Blood Sugar lowering effect of zinc and multi vitamin/ mineral supplementation is dependent on initial fasting blood glucose

P . Gu na se ka ra 1 , M. H ettia ra ch ch i 2 , C. Li ya na ge 3 , S. Le ka mw as am 4 C or re sp o n di n g au th o r: M an ju la H etti ar ac hc hi , manjula.hett iarachchi@gmail.co m Au th o r Af fili ati on s: 1- C o ro na ry Ca re U ni t, Te ac hi ng Ho spit al, Ka ra piti ya , Ga lle. Sr i La nk a. 2- N uc le ar Medi ci ne Un it, Fa cu lt y o f M edic in e, Un iv er sit y o f R uh u na , Ga lle, S ri L an ka . 3 - Dep art men t o f Co mm un it y M edi cin e, F ac ul ty o f M edi cin e, U ni ve rsit y o f R uh u n a, G alle , S ri Lan ka . 4 - Dep art men t o f Me dici ne , Fa cu lty o f Me dici ne , Un iv er sity o f Ru hu n a , Gal le, Sr i La nk a.

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ABSTRACT
To evaluate the effect of zinc and other multi vitamin mineral supplementation on glycemic control and lipid profile in patients with type 2 diabetes mellitus. Diabetes patients (n=96) were randomly allocated to 3 groups; 29 subjects were supplemented with oral zinc sulfate (22 mg/day) and multivitamin mineral (zinc+MVM) preparation; 31 subjects were supplemented with the same preparation, without zinc (MVM) and 36 were given a matching placebo, for a period of 4 months in a single blinded study. Blood samples were taken at the baseline and after 4 months of supplementation, to assess blood glucose (fasting and post prandial) and glycosylated haemoglobin % (HbA1C %)levels. The Zinc+MVM group had a mean change of fasting blood sugar (FBS) -0.33 (SEM, 0.21) mmol/l and was significant (p =0.05) when compared with the other two groups (mean change in the MVM group +0.19 (0.31) and +0.43 (0.23) mmol/l in the control group respectively). When the baseline FBS was >5.56 (>100mg/dL) mmol/l, supplementation either with Zinc+MVM (p<0.001) or MVM (p=0.02) resulted in a significant favourable effect on blood glucose parameters. The HbA1C% level was lowered significantly irrespective of the baseline HbA1C% concentration, only in Zinc+MVM (p=0.01) supplemented individuals. In the other two groups, the change of HbA1C% level was not significant. Zinc supplementation with other multi vitamins daily to adult diabetes patients who had high baseline FBS levels demonstrated better glycemic control. Keywords: Diabetes, Glycemic control, Multi vitamin mineral supplementation, Zinc supplementation

INTRODUCTION
Zinc plays an important role in insulin action and carbohydrate and protein metabolism (1). Type 2 diabetes mellitus is a metabolic disorder, with hyperglycemia as the dominant feature, as a result of reduced secretion of insulin and increased insulin resistance (2). It has not been clearly elucidated whether zinc deficiency is a consequence of hyperglycemia or alternatively, whether zinc deficiency contributes to the pathogenesis of diabetes. Several investigators have shown the perturbation of zinc metabolism in diabetics (3-6). Few studies have been conducted on the effects of zinc supplementation on hyperglycemia of diabetics and their results are inconsistent. Zinc supplementation has improved fasting insulin level and fasting glucose in genetically obese mice models (7). Improved fasting glucose levels up to 30% in patients with cirrhosis through supplementation of zinc for 2 months have been reported (8). Supplementation of 30 mg zinc for 3 months in type 2 diabetics showed a decrease in HbA1C% concentration. Meanwhile, others reported that neither fasting glucose nor HbA1C% levels were changed by a short-term supplementation with zinc for type 1 diabetics (9, 10). The inadequate efficacy of oral hypoglycemic agents seen at times, may be attributed to inadequate post-receptor events associated with oxidative stress induced by long-term hyperglycemia. The administration of antioxidants such as zinc, magnesium, selenium, vitamin A and vitamin E may improve the tissue response to insulin and increase the

efficacy of drugs which act through this pathway. Previous studies indicated that marginal zinc deficiency is more prevalent among diabetic adults, compared to the normal adult population (11, 12). Abnormal zinc and lipid plasma levels occur more frequently in metabolically uncontrolled diabetic patients (13). Yet, zinc sulfate supplementation may be a therapeutical resource to recover some functions and improve life span (14). Therefore, we intended to determine whether zinc (and other vitamins/minerals with similar antioxidant properties) supplementation for a short-term could improve the levels of fasting glucose and HbA1C% as well as the insulin levels in Sri Lankan type 2 diabetic patients. Moreover, this study aimed to find out whether the changes in glycemic control by zinc and other multi vitamin mineral supplementation are associated with changes in lipid profile of type 2 diabetes mellitus patients.

METHODOLOGY

The study received approval from the Ethical Review Committee of the Faculty of Medicine, University of Ruhuna. Previously diagnosed (for at least 2 years) patients of adult onset (type II) diabetes mellitus attending medical clinics of the teaching hospital, Karapitiya, Galle, were considered as the study population. Following were the exclusion criteria: 1. Those, who were diagnosed to have renal or liver failure or any other chronic disease except essential hypertension and dyslipidemia. 2. Those, who were taking vitamin-mineral supplements or any hormone replacement therapy. 3. Those, with a history of a recent surgery or acute infection. 4. Pregnant or lactating women. 5. Those, who were receiving insulin preparations as a part of diabetes management. All subjects (n=96) were stratified by sex, age and duration of treatment and randomly assigned to three treatment groups, using the block randomization procedure. Group A (Zinc+MVM) was supplemented with oral zinc sulfate (22mg of elemental zinc/day) and multivitamin/mineral preparation (vitamin A, Vitamin D3, Vitamin E, magnesium, manganese, copper and selenium) Group B (MVM) was supplemented with the same preparation but without zinc. Group C was supplemented with an identical placebo tablet. The study was registered in the Sri Lanka Clinical Trial Registry (SLCTR/2008/017), managed by Sri Lanka Medical Association, linked to the Registry Network of the International Clinical Trials Registry Platform of WHO. Table 1: Nutrient composition of the preparation 1

The composition of the supplement is shown in Table 1. All subjects were instructed to take one tablet per day for a period of 4 months along with their usual treatment regimen. The supplement and placebo tablets looked identical in colour and shape. Since the reported mean daily intake of zinc in our population is low (15), the supplemented zinc dose was set at the recommended dietary intake (20-25 mg/day) for Sri Lankan population. All other minerals and vitamins that were included in the tablet were more or less equal to the dietary reference intake (DRI) levels set by the Nutrition coordination division of the Ministry of Health, Sri Lanka (16). Once enrolled, all subjects were advised to attend a phlebotomy session at 0800 hours, after overnight fasting. A sample of 5mL of venous blood was drawn before the patient has taken any oral hypoglycemic agent(s) or other drugs and aliquots were made for FBS and HbA1C% in sugar and EDTA bottles, respectively. Remaining portion of the blood sample was centrifuged at 5000 rpm for 10 minutes and serum was separated and stored in acid washed polystyrene tubes at -80oC until analysis for serum insulin, zinc and creatinine was done. In addition, urine creatinine was measured from an aliquot of fresh urine sample obtained from each subject. Another portion of urine was stored at -80oC for analysis of urinary zinc. Following this, the patients were served with breakfast and two hours later another blood sample (2ml) was drawn for the assessment of post prandial blood sugar (PPBS). Another blood sample was collected on the next day, after 14 hours fasting for the assessment of lipid profile. Further, their dietary

1 Gr ou p A su bj ec ts g ot th e t ab le ts pr ep ar ed w it h f ull fo rm ul ae wh er e as gr ou p B s ub je ct s go t th e sam e wi th ou t zi nc s ul ph at e m on o hy d ra t e an d t he g ro u p C su bj ec ts g Ot t he p la ce bo t ab le t c on si sti ng of fill in g b ase m ag ne si um ste ar at e. 2 T he el em en ta l zin c co nt en t i n th e pr ep ar at io n w as 2 2 mg

intakes were assessed using a 24-hr dietary recall questionnaire at their entry into the study. They were advised to take the trial medication once daily, along with other medication and a monthly follow-up was arranged. Clinicians were blinded to the trial medication and they were allowed to continue the usual care given for diabetic subjects during the trial period. Changes in pharmacological or non-pharmacological management occurred during the trial period were recorded. Compliance on the supplementation was assessed by counting the remaining tablets at weeks 4, 8 and 12 and all side effects of the trial medication were noted. After 4 months of supplementation, blood and urine samples were collected under the same conditions as described before, for the same biochemical analyses. Laboratory analysis The zinc content in serum and urine was measured using enzyme linked immunosorbant assay (ELISA) kit of BioAssay Systems, USA, by the quantitative colorimetric zinc determination at 425nm. Serum Insulin level was measured by ELISA kit provided by BIOSOURCE, Europe S.A., Belgium. Fasting and post prandial blood glucose was enzymatically measured. HbA1C% was measured by chromatography method, using commercial kit (Sigma), and the normal range of HbA1C% was 5.58.5%. Serum triglycerides and total cholesterol were enzymatically measured. HDL cholesterol was determined after precipitation with phosphotangestate/ magnesium and LDL cholesterol after precipitation with heparin/sodium citrate. Intra- and inter- assay coefficient of variation was < 5% for all laboratory tests. Statistical analysis Number of subjects per group was calculated, based on the mean improvement in FBS (0.701.10mmol/L), shown by Farvid et al., (17), when supplemented with multi vitamin-mineral supplementation. Twenty five subjects per group would provide adequate power (80%) at the alpha error of 5% to detect the treatment effect of zinc supplementation. The number of patients per group was increased by 3 in each group, with an assumption of 10% dropout rate. A one-sample KolmogorovSmirnov test was used to determine whether the baseline biochemical markers were normally distributed. Because the distributions of HbA1C%, triglycerides and zinc, urine zinc and creatinine values were skewed, they were logtransformed in all calculations. For presentation, these variables were transformed back to the original scale. The difference between the three groups at baseline was determined by one-way ANOVA, for continuous data and the Chi test, for categorical data. Paired-t-tests were used to study within-group treatment effects. The effect of intervention was determined by the univariate analysis - repeated measures design of the general linear model using SPSS version 10.0 (SPSS Inc., Chicago, USA). Baseline values of respective parameters were also included in the analysis as a covariant for between-subject factors to correct for their possible confounding influence on the change in each parameter. Of the 96 subjects randomized, only 86 completed the study and their data were included in the final analysis (Per-protocol analysis). Spearmans rho correlation was used to analyze the mean change of treatment effect with the respective baseline parameter. Two-tailed p<0.05 was considered to be statistically significant. Table 2 Baseline characteristics of the three study groups 1 , 2

Res ul ts pr es en te d as me an (S D) ex ce pt th e du ra ti o n of t re at me nt wh ic h is g ive n as me an ( ra ng e) . T re at me nt ca te go ri es a re g ive n as nu mb er of su bj ec ts ( %) 2 T he re w er e no si gn ifi ca nt diff er en ce s i n ag e an d a nt hr o p o me t ri c pa ra me te rs be tw ee n g ro u ps at t he st ud y e nt ry .

1

Table 3 Effect of supplementation on blood glucose and other serum/urine parameters 1

a, b, c

RESULTS

Res ul ts ex pr ess ed as me an (S D) ex ce pt fo r me an ch an ge ; me an (S EM ). On ly th os e wh o ha d co mp le te d th e tr ia l ( n= 86 ) w er e in cl ud ed in t he a na lysis S up er sc ri pt s w it hi n a r ow in di ca te sig nif ic an t d iffe re nc e (p <0 .0 5) ; un iv ar ia te a na lys is w it h b as elin e val ue o f th e res pe ct iv e p ar am et er w as in cl ud ed as a co fa ct or .

There were 96 diabetic individuals (33 males and 63 females) recruited for the study. The baseline characteristics of the three groups are shown in the Table 2. There was no

difference in age distribution of the patients in the three groups (males had mean age of 54.6 7.0 years, whereas females had mean age of 54.9 9.0 years). Group A consisted of 29 subjects (12 males, 17 females), Group B; 31 subjects (11 males, 20 females) and Group C; 36 subjects (10 males, 26 females). However, only 86 patients completed the study (28 out of 29 subjects from the group A, 31 subjects from the group B and 32 out of 36 from the group C). Of the 10 patients who did not complete the trial, 6 were lost to follow up, 3 did not take tablets for more than 2 weeks continuously, and diabetes treatment regimen was interrupted in the other individual. Those who completed the trial had drug compliance of >98%. Majority of the patients were receiving a combination of medication for their diabetes complications. There were no differences in the cholesterol lowering treatment (statins) and other cardio protective medications between the groups. Further, dietary intake of trace mineral and vitamins were not different and the dietary zinc intake was 3.96 (SD 1.68) mg per day (Table 2). Compared to the baseline, the Zinc+MVM group had a mean reduction in FBS by -0.33 (SEM, 0.21) mmol/L (p =0.05). The FBS changes observed in the MVM (+.19 (0.31) mmol/L) and the control groups (+0.43 (0.23) mmol/L) were not statistically significant (p=0.89). The PPBS level also reduced significantly (p=0.04) in the Zinc+MVM group with a mean change of -1.55 (0.56) mmol/L, after the intervention. The MVM group had a significant increase (p=0.002) in HbA1C% level (+1.28 (0.21) %) from the baseline, whereas in the Zinc+MVM group a significant reduction of HbA1C% was seen (-0.01 (0.25)%, p<0.001), following intervention (Table 3). The mean changes observed in FBS, PPBS and HbA1C% after supplementation were found to be negatively correlated with their respective baseline values of FBS (r=-0.46, p<0.001), PPBS (r=-0.52, p<0.001) and HbA1C% concentration (r=-0.48, p<0.001), raising the possibility of regression to mean effect in data. Data were reanalyzed, using parameter estimate function of univariate analysis of general linear model, where the final values were used as the dependant variable and the baseline values as a covariate. The observed (mean difference) and the 95% CI for 3 groups were more or less similar to the previous results. Therefore, we categorized the subjects according to the degree of glycaemic control (optimum control and partially controlled) based on their baseline levels of FBS, PPBS and HbA1C% and the treatment effect was compared (Table 4). When the baseline FBS was <5.56 mmol/L (<100.0mg/dL), there was no significant treatment effect with the intervention. However, when FBS was >5.56 (>100mg/DL), supplementation, with or without Zinc, showed a significant effect on FBS. In Zinc+MVM group, FBS level dropped to 6.21 (0.85) mmol/L from the baseline 6.76 (0.58) mmol/L, whereas in the MV group baseline FBS level of 6.96 (1.20) mmol/L dropped to 6.61 (1.07) mmol/L. When the PPBS level was > 8.89 mmol/L (>160.0 mg/dL) all the groups showed a reduction in mean PPBS with the intervention (Table 4). The HbA1C% level reduced irrespective of the baseline levels only in Zinc+MVM supplemented individuals. When the baseline HbA1C% level was <6.0, the Zinc+MVM group had a significant net reduction (mean level of 5.63 (0.15) at the baseline and 5.53 (1.27), after the intervention, p<0.05). There was no significant effect on either serum or urine creatinine with the intervention (Table 3). There was a significant increase in (mean change 12.44, SEM 1.3 mol/L; p<0.001) in serum zinc concentration in the Zinc+MVM group, but there was no significant change in urinary zinc excretion indicating that zinc supplementation improved only the body stores. Zinc+MVM and MVM supplementations had significant effect on serum lipids (Table 5). Total cholesterol level in Zinc+MVM group dropped from 4.51 (0.99) mmol/L at baseline to 3.73 mmol/L after 4 months of intervention (p<0.05). In the MVM group it dropped from 4.36 at baseline to 4.22 (0.47) mmol/L (p<0.05). Although a reduction in triglyceride level was seen in all the three groups, only in the MVM group the change reached statistical significance (mean change of -0.07, SEM 0.08

mmol/L, p<0.05). Low density cholesterol (LDL) level also reduced in the Zinc+MVM group from 2.60 (0.84) mmol/L to 2.54 (0.77) mmol/L. (p<0.05). Furthermore, cholesterol:HDL ratio reduced in both Zinc+MVM (from 3.39 to 3.21, p<0.05 ) and in MVM (from 3.37 to 3.20, p<0.05) groups, whereas in the placebo group the baseline ratio of 3.42 increased to 3.92 (p<0.05), after the intervention. Table 4 Effect of supplementation on blood glucose parameters stratified by baseline values 1

a, b, c

1 1 Res ul ts ex pr ess ed as me an (S D) . S up er sc ri pt s w it hi n a r ow in di ca te sig nif ic an t d iffe re nc e (p <0 .0 5) ; un iv ar ia te a na lys is.

Table 5 Supplementary effects on Lipid profile of study subjects 1

a, b,

DISCUSSION

Res ul ts ex pr ess ed as me an (S D) ex ce pt fo r me an ch an ge m ea n (S EM ); r at io = Ch ol es te ro l/ HD L c S up er sc ri pt s w it hi n a r ow in di ca te sig nif ic an t d iffe re nc e (p <0 .0 5) ; un iv ar ia te a na lys is.

1

In this study, the average level of HbA1C% for diabetes subjects (7.08 1.2%) was higher than normal range (5.0~6.0%), whereas 53% (n=51) of the subject did not have an optimal fasting glucose level (>5.56 mmol/L) while on treatment. These results indicated that most of diabetic participants in this study were not under a tight glycemic control. It seems that they were not aware of the importance of dietary compliance to control the blood glucose level. Thus, inappropriate food choices might have resulted in the deterioration of zinc status. Both HbA1C % and fasting glucose were significantly improved, after the supplementation in this subset of individuals. Sri Lanka Diabetes, Cardiovascular Study (SLDCS) indicated that the prevalence of diabetes mellitus has continuously increased in the Island (18) and the age-sex standardized prevalence (95% confidence interval) of diabetes for Sri Lankans aged >or= 20 years was 10.3% (9.4-11.2%) [males 9.8% (8.4-11.2%), females 10.9% (9.7-12.1%), P = 0.129). It has been concluded that one in five adults in Sri Lanka has either diabetes or pre-diabetes and one-third of those with diabetes are undiagnosed. Further, the projected diabetes prevalence for the year 2030 is 13.9%.

There is experimental and clinical evidence, especially in developed countries, supporting an alteration of zinc metabolism in patients with diabetes (1, 19). However, only few studies have examined the relationship between zinc and diabetes in developing countries (20). The mean serum zinc level in healthy individuals has varied from 11.4 to 17.8 mol/L (21). A study in Iran by Al-Maroof and Al-Sharbatti (22) reported a lower mean serum zinc value (9.40 mol/L) among diabetic patients. We are reporting a mean zinc level of 10.29 mol/L (SD 7.50 mol/L) in 96 diabetic patients who have been on treatment over a period ranging from 2- 22 years. This level is lower than that (11.30 mol/L) reported in a study of 110 Tunisian adult diabetic subjects (both type 1 and type 2) (23). When the cutoff level of <10.7 mol/L was used to define zinc deficiency, 65% (n=63) of patients had serum zinc levels below this cutoff level, in the present study. Our data showed that zinc supplementation causes an increase in serum zinc level, as reported in other studies (24, 25). This finding proved the good compliance shown by our patients in taking zinc supplementation while having relatively good lipid control. Our results are comparable with previous reports (14, 17) on the effects of zinc with or without other anti-oxidants, on blood sugar, serum insulin, and lipid profile in adult type II diabetes patients, who are already on cholesterol lowering medications. Coronary heart disease (CHD) is a major cause of death in type 2 diabetes patients, and the risk of CHD is two- to four folds higher among patients with type 2 diabetes than in non-diabetic subjects (13). We have observed that zinc+MVM group had mean change of -0.33mmol/l in blood glucose level, where as placebo controlled group had +0.43 mmol/l change in blood glucose after 4 months of supplementation. Therefore, we can speculate an overall +0.76 mmol/l (14mg/100ml) difference with Zinc+MVM supplementation among diabetes patients. When we attempted to compare the magnitude of the supplementation effects by the status of glycemic control, we observed that more significant effects occurred in less controlled group (FBS >100mg/dL), who are on long-term follow up. Furthermore, supplementation significantly improved PPBS and HbA1C% level when compared with placebo. This was similar to the results reported by Hussain et al., (26) where they showed a 25% reduction in FBS and 17% reduction in HbA1C%, with supplementation with zinc and melatonin for 3 month. The mean HbA1C% of the supplemented groups (Zinc+MVM and MVM groups) reduced significantly by 0.3 0.8% at the end of 4 months of follow up, while no significant change was found in the control group. Higher levels of HbA1C% and fasting glucose with longer diabetic duration have been noted by other investigators (27). The significant changes observed in the supplemented groups refer to the effective improvement in their glycemic control in response to zinc and other multivitamin and mineral supplementation. Our observations are consistent with the results of other trials that examined the effect of zinc supplementation on patients with type 2 diabetes (22, 23). Therefore, we suggest that a short- term zinc supplementation may be effective on glycaemic control for diabetic patients with poorly manageable fasting blood glucose. The significant negative correlation we, observed between serum zinc level change after supplementation and the baseline serum zinc level suggests that zinc deficient individuals had greater absorption of zinc than individuals with higher zinc level at the baseline. This finding is consistent with the results of other studies suggesting that zinc treatment will have a high chance of success in changing the zinc status in zincdeficient subjects (28). In addition we observed that baseline serum Insulin levels were positively correlated with final serum Insulin levels and negatively with mean change after the intervention (data not shown). Previous studies examining the effect of zinc supplementation on insulin level are sparse. Hussain et al, (26) found no change in serum C-peptide level with zinc supplementation. Our findings may indicate a direct effect of zinc on insulin secretion by pancreatic cells. However, studies with longer follow up are required to see whether increase in insulin secretion is sustained over a longer time period. Zinc treatment has also been shown to raise body defenses in immune suppressed patients (29). It is important to note that a large proportion of subjects in our study had a low serum zinc level at the baseline and the 4 months daily

administration resulted in almost doubling of the baseline value (mean improvement +12.44 mol/L). We have not addressed issues related to immune status, in the current study and the clinical importance of high prevalence of zinc deficiency among adult diabetics is not known to us. Due to zinc+MVM supplementation, a significant decrease occurred in triglycerides (p=0.02), total cholesterol (p=0.005), LDL (p=0.05) and significant improvement in HDL (p=0.002), when compared with MVM and placebo supplementation. Some investigations indicated that a zinc-enriched diet has beneficial effects on basal and postprandial glycaemia, the content of cholesterol and triglycerides (30). Cunnane (31) believed that zinc intimately affects many aspects of lipid metabolism, through established enzymes, but also has a modulator effects, the mechanism of which is not obvious or established. Our observations are consistent with those reported by Kadhim et al., (32), where a group of poorly controlled diabetic patients were supplemented with zinc and melatonin for a period of 3 months. El-Hendy et al., (33) have shown increased cholesterol, TG and LDL in the serum of zinc-deficient rats. In the current study, we observed a significant decrease in triglyceride concentration (mean change of -0.07mmol/L; P<0.05) only in MVM group. Further, results of randomized controlled trials of Hughes and Samman (34) showed that LDL, total cholesterol and triglycerides in plasma are unaffected by supplementation with up to 150 mg zinc/day. In contrast, HDL concentrations decline when zinc supplements provide a dose >50 mg/d (34). Hooper et al. (35) examined the effect of zinc administration on serum lipoprotein values when 12 healthy adult men ingested 440 mg of zinc sulfate per day for five weeks. He observed that HDL concentration decreased 25% below baseline values (40.5 to 30.1 mg/dL), without changing the total cholesterol, triglyceride and LDL cholesterol levels. In Freeland-Graves et al. (36) study, four levels of zinc supplements (0, 15, 50, or 100 mg/day) were given to 32 women for 8 weeks. No significant differences were seen in HDL-cholesterol over, 8 week except in the 100 mg group at week 4 (36). Partida-Hernndez et al, (14) however, showed a significant decrease in triglyceride concentration following 12 week supplementation with 100 mg zinc sulfate among diabetics who were not on cholesterol lowering treatment. Furthermore, they showed a significant reduction in total cholesterol and an increase in HDL cholesterol indicating that supplementation, in addition to improving glycaemic indices, have favorable effects on other cardiovascular risk factors. Even though, we have shown a similar trend, the reduction in total cholesterol and reduction in HDL levels were not as great as reported. Most of our patients were already on cholesterol lowering drugs and as such they may have achieved the desired lipid targets at the study entry. Additionally, hyperglycemia increases glycation of lipoprotein, including LDL and HDL, associated with the elevation of triglyceride levels in the blood through increased synthesis from glucose and impaired lipid metabolism. Garber and Karlsson (37) showed that the treatment of dyslipidemia in diabetes must be focused on several targets involving glycemic control and bulk reductions of LDL levels. Results of the present study are agreeable to these suggestions. There is a strong evidence suggesting that zinc can act as an endogenous protective factor against atherosclerosis by inhibiting the oxidation of LDL in the presence of transition metals (38), and that adequate zinc nutrition may protect against inflammatory diseases such as atherosclerosis by inhibiting the activation of oxidative stress responsive transcription factors as well as the expression of inflammatory cytokines. The present intervention did not show any significant effect on renal functions measured by serum as well as urine creatinine. Similar observations were made by Khadim et al, (32) and El-Hendy et al, (33). In contrast, a significant improvement in renal function has been observed in a previous study (39) after 60 days of supplementation. Further, Parham et al., (40) showed that zinc supplementation has beneficial effects on microalbuminuria and serum lipid profile in type 2 DM with microalbuminuria and reduced serum homocysteine and increased vitamin B12 and folate concentrations which

may exert vascular-protective effects in type 2 diabetes patient (25). A limitation of our study is that we did not measure urinary micro-albumin level of these patients. We performed this study in single-blinded manner for logistic reasons and this may reduce the validity of data. This may one of the limitations in the study, as alternative designs are possible which are likely to have expectation bias or change in the behavior of the study subjects. However, only the FBS results were available at the follow-up sessions at their respective clinics. Secondly, we did not measure the change in physical activity and anthropometry because of short duration (4 months) of follow up. We are unable to generalize the generated results to the diabetes patient community in Sri Lanka as a whole because of small sample size that may have contributed to inconclusive results seen in some analyses. To minimize the effects of disparity in baseline values between the groups (i.e. high HbA1C% levels in Zinc+MVM group over the other two groups), we included baseline measurements in each analysis as a co factor (see statistical analysis). Although, physicians were free to make changes in the management of these patients, no changes in the major drugs such as anti-diabetics, anti-hypertensives or lipid lowering therapy, were made during the trial period. Apart from the trial drug we inquired about the compliance of their regular medication as well. However, there is a possibility that patients might have changed their behavior (i.e., diet and physical activity) during the trial period. However we expected this change to be uniform in all three groups. This altered behavior can considerably limit the study outcome. In summary, this study indicated significant improvement of fasting glucose as well as HbA1C% in zinc supplemented diabetic patients with shorter diabetic duration, poorer glycemic control, and marginal zinc status. However, further investigation is needed before a firm conclusion could be drawn for the relationship between zinc supplementation and glycemic control. At present, more attention must be paid to improve the zinc status and glycemic control of Sri Lankan diabetes patients, in order to prevent or minimize complications.

CONCLUSION
The supplementation of zinc with other multi vitamins minerals daily, to adult diabetes patients with glycemic and lipid control, for a period of 4 months, demonstrated favorable changes in metabolic profile, including a better glycemic control and desirable changes in lipid profile. Based on the results presented in this report, it seems that zinc is a beneficial mineral for diabetic patients. Consumption of zinc sulfate, in addition to other nutritional and pharmacological treatments in type 2 diabetes patients could be effective in improving the lipid profile.

ACKNOWLEDGEMENTS

The study was funded by the International Atomic Energy Agency (IAEA-SRL-11958). Multi vitamin-mineral Supplements were provided by the Apex Laboratories, India through their local partner, A Baur & Co Ltd, Sri Lanka. We would like to thank Drs. M D Manjula and N M Kaluthantri and other technical staff of the Nuclear Medicine Unit for helping in data collection and sample analysis respectively. The cooperation extended by the Cardiology Unit and the physicians of the Teaching Hospital, Karapitiya in subject recruitment and follow up is also appreciated.

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