John R. Williamson

[65] Assays of intermediates of the citric acid cycle and related compounds by fluorometric enzyme methods☆

Publisher Summary This chapter is based on the assays of intermediates of the citric acid cycle and related compounds by fluorometric enzyme methods. An eppendorf fluorometer or a metabolite fluorometer are instruments capable of giving a full-scale deflection of the recorder with 0.25μM NADH, with a noise level less than 2%. At such high sensitivities, the full progress of each enzymatic reaction is recorded. The following accounts for the majority of difficulties and inaccuracies commonly encountered with fluorometrie enzyme methods: All solutions should be dust and particle free; the cuvettes should be temperature equilibrated; Particular care should be taken to avoid contamination of solutions with enzymes, or cross-contamination; Fresh enzyme solution must be made each day; Solutions of pyridine nucleotides are best prepared each day and stored on ice. NAD+ and NADP+ are most stable in a slightly acid solution, and may be diluted with distilled water; and all standard solutions should be neutralized, and assayed spectrophotometrically on the day of use. Metabolic intermediates other than reduced pyridine nueleotides, total CoA, fatty acyl-CoA, and fatty acylearnitine compounds are measured in neutralized perchloric acid extracts of tissues. Perchloric acid is generally more convenient to use than trichloroacetic acid for the extraction, because it may be removed by precipitation as the potassium salt.

Effects of acidosis and ischemia on contractility and intracellular pH of rat heart.

SUMMARYThe effects of respiratory and metabolic acidosis on myocardial contractility and energy production have been investigated in the perfused rat heart. Respiratory acidosis, produced by increasing the Pco2, caused an 80% inhibition of pressure development at pH 6.7. When artificial buffers (plu

Diacylglycerol accumulation and microvascular abnormalities induced by elevated glucose levels.

The present experiments were undertaken to examine the hypothesis that glucose-induced increased de novo synthesis of 1,2-diacyl-sn-glycerol (which has been observed in a number of different tissues, including retinal capillary endothelial cells exposed to elevated glucose levels in vitro) and associated activation of protein kinase C may play a role in mediating glucose-induced vascular functional changes. We report here that twice daily instillation of 30 mM glucose over 10 d in a rat skin chamber granulation tissue model induces approximately a 2.7-fold increase in diacylglycerol (DAG) levels (versus tissues exposed to 5 mM glucose) in association with marked increases in vascular clearance of albumin and blood flow. The glucose-induced increase in DAG levels as well as the vascular functional changes are prevented by addition of 3 mM pyruvate. Pharmacological activation of protein kinase C with the phorbol ester TPA in the presence of 5 mM glucose increases microvascular albumin clearance and blood flow, and similar effects are observed with 1-monoolein (MOG), a pharmacological inhibitor of the catabolism of endogenous DAG. A pharmacological inhibitor of protein kinase C (staurosporine) greatly attenuates the rise in microvascular albumin clearance (but not the rise in blood flow) induced by glucose or by MOG. These findings are compatible with the hypothesis that elevated concentrations of glucose increase tissue DAG content via de novo synthesis, resulting in protein kinase C activation, and that these biochemical events are among the factors that generate the increased microvascular albumin clearance.

Medium-chain acyl-CoA dehydrogenase deficiency in children with non-ketotic hypoglycemia and low carnitine levels.

Summary: Three children in two families presented in early childhood with episodes of illness associated with fasting which resembled Reyes syndrome: coma, hypoglycemia, hyperammonemia, and fatty liver. One child died with cerebral edema during an episode. Clinical studies revealed an absence of ketosis on fasting (plasma beta-hydroxybutyrate < 0.4 mmole/liter) despite elevated levels of free fatty acids (2.6–4.2 mmole/liter) which suggested that hepatic fatty acid oxidation was impaired. Urinary dicarboxylic acids were elevated during illness or fasting. Total carnitine levels were low in plasma (18–25 μmole/liter), liver (200–500 nmole/g), and muscle (500–800 nmole/g); however, treatment with L-carnitine failed to correct the defect in ketogenesis. Studies on ketone production from fatty acid substrates by liver tissue in vitro showed normal rates from short-chain fatty acids, but very low rates from all medium and long-chain fatty acid substrates. These results suggested that the defect was in the mid-portion of the intramitochondrial beta-oxidation pathway at the medium- chain acyl-CoA dehydrogenase step. A new assay for the electron transfer flavoprotein-linked acyl-CoA dehydrogenases was used to test this hypothesis. This assay follows the decrease in electron transfer flavoprotein fluorescence as it is reduced by acyl-CoA-acyl-CoA dehydrogenase complex. Results with octanoyl-CoA as substrate indicated that patients had less than 2.5% normal activity of medium-chain acyl-CoA dehydrogenase. The activities of short-chain and isovaleryl acyl-CoA dehydrogenases were normal; the activity of long-chain acyl-CoA dehydrogenase was one-third normal.These results define a previously unrecognized inherited metabolic disorder of fatty acid oxidation due to deficiency of medium-chain acyl-CoA dehydrogenase. The carnitine deficiency in these patients appears to be a secondary consequence of their defect in fatty acid oxidation. It is possible that other patients with “systemic carnitine deficiency,” who fail to respond to carnitine therapy, may also have defects in fatty acid oxidation similar to medium-chain acyl-CoA dehydrogenase deficiency.

Heterogeneity of the hypoxic state in perfused rat heart.

SUMMARY Tissue oxygen gradients were examined in the saline-perfused rat heart by NADH fluorescence photography. In high flow hypoxia, where the coronary flow was maintained and the arterial oxygen tension was gradually reduced, oxygen extraction was virtually complete before oxygen consumption was significantly diminished. Inadequate oxygen delivery resulted in a well defined pattern of anoxic zones. The anoxic zones were several hundred microns in width, an order of magnitude greater than intercapillary distances. In low flow hypoxia (ischemia), where the arterial oxygen tension remained at its control value and the coronary flow was diminished, anoxic zones also developed, following the same pattern as in high flow hypoxia. However, in ischemia, the anoxic areas developed while the effluent oxygen tension was significantly greater than zero. Whereas respiratory acidosis between pH 7.3 and 6.9 resulted in vasodilation, below pH 6.8 there was a marked increase in vascular resistance. Anoxic zones appeared despite only a slight change in effluent oxygen tension from the control. In high flow hypoxia, ischemia, and acidosis-induced ischemia, the anoxic zones disappeared when control perfusion conditions were restored. The data demonstrate that tissue oxygen gradients are very steep in the hypoxic state, so that ischemia and hypoxia result in discrete heterogeneous areas of anoxic tissue bounded by sharp areas where the oxygen supply is sufficient to maintain normal mitochondrial oxidative function. In these states in which oxygen delivery is less than oxygen demand, coronary perfusion appears to be regulated at the level of the arterioles rather than the capillaries.

Pyridine nucleotide distributions and enzyme mass action ratios in hepatocytes from fed and starved rats

Abstract Hepatocytes isolated from fed or starved rats were rapidly lysed using the recently described technique of turbulent flow (M. E. Tischler, P. Hecht, and J. R. Williamson, 1977, Arch. Biochem. Biophys. , 181 , 278–292) . Pyridine nucleotide and metabolite contents were measured in the particulate fraction of both whole and disrupted cells after centrifugation through silicone oil. Lactate/pyruvate, β-hydroxybutyrate/acetoacetate, isocitrate/α-ketoglutarate, and malate/pyruvate ratios were determined for calculation of the free NADH NAD + and NADPH NADP + ratios in the cytosol and mitochondria. Lactate/pyruvate ratios measured in the extracellular and cytosolic compartments were in good agreement. Ratios of β-hydroxybutyrate/acetoacetate measured in the extracellular, cytosolic, and mitochondrial compartments also agreed well. Addition of ammonia to fed or starved rat liver cells incubated with lactate, pyruvate, β-hydroxybutyrate, and acetoacetate caused an oxidation of both the NAD and NADP redox states in the mitochondria and cytosol, although the NADP system was oxidized to a greater extent. Calculation of the free NADH and NAD concentrations in the cytosol provided values of about 1 and 400 to 500 μ m , respectively, under control conditions. The concentrations of free NADH and NAD in the mitochondria were considerably higher, being 300 to 400 μ m and 4 to 6 m m , respectively. The free andm bound NAD systems in both the cytosol and mitochondria were more oxidized in the presence of ammonia. NAD and NADP redox potential differences across the mitochondrial membrane (Δ E h ) were not significantly affected by ammonia addition and were generally similar in cells from both fed and starved rats: −52 and −56 mV for the NAD system and −19 to −29 mV for the NADP system. For the NAD system the cytosolic potential was −260 mV in the absence of ammonia and −250 mV in its presence, the mitochondrial values being −315 and −303 mV, respectively. The average cytosolic NADP potential, on the other hand, was −400 mV in the absence and −384 mV in the presence of ammonia. The mitochondrial fractions yielded NADP potentials of −420 mV in the absence of ammonia with both fed and starved rats. Ammonia decreased the mitochondrial NADP potential to −404 mV in fed rats and to −415 mV in starved rats. The calculated free NADH NAD + and NADPH NADP + ratios as well as metabolite concentrations were used to evaluate the mass action ratios of both cytosolic and mitochondrial enzymes. Cytosolic alanine aminotransferase remained near equilibrium in the absence and presence of ammonia, while cytosolic and mitochondrial aspartate aminotransferase reactions deviated up to fivefold. The glutamate dehydrogenase reaction was in near equilibrium with the NAD system, but deviated by three to four orders of magnitude from equilibrium with the NADP system in the direction favoring glutamate synthesis rather than deamination. Cytosolic malate dehydrogenase deviated from equilibrium by about one order of magnitude, while mitochondrial malate dehydrogenase and citrate synthase deviated by two to six orders of magnitude. These data emphasize the importance of regulation of the citric acid cycle at the citrate synthase step.

[23] Assay of citric acid cycle intermediates and related compounds—Update with tissue metabolite levels and Intracellular Distribution☆

Publisher Summary This chapter discusses new methods that have been developed in various laboratories for the assay of specific intermediates related to the citric acid cycle. Metabolite contents of different organs maintained in vitro under specified conditions are reviewed together with summaries of measured metabolite distributions between cytosol and mitochondria in liver. Considerable improvements have been made in commercial, relatively inexpensive, filter fluorometers and any one may be considered adequate for enzyme assays of metabolites in extracts of tissues and mitochondria at high sensitivity. The basic requirements are a stable light source, good mechanical placement of the cuvette in the machine, and an extended-range, and a zero-offset potentiometer. Several commercial fluorometers are available with automatic features for simultaneous assay of 20 or more samples. Commercial double-beam spectrophotometers are also capable of sensitivity similar to that of fluorometers for metabolite assay purposes, with an equivalent signal-to-noise ratio. Spectrophotometry also has advantages over fluorometry when the sample contains high concentrations of unwanted fluorochromes, as with perchloric acid extracts of tissues containing blood.

Regulation of the citric acid cycle in mammalian systems

Whilst the reactions of the citric acid cycle [l] and related pathways are well established, the manner in which the flux through the pathway is regulated in different tissues remains an area of active research and debate. The primary focus during the last decade has been on enzyme mechanisms, the intracellular compartmentation of enzymes and intermediates of the cycle, and on the interrelations with the specific anion translocating systems of the inner mitochondrial membrane. A number of recent reviews have been concerned with relating citric acid cycle activity to the regulation of carbohydrate and fatty acid oxidation [2-41, metabolite transport across the mitochondrial membrane for gluconeogenesis, ureogenesis and fatty acid synthesis [5-l 11, cellular energy metabolism [12-l 61, as well as with more specialized topics such as pyruvate [ 17,181 and ethanol [ 191 metabolism. The concept that the enzymes of the citric acid cycle are organized as a functional multienzyme complex has also been advocated [20,21]. Until recently it has been generally’accepted that turnover of the citric acid cycle per se is regulated at the dehydrogenase level primarily by the mitochondrial NAD’/NADH ratio. This ratio is linked to the * mitochondrial ATP/ADP ratio and ultimately to the cytosolic phosphorylation potential, which is a primary factor in the control of tissue respiration. However, since the different NAD’substrate dehydrogenases are regulated with different sensitivities by the NAD’INADH ratio, the absolute NAD’ redox potential for a given flux depends on the concentration and nature of the substrate supply to the mitochondria. On the other hand, the anaplerotic functions of the citric acid cycle are regulated by ancillary enzymes such as pyruvate dehydrogenase, pyruvate

Interrelationships between malate-aspartate shuttle and citric acid cycle in rat heart mitochondria☆

Abstract The control of substrate utilization was investigated using rat heart mitochondria incubated under conditions of state 4, state 3, oligomycin-inhibited and uncoupled respiration. A comparison of the changes in metabolite levels after addition of pyruvate or acetylcarnitine showed that cycle flux was controlled primarily at citrate synthase, which appeared to be regulated by the intramitochondrial oxalacetate concentration. A secondary control site located between α-ketoglutarate and succinate was revealed by addition of oligomycin which caused an increased flux through α-ketoglutarate dehydrogenase and thereby a decreased efflux of α-ketoglutarate from the mitchondria. The increased α-ketoglutarate dehydrogenase activity was caused by the fall of the ATP/ADP ratio, which by increasing the availability of GDP for substrate level phosphorylation produced diminished product inhibition by succinyl CoA. Because transport of NADH into mitochondria by the malate-aspartate shuttle requires a stoichiometric influx of malate and glutamate and efflux of aspartate and α-ketoglutarate from the mitochondria, alterations in the rate of efflux of α-ketoglutarate can significantly alter flux through the shuttle and the rate of utilization of cytosolic NADH. Studies with mitochondria oxidizing glutamate and malate in the presence and absence of acetylcarnitine or octanoate showed that α-ketoglutarate efflux could be strongly effected by the intramitochondrial ATP/ADP ratio. Rates of α-ketoglutarate efflux were also increased by extramitochondrial malate (half maximal stimulation at 0.6 mM). Glutamate transamination was inhibited by uncoupling agents and subsequent studies, measuring intramitochondrial aspartate levels, showed that this was due to an inhibition of aspartate efflux from the mitochondria in the uncoupled state. Conclusions drawn regarding regulation of the transport of reducing equivalents were verified using mitochondria supplemented with the extramitochondrial components of the malate aspartate shuttle.

Determination of mitochondrial/cytosolic metabolite gradients in isolated rat liver cells by cell disruption

Abstract A new technique is described for determining the distribution of metabolites between the cytosol and mitochondria. Rapid lysis of the cell plasma membrane is obtained by forcing isolated liver cells under high pressure through a small diameter needle. The cells, after disruption by the shearing forces generated during the turbulent flow through the needle, are exposed to mitochondrial anion transport inhibitors to prevent efflux of mitochondrial metabolites. Maximal release of cytosolic metabolites was obtained when release of lactate dehydrogenase was greater than 70%, which corresponded with minimal release of mitochondrial enzymes (5–9%). A measured Reynolds number between 7600 and 8000 was indicative of optimal disruption. Mitochondria in the disrupted cells were still functional, as shown by the ability of ADP to stimulate respiration when glutamate plus malate were provided as substrates. Measurement of the subcellular volumes yielded values of 2.0 and 0.2 ml/g dry wt, respectively, for the cytosol and mitochondria. Calculation of the mitochondrial ΔpH (pHin-pHout) in the isolated liver cell based on 22 individual measurements of mitochondria/cytosol gradients for citrate, isocitrate, α-ketoglutarate, malate, glutamate, and pyruvate yielded a value of 0.41 ±0.03. The excellent relationship of these gradients to a common ΔpH lends credence to the technique. Cytosolic and mitochondrial ATP ADP ratios were similar in liver cells isolated from starved and fed rats. Fed rat liver cells, however, had a higher cytosolic adenine nucleotide content (16.8 μmol/g dry wt) than those from starved rats (14.5 μmol/ g dry wt) whereas the mitochondrial content was the same (16 nmol/mg of mitochondrial protein). Data obtained by the disruption technique are compared with other previously published data obtained using either digitonin treatment of isolated hepatocytes or nonaqueous solvent extraction of lyophilized freeze-clamped perfused livers.