13C-n.m.r. study of citrate metabolism in rabbit renal proximal-tubule cells.

U.v.-visible-absorption and e.p.r. spectroscopy were used to study the type 2 and type 3 copper centres in the mercury derivative of laccase. After treatment with peroxide the mercury derivative of laccase exhibits a fully developed absorption band at 330 nm (delta epsilon = 2900 +/- 100 M-1.cm-1, which is characteristic of type 3 copper in the oxidized state. In addition, there is a weak ligand-field absorption at 740 nm (epsilon = 380 +/- 30 M-1.cm-1), which can be assigned to the type 3 pair. Because the e.p.r. spectrum of the type 2 copper is well resolved in the case of the mercury derivative of laccase, for the first time we have been able to observe spectroscopic evidence for a pH-dependent structural transition that has been invoked to explain the kinetics of enzyme reduction [Andréasson & Reinhammar (1979) Biochim. Biophys. Acta 568, 145-156]. According to the e.p.r. data the pKa lies in the range 6-7, and comparisons with a model compound show that the spectral changes can plausibly be interpreted in terms of the deprotonation of a water molecule in the co-ordination sphere of the type 2 copper.     

A 13C-n.m.r. investigation of the metabolism of amino acids in renal proximal convoluted tubules of normal and streptozotocin-treated rats and rabbits.

13C-n.m.r. spectroscopy was used to determine the metabolic fate of alanine and aspartate in rat and rabbit kidney proximal tubules. The main purpose of the present study was to investigate the effect of streptozotocin-induced diabetes on the influx of 13C label from [3-13C]alanine into the tricarboxylic acid cycle and through the fructose-1,6-bisphosphatase pathway. This influx was calculated from the relative enrichment of 13C in the various glutamate and glutamine carbon atoms. The relative proportion of 13C label which entered the tricarboxylic acid cycle via pyruvate carboxylase relative to the proportion that entered via pyruvate dehydrogenase was 1.92 +/- 0.02 in fed control rats and 2.27 +/- 0.04 in streptozotocin-treated rats. However, streptozotocin-induced diabetes did not significantly affect this ratio in rabbit proximal convoluted tubular cells. Only in rat proximal convoluted tubular cells did we observe an increase in flux through the fructose-1,6-bisphosphatase pathway by streptozotocin treatment compared with fed controls. The data suggest that streptozotocin-induced diabetes in rats causes the same metabolic changes as does chronic acidosis.     

Formation of n.m.r.-invisible ADP during renal ischaemia in rats.

Measurement of the adenine nucleotide and inorganic phosphate content of normoxic and ischaemic kidney in vivo has been made, comparing enzymic assay (after freeze-clamping and acid extraction) with quantification by 31P-n.m.r. Both methods give similar results for ATP, and n.m.r. quantification of Pi gives a value 25-50% of that obtained by enzymic assay. ADP, which is largely invisible to n.m.r. in the normoxic kidney, remains invisible during ischaemia despite a 2-3 fold rise in enzymically assayed ADP. N.m.r. and enzymic assay of the acid extracts give similar values for all metabolites measured. The question of ADP binding in the kidney is discussed, as are the implications for the metabolic regulation of ADP-dependent reactions.     

31P-n.m.r. studies on cerebral energy metabolism under conditions of hypoglycaemia and hypoxia in vitro.

A system has been developed for performing 31P-n.m.r. studies on cerebral tissues superfused in vitro, and gives results comparable with those reported from studies in vivo. Under optimal superfusion conditions [10 mM-glucose and O2/CO2 (19:1)] the tissue concentrations of phosphocreatine and ATP were calculated to be approx. 3.1 and 1.3 mumol/g respectively. When the glucose of the superfusing medium was lowered to 0.5 mM, slightly decreased sugar phosphate peaks were observed, but there was no detectable change in [ATP] or [phosphocreatine]. At 0.2 mM-glucose, significantly decreased concentrations of phosphocreatine and ATP were observed. Substitution of pyruvate plus malate for glucose did not decrease levels of phosphocreatine and ATP. When the superfusing medium was gassed with air/CO2 (19:1; 'mild hypoxia'), there was an appreciable fall in sugar phosphates and phosphocreatine with no detectable effect on ATP. In the presence of N2/CO2 (19:1; 'severe hypoxia', since O2 was not completely excluded), concentrations of phosphocreatine fell considerably, but with little effect on ATP. The results demonstrate the feasibility of studying cerebral energy metabolism in vitro using the non-invasive 31P-n.m.r. technique and are discussed in relation to the sensitivity of cerebral tissues to metabolic insults in vitro and in vivo.     

N.m.r. studies of metabolism in perfused organs

Several metabolites and intracellular pH in intact organs can be studied in a non-destructive manner by phorphorus nuclear magnetic resonance (31P n.m.r.). This possibility was demonstrated by us nearly five years ago. Since then we have developed the appropriate physiological techniques and improved the n.m.r. method for the study of animal hearts and kidneys. Here we described measurements aimed at clarifying three problesm. (1) Having measured the enzyme-catalysed fluxes between phosphocreatine and ATP by the method of saturation transfer n.m.r., we examine the relations between energy supply and heart rate in the isolated perfused rat heart. (2) We describe experiments to establish the validity of the perfusion model. For the first time, we report 31P n.m.r. measurements of an in vivo rat heart and compare the results with those obtained for the perfused rat heart. (3) Ischaemia and metabolism in rabbit kidneys is investigated to establish the relation between functional and metabolic recovery after a renal transplant operation.     

15N n.m.r. spectroscopy: 36. 1H n.m.r. and 15N n.m.r.spectroscopic investigation on the protonation of polypeptides

¹⁵N n.m.r. (9.12 MHz) spectra of acetamide, polyglycine, poly([l-alanine) and poly(l-leucine) were measured in various acidic solvents. These solvents include dichloroacetic acid (DCA), trifluoroacetic acid (TFA), methane sulphonic acid (MSA) and fluorosulphonic acid (FSA). Full protonation of both amides and polypeptides causes downfield shifts of 17–20 ppm. Furthermore, the concentration dependence of the chemical shift was measured. In solvents which cause partial protonation, decreasing concentration of amide groups may cause downfield shifts up to 8.5 ppm, while in the case of full protonation or in the absence of protonation no concentration dependence is observable. The protonation of peptide groups induces H/D-exchange of the αC proton which was monitored by ¹H n.m.r. spectroscopy. The mechanism of this H/D-exchange is discussed.     

Kinetic analysis of the cerebral creatine kinase reaction under hypoxic and hypoglycaemic conditions in vitro. A 31P-n.m.r. study.

1. The tissue concentration of phosphocreatine (PCr) and the pseudo-first-order rate constant of creatine kinase (kf) were monitored in superfused guinea-pig brain slices in vitro by using 31P-n.m.r. techniques. 2. Superfusion of slices in low oxygen partial pressure (pO2 approx. 16 kPa) decreased tissue PCr concentrations by 48% but ATP concentrations were unchanged. Regression analysis revealed a significant negative correlation between the PCr concentration in hypoxic tissue and the increase in the rate constant, kf. Nevertheless the forward flux through the enzyme (Jf = kf.[PCr]) declined under these conditions. 3. Lowering the glucose concentration to 0.2 or 0.1 mM decreased PCr concentrations by 29% and 48% respectively; here ATP concentrations as well as PCr concentrations also decreased. Only in the presence of the lower glucose concentration (0.1 mM) was kf increased. However, unlike the situation in hypoxic tissue, Jf was maintained at control rates. 4. In spectra obtained in the presence of low oxygen or low glucose concentrations, a resonance attributable to tissue inorganic phosphate became dectectable. This observation is discussed in terms of known changes in tissue phosphate concentrations and possible alterations in cytoplasmic pH.     

A study of hepatic metabolism of thyroxine in BB/W rats treated with L-thyroxine

The effect of thyroxine (T4) on T4 conversion to triiodothyronine (T3) and reverse T3 (rT3) was studied in BB/W rats. A colony of 38 BB/W rats was obtained and half were treated with thyroxine (T4), 1 mg per liter of drinking water. At 106 days of age the following groups were identified: nondiabetic, no T4 treatment, 8 rats; nondiabetic, T4 treated, 8 rats; diabetic, no T4 treatment, 10 rats; diabetic, T4 treated, 7 rats. All animals with diabetes were treated with insulin. T4 conversion to T3 and rT3 was assessed in liver homogenates in 0.1 M Tris-HCl buffer, pH 7.4, with or without 5 mM dithiothreitol (DDT). Serum T4 and rT3 were significantly elevated in both T4-treated groups (P less than 0.001), while serum T3 was not affected in either. Basal T4 deiodination to T3 by the liver homogenate did not change on treatment with T4; the addition of DTT increased T3 production in the homogenate from T4 treated nondiabetic animals (P less than 0.05). In both nondiabetic and insulin-treated diabetic rats there was no effect of T4 on the rate of rT3 production. Since, in the rat, 30-40% of circulating T3 is a direct contribution of thyroid gland secretion, and that would be absent in our T4-suppressed animals, the normal serum T3 may reflect increased absolute peripheral T3 production from the greater concentration of circulating T4.     

Validation of lactose[15N,15N]ureide as a tool to study colonic nitrogen metabolism

In vitro experiments have shown that fermentation of carbohydrates prevents accumulation of nitrogen in the colon. Variable results have been obtained on modulation of dietary intakes in vivo. Lactose[15N,15N]-labeled ureide has been proposed as a tool to study colonic nitrogen metabolism. However, on oral administration of the marker, different urinary excretion patterns of the 15N label have been found. In this study, 50 mg lactose[15N,15N]ureide was directly instilled in the colon through an orocecal tube to investigate the colonic handling of this molecule in a direct way. In basal conditions, 42% (range, 37-48%) of labeled nitrogen administered as lactose[15N,15N]ureide was retrieved in urine after 72 h. A substantial variability in total urinary excretion of the label was found, but the urinary excretion pattern of the label was similar in all volunteers. When inulin, a fermentable carbohydrate, was administered together with the labeled marker, a significant decrease in urinary excretion of 15N after 72 h was found, to 29% (range, 23-34%). The effect of a smaller dose of inulin (250 mg) on colonic handling of lactose[15N,15N]ureide (50 mg), was investigated in another group of volunteers, and this time, fecal excretion of the marker was also evaluated. The results seem to indicate that fermentation of inulin causes an increased fecal excretion of the marker, thereby reducing urinary excretion but not retention in the human nitrogen pool. This instillation study shows that lactose[15N,15N]ureide is a tool with good properties to investigate the effect of different types of carbohydrates on nitrogen metabolism in the proximal colon in vivo.     

Nitrogen-15 NMR studies of nitrogen metabolism in Picea glauca buds.

In vivo (15)N nuclear magnetic resonance (NMR) as well as (15)N solid-state magic angle spinning (MAS) NMR spectroscopy were used to investigate nitrogen metabolism in cultured white spruce (Picea glauca) buds. Long-term as well as short-term experiments were carried out involving the use of inhibitors of the nitrogen pathways such as methionine sulfoximine (MSO), azaserine (AZA) and aminooxyacetate (AOA). Both in vivo and solid-state NMR showed that when MSO blocked glutamine synthetase (GS) no NH(4)(+) is incorporated. When glutamate synthase (GOGAT) is blocked by AZA there is some incorporation into glutamine (Gln), but very little into alpha-amino groups (glutamate, Glu). The transamination inhibitor AOA does not affect the metabolism of (15)NH(4)(+) into Gln and Glu, but blocks the production of arginine (Arg), as would be expected. Proline (Pro) and gamma-aminobutyric acid (GABA), which are produced directly from Glu without a transamination step, were not affected. The solid-state NMR experiments showed that protein synthesis occurred. Collectively, our results show that NH(4)(+) can only be assimilated through the GS/GOGAT pathway in P. glauca buds.     

A window into cellular metabolism: hepatic metabolism of (15)N-labelled substrates

It is now apparent that many of the subtleties of cellular metabolism are intrinsically associated with cell structure and that their physiological study requires techniques that respect the integrity of cells and organs. We have used 15N-substrates to examine urea synthesis in the intact perfused rat liver. This work permits us to determine the extent to which different amino acids donate nitrogen atoms to the two nitrogens of urea. It is apparent that alanine and the amino group of glutamine provide nitrogen for urea synthesis primarily via cytoplasmic aspartate, whereas mitochondrial ammonia is the preferred route of entry for nitrogen from pre-formed ammonia or from the amide nitrogen of glutamine. Most importantly, this methodology permits us to explore for the occurrence of metabolic channels in such a highly organised, physiological system. Our studies indicate that a metabolic channel does not exist between glutaminase and carbamoylphosphate synthetase 1.     

13C-n.m.r. study of C hordein.

Insoluble xylan was prepared from ground birch (Betula pubescens) pulp by alkali extraction and precipitation with ethanol. The only sugar detected after acid hydrolysis of the preparation was xylose. The insoluble xylan was used as substrate in a nephelometric assay to determine the xylanase (EC 3.2.1.8, 1,4-beta-D-xylan xylanohydrolase and EC 3.2.1.37, 1,4-beta-D-xylan xylohydrolase) activities of Aspergillus and Trichoderma enzymes. The nephelometric method is reliable in evaluating xylanase hydrolysis of insoluble xylan.     

Quantitative analysis of metabolism of hepatic triglyceride in ethanol-treated rats.

An acute intraperitoneal injection of ethanol (0.7 or 2.1g/kg body wt.) causes the reversible, dose-dependent accumulation of hepatic triglyceride in rats. By using a pulse of [14C]palmitate injected into a tail vein, it was found that ethanol (2.1g/kg)had no effect on the flux of unesterified fatty acid of serum (4.3mumol/min per 100g body wt.). However, either dose increased the fraction of the total flux going to liver from 0.16 to0.27 as rapidly as could be measured (30s), and it remained elevated until all ethanol had been cleared from the blood. The fraction of the total radioactivity in lipids of liver that was in triglyceride increased linearly for 1 h from 30 to 50% and there was a simultaneous decrease in phospholipid from 60 to 40%. The rate of synthesis of hepatic triglyceride derived directly from unesterified fatty acid of serum was calculated by using the flux rate of unesterified fatty acid in serum, the fractional hepatic uptake of this flux, and the percentage of liver fatty acid esterified to triglyceride. This contribution is related to the total synthetic rate of hepatic triglyceride (rate of accumulation+rate of release) to determine quantitatively how much of the developing fatty liver is attributable to increased uptake of unesterfied fatty acid of serum. At the higher dose of ethanol, about half of the accumulating triglyceride is derived from this source, whereas with the lower dose of ethanol it can account for all of the build-up.