RESEARCH ARTICLES / BfLİMSEL ARAŞTIRMALAR
Erythrocyte Glutathione 5-Transferase
Activity in Diabetes Mellitus: The Effect of the Treatment
Hilmi ORHAN*, Gönül ŞAHiN*0
Erythrocyte Glutathione S-Transferase Activity in Diabetes Mellitus: The Effectofthe Treatment
Summary : Diabetes mellitus is a chronic disease character- ized by abnormal metabolism, most prominently of car- bohydrates, resulting in elevated blood glucose level in as- sociation with long-term vascular and neurological com- plications. The disorder may be related to the hyper- production of free radicals and/or to a dysfunction of bio- lo gical antioxidant systems because of low antioxidant en- zymes activity or deficient micronutrients status.
Glutathione S-transferase (GST) is a patent intracellular de- toxifier as it catalyzes the conjugations of the thiol group of glutathione (GSH) to foreign or biogenic cytotoxins. Al- though there are a number of investigations concerning the alterations of GSH system and GST activity in both chem- ically induced diabetic animals and in diabetic patients, the results are contradictory.
in this regard, the aim of the present study was to assess the erythrocyte GST activity in diabetics both treated with in- sulin (DTI) and not-treated with insulin (DNTI).
We found that the mean GST activity of the DNT1 group was significantly lower than those of the healthy control group (p=0.0073).
Key words: Glutathione S-transferase, diabetes, erythrocyte, human
Received Revised Accepted
21.04.1999 13.09.1999 15.09.1999
INTRODUCTION
The activity of antioxidant enzymes in the pancreas is low relative to that in other tissues, riı.aking it par- ticularly vulnerable to oxygen attack. It has, in facı,
been suggested !hat cumulative radical rnediated ~
celi darnage induced by a variety of unrelated nox- ious agents rnay be iınportant in the etiology of in- sulin-dependent diabetes. These events rnay be re-
Diabetes Mellitus'da Glutation S-transferazAktivitesi:
Tedavinin Etkisi
Özet : Diabetes Mellitus organizmada özellikle kar-
bonhidratların anormal metabolizmasıyla karakterize kronik bir hastalık olup kan glukoz düzeylerinin yükselmesi uzun vadede vasküler ve nô"rolojik komplikasyonlara yol açar.
Hastalık serbest radikallerin aşırı üretimi ve/veya an- tioksidan enzimlerin düşük aktivite göstermeleri ya da eksik beslenme sonucu biyolojik antioksidan sistemlerde ortaya
çıkan bir düzensizliğe bağlı olabilir.
Glutation S-transferazlar (GST) güçlü hücre içi de- toksifikasyon enzim/eridir; yabancı ya da biyojenik hücresel toksinlerle glutation'un (GSH) tiyol gurubunun kon- jugasyonunu katalize ederler. Literatürde hem deney hay-
vanlarında kimyasal yolla indüklenmiş diyabette, hem de di- yabetik hastalarda GSH sisteminde ve GST aktivitesinde
değişiklik olduğuna ilişkin araştırmalar olmasına rağmen
sonuçlar çelişkilidir.
Bu nedenle bu çalışmanın amacı insülin tedavisi alan (DT!) ve almayan (DNTI) hastalarda eritrosit GST izoziminin ak- tivitesini değerlendirmektir.
DNTI grubunda eritrosit GST ortalama aktivitesinin sağlıklı kontrol gurubuna oranla daha düşük olduğunu saptadık
(p=0.0073 ).
Anahtar kelimeler: Glutation S-transferaz, diyabet, eritrosit, insan
lated to either increased oxidative stress or de- creased defense systern against increased oxidative stress in diabetics. Indeed, plasrna lipid peroxide levels have been found to be increased in diabetic individuals and this increase was rnore pronou;,ced in diabetics with retinopathyl.
Uzel and coworkers have suggested !hat i.ncreased plasrna lipid peroxide levels rnight originate frorn
* Department of Toxicology, Faculty of Phannacy, Hacettepe University, 06100 Ankara I TURKEY.
°
CorrespondenceOrhan, Şahin
the peroxidative destruction of erythrocyte mem- brane lipids2. This has also been demonstrated in vi- tro in human red blood cells (RBCs)3. They in- cubated RBCs with varying concentrations of glu- cose and demonstrated that elevated glucose levels can cause peroxidation of membrane lipids in hu- man RBC. in studying the role of antioxidant de- fenses in diabetes, attention has been given to the role of superoxide dismutase (SOD), catalase (CAT), selenium dependent glutathione peroxidase (Se- GPx) and glutathione (GSH) in various tissues and especially in erythrocyte in hurnan studies4-11.
However, erythrocyte glutathione S-transferase (GST) is another important antioxidant enzyme re- ducing lipid hydroperoxides (so-called Se- independent GPx) and catalysing the conjugation of numerous electrophilic substances with the thiol group of GSH. There are limited and contradictory studies about erythrocyte GST activity in diabetics both in hurnansl2 and in animalsl3-25. Moreover the main GST in erythrocytes (GST-P, accounts for 90- 95% of the total activity towards the common sub- strate 1-chloro-2,4-dinitrobenzene -CDNB-) is shown to be inactivated by oxidative stress prod- ucts, i.e. H202, and could be used a sensitive marker of oxidative stress. Therefore the aim of the present study was to evaluate the change in erythrocyte GST activity in diabetics both treated with insulin (DTI), and not-treated with insulin (DNTI). The present paper, to our knowledge, is the first de- scribing the changes in the activity of erythrocyte GST in the DNTI group and comparing the re- storing effect of both antiglycemic drugs and in- sulin.
MATERIALS AND METHODS
Cheınicals
Ali chemicals used in this study were of analytical grade. GSH, 1-chloro-2,4-dinitrobenzene (CDNB), disodium ethylenediaminetetraacetic acid (Na2EDTA), Folin-phenol reagent and standard bo- vine serum albumin were purchased from SIGMA Co. (St. Louis, Mü., USA). Ethanol, sodiurn chlo- ride, sodiurn hydroxide, sodium carbonate, so-
dium/potassium tartarate, dipotassium hydrogen phosphate and potassium dihydrogen phosphate were obtained from MERCK Co. (Darmstadt, Ger- many). Ali other chemicals were purchased from common commercial sources.
Subjects
Blood samples were obtained from diabetic patients who came to the outpatient clinic of the Endo- crinology Division of Hacettepe University Hos- pital. Only nonsmoking patients without other se- rious diseases and not treated with any drugs except oral antiglycemics or insulin were included in the study. They were divided into groups of diabetics treated with insulin (DTI; n:41, ages 17-78 years,10 men and 31 women) and diabetics not-treated with insulin (DNTI; n:28, ages 18-70 years, 12 men and 16 women). The latter group consisted of 12 patients treated with sulphonylureas, 5 patients treated with a combination of sulphonylureas, biguanides and acarbose, 2 patients treated with acarbose alone, 1 patient treated with biguanides alone and 8 patients without medication (under diet control). The control group consisted of healthy nonsmoking volunteers (n:25, ages 23-56 years, 15 men and 10 women).
Blood Sarnples
Blood samples were collected by venopuncture, im- mediately transferred into polypropylene tubes con- taining heparin and centrifugated for 15 min. at 3,000 xg and 4°C. After removal of plasma and buffy coats, the red cells were washed twice with two vol- umes of phosphate buffered saline of pH 7.00. He- molysates were prepared by addition of two vol- urnes of ice-cold distilled deionized water to the erythrocytes. Cellular debris was removed by cen- trifugation at 4,000 xg and 4°C for 30 rninutes.
Analytical Procedures
Hemolysates were promptly analyzed in triplicate for GST activity towards CDNB using the method of Habig et al26. Fasting blood glucose values were de- terrnined using the method of Somogyi-Nelson27_
Protein content of the samples was determined ac- cording to the Lowry method as rnodified by Mil- ler28 using bovine serum albumin as standard. Al!
measurements were carried out with Shimadzu UV- 160A spectrophotometer except fasting blood glu- cose, the glucose levels were measured color- imetrically by the routine biochemistry laboratory of the university.
Statistical Analysis
The results were expressed as mean ± SE. Between DTI and control groups, statistical significance was as- sessed using student's t test. For the difference be- tween DNTI and control group, Mann-Whitney U test was used, since the variances were not homogen. Cor- relation coefficients between fasting blood glucose and GST activities of DTI and DNTI groups were calculat- ed using the Spearman Rank Correlation test.
·RESULTS
We found a statistically significant difference be- tween diabetics non-treated with insulin and the control group (p=0.0073), but !here was no sig- nificant difference between diabetics treated with insulin group and control group, as seen in Table !.
Table !. The Erythrocyte GST Activity in DTI and DNTIGroups
SUBJECTS GST Activity
Control (n:25) 4.3 ± 0.2 Diabetics treated
with insulin (n:41) 4.1 ± 0.2 Diabetics not-treated
with insulin (n:28) 3.4 ± 0.2* (p:0.0073) Results are expressed ın terms of corresponding mean en- zyme activity (nmol conjugate/min/mg of protein), and represent the mean ± SE (n: 3). Key: (*) significantly differ- ent from the corresponding control activity.
In a subdivision of diabetics not-treated with insulin group, the diabetic group under control by diet was significantly different from the control group (Table Il). The subdivided groups were not significantly different from each other.
Table Il. The Erythrocyte GST Activity in DNTI Subdivided Groups
SUBJECTS
GST ActivityControl (n:25) 4.3 ± 0.2
Diabetics treated with
sulphonylureas (n:l2 3.7± 0.4 Diabetics treated with
combination of sulphonylureas 3.2± 0.7 biguanides and acarbose (n:5)
Diabetics under control
with diet (n: 8) 3.1 ± 0.4* (p:0.0076) Results are expressed in terms of corresponding mean en- zyme activity (nmol conjugate/min/mg of protein), and represent the mean ± SE (n: 3). Key: (*) significantly differ- ent from the corresponding control activity.
Fasting blood glucose values of the patients were measured. They were 157.62 ± 10.33 mg % (mean ± SE) and 126.28 ± 8.59 mg% (mean ± SE) in patients treated with insulin and nontreated with insulin, re- spectively. It was found that !here was no correla- tion between fasting blood glucose values and the erythrocyte GST activity.
DISCUSSION
Glutathione S-transferases are directly involved in the protection of erythrocytes by catalysing the con- jugation of oxygenated derivates with glutathione.
Additionally, erythrocytes may be an important
"sink" far reactive oxygen species such as super- oxide anion and hydrogen peroxide, which are gen- erated at the different sites of organism and can cross the red celi membrane29. Since it is suggested that the major type erythrocyte GST (GST-p) is in- hibited by hydrogen peroxide30,3l, a reduced activ- ity in pathological conditions can be expected, e.g.
in diabetes, and this causes an excess production of reactive oxygen radicals via glucose autoxidation32 and alsa via non-enzymatic protein glycation33.
It has been suggested that the slight suppression of rat liver GST activity deserves attention particularly . with respect to a potentially increased risk of forma- tion of reactive metabolites under these conditions of impaired detoxification capacities18. Carnovale
Orhan, Şahin
and coworkers have reported that GST from differ- ent tissues of rat can be regulated by blood insulin and/ or glucose. Insulin administration to diabetic animals lowered the increased GST activity but could not bring it to the control levell9. Younes et al.
have shown that rat liver cytosolic GST's in short term (4 days) diabetes were decreased, and later re- stored by insulin treatment34, whereas according to Aniya et al., the decrease in GST activity for CDNB was not restored by insulin treatment. They have explained this difference with the starting time of insulin treatment2D. Although there are isozyme and species differences, according to our resul! of the pa- tients treated with insulin, there was no difference between GST-p activities when compared to the control activity, and !his may be due to regular in- sulin therapy (Table I). We were unable to in- vestigate the difference in erythrocyte GST activity before and after insulin therapy in the present study, since it is difficult to establish the groups - especially for the pre-treatment patients.
As previously mentioned, oxidative stress and sub- sequently lipid peroxidation, at least in part, can play a role in the pathogenesis of diabetes. Addi- tionally, erythrocyte GST-p was reported to be in- hibited by reactive oxygen species. Decreased GST activity in the DNTI group in the present study sup- ports this theory. Gliclazide, the relatively novel sul- phonylurea, was suggested to have a powerful free radical scavenging activity35 and another member of the sulphonylureas, glyburide, increased Jiver GSH concentrations in diabetic rats36. Our result suggests that sulphonylureas have a weak res- torative effect on GST activity when compared to the other two subdivided groups, although the number of subjects in the subdivided groups in the DNTI group is not sufficient statistically (Table II).
Whether or not the other members of the sul- phonylurea group have the same scavenging effect and/ or cause GSH increase in erythrocytes, it is rea- sonable to observe higher erythrocyte GST activity in the sulphonylurea-treated group than the other subdivided groups of DNTI.
Further studies are needed to clarify the regulatory
mechanism of erythrocyte GST in diabetics. More- over it is important to dwell on the decrease in GST activity in diabetes and this may contribute to al- tering xenobiotics including drug metabolism and toxicity in diabetes.
Acknowledgement
The authors would thank Prof. Dr. Sema Akalın,
MD, for the blood samples of the patients.
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