Water-Soluble Fish Protein Intake Led to Lower Serum and Liver Cholesterol Concentrations in Obese Zucker fa/fa Rats.

Proteins from different fish species and different raw materials such as fish fillets and by-products have shown promising cardioprotective effects in rodents and humans, including effects on cholesterol metabolism. Blue whiting is used mainly to produce fish meal for the feed industry and during this production, a water-soluble protein fraction, containing small peptides that are easily absorbed and may hold bioactive properties, is isolated. The effects of water-soluble fish protein on cholesterol metabolism were investigated in twelve male obese Zucker fa/fa rats. Rats were fed diets with water-soluble protein from blue whiting (BWW) as 1/3 of the total protein and the remaining 2/3 as casein (BWW group) or with casein as the sole protein source (control group). After 5 weeks intervention, the BWW group had lower serum total, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) cholesterol concentrations and lower cholesteryl ester concentration compared to controls. Hepatic concentrations of cholesterol, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and LDL receptors were also lower in the BWW group. The groups had a similar concentration of serum total bile acids and similar fecal excretions of cholesterol and bile acids. To conclude, the BWW diet led to lower concentrations of serum and liver cholesterol in obese Zucker fa/fa rats, probably due to lower hepatic cholesterol synthesis.

Multiple acyl-CoA dehydrogenation deficiency as decreased acyl-carnitine profile in serum.

We report a case with late onset riboflavin-responsive multiple acyl-CoA dehydrogenation deficiency (MADD) characterized by decreased acyl-carnitine profile in serum which is consistent with primary systemic carnitine deficiency (CDSP) while just the contrary to a typical MADD. This patient complained with muscle weakness, muscle pain and intermittent vomiting, and was diagnosed as polymyositis, received prednisone therapy before consulted with us. Muscle biopsy revealed mild lipid storage. The findings of serum acyl-carnitines were consistent with CDSP manifesting as decreased free and total carnitines in serum. But oral L-carnitine supplementation was not very effective to this patient and mutation analysis of the SLC22A5 gene for CDSP was normal. Later, another acyl-carnitine analysis revealed a typical MADD profile in serum, which was characterized by increased multiple acyl-carnitines. Compound heterozygous mutations were identified in electron transferring-flavoprotein dehydrogenase (ETFDH) gene which confirmed the diagnosis of MADD. After administration of riboflavin, he improved dramatically, both clinically and biochemically. Thus, late onset riboflavin-responsive MADD should be included in the differential diagnosis for adult carnitine deficiency.

Increased IL-4 mRNA expression and poly-aromatic hydrocarbon concentrations from children with asthma.

BACKGROUND: Asthma is the most common chronic childhood disease. Imbalance of cytokines released from T helper cells and environmental factors, such as exposure to poly-aromatic hydrocarbon (PAH), play pivotal roles in the pathogenesis of asthma. The aim of this study was to compare the mRNA expression patterns of Interleukin (IL)-4, interferon (IFN)-γ and Acyl Co A long chain 3 (ACSL3) in peripheral blood leukocytes of children with and without asthma. To correlate the obtained mRNA data with serum IL-4, IFN-γ and PAH levels. Further, to determine the effect of in vivo exposure to PAH on mRNA expression of IL-4, IFN-γ and ACSL3 genes in a rat model. METHODS: A total of 170 children below 16 years (85 pediatric asthma patients and 85 matched healthy controls) were randomly selected from the Riyadh Cohort, Saudi Arabia. Gene expression analysis was performed using qRTPCR. Serum IL-4, IFN-γ and PAH were measured using LINCOplex (human multiplex immunoassay kit) and HPLC respectively. RESULTS: IL-4 mRNA expression was significantly increased (P < 0.05) in children with asthma compared to healthy control group whereas no differences were observed for either IFN-γ or ACSL3 mRNA. Similarly, serum IL- 4 and PAHs concentration was significantly higher as well in children with asthma in whom IFN-γ was also significantly lower. Results obtained in rats showed that exposure to the benzopyrene prototype PAH resulted in a 2.6 fold (P < 0.001) increased IL-4 mRNA expression in blood. CONCLUSION: Peripheral blood IL-4 mRNA levels, serum concentration of this cytokine are elevated in children with asthma. Also, elevated levels of PAH were observed in children with asthma. Additionally, PAH administration in rodents resulted in an increased IL-4 mRNA which is supposed to partly mediate the inflammatory response noted in asthma.

Lack of acyl-CoA:diacylglycerol acyltransferase 1 reduces intestinal cholesterol absorption and attenuates atherosclerosis in apolipoprotein E knockout mice.

Triacylglycerols (TG) are the major storage molecules of metabolic energy and fatty acids in several tissues. The final step in TG biosynthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. Lack of whole body DGAT1 is associated with reduced lipid-induced inflammation. Since one major component of atherosclerosis is chronic inflammation we hypothesized that DGAT1 deficiency might ameliorate atherosclerotic lesion development. We therefore crossbred Apolipoprotein E-deficient (ApoE(-/-)) mice with Dgat1(-/-) mice. ApoE(-/-) and ApoE(-/-)Dgat1(-/-) mice were fed Western-type diet (WTD) for 9weeks and thereafter examined for plaque formation. The mean atherosclerotic lesion area was substantially reduced in ApoE(-/-)Dgat1(-/-) compared with ApoE(-/-) mice in en face and aortic valve section analyses. The reduced lesion size was associated with decreased cholesterol uptake and absorption by the intestine, reduced plasma TG and cholesterol concentrations and increased cholesterol efflux from macrophages. The expression of adhesion molecules was reduced in aortas of ApoE(-/-)Dgat1(-/-) mice, which might be the reason for less migration capacities of monocytes and macrophages and the observed decreased amount of macrophages within the plaques. From our results we conclude that the lack of DGAT1 is atheroprotective, implicating an additional application of DGAT1 inhibitors with regard to maintaining cholesterol homeostasis and attenuating atherosclerosis.

Biochemical, molecular, and clinical characteristics of children with short chain acyl-CoA dehydrogenase deficiency detected by newborn screening in California.

BACKGROUND: Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is an autosomal recessive inborn error of mitochondrial fatty acid oxidation with highly variable biochemical, genetic, and clinical characteristics. SCADD has been associated with accumulation of butyryl-CoA byproducts, including butyrylcarnitine (C4), butyrylglycine, ethylmalonic acid (EMA), and methylsuccinic acid (MS) in body fluid and tissues. Differences in genotype frequencies have been shown between patients diagnosed clinically versus those diagnosed by newborn screening. Moreover, while patients diagnosed clinically have a variable clinical presentation including developmental delay, ketotic hypoglycemia, epilepsy and behavioral disorders, studies suggest patients diagnosed by newborn screening are largely asymptomatic. Scant information is published about the biochemical, genetic and clinical outcome of SCADD patients diagnosed by newborn screening. METHODS: We collected California newborn screening, follow-up biochemical levels, and ACADS mutation data from September, 2005 through April, 2010. We retrospectively reviewed available data on SCADD cases diagnosed by newborn screening for clinical outcomes. RESULTS: During the study period, 2,632,058 newborns were screened and 76 confirmed SCADD cases were identified. No correlations between initial C4 value and follow-up biochemical markers (C4, EMA or MS levels) were found in the 76 cases studied. We found significant correlation between urine EMA versus MS, and correlation between follow-up C4 versus urine EMA. Of 22 cases where ACADS gene sequencing was performed: 7 had two or more deleterious mutations; 8 were compound heterozygotes for a deleterious mutation and common variant; 7 were homozygous for the common variant c.625G>A; and 1 was heterozygous for c.625G>A. Significant increases in mean urine EMA and MS levels were noted in patients with two or more deleterious mutations versus mutation heterozygotes or common polymorphism homozygotes. Clinical outcome data was available in 31 patients with follow-up extending from 0.5 to 60 months. None developed epilepsy or behavioral disorders, and three patients had isolated speech delay. Hypoglycemia occurred in two patients, both in the neonatal period. The first patient had concomitant meconium aspiration; the other presented with central apnea, poor feeding, and hypotonia. The latter, a c.625G>A homozygote, has had persistent elevations in both short- and medium-chain acylcarnitines; diagnostic workup in this case is extensive and ongoing. CONCLUSIONS: This study examines the largest series to date of SCADD patients identified by newborn screening. Our results suggest that confirmatory tests may be useful to differentiate patients with common variants from those with deleterious mutations. This study also provides evidence to suggest that, even when associated with deleterious mutations, SCADD diagnosed by newborn screening presents largely as a benign condition.

Similar changes in muscle lipid metabolism are induced by chronic high-fructose feeding and high-fat feeding in C57BL/J6 mice.

The aim of the present study was to investigate the effects of high fructose and high fat feeding on muscle lipid metabolism and to illustrate the mechanisms by which the two different dietary factors induce muscle lipid accumulation. C57BL/J6 mice were fed either a standard, high-fructose (HFru) or high-fat diet. After 16 weeks feeding, mice were killed and plasma triglyceride (TG) and free fatty acid (FFA) levels were detected. In addition, muscle TG and long chain acyl CoA (LCACoA) content was determined, glucose tolerance was evaluated and the protein content of fatty acid translocase CD36 (FATCD36) in muscle was measured. Mitochondrial oxidative function in the muscle was evaluated by estimating the activity of oxidative enzymes, namely cytochrome oxidase (COx), citrate synthase (CS) and ß-hydroxyacyl CoA dehydrogenase (ß-HAD), and the muscle protein content of carnitine palmitoyltransferase-1 (CPT-1), cyclo-oxygenase (COX)-1 and proliferator-activated receptor coactivator (PGC)-1α was determined. Finally, sterol regulatory element-binding protein-1c (SREBP-1c) gene expression and fatty acid synthase (FAS) protein content were determined in muscle tissues. After 16 weeks, plasma TG and FFA levels were significantly increased in both the HFru and HF groups. In addition, mice in both groups exhibited significant increases in muscle TG and LCACoA content. Compared with mice fed the standard diet (control group), those in the HFru and HF groups developed glucose intolerance and exhibited increased FATCD36 protein levels, enzyme activity related to fatty acid utilization in the mitochondria and protein expressions of CPT-1, COX-1 and PGC-1α in muscle tissue. Finally, mice in both the HFru and HF groups exhibited increase SREBP-1c expression and FAS protein content. In conclusion, high fructose and high fat feeding lead to similar changes in muscle lipid metabolism in C57BL/J6 mice. Lipid accumulation in the muscle may be associated with increased expression of proteins related to lipid transportation and synthesis.

Alpha methyl acyl CoA racemase deficiency: Diagnosis with isolated elevated liver enzymes.

Gündüz M, Ünal Ö, Küçükçongar-Yavas A, Kasapkara Ç. Alpha methyl acyl CoA racemase deficiency: Diagnosis with isolated elevated liver enzymes. Turk J Pediatr 2019; 61: 289-291. Alpha methy acyl CoA racemase (AMACR) deficiency is a rare autosomal recessive peroxisomal disorder characterized by cholestatic liver disease in the neonatal period, and variable neurologic symptoms affecting central and peripheral nervous systems in the following years. We report a Turkish patient who was diagnosed with AMACR deficiency with presentation of isolated elevated liver enzymes. The patient was referred for elevated liver enzymes when he was 10 months old. He had no cholestasis history in the neonatal period. Initially, an etiology could not be identified. Ultimately, the patient was diagnosed with AMACR deficiency with previously unreported p.Cys20Tyr (c.596G > A) homozygous pathogenic variant. At last visit, when he was 7.5 years old, his growth, development and neurologic examination were all normal. Biochemical analysis was normal except for mildly elevated AST levels. We suggest that checking VLCFA analysis may be useful in isolated elevated liver enzymes with unknown etiology.

Association of Anti-3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase Autoantibodies With DRB1*07:01 and Severe Myositis in Juvenile Myositis Patients.

OBJECTIVE: Autoantibodies recognizing 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) are associated with statin exposure, the HLA allele DRB1*11:01, and necrotizing muscle biopsies in adult myositis patients. The aim of this study was to characterize the features of juvenile anti-HMGCR-positive myositis patients. METHODS: The sera of 440 juvenile myositis patients were screened for anti-HMGCR autoantibodies. Demographic and clinical features, responses to therapy, and HLA alleles were assessed. The features of anti-HMGCR-positive patients were compared to those of previously described adult patients with this autoantibody and to children with other myositis-specific autoantibodies (MSAs). RESULTS: Five of 440 patients (1.1%) were anti-HMGCR-positive; none had taken statin medications. Three patients had rashes characteristic of juvenile dermatomyositis and 2 patients had immune-mediated necrotizing myopathies. The median highest creatine kinase (CK) level of anti-HMGCR-positive subjects was 17,000 IU/liter. All patients had severe proximal muscle weakness, distal weakness, muscle atrophy, joint contractures, and arthralgias, which were all more prevalent in HMGCR-positive subjects compared to MSA-negative patients or those with other MSAs. Anti-HMGCR-positive patients had only partial responses to multiple immunosuppressive medications, and their disease often took a chronic course. The DRB1*07:01 allele was present in all 5 patients, compared to 26.25% of healthy controls (corrected P = 0.01); none of the 5 juvenile patients had DRB1*11:01. CONCLUSION: Compared to children with other MSAs, muscle disease appears to be more severe in those with anti-HMGCR autoantibodies. Like adults, children with anti-HMGCR autoantibodies have severe weakness and high CK levels. In contrast to adults, in anti-HMGCR-positive children, there is a strong association with HLA-DRB1*07:01.

L-Arginine in the treatment of valproate overdose - five clinical cases.

BACKGROUND: Valproic acid and its metabolites - particularly valproyl-CoA - are inhibitors of the enzyme N-acetylglutamate synthetase. The amino acid l-arginine can stimulate N-acetylglutamate synthetase activity and could be potentially used therapeutically to correct hyperammonemia caused by valproate therapy or overdose. Severely valproic-acid-poisoned patients are usually treated with l-carnitine or hemodialysis in order to decrease hyperammonemia. We herein report of five cases, in which l-arginine was administered. METHODS: Observational study on five cases. Patients with hyperammonemia (i.e., ammonia 80 > µg/dL) and symptoms consistent with valproate overdose (i.e., drowsiness, coma) were selected for treatment with l-arginine. Data was collected retrospectively. RESULTS: l-Arginine decreased ammonia levels in a close temporal relation (case I ammonia in EDTA-plasma [µg/dL] decreased from 381 to 39; case II from 281 to 50; case III from 669 to 74; case IV from 447 to 56; case V from 202 to 60). In cases I and II, hemodialysis was performed and l-carnitine was given before the administration of l-arginine. In case III, hemodialysis was performed after the administration of l-arginine was already started. In cases IV and V, treatment with l-arginine was the sole measure to decrease ammonia levels in plasma. CONCLUSION: The results suggest that l-arginine may be beneficial in selected cases of valproate overdose complicated by hyperammonemia. l-Arginine could extend our conventional treatment options for valproic acid overdose.

Management and long-term evolution of a patient with 3-hydroxy-3-methylglutaryl-coenzyme A lyase deficiency.

BACKGROUND: 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase deficiency is a rare inborn error of metabolism characterized by recurrent metabolic crises caused by fasting, intercurrent illness and excessive physical exercise. Non ketotic hypoglycemia is normally the cause of primary symptoms but without an immediate treatment the illness can evolve into a worsening metabolic state resembling the Reye's syndrome that may cause the patient's death. We report a case with some clinical and therapeutic features not previously described. CASE PRESENTATION: Patient with HMG-CoA lyase deficiency whom after diagnosis at 2 years of age was re-admitted 12 years later, after severe metabolic decompensation following consumption of alcohol. Despite a quick correction of hypoglycemia, within the following few hours, the patient fell into a coma. Suspecting intracranial hypertension (ICH), the patient required mechanical ventilation. Although liver cytolysis was minimal, hyperamoniemia reached 1394 µmol/L, returning to normal, a few hours after administering sodium phenylacetate and sodium benzoate, whose use has not been reported in these patients. Brain edema was evidenced in the computed tomography and by the magnetic resonance imaging that determined that the edema was cytotoxic, as quantified with the restriction of diffusion in the apparent diffusion coefficient map. During the recovery of the ICH, we belatedly, detected vasospasm moderate-severe that was treated with nimodipine. Currently, the patient maintains clinical normality. CONCLUSIONS: The alcohol consumption must be avoided in patients with HMG-CoA lyase deficiency. In our patient hyperamoniemia was effectively treated with sodium phenylacetate and sodium benzoate. Magnetic resonance imaging showed and quantified the cytotoxic brain edema. Belatedly, a cerebral vasospasm was an additional mechanism of cerebral injury. None of these observations has been previously reported.

Rice bran enzymatic extract reduces atherosclerotic plaque development and steatosis in high-fat fed ApoE-/- mice.

OBJECTIVE: Rice bran is a by-product of rice milling and is rich in bioactive molecules such as γ-oryzanol, phytosterols, and tocotrienols. The rice bran enzymatic extract (RBEE) previously showed vessel remodeling prevention and lipid-lowering, antioxidant, anti-inflammatory, and antiapoptotic activities. The aim of this study was to identify RBEE hypolipidemic mechanisms and to study the effects of RBEE on the progression of atherosclerosis disease and linked vascular dysfunction and liver steatosis in apolipoprotein E-knockout (ApoE-/-) mice fed low- or high-fat (LFD, HFD, respectively) and cholesterol diets. METHODS: ApoE-/- mice were fed LFD (13% kcal) or HFD (42% kcal) supplemented or not supplemented with 1 or 5% RBEE (w/w) for 23 wk. Then, serum, aorta, liver, and feces were collected and flash frozen for further analysis. RESULTS: RBEE supplementation of HFD improved serum values by augmenting high-density lipoprotein cholesterol and preventing total cholesterol and aspartate aminotransferase increase. 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity was attenuated (1 and 5% RBEE) and cholesterol excretion increased (5% RBEE). Diet supplementation with 5% RBEE reduced plaque development regardless of the diet. In HFD-fed mice, both doses of RBEE reduced lipid deposition and macrophage infiltration in the aortic sinus and downregulated intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression. None of these effects was observed in mice fed LFD. Liver steatosis was reduced by RBEE supplementation of LFD (1% RBEE) and HFD (1 and 5% RBEE) and nuclear peroxisome proliferator-activated receptor-α expression upregulated in the HDF 5% RBEE group. CONCLUSION: Regular consumption of RBEE-supplemented HFD reduced plaque development and liver steatosis by decreasing inflammation and hyperlipidemia through an HMG-CoA reductase activity and lipid excretion-related mechanism.

Incorporation of arachidonic acid into 1-acyl-2-lyso-sn-glycero-3-phosphocholine of the human neutrophil.

In this study, the initial incorporation of arachidonic acid into human neutrophils has been examined. Neutrophils pulse labeled for 5 min with [3H]arachidonic acid rapidly incorporated this fatty acid into 1,2-diacylglycerophosphocholine. However, when neutrophils were pulse labeled with [3H]arachidonic acid for 5 min, washed, and allowed to incubate for an additional 120 min, the relative amount of [3H]arachidonic acid increased in alkylacylglycerophosphocholine molecular species. Similar, when neutrophils were pulse labeled, washed, and allowed to incubate in the presence of 30 microM unlabeled arachidonic acid for 120 min, [3H]arachidonic acid was also remodeled into alkylacylglycerophosphocholine. These results implied that the initial incorporation of [3H]arachidonic acid proceeded via a free fatty acid intermediate into 1,2-diacyl-GPC, while the subsequent remodeling of arachidonate-containing glycerophospholipids did not. This initial incorporation was further investigated in a number of cell-free systems. Disrupted neutrophils incubated with [14C]arachidonoyl-CoA incorporated [14C]arachidonic acid into 1,2-diacyl-GPC containing 16:0, 18:0, and 18:1 at their sn-1 position in a pattern similar to that seen when whole neutrophils were incubated with arachidonic acid for 5 min. A small percentage of [14C]arachidonate from [14C]arachidonoyl-CoA was incorporated into 1-alkyl-2-acyl-GPC. The enzymatic activity responsible was found predominately in the membrane fraction of the broken cell preparation. This selectivity of the CoA-dependent acyltransferase for 1-acyl-linked glycerophosphocholine was further examined by adding [14C]arachidonoyl-CoA and various 1-radyl-2-lyso-GPC to neutrophil membrane preparations. These studies provide evidence that the initial incorporation of arachidonic acid into sn-glycero-3-phosphocholine takes place by an arachidonoyl-CoA: lysophosphatidylcholine acyltransferase(s) which is selective for the 1-acyl-2-lyso-GPC.

Possible involvement of 1-acyl-glycerophosphorylinositol acyltransferase in arachidonate enrichment of phosphatidylinositol in human platelets.

Microsomes isolated from human platelets synthesize phosphatidylinositol by the action of acyl-CoA: 1-acyl-sn-glycerol-3-phosphorylinositol(1-acyl-GPI) acyltransferase. The properties of 1-acyl-GPI acyltransferase were compared with those of 1-acyl-glycerophosphorylcholine (1-acyl-GPC) acyltransferase. Apparent Km values of 1-acyl-GPI and 1-acyl-GPC acyltransferases for the corresponding acyl acceptor (lysophospholipid) were 22 and 20 microM, respectively, in the presence of arachidonoyl-CoA as fatty acyl donor. However, the Km value (1.3 microM) of 1-acyl-GPI acyltransferase for arachidonoyl-CoA was much lower than that (5.0 microM) of 1-acyl-GPC acyltransferase. Under optimal conditions, the acylation rate of 1-acyl-GPI with arachidonoyl-CoA was 2-6 times higher than with oleoyl-CoA and linoleoyl-CoA, and was very low with saturated fatty acyl-CoAs. The acylation rates with various acyl-CoAs for 1-acyl-GPI were different from those for 1-acyl-GPC. These results suggest that the reacylation pathway of 1-acyl-GPI participates in the incorporation of arachidonic acid to phosphatidylinositol in platelet microsomes. Furthermore, there were no significant effects of thrombin-activation on acyl-CoA specificity for 1-acyl-GPI and 1-acyl-GPC acyltransferase in human platelets.

Translocation of oleic acid across the erythrocyte membrane. Evidence for a fast process.

To clarify divergent views concerning the mechanism of fatty acid translocation across biomembranes this issue was now investigated in human erythrocytes. Translocation rates of exogenously inserted radioactive oleic acid across the membrane of native cells were derived from the time-dependent increase of the fraction of radioactivity becoming non-extractable by albumin. No accumulation of non-extractable unesterified oleic acid occurred. The rate of transfer was markedly suppressed by SH-reagents and by ATP-depletion. The suppression, however, resulted from a mere decrease of incorporation of oleic acid into phospholipids and was not accompanied by an increase of non-extractable unesterified oleic acid. These findings were reconcilable with the concept of a slow, possibly carrier-mediated fatty acid transfer as well as a very fast presumably, diffusional process not resolvable by the albumin extraction procedure. This ambiguity was resolved by using resealed ghosts, which are unable to incorporate oleic acid into phospholipids. In such ghosts all of the oleic acid inserted into the membrane remains extractable by albumin even after prolonged incubation. On the other hand, ghosts containing albumin accumulated non-extractable oleic acid. The rate of accumulation was beyond the time resolution of the albumin extraction procedure at 4 degrees C. Oleic acid uptake into albumin-containing ghosts became kinetically resolvable when the fatty acid was added as a complex with albumin. Correspondingly, time-resolvable release of oleic acid, originally complexed to internal albumin, into an albumin-containing medium was demonstrated at 4 degrees C. Rate and extent of these redistributions of oleic acid were dependent on the concentrations of internal and external albumin. This indicates limitation by the dissociation of oleic acid from albumin and not its translocation across the membrane. Translocation of oleic acid, which is probably a simple diffusive flip-flop process, must therefore occur with a half-time of less than 15 s. These findings raise doubts on the physiological role of presently discussed concepts of a carrier-mediated translocation of fatty acids across plasma membranes.

Preparation of inside-out vesicles from erythrocyte membranes inactivates the pathway for oleic acid incorporation into phospholipid.

The pathway for membrane phospholipid fatty acid turnover in situ may be important in the regulation of the composition and turnover of the lipid microenvironment of membrane proteins. This pathway has been characterized further by studying the activation and incorporation of [9,10(n)-3H]oleic acid and transesterification of [1-14C]oleoyl-CoA into membrane phospholipids by isolated erythrocyte membrane ghosts and inside-out vesicles derived from these ghosts. Erythrocyte ghosts and sealed vesicles of defined orientation prepared from them have been widely employed in studies of the function of membrane proteins, particularly those which mediate the transport of ions and sugars. Preparation of inside-out vesicles from ghosts by exposure to alkaline hypotonic conditions results in elution of some membrane proteins but no loss of membrane phospholipid. Compared to ghosts, the ability of inside-out vesicles to activate and incorporate [9,10(n)-3H]oleic acid into phospholipid is diminished by over 90% and the ability of inside-out vesicles to transesterify [1-14C]oleoyl-CoA to phospholipid is diminished by over 50%. These findings indicate that exposure of erythrocyte membranes to the alkaline hypotonic conditions required for inside-out vesicle preparation results in loss or inactivation of both acyl-CoA ligase and acyl-CoA-lysophospholipid acyltransferase activities. This lability of the enzymes for in situ phospholipid fatty acid turnover should be considered in the design and interpretation of studies concerned with elucidation of the relationship between phospholipid fatty acid turnover and the regulation of membrane protein function in this membrane preparation.

Effects of lovastatin in diabetic patients treated with chlorpropamide.

Patients with non-insulin dependent diabetes mellitus (NIDDM) have a higher risk of atherosclerotic cardiovascular disease than nondiabetic subjects. In seven patients with both hypercholesterolemia and NIDDM controlled by chlorpropamide, lovastatin (20 mg b.i.d. for 6 weeks) lowered low-density lipoprotein cholesterol by 28%, total cholesterol by 24%, and apolipoprotein B by 24%. Lovastatin levels for a 4-hour period (measured as 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitory activity) were similar to those measured previously in nondiabetic patients. Lovastatin did not alter chlorpropamide kinetics or glycemic profiles. No patient had an elevation in serum transaminases or creatinine phosphokinase, and no patient had any other laboratory or clinical drug-related adverse experience during the study. Lovastatin was as effective in reducing low-density lipoprotein cholesterol in patients with NIDDM as in nondiabetic subjects. Diabetic control was unaltered, and no evidence of alteration in lovastatin or chlorpropamide blood levels was noted.

Effects of L-carnitine on erythrocyte acyl-CoA, free CoA, and glycerophospholipid acyltransferase in uremia.

We studied the effects of L-carnitine treatment in the acyl flux of erythrocyte membranes from uremic patients. We found a significantly lower relative proportion of long-chain acyl-CoA (LCCoA) to free CoA (FCoA) in patients than in control subjects. In addition, patients had reduced activities of both carnitine palmitoyltransferase (CPT) and glycerophospholipid acyltransferase (LAT; CoA dependent), and increased ratios of long-chain acylcarnitine (LCAC) to free carnitine in their erythrocytes. These data support the hypothesis that acyl-trafficking is altered in erythrocytes in uremia. After treatment with L-carnitine, we observed a significant increase in CPT and LAT activities as well as in the LCCoA-FCoA ratio, and a significant decrease in the ratio of LCAC to free carnitine. These results support the conclusion that L-carnitine supplementation improves erythrocyte flux in uremic patients.

Effect of 4-(4'-chlorobenzyloxy)benzyl nicotinate (KCD-232) on triglyceride and fatty acid metabolism in rats.

The effects of KCD-232, a new hypolipidemic agent with a structure of 4-(4'-chlorobenzyloxy) benzyl nicotinate, on triglyceride (TG) and fatty acid (FA) metabolism were studied in rats. KCD-232 dose-dependently reduced both liver and serum TG levels. From in vivo and in vitro studies, the hypotriglyceridemic action of KCD-232 was shown to be based on the inhibition of hepatic TG synthesis due to both decreased FA synthesis and increased FA oxidation in the liver. Of two metabolites of KCD-232, i.e. 4-(4'-chlorobenzyloxy)benzoic acid (MII) and nicotinic acid, MII was found to be responsible for the decreased synthesis and increased oxidation of FA in the liver, the latter apparently being due to increased mitochondrial oxidation activated by MII. MII was demonstrated to form a xenobiotic TG in which one fatty acid moiety was substituted by MII and to form a thioester with CoA by rat liver microsomes. This thioester, MII-CoA, inhibited fatty acid syntheses from [14C]acetate, [14C] acetyl-CoA and [14C]malonyl-CoA in cell-free enzyme systems from rat liver both with and without an NADPH-generating system, whereas MII as such showed no effect. MII-CoA were therefore considered to be a chemical entity for the inhibition of hepatic fatty acid synthesis by KCD-232 and was suggested to inhibit fatty acid synthetase directly.

The content of coenzyme A, acetyl-CoA and long-chain acyl-CoA in human blood platelets.

The amounts of coenzyme A (CoASH) and acetyl-CoA in the acid-soluble extract of human blood platelets were quantitated by an isocratic reversed-phase liquid-chromatographic system. The analytical column was Supelcosil LC-18, and the solvent was 220 mmol/l potassium phosphate, pH 4.0, and 120 ml/l methanol. Long-chain acyl-CoA was estimated by the released coenzyme A after an alkaline hydrolysis of the acid-insoluble material of the blood platelets. The median values in platelets from fourteen healthy persons were 30 pmol CoASH/mg platelet protein, 45 pmol acetyl-CoA/mg platelet protein, and 34 pmol long-chain acyl-CoA/mg platelet protein.

Rapid differential diagnosis of carboxylase deficiencies and evaluation for biotin-responsiveness in a single blood sample.

We have developed a method for rapid differential diagnosis of isolated or multiple deficiencies of the 3 mitochondrial biotin-dependent carboxylases: propionyl-CoA (PCC), 3-methylcrotonyl-CoA (MCC) and pyruvate carboxylase (PC), and for simultaneous evaluation of biotin-responsiveness using a single blood sample. Lymphocytes were isolated from heparinized blood and preincubated without and with 10(-5) mol/l biotin in medium before determination of PCC, MCC and PC activities. Plasma was used for estimation of biotin concentration and biotinidase activity. A definitive diagnosis could be made in 7 of 9 patients studied up to now: 4 patients suffered from biotin-nonresponsive isolated PCC-deficiency, and 3 patients from biotin-responsive multiple carboxylase deficiency caused by deficient biotinidase activity. In two patients, a carboxylase deficiency was excluded. These results were confirmed in studies using fibroblasts. In addition, a simple method for detection of deficiency in holocarboxylase synthesis is described.