Mikael Farstad

Determination of adenine nucleotides and inosine in human myocard by ion-pair reversed-phase high-performance liquid chromatography

An isocratic high-performance liquid chromatographic system for the quantitation of AMP, ADP and ATP is presented. The separations were achieved at room temperature by reversed-phase chromatography (Supelcosil LC-18). The standard solvent was 220 mM potassium phosphate, pH 6.9, 1% (v/v) methanol and 0.3 mM tetrabutylammonium hydrogen sulphate. A selective retention of the adenine nucleotides as a group relative to the mono-, di- and triphosphates of guanosine, uridine and cytidine was observed under these experimental conditions. The adopted procedure was applied to the separation of adenine nucleotides in biological extracts, i.e., human myocard. The adenine nucleotides in an extract of myocard were quantitated in less than 20 min. Only 5-10 mg (wet weight) of myocard were needed in order to determine the energy charge of a myocardial sample. Also inosine was easily quantitated in this liquid chromatographic system.

Sixty minutes of normothermic ischemia in the rat liver: Correlation between adenine nucleotides and bile excretion

The effect of 60 min of normothermic liver ischemia on cellular levels of adenine nucleotides (ATP, ADP, and AMP), energy charge (EC), and bile excretion was studied. Following ischemia the concentration of ATP was reduced to 12% of preischemic and control values within the first 10 min and remained low during the remaining ischemic period. EC values were also low. During 120 min of reperfusion, ATP increased to 34% of the ATP level found in the control group. EC values increased immediately to reach values not significantly different from those of control animals. Bile flow was nonexistent during ischemia and increased during reperfusion. The increase paralleled those of ATP and EC. Bile flow seems to reflect the degree of liver ischemia and may be used as a functional parameter.

Hepatic enzymes, coash and long-chain acyl-coa in subcellular fractions as affected by drugs inducing peroxisomes and smooth endoplasmic reticulum

1. The activities of acyl-CoA hydrolase, catalase, urate oxidase and peroxisomal palmitoyl-CoA oxidation as well as the protein content and the level of CoASH and long-chain acyl-CoA were measured in subcellular fractions of liver from rats fed diets containing phenobarbital (0.1% w/w) or clofibrate (0.3% w/w). 2. Whereas phenobarbital administration resulted in increased microsomal protein, the clofibrate-induced increase was almost entirely attributed to the mitochondrial fraction with minor contribution from the light mitochondrial fraction. 3. The specific activity of palmitoyl-CoA hydrolase in the microsomal fraction was only slightly affected while the mitochondrial enzyme was increased to a marked extent (3-4-fold) by clofibrate. 4. Phenobarbital administration mainly enhanced the microsomal palmitoyl-CoA hydrolase. 5. The increased long-chain acyl-CoA and CoASH level observed after clofibrate treatment was mainly associated with the mitochondrial, light mitochondrial and cytosolic fractions, while the slight increase in the levels of these compounds found after phenobarbital feeding was largely of microsomal origin. 6. The findings suggest that there is an intraperoxisomal CoASH and long-chain acyl-CoA pool. 7. The specific activity of palmitoyl-CoA hydrolase, catalase and peroxisomal palmitoyl-CoA oxidation was increased in the lipid-rich floating layer of the cytosol-fraction. 8. The changes distribution of the peroxisomal marker enzymes and microsomal palmitoyl-CoA hydrolase after treatment with hypolipidemic drugs may be related to the origin of peroxisomes.

Direct measurement of free coenzyme A in biological extracts by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic system was developed with baseline separation of coenzyme A (CoASH) from dephospho-coenzyme A and acetyl-coenzyme A using isocratic elution. The chromatographic separation was achieved in a reversed-phase system with a high concentration (220 mM) of potassium phosphate buffer at pH 4.0 and appropriate amounts of methanol (ca. 12%). The eluate was monitored with a UV detector at 254 nm with the limit of detection at ca. 5 pmoles. The system could be used without modification for the estimation of the content of CoASH in biological extracts, e.g. mitochondria.

Effects of clofibrate on the intracellular localization of palmitoyl-CoA hydrolase and palmitoyl-L-carnitine hydrolase in rat liver

Feeding clotibrate to rats will induce a prominent increase in the number of peroxisomes [ 1 ] and of the capacity for fatty-acid oxidation [2,3]. Clofibrate feeding also results in a great increase in the hepatic content of free CoA and long-chain acyl- CoA [4--61 and an increase of the acylCoA hydrolase activity (71. We now report that clofibrate treatment of rats results in an increase of the activity of both palmitoyl- Lcarnitine hydrolase and palmitoylCoA hydrolase. Also a subcellular redistribution of the latter enzyme is found, possibly due to release from structurally changed microsomal membranes (endoplasmatic retic- ulum) to the particle-free supernatant. 2.

Enzymatic changes in rat liver associated with low and high doses of a peroxisome proliferator.

The activities of a number of lipid-metabolizing and subcellular marker enzymes were measured in total homogenates and subcellular fractions prepared from the livers of male rats fed diets containing 0.05, 0.1, 0.3, and 0.5% of the hypolipidemic drug tiadenol, resulting in mean drug intake of 45, 90, 330, and 530 mg/day/kg body wt, respectively. In the total homogenates, a massive induction of palmitoyl-CoA hydrolase and peroxisomal palmitoyl-CoA oxidation accompanied by increased free CoASH and long-chain acyl-CoA content was observed at the highest dose levels whereas little change occurred up to 90 mg/day/kg/body wt. The palmitoyl-CoA synthetase activity increased slightly up to 90 mg/day/kg body wt, but higher doses resulted in decreased enzyme activity. Catalase activity increased with the dose to be elevated by a factor of approximately 1.6 at 330 mg/day/kg, whereas the activities of urate oxidase decreased. The specific activities of palmitoyl-CoA hydrolase and peroxisomal palmitoyl-CoA oxidation increased in all fractions, but most markedly in the cytosol. The changes in the activities and the distribution of subcellular marker enzymes and the increase of the peroxisome-associated polypeptide (PPA-80) are in keeping with a peroxisome proliferating effect resulting in formation of premature organelles with altered properties. Since high doses of many hypolipidemic drugs produce hepatic tumors and peroxisomal proliferation in rodents and since no increase in peroxisomes is found in human liver after therapeutic use of lower doses, the dose-response relationship is of interest for the evaluation of the toxicology of this class of agents.

Influence of dietary status on liver palmitoyl-coa hydrolase, peroxisomal enzymes, coash and long-chain acyl-coa in rats

In the livers of fasted rats, the activity of mitochondrial palmitoyl-CoA hydrolase was increased whereas the microsomal palmitoyl-CoA hydrolase activity decreased. Refeeding with a high-carbohydrate diet (glucose), the corresponding enzyme activities were decreased while refeeding with a high-fat diet (sheep tallow) increased the enzyme activities over the control values. The increased content of long-chain acyl-CoA and free CoASH under fasting and fat-refeeding was mainly attributed to the mitochondrial fraction with the remainder in the light mitochondrial fraction which contains peroxisomes. The results suggest a correlation of the compartmentation of the palmitoyl-CoA hydrolase and the content and compartmentation of the CoA derivatives in the liver under different nutritional states. The peroxisomal palmitoyl-CoA oxidase activity was increased by fasting. Fat-refeeding increased the activity even more; 1.8-fold as compared to the fasting animals. On the other hand, the activities of other peroxisomal enzymes which are not directly involved in the fatty acid metabolism such as urate oxidase were decreased to approximately the same extent by fasting. Re-feeding with glucose and fat further decreased the corresponding enzyme activity, particularly seen in the glucose-refed group.

S-Adenosylhomocysteine hydrolase in human and rat liver is localized to the cytosol fraction of the tissue homogenate

S-Adenosylhomocysteine (SAH) is formed from S-adenosylmethionine (SAM) upon transmethylation using SAM as a methyl donor [ 11. SAH is a potent inhibitor to a wide group of methyl-transfer reactions and has been suggested to be a regulator of biological methylations [2-71. The tissue level of SAH is regulated by the enzyme S-adenosylhomocysteinase (EC 3.3 .I. 1). This enzyme catalyzes the reversible synthesis of SAH from adenosine and L-homocysteine and the equilibrium of the reaction is far in the direction of condensation [8]. The metabolic flow has been suggested to be in the hydrolytic direction [9] and the enzyme catalyzes the first step in the degradation of SAH. No study has been devoted to the subcellular localization of S-adenosylhomocysteinase. Data have been presented [lo] suggesting that the post-microsomal supernatant fraction contains almost all of the S-adenosylhomocysteine synthase activity, but a substantial amount was associated with the ‘nuclear’ fraction. This finding seems interesting in the light of the finding that SAH is a potent inhibitor of DNA methylation in isolated rat liver nuclei [ 1 I]. However, in this report data are presented showing that S-adenosylhomocysteine hydrolase activity is localized exclusively to the soluble fraction of tissue homogenates from human and rat liver.

Sixty-minute normothermic liver ischemia in rats : evidence that allopurinol improves liver cell energy metabolism during reperfusion but that timing of drug administration is important

Allopurinol (ALL) improves energy metabolism in organs subjected to ischemia-reperfusion injury. The importance of different administration schedules of ALL has been studied in a rat liver model exposed to 60 min of normothermic ischemia followed by reperfusion. ALL (100 mg/kg) that administered in two doses, one prior to ischemia and one prior to reperfusion, improved production of adenosine triphosphate in the liver as well as bile flow during reperfusion. ALL administered in a single dose, either prior to ischemia or prior to reperfusion, was less effective. The concentration of hypoxanthine during ischemia increased in the groups given ALL prior to induction of ischemia. Based on the present findings, we argue that the beneficial effect of ALL administration can be potentiated by different drug-administration schedules. Our data also suggest that the prime mechanism of action for ALL is not only related to inhibition of free-oxygen-radicals production but that preservation of hypoxanthine, which can be used for ATP resynthesis and the scavenging properties of ALL itself, may be equally important.

The existence of separate peroxisomal pools of free coenzyme a and long-chain acyl-CoA in rat liver, demonstrated by a specific high performance liquid chromatography method

1. Tiadenol administration of rats lead to an increased hepatic content of unesterified and esterified CoA. 2. Liver homogenates from normal tiadenol treated rats were fractionated by differential centrifugation and fractions enriched in peroxisomes were subfractionated by isopycnic density gradient centrifugation. 3. The analysis demonstrated that purified peroxisomes contained a separate pool of free CoASH and long-chain acyl-CoA. 4. The data also provides indications of the presence of palmitoyl-CoA synthetase in the peroxisomes.