Tricarboxylic Cycle Intermediates Research Articles

Tricarboxylic Cycle Intermediates in Combination with Calcium Phosphate Chelators and Sodium Bicarbonate Increases eGFR in Patients with Stages 3b, 4 and 5 CKD: A Retrospective Observational Study

Introduction: Currently, the conservative management of chronic kidney disease (CKD) involves treating the primary disease and its consequences, as well as improving altered biochemical markers. Evidence suggests that the key management strategy for slowing down the progression of the disease is nephroprotection. Objective: This study aimed to evaluate the effect of the supply of TCA cycle intermediates in combination with calcium carbonate, calcium lactate, and sodium bicarbonate in patients with CKD. Methodology: A retrospective observational study was undertaken in nephrology and internal medicine clinics in Mexico. The study enrolled patients aged over 18 with stages 3b, 4, and 5 chronic kidney disease (CKD) who were not undergoing renal replacement therapy (RRT) and had received treatment based on TCA therapy. Results: The study included a total of 55 patients with CKD. The results showed an increase in eGFR from a baseline average of 16.73 ± 1,374 mL/min to a final average of 19.18 ± 1,516 mL/min and a decrease in creatinine values from a baseline average of 4.26 ± 2.44 mg/dL to a final average of 3.77 ± 2.23 mg/dL. These changes had a statistical significance P?0.05.Conclusions: The observed benefits of TCA in combination with sodium bicarbonate, calcium carbonate, and calcium lactate include: 1) Increased eGFR, 2) Decreased serum creatinine, 3) Decreased serum urea, 4) Decreased serum phosphorus, 5) Increased serum hemoglobin, and 6) Maintenance of albumin levels within normal ranges. Adjuvant therapy with the combination of TCA could be a useful tool as a new therapeutic option in patients with CKD.

Differential hepatic mitochondrial function and gluconeogenic gene expression in 2 Holstein strains in a pasture-based system

The objective of this study was to assess hepatic ATP synthesis in Holstein cows of North American and New Zealand origins and the gluconeogenic pathway, one of the pathways with the highest ATP demands in the ruminant liver. Autumn-calving Holstein cows of New Zealand and North American origins were managed in a pasture-based system with supplementation of concentrate that represented approximately 33% of the predicted dry matter intake during 2017, 2018, and 2019, and hepatic biopsies were taken during mid-lactation at 174 ± 23 days in milk. Cows of both strains produced similar levels of solids-corrected milk, and no differences in body condition score were found. Plasma glucose concentrations were higher for cows of New Zealand versus North American origin. Hepatic mitochondrial function evaluated measuring oxygen consumption rates showed that mitochondrial parameters related to ATP synthesis and maximum respiratory rate were increased for cows of New Zealand compared with North American origin. However, hepatic gene expression of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and pyruvate dehydrogenase kinase was increased in North American compared with New Zealand cows. These results altogether suggest an increased activity of the tricarboxylic cycle in New Zealand cows, leading to increased ATP synthesis, whereas North American cows pull tricarboxylic cycle intermediates toward gluconeogenesis. The fact that this occurs during mid-lactation could account for the increased persistency of North American cows, especially in a pasture-based system. In addition, we observed an augmented mitochondrial density in New Zealand cows, which could be related to feed efficiency mechanisms. In sum, our results contribute to the elucidation of hepatic molecular mechanisms in dairy cows in production systems with higher inclusion of pastures.

Metabolomics-Based Identification of Metabolic Dysfunction in Frailty.

Dysregulation of energy producing metabolic pathways has been observed in older adults with frailty. In this study, we used liquid chromatography-mass spectrometry technology to identify aging- and frailty-related differences in metabolites involved in glycolysis, the tricarboxylic (TCA) cycle, and other energy metabolism-related pathways in the serum of a cohort of community-dwelling adults aged 20-97 (n = 146). We also examined the relationship between serum levels of metabolites and functional measures, physical frailty, and risk status for adverse health outcomes. We observed elevated levels of TCA cycle and glycolytic intermediates in frail subjects; however, the differences in the levels of ATP and other energy metabolites between young, nonfrail, and frail adults were not significant. Instead, we found that serum levels of neurotransmitters N-acetyl-aspartyl-glutamate, glutamate, and γ-aminobutyric acid were significantly elevated in older adults with frailty. These elevations of glycolytic and TCA cycle intermediates, and neurotransmitters may be part of the biological signature of frailty.

Physiological Perspectives on the Use of Triheptanoin as Anaplerotic Therapy for Long Chain Fatty Acid Oxidation Disorders.

Inborn errors of mitochondrial fatty acid oxidation (FAO) comprise the most common group of disorders identified through expanded newborn screening mandated in all 50 states in the United States, affecting 1:10,000 newborns. While some of the morbidity in FAO disorders (FAODs) can be reduced if identified through screening, a significant gap remains between the ability to diagnose these disorders and the ability to treat them. At least 25 enzymes and specific transport proteins are responsible for carrying out the steps of mitochondrial fatty acid metabolism, with at least 22 associated genetic disorders. Common symptoms in long chain FAODs (LC-FAODs) in the first week of life include cardiac arrhythmias, hypoglycemia, and sudden death. Symptoms later in infancy and early childhood may relate to the liver or cardiac or skeletal muscle dysfunction, and include fasting or stress-related hypoketotic hypoglycemia or Reye-like syndrome, conduction abnormalities, arrhythmias, dilated or hypertrophic cardiomyopathy, and muscle weakness or fasting- and exercise-induced rhabdomyolysis. In adolescent or adult-onset disease, muscular symptoms, including rhabdomyolysis, and cardiomyopathy predominate. Unfortunately, progress in developing better therapeutic strategies has been slow and incremental. Supplementation with medium chain triglyceride (MCT; most often a mixture of C8–12 fatty acids containing triglycerides) oil provides a fat source that can be utilized by patients with long chain defects, but does not eliminate symptoms. Three mitochondrial metabolic pathways are required for efficient energy production in eukaryotic cells: oxidative phosphorylation (OXPHOS), FAO, and the tricarboxylic (TCA) cycle, also called the Krebs cycle. Cell and mouse studies have identified a deficiency in TCA cycle intermediates in LC-FAODs, thought to be due to a depletion of odd chain carbon compounds in patients treated with a predominantly MCT fat source. Triheptanoin (triheptanoyl glycerol; UX007, Ultragenyx Pharmaceuticals) is chemically composed of three heptanoate (seven carbon fatty acid) molecules linked to glycerol through ester bonds that has the potential to replete TCA cycle intermediates through production of both acetyl-CoA and propionyl-CoA through medium chain FAO. Compassionate use, retrospective, and recently completed prospective studies demonstrate significant reduction of hypoglycemic events and improved cardiac function in LC-FAOD patients, but a less dramatic effect on muscle symptoms.

Metabolic reprogramming as a feast for virus replication.

Viral replication depends entirely on the energy and biosynthetic precursors supplied by the host cell metabolic network. Viruses actively reprogram host cell metabolism to establish optimal environment for their replication and spread. They stimulate the uptake of extracellular nutrients and predominantly modulate glucose, glutamine, and fatty acid metabolism to support anabolic metabolic pathways. Some viruses activate the process of aerobic glycolysis, divert the glycolytic carbon for biosynthetic reactions, and stimulate glutamine utilization to replenish tricarboxylic cycle intermediates. Others use glutamine carbon to promote de novo fatty acid synthesis, amino acid supply or glutathione production. The unique metabolic signature and different dependence of viral life cycle on the individual metabolic processes is therefore characteristic feature of almost each virus. Deeper understanding of how viruses alter cellular metabolic pathways or their upstream regulatory circuits may lead to development of more effective antiviral treatment strategies based on targeted metabolic inhibition. Keywords: virus infection; metabolism; glycolysis; glutamine metabolism; fatty acid synthesis; metabolic reprogramming; virus-host interaction.

Nudix Hydrolase Controls Nucleotides and Glycolytic Mechanisms in HypoxicAspergillus nidulans

Nucleoside diphosphates linked to moiety X (Nudix) hydrolase functions were investigated in hypoxic Aspergillus nidulans cells. Among three nudix hydrolase isozymes, NdxA transcription was up-regulated under oxygen (O2)-limited conditions. A gene disruptant of the NdxA-encoding gene (NdxAΔ) accumulated more NADH and ADP-ribose than the wild type (WT) under the same conditions. These results indicate that NdxA hydrolyzes these nucleotides in hypoxic fungal cells, which accords with the thesis that NdxA hydrolyzes NADH and ADP-ribose. Under O2-limited conditions, NdxAΔ decreased glucose consumption, the production of ethanol and lactate, cellular ATP levels, and growth as compared with WT. WT cultured under hypoxia converted exogenously added fructose 1,6-bisphophate, a glycolytic intermediate, to glyceraldehyde 3-phosphate (GAP). The hypoxic NdxAΔ cells accumulated 3.0- to 4.2-fold more GAP than WT under the same conditions, indicating that NdxA increased GAP oxidation by a glycolytic mechanism. Steady-state kinetics indicated that NADH and ADP-ribose competitively inhibited fungal GAP dehydrogenase (GAPDH) with Ki values of 34- and 55-µM, respectively. These results indicate that NdxA hydrolyzes cellular NADH- and ADP-ribose, derepresses GAPDH activity, and hence up-regulates glycolysis in hypoxic A. nidulans cells. That NdxAΔ consumed less pyruvate and tricarboxylate cycle intermediates than WT suggests that NdxA-dependent hydrolysis of nucleotides controls the catabolism of these carbon sources under O2-limited conditions.

Enhancing itaconic acid production by Aspergillus terreus

Aspergillus terreus is successfully used for industrial production of itaconic acid. The acid is formed from cis-aconitate, an intermediate of the tricarboxylic (TCA) cycle, by catalytic action of cis-aconitate decarboxylase. It could be assumed that strong anaplerotic reactions that replenish the pool of the TCA cycle intermediates would enhance the synthesis and excretion rate of itaconic acid. In the phylogenetic close relative Aspergillus niger, upregulated metabolic flux through glycolysis has been described that acted as a strong anaplerotic reaction. Deregulated glycolytic flux was caused by posttranslational modification of 6-phosphofructo-1-kinase (PFK1) that resulted in formation of a highly active, citrate inhibition-resistant shorter form of the enzyme. In order to avoid complex posttranslational modification, the native A. niger pfkA gene has been modified to encode for an active shorter PFK1 fragment. By the insertion of the modified A. niger pfkA genes into the A. terreus strain, increased specific productivities of itaconic acid and final yields were documented by transformants in respect to the parental strain. On the other hand, growth rate of all transformants remained suppressed which is due to the low initial pH value of the medium, one of the prerequisites for the accumulation of itaconic acid by A. terreus mycelium.

Osmoprotectant-dependent expression of plcH, encoding the hemolytic phospholipase C, is subject to novel catabolite repression control in Pseudomonas aeruginosa PAO1.

Expression of the hemolytic phospholipase C (PlcH) of Pseudomonas aeruginosa is induced under phosphate starvation conditions or in the presence of the osmoprotectants choline and glycine betaine. Because choline and glycine betaine may serve as carbon and energy sources in addition to conferring osmoprotection to P. aeruginosa, it seemed possible that induction of plcH is subject to catabolite repression control (CRC) by tricarboxylic cycle intermediates such as succinate. Total phospholipase (PLC) activity in osmoprotectant-induced cultures of P. aeruginosa PAO1 supplemented with 20 mM succinate was three- to fourfold lower than the levels in cultures supplemented with the non-catabolite-repressive substrate lactate. Analyses of osmoprotectant-dependent plcH expression in a derivative of strain PAO1 containing a plcH::lacZ operon fusion showed that (i) succinate prevented induction of plcH expression by osmoprotectants; and (ii) addition of succinate reduced or shut down further expression of plcH in osmoprotectant-induced bacteria, while cultures supplemented with lactate had little or no change in plcH expression. RNase protection analysis confirmed that repression of plcH occurs at the transcriptional level. However, a P. aeruginosa mutant decoupled in CRC exhibited a phenotype similar to that of the wild-type strain (PAO1) with respect to succinate-dependent repression of plcH expression. Osmoprotectant-induced total PLC activities, levels of expression of plcH measured with the same plcH::lacZ fusion, and levels of plcH transcription in a CRC-deficient strain reflected those seen in strain PAO1. This indicates that CRC of plcH functions by a distinct mechanism which differs from that regulating the glucose or mannitol catabolic pathway. A strain carrying a mutation in vfr, which encodes the Escherichia coli Crp homolog in P. aeruginosa, still exhibited a wild-type phenotype with respect to osmoprotectant-dependent expression and CRC of plcH. These data indicate that there is a novel CRC system that regulates the expression of plcH in P. aeruginosa.

Determination of the orientation of membrane vesicles derived from mitochondria.

Membrane vesicles of physiological as well as inverted orientation can be isolated from mitochondria. The presence of these vesicles in a membrane can be determined and quantitated by determining the differences between the two vesicle types in terms of rates of NADH oxidation, rates of oxidation of tricarboxylate cycle intermediates, rates of ATP hydrolysis and sensitivity to inhibitors, stimulation of respiration by exogenous cytochrome c, inhibition of respiration by polycationic proteins, and visualization of the ATPase by electron microscopy. Procedures to isolate the two membrane types and characteristics of homogeneously oriented preparations are described. Differences in data obtained with homogeneous vesicle preparations and with vesicles of mixed orientation are illustrated. Nonhomogeneously oriented preparations can be enriched in the desired vesicular type by the use of immunoprecipitation, affinity chromatography, and differential centrifugation. The concept of a hybrid vesicle containing oppositely oriented regions is not supported by experimental data.

Heterotrophic (Dark) CO2 Fixation by Euglena gracilis. Possible regulation by tricarboxylic acid cycle intermediates

SYNOPSIS. Heterotrophic (dark) CO2 fixation by Euglena gracilis strain Z varies with phase of batch culture and mode of nutrition. Dark CO2 fixation increased transiently during the growth of cells under photoautotrophic (CO2, light) and heterotrophic (glucose, dark) conditions. Cells grown heterotrophically with acetate or ethanol had no transient increase in fixation. The addition of acetate to a heterotrophically growing culture during the period of increasing dark CO2 fixation resulted in rapid elimination of this fixation. The results suggest that dark CO2 fixation in Euglena functions in anaplerotic feeding of the tricarboxylic acid cycle, drained by biosyntheses during growth. Induction of the glyoxylate cycle by acetate may provide an alternate source of tricarboxylic cycle intermediates, obviating the requirement for dark CO2 fixation as a source of the intermediates.

Active oxidative decarboxylation of malate by mitochondria isolated from L-1210 ascites tumor cells

Abstract Mitochondria from L-1210 mouse ascites tumor show a very high rate of oxidation of L-malate in comparison with mitochondria from normal tissues. They were found to contain large amounts of malic enzyme (E.C.1.1.1.39) catalyzing oxidative decarboxylation of L-malate to pyruvate. Malic enzyme in extracts of tumor mitochondria requires Mn2+ or Mg2+, utilizes either NAD+ or NADP+ as electron acceptor, and shows positive cooperativity in binding of L-malate. These observations suggest that L-1210 tumor mitochondria actively convert excess tricarboxylate cycle intermediates and their precursors into pyruvate for further oxidation.

Biochemical effects of the anticonvulsants trimethadione, ethosuximide and chlordiazepoxide in rat brain.

Abstract— The concentrations of several metabolites, including glucose, glycogen, hexose phosphates, adenine nucleotides phosphocreatine, amino acids and some tricarboxylate cycle intermediates, have been estimated in cerebral tissues of rats treated with anticonvulsant doses of trimethadione, ethosuximide and chlordiazepoxide.Anticonvulsant administration, in each case, produced an increase in brain glucose, but only trimethadione and ethosuximide resulted in elevated brain/blood glucose ratios. It was concluded that the apparent rise in intracellular glucose with the latter drugs may, in part, be the result of a stimulation of glucose transport from blood into the brain. Anticonvulsant administration was also shown to result in a depression of some tricarboxylate cycle intermediates. The pattern of these metabolite changes was in effect similar to those reported independently in mice treated with anaesthetics and it was therefore concluded that these differences probably reflected a depression in metabolic rate.Metabolic alterations in general do not indicate aetiology but rather effects of the drug activities. However, a role implicating increased intracellular glucose levels with membrane stabilization is discussed.

The effects of increased heart work on the tricarboxylate cycle and its interactions with glycolysis in the perfused rat heart.

1. The work of the perfused rat heart was acutely increased by raising the aortic pressure in the Langendorff preparation from 50 to 120mmHg; within 1 min in perfusions with media containing glucose or glucose+acetate, rates of oxygen consumption and tricarboxylate-cycle turnover increased 2.5-fold, glycolysis rate doubled and oxidation of triglyceride fatty acid was strikingly enhanced. 2. Increased cardiac work had no significant effects on the heart concentrations of creatine phosphate, ATP, ADP or 5'-AMP. The only significant changes in tricarboxylate-cycle intermediates were a decrease in malate in perfusions with glucose and decreases in acetyl-CoA and citrate and an increase in aspartate in perfusions with glucose+acetate. 3. Measurements of intracellular concentrations of hexose phosphates, glucose and glycogen indicated that work accelerated glycolysis by activation of phosphofructokinase and subsequently hexokinase; the activation could not be accounted for by changes in the known effectors of phosphofructokinase. 4. Acetate at either perfusion pressure increased heart concentrations of acetyl-CoA, citrate, glutamate and malate and decreased that of aspartate; acetate increased tricarboxylate-cycle turnover by 50-60% and inhibited glycolysis and pyruvate oxidation. 5. In view of the markedly different effects of acetate and of cardiac work on the concentrations of cycle intermediates the changes that accompany acetate utilization may be specifically concerned with the regulatory functions of the cycle in control of glycolysis and pyruvate oxidation and not with the associated increase in cycle turnover. It is suggested that the concentrations of key metabolites controlling the rate of cycle turnover may fluctuate with each heart beat and that this may explain why no significant changes (for example, in adenine nucleotide concentrations) have been detected with increased work in the present study.

The influence of intraventricularly injected amino acid excitants on the labelling of endogenous brain amino acids from (U- 14 C)acetate in nembutalized mice.

AbstractMice were anaesthetized with nembutal and the effects of intraventricularly injected excitant amino acids on [U‐14C]acetate metabolism were investigated. The natural excitant amino acids, l‐glutamate and l‐aspartate, reduced the incorporation of 14C from [U‐14C]acetate into glutamine, GAB A and possibly alanine. The synthetic excitant amino acid, N‐methyl‐d‐aspartate caused a reduction in the incorporation of 14C from intraventricularly injected [U‐14C]acetate into all of the brain amino acids labelled by [U‐14C]acetate within 5 min. It is suggested that these effects may be due to changes in pool sizes of tricarboxylic cycle intermediates, to inhibition of acetyl‐CoA formation, or both. Differences in the metabolic effects of the synthetic and natural excitants are interpreted in terms of the uptake of the natural amino acids into glutamine‐forming pool(s) of glutamate metabolism.

Some neurochemical aspects of pentamethylenetetrazole convulsive activity in rat brain.

Abstract— The concentrations of several metabolites, including glucose, glycogen, hexose phosphates, adenine nucleotides, amino acids and some tricarboxylic cycle intermediates were estimated in cerebral tissue of rats killed at various stages of pentamethylenetetrazole convulsions. Prior to and during the seizure there was an increase in glycolytic flux and depletion of energy reserves. An accumulation of alanine concomitant with a decreased citrate level observed before the convulsion indicated that a possible interference of pyruvate entry into the tricarboxylic cycle had occurred. At the tonic extensor phase of the seizure and during post‐convulsive sedation a small but significant rise in GABA and an attempt to restore some of the labile constituents to more normal values was apparent. The significance of these results in relation to cerebral metabolic control and possible mechanisms of initiation and arrest of the seizure is discussed.

Inhibition of Pyruvate Carboxylation by Fluorocitrate in Rat Kidney Mitochondria

Rat kidney mitochondria, in the presence of ATP, Pi, and magnesium ions, converted pyruvate and bicarbonate to malate, fumarate, and citrate and, in the presence of glutamate, also to aspartate. The addition of fluorocitrate strongly inhibited pyruvate utilization and 14CO2 incorporation into organic acids. The inhibition of 14CO2 incorporation into organic acids in the presence of fluorocitrate appeared to be caused by malonyl coenzyme A produced by fluorocitrate stimulation of acetyl-CoA carboxylase. This inhibition by fluorocitrate of 14CO2 incorporation was reversed by carnitine, acetylcarnitine, acylcarnitines, isocitrate, α-ketoglutarate, succinate, fumarate, and glutamate, but not by citrate, β-hydroxybutyrate, or caprylate. The ratio of carnitine to fluorocitrate determined the degree of reversal of this inhibition. The reversal of inhibition by carnitine or acylcarnitine resulted in greatly increased citrate formation, with nearly all of the 14CO2 being incorporated into citrate, and in nearly complete inhibition of malate synthesis. Fluorocitrate inhibited oxidation of citrate. In the presence of tricarboxylic cycle intermediates, the incorporation of bicarbonate-14C into malate was greatly increased. With fluorocitrate in the system, the incorporation of radioactive bicarbonate into malate in the presence of α-ketoglutarate and succinate did not change, but decreased in the presence of fumarate.

Regulation of gluconeogenesis and lipogenesis: Inhibition of pyruvate carboxylation in rat kidney mitochondria by malonate and malonyl CoA

Abstract Rat kidney mitochondria in the presence of ATP, P i and magnesium ions, converted pyruvate and bicarbonate to malate, fumarate and citrate. Pyruvate carboxylase activity was estimated by measuring the incorporation of bicarbonate- 14 C into tricarboxylic acid cycle intermediates. The addition of malonate decreased utilization of pyruvate and strongly inhibited bicarbonate- 14 C incorporation into the organic acids. This inhibition of bicarbonate- 14 C incorporation into organic acids in the presence of malonate was reversed by carnitine, oxalacetate, malate, fumarate, succinate and α-ketoglutarate, but not by citrate, isocitrate, aspartate, glutamate or acetylcarnitine. The ratio of concentration of carnitine to concentration of malonate determined the degree of reversal of this inhibition. Malonate-1- 14 C was metabolized by rat kidney mitochondria to acetyl CoA and 14 CO 2 . Carnitine stimulated malonate-1- 14 C decarboxylation presumably by removing acetyl CoA from the system and forming acetylcarnitine. In the presence of tricarboxylic cycle intermediates the incorporation of bicarbonate- 14 C into malate was greatly increased. The inhibition of 14 CO 2 incorporation into organic acids in the presence of malonate appears to be caused by malonyl CoA. Externally added malonyl CoA was shown to be a potent inhibitor of 14 C0 2 incorporation into organic acids. This inhibition by malonyl CoA was partially reversed by carnitine. The conversion of pyruvate to dicarboxylic acids is one of the key steps in the pathway of gluconeogenesis. The inhibition of this step by malonate may exercise regulatory effects on gluconeogenesis. It is postulated in this paper that changes in the ratio of concentration of acetyl CoA to malonyl CoA may play a regulatory role, since malonyl CoA is a key intermediate in the pathway of lipogenesis this mechanism may be of physiologic significance.

Groupes sulfhydriles et morphogénèse: I. Effets du β-mercaptoéthanol, de l'acide α-lipoïque, de l'acide adénosinetriphosphorique et de l'oxaloacétate sur le développement embryonnaire des Batraciens

Groupes sulfhydriles et morphogénèse: I. Effets du β-mercaptoéthanol, de l'acide α-lipoïque, de l'acide adénosinetriphosphorique et de l'oxaloacétate sur le développement embryonnaire des Batraciens

A METHOD FOR THE SEPARATION OF TRICARBOXYLIC CYCLE INTERMEDIATES BY CELITE-COLUMN CHROMATOGRAPHY AND ITS APPLICATION TO THE TISSUES OF ASCARIS LUMBRICOIDES VAR. SUIS*

Journal Article A METHOD FOR THE SEPARATION OF TRICARBOXYLIC CYCLE INTERMEDIATES BY CELITE-COLUMN CHROMATOGRAPHY AND ITS APPLICATION TO THE TISSUES OF ASCARIS LUMBRICOIDES VAR. SUIS Get access YOSHIO UENO, YOSHIO UENO Faculty of Medicine From the Department of Pharmacology, University of TokyoTokyo Search for other works by this author on: Oxford Academic PubMed Google Scholar HIROSHI OYA, HIROSHI OYA Faculty of Medicine From the Department of Pharmacology, University of TokyoTokyo Search for other works by this author on: Oxford Academic PubMed Google Scholar TAKEO BANDO TAKEO BANDO Faculty of Medicine From the Department of Pharmacology, University of TokyoTokyo Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Biochemistry, Volume 47, Issue 6, 25 June 1960, Pages 771–776, https://doi.org/10.1093/oxfordjournals.jbchem.a127123 Published: 25 June 1960 Article history Received: 20 November 1959 Published: 25 June 1960