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Biomedical subjects

G K Radda

Publications and source records attributed to G K Radda.

At least 235 records · Page 13Linked to original sources

Imaging of human brain creatine kinase activity in vivo.

Creatine kinase activity and high-energy phosphate concentration have been investigated using localized 31P spectroscopy in the human brain in vivo. The phase-modulated rotating frame imaging technique, incorporating magnetization transfer and inversion recovery, has been used to produce a 1-dimensional rate profile map of steady-state enzyme activity. Large differences in the flux from phosphocreatine (PCr) to ATP have been discovered between volumes of human brain consisting of predominantly gray (2.0 cm) and white (4.5 cm) matter. The concentration of PCr changes slightly (2.0 cm = 5.20 +/- 0.45 mmol.l-1, 4.5 cm = 4.63 +/- 0.31 mmol.l-1), while the ATP concentration remains within limits (3.30 +/- 0.4 mmol.l-1). No change in pHi was detected between the two regions in normal volunteers (n = 6). The forward rate constant of the PCr----ATP reaction in regions of predominantly gray matter (0.30 +/- 0.04 s-1) was twice that of white matter (0.16 +/- 0.02 s-1) in vivo.

Adenosine Triphosphate↗

Bioenergetic changes during contraction and recovery in diabetic rat skeletal muscle.

Phosphorus nuclear magnetic resonance (31P-NMR) spectroscopy was used to assess the effects of hypoinsulinemia on skeletal muscle during contraction in vivo. Five groups of rats were studied: age-matched (CONA) and weight-matched (CONW) nondiabetic controls; rats given streptozotocin 21 days before study (UD); diabetic rats treated with insulin for 21 days (ITD); and insulin-treated diabetic rats with insulin treatment withheld for 72 h before study (IWD). Both UD and IWD had similar alterations in plasma substrate concentrations and an impairment in the rate of glycogen resynthesis after the stimulation protocol compared with ITD, CONA, and CONW. Pyruvate oxidation was decreased by 30-40% in mitochondria isolated from gastrocnemius of the UD group, whereas no significant decrease was observed for mitochondria from the IWD (or ITD) group(s). In UD, maintenance of gastrocnemius muscle isometric twitch tension at 1 Hz required exaggerated decreases in phosphocreatine (PCr) concentration and pH; at 5 Hz, muscle performance declined significantly, and intracellular pH decreased to lower values than observed for the control groups; during recovery, no impairment of PCr resynthesis was observed. We conclude that in skeletal muscle of UD 1) at 1 Hz there is an increased reliance on glycolytic mechanisms of ATP resynthesis and 2) at 5 Hz force failure may occur because of the decreased rate of pyruvate utilization.

Animals↗

Timing of the onset of changes in renal energetics in relation to blood pressure and glomerular filtration in haemorrhagic hypotension in the rat.

The timing and circumstances of changes in renal energetics during the gradual induction of haemorrhagic hypotension were studied in anaesthetised rats by phosphorus-31 nuclear magnetic resonance. Animals were bled at a constant rate of 0.1 ml/min via the femoral artery. Whenever changes in renal energetics were seen, a similar pattern was observed. A decrease in adenosine triphosphate occurred rapidly and was always associated with accumulation of inorganic phosphate and tissue acidosis. Profound oliguria, reflecting a markedly decreased rate of glomerular filtration preceded the changes in metabolite levels. Such a fall in glomerular filtration rate and consequently in the energy requirement for tubular reabsorption could be viewed as a mechanism by which energy demands of the kidney are reduced before a critical limitation of energy supply is reached. During uncomplicated haemorrhage in Wistar rats, mean arterial pressures as low as 25-40 mm Hg were reached before changes in renal energetics developed. In contrast, spontaneously hypertensive rates subjected to uncomplicated haemorrhage, and Wistar rats subjected to haemorrhage during concurrent stimulation of the ipsilateral sciatic nerve, developed changes in renal energetics at higher and more variable blood pressures and in response to the withdrawal of lesser but more variable quantities of blood. The sudden onset and severe degree of energy depletion at varying blood pressures during bleeding and its more ready occurrence in animals in which sympathetic nervous activity could be expected to be increased, suggests that sudden renal vasoconstriction is responsible for the unpredictable occurrence of tubular ischaemia in haemorrhagic hypotension.

Adenosine Triphosphate↗

A 31P-NMR study of the acute effects of altered beta-adrenoceptor stimulation on the bioenergetics of skeletal muscle during contraction.

The effects of acute administration of a beta-adrenoceptor agonist (isoprenaline) or antagonist (propranolol) on skeletal muscle contraction and metabolism in the rat have been studied in vivo using 31P-nuclear magnetic resonance spectroscopy and conventional metabolite analysis. In resting muscle, isoprenaline caused a three-fold increase in cyclic AMP concentration, whereas propranolol decreased cyclic AMP concentration by 40%. Isometric contraction of gastrocnemius muscle at a frequency of 4 Hz was caused by supramaximal sciatic nerve stimulation. Altered beta-adrenoceptor stimulation had no effect on contractile performance at any time during the 30 min stimulation period. During the initial stimulation period (0-4 min) intracellular pH decreased to significantly lower values in the isoprenaline-treated animals (6.24 +/- 0.03) compared to either the control (6.44 +/- 0.08) or propranolol-treated (6.42 +/- 0.08) groups. During the subsequent stimulation period (after 15-30 min stimulation at 4 Hz), pH recovered in all experimental groups to values greater than 6.90 and phosphocreatine concentration achieved a constant level at 35-40% of resting values. Calculation of free ADP concentrations using 31P-NMR determined metabolite concentrations and the creatine phosphokinase equilibrium showed that at similar tension development, [ADP]free varied between the three experimental groups; with the lowest (47 +/- 4 microM) and highest (73 +/- 4 microM) values being calculated for the beta-adrenoceptor agonist- and antagonist-treated groups respectively. Upon termination of stimulation, recovery of phosphocreatine concentration to pre-stimulation values was rapid and similar in all experimental groups. However, gastrocnemius muscle ATP concentration, determined by 31P-NMR and analysis of freeze-clamped muscle, was lower in the isoprenaline-treated group. This study has shown that although altered beta-adrenoceptor stimulation had no effect on contractile performance, significant changes in muscle metabolism were observed in vivo; these effects are discussed with respect to the role of beta-adrenoceptors in skeletal muscle.

Adenine Nucleotides↗

Inhibition of glucose phosphorylation by fatty acids in the perfused rat heart.

The flux of glucose entering the glycolytic pathway under various metabolic conditions has been indirectly monitored in the Langendorff perfused rat heart using 31P-NMR spectroscopy. By totally inhibiting (greater than 95%) glyceraldehyde-3-phosphate dehydrogenase with low concentrations of iodoacetic acid (0.2 mM) in the perfusion medium, active glycolysis results in the accumulation of sugar phosphate species (fructose 1,6-bisphosphate, dihydroxyacetone phosphate, and glyceraldehyde 3-phosphate) which can be observed in the 31P-NMR spectrum. Using this technique, it has been shown that butyrate (10 mM) in the perfusion medium decreases the flux through the initial steps of the glycolytic pathway by at least 6-fold and that both glucose phosphorylation and glycogenolysis are inhibited. Upon total global ischemia in the presence of both glucose and butyrate, the glycolysis rate is stimulated approx. 100-fold.

Adenosine Triphosphate↗

Alterations of skeletal muscle metabolism in humans studied by phosphorus 31 magnetic resonance spectroscopy in congestive heart failure.

Muscle metabolism of the finger flexor muscle of the dominant arm was examined in patients with congestive heart failure and age-related controls. Phosphocreatine utilization and decrease in pH during exercise was significantly greater in patients than in controls. Recovery rates were slow in some of these patients. Measurements of forearm blood flow at rest or during exercise were not significantly reduced in patients compared with controls. The inorganic phosphate peak was split during exercise in some of the patients, suggesting the presence of more glycolytic fibers in their muscles. These observations suggest that metabolic abnormalities may be present in the skeletal muscles of patients with heart failure that are not due to reduced nutritive blood flow. This may, in part, explain the lack of correlation between measurements of cardiac function and exercise tolerance in these patients.

Adenosine Triphosphate↗

Determination of buffering capacity of rat myocardium during ischemia.

To determine the buffering capacity of ischemic rat myocardium, lactate production was altered by glycogen depletion prior to total global ischemia. Lactate production was monitored by 1H-NMR spectroscopy in perfused rat hearts and determined by enzymatic assay of freeze-clamped tissue extracts. Intracellular pH was measured by 31P-NMR spectroscopy. The relationship between total lactate produced and pH varied considerably, depending on the final pH reached. At pH greater than 6.4 this relationship is linear with a total buffering capacity (delta lactate/delta pH) of 25 mumol H+/g wet weight per pH unit. At lower pH values (pH less than 6.4), the total buffering capacity increases progressively. Since ischemia is invariably accompanied by ATP and phosphocreatine (PCr) hydrolysis, the proton production/consumption during high-energy phosphate hydrolysis must be considered when evaluating the intrinsic buffering capacity of the myocardium against proton loads produced by lactate production from glucose and glycogen. Schemes are presented which allow an estimation of the contribution of ATP and PCr hydrolysis and the buffering by the CO2/HCO3- system during ischemia. At pH greater than 6.4, the majority (about 60%) of buffering is due to hydrolysis of adenosine triphosphate, phosphocreatine in the heart, and neutralization of sodium bicarbonate in the perfusate. At pH less than 6.4 an increasing proportion of cardiac buffering is from intrinsic cardiac buffers, most likely from intracellular proteins. After correction for these contributions to the observed total cardiac buffering capacity, the intrinsic buffering capacity of the myocardium can be accounted for by a high capacity (170 mumol/g wet weight) but low pKa (5.2) buffering system.

Adenosine Triphosphate↗

31P-NMR saturation transfer studies of aerobic Escherichia coli cells.

31P-NMR measurements of saturation transfer have been used to measure the flux between Pi and ATP in Escherichia coli cells respiring on an endogenous carbon source. Measurements were made in the wild type and in cells genetically modified to give a 5-fold higher concentration of the F1F0-ATP synthase. The flux in the two cell types was not significantly different. This, together with studies using inhibitors specific for the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase and the ATP synthase, suggests that the observed flux arises predominantly from glycolytic rather than ATP synthase activity. Although this conclusion is in disagreement with previous experiments on E. coli, it is in agreement with recent experiments on yeast.

Adenosine Triphosphate↗

31P NMR measurements of myocardial pH in vivo.

A 31P NMR magnetization transfer method for measuring myocardial pH in vivo is demonstrated in the lamb, dog and cat. The method involves measuring the difference in chemical shift between the resonances of phosphocreatine and inorganic phosphate in magnetization transfer difference spectra in which the gamma-phosphate resonance of ATP has been saturated. The method has been verified by measuring the chemical shift difference between the resonances of 2-deoxyglucose 6-phosphate and phosphocreatine following infusion of the animals with 2-deoxyglucose. The measured pH values are significantly lower than those obtained in previous studies on the heart in vivo.

Animals↗

An animal model of mitochondrial myopathy: a biochemical and physiological investigation of rats treated in vivo with the NADH-CoQ reductase inhibitor, diphenyleneiodonium.

Chronic administration of the NADH-CoQ reductase inhibitor, diphenyleneiodonium to rats at two dose levels, 1.0 and 1.5 mg/kg per day, caused a 40% and 60% reduction, respectively, in the in vitro rate of NAD-linked respiration by skeletal muscle mitochondria. At the highest dose, muscle fatigue, lactic acidosis and an over-utilization of phosphocreatine was observed in the gastrocnemius muscle during mild stimulation of 1 Hz frequency. The resynthesis of phosphocreatine following muscle stimulation was about 2 fold slower in the treated animal group. At the low dose, no significant biochemical changes were observed during muscle stimulation at 4 Hz. The results are discussed in terms of skeletal muscle "oxidative reserve", twitch tension maintenance and the relevance to the human diseased state of mitochondrial myopathy.

Acidosis↗

31P nuclear magnetic resonance study of steady-state adenosine 5'-triphosphate levels during graded hypoxia in the isolated perfused rat kidney.

1. A model of controlled hypoxia in the isolated perfused rat kidney has been used to compare the extent of reduction in the steady-state level of adenosine 5'-triphosphate (ATP) from that initially observed with alterations in renal function and with the development of tubular cell injury. 2. ATP depletion was observed in response to decreased total oxygen delivery even when delivery greatly exceeded consumption and the venous oxygen tension remained in excess of 150 mmHg. 3. Increases in the fractional excretion of sodium occurred progressively below an apparent threshold value of whole kidney ATP of approximately 80% of the baseline. 4. With modestly decreased oxygen delivery, cellular injury was confined to deep proximal tubule and medullary thick ascending limb of Henle's loop. Severely decreased oxygen delivery rates were associated with cellular damage spreading throughout the cortex. 5. Even the smallest reductions in whole kidney ATP were associated with morphological damage to tubular cells. The extent of reduction in whole kidney ATP was closely correlated and approximately equivalent to the calculated volume of injured cells. 6. Our results indicate that reduction in whole kidney ATP during decreased oxygen delivery is a valid marker of the extent of injurious cellular hypoxia and are consistent with the view that cellular ATP concentrations in hypoxia are markedly inhomogeneous. They support the hypothesis that specific regions of the perfused kidney become critically hypoxic and develop cellular injury while overall oxygen delivery remains high. Areas at risk include deep proximal tubule as well as the medullary thick ascending limb of Henle's loop.

Adenosine Triphosphate↗

Depletion of myocardial glutathione: its effects on heart function and metabolism during ischaemia and reperfusion.

To determine the importance of intracellular reactive oxygen metabolites in ischaemia and reperfusion, rat hearts were depleted of glutathione, a major intracellular antioxidant, prior to total global or low flow ischaemia. Chronic pretreatment of rats with up to 7 mmol.kg-1 L-buthionine-S,R-sulphoximine over a period of 96 hours resulted in 85% depletion of total myocardial glutathione. Subsequent recovery of glutathione after treatment with the sulphoximine was slow, reaching only 54% of control levels, 96 hours after the final dose. Depletion of myocardial glutathione by 70% did not increase the metabolic consequences of either total global or low flow ischaemia in the isolated perfused heart, as determined by 31P NMR spectroscopy and high performance liquid chromatographic analysis of purine release. During low flow ischaemia glutathione depletion caused a significant reduction in purine release. On reperfusion, functional recovery was depressed compared to controls. Hearts depleted of glutathione also showed a decrease in developed tension during normoxic perfusion prior to low flow ischaemia. There was no difference between the two groups before or after total global ischaemia. These results do not support the hypothesis of free radical mediated reperfusion injury; however, the method described here for the depletion of glutathione should prove a useful tool for further investigation into the role of glutathione in cardiac metabolism and function.

Adenosine Triphosphate↗

Ca2+-ATPase deficiency in a patient with an exertional muscle pain syndrome.

31P Magnetic resonance spectroscopy studies were carried out in vivo on skeletal muscle of a patient with verapamil-responsive, chronic, progressive post-exertional muscle pain. A sister suffered from a similar complaint. The results showed that the muscle: (1) decreased its high energy phosphate content more rapidly than normal during exercise, indicating either increased utilisation or decreased production of ATP; (2) acidified more rapidly than normal during exercise suggesting an increased glycolytic rate; (3) continued in some studies to acidify markedly during the first minute after exercise, indicating that glycolysis remained active into the recovery period; (4) had phosphocreatine and ADP recovery rates consistent with normal rates of oxidative phosphorylation. On the basis of these results, it was proposed that the patient suffers from a defect in Ca2+ handling in the muscle. Subsequently, direct measurement of Ca2+-ATPase activity in the sarcoplasmic reticulum fraction from a muscle biopsy sample showed that the activity of this enzyme was reduced by about 90%.

Adult↗

Spatial heterogeneity of metabolism in skeletal muscle in vivo studied by 31P-NMR spectroscopy.

Phase-modulated rotating-frame imaging, a localization technique for phosphorus nuclear magnetic resonance spectroscopy, has been applied to obtain information on heterogeneity of phosphorus-containing metabolites in skeletal muscle of the rat in vivo. The distal muscles of the rat hindlimb have been studied at rest and during steady-state isometric twitch contraction; the use of a transmitter surface coil and an electrically isolated, orthogonal receiver Helmholtz coil ensure accurate spatial assignment (1 mm resolution). At rest, intracellular pH was higher and PCr/(PCr + Pi) was lower in deeper muscles compared with superficial muscle of the distal hindlimb. Upon steady-state stimulation, the relatively more alkaline pH of deep muscle was maintained, whereas greater changes in PCr/(PCr + Pi) and Pi/ATP occurred in the superficial muscle layer. This method allows rapid (75 min for each spectral image) acquisition of quantitative information on metabolic heterogeneity in vivo.

Adenosine Triphosphate↗

Metabolic changes during experimental cerebral ischemia in hyperglycemic rats, observed by 31P and 1H magnetic resonance spectroscopy.

Progressive cerebral ischemia was induced in seven anesthetized hyperglycemic rats by carotid artery ligation and hemorrhagic hypotension. Phosphorus metabolites, intracellular pH, and lactate in the brain were monitored by 31P and 1H magnetic resonance spectroscopy. Under conditions in which blood flow was low, phosphocreatine (PCr) concentration and intracellular pH decreased and the concentration of lactate increased. The decrease in ATP was approximately one-third that of PCr until only 25% PCr remained, after which ATP was lost more rapidly than PCr. These changes were interpreted in terms of three regions observed by the magnetic resonance coil, one of complete ischemia, one of partial ischemia, and one of perfusion sufficient to maintain normal metabolite levels. The extent of the three regions was estimated quantitatively. Broadening and splitting of the inorganic phosphorus (Pi) peak into two components provided further evidence of distinct populations of cells, one very acidic and another less so. Apparent intracellular buffering capacity was calculated as 23.6 +/- 1.3 mumol lactate/g wet wt/pH.

Acidosis, Lactic↗

Study of liver metabolism in glucose-6-phosphatase deficiency (glycogen storage disease type 1A) by P-31 magnetic resonance spectroscopy.

Liver metabolism of two patients (aged 15 and 23 yr) was studied by P-31 magnetic resonance spectroscopy at 1.9 tesla. The P-31 spectra of liver showed the resonances of phosphomonoesters (including sugar phosphates), inorganic phosphate (Pi), phosphodiesters (e.g. glycerophosphorylcholine, glycerophosporylethanolamine), and ATP. These resonances were quantified by expressing their peak areas in mM (assuming that ATP concentrations in normal liver is 2.5 mM) or as a ratio relative to the area of the phosphodiester resonance. After an overnight fast liver phosphomonoesters in patients were 2.6 and 1.6 AU, respectively (controls 1.1 +/- 0.5, mean +/- 2 SD, n = 17). At the same time liver Pi was decreased in patients to 1.3 and 1.0, respectively (controls 1.8 +/- 0.8). Based on chemical shift measurements the increase in phosphomonoesters could be attributed to accumulation of sugar phosphates (mainly glycolytic intermediates). After 1 g/kg oral glucose, hepatic sugar phosphates decreased in patients by 64 and 40%, respectively, and reached normal levels (on the absolute intensity scale); whereas liver Pi increased by 130 and 40%, respectively. Liver Pi levels remained elevated in both patients 30 min after ingestion of glucose. Liver sugar phosphates and Pi did not change in control subjects (n = 4) after glucose. In contrast to some previous reports, we have found accumulation of glycolytic intermediates in the liver of glucose-6-phosphatase-deficient patients during fasting. In these patients high levels may enhance the activity of residual glucose-6-phosphatase thus increasing hepatic glucose production and reducing the degree of hypoglycemia during fasting.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗