Effect of food intake on the activity of liver enzymes in partially hepatectomized rats treated with tumor necrosis factor.

After partial hepatectomy (PHx), there are significant changes in the activity of a number of enzymes in the regenerating rat liver. Administration of low doses of recombinant human tumor necrosis factor-alpha (rHu-TNF) to normal rats induces similar changes in some of the enzymes but not in others. Because certain observations suggest that TNF may play a dominant role in liver regeneration, we speculated that the discrepancies in enzyme activities may be due to the decrease in food intake caused by PHx. Accordingly, the activities of eleven liver enzymes of 70% PHx rats additionally treated i.p. with rHu-TNF (20-50 micrograms/kg/day for 3-4 days) were compared with those of (i) PHx controls fed ad lib., and (ii) PHx controls pair-fed the same amount of food. When pair-fed controls were used, the discrepancies in the activities of the enzymes that are affected by fasting tended to disappear, suggesting that the decrease in the food intake was responsible for the differences.

Tumor necrosis factor induces enzymatic changes in liver comparable to those in extrahepatic cancer.

Human recombinant tumor necrosis factor was administered to rats in small doses to determine whether it causes changes in the activity of liver enzymes similar to those observed in cancer growing extrahepatically. Intraperitoneal injection of increasing doses of tumor necrosis factor (20-100 micrograms/kg/day for 5 days) resulted in a 20-50% decrease in hepatic alanine aminotransferase (P < or = 0.05), a 10-20% decrease in aspartate aminotransferase (P < or = 0.04), and a 50-200% increase in alkaline phosphatase (P < or = 0.02). The activity of hepatic 5'-nucleotidase was unchanged. In the serum, there was no significant change in the activity of any of the enzymes. Histologically, there was no damage detectable by light or electron microscopic examination of the liver, and no evidence of biliary obstruction. However, in frozen liver sections stained histochemically for alkaline phosphatase, there was a dramatic increase in the activity of this enzyme in hepatocytes, which was confined to the bile canaliculi. There was also a 3- to 9-fold increase in the mitotic activity of hepatocytes. Comparable changes have been reported in the tumor-free liver of animals with cancer.

Homogeneous trinder-coupled assay for the determination of glucose-6-phosphatase activity in tissue extracts.

We describe an automated, homogeneous, glucose oxidase-coupled method for the determination of glucose-6-phosphatase activity in tissue extracts. The method is based on measurement of the rate of glucose formation by the Trinder reaction, in which the end product is a quinoneimine dye which absorbs maximally at 505 nm and has a molar extinction coefficient of 5700. The incubation mixture contains 20 microL of tissue extract, 25 microL of 0.5 M phosphate buffer, pH 7.0, 175 microL of Trinder/glucose-6-phosphate reagent, and 30 microL of distilled water. After a delay period of 15 min, to exhaust any glucose endogenously present in the extract, glucose production from glucose-6-phosphate is monitored at 505 nm for 5 min in a centrifugal analyzer. The Km was 13 mM over a 10-fold range in glucose-6-phosphate concentration and the reaction was linear up to about 250 U/L. Within-run CV of the assay at activities of 48 and 190 U/L ranged between 2.5-5.0%. The between-run CV at 190 U/L was 5.1%.

Determination of serum catalase activity on a centrifugal analyzer by an NADP/NADPH coupled enzyme reaction system.

We developed a simple, kinetic method for the determination of catalase activity in which i) the enzyme catalyzes the peroxidation of ethanol by hydrogen peroxide to acetaldehyde and water, and ii) the acetaldehyde so formed is rapidly oxidized to acetic acid and NADPH by the addition of an excess of NADP+ and aldehyde dehydrogenase. The rate of NADPH production was monitored at 340 nm in a COBAS centrifugal analyzer. The reaction was linear to 800 U/L or a delta A of 0.020/min. Using human serum pools containing 80 and 460 U/L of peroxidase activity, the within-run coefficients of variation (CV) were 1.9 and 1.3%, respectively. Between-run CV values were 5% for both pools. The reference range for sera from 72 males and 52 females was 23 to 158 U/L (mean + 2 SD) by log normal transformation. The activity in red cells was 600 U/g hemoglobin but did not change the reference range appreciably provided that serum without visible hemolysis was used. Preliminary observations on sera from nine patients with various pancreatic disorders showed a poor correlation between the activities of catalase (peroxidase) and amylase in serum. The reasons for this discrepancy are under investigation.

Tumor necrosis factor/cachectin decreases catalase activity of rat liver.

Tumor bearing hosts and animals treated with endotoxin commonly show a decrease in the catalase activity of the liver and kidney. Since tumor necrosis factor (TNF)/cachectin may play a significant role in these conditions, we investigated its effects on the catalatic and peroxidatic activity of catalase in the liver and kidney of the rat. The activities of glucose-6-phosphate dehydrogenase and lactate dehydrogenase were measured simultaneously to monitor the pentose phosphate and glycolytic pathways, respectively. Injection i.p. of 100 micrograms/kg/day human recombinant TNF-alpha for 5 days resulted in a significant (P less than 0.01) decrease in the catalatic activity of the liver when compared to rats fed ad libitum. The decrease in four experiments ranged from 21 to 56%. A significant decrease (18%; P = 0.01) in liver catalatic and peroxidatic activity was also observed in another experiment using pair fed rats as controls. The peroxidatic activity of catalase with ethanol as hydrogen donor closely paralleled the catalatic activity. TNF treatment had no detectable effect on the catalatic or peroxidatic activity of catalase in the kidney. The activity of glucose-6-phosphate dehydrogenase increased (31-80%) significantly (P less than or equal to 0.02) in the liver and, to a lesser extent, in the kidney (5-27%, P = 0.05). Lactate dehydrogenase activity decreased (14-19%) significantly (P less than or equal to 0.05) in the liver and kidney but mainly in rats treated with TNF and additionally fasted for 24 h. Electron microscopic examination of liver sections showed that the hepatocytes of TNF-treated rats were undamaged but contained fewer and smaller peroxisomes than those of the control rats.