The association of age with the activity of alcohol dehydrogenase in human liver.
Ethanol (ethyl alcohol) is principally oxidized in the liver, primarily by the cytosolic enzyme, alcohol dehydrogenase (ADH), to acetaldehyde and then to acetate. In contrast with the decline in clearance of many drugs undergoing microsomal oxidation with ageing, ethanol elimination rates are reported to be unaffected .
ADH exists as multiple isoenzymes in humans, made from combinations of subunits, coded at five gene loci. The typical forms, present in 95% of the white British population, have a pH optimum around 10.5. Atypical forms, present in 5% of the white British population, have a pH optimum of around 8,5.
It has recently been reported that alcohol dehydrogenase (ADH) activity in gastric mucosa contributes substantially to the first-pass metabolism of ethanol, and that activities in the stomach are lower in women than in men. This lower activity may be an important factor in the increased bioavailability of ethanol noted in women and their greater susceptibility to ethanol-induced impairment of muscle and neurological function. Gastric ADH activity has also been reported to fall with age in men, but not in women. The liver contains around 85% of total ADH in humans and is the major site of the systemic clearance of ethanol. Although both sex- and age-associated changes in gastric ADH activities have been reported the situation in human liver has not been classified. We have therefore examined the association of age and sex with alcohol dehydrogenase activities in human liver.
Liver tissue was obtained from 16 subjects (eight men), age range 45-88 years at elective cholecystectomy. All subjects gave informed consent and the study had the approval of the Newcastle Joint Ethics Committee. No subject had hepatic, renal or cardiac disease, nor was any taking drugs known to affect enzyme activity. All subjects reported only occasional ethanol ingestion apart from two subjects drinking 2 units and 4 units ethanol per day, respectively.
All subjects had normal biochemical tests of liver function. Liver biopsies were immediately frozen in liquid nitrogen upon removal, and stored at - 80[degrees]C until use.
Liver tissue (weight 71-271 mg) was thawed and homogenized with ice-cold buffer (0.2 M potassium phosphate, 0.15 M potassium chloride, pH 7.2.5), using a glass to glass homogenizer. Samples were centrifuged for 5 min at 100 g, then 10 min at 18 000 g. The supernatant was centrifuged for 70 min at 120 000 g, the cytosolic fraction retained and stored at - 80[degrees]C until use.
Alhohol dehydrogenase activity was measured by direct spectrophotometry at pH 8.5 and pH 10.5. Incubations were carried out at 25[degrees]C using 500 [micro]1 cytosol, 2.4 mM NAD+ as cofactor and 33 mM ethanol as substrate. The rates of reaction obtained were linear with time and proportional to protein concentration and had substrate concentrations in excess. Activity was expressed in terms of NADH formed per minute, based on an A[sub.340] nm of 6.22 mMcm[sup.-1]. Protein estimations were performed by the method of Lowry et al.. The associations between age, sex and ADH activities were examined bv calculating Kendall's rank order correlation coefficient.
There was no correlation between alcohol dehydrogenase activity and age, whether expressed as activity per mg cytosolic protein (Figures 1 and 2) or per gram wet weight liver (Figures 3 and 4). Mean activities were similar in men and women Table). No subject exhibited the atypical ADH forms.
ADH has broad substrate specificity including oliphatic, aromatic, cyclic, primary and secondary alcohols and their respective aldehydes and ketones. After ethanol ingestion, ADH is the main pathway for its clearance, but is only one of the rate limiting steps in ethanol metabolism. The lack of correlation between hepatic ADH activities in vitro, and oxidation rates in vivo suggests that the rate limiting step of metabolism may depend more upon, for example, competition with other substrates or the rate of reoxidation of NADH. If hepatic ADH activity is reduced, for example by protein deficiency, it may become the main rate-limiting step. Hepatic ADH activity is known to be lower in subjects with alcoholic hepatitis or alcoholic cirrhosis compared with healthy controls but, even in these individuals, ADH activities did not correlate with ethanol clearance in vivo. A significant correlation between gastric ADH activities and first-pass metabolism of ethanol has been reported, suggesting that activities can be rate limiting under certain conditions.
The effect of age on hepatic ADH, activity in the rat has been reported. Activity develops gradually during early life, to stabilize in adulthood and old age[12, 13], although a continual increase in activity in male (but not in female) rats with ageing has been reported. In the latter study, a significant correlation between ADH activity and allylalcohol-induced hepatotoxicity was reported. The age-associated enhancement of hepatotoxicity noted in male rats was felt to be the result of the rise in ADH activity. If this hypothesis has validity in humans, our report of unchanged ADH activity with age is consistent with the clinical observation that, although older patients may present with advanced disease, there is no clear evidence of their having an increased susceptibility to its development.
In contrast with the report the alcohol dehydrogenase activity in gastric mucosa is lower in women than in men, in this small study we noted no significant difference in activities between the sexes. In the group as a whole, we report mean activities of ADH at pH 10.5 of 52.4[+ or -]17.4 nmol/mg cytosolic protein/ minute and of 4.4[+ or -]1.5 [micro]mol/g wet weight liver/ minute. This degree of interindividual variation is lower than that in cadaveric liver biopsy specimens in which a standard deviation of up to 100% from the mean has been reported. Post-mortem effects upon ADH activities in these liver specimens are a probable explanation for these differences. The interindividual variability in ADH activity measured in gastric biopsies from live subjects is consistent with that which we noted in liver tissue collected from a similarly healthy population. Our results suggest that, in common with measurements of mono-oxygenase, esterase, glucuronyl and sulphotransferase activities reported in human liver, age has no effect upon the hepatic activity of ADH.
Table. Hepatic ADH activity (mean [+ or -]SEM) nmol/mg cytosolic pH protein/min [micro]mol/g liver/min Men 10.5 50.7[+ or -]2.8 4.7[+ or -]0.4 8.5 26.3[+ or -]1.7 2.4[+ or -]0.7 Women 10.5 54.0[+ or -]8.0 3.7[+ or -]0.5 8.5 30.3[+ or -]5.7 1.9[+ or -]0.2
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|Author:||Wynne, H.A.; Wood, P.; Herd, B.; Wright, P.; Rawlins, M.D.; James, O.F.W.|
|Publication:||Age and Ageing|
|Date:||Nov 1, 1992|
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