Total Coenzyme A Research Articles

Emerging variants, unique phenotypes, and transcriptomic signatures: an integrated study of COASY-associated diseases.

COASY, the gene encoding the bifunctional enzyme CoA synthase, which catalyzes the last two reactions of cellular de novo coenzyme A (CoA) biosynthesis, has been linked to two exceedingly rare autosomal recessive disorders, such as COASY protein-associated neurodegeneration (CoPAN), a form of neurodegeneration with brain iron accumulation (NBIA), and pontocerebellar hypoplasia type 12 (PCH12). We aimed to expand the phenotypic spectrum and gain insights into the pathogenesis of COASY-related disorders. Patients were identified through targeted or exome sequencing. To unravel the molecular mechanisms of disease, RNA sequencing, bioenergetic analysis, and quantification of critical proteins were performed on fibroblasts. We identified five new individuals harboring novel COASY variants. While one case exhibited classical CoPAN features, the others displayed atypical symptoms such as deafness, language and autism spectrum disorders, brain atrophy, and microcephaly. All patients experienced epilepsy, highlighting its potential frequency in COASY-related disorders. Fibroblast transcriptomic profiling unveiled dysregulated expression in genes associated with mitochondrial respiration, responses to oxidative stress, transmembrane transport, various cellular signaling pathways, and protein translation, modification, and trafficking. Bioenergetic analysis revealed impaired mitochondrial oxygen consumption in COASY fibroblasts. Despite comparable total CoA levels to control cells, the amounts of mitochondrial 4'-phosphopantetheinylated proteins were significantly reduced in COASY patients. These results not only extend the clinical phenotype associated with COASY variants but also suggest a continuum between CoPAN and PCH12. The intricate interplay of altered cellular processes and signaling pathways provides valuable insights for further research into the pathogenesis of COASY-associated diseases.

Elucidating the role of NUDT19 in renal lipid metabolism

Coenzyme A (CoA) is an essential cofactor that plays a key role in fuel metabolism, as it is required for glucose oxidation and the synthesis and oxidation of fatty acids, amino acids, and ketone bodies. Three Nudix hydrolases, NUDT7, NUDT8, and NUDT19, hydrolyze CoA species at the phosphodiester bond, potentially contributing to the regulation of specific metabolic processes in the subcellular compartments where they reside. NUDT19 is found in the peroxisomes and is highly expressed in kidney proximal tubule cells (PTC). In vitro, NUDT19 readily hydrolyzes short and medium chain acyl‐CoAs, malonyl‐ and succinyl‐CoA, as well as free CoA. This substrate preference, combined with the peroxisomal localization of NUDT19, suggests a potential role in the regulation of peroxisomal lipid metabolism. To investigate the physiological function of NUDT19, we obtained global Nudt19‐/‐ mice and fed them a high fat diet (HFD) or a low fat control diet (CD) for 15 weeks. On either diet, Nudt19‐/‐ mice exhibit higher total CoA levels compared to wild type mice, indicating that NUDT19 contributes to the regulation of this cofactor. HFD feeding led to a higher albumin‐to‐creatinine ratio in the Nudt19‐/‐ mice compared to wild type mice, indicating a decrease in kidney function. Nudt19‐/‐ mice also exhibited elevated serum triglycerides and a higher body weight, not associated with changes in bone density or adiposity. In mice fed the HFD, deletion of Nudt19 caused only a few changes to the kidney cortex proteome, with only 8 proteins significantly altered, including 3 involved in peroxisomal and mitochondria fatty acid oxidation, which were increased in the kidneys of the Nudt19‐/‐ mice. Measurement of mitochondrial and peroxisomal fatty acid oxidation using 14C‐palmitate in kidney cortex slides did not reveal any differences between genotypes. Conversely, untargeted metabolomics analysis on whole kidneys revealed a general decrease in multiple lipid classes including lysophospholipids, monoacylglycerols, and long chain free fatty acids, without changes in triglycerides levels. Combined, the data support the conclusion that deletion of Nudt19 alters lipid metabolism.

Turnover rate of coenzyme A in mouse brain and liver.

Coenzyme A (CoA) is a fundamental cofactor involved in a number of important biochemical reactions in the cell. Altered CoA metabolism results in severe conditions such as pantothenate kinase-associated neurodegeneration (PKAN) in which a reduction of the activity of pantothenate kinase isoform 2 (PANK2) present in CoA biosynthesis in the brain consequently lowers the level of CoA in this organ. In order to develop a new drug aimed at restoring the sufficient amount of CoA in the brain of PKAN patients, we looked at its turnover. We report here the results of two experiments that enabled us to measure the half-life of pantothenic acid, free CoA (CoASH) and acetylCoA in the brains and livers of male and female C57BL/6N mice, and total CoA in the brains of male mice. We administered (intrastriatally or orally) a single dose of a [13C3-15N-18O]-labelled coenzyme A precursor (fosmetpantotenate or [13C3-15N]-pantothenic acid) to the mice and measured, by liquid chromatography-mass spectrometry, unlabelled- and labelled-coenzyme A species appearance and disappearance over time. We found that the turnover of all metabolites was faster in the liver than in the brain in both genders with no evident gender difference observed. In the oral study, the CoASH half-life was: 69 ± 5 h (male) and 82 ± 6 h (female) in the liver; 136 ± 14 h (male) and 144 ± 12 h (female) in the brain. AcetylCoA half-life was 74 ± 9 h (male) and 71 ± 7 h (female) in the liver; 117 ± 13 h (male) and 158 ± 23 (female) in the brain. These results were in accordance with the corresponding values obtained after intrastriatal infusion of labelled-fosmetpantotenate (CoASH 124 ± 13 h, acetylCoA 117 ± 11 and total CoA 144 ± 17 in male brain).

The Role of Peroxisomal CoA Degradation in the Regulation of Bile Acid Metabolism

Coenzyme A (CoA) is an essential metabolic cofactor that activates fatty acids and other organic acids as CoA thioesters for a multitude of metabolic pathways in different subcellular compartments. For example, CoA is utilized in the bile acid synthesis pathway and for shortening of very long chain fatty acids in the peroxisomes. In the liver, CoA levels are tightly regulated and dynamically adjusted in response to changes in nutritional status and energy demands. Balance between CoA synthesis and degradation is responsible for the regulation of CoA. Three intracellular CoA diphosphohydrolases, Nudt7, Nudt8, and Nudt19, catalyze the degradation of CoA by catalyzing the hydrolysis of its pyrophosphate bond. Nudt7 is the major CoA-degrading enzyme in the liver and resides in the peroxisomes. This enzyme displays a broad substrate specificity; it is active against free CoA, short- and medium-chain acyl-CoAs, including acetyl-CoA, and the acyl-CoA derivatives of bile acids.Nudt7 expression and activity are regulated by nutritional state, as they both decrease upon fasting. Overexpression of Nudt7 in the liver was recently found to decrease the levels of a variety of short-chain acyl-CoAs and choloyl-CoA, and to decrease both the rate of peroxisomal fatty acid oxidation and the concentration of bile acids in the liver. To gain further insight into the role of Nudt7 in vivo, we generated Nudt7-/- mice and analyzed their lipid metabolism in response to a dietary challenge with a high fat, high cholesterol (western) diet. Deletion of Nudt7 caused a significant increase in total hepatic CoA levels in Nudt7-/- males compared to control mice.Targeted and untargeted metabolomics analysis of whole liver extracts revealed an increase in glutaryl- and adipoyl-CoA in the Nudt7-/- males, associated with an accumulation of glutaryl-carnitine and other short-chain dicarboxylic acylcarnitines and with a decrease in long-chain acyl carnitines, suggesting an increase in the rate of fatty acid oxidation. Pathway enrichment analysis revealed changes in bile acid metabolism in both male and female Nudt7-/- mice compared to gender-matched controls.Specifically, deletion of Nudt7 led to an accumulation of bile acids in the liver, gallbladder, and small intestine, which also correlated with higher serum levels of Fgf15. This increase in bile acids occurred without changes in the expression of genes involved in bile acid synthesis or transport and without a correspondent increase in the concentration of bile acid precursors 7α-hydroxycholesterol and 7α-hydroxycholestenone. These results suggest that higher CoA levels in the peroxisomes may mediate an increase in flux through the bile acid biosynthetic pathway by activating the last, peroxisomal steps in the pathway. Overall, analysis of lipid metabolism following manipulation of Nudt7 expression in mouse liver indicates that this enzyme contributes to the regulation of peroxisomal fatty acids and bile acid metabolism.

Enhancement of fatty acid biosynthesis by exogenous acetyl-CoA carboxylase and pantothenate kinase in Escherichia coli

To establish a technique for efficient fatty acid production through enhancement of coenzyme A (CoA) biosynthesis and malonyl-CoA supply by introducing exogenous pantothenate kinase (coaA) and acetyl-CoA carboxylase (acc) in Escherichia coli. The expression of acc, obtained from Corynebacterium glutamicum, accumulated 2.2-fold more fatty acids in E. coli. The addition of coaA from Pseudomonas putaida or fatty acid synthase (fasA) from C. glutamicum resulted in a 3.1- and 3.6-fold increase in fatty acid synthesis in E. coli cells, which expressed acc and coaA, or acc and fasA, respectively. The transformants, simultaneously possessing all three genes, produced 5.6-fold more fatty acids. The strain possessing acc, coaA, and fasA stored 691mg/L of fatty acids, primarily as phospholipids, inside the inner membrane after 72-h cultivation. In addition, 19% of the total CoA pool was occupied by malonyl-CoA. Increased malonyl-CoA significantly contributed to fatty acid production, and the effect was boosted by the expanded total CoA pool. Manipulation of the intracellular CoA species is effective for fatty acid production in E. coli.

The Role of Coenzyme A Degradation in the Regulation of Peroxisomal Metabolism

Coenzyme A (CoA) is an essential metabolic cofactor that activates fatty acids and other organic acids as CoA thioesters for a multitude of metabolic pathways in different subcellular compartments. For example, CoA is utilized in the bile acid synthesis pathway and for shortening of very long chain fatty acids in the peroxisomes. The regulation of CoA levels is essential to maintain metabolic flexibility in response to changes in nutritional status and energy demands. Balance between CoA synthesis and degradation is responsible for the regulation of CoA. While CoA synthesis is controlled at the pantothenate kinase step, the intracellular degradation of CoA is controlled by Nudix hydrolases. Nudt7 is a hepatic, peroxisomal Nudix hydrolase that specifically hydrolyzes CoA species into 3’,5’‐ADP and (acyl)phosphopantetheine. Nudt7 displays a broad substrate specificity; it is active against free CoA, short‐ and medium‐chain acyl‐CoAs, including acetyl‐CoA, and the acyl‐CoA derivatives of bile acids. Nudt7 expression and activity are regulated by nutritional state, as they both decrease upon fasting. Overexpression of Nudt7 in the liver was recently found to decrease the levels of a variety of short‐chain acyl‐CoAs and choloyl‐CoA in the liver, and to decrease the rate of peroxisomal β‐oxidation. To gain further insight into the role of Nudt7 in vivo, Nudt7−/− mice were generated and their lipid metabolism assessed. Deletion of Nudt7 caused a small but significant increase in total hepatic CoA levels in the fasted state. In particular, higher concentrations of malonyl‐and acetyl‐CoA were observed. Furthermore, livers from Nudt7−/− males contained higher levels of short‐chain fatty acids and short‐chain acylcarnitines, which, combined, suggest an increase in fatty acid oxidation. In the fed state, female Nudt7−/− mice exhibited a higher concentration of selected bile acids in the intestine, while feeding of a high fat high/cholesterol diet resulted in higher bile acid levels in the liver. This increase in bile acids occurred without changes in the expression of genes involved in bile acid synthesis or transport and without a correspondent increase in the concentration of bile acid precursors 7α‐hydroxycholesterol and 7α‐hydroxycholestenone. These results suggest that higher CoA levels in the peroxisomes may mediate an increase in flux through the bile acid biosynthetic pathway by activating of the last, peroxisomal steps in the pathway.Overall, analysis of lipid metabolism following manipulation of Nudt7 expression in mouse liver indicates that this enzyme contributes to the regulation of peroxisomal fatty acids and bile acid metabolism.Support or Funding InformationThis work was supported by West Virginia University’s School of Medicine startup foundation funding and NIH grant GM119528.

Changes in acetyl CoA levels during the early embryonic development of Xenopus laevis.

Coenzyme A (CoA) is a ubiquitous and fundamental intracellular cofactor. CoA acts as a carrier of metabolically important carboxylic acids in the form of CoA thioesters and is an obligatory component of a multitude of catabolic and anabolic reactions. Acetyl CoA is a CoA thioester derived from catabolism of all major carbon fuels. This metabolite is at a metabolic crossroads, either being further metabolised as an energy source or used as a building block for biosynthesis of lipids and cholesterol. In addition, acetyl CoA serves as the acetyl donor in protein acetylation reactions, linking metabolism to protein post-translational modifications. Recent studies in yeast and cultured mammalian cells have suggested that the intracellular level of acetyl CoA may play a role in the regulation of cell growth, proliferation and apoptosis, by affecting protein acetylation reactions. Yet, how the levels of this metabolite change in vivo during the development of a vertebrate is not known. We measured levels of acetyl CoA, free CoA and total short chain CoA esters during the early embryonic development of Xenopus laevis using HPLC. Acetyl CoA and total short chain CoA esters start to increase around midblastula transition (MBT) and continue to increase through stages of gastrulation, neurulation and early organogenesis. Pre-MBT embryos contain more free CoA relative to acetyl CoA but there is a shift in the ratio of acetyl CoA to CoA after MBT, suggesting a metabolic transition that results in net accumulation of acetyl CoA. At the whole-embryo level, there is an apparent correlation between the levels of acetyl CoA and levels of acetylation of a number of proteins including histones H3 and H2B. This suggests the level of acetyl CoA may be a factor, which determines the degree of acetylation of these proteins, hence may play a role in the regulation of embryogenesis.

Amino-4-Imidazolecarboxamide Ribotide Directly Inhibits Coenzyme A Biosynthesis in Salmonella enterica

Aminoimidazole carboxamide ribotide (AICAR) is a purine biosynthetic intermediate and a by-product of histidine biosynthesis. In bacteria, yeast, and humans, accumulation of AICAR has been shown to affect an array of cellular processes by both direct and indirect mechanisms. In purine biosynthesis, AICAR is the substrate of the bifunctional protein phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase (PurH, EC 2.1.2.3/3.5.4.10). Strains lacking PurH accumulate AICAR and have a defect in the synthesis of the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) moiety of thiamine. The formation of HMP is also compromised in vivo when coenzyme A (CoA) levels are reduced. Our results show that the in vivo accumulation of AICAR decreased total CoA pools and, further, that AICAR inhibited the activity of pantoate β-alanine ligase in vitro (PanC, EC 6.3.2.1). These results demonstrated a mechanism of AICAR action and provide new insights into the metabolic consequences of disrupting purine metabolism.

Effect of diet composition on coenzyme A and its thioester pools in various rat tissues

Three coenzyme A (CoA) molecular species, i.e., acetyl-CoA, malonyl-CoA, and nonesterified CoA (CoASH), in 13 types of fasted rat tissue were analyzed. A relatively larger pool size of total CoA, consisting of acetyl-CoA, malonyl-CoA, and CoASH, was observed in the medulla oblongata, liver, heart, and brown adipose tissue. Focusing on changes in the CoA pool size in response to the nutrient composition of the diet given, total CoA pools in rats continuously fed a high-fat diet for 4weeks were significantly higher in the hypothalamus, cerebellum, and kidney, and significantly lower in the liver and skeletal muscle than those of rats fed a high-carbohydrate or high-protein diet. In particular, reductions in the liver were remarkable and were caused by decreased CoASH levels. Consequently, the total CoA pool size was reduced by approximately one-fifth of the hepatic contents of rats fed the other diets. In the hypothalamus, which monitors energy balance, all three CoA molecular species measured were at higher levels when rats were fed the high-fat diet. Thus, it was of interest that feeding rats a high-fat diet affected the behaviors of CoA pools in the hypothalamus, liver, and skeletal muscle, suggesting a significant relationship between CoA pools, especially malonyl-CoA and/or CoASH pools, and lipid metabolism in vivo.

Coenzyme A and its thioester pools in fasted and fed rat tissues

Levels of three coenzyme A (CoA) molecular species, i.e., nonesterified CoA (CoASH), acetyl-CoA, and malonyl-CoA, in fasted and fed rat tissues were analyzed by the acyl-CoA cycling method which makes detection possible at the pmol level. Malonyl-CoA in brain tissues readily increased with feeding, and inversely, acetyl-CoA decreased. This phenomenon occurred in the cerebral cortex, hippocampus, cerebellum, and medulla oblongata, as well as in the hypothalamus which controls energy balance by monitoring malonyl-CoA. In the non-brain tissues, the sizes of the acetyl-CoA, malonyl-CoA, and CoASH pools depended on the tissues. The total CoA pools consisting of the above three CoA species in the liver, heart, and brown adipose tissue were larger and those of the perirenal, epididymal, and ovarian adipose tissues were much smaller, compared with those of other tissues including brain tissues. In addition, the response of each CoA pool to feeding was not uniform, suggesting that the tissue-specific metabolism individually functions in the non-brain tissues. Thus, a comprehensive analysis of thirteen types of rat tissue revealed that CoA pools have different sizes and showed a different response to fasting and feeding depending on the tissue.

Pantothenate kinase 1 is required to support the metabolic transition from the fed to the fasted state.

Coenzyme A (CoA) biosynthesis is regulated by the pantothenate kinases (PanK), of which there are four active isoforms. The PanK1 isoform is selectively expressed in liver and accounted for 40% of the total PanK activity in this organ. CoA synthesis was limited using a Pank1 −/− knockout mouse model to determine whether the regulation of CoA levels was critical to liver function. The elimination of PanK1 reduced hepatic CoA levels, and fasting triggered a substantial increase in total hepatic CoA in both Pank1 −/− and wild-type mice. The increase in hepatic CoA during fasting was blunted in the Pank1 −/− mouse, and resulted in reduced fatty acid oxidation as evidenced by abnormally high accumulation of long-chain acyl-CoAs, acyl-carnitines, and triglycerides in the form of lipid droplets. The Pank1 −/− mice became hypoglycemic during a fast due to impaired gluconeogenesis, although ketogenesis was normal. These data illustrate the importance of PanK1 and elevated liver CoA levels during fasting to support the metabolic transition from glucose utilization and fatty acid synthesis to gluconeogenesis and fatty acid oxidation. The findings also suggest that PanK1 may be a suitable target for therapeutic intervention in metabolic disorders that feature hyperglycemia and hypertriglyceridemia.

Pank1 plays an important role in coenzyme A homeostasis during fasting

Coenzyme A (CoA) is an essential cofactor synthesized in five steps from pantothenate, and pantothenate kinase (PanK) catalyzes the first and most regulated step. The Pank1 gene encodes two of the four PanK isoforms, PanK1α and 1β. Deletion of exon 3 in the mouse Pank1 gene resulted in loss of expression of both isoforms. The liver of the Pank1 knockout animals contained 40% less total CoA, and the knockout mice showed a blunted response to a pyruvate challenge, indicating impairment in gluconeogenesis. Fasting increases CoA levels to support β‐oxidation and gluconeogenesis. Following a 48h fast, the CoA levels increased by 70% (from 122 ± 4 to 207 ± 23 nmoles/mg of tissue) in livers from wild type and by 50% (from 71 ± 7 to 107 ± 11 nmole/mg of tissue) in livers from knockout mice. Concomitantly, the knockout livers accumulated five times more triglycerides than controls, and the knockout liver homogenates exhibited a decreased rate of palmitic acid oxidation. These results indicate that the reduction in CoA decreases the efficiency of fatty acid β‐oxidation, and that in addition to contributing a large portion of the cofactor pool, Pank1 plays an important role in responding to the increased demand for CoA during fasting. We also observed that the Pank1 knockout males are hyperphagic and 15% heavier than controls, which correlated with a 50% lower malonyl‐CoA in the hypothalamus. Supported by NIH GM062896, CA21765, and ALSAC.

Overproduction of a functional fatty acid biosynthetic enzyme blocks fatty acid synthesis in Escherichia coli.

beta-Ketoacyl-acyl carrier protein (ACP) synthetase II (KAS II) is one of three Escherichia coli isozymes that catalyze the elongation of growing fatty acid chains by condensation of acyl-ACP with malonyl-ACP. Overexpression of this enzyme has been found to be extremely toxic to E. coli, much more so than overproduction of either of the other KAS isozymes, KAS I or KAS III. The immediate effect of KAS II overproduction is the cessation of phospholipid synthesis, and this inhibition is specifically due to the blockage of fatty acid synthesis. To determine the cause of this inhibition, we examined the intracellular pools of ACP, coenzyme A (CoA), and their acyl thioesters. Although no significant changes were detected in the acyl-ACP pools, the CoA pools were dramatically altered by KAS II overproduction. Malonyl-CoA increased to about 40% of the total cellular CoA pool upon KAS II overproduction from a steady-state level of around 0.5% in the absence of KAS II overproduction. This finding indicated that the conversion of malonyl-CoA to fatty acids had been blocked and could be explained if either the conversion of malonyl-CoA to malonyl-ACP and/or the elongation reactions of fatty acid synthesis had been blocked. Overproduction of malonyl-CoA:ACP transacylase, the enzyme catalyzing the conversion of malonyl-CoA to malonyl-ACP, partially relieved the toxicity of KAS II overproduction, consistent with a model in which high levels of KAS II blocks access of the other KAS isozymes to malonyl-CoA:ACP transacylase.

Mitochondrial, but not peroxisomal, beta-oxidation of fatty acids is conserved in coenzyme A-deficient rat liver.

Hepatic coenzyme A (CoA) plays an important role in cellular lipid metabolism. Because mitochondria and peroxisomes represent the two major subcellular sites of lipid metabolism, the present study was designed to investigate the specific impact of hepatic CoA deficiency on peroxisomal as well as mitochondrial beta-oxidation of fatty acids. CoA deficiency (47% decrease in free CoA and 23% decrease in total CoA) was produced by maintaining weanling male Sprague-Dawley rats on a semipurified diet deficient in pantothenic acid (the precursor of CoA) for 5 weeks. Hepatic mitochondrial fatty acid oxidation of short-chain and long-chain fatty acids were not significantly different between control and CoA-deficient rats. Conversely, peroxisomal beta-oxidation was significantly diminished (38% inhibition) in livers of CoA-deficient rats compared to control animals. Peroxisomal beta-oxidation was restored to normal levels when hepatic CoA was replenished. It is postulated that since the role of hepatic mitochondrial beta-oxidation is energy production while peroxisomal beta-oxidation acts mainly as a detoxification system, the mitochondrial pathway of beta-oxidation is spared at the expense of the peroxisomal pathway when liver CoA plummets. The present study may offer an animal model to investigate mechanisms involved in peroxisomal diseases.

Influence of pantothenic acid deficient diet on the metabolic changes in rats exposed to bezafibrate and di-(2-ethylhexyl)phthalate

The present study was undertaken to examine the influence of pantothenic acid deficient diet on the metabolic responses to peroxisome proliferators including bezafibrate and di-(2-ethylhexyl)phthalate (DEHP). The growing rats were fed on the pantothenic acid deficient diet with or without addition of 0.1% bezafibrate or 0.5% DEHP for 14 days. Feeding of pantothenic acid deficient diet decreased food intake and growth rate. Dietary addition of these chemicals caused the earlier onset of growth retardation by pantothenic acid deficient diet. These chemicals increased hepatic total Coenzyme A (CoA) levels, associated with increased activities of hepatic peroxisomal β-oxidation and catalase. These increments in hepatic total CoA and activities of these enzymes were all depressed by pantothenic acid deficient diet. The results suggest that an increased hepatic CoA pool by the proliferators might relate to the induction of peroxisome β-oxidation and catalase, and be an adaptive response to exposure to the proliferators.

ヒ酸鉛がウンシュウミカン果肉中のコエンザイムAに及ぼす影響

The effect of lead arsenate on coenzyme A (CoA) was examined in order to investigate the involvement of CoA in the reduction of free acidity of juice by the chemical in hopes of finding non-toxic acid-reducing chemicals.0.3% lead arsenate solution was sprayed on satsuma mandarin (Citrus unshiu) trees 3 to 5 weeks after full bloom.Total CoA concentration was not affected apparently, but acetyl coenzyme A (AcCoA) concentration decreased meaning a proportional decrease of AcCoA to total CoA. Meanwhile the free acidity of juice in lead arsenate sprayed fruits was 16 to 22% lower than that in unsprayed fruits.The decrease in proportion of AcCoA in total CoA about five weeks after lead arsenate spraying showed a high correlation with the decrease of free acidity of juice at harvest in sprayed fruits. It was suggested that arsenolysis could cause acidity of juice to decrease by reducing production of citric acid.

Metabolism of pantothenic acid in hearts of diabetic rats

The metabolism of pantothenic acid (Pa) by cardiac muscle was studied in normal and diabetic rats. Tissue levels of Coenzyme A (CoA) are elevated in the heart during early (6 to 12 h) diabetes, remains at a high level for several days, and then returns to normal or below normal levels. The increase in total tissue CoA mainly occurs in myocytes as indicated by isolation of cardiac myocytes from control and diabetic animals and measuring their content of CoA. The CoA concentration increased from 37 to 93 μ m in the cytosolic compartment and from 2.0 to 2.6 m m in the mitochondrial matrix. These effects of diabetes were reversed by insulin treatment. CoA synthesis in hearts removed from control rats and perfused in vitro was stimulated by including in the perfusate Pa, cysteine and dithiothreitol, but no exogenous energy substrate. This stimulated in vitro rate of CoA synthesis was reduced in hearts removed from diabetic animals, and the reduction increased with duration of diabetes. The reduced rate in diabetic hearts resulted from both a decreased rate of Pa phosphorylation and decreased Pa transport. Transport of Pa into myocytes was decreased by as much as 80% in hearts from diabetic animals. The low transport rate was due to a decrease in V max with no apparent change in K m. Treatment of the isolated heart with insulin did not correct the diabetic-induced reduction in Pa transport. The transport rate in normal and diabetic hearts was not influenced by the type of energy substrate provided to the heart. These data indicate that major modifications occur in the rates of Pa transport and CoA synthesis in hearts of diabetic rats.

Biosynthesis and degradation both contribute to the regulation of coenzyme A content in Escherichia coli.

Escherichia coli mutants [coaA16(Fr); Fr indicates feedback resistance] were isolated which possessed a pantothenate kinase activity that was refractory to feedback inhibition by coenzyme A (CoA). Strains harboring this mutation had CoA levels that were significantly elevated compared with strains containing the wild-type kinase and also overproduced both intra- and extracellular 4'-phosphopantetheine. The origin of 4'-phosphopantetheine was investigated by using strain SJ135 [panD delta(aroP-aceEF)], in which synthesis of acetyl-CoA was dependent on the addition of an acetate growth supplement. Rapid degradation of CoA to 4'-phosphopantetheine was triggered by the conversion of acetyl-CoA to CoA following the removal of acetate from the media. CoA hydrolysis under these conditions appeared not to involve acyl carrier protein prosthetic group turnover since [acyl carrier protein] phosphodiesterase was inhibited equally well by acetyl-CoA or CoA. These data support the view that the total cellular CoA content is controlled by modulation of biosynthesis at the pantothenate kinase step and by degradation of CoA to 4'-phosphopantetheine.

Effects of Acute and Chronic Ethanol Ingestion on Pantothenate and CoA Status of Rats

Total coenzyme A (CoA) and pantothenate levels in tissues were compared in three studies in which rats were fed Lieber-DeCarli formula diet with either 5% ethanol or maltose-dextrin (controls) for 19–36 d. The heart pantothenate levels of ethanol-fed rats were 51–78% of that in controls. The heart total CoA levels of ethanol-fed rats were 78–90% of that in controls. Liver total CoA and pantothenate levels were lower in ethanol-fed rats than in controls in one and two studies, respectively. Differences were diminished in rats fasted for 24 h. Control and chronic ethanol-treated rats given no ethanol for 24 h were intubated with [1-14C]pantothenate. Accumulation of radioactive pantothenate in hearts of ethanol-treated rats was 58% and accumulation in livers was 75% of that in controls. An acute effect of ethanol on pantothenate conversion to CoA in liver was found in naive rats intubated with 0.5–2.5 g ethanol/kg body weight and given [1-14C]pantothenate by intubation or intraperitoneal injection. In ethanol-treated rats, conversion of [1-14C]pantothenate to CoA was 4–28% of that in controls. These studies demonstrated an effect of chronic ethanol ingestion on pantothenate and total CoA contents of heart and possibly other tissues and an acute effect of ethanol ingestion on [1-14C]pantothenate conversion to CoA in liver.

Coenzyme a degradation in the heart: Effects of diabetes and insulin

Coenzyme A (CoA) degradation was studied in isolated working hearts from acutely diabetic rats (48 h). Hearts from diabetic rats had elevated levels of total CoA (752 +/- 15 nmol/g dry) compared to control (537 +/- 14 nmol/g dry). When hearts from diabetic animals were perfused for 5 mins with perfusate containing pyruvate, (5 mM) and glucose (11 mM) CoA levels remained unchanged. Addition of palmitate, (1.2 mM) and glucose (11 mM) to the perfusate, however, resulted in a rapid drop in CoA levels to 672 +/- 19 nmol/g dry. Palmitate had no effect on CoA levels in control hearts which did not have elevated levels of CoA. Addition of insulin to the buffer containing glucose and palmitate prevented the decrease in CoA levels in diabetic hearts. The level of long chain acyl CoA in diabetic hearts perfused with pyruvate was 105 +/- 11 nmol/g dry, and did not change when insulin was present in the perfusate. In the presence of palmitate, levels of long chain acyl CoA increased from 76 +/- 16 to 149 +/- 13 nmol/g dry, and, in this case, addition of insulin caused a further increase to 192 +/- 18 nmol/g dry. Thus, the lower rate of CoA degradation in the presence of insulin was associated with a rise in long chain acyl CoA levels. In a separate series of experiments, CoA levels were increased in control hearts in vitro (from 537 +/- 14 to 842 +/- 19 nmol/g dry). Subsequent perfusion of these hearts that contained elevated CoA with palmitate also resulted in a rapid drop of CoA to 655 +/- 17 nmol/g dry.(ABSTRACT TRUNCATED AT 250 WORDS)