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.

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.

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.

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.

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.

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.

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.

Fasting and postprandial acyl and desacyl ghrelin levels in obese and non-obese subjects.

INTRODUCTION: The potentially differential roles of both forms of ghrelin in obesity are undefined, and little is known about desacyl ghrelin's (DAG) regulation by meals. We aimed to assess changes in acyl ghrelin (AG) and DAG in response to mixed-meal consumption in obese and non-obese subjects. MATERIAL AND METHODS: Venous blood for plasma glucose, AG and DAG assays were collected in both groups after an overnight fast and two hours after the consumption of a standard 300 kcal-mixed meal (Nutridrink, Nutricia). RESULTS: Mean fasting values of both AG and DAG were significantly lower in the obese individuals. On the other hand, among non-obese controls, the mean postprandial DAG levels did not change and AG levels decreased, whereas in obese individuals the mean DAG levels after a mixed-meal diminished and AG levels were unchanged. CONCLUSIONS: It is necessary to distinguish between the desacylated and acylated forms of ghrelin, as we have shown differential postprandial AG and DAG responses in obese and non-obese individuals. Whether targeting changed proportions between AG and DAG could be a successful strategy in obesity treatment remains a question for future studies.

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.

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.

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.

Serum 2-methoxyestradiol, an estrogen metabolite, is positively associated with serum HDL-C in a population-based sample.

Serum HDL cholesterol (HDL-C) is inversely associated with coronary artery disease, ischemic stroke, and atherosclerosis in men and women. Among postmenopausal women, oral conjugated equine estrogen (CEE) increases serum HDL-C. This is due to activation of hepatic nuclear estrogen receptors, resulting in increased HDL-C expression, as well as modulation of proteins which metabolize HDL-C. 2-methoxyestradiol (2-MeOE2), an estrogen metabolite, has several vasculoprotective effects and may play a role in HDL-C production. 2-MeOE2 inhibits HMG-CoA reductase in vitro but no study has examined the relationship between serum 2-MeOE2 and serum HDL-C. A population-based sample provided information regarding demographic characteristics and use of antihyperlipidemic medications. Serum was analyzed for 17ß-estradiol (E2), estrogen metabolites (EMs), and lipoproteins. Results included serum EM data from 51 men and 47 postmenopausal women. Preliminary analysis revealed no correlation between 2-MeOE2 and serum HDL-C in men so the current analysis includes only women (N = 40) with no missing demographic, medication, EM, or lipoprotein data. Linear regression revealed that serum 2-MeOE2 and antihyperlipidemic medications were positively associated with serum HDL-C (ß = 0.276, P = 0.043, and ß = 0.307, P = 0.047, respectively) when age, race/ethnicity, and body mass index were held constant. Prospective studies are needed to determine if 2-MeOE2 is causally related to HDL-C in women.

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.

Déficit de acil-coenzima A deshidrogenasa de ácidos grasos de cadena media / Medium-chain acyl-coenzyme A dehydrogenase deficiency

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Assay of the concentration and (13)C isotopic enrichment of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA by gas chromatography-mass spectrometry.

We developed gas chromatography-mass spectrometry assays for the concentration and mass isotopomer distribution of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA in tissues. The assays involves perchloric acid extraction of the tissue, spiking the extract with [(2)H(5)]propionyl-CoA and [(2)H(4)]succinyl-CoA internal standards, and isolation of short-chain acyl-CoA fraction on an oligonucleotide purification cartridge. Propionyl-CoA is reacted with sarcosine and the formed N-propionylsarcosine is assayed as its pentafluorobenzyl derivative. Methylmalonyl-CoA and succinyl-CoA are hydrolyzed and the corresponding acids assayed as tert-butyl dimethylsilyl derivatives. The assay was applied to a study of [U-(13)C(3)]propionate metabolism in perfused rat livers. While propionyl-CoA is only M3 labeled, succinyl-CoA is M3, M2, and M1 labeled because of isotopic exchanges in the citric acid cycle. Methylmalonyl-CoA is M3 and M2 labeled, reflecting reversal of S-methylmalonyl-CoA mutase. Thus, our assays allow measuring the turnover of the coenzyme A derivatives involved in anaplerosis of the citric acid cycle via precursors of propionyl-CoA, i.e., propionate, odd-chain fatty acids, isoleucine, threonine, and valine.

Neonatal seizures: unusual causes.

Seizures are the most common manifestation of neurological dysfunction in the newborn. The causes of newborn seizures are manifold with etiological determination important with respect to treatment, prognosis, and recurrence risk perspectives. This article highlights two cases with unusual, genetically based causes for newborn seizures. These cases are used to highlight the diagnostic approach to this clinical problem.

A child with reading impairment and a family history of adrenoleukodystrophy.

The clinical course of a 6-year-old boy with adrenoleukodystrophy (ALD) who underwent allogeneic stem-cell transplantation during an early clinical stage is described. Twenty-three months after transplant, he remains neurologically stable, but with moderate neurological sequelae; the serum very long chain fatty acid profile has improved, but not normalized. The indications, mechanism of action, and complications of bone marrow transplantation in ALD are discussed briefly, along with other potential therapies.

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.

Long chain acyl coenzyme A and signaling in neutrophils. An inhibitor of acyl coenzyme A synthetase, triacsin C, inhibits superoxide anion generation and degranulation by human neutrophils.

Ligand-initiated activation of neutrophils triggers O2- generation, degranulation, phospholipid remodeling, and release of fatty acids such as arachidonate, oleate, and palmitate. Long chain acyl-CoA synthetase converts free fatty acids to acyl-CoA esters; a role for acyl-CoA esters as positive modulators of neutrophil functions is proposed. Physiologically relevant concentrations (1-10 microM) of acyl-CoA esters such as palmitoyl-CoA, enhanced O2- generation triggered by fMet-Leu-Phe or guanosine 5'-O-(thiotriphosphate) (GTP gamma S) but did not act as a trigger per se. Triacsin C, an inhibitor of acyl-CoA synthetase, inhibited fMet-Leu-Phe-elicited O2- generation and degranulation in a concentration-dependent manner. Triacsin C inhibited O2- generation elicited by fMet-Leu-Phe and GTP gamma S in electroporated neutrophils, indicating that acyl-CoA acted downstream from the receptor. Palmitoyl-CoA reversed the Triacsin C-induced inhibition of O2- generation. fMet-Leu-Phe elicited a prompt increase in total long chain acyl-CoA esters. Arachidonoyl-CoA and oleoyl-CoA were elevated 5 s after addition of fMet-Leu-Phe, while palmitoyl-CoA was not elevated until 60 s. Triacsin C inhibited fMet-Leu-Phe-initiated increases in arachidonoyl-CoA, oleoyl-CoA, and palmitoyl-CoA. These results suggest a role for acyl-CoA esters in regulating activation of O2- generation and degranulation at the G protein or subsequent step(s).

Enzymatic studies of lyso platelet-activating factor acylation in human neutrophils and changes upon stimulation.

Resting human neutrophils acylate 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (1-O-alkyl-2-lyso-GPC; lyso-PAF) specifically with arachidonate (AA); upon stimulation, however, the specificity is lost and other fatty acid residues are added. The major goals of this study were to compare the various acylation reactions present in the cells and to determine the cause of the specificity loss upon stimulation. The CoA-independent transacylase was active in neutrophil homogenates and was found to be both highly specific for AA and stereospecific, requiring 1-O-alkyl-2-lyso-GPC for activity. Homogenates also contained acyl-CoA:1-radyl-2-lyso-sn-glycero-3-phosphocholine acyltransferase activity, which transferred acyl chains from oleoyl-, linoleoyl-, or linolenoyl-CoA to both 1-alkyl and 1-acyl acceptors, but preferred the 1-acyl acceptor when arachidonoyl-CoA was used. The CoA-dependent and -independent activities co-sedimented on a discontinuous Percoll gradient in a single band containing plasma membrane and possibly other membranes. CoA alone promoted nonspecific acylation in the homogenates. The AA-specific acylation was attenuated up to 80% in sonicates of ionophore-stimulated cells, whereas the CoA-dependent acyltransferase remained unchanged. Potential phospholipid AA donors for the transacylase were substantially depleted in the stimulated cells but could not account for the large decrease in acylation. An accumulation of 1-O-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine (alkenyl-2-lyso-GPE), which acts as a competing substrate, appeared to be the major cause of the reduced AA-specific acylation of lyso-PAF observed in the stimulated preparations. Removal of the alkenyl-2-lyso-GPE restored the activity, whereas the addition of alkenyl-2-lyso-GPE (2 microM) to resting membrane preparations resulted in a marked decrease in transacylation of lyso-PAF.