Asle Aarsland

Alkylthioacetic acid (3-thia fatty acids) ― a new group of non-β-oxidizable, peroxisome-inducing fatty acid analogues. I: A study on the structural requirements for proliferation of peroxisomes and mitochondria in rat liver

The induction of peroxisome proliferation was examined in rat liver after administration of equal concentrations (1 mmol/kg body weight) of 1,10-bis(carboxymethylthiodecane) (BCMTD), 1-mono(carboxymethylthiotetradecane) (CMTTD), 1-mono(carboxymethylthiooctane) (CMTO), 1-mono(carboxyethylthiotetradecane) (CETTD), palmitic acid and hexadecanedioic acid (HDDA). BCMTD, a non-beta-oxidizable and non-omega-oxidizable sulphur-substituted fatty acid analogue was considerably more potent than CMTTD (only non-beta-oxidizable) in inducing enlargement of the liver and increasing peroxisomal activities (monitored by peroxisomal beta-oxidation, palmitoyl-CoA hydrolase and catalase activities). Morphometric analysis of randomly selected hepatocytes revealed that BCMTD and CMTTD treatment increased the number and size of peroxisomes and the relative volume fraction of the peroxisomes. All these cellular responses were more marked with BCMTD than compared with CMTTD. CMTO, a non-beta-oxidizable fatty acid analogue containing a lower hydrophobic alkyl-end than CMTTD and CETTD (a beta-oxidizable fatty acid analogue), showed a slight increase (1.4-1.8-fold) of peroxisomal beta-oxidation and caused marginally morphological changes of peroxisomes compared with CMTTD and BCMTD. The most striking effect of the alkylthiopropionic acid (CETTD) was an enhancement of the hepatic triacylglycerol level. Palmitic acid and hexadecanedioic acid only marginally affected the peroxisomal activities, but no morphological changes of peroxisomes and fat droplets were observed. The presented data strongly suggest that a minimal structural requirement for a peroxisome proliferator may be (1) a carboxylic acid group linked to (2) a hydrophobic backbone which (3) cannot be beta-oxidized i.e., the fatty acid analogues have a sulphur atom in the beta-position. It is also conceivable that blockage for omega-oxidation may potentiate the peroxisome-proliferating activities in as much as BCMTD was more potent than CMTTD. Two mitochondrial marker enzymes, carnitine palmitoyltransferase and succinate phenazine methosulphate oxidoreductase were differently affected after administration of the investigated compounds. Furthermore, BCMTD and CMTTD as well as HDDA treatments increased the number of mitochondria, but the mitochondria tended to be smaller. The overall results presented here indicate that the structural requirements for proliferation of mitochondria are not identical to those for proliferation of peroxisomes.

Redox status and protein binding of plasma homocysteine and other aminothiols in patients with homocystinuria.

Elevations of homocyst(e)ine levels in the blood of patients with homocystinuria may explain the high cardiovascular morbidity. We determined levels of reduced, oxidized, and protein-bound homocyst(e)ine, cyst(e)ine, and cyst(e)inylglycine in plasma from eight patients with homocystinuria. The technique used involved trapping of reduced thiols by collecting blood directly into tubes containing sulfhydryl-reactive reagents. All patients had high levels of homocysteine (range, 1.9 to 91.2 mumol/L), and among the aminothiols investigated, this species showed the most drastic elevation compared with trace levels (< 0.4 mumol/L) found in healthy subjects. The ratio between free homocysteine and total homocyst(e)ine (reduced to total ratio) was above normal and positively correlated to the reduced to total ratio for cyst(e)ine, suggesting that an equilibrium exists between these species through sulfhydryl disulfide exchange. The other homocyst(e)ine species (oxidized and protein-bound) were also markedly increased in patients with homocystinuria. Plasma cysteine and cysteinylglycine levels were moderately increased, whereas plasma concentrations of protein-bound cyst(e)ine, protein-bound cyst(e)inylglycine, and free cystine were below normal. Homocysteine in particular and other homocyst(e)ine species are markedly increased in plasma of homocystinurics, and these changes are associated with pronounced alterations in the level and the redox status of other aminothiols. This should be taken into account when considering homocyst(e)ine as an atherogenic agent, and the role of various homocyst(e)ine species in the pathogenesis of homocystinuria.

On the effect of peroxisomal β-oxidation and carnitine palmitoyltransferase activity by eicosapentaenoic acid in liver and heart from rats

Repeated administration of highly purified eicosapentaenoic acid (as ethyl ester) resulted in a decrease in plasma triglycerides and high density lipoprotein (HDL) cholesterol. This was accompanied by a stimulation in the activities of carnitine palmitoyltransferase, fatty acyl-CoA oxidase and peroxisomal β-oxidation in the liver. The results suggest that the triglyceride-lowering effect observed with eicosapentaenoic acid may be due to a reduced supply of fatty acids for hepatic triglyceride synthesis because of increased fatty acid oxidation. Eicosapentaenoic acid feeding marginally affected the triglyceride content of heart and mitochondrial and peroxisomal enzyme activities.

Peroxisome proliferating sulphur-and oxy-substituted fatty acid analogues are activated to acyl coenzyme a thioesters

In liver homogenates from untreated rats the sulphur-substituted fatty acid analogues tetradecylthioacetic acid (CMTTD) was activated to its acyl-coenzyme A thioester. The activation was found to take place in the mitochondrial, microsomal and peroxisomal fractions. The activity of CMTTD-CoA synthetase was 50% compared to palmitoyl-CoA synthetase in all cellular fractions. When rats were treated with the peroxisome proliferating sulphur-substituted fatty acid analogues CMTTD and 3-dithiahexadecanedioic acid (BCMTD), the CMTTD-CoA synthetase activity was induced in mitochondrial, peroxisomal and microsomal fractions. Palmitoyl-CoA synthetase was induced proportionally. In rats treated with tetradecylthiopropionic acid (CETTD) of low peroxisome proliferating potency, the activities of CMTTD-CoA synthetase and palmitoyl-CoA synthetase were inhibited in mitochondrial and microsomal fractions. In contrast, all three sulphur-substituted acids induced the activity of palmitoyl-CoA synthetase and CMTTD-CoA synthetase in peroxisomes. Both the CMTTD-CoA and palmitoyl-CoA synthetase activities were induced by CMTTD and BCMTD, in close correlation to the induction of peroxisomal beta-oxidation. During the three treatment regimes, CMTTD-CoA synthetase activity ran parallel to the palmitoyl-CoA synthetase activity at a rate of 50% in all cellular fractions. Thus, CMTTD is assumed to be activated by the long-chain acyl-CoA synthetase enzyme. Rats were treated for 5 days with sulphur- and oxy-substituted fatty acid analogues, clofibric acid and fenofibric acid. All compounds which induced peroxisomal beta-oxidation activity in vivo could be activated to their respective CoA thioesters in liver homogenate. CETTD which induced peroxisomal beta-oxidation only two-fold, was activated at a rate of 50% compared to palmitate. Fenofibric acid induced peroxisomal beta-oxidation 9.6-fold, while it was activated at a rate of only 10% compared to palmitate. Thus, no correlation was found between rate of activation in vitro and induction of peroxisomal activity in vivo. On the other hand, tetradecylsulfoxyacetic acid (TSOA) and tetradecylsulfonacetic acid (TSA) (sulphuroxygenated metabolites of CMTTD) with no inductive effects, were not activated to their respective CoA derivatives. Altogether the data suggest that the enzymatic activation of the peroxisome proliferating compounds is essential for their proliferating activity, but the rate of activation does not determine the potency of the proliferators. The role of the xenobiotic-CoA pool in relation to the whole coenzyme A profile during peroxisome proliferation is discussed.

Correlation between the cellular level of long-chain acyl-coa, peroxisomal β-oxidation, and palmitoyl-CoA hydrolase activity in rat liver. Are the two enzyme systems regulated by a substrate-induced mechanism?

Data obtained in earlier studies with rats fed diets containing high doses of peroxisome proliferators (niadenate, tiadenol, clofibrate, or nitotinic acid) are used to look for a quantitative relationship between peroxisomal beta-oxidation, palmitoyl-CoA hydrolase, palmitoyl-CoA synthetase and carnitine palmitoyltransferase activities, and the cellular concentration of their substrate and reaction products. The order of the hyperlipidemic drugs with regard to their effect on CoA derivatives and enzyme activities was niadenate greater than tiadenol greater than clofibrate greater than nicotinic acid. Linear regression analysis of long-chain acyl-CoA content versus palmitoyl-CoA hydrolase and peroxisomal beta-oxidation activity showed highly significant linear correlations both in the total liver homogenate and in the peroxisome-enriched fractions. A dose-response curve of tiadenol showed that carnitine palmitoyltransferase and palmitoyl-CoA synthetase activities and the ratio of long-chain acyl-CoA to free CoASH in total homogenate rose at low doses before detectable changes occurred in the peroxisomal beta-oxidation and palmitoyl-CoA hydrolase activity. A plot of this ratio parallelled the palmitoyl-CoA synthetase activity. The specific activity of microsomally localized carnitine palmitoyl-transferase was low and unchanged up to a dose where no enhanced peroxisomal beta-oxidation was observed, but over this dose the activity increased considerably so that the specific of the enzyme in the mitochondrial and microsomal fractions became comparable. The mitochondrial palmitoyl-CoA synthetase activity decreased gradually. The correlations may be interpreted as reflecting a common regulation mechanism for palmitoyl-CoA hydrolase and peroxisomal beta-oxidation enzymes, i.e., the cellular level of long-chain acyl-CoA acting as the metabolic message for peroxisomal proliferation resulting in induction of peroxisomal beta-oxidation and palmitoyl-CoA hydrolase activity. The findings are discussed with regard to their possible consequences for mitochondrial fatty acid oxidation and the conversion of long-chain acyl-L-carnitine to acyl-CoA derivatives.

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.

Fatty acid metabolism in liver of rats treated with hypolipidemic sulphur-substituted fatty acid analogues

The purpose of this study was to investigate early biochemical changes and possible mechanisms via which alkyl(C12)thioacetic acid (CMTTD, blocked for beta-oxidation), alkyl(C12)thiopropionic acid (CETTD, undergo one cycle of beta-oxidation) and a 3-thiadicarboxylic acid (BCMTD, blocked for both omega- (and beta-oxidation) influence the peroxisomal beta-oxidation in liver of rats. Treatment of rats with CMTTD caused a stimulation of the palmitoyl-CoA synthetase activity accompanied with increased concentration of hepatic acid-insoluble CoA. This effect was already established during 12-24 h of feeding. From 2 days of feeding, the cellular level of acid-insoluble CoA began to decrease, whereas free CoASH content increased. Stimulation of [1-14C]palmitoyl-CoA oxidation in the presence of KCN, palmitoyl-CoA-dependent dehydrogenase (termed peroxisomal beta-oxidation) and palmitoyl-CoA hydrolase activities were revealed after 36-48 h of CMTTD-feeding. Administration of BCMTD affected the enzymatic activities and altered the distribution of CoA between acid-insoluble and free forms comparable to what was observed in CMTTD-treated rats. It is evident that treatment of peroxisome proliferators (BCMTD and CMTTD), the level of acyl-CoA esters and the enzyme activity involved in their formation precede the increase in peroxisomal and palmitoyl-CoA hydrolase activities. In CMTTD-fed animals the activity of cyanide-insensitive fatty acid oxidation remained unchanged when the mitochondrial beta-oxidation and carnitine palmitoyltransferase operated at maximum rates. The sequence and redistribution of CoA and enzyme changes were interpreted as support for the hypothesis that substrate supply is an important factor in the regulation of peroxisomal fatty acid metabolism, i.e., the fatty acyl-CoA species appear to be catabolized by peroxisomes at high rates only when uptake into mitochondria is saturated. Administration of CETTD led to an inhibition of mitochondrial fatty acid oxidation accompanied with a rise in the concentration of acyl-CoA esters in the liver. Consequently, fatty liver developed. The peroxisomal beta-oxidation was marginally affected. Whether inhibition of mitochondrial beta-oxidation may be involved in regulation of peroxisomal fatty acid metabolism and in development of fatty liver should be considered.

The hypolipidemic peroxisome-proliferating drug, bis(carboxymethylthio)-1.10 decane, a dicarboxylic metabolite of tiadenol, is activated to an acylcoenzyme A thioester

Bis(carboxymethylthio)-1.10 decane (BCMTD), a thiodicarboxylic acid, was shown to be a hypolipidemic peroxisome-proliferating drug as it: (a) decreased the total serum triacylglycerols and cholesterol; (b) induced hepatomegaly; (c) increased the peroxisomal beta-oxidation and catalase activity and the activities of the multiorganelle localized enzymes: palmitoyl-CoA synthetase, palmitoyl-CoA hydrolase, glycerophosphate acyltransferase; (d) decreased the carnitine palmitoyltransferase and urate oxidase activities; and (e) induced the bifunctional eonyl-CoA hydratase in peroxisomes. The present study has confirmed the effect of tiadenol administration on the activities of key enzymes involved in hepatic fatty acid metabolism in male rats. However, the hepatic pleiotropic response was more marked with the dicarboxylic acid than with its alcohol. In a separate dose-response study BCMTD was found to be a more potent inducer of peroxisomal beta-oxidation compared to tiadenol. BCMTD can be activated in vitro to its coenzyme A thioester by a dicarboxyl-CoA synthetase. In control and BCMTD-treated animals, the synthetase activity was found in all cellular fractions except the cytosolic. Whether the acyl-CoA thioesters of peroxisome-proliferating drugs may be mediators of peroxisomal proliferation should be considered.

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.

Separation and measurement of clofibroyl coenzyme a and clofibric acid in rat liver after clofibrate administration by reversed-phase high-performance liquid chromatography with photodiode array detection

A method to identify and quantitate clofibric acid and clofibroyl coenzyme A (CoA) products in rat liver was developed using reversed-phase high-performance liquid chromatography. The system was developed with baseline separation of clofibroyl-CoA from clofibric acid using isocratic elution, with a mobile phase consisting of 52% methanol and 28 mM potassium phosphate buffer (pH 4.2). With this high methanol concentration, the large amount of UV-absorbing materials present in the liver extracts were eluted earlier than the investigated compounds. Clofibroyl-CoA has a characteristic absorbance spectrum with distinct peaks at 260 and 230 nm, while clofibric acid showed only a distinct peak at 230 nm. Using an on-line photodiode array detector, the spectra could be recorded during analysis without interrupting the flow of the mobile phase. This spectral analysis identification possibilities and evaluation of the purity of the chromatographic peaks. In a perchloric extract of rat liver, the recovery of clofibric acid and clofibroyl-CoA added to the liver extract ranged from 70 to 80%. A linear relationship was observed between clofibric acid and clofibroyl-CoA concentration and the area of their peaks in the chromatogram. The detection limit of the method was lower than 5 pmol for both compounds when the absorbance was recorded at 230 nm. The method could be used without modification for the estimation of clofibroyl-CoA and clofibric acid in biological extracts.