Department of Clinical Chemistry,
1 Central Laboratory and
2 Department of Pathology, University Medical School of Pécs, 7624 Pécs, Hungary
Received 4 March 1999; in revised form 22 September 1999; accepted 7 October 1999
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ABSTRACT |
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INTRODUCTION |
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In the course of our preliminary work, we demonstrated glycohistone in histones of hepatic nuclei of patients dying of decompensated diabetes (Jobst et al., 1991; Jobst and Lakatos, 1996
). At the same time, the spread of alcoholism in Hungary prompted us to examine the effect of EtOH and its metabolite, acetaldehyde (ACAD), on the glycation of histone proteins of diabetic rats.
Based on earlier experiments (Donohue et al., 1983; Tuma et al., 1991
), we have reported a difference in the kinetics of separate and combined addition of glucose (Glu) and ACAD to histone proteins in vitro (Lakatos et al., 1994b
). In the present experiments, we were able to measure fluorescence in histone proteins when incubated with ACAD but not with Glu.
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ANIMALS AND METHODS |
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Group 1. These were diabetic animals: during the first month, they received 10 mg of Streptozotocin (Stz) (Sigma) in 0.5 ml of citrate buffer subcutaneously twice. Five survived until the end of the experiment.
Group 2.
These were EtOH-treated animals: EtOH 15% (v/v) was added to the drinking water supplied to the rats ad libitum, 4 days a week (Nicholls et al., 1994). Five rats survived until the end of the experiment.
Group 3. In addition to inducing diabetes with Stz as in group 1, the rats also received 15% EtOH in the drinking water ad libitum, as in group 2. Six rats survived until the end of the experiment.
Group 4. This was the control group of untreated rats maintained on a normal standard diet and tap water ad libitum. Six rats survived until the end of the experiment.
The experiments were performed with the permission of the Scientific Council (ETT) of the Hungarian Ministry of Health and followed their ethical guidelines.
Laboratory procedures
Determinations were made once a week in the first month and then once a month for 9 months. After month 9, the rats were killed. Histones were isolated from the liver using the acid extraction method (Johns, 1971). In addition to body weight, Glu was also measured using the GODPOD method (Trinder, 1969
). Glycated haemoglobin (GHgb) and HgbA1C were determined by the Abbot IMX method (Wilson et al., 1993
). Fructosamine was assayed by the NBT Roche method (Baker et al., 1991
). Total protein was determined using the biuret method (Tietz, 1986
). Serum ACAD was measured by gas chromatography (Duritz and Truitt, 1964
). Glycohistone was determined by the fructosamine NBT test (fram) and by the fluorimetric method (flsc) described earlier (Lakatos and Jobst, 1989
). Fluorimetry is the usual method for determining the advanced glycation end-product (AGE) of proteins (Gugliucci and Bendayan, 1995
).
Tissue samples from livers and kidneys were fixed in formalin and alcohol. Alterations were evaluated in frozen sections and after paraffin embedding using routine histological staining.
The paired t-test was used for statistical analysis.
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RESULTS |
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In addition to a slight elevation in blood Glu (P = 0.09) in group 2 (alcoholic rats), the analytes indicative of glycation decreased slightly. The decrease may be ascribed to the high value of ACAD in alcohol-treated rats. The lower level of fructosamine also shows this. At the same time, a 50% higher value was obtained for H-flsc (P = 0.06).
In group 3 (diabetic rats given EtOH), all parameters indicative of the development of diabetes were also observed, as in group 1, but to a lesser extent. Contrary to our expectations and in spite of moderately higher ACAD, no values indicative of increased histone glycation were observed. We cannot explain why the level of the characteristic values of diabetes are lower than in group 1.
Microscopically, decreased glycogen, fatty degeneration and fibrosis were observed in the liver of rats treated with Stz with or without EtOH. The kidneys showed signs of heavy proteinuria. The thickened glomeruli were slightly lobulated and numerous loops were thickened. In EtOH-treated animals, diffuse fatty degeneration of the liver was dominant.
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DISCUSSION |
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Our answer to the first question is this: the conditions of glucose-protein addition are more favourable in serum and in erythrocytes than within the nucleus. This is proved by the elevation in the values of serum fructosamine and glycated haemoglobin. It is not known whether, during their synthesis, histones are glycated and enter the nucleus as glycohistones, or if they take up glucose non-enzymatically, i.e. glycation takes place in the nucleus. However, after comparison with control animals, we failed to observe increased glycation. This finding is not in agreement with what was observed earlier in human material, where we had noted a moderate increase in liver histone in patients dying of diabetes. (This would not be the first discrepancy between findings in human and animal experiments.)
In the second group, treated with EtOH only, the drug did not greatly influence the glycation of histones. This finding agrees with the observation made by Al-Abed et al. (1999) on haemoglobin in circulating blood. At the same time, the level of serum ACAD was elevated, which resulted in considerable fluorescence. It is of interest that we also found elevated ACAD values and elevated histone fluorescence, in the diabetic group. Since it is known from our earlier investigations (Lakatos et al., 1994b) that, in contrast to the histoneACAD reaction product, the histoneGlu adduct does not fluoresce; a secondary reaction in response to the effect of ACAD, the formation of an AGE, may be an explanation.
An answer to question 3 is given by the result obtained for group 3. This agrees with our earlier in vitro data: the simultaneous effect of diabetes and EtOH, i.e. the reaction of Glu and ACAD together gives lower values for glycohistone than do the two aldehydes separately. We do not know why this is so; however, our finding is in good agreement with the latest results of Al-Abed et al. (1999).
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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REFERENCES |
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