Mahdieh Abbasalizad Farhanghi  ( Nutritional Research Center, Tabriz University of Medical Sciences. )
Soltanali Mahboob, Alireza Ostadrahimi   ( Nutritional Research Center, Tabriz University of Medical Sciences. )

 Abstract
Objective: To determine the effect of vitamin D injection on Serum Magnesium concentration in obese and non obese women.
Method: This Interventional study was performed on 82 women (17-50 years) which were randomly selected from general population of Tabriz city. They were assigned into two experimental groups. Obese group with stage 1 and 2 obesity and non obese group with normal weight. Weight was measured to the nearest 0.1 kg using a calibrated Seca scale. Height was measured using a cotton ruler which was pasted on the wall. Body mass index was calculated based on weight and height results. Biochemical parameters were measured before and after injection of 600000 IU doses of vitamin D. Serum Magnesium was measured calorimetrically and Serum 25 hydroxy vitamin D was estimated by Chemiluminescence Immuno Assay method (CLIA).
Results: Baseline concentrations of serum Magnesium and 25 hydroxy vitamin D in obese individuals was lower than non obese individuals, the former being significant. Twenty seven percent of obese women versus 15% of non obese women were Magnesium deficient. Vitamin D injection caused a significant increase in serum Magnesium concentration in obese subjects but not in non obese subjects. There was also a significant increase of serum 25 hydroxy vitamin D in both groups. Mean elevation in serum Magnesium level among women who had Magnesium deficiency was higher than women with Magnesium adequacy (P<0.05).
Conclusion: Low serum Magnesium concentration in obese individuals can be modified by vitamin D injection (JPMA 59:258; 2009).
Introduction
Magnesium a divalent ion, is widespread throughout the mammalian organism.¹ The physiological role of Magnesium is achieved through its ability to form chelates with important intracellular ligands especially ATP.² Magnesium is therefore essential for the synthesis of nucleic acids and proteins, for intermediary metabolism and for specific actions in different organs such as neuromuscular and cardiovascular system.³ Magnesium deficiency can be divided to primary and secondary deficiency. Primary Magnesium deficiency is entirely due to reduced dietary intake and secondary Magnesium deficiency is a result of increased gastrointestinal or renal loss of Magnesium.^4,5
Recent studies linked obesity with low serum magnesium. Obesity is characterized by a high risk for glucose intolerance, cardiovascular disease, dyslipidaemia and insulin resistance.⁶ It is speculated that one of the causes for the aforementioned disorders in obese individuals, is Magnesium deficiency.6-10 Several studies have identified low serum Magnesium in obesity. Huerta et al⁷ and Song et al¹¹ found a negative correlation between serum magnesium and BMI in healthy children and adults(P<0.05). In some of studies low serum and intracellular Magnesium has been reported in obesity.^12,13

On the other hand, if Magnesium deficiency is detected, it should be treated because non treated Hypomagnesemia can lead to chronic disease as atherosclerosis, myocardial infarction, hypertension and renal calculi.⁵ Although no hormone or factor has been described that regulates Magnesium absorption ,the effect of vitamin D on serum Magnesium concentration has been confirmed in some of studies.¹⁴ In this study, the active metabolite of vitamin D,1,25 (OH)2 -vitamin D, increased intestinal absorption of Magnesium in physiologically normal human subjects.¹⁴ In another study by Heaton and his colleagues¹⁵ vitamin D enhanced intestinal absorption of both Magnesium and calcium, however these results were not confirmed by some other studies.

Wilz et al¹⁶ found no relationship between plasma 1,25 (OH)2-viatmin D and net intestinal Magnesium absorption. Gray TK et al¹⁷ also reported that intravenous injection of 1,25 (OH)2-viatmin D had no significant effect on intestinal Magnesium absorption. No interventional study examining the effect of intramuscular injection of vitamin D in treatment of Magnesium deficiency in obesity was found in literature search. The aim of this interventional study was to determine the effect of vitamin D supplementation on serum Magnesium concentration in obese and non obese women.
Subjects and Methods
This study was carried out in northwestern region of Tabriz- Iran ,with the approval of the Tabriz University of Medical Science Ethics Committee and the consent of the volunteers. The study was performed during winter ( January through March, 2007) on 82 reproductive age women (17-50 years), 40 of them had normal BMI ( 18.5-24.9 kg/m2) and 42 had stage 1 and stage 2 obesity ( BMI: 30-34.9 and 35-39.9 kg/m2 respectively). Women with a history of Hepatic or Renal Disorders, Cardiovascular disease, Diabetes and Hypertension were excluded from the study. None of subjects was taking vitamin D and calcium supplements, anticonvulsant medications, Hormone Replacements such as estrogen, oral contraceptives (OCP), loop diuretics or corticosteroids. Fasting blood samples were collected in the morning and stored at -70 0C until assay. Each subject received a dose of 6000000 IU of vitamin D intramuscularly and after 2 weeks blood samples were collected again to determine the effect of vitamin D injection on serum Magnesium level.
Serum Magnesium was measured colorimetrically (Darman Kave , Res). CV of this test was 6%. Our reference range for serum Magnesium was 1.5-2.1 meq/l and Magnesium deficiency was identified as serum magnesium lower than 1.5meq/l.18 Serum 25 hydroxy vitamin D was measured using a Chemiluminescence Immuno Assay method or CLIA (Diasorin, Still water, MN).¹⁹ Sensitivity of this assay was 7.0 ng/ml. Mean inter and intra assay coefficient of variation (CV) were 13.2 and 10.8 respectively.
Weight was measured to the nearest 0.1 kg using a calibrated Seca scale while subjects had light clothes and no shoes. Height was measured using a cotton ruler which was pasted to the wall. Body mass index was calculated based on weight and height BMI: Weight (kg)/Height (m2). Demographic characteristic of participants were measured using an interview questionnaire. A 3-day diet record was obtained from participants. Average daily nutrient intakes were calculated by Nutritionist III software. It should be noted that all of participants had been educated to have their usual diet during 15 days follow up period.
Data was reported as mean ± SD. To compare Serum Magnesium and 25 hydroxy vitamin D before and after injection and between groups, paired t-test, independent sample t-test and one way ANOVA (analysis of variance) were used. P value less than 0.05 was considered significant.
Results
The demographic characteristics of subjects are shown in Table 1. The mean age of obese and non obese women were 32.54 ±7.79 and 31.38 ± 6.68 years respectively. There was no significant differences between dietary energy and macronutrients intake between obese and non obese women. Dietary vitamin D and Magnesium  intake was higher in non-obese women than in obese women (Vitamin D: 12.83 ± 19 vs 8.08 ±10 IU/d) (Magnesium: 144.66±17.48 vs 122.43±17mg/d) and   Dietary calcium intake was slightly higher in obese women although these differences in both vitamin D and calcium intake were not statistically significant. Twenty seven percent of obese women versus 15% of non obese women were Magnesium deficient. Table 2 summarizes the biochemical characteristic of participants. There was no significant difference in serum 25 hydroxy vitamin D between obese and non-obese subjects. Mean serum Magnesium in obese women was significantly lower than non obese women (P<0.05).
After vitamin D injection there was a significant increase in serum Magnesium concentration in obese individuals (P<0.05) but serum Magnesium concentration in non obese individuals before and after intervention was not significantly different. Serum 25 hydroxy vitamin D has increased significantly in both obese and non-obese subjects (p<0.01).
Mean elevation in serum Magnesium level after vitamin D injection among women who had Magnesium deficiency (Serum Magnesium <1.5meq/l) was ±0.37 meq/l whereas women with Magnesium adequacy (Serum Magnesium 1.5-2 meq/l) showed ±0.26 meq/l elevation in serum Magnesium. Women with Serum Magnesium higher than 2.1 meq/l showed ±0.86 meq/l decrease in serum Magnesium (P<0.01) (Figure 1).
Discussion
In the present Interventional study, we found a significant increase in serum magnesium concentration in obese individuals after vitamin D supplementation (p<0.05). There was no significant difference in serum Magnesium concentration in non obese individuals before and after intervention. Baseline concentration of serum magnesium in obese subjects was significantly lower than non obese subjects (p = 0.005). Some of other previously published studies have also reported lower serum Magnesium concentration in obese individuals.^7,11
Some of the factors that can cause low serum magnesium in obesity, are lower dietary intake of Magnesium,⁷ reduced Magnesium intestinal absorption because of high fat or calcium intake,²⁰ and high inflammatory factors in obesity.²¹
The effect of vitamin D on serum Magnesium is controversial. Some of studies reported increasing in serum Magnesium concentration in response to vitamin D supplementation. Schmulen et al²² found that vitamin D increases intestinal absorption of Magnesium in normal human subjects and in patients with chronic renal failure.
On the other hand, Wilz et al¹⁶ could not find any relationship between plasma 1,25 (OH)2vitamin D concentration and net intestinal Magnesium absorption. Wiltz et al¹⁶ also found that Magnesium is absorbed by persons with no detectable plasma 1,25 (OH)2 vitamin D and in contrast to calcium absorption, no significant correlation exists between plasma   1,25 (OH)2 vitamin D and Magnesium absorption.
In addition to intestinal Magnesium absorption renal excretion is also another important key in regulating Magnesium homeostasis. The kidney is the critical organ regulating serum magnesium concentration. The 1,25 (OH)2 D- dependent calcium binding proteins, Calbindin -D 9K and Calbindin -D 28K in the kidney, are localized in the distal tubule where a significant portion of calcium and Magnesium is reabsorbed²³ Calbindin -D 9K has a relatively high affinity for Magnesium²⁴ suggesting a role for these binding proteins in renal Magnesium control. Dai LJ et al25 in their study demonstrated that 1,²⁵ (OH)2 vitamin D increases magnesium entry rates in distal tubule cells and the response is concentration dependent. This means that the higher Magnesium reaches to distal tubule, the higher is renal Magnesium excretion.  
In our study increase in serum Magnesium concentration in response to vitamin D supplementation in obese women, but not in non obese women, can also be explained by this mechanism. Lower baseline concentration of serum Magnesium in obese subjects can induce higher renal Magnesium retention after vitamin D supplementation. Another evidence to this issue is that subjects with baseline serum Magnesium concentration lower than 1meq/l showed higher increase in serum Magnesium in response to vitamin D injection in comparison with persons who baseline serum Magnesium concentration was higher than 1meq/l (P<0.05).
In conclusion, we found a strong modifier effect of vitamin D injection on serum Magnesium concentration in obese women, so it is suggested that vitamin D injection can be used as a good method for treatment of magnesium deficiency in obese individuals.
Acknowledgement
This research was financially supported by a grant from Nutritional Research Center of Tabriz University of Medical Sciences. The authors gratefully acknowledge all participants of this study.
References
1.Briscoe AM, Ragan C. Effect of Magnesium on Calcium Metabolism in Man, Am J Clin Nut 1966; 19:296-306.
2.Altura BM. Basic biochemistry and physiology of Magnesium:A brief review. Magnesium and Trace Elements1991;10:167-71.
3.Elin RJ:Magnesium metabolism in health and disease. Dis Mon 1988; 34:161-218.
4.Katleen ML, Stump S. In: Krauses food nutrition and diet therapy. 11th ed. John Rogers. Philadelphia 2004; p 131-3.
5.Nadler JL, Rude RK. Disorders of magnesium metabolism. Endocrinol Metab Clin North Am 1995; 24:623-41.
6.De Leeuw I, Vansant G, Van Gaal L. Magnesium and obesity: influence of gender, glucose, tolerance, and body fat distribution on circulating magnesium concentration. Magnes Res 1992; 5:183-7.
7.Huerta MG, Roemmich JN, Kington ML, Bovbjerg VE, Wettman AL, Holmes YF, et al. Magnesium deficiency is associated with insulin resistance in obese children: Diabetes Care 2005; 28:1175-81.
8. Inoue I. Lipid metabolism and magnesium. Clin Calcium 2005; 15:65-79. 
9.Song Y, Manson JE, Buring JE, Liu S. Dietary magnesium intake in relation to plasma insulin levels and risk of type 2 diabetes in women. Diabetes Care 2004; 27:59-65.
10.Lopez- Ridaura R. Willet WC, Rimm EB, Liu S, Stampfer MJ, Manson JE, et al. Magnesium intake and risk of type 2 diabetes in men and women. Diabetes Care 2004; 27:134-40.
11.Song CH. Associations of serum minerals with body mass index in adult women, Eur J Clin Nutr 2007; 61:682-5.
12.Ma J, Falson AR, Melnick SL, Eckfeldt JH, Sharret AR, Nabulsi AA, et al. Associations of serum and dietary magnesium with cardiovascular disease, hypertension, diabetes, insulin and carotid wall thickness: the ARIS study; J Clin Epidemiol 1998; 48:927-40.
13.Resnick LM, Gupta RK, Gruenspan H, Alderman MH, Laragh JH. Hypertension and peripheral insulin resistance. Possible mediating role of intracellular free magnesium. Am J Hypertens 1990; 3:373-9.
14.Shils ME, Shike M. Modern Nutrition In Health   And Disease, 10th ed. Philadelphia Williams & Wilkins 2006; pp:223-48.
15.Heaton FW, Hodgkinson A, Rose GA. Observation on the relation between calcium and magnesium metabolism in man. Clin Sc 1964; 27:31-40.
16.Wilz DR, Gray RW, Dominguez JH, et al. Plasma 1,25 -(OH)2 -vitamin D concentrations and net intestinal calcium, Phosphate and Magnesium absorption in humans. Am J Clin Nut 1979; 32: 2052-60.
17.Gray TK, Walton J, Wiliams ME, PooL D. 1,25 Dihydroxyvitamin D and jejunal absorption of calcium and phosphate in normal subjects .In: Vitamin D: Biochemical, Chemical and Clinical Aspects Related to Calcium Metabolism. Walter de Gruyter 1977; pp 623.
18.Gibb MA, Wolfson AB, Tayal VS. Electrolyte disturbances. Emergency Medicine Concepts and Clinical Practice 1998; 3:2445-6.
19.Hollis B, Macfarlane G, Valcour A. Clinical significance and assessment of 25 hydroxy vitamin D, Advance/Laboratory 2005; 72-8.
20.Ziegler E, Filer LJ. In: Present knowledge of Nutrition. 7th ed. Washington DC 1996; pp 256-64.
21.Rodriguez-Moran M, Guerrero - Romero F. Elevated concentrations of TNF -alpha are related to low serum magnesium levels in obese subjects. Magnes Res 2004; 17:189-98.
22.Schmulen AC, Lerman M, Pak CY, Zerwekh J, Morawski S, Fordtran JS et al. Effect of 1,25-(OH) 2D3 on jejunal absorption of magnesium in patients with chronic renal diseases. Am J Physiol Gastrointest Liver Physiol 1980; 238: 4349-52.
23.Liu LT, NG M, Iacopino AM, Dunn ST, Hughes MR, Bouredau JE. Vitamin