urinary selenium in healthy and diabetic saudi arabians

OCopyright 1996 by Humana Press Inc. All rights of any nature whatsoever reserved. 01634984/96/520143055 $07.25

Urinary Selenium in Healthy and Diabetic Saudi Arabians

ADNAN EL-YAZIGI* AND ERLINDA LEGAYADA

Biological and Medical Research, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh,

11211 Saudi Arabia

Received January 27, 1995; Accepted March 3, 1995

ABSTRACT

We examined Se in urine of 170 Saudi Arabian diabetics (19 insulin-dependent [type 1] and 151 insulin-independent [type 2]) and in an equal number of control subjects of the same origin by measuring the ratio of the concentration of this metal (CSe) to that of creatinine in urine (Ccreat) for each subject. The mean (and SEM) of CSe/Ccreat for the control subjects was 56 (2.9) ~tmol/mol creat, whereas, the value for the diabetics combined or separated into type 1 and type 2 was 56.7 (3.2), 51.5 (6.3), and 57.4 (3.5) ~tmol/mol creat, respectively. With the exception of type 2 diabetics who were treated with insulin in addition to oral hypoglycemic and diet (35 patients) (mean [SEM] -- 43 (4.3) ~tmol/mol creat), there was no significant difference in Cse/Ccreat between the diabetics and control subjects. Also, there was no significant correlation between CSe/Ccreat and age, sex, or weight of diabetics, whereas, the correlation with the degree of diabetic control was significant (p _< 0.0136). Of all diabetes-associated disorders (cardiovascular diseases, neuropathy, ophthalmologic diseases, infections, and hepatic disease), only ophthalmologic diseases appears to cause a significant (p < 0.05) reduction in CSe/Ccreat, but only among type 2 diabetics. Inasmuch as Se status is reflected by urinary Se, healthy Saudi Arabians appear to have Se status that is comparable or higher than those reported for other populations.

Index Entries: Selenium; diabetes mellitus; urinary excretion; spectrofluometry; insulin treatment; diabetes-associated disorders.

INTRODUCTION

Selenium is an essential trace metal wi th a protective capacity against cell membrane damage caused by peroxides generated from lipid

*Author to whom all correspondence and reprint requests should be addressed.

Biological Trace Element Research 5 5 Vol. 52, 1996

56 El-Yazigi and Legayada

metabolism. This antioxidant role is directly linked to its essential involvement in the glutathione peroxidase enzyme system. A deficiency of Se has been directly associated with cardiomyopathy known as Keshan diseases (1,2), cancer (3,4) chronic liver diseases (5), alcoholism (6,7), ischemic heart disease (8,9), and a host of other diseases.

There is growing but conflicting evidence of an association between Se status and diabetes mellitus. Compared to matching control groups, a decrease (10), an increase (11,12), and no significant difference (13,14) in the concentration of plasma or serum Se in diabetic children (11,12) or adults (10,13,14) have been reported by various researchers from different countries. Plasma Se increased markedly in streptozotocin-induced diabetic rats, but this abnormality improved after insulin administration (15). Furthermore, rats maintained on a diet deficient of vitamin E and Se, i.e., poor antioxidant state, exhibited an enhanced susceptibility to diabetes following normally nondiabetogenic doses of streptozotocin (16). Administration of Se in the form of selenite to streptozotocin- induced diabetic rats drastically diminished the high level of serum glucose which returned to normal after repeated doses of selenite (1 7).

This study was conducted to investigate the urinary Se in the diabetic state, and to examine the influence of diabetes types and associated disorders on the urinary excretion of this metal. Further, as the presence of Se is dependent upon the soil composition, water, and types of food ingested, etc., we aimed at establishing the Se status in healthy Saudi Arabians using its urinary concentration as an indicator and comparing our data with those generated for similar subjects of different countries.

MATERIALS AND METHODS

Subjects Diabetics

Included in this group were 170 Saudi Arabian diabetics (103 male, 67 female) attending the Diabetes Clinic of King Faisal Specialist Hospi- tal and Research Centre. They were classified as type 1 (19 patients) or type 2 (151 patients) according to established criteria. Some of the type 2 diabetics (35 patients) received insulin in addition to oral hypoglycemics and a special diet (type 2i), and the remainder did not (type 2hi).

Control Subjects This group consisted of 170 nondiabetic volunteers (mean and [fiEM]

of age = 37.2 (0.9) y) from the same region of Saudi Arabia, i.e., Riyadh and its surroundings. Eighty two of these 94 males and 76 females were apparently healthy individuals and the rest were seen at this institution for medically unfounded general complaints, orthopedic, or infertility prob- lems. The biochemical blood and urine profiles of these subjects were normal with normal electrocardiogram and radiological findings. We

Biological Trace Element Research Vol. 52, 1996

Selenium in Diabetes in Saudi Arabia 57

reconfirmed the nondiabetic status of all these subjects by use of the Dip & Read Test for glucose in urine (Boehringer Mannheim, Indianapolis, IN).

From each of the diabetics and control subjects, a urine sample was collected in a plastic urine container and immediately frozen (i.e., -80 ~ until analysis.

Reagents The concentrated hydrochloric acid, concentrated perchloric acid

(both Baker Instra Analyzed Reagents for Trace Metal Analysis), and Se standard (1 mg/mL) used were purchased from J.T. Baker, Phillisberg, NJ. The concentrated nitric acid, cyclohexane, and ethylenediamine tetraacetic acid tetra sodium salt (EDTA) were either of Spectrosol (first two) or analytical grade from BDH Laboratory, Poole, England. The derivatization reagent, i.e., 2-3 diaminonaphtalene hydrochloride (DAN) was high purity (> 99%) reagent from Aldrich Chemical, Milwaukee, WI.

Analysis of Se in Urine The fie level was measured in urine according to the simplified flu-

orometric assay of Sheehan and Gao (18). A fluorescence spectropho- tometer (Model MPF-44 from Perkin Elmer, Beaconsfield, U.K.) set at excitation and emission wavelengths of 366 and 520 nm, respectively, was used.

The urine sample was centrifuged at 3500 rpm for 5 min to remove debris, and a 100 ~tL portion was transferred into a test tube containing 500 ~tL of the digestion acid mixture (concentrated nitric acid:perchloric acid; 4:1, by volume) and several boiling chips. After shaking, the tube was heated for 90 min at 190 ~ by placing it in a thermal block (Hycel, Houston, TX) hole. The tube was then removed and the heating block temperature was adjusted to 150 ~ After 10 min cooling at room temperature, 500 ~tL of concentrated hydrochloric acid was added to the tube which was replaced in the heating block hole for 30 min. During this time, the derivatization reagent, i.e., 2-3 diaminonaphtalene hydrochloride (DAN) in distilled water (6.3 mmol/L) was prepared by ultrasonification. The tube was removed from the block, and 2 mL of EDTA aqueous solution (2.5 mmol/L), 500 ~tL of the DAN solution, and 2 mL of cyclohexane were immediately added. The tube was capped, and was left at 60 ~ for 30 min prior to vortex-mixing for 2 min. After centrifugation for 10 min at 3000 rpm, the cyclohexane layer was removed and transferred into the spectrofluorometric cell, and the fluorescence intensity was read at the above-mentioned excitation and emission wavelengths. The concentration of total Se in the sample was calculated by use of a standard curve produced on the same day under identical conditions.

The reliability of the data generated was established daily by exam- ining the linearity of the assay, and measuring Se in quality control urine samples (Bio-Rad, ECS Division, Anaheim, CA). The assay was highly



linear (r = 0.9921-0.9997; mean = 9968; CV = 0.19%) in the range 0.1-3.17 ~t mole/L used, and the concentrations of this metal in the quality control samples were consistently within the range specified by the supplier.

Analysis of Creatinine in Urine We analyzed creatinine in urine using a Jaffe based reaction

(Boehringer Mannheim Automated Analysis for BM Hitachi Systems 747/737) on a Hitachi 737 analyzer.

Statistical Analysis

Both parametric and nonparametric statistical tests [STATGRAPH- ICS Statistical Graphic System package, Statistical Graphics Co., Rockville, MD) were used to analyze the data generated. The test employed was specified where appropriate, and the difference or correlation was considered statistically significant if p < 0.05.

RESULTS

The mean and (SEM) of age and urinary creatinine concentration (Ccreat) of the Saudi Arabian control subjects were 37.2 (0.9) y and 8.79 raM, respectively, and the mean and (SEM) of the urinary Se concentration (CSe), and the normalized urinary Se concentration which is the urinary content of Se/mol of creatinine (i.e., urinary Se concentration/ urinary creatinine concentration [CSe/Ccreat]) observed for these 94 men and 76 women were 0.394 (0.017) ~tM (31.1 [1.3] ~tg/L) and 56 (2.9) ~t rnole/mol creat (39.1 [2] ~tg/g creat), respectively. There was a highly significant correlation (p < 0.0000) between CSe and Ccreat (Fig. 1), but no significant correlation was observed between CSe/Ccreat and age or sex of the control subjects.

The clinical and vital characteristics of the various groups of diabetics (type 1, type 2i, type 2ni, and type 2 [i and ni combined]) investigated are summarized in Table 1. The overall means and (SEM) of the age, weight, hemoglobin Alc (HbAlc), C-peptide, and urinary creatinine concentration in the diabetics were 52.6 (0.9) y, 73.4 (1.2) kg, 11.2 (0.2) %, and 7.04 (0.38) raM, respectively. Of all these parameters, only C-peptide concentration showed by analysis of variance a significant difference among the groups investigated, presumably due to its expectedly low value in type 1 diabetics. A highly significant correlation (p ___ 0.0000) between CSe and Ccreat was observed for the overall diabetics (Fig. 1), as was the case for the control subjects. There was no significant correlation between CSe/Ccreat and age or sex of these patients, however, a positive, significant correlation was observed between this parameter and C-peptide concentration (p < 0.0155) or HBAlc (p -< 0.0136) according to linear regression analysis.



Control Diabetics ' ' ' ' I ' ' * ' I . . . . I ' ' ' ' I /

1.5 r = O . ~

1 1.2

.o

.~ 0.9

~ u a a ~ o a a

0.6 Oo~ .~o

o ~ ~~

~ o.3 rJl

�9 i , i I i , i i I , �9 �9 . I . . . .

0 1 2 3 4

1 . S * * , ' I , ' ' ' I . . . . I . . . . I .

r = 0.6788 p = 0.0000

1.5

1.2

0.9

0.6

g ~ Q

0.3 ,.~o ~

;o , , , , I , * , , I . . . . I . . . .

0 1 2 3 4

(x loooo) Creatinine Concentration, pM

Fig. 1. Relationship between urinary Se concentration and urinary creatinine concentration for the diabetics and control subjects.

With the exception of patients with type 1 diabetes, there were significant (p < 0.05) or near-significant (0.05 < p < 0.1) differences in the urinary concentration of Se between the control group and the diabetics combined or separated into groups (Table 2). However, when the CSe is normalized by dividing by Ccreat (i.e., C S e / C c r e a t ) for each subject, the differences become nonsignificant with the exception of type 2i, in which the value was smaller (p < 0.056) than that in the control subjects. The normalization of CSe was necessary to eliminate the impact of the increased urine volume effected by diabetes due to polyurea. This was clearly evident from the significant (p < 0.00184) difference in Ccreat

between the diabetics [mean (SEM) = 7.04 (0.38) mM] and control subjects [8.79 (0.41) mM].

The differences in CSe and CSe /Cc re a t among the various diabetic groups investigated and between patients with or without a diabetes-associated disorder are presented in Table 3. As demonstrated in this table, with the exception of the difference (p <_ 0.0222 by t-test or p < 0.00995 by Willcox rank test) in the CSe/Ccreat between type 2 diabetics who received insulin (type 2i) and those who did not (type 2ni), there was no significant difference in either Cse or CSe/Ccreat among the diabetic groups investigated. Of all diabetes associated disorders, only ophthalmologic diseases appear to cause a significant (p < 0.05) reduction in CSe/Ccreat, but only among type 2 diabetics which represent 88.8% of our patients.



Table 1 Mean (and SEM) of Clinical Parameters for the Diabetics

Overall Type 1 Type 2 Type 2i a Type 2hi b

Sex (M/F) 103/67 16/3 87/64 23/12 64/52

Age (yr) 52.6 (0.9) 48.4 (3) 53.1 (0.9) 53.6 (2) 52.9 (1.1)

Weight (kg) 73.4 (1.2) 72.1 (3.6) 73.5 (1.3) 74.6 (2.5) 73.5 (1.6)

HbAlc (%) 11.2 (0.2) 10.9 (0.8) 11.3 (0.2) 10.7 (0.4) 11.4 (0.3)

C-peptide 3.00 (0.22) 0.7 (0.1) 3.36 (0.24) 2.67 (0.37) 3.66 (0.3)

Urinary 7.04 (0.38) 8.59 (1.91) 6.84 (0.35) 7.40 (0.49) 6.67 (0.43) Creatinine (mM)

aType 2 treated with insulin in addition to diet and oral hypoglycemics. bType 2 treated with diet and oral hypoglycemics only.

Table 2 Urinary Se in the Diabetics and Control Subjects

CSe CSe/Ccrea t

Group Mean (SEM) pa Mean (SEM) p M) (~ mole/mole Creat)

Control 0.394 (0.017) 56.0 (2.9)

Diabetics 0.339 (0.02)

Type 1 0.383 (0.074) Type 2 0.334 (0.02) Type 2ni 0.344 (0.025) Type 2i 0.301 (0.032)

CVD c 0.311 (0.022) Infections 0.290 (0.041) Ophth',dmologic diseases d 0.278 (0.032) Neuropathy 0.274 (0.03) Hepatic disorders 0.310 (0.028)

0.037 56.7 (3.2) NS b

NS 51.5 (6.3) NS b 0.023 57.4 (3.5) NS 0.084 61.7 (4.3) NS 0.019 43.0 (4.3) 0.056

0.0025 54.5 (4.2) NS 0.017 55.2 (6.4) NS 0.0016 49.3 (5.2) NS 0.0016 54.0 (6.1) NS 0.017 65.4 (7.9) NS

aLevel of significance of the difference from the control group according to the two- tailed t-test.

bNonsignificant. cCardiovascular diseases including hypertension. dRetinopathy and other diseases of the eye.


Selenium in Diabetes in Saudi Arabia

Table 3 Ratio of the Means of Urinary fie in the Diabetic Groups Examined

61

Group CSe CSe/Ccrea t

Type 1 / Type 2 1.15 0.90

Type 1 / Type 2i 1.27 1.20

Type 1 / Type 2ni 1.ll 0.84

Type 2ni / Type 2i 1.14 1.44 a

CVD / No-CVD 0.80 0.88

Retinopathy / No-Retinopathy 0.74 a 0.82

Neuropathy / No-Neuropathy 0.73 b 0.98

Infections / No-Infections 0.83 0.95

Liver disease / No-liver disease 0.88 1.21

a0.0174 <_ p _< 0.0222 bp _< 0,0712

DISCUSSION

The use of urinary Se for assessing the status of Se in different populations has been recently reviewed by Alaejos and Romero (19). To our knowledge, no such data have been reported for Saudi Arabians. Because of differences in genetics, environment, soil composition, and food habits, one may assume that Se status in healthy Saudi Arabians may be different from those reported for other populations. However, the mean urinary concentration of Se, both before (i.e., 0.394 ~M or 31 gg/L) and after normalization to the content of creatinine (56 g tool/tool creat or 39.1 ~tg Se/g creat) obtained in this study for healthy Saudi Arabians is similar to those reported for several European, North American, South American, and Asian populations (19), but somewhat higher than those reported for others, i.e., Italians, French, British, and Poles (19). This may be attributed to the highly enriched food (wheat, meat, milk, etc.) cur- rently produced and consumed in Saudi Arabia, and the importation of other quality foodstuffs from the West.

Although several studies have examined the effect of diabetes mellitus on the concentration of Se in serum, none, to our knowledge, investigated the urinary Se in these patients. Thus, our study is apparently the first to address this point. Based on the results obtained it appears that



regardless of the type of diabetes and the disorders associated with it, the urinary excretion of Se in diabetics is not significantly different from that in control subjects of the same origin. This seems to concur with previous reports (13,14) indicating a lack of influence of diabetes on Se status as determined by plasma concentration, but disagree with others with opposing results (10-12). It is noteworthy that our study is by far the largest (i.e., 170 diabetics and 170 controls) that have been reported to date. The disagreement between results of different studies, including ours, on the impact of diabetes on Se status may be ascribed to dissimi- larities in the intake and metabolism of Se between the various diabetic populations examined for the reasons indicated above.

The lack of significant correlation between CSe/Ccreat and age, sex, or weight observed in this study among Saudi diabetics agrees with the finding of Gebre-Medhin et al. (12) in diabetic Swedish children, however, this was not the case with hemoglobin Alc (HbAlc). Indeed, we found a significant, positive association between this parameter and the urinary excretion of Se, indicating that urinary Se is inversely related to the degree of diabetes control.

Our data indicate that there was a significant inverse association between insulin administration and urinary Se in type 2 diabetics. Indeed, patients who received insulin as part of their treatment excreted 44% less Se than those who did not. Inasmuch as urinary Se reflects that of the plasma, this finding in patients concurs with a previous report (15) indicating a drop in plasma Se following the administration of insulin to streptozotocin-induced diabetic rats. On the other hand, the significant reduction in the urinary excretion of Se observed in this study among type 2 diabetics with ophthalmologic disorders suggests that Se depriva- tion which reflects a poor antioxidant state, may be involved in the pathogeneis of these disorders.

In conclusion, the urinary Se data presented in this report are the first to be reported on Se status for healthy and diabetic Saudi Arabians. The values obtained are comparable or higher than those reported for similar healthy subjects from different European, Asian, North American, or South American countries. There was no significant difference in urinary content of Se/mol of creatinine between diabetics and control subjects; however, among type 2 diabetics there seems to be significant associations between this parameter and the degree of diabetic control, treatment with insulin, or diabetes associated ophthalmologic disorders.

ACKNOWLEDGMENT

We thank the Administration of the King Faisal Specialist Hospital and Research Centre for their support and encouragement of the diabetes research program in this institution.



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urinary selenium in healthy and diabetic saudi arabians

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