripoli.

Table 1 Baseline characteristics of study participants

Variable	Participants
N	100
Gender (n [%])
Male	50 (50%)
Female	50 (50%)
Age (y)	45.6 ± 8.5
BMI (kg/m2)	26.1 ± 1.9
Marital status
Single	21
Married	79
Education level
Illiterate	13
High school	0
University and above	87
Smoking status
Yes	37
No	63
Physical activity
Yes	2
No	84
Sometimes	14
City of residence
Tripoli	58
Ghadames	40
Al-Zawiya	2

Biochemical Parameters

The biochemical parameters were measured at the baseline (pre-treatment values) and 1 month after the last dose of vitamin D injection (post-treatment values) ([Table 2] and [Table 3]). At baseline all participants had severe vitamin D deficiency (25(OH)D <10 ng/mL-Table 2). Comparing 25(OH)D baseline levels, females showed significant lower levels than males (p < 0.02; [Table 3]). 25(OH)D levels were increased significantly in both sexes 12 weeks after treatment (p < 0.001; [Table 2]), with no significant differences between males and females (p < 0.66; [Table 3]). Post-treatment values for FBG were decreased significantly in both sexes (p < 0.001; [Table 2]) with no significant differences between male and female either before or after vitamin D treatments. The post-treatment values for the liver enzymes AST and ALT were increased significantly from their pre-treatment values, while ALP values decreased significantly in both sexes after 12 weeks of treatments (p < 0.001; [Table 2]). However, there was no significant difference in liver enzymes measured between males and females neither pre nor post-vitamin D injection ([Table 3]).

Table 2 Comparison between the pre- and post-treatments values of 25(OH)D, FBG, and liver enzymes in all patients (n = 100)

Variable	Range		Mean ± SE		p-Value
	Pre-treatment	Post-treatment	Pre-treatment	Post-treatment
25(OH)D (ng/mL)	3.76–10	19.02–30.81	7.45 ± 1.82	26.69 ± 2.40	<0.001
FBG (mg/dL)	122–197	80.01–94.81	144.68 ± 18.38	85.96 ± 3.44	<0.001
AST (U/L)	7.99–19.78	13.84–26.08	11.23 ± 2.08	20.57 ± 2.18	<0.001
ALT (U/L)	5.14–17.14	13.99–33.89	10.24 ± 1.66	20.34 ± 3.66	<0.001
ALP (U/L)	95.99–180.21	58.07–162.01	124.95 ± 11.43	111.17 ± 12.17	<0.001

Table 3 Comparison between the pre- and post-treatments values of 25(OH)D, and liver enzymes between males and females (n = 50 patients each)

Variable	Males Mean ± SE	Females Mean ± SE	Independent t-test	p-Value
Pre-treatment 25(OH)D	7.86 ± 0.26	7.03 ± 0.25	1.97	0.02
Post-treatment 25(OH)D	26.79 ± 0.26	26.58 ± 0.36	1.98	0.66
Pre-treatment ALT	10.06 ± 0.22	10.43 ± 0.26	−1.10	0.27
Post-treatment ALT	19.69 ± 0.39	20.99 ± 0.61	1.79	0.08
Pre-treatment AST	10.94 ± 0.25	11.53 ± 0.29	−1.42	0.16
Post-treatment AST	20.50 ± 0.31	20.64 ± 0.32	10.32	0.75
Pre-treatment ALP	126.29 ± 1.67	123.63 ± 1.56	1.16	0.25
Post-treatment ALP	113.55 ± 1.61	108.79 ± 1.93	1.9	0.06

Discussion

Vitamin D deficiency is prevalent worldwide and its supplementation is simple, safe, and inexpensive.[13] [14] The recent renewed interest in vitamin D results from a worsening trend of worldwide deficiency as well as novel insights regarding its effects on glucose metabolism.[1] [15] [16] [17] [18] [19] Vitamin D deficiency was defined as a serum circulating 25(OH)D levels of <20 ng/mL based on the Endocrine Society Clinical Practice Guidelines.[20] Normal FBG was defined as blood glucose of < 110 mg/dL,[21] and elevated liver enzymes were defined as one or more measurement of AST (>34 U/L), ALT (>41 U/L),[22] and ALP (>115 U/L) in both genders.[23]

The diabetic patients included in this study had a significant vitamin D deficiency, (25(OH)D levels lower than 20 ng/mL), with female still have a significantly lower levels than males. These results are in agreement with many international reported results regarding vitamin D deficiency in T2DM.[19] [24] [25] [26] [27] [28] [29] Vitamin D deficiency was suggested to be associated with a decreased insulin release and insulin resistance.[28] [30] [31] [32] The relationship between vitamin D deficiency and insulin resistance could develop through inflammation, as vitamin D deficiency was associated with increased inflammatory markers.[33] In addition, genetic polymorphisms of vitamin D-related genes may predispose to impaired glycemic control and T2DM.[34] However, these speculations about the mechanism responsible for the development of T2DM in vitamin D deficiency should be proven by randomized clinical trials on a large number of patients.

The administration of vitamin D to our patients resulted in a statistically significant increase in 25(OH)D levels after 12 weeks of treatment. This finding nicely agreed with previously reported results,[24] [25] [26] suggesting that the treatment protocol is successfully effective. The increase in the levels of 25(OH)D was accompanied by a significant decrease in FBG concentration, returning the FBG to the normal accepted range. Similar results were reported in a meta-analysis of Iranian diabetics.[35] However, high doses of vitamin D failed to produce any improvements in the glycemic indices in prediabetes individuals with impaired fasting glucose.[36] The decrease in FBG concentration produced by vitamin D administration might have resulted from the stimulation of insulin secretion from the pancreatic β-cell, or a decrease in insulin resistance.[37]

Liver function tests are used to determine the presence of hepatic damage or impaired function. This is done by measuring ALT, AST, and ALP levels. We found that both ALT and AST were increased by the administration of vitamin D. These increases did not exceed the normal limits allowed for both enzymes (7–55 U/L for ALT and 8–48 U/L for AST). This finding is not in agreement with other studies that vitamin D supplementation in subjects with T2DM was associated with statistically significant decreases in ALT.[8] [24] [38] However, another study that investigated the effect of high-dose vitamin D supplementation on serum markers of liver function among normal individuals showed that there was no significant difference in ALT and AST activity between pre- and post-treatment with vitamin D.[39]

The pre-treatment values of ALP levels in our patients were at the upper allowed limit (40–129 U/L). These values were decreased after treatment with vitamin D. Several previous studies showed that patients with vitamin D deficiency have high ALP levels.[40] [41] Our result was in accordance with the findings of previous studies that showed that ALP decreased post-treatment with vitamin D.[38] [42] It has been suggested that serum alkaline phosphatase might be used as a tool to screen for hypovitaminosis D,[43] but others documented that it was not a useful screening tool.[44] [45] In general, treatment with vitamin D did not affect liver function considerably, which confirm the safety of the doses used of vitamin D.

Strengths and Limitations

This study had several strengths: no change in the patients' antidiabetic medications and diet during vitamin D treatment were made, as doing so could have affected the results. Moreover, all participants were vitamin D-deficient. However, this study had some limitations: first, it included relatively few participants; second, it included only the western region of Libya; and third, no placebo group was included.

Conclusion

This study suggests that with the doses used, vitamin D supplementation may improve glycemic control without affecting liver functions in patients with T2DM and vitamin D deficiency. Future longer-term, high-dose vitamin D intervention trials using a larger number of T2DM patients with vitamin D deficiency are needed to validate these results.

Conflicts of Interest

None declared.

Authors' Contributions

Hafsa M. Alemam, Mouna M. ElJilani, and Abdulla M. Bashein contributed to study design and concept, definition of intellectual content, literature search, data and statistical analysis, manuscript preparation. Hafsa M. Alemam and Mouna M. ElJilani contributed to experimental studies and data acquisition. Abdulla M. Bashein contributed to manuscript editing and reviewing.

• References

• 1 Mitri J, Pittas AG. Vitamin D and diabetes. Endocrinol Metab Clin North Am 2014; 43 (01) 205-232
• 2 Johnson JA, Grande JP, Roche PC, Kumar R. Immunohistochemical localization of the 1,25(OH)2D3 receptor and calbindin D28k in human and rat pancreas. Am J Physiol 1994; 267 (3 Pt 1): E356-E360
• 3 Maestro B, Dávila N, Carranza MC, Calle C. Identification of a vitamin D response element in the human insulin receptor gene promoter. J Steroid Biochem Mol Biol 2003; 84 (2-3): 223-230
• 4 Maestro B, Molero S, Bajo S, Dávila N, Calle C. Transcriptional activation of the human insulin receptor gene by 1,25-dihydroxyvitamin D(3). Cell Biochem Funct 2002; 20 (03) 227-232
• 5 Hitman GA, Mannan N, McDermott MF. et al. Vitamin D receptor gene polymorphisms influence insulin secretion in Bangladeshi Asians. Diabetes 1998; 47 (04) 688-690
• 6 Targher G, Bertolini L, Scala L. et al. Associations between serum 25-hydroxyvitamin D3 concentrations and liver histology in patients with non-alcoholic fatty liver disease. Nutr Metab Cardiovasc Dis 2007; 17 (07) 517-524
• 7 Gad AI, Elmedames MR, Abdelhai AR, Marei AM, Abdel-Ghani HA. Efficacy of vitamin D supplementation on adult patients with non-alcoholic fatty liver disease: a single-center experience. Gastroenterol Hepatol Bed Bench 2021; 14 (01) 44-52
• 8 Zelber-Sagi S, Zur R, Thurm T. et al. Low serum vitamin D is independently associated with unexplained elevated ALT only among non-obese men in the general population. Ann Hepatol 2019; 18 (04) 578-584
• 9 Sattar N, Forrest E, Preiss D. Non-alcoholic fatty liver disease. BMJ 2014; 349: g4596
• 10 Fazel Y, Koenig AB, Sayiner M, Goodman ZD, Younossi ZM. Epidemiology and natural history of non-alcoholic fatty liver disease. Metabolism 2016; 65 (08) 1017-1025
• 11 Satapathy SK, Sanyal AJ. Epidemiology and natural history of nonalcoholic fatty liver disease. Semin Liver Dis 2015; 35 (03) 221-235
• 12 ElJilani MM, Alemam HA, Bashein A. Vitamin D and liver enzymes' levels in Libyans with type 2 diabetes. Libyan J Med Sci 2021; 5(3): 116-120
• 13 Palacios C, Gonzalez L. Is vitamin D deficiency a major global public health problem?. J Steroid Biochem Mol Biol 2014; 144 (Pt A): 138-145
• 14 Annweiler C, Souberbielle J-C. Vitamin D supplementation and COVID-19: expert consensus and guidelines [article in French]. Geriatr Psychol Neuropsychiatr Vieil 2021; 19 (01) 20-29
• 15 Xuan Y, Zhao HY, Liu J-M. Vitamin D and type 2 diabetes mellitus (D2). J Diabetes 2013; 5 (03) 261-267
• 16 Issa CM. Vitamin D and Type 2 diabetes mellitus. Adv Exp Med Biol 2017; 996: 193-205
• 17 Li Y-X, Zhou L, Vitamin D. Vitamin D deficiency, obesity and diabetes. Cell Mol Biol 2015; 61 (03) 35-38
• 18 Sacerdote A, Dave P, Lokshin V, Bahtiyar G. Type 2 diabetes mellitus, insulin resistance, and vitamin D. Curr Diab Rep 2019; 19 (10) 101
• 19 Boucher BJ. Vitamin D insufficiency and diabetes risks. Curr Drug Targets 2011; 12 (01) 61-87
• 20 Holick MF, Binkley NC, Bischoff-Ferrari HA. et al; Endocrine Society. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2011; 96 (07) 1911-1930
• 21 Genuth S, Alberti KGMM, Bennett P. et al; Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. Follow-up report on the diagnosis of diabetes mellitus. Diabetes Care 2003; 26 (11) 3160-3167
• 22 Ceriotti F, Henny J, Queraltó J. et al; IFCC Committee on Reference Intervals and Decision Limits (C-RIDL); Committee on Reference Systems for Enzymes (C-RSE). Common reference intervals for aspartate aminotransferase (AST), alanine aminotransferase (ALT) and γ-glutamyl transferase (GGT) in serum: results from an IFCC multicenter study. Clin Chem Lab Med 2010; 48 (11) 1593-1601
• 23 Strømme JH, Rustad P, Steensland H, Theodorsen L, Urdal P. Reference intervals for eight enzymes in blood of adult females and males measured in accordance with the International Federation of Clinical Chemistry reference system at 37 degrees C: part of the Nordic Reference Interval Project. Scand J Clin Lab Invest 2004; 64 (04) 371-384
• 24 Nwosu BU, Maranda L. The effects of vitamin D supplementation on hepatic dysfunction, vitamin D status, and glycemic control in children and adolescents with vitamin D deficiency and either type 1 or type 2 diabetes mellitus. PLoS One 2014; 9 (06) e99646
• 25 Gupta N, Farooqui KJ, Batra CM, Marwaha RK, Mithal A. Effect of oral versus intramuscular vitamin D replacement in apparently healthy adults with Vitamin D deficiency. Indian J Endocrinol Metab 2017; 21 (01) 131-136
• 26 Tellioglu A, Basaran S, Guzel R, Seydaoglu G. Efficacy and safety of high dose intramuscular or oral cholecalciferol in vitamin D deficient/insufficient elderly. Maturitas 2012; 72 (04) 332-338
• 27 Pittas AG, Lau J, Hu FB, Dawson-Hughes B. The role of vitamin D and calcium in type 2 diabetes. A systematic review and meta-analysis. J Clin Endocrinol Metab 2007; 92 (06) 2017-2029
• 28 Alvarez JA, Ashraf A. Role of vitamin d in insulin secretion and insulin sensitivity for glucose homeostasis. Int J Endocrinol 2010; 2010: 351385
• 29 Mirhosseini N, Vatanparast H, Mazidi M, Kimball SM. The effect of improved serum 25-hydroxyvitamin D status on glycemic control in diabetic patients: a meta-analysis. J Clin Endocrinol Metab 2017; 102 (09) 3097-3110
• 30 Pilz S, Kienreich K, Rutters F. et al. Role of vitamin D in the development of insulin resistance and type 2 diabetes. Curr Diab Rep 2013; 13 (02) 261-270
• 31 Szymczak-Pajor I, Drzewoski J, Śliwińska A. The molecular mechanisms by which vitamin D prevents insulin resistance and associated disorders. Int J Mol Sci 2020; 21 (18) E6644
• 32 Szymczak-Pajor I, Śliwińska A. Analysis of association between vitamin D deficiency and insulin resistance. Nutrients 2019; 11 (04) E794
• 33 de Oliveira C, Biddulph JP, Hirani V, Schneider IJC. Vitamin D and inflammatory markers: cross-sectional analyses using data from the English Longitudinal Study of Ageing (ELSA). J Nutr Sci 2017; 6: e1
• 34 Baier LJ, Dobberfuhl AM, Pratley RE, Hanson RL, Bogardus C. Variations in the vitamin D-binding protein (Gc locus) are associated with oral glucose tolerance in nondiabetic Pima Indians. J Clin Endocrinol Metab 1998; 83 (08) 2993-2996
• 35 Sahebi R, Rezayi M, Emadzadeh M. et al. The effects of vitamin D supplementation on indices of glycemic control in Iranian diabetics: a systematic review and meta-analysis. Complement Ther Clin Pract 2019; 34: 294-304
• 36 Sollid ST, Hutchinson MY, Fuskevåg OM. et al. No effect of high-dose vitamin D supplementation on glycemic status or cardiovascular risk factors in subjects with prediabetes. Diabetes Care 2014; 37 (08) 2123-2131
• 37 Rafiq S, Jeppesen PB. Insulin resistance is inversely associated with the status of vitamin D in both diabetic and non-diabetic populations. Nutrients 2021; 13 (06) 1742
• 38 Sangatrashani SJH, Habibian M. Moosavi SJ. Effect of core stability exercise and vitamin D intake on liver enzymes activities in women with chronic low back pain. The Journal of Qazvin University of Medical Sciences 2020; 24 (04) 332-345
• 39 Tavakoli H, Rostami H, Avan A. et al. High dose vitamin D supplementation is associated with an improvement in serum markers of liver function. Biofactors 2019; 45 (03) 335-342
• 40 Sharma U, Pal D, Prasad R. Alkaline phosphatase: an overview. Indian J Clin Biochem 2014; 29 (03) 269-278
• 41 Saraç F, Saygılı F. Causes of high bone alkaline phosphatase. Biotechnol Equip 2007; 21: 194-197
• 42 Bellastella G, Scappaticcio L, Longo M. et al. New insights into vitamin D regulation: is there a role for alkaline phosphatase?. J Endocrinol Invest 2021; 44 (09) 1891-1896
• 43 Masood H, Narang AP, Bhat IA, Shah GN. Persistent limb pain and raised serum alkaline phosphatase the earliest markers of subclinical hypovitaminosis D in Kashmir. Indian J Physiol Pharmacol 1989; 33 (04) 259-261
• 44 Vasudevan J, Jenifer A, Reddy GMM, Thayumanavan S. Serum alkaline phosphatase for screening of hypovitaminosis D. Indian Pediatr 2014; 51 (01) 60-61
• 45 Shaheen S, Noor SS, Barakzai Q. Serum alkaline phosphatase screening for vitamin D deficiency states. J Coll Physicians Surg Pak 2012; 22 (07) 424-427

Publication History

Article published online:
02 August 2022