The effect of alcohol, isoniazid, rifampicin, paracetamol and hexane on hepatic gluconeogenesis and bromosulphthalein clearance.

Abstract

The first workers to use the isolated perfused rat liver for the study of gluconeogenesis were Corey and Britton (1941). Subsequently, other investigators found the modified method of Miller et al (1951) to be more suitable. This technique, with modifications introduced by Mortimore (1961) and Hems et al (1966) was used in the present study. The isolated liver is perfused through the portal vein with saline, supplemented by bovine serum albumin and washed human erythrocytes, under a pressure of about 20cm of water, maintained by a reservoir of adjustable height. The perfusate which passes through the liver enters the inferior vena cava and passes, via a cannula, to a collecting vessel from which it is pumped to the top of a multiple bulb oxygenator and then returned to the liver for re-perfusion. This technique has proved to be a satisfactory means of assessing changes in the metabolic status of hepatic cells in response to starvation and exposure to halothane. The study described here was performed to determine whether the isolated liver perfusion technique can be used to measure the effects on liver perfusion of therapeutic and supratherapeutic doses of various drugs, some of which have been reported to affect liver metabolism adversely in the intact animal. Liver function was assessed by studying gluconeogenesis and bromosulphthalein clearance. Alcohol and hexane were administered in toxic doses, rifampicin and isoniazid in high doses and paracetamol in therapeutic doses. Inbred male Wistar rats were used for these studies. Hexane was injected subcutaneously, while the other drugs were given per os on 7 consecutive days each week for a period of 90 days; with the exception of the control group in the hexane study, all the control groups were untreated. Pyruvate, a precursor for gluconeogenesis (synthesis of glucose from non-carbohydrate sources) is an excellent substrate for the formation of oxaloacetate, which is probably an obligatory intermediate in the pathway to glucose synthesis. It has been used over a number of years by different investigators who have .studied gluconeogenesis using the isolated liver perfusion technique. It was used for the same purpose in the present study. Methylene blue, a redox dye, capable of oxidising NADH to NAD+, was used to determine whether an altered NADH : NAD+ ratio would have any effect on the output of glucose in the ethanol, paracetamol and hexane studies. Fructose, a non-NAD+ dependent precursor of glucose. was also used for this purpose in the ethanol study. All the drugs studied were found to inhibit gluconeogenesis. This was shown by a decrease in glucose levels and an increase in lactate : pyruvate ratios in the perfusion medium of experimental livers. The decreased glucose production by the experimental livers, which occurred pari passu with an increased pyruvate utilization, indicates that in these animals pyruvate was used for the production of other compounds such as lactate. In contrast. glucose production and pyruvate utilization were increased in the control group indicating that pyruvate was used mainly for the production of glucose. In the ethanol group, impaired gluconeogenesis was probably due to a change in the NADH : NAD+ ratio; when methylene blue was introduced into the perfusion medium of this group the output of glucose was high. Impaired gluconeogenesis in the paracetamol and hexane-treated groups was probably related to the non-availability of oxaloacetate or impairment of the activity of key enzymes involved in gluconeogenesis; when methylene blue was added to the perfusion medium of these animals the glucose output remained low. Except for the rifampicin study. bromosulphthalein clearance was impaired in all the experimental groups. Histological examination of liver tissue obtained from the hexane-treated animals demonstrated severe fatty change. In conclusion, these studies have demonstrated that the isolated liver perfusion technique is a suitable method of evaluating the effect of therapeutic and supra-therapeutic doses of some drugs which affect hepatic function. Ethanol, isoniazid, rifampicin, paracetamol (in therapeutic doses) and hexane were found to alter liver function as evidenced by impaired gluconeogenesis and bromosulphthalein clearance. In addition, histological evidence of liver damage was noted in rats treated with hexane.