Circulating trimethyllysine and risk of acute myocardial infarction in patients with suspected stable coronary heart disease.

BACKGROUND: The carnitine precursor trimethyllysine (TML) is associated with progression of atherosclerosis, possibly through a relationship with trimethylamine-N-oxide (TMAO). Riboflavin is a cofactor in TMAO synthesis. We examined prospective relationships of circulating TML and TMAO with acute myocardial infarction (AMI) and potential effect modifications by riboflavin status. METHODS: By Cox modelling, risk associations were examined amongst 4098 patients (71.8% men) with suspected stable angina pectoris. Subgroup analyses were performed according to median plasma riboflavin. RESULTS: During a median follow-up of 4.9 years, 336 (8.2%) patients experienced an AMI. The age- and sex-adjusted hazard ratio (HR) (95% CI) comparing the 4th vs. 1st TML quartile was 2.19 (1.56-3.09). Multivariable adjustment for traditional cardiovascular risk factors and indices of renal function only slightly attenuated the risk estimates [HR (95% CI) 1.79 (1.23-2.59)], which were particularly strong amongst patients with riboflavin levels above the median (Pint  = 0.035). Plasma TML and TMAO were strongly correlated (rs  = 0.41; P < 0.001); however, plasma TMAO was not associated with AMI risk in adjusted analyses [HR (95% CI) 0.81 (0.58-1.14)]. No interaction between TML and TMAO was observed. CONCLUSION: Amongst patients with suspected stable angina pectoris, plasma TML, but not TMAO, independently predicted risk of AMI. Our results motivate further research on metabolic processes determining TML levels and their potential associations with cardiovascular disease. We did not adjust for multiple comparisons, and the subgroup analyses should be interpreted with caution.

Microbiota-dependent metabolite trimethylamine-N-oxide is associated with disease severity and survival of patients with chronic heart failure.

OBJECTIVES: Recent metabolomic, experimental and clinical studies have demonstrated that trimethylamine-N-oxide (TMAO), a microbiota-dependent metabolite from dietary phosphatidylcholine and carnitine, is a strong predictor of coronary artery disease (CAD). This finding suggests a link between the gut microbiota and atherosclerosis. The potential impact of TMAO in chronic heart failure (HF) is unknown. We hypothesized that TMAO levels would provide prognostic information about adverse outcomes in chronic HF. DESIGN: Prospective, observational study including 155 consecutive patients with chronic HF. In addition, 100 patients with stable CAD without HF and 33 matched healthy individuals were included as controls. Plasma levels of TMAO and its precursors choline and betaine were measured, and associations with symptoms, aetiology and transplant-free survival in the patients with HF were explored. RESULTS: Plasma levels of TMAO (P = 0.01), choline (P < 0.001) and betaine (P < 0.001) were elevated in patients with chronic HF compared to control subjects, with the highest levels in patients with New York Heart Association (NYHA) classes III and IV. Furthermore, TMAO levels were highest in individuals with ischaemic HF, followed by those with stable CAD and nonischaemic HF. TMAO, but not choline or betaine, was associated with reduced transplant-free survival: approximately 50% of patients in the upper tertile of TMAO levels died or received a heart transplant during 5.2 years of follow-up (unadjusted Cox-regression: hazard ratio 2.24, 95% confidence interval 1.28-3.92, P = 0.005). CONCLUSIONS: TMAO levels were elevated in patients with HF and associated with NYHA class, ischaemic aetiology and adverse outcomes. Future studies should focus on gut microbiota, dietary composition and intestinal dysfunction in relation to TMAO levels and clinical outcome in HF.

Fatty acid composition in chronic heart failure: low circulating levels of eicosatetraenoic acid and high levels of vaccenic acid are associated with disease severity and mortality.

OBJECTIVES: Free fatty acids (FFAs) are the major energy sources of the heart, and fatty acids (FAs) are active components of biological membranes. Data indicate that levels of FAs and their composition may influence myocardial function and inflammation. The aim of this study was to investigate whether total levels and composition of FAs and FFAs in plasma are altered in clinical heart failure (HF) and whether any alterations in these parameters are correlated with the severity of HF. SUBJECTS: Plasma from 183 patients with stable HF was compared with plasma from 44 healthy control subjects. RESULTS: Our main findings are as follows: (i) patients with HF had decreased levels of several lipid parameters and increased levels of FFAs in plasma, compared with controls, which were significantly correlated with clinical disease severity. (ii) Patients with HF also had a decreased proportion in the plasma of several n-3 polyunsaturated FAs, an increased proportion of several monounsaturated FAs, and a decreased proportion of some readily oxidized long-chain saturated FAs. (iii) These changes in FA composition were significantly associated with functional class, impaired cardiac function (i.e., decreased cardiac index and increased plasma N-terminal pro-B-type natriuretic peptide levels) and enhanced systemic inflammation (i.e., increased high-sensitivity C-reactive protein levels). (iv) Low levels of C20:4n-3 (eicosatetraenoic acid) and in particular high levels of C18:1n-7 (vaccenic acid) were significantly associated with total mortality in this HF population. CONCLUSIONS: Our data demonstrate that patients with HF are characterized by a certain FA phenotype and may support a link between disturbed FA composition and the progression of HF.

Tetradecylthioacetic acid attenuates dyslipidaemia in male patients with type 2 diabetes mellitus, possibly by dual PPAR-alpha/delta activation and increased mitochondrial fatty acid oxidation.

AIM: We previously demonstrated that a modified fatty acid, tetradecylthioacetic acid (TTA), improves transport and utilization of lipids and increases mitochondrial fatty acid oxidation in animal and cell studies. We conducted an exploratory study of safety and effects of this novel drug in patients with type 2 diabetes mellitus and investigated the mechanism of action in human cell lines. METHODS: Sixteen male patients with type 2 diabetes mellitus received 1 g TTA daily for 28 days in an open-labelled study, with measurement of parameters of lipid metabolism, glucose metabolism and safety (ClinicalTrials.gov NCT00605787). The mechanism of action was further investigated in a human liver cell line (HepG2) and in cultured human skeletal muscle cells (myotubes). RESULTS: Mean LDL cholesterol level declined from 4.2 to 3.7 mmol/l (p < 0.001), accompanied by increased levels of the HDL apolipoproteins A1 and A2, and a decline in LDL/HDL ratio from 4.00 to 3.66 (p = 0.008). Total fatty acid levels declined, especially the fraction of the polyunsaturated n-3 fatty acids docosahexaenoic acid (-13%, p = 0.002) and eicosapentaenoic acid (-10%, p = 0.07). Glucose metabolism was not altered and the drug was well tolerated. In cultured liver cells, TTA acted as a pan-PPAR agonist with predominant PPAR-alpha and PPAR-delta activation at low TTA concentrations. In myotubes, TTA and a PPAR-delta agonist, but not the PPAR-alpha or PPAR-gamma agonists, increased the fatty acid oxidation. CONCLUSIONS: We demonstrate for the first time that TTA attenuates dyslipidaemia in patients with type 2 diabetes mellitus. These effects may occur through mechanisms involving PPAR-alpha and PPAR-delta activation, resulting in increased mitochondrial fatty acid oxidation.

Dietary supplementation of tetradecylthioacetic acid increases feed intake but reduces body weight gain and adipose depot sizes in rats fed on high-fat diets.

AIM: The pan-peroxisome proliferator-activated receptor (PPAR) ligand and fatty acid analogue tetradecylthioacetic acid (TTA) may reduce plasma lipids and enhance hepatic lipid metabolism, as well as reduce adipose tissue sizes in rats fed on high-fat diets. This study further explores the effects of TTA on weight gain, feed intake and adipose tissue functions in rats that are fed a high-fat diet for 7 weeks. METHODS: The effects on feed intake and body weight during 7 weeks' dietary supplement with TTA ( approximately 200 mg/kg bw) were studied in male Wistar rats fed on a lard-based diet containing approximately 40% energy from fat. Adipose tissue mass, body composition and expression of relevant genes in fat depots and liver were measured at the end of the feeding. RESULTS: Despite higher feed intake during the final 2 weeks of the study, rats fed on TTA gained less body weight than lard-fed rats and had markedly decreased subcutaneous, epididymal, perirenal and mesenteric adipose depots. The effects of TTA feeding with reduced body weight gain and energy efficiency (weight gain/feed intake) started between day 10 and 13. Body contents of fat, protein and water were reduced after feeding lard plus TTA, with a stronger decrease in fat relative to protein. Plasma lipids, including Non-Esterified Fatty Acids (NEFA), were significantly reduced, whereas fatty acid beta-oxidation in liver and heart was enhanced in lard plus TTA-fed rats. Hepatic UCP3 was expressed ectopically both at protein and mRNA level (>1900-fold), whereas Ucp1 mRNA was increased approximately 30-fold in epididymal and approximately 90-fold in mesenteric fat after lard plus TTA feeding. CONCLUSION: Our data support the hypothesis that TTA feeding may increase hepatic fatty acid beta-oxidation, and thereby reduce the size of adipose tissues. The functional importance of ectopic hepatic UCP3 is unknown, but might be associated with enhanced energy expenditure and thus the reduced feed efficiency.

Increased survival by feeding tetradecylthioacetic acid during a natural outbreak of heart and skeletal muscle inflammation in S0 Atlantic salmon, Salmo salar L.

We have previously documented increased survival by feeding tetradecylthioacetic acid (TTA) during a natural outbreak of infectious pancreatic necrosis in post-smolt S1 Atlantic salmon. The aim of the present study was to test the effects of dietary TTA in S0 smolt at a location where fish often experience natural outbreaks of heart and skeletal muscle inflammation (HSMI) during their first spring at sea. The experimental groups were fed a diet supplemented with 0.25% TTA for a 6-week period prior to a natural outbreak of HSMI in May 2007. Relative percent survival for the groups fed TTA was 45% compared with control diets, reducing mortality from 4.7% to 2.5%. Expression of genes related to lipid oxidation was higher in cardiac ventricles from salmon fed TTA compared with controls. In addition, salmon fed TTA had periodically reduced levels of plasma urea, and increased cardiosomatic index and growth. Reduced mortality and increased growth after administration of TTA may be related to a combination of anti-inflammatory effects, and an altered metabolic balance with better protein conservation because of increased lipid degradation.

Rifaximin alters gut microbiota profile, but does not affect systemic inflammation - a randomized controlled trial in common variable immunodeficiency.

Common variable immunodeficiency (CVID) patients have reduced gut microbial diversity compared to healthy controls. The reduced diversity is associated with gut leakage, increased systemic inflammation and ten "key" bacteria that capture the gut dysbiosis (dysbiosis index) in CVID. Rifaximin is a broad-spectrum non-absorbable antibiotic known to reduce gut leakage (lipopolysaccharides, LPS) in liver disease. In this study, we explored as a 'proof of concept' that altering gut microbial composition could reduce systemic inflammation, using CVID as a disease model. Forty adult CVID patients were randomized, (1:1) to twice-daily oral rifaximin 550 mg versus no treatment for 2 weeks in an open-label, single-centre study. Primary endpoints were reduction in plasma/serum levels of soluble (s) CD14, sCD25, sCD163, neopterin, CRP, TNF, LPS and selected cytokines measured at 0, 2 and 8 weeks. Secondary endpoint was changes in intra-individual bacterial diversity in stool samples. Rifaximin-use did not significantly change any of the inflammation or gut leakage markers, but decreased gut microbial diversity compared with no treatment (p = 0.002). Importantly, the gut bacteria in the CVID dysbiosis index were not changed by rifaximin. The results suggest that modulating gut microbiota by rifaximin is not the chosen intervention to affect systemic inflammation, at least not in CVID.

Altered gut microbiota profile in common variable immunodeficiency associates with levels of lipopolysaccharide and markers of systemic immune activation.

Common variable immunodeficiency (CVID) is the most common symptomatic primary immunodeficiency characterized by low immunoglobulin (Ig)G and IgA, and/or IgM. In addition to bacterial infections, a large subgroup has noninfectious inflammatory and autoimmune complications. We performed 16S ribosomal RNA-based profiling of stool samples in 44 CVID patients, 45 patients with inflammatory bowel disease (disease controls), and 263 healthy controls. We measured plasma lipopolysaccharide (LPS) and markers of immune cell activation (i.e., soluble (s) CD14 and sCD25) in an expanded cohort of 104 patients with CVID and in 30 healthy controls. We found a large shift in the microbiota of CVID patients characterized by a reduced within-individual bacterial diversity (alpha diversity, P<0.001) without obvious associations to antibiotics use. Plasma levels of both LPS (P=0.001) and sCD25 (P<0.0001) were elevated in CVID, correlating negatively with alpha diversity and positively with a dysbiosis index calculated from the taxonomic profile. Low alpha diversity and high dysbiosis index, LPS, and immune markers were most pronounced in the subgroup with inflammatory and autoimmune complications. Low level of IgA was associated with decreased alpha diversity, but not independently from sCD25 and LPS. Our findings suggest a link between immunodeficiency, systemic immune activation, LPS, and altered gut microbiota.

Purification and characterization of a long-chain acyl-CoA hydrolase from rat liver microsomes.

A long-chain acyl-CoA hydrolase from rat liver microsomes has been purified by solvent extraction and gel chromatography to homogeneity as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The enzyme was a monomer of molecular weight 59 000. In a sucrose gradient it sedimented at 4.3 S. The isoelectric point, pI was 6.9, and the Stokes radius was approx. 31 A. The enzyme hydrolyzed long-chain fatty acyl-CoA (C7--C18) with maximum activity for palmitoyl-CoA. Bovine serum albumin activation of the enzyme was related to the ratio acyl-CoA/bovine serum albumin, and at high ratios, acyl-CoA inhibited the enzyme activity. Disregarding the substrate inhibition, an apparent Km of 65 nmol/mg protein or 1-10(-7) M and a V of 750 nmol/mg protein per min were calculated. The enzyme was inhibited by p-hydroxymercuribenzoate and N-ethylmaleimide. Reactivation by means of dithiothreitol was not complete.

The hypocholesterolemic effect of sulfur-substituted fatty acid analogues in rats fed a high carbohydrate diet.

Sulfur-substituted fatty acid analogues have been administered to rats fed a high carbohydrate diet, and the effect on plasma and hepatic lipid metabolism was investigated. Two of the analogues studied, 3-thiadicarboxylic acid and tetradecylthioacetic acid, reduced the plasma cholesterol level significantly, whereas the effect on plasma triacylglycerol level was only marginal. 3-Thiadicarboxylic acid was the most potent, decreasing the cholesterol level faster and at a lower dose than tetradecylthioacetic acid. The relative effects on plasma cholesterol and triacylglycerol levels were different from what have been observed in rats fed a conventional pellet diet. Tetradecylthiopropionic acid had no hypocholesterolemic effect. The activities of three lipogenic enzymes: ATP-citrate lyase, acetyl-CoA carboxylase and fatty acid synthase was measured. The two hypocholesterolemic analogues reduced the activities of these enzymes in a coordinated manner. The enzyme activities was found to correlate with the the plasma cholesterol level, indicating a coordinated regulation of these enzymes and cholesterol synthesis or secretion. The effect on two enzymes involved in cholesterol metabolism was also studied. The activity of acyl-CoA:cholesterol acyltransferase (ACAT) was reduced by the two hypocholesterolemic analogues, in contrast to the rate-limiting enzyme in cholesterol biosynthesis, HMG-CoA reductase, which tended to increase. The cholesterol lowering effect of 3-thiadicarboxylic acid and tetradecylthioacetic acid can probably be ascribed to diminished cholesterol synthesis due to a reduced availability of acetyl-CoA. A reduction in the esterification of hepatic cholesterol may be a contributing factor.

Correlation between the cellular level of long-chain acyl-CoA, peroxisomal beta-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.

Subcellular localisation and induction of NADH-sensitive acetyl-CoA hydrolase and propionyl-CoA hydrolase activities in rat liver under lipogenic conditions after treatment with sulfur-substituted fatty acids.

The effects of sulfur-substituted fatty acid analogues on the subcellular distribution and activities of acetyl-CoA and propionyl-CoA hydrolases in rats fed a high carbohydrate diet were studied. Among subcellular fractions of liver homogenates from rats fed a high carbohydrate diet (20%), the acetyl-CoA and propionyl-CoA hydrolase activities are found in the mitochondrial, peroxisome-enriched and cytosolic fractions. We have shown that the subcellular distribution of acetyl-CoA hydrolase appears to be different from the distribution propionyl-CoA hydrolase activity. Thus, the highest specific activity of acetyl-CoA hydrolase was found in the mitochondrial fraction, whereas the highest specific activity of propionyl-CoA hydrolase was found in the peroxisome-enriched fraction. Rats treated with sulfur-substituted fatty acids, i.e., 3-thiadicarboxylic acid (400 mg/day per kg body weight), showed a significant increase in acetyl-CoA hydrolase activity where the peroxisomal and cytosolic hydrolases were increased 3.9- and 2.7-fold, respectively, compared to palmitic acid treated rats. Similar results were obtained with tetradecylthioacetic acid treated rats. Propionyl-CoA hydrolase activities, in rats treated with these two peroxisome proliferating fatty acid analogues showed increased activity mainly in the mitochondrial and the cytosolic subcellular fractions. Acetyl-CoA hydrolase activity was sensitive to NADH, whereas no stimulation of the propionyl-CoA hydrolase activity was observed in the presence of NADH. The hepatic amounts of acetyl-CoA, propionyl-CoA, and free CoASH were elevated after sulfur-substituted fatty acid treatment. Sulfur-substituted fatty acids also elevated the specific acetyl-CoA hydrolase activity in the mitochondrial fraction and the propionyl-CoA hydrolase activity in the light-mitochondrial fraction. These results, therefore, suggest that acetyl-CoA hydrolase and propionyl-CoA hydrolase are two distinct proteins and that these two enzymes have a multiorganelle localisation.

Eicosapentaenoic acid causes transient accumulation of lipids in rat myocardium.

Rats were given eicosapentaenoic acid (EPA) or palmitic acid (PALM) up to 15 days, and control animals were given carboxymethylcellulose. All suspensions which were given by gastric intubation contained tocopherol. Heart triacylglycerols, heart cholesterol and heart phospholipids significantly increased after one day of EPA treatment, but they were normalized within 15 days. Both after 2 and 10 days of treatment with palmitic acid the heart triacylglycerols were significantly greater than control. The heart cholesterol and heart phospholipids were significantly greater than control after 10 days of treatment with palmitic acid. Total carnitine palmitoyltransferase (CPT) activity in heart was significantly greater in rats treated with EPA for 15 days compared to control, but treatment with palmitic acid had no effect. The fatty acyl-CoA oxidase activity was greater in rats treated with EPA for 15 days and palmitic acid for 10 days compared to control. The fractional volume of lipid droplets in myocardial cells was calculated from electronmicrographs and was 0.112 +/- 0.016% after 1 day of EPA treatment compared to 0.035 +/- 0.016% in the control group. After 5 and 15 days the fractional volume was the same as control. The fractional volume of lipid droplets in rats treated with palmitic acid for 10 days was 0.120 +/- 0.023%. Treatment with EPA caused an immediate accumulation of lipids and lipid droplets in the rat heart which after few days normalized in parallel with an increased activity of total CPT in the heart.

Rapid stimulation of liver palmitoyl-CoA synthetase, carnitine palmitoyltransferase and glycerophosphate acyltransferase compared to peroxisomal beta-oxidation and palmitoyl-CoA hydrolase in rats fed high-fat diets.

Key enzymes involved in oxidation and esterification of long-chain fatty acids were investigated in male rats fed different types and amounts of oil in their diet. A diet with 20% (w/w) fish oil, partially hydrogenated fish oil (PHFO) and partially hydrogenated soybean oil (PHSO) was shown to stimulate the mitochondrial and microsomal palmitoyl-CoA synthetase activity (EC 6.2.1.3) compared to soybean oil-fed animals after 1 week of feeding. Rapeseed oil had no effect. Partially hydrogenated oils in the diet resulted in significantly higher levels of mitochondrial glycerophosphate acyltransferase compared to unhydrogenated oils in the diet. Rats fed 20% (w/w) rapeseed oil had a decreased activity of this mitochondrial enzyme, whereas the microsomal glycerophosphate acyltransferase activity was stimulated to a comparable extent with 20% (w/w) rapeseed oil, fish oil or PHFO in the diet. Increasing the amount of PHFO (from 5 to 25% (w/w)) in the diet for 3 days led to increased mitochondrial and microsomal palmitoyl-CoA synthetase and microsomal glycerophosphate acyltransferase activities with 5% of this oil in the diet. The mitochondrial glycerophosphate acyltransferase was only marginally affected by increasing the oil dose. Administration of 20% (w/w) PHFO increased rapidly the mitochondrial and microsomal palmitoyl-CoA synthetase, carnitine palmitoyltransferase and microsomal glycerophosphate acyltransferase activities almost to their maximum value within 36 h. In contrast, the glycerophosphate acyltransferase and palmitoyl-CoA hydrolase (EC 3.1.2.2) activities of the mitochondrial fraction and the peroxisomal beta-oxidation reached their maximum activities after administration of the dietary oil for 6.5 days. This sequence of enzyme changes (a) is in accordance with the proposal that an increased cellular level of long-chain acyl-CoA species act as metabolic messages for induction of peroxisomal beta-oxidation and palmitoyl-CoA hydrolase, i.e., these enzymes are regulated by a substrate-induced mechanism, and (b) indicates that, with PHFO, a greater part of the activated fatty acids are directed from triacylglycerol esterification and hydrolysis towards oxidation in the mitochondria. It is also conceivable that the mitochondrial beta-oxidation is proceeding before the enhancement of peroxisomal beta-oxidation.

Variations in the activity of microsomal palmitoyl-CoA hydrolase in mixed micelle solutions of palmitoyl-CoA and non-ionic detergents of the triton X series.

The kinetics of palmitoyl-CoA hydrolase were influenced by both the availability of the substrate and formation of micelles. At palmitoyl-CoA concentrations below the critical micelle concentration, addition of non-ionic detergent increased the activity until the critical micelle concentration of the mixed micelles was reached. At palmitoyl-CoA concentrations above the critical micelle concentration, inhibitor of the activity was observed, but addition of detergents of the Triton X series reversed the inhibition. Maximum palmitoyl-CoA hydrolase activity was found when the ratios (w/v) of palmitoyl-CoA: Triton X-100 and palmitoyl-CoA: Triton X-405 were approximately 0.35 and 0.05, respectively. At these above the mixed critical micelle concentration. The results indicate that monomer palmitoyl-CoA is the substrate and that monomer forms of the non-ionic detergents of the Triton X series activate the enzyme. Isolated microsomal lipids activated the microsomal palmitoyl-CoA hydrolase, suggesting that a hydrophobic environment is advantageous for interaction between enzyme and substrate in vivo. The maximum activity in the presence of mixed micelles is discussed in relation to a model where mixed micelles are regarded as artificial membranes to which the enzyme may adhere in an equilibrium with the monomer substrate and detergent in the monomer form. It is suggested that intracellular membranes may resemble mixed micelles in equilibrium with detergent-active substrates such as palmitoyl-CoA.

Early effects on mitochondrial and peroxisomal beta-oxidation by the hypolipidemic 3-thia-fatty acids in rat livers.

A single administration of 3-thiadicarboxylic and tetradecylthioacetic acids stimulates both mitochondrial and peroxisomal beta-oxidation and lowers plasma triacylglycerol levels. An increased rate of mitochondrial beta-oxidation and carnitine palmitoyl-transferase activity was established after 3 h and this was accompanied by a lowering of plasma triacylglycerol. Peroxisomal beta-oxidation, however, remained unchanged up to 8 h and was significantly increased after 12 h. These results suggest that after a single administration of 3-thia fatty acids mitochondrial beta-oxidation precedes peroxisomal beta-oxidation. Furthermore, they show that the observed tricylglycerol-lowering effect, which is established early (3-4 h) after the administration of 3-thia fatty acids, is initially due to an increased mitochondrial beta-oxidation.

Effect of methotrexate on long-chain fatty acid metabolism in liver of rats fed a standard or a defined, choline-deficient diet.

The effect of methotrexate on lipids in serum and liver and key enzymes involved in esterification and oxidation of long-chain fatty acids were investigated in rats fed a standard diet and a defined choline-deficient diet. Hepatic metabolism of long-chain fatty acids were also studied in rats fed the defined diet with or without choline. When methotrexate was administered to the rats fed the standard diet there was a slight increase in hepatic lipids and a moderate reduction in the serum level. The palmitoyl-CoA synthetase activity and the microsomal glycerophosphate acyltransferase activity in the liver of rats were increased by methotrexate. The data are consistent with those where the liver may fail to transfer the newly formed triacylglycerols into the plasma with a resultant increase in liver triacylglycerol content and a decrease in serum lipid levels. Fatty liver of methotrexate-exposed rats can not be attributed simply to a reduction of fatty acid oxidation as the carnitine palmitoyltransferase activity was increased. The methotrexate response in the rats fed the defined choline-deficient diet was different. There was a reduction in both serum and hepatic triacylglycerol and the glycerophosphate acyltransferase and palmitoyl-CoA synthetase activities. The carnitine palmitoyltransferase activity was unchanged. Hepatomegaly and increased hepatic fat content, but decreased serum triacylglycerol, total cholesterol and HDL cholesterol were found to be related to the development of choline deficiency as the pleiotropic responses were almost fully prevented by addition of choline to the choline-deficient diet. Addition of choline to the choline-deficient diet normalized the total palmitoyl-CoA synthetase and carnitine palmitoyltransferase activities. In contrast to methotrexate exposure, choline deficiency increased the mitochondrial glycerophosphate acyltransferase activity. The data are consistent with those of where fatty liver induction of choline deficiency may be related to an enhanced esterification of long-chain fatty acids concomitant with a reduction of their oxidation.

On the effect of 2-deuterium- and 2-methyl-eicosapentaenoic acid derivatives on triglycerides, peroxisomal beta-oxidation and platelet aggregation in rats.

A series of 2-substituted eicosapentaenoic acid (EPA) derivatives (as ethyl esters) have been synthesized and evaluated as hypolipidemic and antithrombotic agents in feeding experiments in rats. Repeated administration of purified 2-methyl-eicosapentaenoic acid and its deuterium analogues (all as ethyl esters) to rats resulted in a decrease in plasma triglycerides and high density lipoprotein cholesterol. The 2-methyl-EPA analogues were, apparently, four times more potent than EPA in inducing the triglyceride lowering effect. The 2-deuterium-2-methyl-EPA decreased plasma cholesterol level to approximately 40%. A moderate enlargement of the liver was observed in 2-methyl-EPA treated rats. This was accompanied with an acute reduction in the liver content of triglycerides and a stimulation of peroxisomal beta-oxidation and fatty acyl-CoA oxidase activity. The results suggest that the triglyceride-lowering effect of 2-methyl-EPA may be due to a reduced supply of fatty acids for hepatic triglyceride biosynthesis because of increased fatty acid oxidation. Platelet aggregation with ADP and A23187 was performed ex vivo in platelet-rich plasma, after administration of different doses of the EPA-derivatives for five days. EPA and 2,2-dideuterium EPA had no effect on ADP-induced aggregation, while 2-deuterium-, 2-methyl- and 2-deuterium-2-methyl EPA produced a biphasic effect, i.e. potentiation and inhibition at low (250 mg/day kg body weight) and higher doses (600-1300 mg/day kg body weight), respectively. A23187-induced platelet aggregation was affected in a similar way by feeding the 2-substituted EPA derivatives, except that 2-deuterium-2-methyl EPA had no effect relative to EPA itself and that the inhibition was far greater than that for ADP-induced aggregation (approximately 100% inhibition with 600 mg 2-methyl-EPA/day kg body weight). The ranking order of the EPA-derivatives to affect platelet aggregation and to cause hypolipidemia was different, suggesting different mechanisms. Our observations suggest that the effects of the EPA derivatives on platelet aggregation could be related to the degree of bulkiness around C2 and that an asymmetric substitution at C2 caused inhibition of platelet aggregation while a symmetric substitution did not. It is suggested that the bulky, asymmetric derivatives inhibit platelet aggregation by altering platelet membrane phospholipid packing.

Effects of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate on peroxisomal activities and enzyme activities involved in lipid metabolism in rat liver.

The effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on hepatic lipids and key enzymes involved in esterification, hydrolysis and oxidation of long-chain fatty acids at increasing doses were investigated in rats. TPA administration tended to decrease the mitochondrial activities of palmitoyl-CoA synthetase and carnitine palmitoyltransferase. The microsomal palmitoyl-CoA synthetase activity was increased. TPA administration was also associated with a dose-dependent increase of glycerophosphate acyltransferase activity both in the mitochondrial and microsomal fractions in particular. The data are consistent with a decreased catabolism of long-chain fatty acids at the mitochondrial level, and an increased capacity for esterification of fatty acids in the microsomal fraction. Peroxisomal beta-oxidation was increased about 2-fold in the peroxisome-enriched fraction of TPA-treated rats while the catalase and urate oxidase activities were only marginally affected. TPA administration revealed elevated capacity for hydrolysis of palmitoyl-CoA and palmitoyl-L-carnitine in the microsomal fraction. Neither increased cytosolic palmitoyl-CoA hydrolase activity nor increased hydroxylation of lauric acid nor changes of the hepatic content of cytochrome P-450 isoenzymic forms were observed in the TPA-treated animals. There was no induction of the protein content of the bifunctional enoyl-CoA hydratase. Thus, TPA behaves more like choline-deficient diet and ethionine treatment than well-known peroxisome proliferators. It seems possible that TPA selectively stimulated the peroxisomal activities, i.e., peroxisomal beta-oxidation rather than evoking a peroxisome proliferation capacity.