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ASSOCIATION OF MAGNESIUM LEVELS WITH LIPID PROFILE AND INFLAMMATORY MARKERS IN THIAZIDE INDUCED HYPOMAGNESEMIC RATS.

Byline: Sarah Sadiq, Abdul Khaliq Naveed, Tausif Ahmed and Shakir Khan

Abstract

Objective: To study the effect of Magnesium supplementation on lipid profile (TC, HDL, LDL, TG, VLDL) and inflammatory markers (CRP s-VCAM 1) in thiazide administered rats.

Study Design: Randomized control trial.

Place and Duration of Study: Department of Biochemistry and Molecular Biology, Army Medical College Rawalpindi from Feb 2010 to Feb 2011.

Material and Methods: Ninety male Sprague Dawley rats, of age 90 to 120 days, divided into three groups: Control, Hydrochlorothiazide (HCTZ) administered control and Hydrochlorothiazide + Magnesium Oxide (HTCZ + Mg2+), administered experimental groups. Levels of inflammatory markers, Magnesium, lipid profile were measured in blood, after three weeks.

Results: Mean values in control group were: Serum TG 104.39 mg/dl,TC151.86 mg/dl, HDL76.91 mg/dl, LDL 52.52 mg/dl, VLDL 19.78mg/dl, CRP 1784.27 mg/L, sVCAM-1 564.33ng/ml and serum Magnesium0.85mmol/l. Mean lipid profile values in HCTZ administered control group were Serum TG 150.04 mg/dl, TC 182.92 mg/dl, HDL 71.77 mg/dl, LDL 76.40 mg/dl,VLDL 32.56 mg/dl, CRP 2923.33 mg/L, sVCAM-1 2003.00 ng/ml and serum Magnesium 0.49 mmol/l. Mean lipid profile values in HCTZ + Magnesium administered experimental group were Serum TG 131.69 mg/dl,TC 157.60 mg/dl, HDL 73.55 mg/dl,LDL 58.2 mg/dl,VLDL 25.05mg/dl, CRP 2149.53 mg/L ,sVCAM-1 1516.60 ng/mland serum Magnesium 0.68mmol/l.

Conclusion: Levels of sVCAM-1, CRP, TG, TC, LDL and VLDL are increased in HCTZ administered group, with negative correlation with serum magnesium levels. Levels of these parameters decreased in HTCZ + Mg2+ administered group, showing that magnesium supplementation helped in normalizing derangements (pless than 0.05).

Keywords: CRP, Hyperlipidemia, Inflammation, Magnesium, sVCAM-1.

INTRODUCTION

The most prevalent type of hypertension is Primary hypertension1. Though no direct cause has been identified, many factors such as sedentary lifestyle, stress, potassium deficiency can be responsible2. Obesity3, kidney diseases and metabolic disorders can cause secondary hypertension4. There has been 40% reduction in risk of stroke and a 15% reduction in risk of myocardial infarction by treatment of hypertension5.

A thiazide-type diuretic is usually prescribed as first-line pharmacotherapy6. For long term treatment of hypertension, thiazides are often indicated7. This helps in reducing BP and cardiovascular events8. Use of hydrochlorthiazide, can lead to several unfavorable effects such as electrolyte disorders (hyponatremia, hypokalemia and hypomagnesemia), hyperlipidemia, hyperuricemia and impairment of glucose metabolism9.

The main intracellular divalent cat-ion is Magnesium10. involved in a largenumber of biochemical processes11. Normal serum magnesium has the range between 1.7 to 2.3 mg/dl (0.75-0.95 mmol/L)12. Hypomagnesaemia is associated with considerable morbidity13.

Table-1: Mean levels of lipid profile, inflammatory markers and serum magnesium in all the groups.

###Sample###Group 1###Group 2###Group 3

Parameters

###Size###Mean SD###Mean SD###Mean SD

###Serum TG (mg/dl)###104.39 8.55###150.04 14.00###131.69 7.77

###Total Cholesterol (mg/dl)###151.86 14.53###182.92 6.22###157.60 6.05

Lipid

###HDL (mg/dl)###76.91 4.90###71.77 3.59###73.55 3.90

Profile

###LDL (mg/dl)###52.52 4.47###76.40 5.43###58.25 4.07

###VLDL (mg/dl)###30###9.78 2.39###32.56 4.31###25.05 4.09

Inflammato###sVCAM-1 (ng/ml)###564.33 67.91###2003.00 927.92 1516.60 300.36

ry Markers###CRP (ng/ml)###1784.27 731.97###2923.33 702.37 2149.53 566.61

Serum###Serum Magnesium

###0.85 0.11###0.49 0.13###0.68 0.10

Mg2+###(mmol/l)

Table-2: Pearson's product moment correlation between serum magnesium levels and various markers of lipid profile and inflammation of HCTZ + Mg2+ administered group-3

###Pearson's product moment correlation

###Correlation Coffecient "r"

Correlation Between

###Positive###Negetive

Serum TG###-0.257

Total Cholesterol###-0.241

HDL###0.109

LDL###Serum Magnesium###-0.112

VLDL###-0.199

sVCAM-1###-0.155

CRP###-0.051

More recently, Mair et al discovered that low Mg2+ concentrations influence the inflammatory response and affect the endothelial proliferation,by up regulation of interleukin-1 (IL1) and sVCAM-1 (pro inflammatory cytokines)14. People who consume only slightly below the RDA of Mg2+ had a higher likelihood of elevation of CRP15. Studies demonstrate that increased intake of Mg2+ in diet may lower blood triglyceride and increase high density lipoprotein (HDL) levels16.

MATERIAL AND METHODS

It was a randomized control trial and duration of study was two years. Ninety Sprague Dawley rats were selected by purposive sampling. They had a weight 220 30g and were kept at animal house of AMSON VACCINES and PHARMA, Pvt. (Ltd). They were divided, through random number table, into Group 1: control group (n=30) and, provided with standard diet and water ad libitum, Group-2 (n=30) rats, administered with Hydrochlorothiazide (10mg/kg body weight) by drenching through gavage needle ,given standard diet and water ad libitum andGroup 3 (n=30), administered with Hydrochlorothiazide (10mg/kg body weight) and Magnesium Oxide supplemented diet (1.075mg/kg body weight/day) with water ad libitum, for three weeks. Blood samples were drawn by intra- cardiac sampling. Levels of inflammatory markers were determined by ELISA and levels of serum Magnesium and lipid profile, by Automated analyzer (SELECTRA), using specific kits.

1.C - Reactive Protein was measured by using RandD Systems, Inc, USA. Quantikine(r) Human C-Reactive protein Immunoassay, Catalog No. DCRP00. Assay was performed on Awareness Technology Inc, USA, Complete system (Shaker incubator Stat Fax 2200, automated washer Stat Fax 2600 andMicroplate reader Stat Fax 2100).

100ul of Assay Diluent RD1F, was added to each well. We added 50 ul of standard, control, sample per well. Wells were covered,incubated and washed. 200 ul of CRP Conjugate was addedto each well. It was covered and incubated. Added 200 ul substratesolution andincubated for 30 minutes. Added 50 ul stop solution. Color in wells changed to yellow. We determined the optical density, at 450 nm.

Soluble Vascular Cell Adhesion Molecule Type-1 was measured by (ELISA), using RandD Systems, Inc, USA. Quantikine(r) Human sVCAM-1 Immunoassay, Catalog No. SVC00. We added 100 ul of sVCAM-1 Conjugate to each well and the added 100 ul of standard, controland sample per well. Covered with the adhesive strip.

Incubated for 1.5 hours at room temperature, washed, with Wash Buffer (400 ul) using an Automated washer. We added 100 ul of Substrate Solution to each well, covered andincubated.

Added 50 ul of Stop Solution to each well. Color in wells changed from blue to yellow. We determined optical density, at 450 nm.

For estimation of Total Cholesterol commercially available kit manufactured by Pioneer Diagnostics, New York, U.S.A. was used.

Three test tubes, labeled blank, standard and sample,with one ml of monoreagent in each tube.10 uL of distilled water, 10 uL of standard and 10 uL of sample were added to each test tube respectively. Mixing and incubation for 5 minutes at 37C was done. The results of standard and samples were measured at 500 nm wavelength against reagent blank by Selectra.

Estimation of TG was done, using Kit (Ready to use) by Pioneer diagnostics New York, U.S.A.

Three test tubes , labeled as blank, standard and sample, with 1 ml of monoreagent,in one tube. 10 uL of distilled water, 10 uL of standard and 10 uL of sample were added by micropipette in each test tube respectively. Thorough mixing and incubation for 5 minutes at 37C. Results of standard and samples measured at 500 nm wavelength against reagent blank by Selectra. Absorbance (A) of the sample and standard was read.

A (sample) / B (standard) x C (standard) = mg/dl Triglycerides.

Estimation of HDL-c was done by kits prepared by Linear Chemicals, Barcelona (Spain), direct enzymatic method. 200 uL sample and 500 uL precipitant were mixed, centrifuged for 10 minutes at 4000 r.p.m. Supernatant separated andcholesterol measured by CHOD-PAP method.

Estimation of (LDL) and VLDLwas measured using Freidwald's formula.

LDL = Total Cholesterol - HDL - Triglycerides / 5

VLDL-c = Triglycerides/5

Statistical Analysis

Data was entered in a database using SPSS version 16. Values were expressed as mean and standard deviation (SD). Independent Sample t-test was applied. A p-value of less than 0.05 and less than 0.01 was considered significant and highly significant respectively. Pearson's product moment correlation (r) was applied.

RESULTS

The mean values of lipid profile (serum TG, Total Cholesterol, HDL, LDL, VLDL), inflammatory markers (sVCAM1, CRP) and serum Magnesium in all three groups are shown in table-1.

Lipid Profile of control group 1 was compared with HCTZ administered control group 2 by using student's t-test. There was a highly significant difference between the levels of Triglyceride (pless than 0.001), Total Cholesterol (pless than 0.001), HDL (pless than 0.001), LDL (pless than 0.001) and VLDL (pless than 0.001) among both the groups. Diseased control group 2 was also compared with Experimental group 3 by using student's t-test. There was a highly significant difference between the levels of Triglyceride (pless than 0.001), Total Cholesterol (pless than 0.001), LDL (pless than 0.001) and VLDL (pless than 0.001) among both the groups and an insignificant CRP (difference between HDL (p=0.228) - Fig-1. State of inflammation for control group 1 was compared with diseased control group 2 by using student's t-test. There was a highly significant difference between the levels of sVCAM - 1 (pless than 0.001) and insignificant difference between CRP (p=0.094).

Diseased control group 2 was also compared with Experimental group 3 by using student's t-test. There was a significant difference between the levels of sVCAM-1 (p=0.004) and highly significant difference between pless than 0.001) fig-2. Pearson's product moment correlation (r) was applied to assess the correlation betweenparameters of serum magnesium levels, lipid profile and inflammatory markers in the experimental HCTZ + Mg2+ administered group.A positive and significant (r=0.109) correlation was found between Magnesium levels andHDL whereas negative and significant correlation was found between Magnesium levels and TG (r=-0.257), TC (r=-0.241), LDL (r=-0.112) and VLDL (r=-0.199). A negative and significant (r=-0.155) correlation was found between Magnesium levelsand sVCAM -1 and CRP (r=-0.051) table-2.

DISCUSSION

In our study serum levels of Magnesium in the control group were in the range of 0.61-1.1mmol/l, with a mean value of 0.85 as compared to the range of 0.24-0.84mmol/l, with a mean value of 0.49 in the HCTZ administered control. There was a significantdecrease in serum Magnesium levels between both groups. This fact is supported by John W in his study which shows that elderly hypertensive patients are at particular risk because of their tendency to have significantly depressed serum magnesium levels, which decrease even further when treated with thiazide diuretics17. There was a significant decrease in serum Magnesium levels between both groups. Animal studies found that serumtriglycerides were significantly higher and HDL cholesterol levels were significantly lower withmagnesium deficiency18. Magnesium administration in our study resulted in the lowering of serum TG, TC,LDL, VLDL and increase in the levels of HDL.

Acute magnesium deficiency leads to aninflammatory response19. Several cross-sectional studies have reported a link between both low dietarymagnesium intake20, and serum magnesium concentrations21 and elevated CRP. Withregard to endothelial dysfunction, it was observed that dietary magnesium was inverselyassociated with plasma concentrations of sVCAM-1 independently of other dietary factors. Levels of inflammatory markers were alsodecreased in group 3 as compared to the HCTZ administered group, in our study.

CONCLUSION

This study demonstrates a significant decrease in inflammatory markers and lipid profile in association with serum magnesium levels.

CONFLICT OF INTEREST

This study has no conflict of interest to declare by any author.

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Publication:Pakistan Armed Forces Medical Journal
Article Type:Clinical report
Date:Sep 30, 2016
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