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1H and 31P nuclear magnetic resonance (NMR) spectroscopy were used to study the role of external pH (pHo) in regulation of intracellular pH (pHi) in the cerebral cortex in vitro. Lowering the pH of the oxygenated superfusion medium (pHb) from 7.5 to 7.0-6.8 decreased pHi from 7.33 +/- 0.08 to approximately 7.00 independently of the presence of HCO3- and medium buffering power. Reduction of medium (pHb 6.8) buffering power during hypoxia severely acidified pHi independently of the presence of HCO3-. Following hypoxia, recovery of pHi was not sensitive to pHb provided that medium buffering was maintained. Lactate removal proceeded independently of pHb, HCO3- and external buffering power. These results indicate that decrease in extracellular buffering capacity has a feed-back effect on pHi in the cerebral cortex.

Spectral editing experiments were used to quantify CH3 groups from macromolecular species in the chemical shift region from 1.2 to 1.4 ppm of rat cerebrum in vivo. Two peaks centred at 1.22 and 1.40 ppm were revealed when irradiation was positioned at 4.35 or 4.30 ppm. These peaks had lower saturation factors (1 vs. 1.72 +/- 0.10) than N-acetyl aspartate (NAA) and shorter T2 [60 +/- 5.8 (1.22 ppm) and 51 +/- 2.2 (1.40 ppm) vs. 123 +/- 12 (NAA) ms]. The concentrations of the peaks at 1.22 and 1.40 ppm were calculated to be 0.65 +/- 0.09 and 1.37 +/- 0.18 mmol of CH3 equivalents/kg brain. Acid extract from cerebral cortices contained macromolecular peaks at the same chemical shifts with approximately the same area ratios to NAA as in vivo. These data show that the macromolecular peaks in the brain at TE > 100 ms arise predominantly from proteins which are acid soluble. The assignment of macromolecular signals in the cerebral spectrum to a given polypeptide (thymosin beta 4 and histone H1) is discussed in the light of protein analyses of brain acid extracts.

We studied a combined utilization of serum tumour markers and proton nuclear magnetic resonance 1H NMR spectroscopy of plasma in the detection of pancreatic cancer. Serum concentrations of carcinoembryonic antigen (CEA), carboanhydrate antigens CA 50 and CA 242, and 1H NMR spectra of plasma were determined in a series of 51 patients, of whom 25 had malignant and 26 benign hepatopancreatobiliary disorder. The measurements in 1H NMR spectra were performed both manually and by computed technique, and both methylene (CH2) and methyl (CH3) peaks were evaluated. In the patients with pancreatic cancer (n = 17, including two cases of cancer of the papilla of Vater), the mean serum values of all tumour markers were significantly (p = 0.001) higher than in the patients with benign disease. The diagnostic sensitivity of the tumour markers alone reached 82-100% with a specificity of 35-82%. In the 1H NMR spectra of sera, the mean linewidth of the CH3-peak both in the manual and computed measurement was significantly narrower in the patients with pancreatic cancer than in the benign disease group. Using the cut-off level of 33 Hz, the sensitivity and specificity of 1H NMR alone was in the manual measurement 53% and 76%, respectively, while in the computed measurement the corresponding figures were 94% and 20%. When the serum tumour markers and 1H NMR spectroscopy were evaluated as combinations, both in the manual and computed measurements the specificities and positive likelihood ratios were clearly better than those of the tumour markers alone, but efficiencies improved only slightly. The results suggest that the combined use of tumour marker tests and 1H NMR of plasma gives only slightly improved accuracy in the diagnosis of pancreatic cancer.

Cerebral glutamate was monitored in a superfused cerebral cortical preparation by 1H NMR spectroscopy using a semiselective spin-echo sequence N-acetyl aspartate (NAA) as an internal concentration reference. During controlled metabolic conditions, the cerebral 1H NMR-detected glutamate-to-NAA ratio was approximately 20-30% lower than expected from the ratio of neutralized perchloric acid extracts of the preparations. Inhibition of respiration in the presence of glucose did not change the 1H NMR glutamate-to-NAA ratio in brain slice preparation. In contrast, either complete depletion of ATP during cyanide poisoning together with 0 mM glucose, anoxia in the absence of glucose, or treatment with nigericin or with a protonophore, carbonyl cyanide-m-fluorophenylhydrazone, increased 1H NMR-detected glutamate/NAA in the cerebral preparations without a change in the relative and absolute concentration ratios determined from the tissue acid extracts. Spin-spin relaxation times of glutamate and NAA peaks in anoxic slices were 749 +/- 89 and 729 +/- 94 ms, respectively, and thus, the portion of glutamate that could not be detected by 1H NMR was quantified in absolute terms. It was calculated that an increase in the glutamate-to-NAA ratio from 0.55 +/- 0.02 to 0.67 +/- 0.02 during aglycemic anoxia corresponded to some 6 mmol/kg of tissue dry weight of glutamate from the total concentration of 28 mmol/kg dry weight. It is suggested that this 22% of total glutamate pool is present in a noncytoplasmic compartment during controlled metabolic state.

The first practical demonstration that nuclear magnetic resonance (NMR) spectroscopy could be applied to the study of brain biochemistry in vivo came in 1980, with the studies of the rat brain using a surface coil. Since then the technique has been rapidly and extensively developed into a versatile, non-invasive tool for the investigation of various aspects of brain biochemistry, physiology and disease. NMR is non-destructive and can be used to examine a wide variety of samples, ranging from localized regions within the whole brain in humans or animals, through tissue preparations (perfused organ, tissue slices and homogenates), to isolated cells and aqueous solutions, such as tissue extracts. 31P and 1H NMR spectra deriving from endogenous compounds of the brain in situ allow assessment of tissue metabolites and provide information about high-energy phosphates, lactate, certain amino acids, intracellular pH and ionic concentrations. Exogenous substrates or probes labelled with stable isotopes can also be introduced into the brain and used to monitor metabolism. Animal models of brain diseases have given some impetus to rapid progress in clinical NMR spectroscopy and also magnetic imaging techniques. The purpose of this article is to highlight the type of information available from these NMR techniques, and to present this in a neuroscience context, emphasizing the biochemical, physiological and pathological information that can be obtained using these methods.

We have used double quantum filtered (DQF) 23Na+ nuclear magnetic resonance (NMR) spectroscopy without shift reagents in order to monitor intracellular Na+ (Na+i) in a cortical brain slice preparation. The external Na+ (Na+o) signal was reduced by 95% by the DQF sequence compared with the directly observed 23Na+. The DQF 23Na+ signal is not exclusively due to Na+i, however, as 40% of this signal appears to arise from Na(+)-ions interacting with extracellular membrane proteins or proteins exposed at the cut surfaces of the slices. Veratridine increased instantly the DQF 23Na+ signal so that it reached 130.4 +/- 5.0% by 12 min. This shows that there was a significant contribution from Na+i in the DQF 23Na+ NMR spectra. Hypoxia of 30 min duration in the presence of 10 nM glucose did not influence intensity of the DQF 23Na+ signal. Aglycaemic hypoxia caused complete collapse of phosphocreatine (PCr) within 7 min whereas DQF 23Na+ first increased 15 min after the insult. This increase reached its maximal value of 125% after 25 min. There was an incomplete recovery of the DQF 23Na+ after aglycaemic hypoxia to 110% of the control value parallel to poor metabolic recovery. The presence of 10 mM extracellular Mg2+ had no apparent effect on the aglycaemic hypoxia-induced rise in Na+i indicating that it was linked to Ca2+ influx. Tetrodotoxin (TTx, 4.7 microM) did not influence the rise of Na+i caused by aglycaemic hypoxia. These results indicate that elevation of Na+i is a late consequence of energy failure in the cerebral cortex.

Between December 1988 and January 1992, 11 men underwent lower urinary tract reconstruction by means of the hemi-Kock ileal reservoir following radical cystoprostatectomy (ten patients) and subtotal cystectomy (one patient). Six of the 11 patients developed an early postoperative complication, with a fatal outcome in two of them. One patient succumbed to subarachnoidal bleeding and one to septic shock. During the follow-up, another two men died of advanced bladder carcinoma. The late complication rate was low. Most patients regained their continence within six months. A good continence was achieved in all the nine remaining patients during the day and in six of them at night. This experience suggests that the hemi-Kock reservoir is a useful method for the urinary tract reconstruction following cystectomy.

The sera of 51 patients with malignant (n = 25) and benign (n = 26) hepatopancreatobiliary disorders were analysed by 1H magnetic resonance spectroscopy (NMR) in order to distinguish between malignant and benign diseases causing jaundice and/or cholestasis. Macromolecular linewidths were determined both manually and automatically with a computed analysis, and both methylene (CH2) and methyl (CH3) resonances were evaluated. The mean linewidth of the CH3 peak was significantly narrower in the patients with malignant disease than in the patients with benign disease both in the manual and computed analyses, but no significant differences in the CH2 peak were detected. Diagnostic sensitivity and specificity of the CH3 peak determined in the computed analysis were 92% and 27% respectively. In the light of the current study, it seems obvious that because overlap between benign and malignant groups was too great, 1H NMR spectroscopy of plasma is not of practical value in distinguishing between benign and malignant causes of jaundice and/or cholestasis.

A radiometric determination of monoaminooxy analogues of naturally occurring polyamines is described in which [2-14C]acetone is employed as reagent. The reagent is volatile while the oxime product is not allowing unreacted reagent to be removed and the oxime formation to be completed by lyophilization in vacuo. The residual radioactive compound is soluble in water and proportional to the reactive aminooxy content of the reaction mixture and can be quantified by liquid scintillation counting. The assay method is inexpensive and simple and has high specificity and flexibility in sample volume enabling reliable quantification of reactive aminooxy amines in biological extracts at concentrations exceeding 0.25 microM. Optimal pH values for oxime formation of five monoaminooxy analogues of polyamines with acetone were resolved. Reactions via reversible intermediates to irreversible oximes were sped up by removal of water. Complete oxime formation and stability was confirmed by 1H NMR studies. Tested drugs readily formed oximes with pyridoxal 5'-phosphate, too. Diaminooxy analogue of cadaverine formed a volatile oxime with acetone. The method was used to monitor the stability of aminooxy analogues of putrescine and spermidine during storage and under culture conditions and to establish their scant accumulation in, but fast catabolism by, cultured baby hamster kidney cells.

In the present study the contribution of cytoplasmic polypeptides thymosins beta 4 and beta 10 to the 1H NMR spectrum during the maturation of the rat cerebral cortex was assessed. In the proton spectrum intense broad peaks at 0.9, 1.22, and 1.40 ppm from thymosins decreased in size relative to the signal at 2.02 ppm in parallel to the reciprocal increase in the concentration of N-acetyl aspartate. Levels of thymosins beta 4 and beta 10 were under developmental regulation. It is concluded that peaks from thymosins may provide extended information for an NMR spectroscopist and thus they have to be taken into account in the interpretation of the newborn cerebral 1H NMR spectrum.

In the present work we have investigated the macromolecules that contribute to the brain 1H NMR spectrum. The cerebral cortex showed distinct resonances at the uncrowded methyl- and methylene chemical shift scale of the spin-echo 1H NMR spectrum. The peaks at 1.22 and 1.40 ppm (relative to the methyl protons of N-acetyl aspartate at 2.02 ppm) arise from cerebral macromolecules without evidence for co-resonances from low molecular weight metabolites as shown by the spin-spin relaxation decays of these resonances. In addition to these NMR signals, peaks at 0.9 and 1.7 ppm from macromolecules were detected. These resonances are from proteins, and we have identified the polypeptides that contributed to the 1H NMR peaks. Two proteins that were present at concentrations of 250 and 350 micrograms/g of dryed tissue showed 1H NMR spectra that resembled the macromolecular pattern in the cerebral 1H NMR spectrum. They were identified as thymosin beta 4 and histone H1, respectively. Thymosin beta 4 was present in soluble high speed cytoplasmic fraction and in P2 pellet, whereas histone H1 was detected in nuclear enriched fraction. A chemical shift-correlated two-dimensional 1H NMR spectrum of thymosin beta 4 in vitro revealed a coupling pattern that matched the macromolecule in the cerebral cortex which we have previously noted (Kauppinen R. A., Kokko, H., and Williams, S. R. (1992) J. Neurochem. 58, 967-974). On the basis of both one- and two-dimensional NMR evidence, subcellular distribution and high concentration, we assign the 1H NMR signals at 0.9, 1.22, 1.40, and 1.7 ppm in the cerebral cortex to thymosin beta 4.

Proton nuclear magnetic resonance (1H NMR) spectroscopy was used to study metabolites of the brain cortex ex vivo. The superfused brain cortex preparation was judged to be metabolically viable on the basis of the 31P NMR spectrum (intracellular pH of 7.23 +/- 0.03 and phosphocreatine/ATP ratio of 1.21 +/- 0.09). Using 1H NMR a group of previously unidentified signals was detectable at 0.94, 1.22, and 1.40 ppm with a water-suppressed spin-echo sequence. These signals had shorter spin-spin relaxation times (51-54 ms) than N-acetylaspartate and lactate (84-93 ms) and also smaller saturation factors, an indication of shorter spin-lattice relaxation times than the latter two low-molecular-weight metabolites. The unidentified signals also displayed homonuclear coupling to other spins in the methine region of the spectrum. Acid extraction of the brain slices or cortex from animals that were killed yielded a mixture of proteins that exhibited NMR properties matching the 1H NMR signals in the brain cortex. The molecular mass of these thermoresistant, "mobile" proteins, which contained proline plus hydroxyproline (9-16% of all amino acids), ranged between 8 and 40 kDa. These "new" assignments of 1H NMR-detectable compounds may influence interpretation of NMR data of some metabolites, as their signals are in the vicinity of the -CH3 1H NMR peaks of lactate and alanine.

We have generated a transgenic mouse line strikingly overexpressing the human ornithine decarboxylase (ODC) gene in their brain. Brain ODC activity was increased in the transgenic animals by a factor of 70 in comparison with their nontransgenic littermates. The content of brain putrescine, the product of ODC, was greater than 60 mumol/g of tissue in the transgenic mice, whereas in the normal animals it was below the level that could be detected by an HPLC method. The concentrations of the higher polyamines (spermidine and spermine) were not significantly different from control values. 31P nuclear magnetic resonance (31P NMR) spectroscopy analyses revealed a significantly reduced (40%) free Mg2+ concentration as calculated from the chemical shift differences of the nucleoside triphosphate alpha and beta peaks in the brains of the transgenic animals. The lower free Mg2+ concentration in the brains of ODC transgenic mice was not a consequence of altered intracellular pH or changes in cellular high-energy metabolites. 1H NMR showed no differences in brain choline/N-acetylaspartate and total creatine/N-acetylaspartate ratios between the two animal groups. These ODC transgenic animals may serve as models in vivo for studies on cerebral postischemic events and on epilepsy, as polyamines are supposed to be involved in these processes.

[31P]- and [1H]nuclear magnetic resonances recorded in an interleaved fashion were used in order to quantify high-energy phosphates, intracellular pH and lactate in cortical brain slices of the guinea-pig superfused in a CO2/HCO3(-)-buffered medium during and after anoxic insults. The volume-averaged intracellular pH and energy status of the preparation following anoxia were determined. In the presence of external Na+, intracellular pH normalized in 3 min and was significantly more alkaline from 10 to 12 min of recovery, but lactate remained elevated for 12 min of reoxygenation following anoxia. The amount of lactate removed was only 40% of the quantity of acid extruded showing operation of H+ neutralizing transmembrane mechanisms other than transport of lactic acid. Amiloride (1 or 2 mM) did not prevent the recovery of intracellular pH, but it blocked the "overshoot" of the alkalinization at 10-12 min of recovery. In a medium containing 70 mM K+, 60 mM Na+ and 0.1 mM Ca2+, the recovery of pH, but not lactate washout, was significantly delayed. Removal of external Na+ caused severe energetic failure, decreases both in oxygen uptake and in N-acetyl aspartate concentration, indicating loss of viable tissue. In Na(+)-free superfusion, lactic acidosis caused a more severe drop in intracellular pH than in the presence of Na+. Complexing of extracellular Ca2+ in the Na(+)-free medium inhibited the acidification by 0.38 pH units during anoxia which is as much as the acidification caused by lactate accumulation in the absence of Na+. In Na(+)-free medium intracellular pH recovered, however, from an anoxic level to a normoxic value in 6 min. Metabolic damage of the slice preparation induced by anoxia in the absence of Na+ was as profound in the presence as in the absence of Ca2+ showing that accumulation of Ca2+ is not the only reason for the damage. It is concluded that recovery of intracellular pH from lactic-acidosis can occur independently of energetic recovery and involves acid extrusion mechanism(s) that is(are) dependent on external Na+ and sensitive to high K+.

31P and 1H nuclear magnetic resonance spectroscopy (NMR) was used to study the metabolism of intact superfused cortical brain slices during normoxia and anoxia. Attention was focused on quantification of 1H NMR-detected glutamate by a water-suppressed spin-echo method, using N-acetyl aspartate as an internal concentration reference. To quantify the 1H NMR signals, the spin-spin relaxation times and saturation effects were estimated for given metabolites. In addition, absolute concentrations of metabolites were determined by biochemical methods from acid extracts of the preparations after NMR experiments. Under aerobic conditions, 1H NMR detected 79% of the glutamate determined biochemically from the brain slice extracts. During anoxia in the absence of glucose when a severe energetic failure was evident, both 1H NMR and biochemical assays gave closely matching levels for glutamate. We conclude that in the brain cortex 21% of glutamate is located in an intracellular compartment in which this amino acid does not contribute to the 1H NMR signal. However, during severe anoxia an intracellular reorganisation occurs increasing the detectability of this amino acid neurotransmitter by NMR.

The effects of antifungal heptaene antibiotics candicidin and amphotericin B were investigated in isolated cerebral cortical nerve terminals (synaptosomes). The synaptosomes were incubated with candicidin or amphotericin B in the presence or absence of external Ca2+. Candicidin (0.4-0.8 I.U./mL) increased intrasynaptosomal free Ca2+ significantly. This increase was not significantly suppressed by 30 microM verapamil or 2 microM nifedipine. In the absence of extrasynaptosomal Ca2+ intrasynaptosomal free Ca2+ was not changed by candicidin. Amphotericin B increased intrasynaptosomal free Ca2+ as well. Candicidin (0.05-0.6 I.U./mL) increased the respiration rate up to 3.5-fold above the basal rate. This response was not affected by the absence of extracellular Ca2+. Ouabain completely blocked the increase of respiration caused by candicidin, whereas tetrodotoxin was ineffective. The plasma membrane depolarized in a dose-dependent manner after candicidin (0.2-0.8 I.U./mL). The mitochondrial membrane potential was little affected and only at the highest concentrations. The results indicate that heptaene polyenes increase synaptosomal ionic permeability, which is reflected in increased Ca2(+)-influx and accelerated respiration. The increment in synaptosomal free calcium takes place probably as a nonspecific leak via typical polyene-cholesterol channels. The respiration is accelerated by increased Na(+)-permeability through the plasma membrane which stimulates the function of Na+, K(+)-ATPase and thus increases the energy demand.

The distribution of glutamate and aspartate and the mitochondrial membrane potential (delta psi) were studied in isolated rat heart mitochondria and in the intact perfused rat heart. The diffusion potential imposed by the glutamate-aspartate exchange through mediation of the electrogenic glutamate-aspartate translocator attained a value close to the mitochondrial delta psi measured from the distribution of triphenylmethylphosphonium ion (TPMP+) both in isolated mitochondria and in intact myocardium. Distributions of the delta psi probe and metabolites were determined by subcellular fractionation of the heart muscle in a non-aqueous medium. The results indicate that the glutamate-aspartate translocator is in near equilibrium in the myocardium. The diffusion potential of the glutamate-aspartate exchange, and the mitochondrial/cytosolic difference in the redox potentials of the free NAD+/NADH pools are equal allowing for experimental error. These data obtained from intact tissue can therefore be interpreted as supporting the notion of the transmembrane uphill transport of reducing equivalent from the cytosolic free NAD+/NADH pool being driven by the malate-aspartate cycle energized by the mitochondrial delta psi.

Metabolically competent isolated cerebral cortical nerve terminals were used to determine the effects of triethyllead (TEL) and triethyltin (TET) on cytosolic free calcium ([Ca2+]c), on plasma and mitochondrial membrane potentials, and on oxidative metabolism. In the presence of physiological concentrations of extracellular ions, 20 microM TEL and 20 microM TET increase [Ca2+]c from 185 nM to 390 and 340 nM, respectively. A simultaneous depolarization of plasma membrane potential (delta psi p) by only 3-4 mV occurs, a drop which is insufficient to open the voltage-sensitive Ca2+ channels. In contrast, an instant and substantial depolarization of mitochondrial membrane potential (delta psi m) upon addition of TEL and TET is evident, as monitored with safranine O fluorescence. At the same concentration, TEL and TET stimulate basal respiration of synaptosomes by 45%, induce oxidation of endogenous NAD(P)H, and reduce the terminal ATP/ADP ratio by 45%. Thus, TEL and TET inhibit ATP production of intrasynaptosomal mitochondria by a mechanism consistent with uncoupling of oxidative phosphorylation. This bioenergetic effect by TEL and TET can be prevented by omitting external chloride, and a concomitant reduction of the increase in [Ca2+]c by about 60% is observed. Uncoupling of mitochondrial ATP synthesis from oxidation by TEL and TET, [corrected] a process that is dependent on external chloride, is the main mechanism by which they [corrected] increase [Ca2+]c.

Synaptosomes from guinea-pig cerebral cortex reveal two distinct Na+ permeabilities when divalent cations are removed from the incubation. In the presence of Mg2+, Ca2+ chelation by EGTA causes a partial activation of a voltage-dependent tetrodotoxin-sensitive pathway, manifested as a ouabain-sensitive respiratory increase, a partial depolarization of the plasma membrane, and a lowered gradient of gamma-amino[14C]butyrate. In addition there is a hyperpolarization of the mitochondrial membrane potential. When Mg2+ is omitted from the incubation, Ca2+ chelation induces a substantially larger permeability which is only partially sensitive to tetrodotoxin. The tetrodotoxin-insensitive component is not associated with a non-specific permeabilization of the plasma membrane and may be reversed by either Mg2+ or Ca2+.

The bioenergetic interaction between glycolysis and oxidative phosphorylation in isolated nerve terminals (synaptosomes) from guinea-pig cerebral cortex is characterized. Essentially all synaptosomes contain functioning mitochondria. There is a tight coupling between glycolytic rate and respiration: uncoupler causes a tenfold increase in glycolysis and a sixfold increase in respiration. Synaptosomes contain little endogenous glycolytic substrate and glycolysis is dependent on external glucose. In glucose-free media, or following addition of iodoacetate, synaptosomes continue to respire and to maintain high ATP/ADP ratios. In contrast to glucose, the endogenous substrate can neither maintain high respiration in the presence of uncoupler nor generate ATP in the presence of cyanide. Pyruvate, but not succinate, is an excellent substrate for intact synaptosomes. The in-situ mitochondrial membrane potential (delta psi m) is highly dependent upon the availability of glycolytic or exogenous pyruvate; glucose deprivation causes a 20-mV depolarization, while added pyruvate causes a 6-mV hyperpolarization even in the presence of glucose. Inhibition of pyruvate dehydrogenase by arsenite or pyruvate transport by alpha-cyano-4-hydroxycinnamate has little effect on ATP/ADP ratios; however respiratory capacity is severely restricted. It is concluded that synaptosomes are valuable models for studying the control of mitochondrial substrate supply in situ.

Aminooxyacetate, an inhibitor of pyridoxal-dependent enzymes, is routinely used to inhibit gamma-aminobutyrate metabolism. The bioenergetic effects of the inhibitor on guinea-pig cerebral cortical synaptosomes are investigated. It prevents the reoxidation of cytosolic NADH by the mitochondria by inhibiting the malate-aspartate shuttle, causing a 26 mV negative shift in the cytosolic NAD+/NADH redox potential, an increase in the lactate/pyruvate ratio and an inhibition of the ability of the mitochondria to utilize glycolytic pyruvate. The 3-hydroxybutyrate/acetoacetate ratio decreased significantly, indicating oxidation of the mitochondrial NAD+/NADH couple. The results are consistent with a predominant role of the malate-aspartate shuttle in the reoxidation of cytosolic NADH in isolated nerve terminals. Aminooxyacetate limits respiratory capacity and lowers mitochondrial membrane potential and synaptosomal ATP/ADP ratios to an extent similar to glucose deprivation. Thus, the inhibitor induces a functional 'hypoglycaemia' in nerve terminals and should be used with caution.

Acute liver failure was induced in rats by a single intragastric dose of carbon tetrachloride. This causes hepatic centrilobular necrosis, as indicated by histological examinations, and produces a large increase in the activity of serum alanine aminotransferase. The plasma NH4+ level (mean +/- SEM) was 123 +/- 10 microM in the control group and 564 +/- 41 microM in animals with acute liver failure (each n = 5). 31P nuclear magnetic resonance (NMR) was used to monitor brain cortical high-energy phosphate compounds, Pi, and intracellular pH. 1H NMR spectroscopy was utilised to detect additional metabolites, including glutamate, glutamine, and lactate. The results show that the forebrain is capable of maintaining normal phosphorus energy metabolite ratios and intracellular pH despite the metabolic challenge by an elevated blood NH4+ level. There was a significant increase in the brain glutamine level and a concomitant decrease in the glutamate level during hyperammonaemia. The brain lactate level increased twofold in rats with acute liver failure. The results indicate that 1H NMR can be used to detect cerebral metabolic changes in this model of hyperammonaemia, and our observations are discussed in relation to compartmentation of NH4+ metabolism.

The metabolism of four short-chain odd-number-carbon fatty acids, pentanoate, pent-4-enoate, propionate and acrylate, was studied in isolated rat heart mitochondria incubated in [14C]bicarbonate buffer. Under these conditions pentanoate was metabolized with a concomitant accumulation of malate and incorporation of 14CO2 into non-volatile compounds. The metabolism of propionate to tricarboxylic acid-cycle intermediates required the addition of ATP and oligomycin. After addition of a small amount of rotenone to the incubation medium, pent-4-enoate was metabolized with an increase in malate from less than 3 nmol/mg of protein to 34.0 +/- 1.5 nmol/mg in 40 min, during which time the amount of 14CO2 fixed in acid-stable compounds increased from 1.56 +/- 0.30 to 41.1 +/- 2.6 nmol/mg of protein. Acrylate was not metabolized under any of the conditions tested. The results show that cardiac mitochondria must have an enzyme system that is capable of reducing the double bond of either pent-4-enoate or its metabolities. That the metabolism of pent-4-enoate occurs through a reductive step and energy-dependent carboxylation is evident from the requirement for NAD+ reduction by partial inhibition of the mitochondrial respiratory chain and the presence of ATP and CO2. The results do not enable us to say whether the compound reduced is pent-4-enoyl-CoA or acryloyl-CoA.

Neurotoxic mechanisms of methylmercury (Met-Hg) on presynaptic nerve terminals were studied using the synaptosomes from the cerebral cortex of guinea pig as a model. Cytosolic free calcium [Ca++)c was determined using intrasynaptically trapped fluorescence indicator, fura-2; the plasma membrane potential (delta Up) by measuring the diffusion potential of 86Rb+ and the mitochondrial membrane potential was monitored using the safranine method. Synaptosomal respiration, glycolysis and concentrations of ATP and ADP in the presence and absence of Met-Hg also were quantified. Met-Hg increased synaptosomal [Ca++]c by two distinctive mechanisms. Moderate elevation of [Ca++]c by 127 nM was observed at 30 microM Met-Hg, at which concentration synaptosomal respiration was inhibited completely, leading to partial depolarization of mitochondria. A 3-fold activation of anaerobic glycolysis upon inhibition of respiration was insufficient to sustain terminal energy levels. The delta Up did not depolarize significantly from the resting potential of--67 mV. Thus, the rise in [Ca++]c was due to the energy failure of the synaptosomes, which has been caused by Met-Hg. With 100 microM Met-Hg, [Ca++]c increased extensively by 882 nM. Upon addition of 100 microM Met-Hg the delta Up depolarized instantly dropping 36 mV within 1 min. Synaptosomes were severely energy-deprived, because anaerobic glycolysis was inhibited by 90% from the aerobic level and mitochondrial membrane potential dropped below the limit that could be detected by the safranine method. The proportion of fura-2 signal quenching by Mn++ also increased, indicating that the plasma membrane had become leaky. Thus, at high concentrations of Met-Hg, the rise in [Ca++]c was ascribed to increased ionic permeability of the plasma membrane. The contribution of presynaptic energy failure by Met-Hg is discussed as a possible biochemical mechanism underlying the neurotoxicity of organic mercury.

The membrane potential (delta psi) and delta pH of the inner mitochondrial membrane were studied in isolated perfused rat hearts using exogenous labelled probes and tissue fractionation in non-aqueous media. The mitochondrial delta psi, measured by means of the subcellular distribution of [3H]triphenylmethylphosphonium (TPMP+), was 125 +/- 7 mV (negative inside) in hearts beating at 5 Hz and 150 +/- 3 mV (negative inside) in hearts beating at 1.5 Hz. The mitochondrial membrane delta pH, measured by means of the subcellular distribution of low concentrations of [1-14C]propionate, was 0.63 +/- 0.06 pH units (alkaline inside) in hearts beating at 5 Hz and 0.53 +/- 0.12 pH units (alkaline inside) in hearts beating at 1.5 Hz. The implication of proton and electron gradients in the regulation of cellular respiration is discussed. In combination with previous evidence on adenylate distribution in the isolated perfused rat heart, the results indicate that the mitochondrial electrogenic adenylate translocator is in near equilibrium with delta psi.

Subcellular fractionation of tissue in nonaqueous media was employed to study metabolite compartmentation in isolated perfused rat hearts. The mitochondrial and cytosolic concentrations of citrate and 2-oxoglutarate, total concentrations of the glycolytic intermediates and rate of glycolysis were measured in connection with changes in the rate of cellular respiration upon modulation of the ATP consumption by changes of the mechanical work load of the heart. The concentrations of citrate and 2-oxoglutarate in the mitochondria were 16- and 14-fold, respectively, greater than those in the cytosol of beating hearts. The cytosolic citrate concentration was low compared with concentrations which have been employed in demonstrations of the citrate inhibition of glycolysis. In spite of the low activities reported for the tricarboxylate carrier in heart mitochondria, the cytosolic citrate concentration reacted to perturbations of the mitochondrial citrate concentration, and inhibition of glycolysis at the phosphofructokinase step could be observed concomitantly with an increase in the cytosolic citrate concentration. The delta pH across the inner mitochondrial membrane calculated from the 2-oxoglutarate concentration gradient and the mitochondrial membrane potential calculated from the adenylate distribution gave an electrochemical potential difference of protons compatible with chemiosmotic coupling in the intact myocardium.

1H NMR spectroscopy of human brain in vivo can be used to detect a number of cerebral metabolites including N-acetylaspartate, creatine + phosphocreatine and choline-containing compounds. We have used 1H NMR spectroscopy to analyse these signals in (i) biopsy material from both normal human brain and astrocytomas, and (ii) primary astrocyte cultures. On the basis of this analysis, we conclude that in vivo 1H NMR spectroscopy could play an important clinical role in the non-invasive assessment of neuronal degeneration and proliferation of non-neuronal cells.

Hypoglycaemia and anoxia both cause massive release of glutamate from the brain in vivo, and the nature of this release was investigated using guinea-pig cerebral-cortical synaptosomes and iodoacetate and rotenone to simulate the energetic consequences of these conditions. Glutamate release (by continuous fluorimetry), cytoplasmic free Ca2+ (by fura-2), membrane potentials, ATP, ADP and creatine phosphate were determined in parallel, following the addition of iodoacetate or rotenone, alone or in combination. Ca2+-dependent glutamate release had a high energy requirement which could only be satisfied by aerobic glycolysis. Respiration using endogenous substrates, or anaerobic glycolysis following rotenone, caused a progressive inhibition of Ca2+-dependent release, correlating with a decline in the total ATP/ADP ratio and creatine phosphate. With rotenone, an increase in Ca2+-independent glutamate release was observed, correlating with a decline in plasma membrane potential. Only a slight increase in free Ca2+ was seen. Rotenone plus iodoacetate caused an almost immediate collapse of ATP/ADP ratio and a parallel loss of Ca2+-dependent glutamate release before free Ca2+ had risen to a level sufficient for exocytosis. In contrast, Ca2+-independent glutamate release increased. The Ca2+-dependent release of L-glutamate had the characteristics of an exocytotic transmitter release mechanism, being energy-dependent and triggered by elevated cytoplasmic free Ca2+ concentration. A distinct Ca2+-independent release of cytoplasmic glutamate occurred by reversal of the Na+-coupled uptake carrier, which was accelerated by a decline in the Na+ gradient. It is concluded that the Ca2+-independent release of cytoplasmic glutamate may make the major contribution to the excitotoxic release of glutamate in hypoglycaemic and anoxic conditions.

The release of radioactive histidine was studied in a superfusion system by using preloaded rat cerebral cortical slices and guinea pig synaptosomes. ATP/ADP ratios in both preparations were used to assess the energy levels and thus the oxygenation of brain slices and synaptosomes. ATP/ADP ratios similar to the intact brain and nerve terminals were found in slices and synaptosomes even after hours of superfusion with oxygenated buffer. Depolarization by 30 or 50 mM K+ induced release of histidine from slices and synaptosomes. The results confirm that under physiological energetic conditions, histidine is released both from slices and synaptosomes after depolarization with potassium.

Porphobilinogen deaminase, the third enzyme of the haem biosynthetic pathway, is encoded by two distinct mRNA species expressed in a tissue-specific manner from a single gene. These two mRNAs are transcribed from two promoters and only differ in their first exon. An inherited deficiency or porphobilinogen deaminase in man is responsible for the autosomal dominant disease acute intermittent porphyria. Different classes of mutations have been described at the protein level suggesting that this is a heterogeneous disease. In the present report, we describe the molecular abnormality responsible for a variant form of acute intermittent porphyria where the enzyme defect is restricted to non-erythroid cells. Upon cloning and sequencing the mutant allele of a patient from a large Finnish kindred, a single-base substitution within the 5'-splice donor sequence of intron 1 was found at the last position of exon 1 (CG----CT). The identification of this mutation allowed us to detect asymptomatic gene carriers among family members using in vitro amplification of DNA and hybridization of the target sequence to allele-specific oligonucleotides.

The significance of Ca2+ is assessed for the activation of pyruvate by intact nerve terminals (synaptosomes). Titration of glucose-depleted synaptosomes with pyruvate in the presence of either veratridine or uncoupler stimulates respiration in a Ca2+-independent manner. Additionally, the ability of exogenous pyruvate to support the mitochondrial membrane potential in situ is independent of Ca2+. It is concluded that Ca2+ does not regulate pyruvate oxidation in intact synaptosomes.

Effects of ruthenium red (RuR) on adenylates, plasma membrane potential (delta psi p) and cytosolic free calcium concentration ([Ca2+]c) in cortical synaptosomes from guinea-pig were investigated. Ten micromoles of RuR did not affect either energy levels as indicated by ATP/ADP ratio or the basal delta psi p. The resting [Ca2+]c in the presence of RuR was unchanged, but above 5 microM it inhibited by more than 50% of the voltage-activated increase in [Ca2+]c by K+-depolarization. In another experiment the potencies of 10 microM RuR and 100 microM verapamil to inhibit high K+-induced increase in [Ca2+]c were compared. It was found that either produced 59% inhibition and this inhibition was not potentiated by the substances together (65% inhibition). The extent of depolarisation of delta psi p by high external K+ was independent of the presence of RuR. RuR blocked only 20% of the increase in [Ca2+]c by veratridine treatment, indicating that Ca2+ accumulation into synaptosomal cytoplasm by veratridine involves some additional mechanisms other than depolarisation of delta psi p. The mechanism of inhibition of evoked release of neurotransmitters by RuR is discussed.

The stoichiometries of glycolysis and pyruvate oxidation were determined in cortical synaptosomes under varying rates of ATP consumption. Glycolysis was measured by using D-3-[3H]glucose as a marker and pyruvate oxidation by using D-3,4-[14C]glucose, which has to be metabolized to 1-[14C]pyruvate before being decarboxylated by the pyruvate dehydrogenase complex of intrasynaptosomal mitochondria. Cytosolic free Ca2+ concentration [( Ca2+]c) was determined in parallel and was manipulated by using EGTA in the incubation. The results show that in nonstimulated synaptosomes glycolysis and pyruvate oxidation are tightly coupled and stoichiometric. In the absence of Ca2+, when [Ca2+]c drops from 260 nM to 40 nM, glucose utilization increases, following the increase in energy demand, which has been shown to be due to elevated Na+ cycling. KCl depolarization, veratridine, and a mitochondrial uncoupler, carbonyl cyanide m-chlorophenylhydrazone, all stimulate glycolysis and pyruvate oxidation stoichiometrically, independently of the presence of external Ca2+. A rise in [Ca2+]c, therefore, is not required to regulate mitochondrial pyruvate metabolism. It is concluded that synaptosomes exhibit a high degree of respiratory control, that they rely on glucose oxidation for their energetics, and that stimulation of energy production can be achieved independently of changes in [Ca2+]c.

[31P]- and [1H]Nuclear magnetic resonance spectroscopy were used to study metabolism in cortical brain slices in the guinea-pig during acute exposure to pathophysiological concentrations of ammonia. Intracellular acidification, measured from the chemical shift of endogenous inorganic phosphate, was observed without any change in cellular energy status or concentrations of lactate, glutamate and glutamine. The initial acidification, which developed over a period of 9 min appeared to be heterogeneous, on the basis of a splitting of the inorganic phosphate resonance in a number of experiments, corresponding to pH changes of 0.07 and 0.27 pH units. Subsequently a homogeneous acidification, of 0.15 pH units, developed by 23 min following exposure to ammonia. Intracellular pH recovered within 6 min after discontinuation of the ammonia load. In the absence of external bicarbonate, intracellular pH was 0.12 units more acidic than in the bicarbonate buffer and ammonia caused a further acidification by 0.16 units. When glutamine synthase inhibitor, methionine sulphoximine, was added, there was a slow fall in intracellular pH. Under these conditions, subsequent addition of ammonia failed to cause acidification directly. Thus acute elevation of ammonia does not lead to a change in cerebral high-energy phosphate or lactate metabolism, but may be associated with a fall in cortical intracellular pH.

Primary cultures of astrocytes were used to investigate the effects of glucose deprivation on plasma membrane potential, on the respiration and on the energy status of these cells. Plasma membrane potential, as monitored with a cyanine dye, 3,3'-diethylthiadicarbocyanine, hyperpolarized by about 100% when glucose was added to substrate-deprived cells. The effect of glucose was prevented by iodoacetate or ouabain. In the absence of glucose, cellular adenosine triphosphate/adenosine diphosphate ratio was extensively reduced and pyruvate was unable either to restore energy status or to hyperpolarize the plasma membrane of astrocytes, although it was the preferential substrate for mitochondria within the cells. Glucose deprivation and inhibition of glycolysis or respiration in the presence of glucose caused dramatic decrease in transmembrane potassium ion and L-glutamate gradients. The gradients were not restored in the presence of pyruvate. Thus, aerobic glycolysis, rather than oxidation of pyruvate, is required to maintain maximal plasma membrane potential, adenosine triphosphate/adenosine diphosphate ratios as well as K+ and L-glutamate gradients. This evidence, together with the unresponsiveness of astrocyte respiration to ouabain, indicates a functional dissociation between energy dissipation at the plasma membrane and mitochondrial synthesis of adenosine triphosphate. The results are discussed with regard to the vulnerability of glia at low levels of blood glucose and the contribution of glial dysfunction to development of hypoglycaemic encephalopathy.

Synaptosomal glycolysis is stimulated eight- to 10-fold when the respiratory chain is inhibited by cyanide or by anoxia. However, the stimulation is transient and after 15 min declines toward the preanoxic rate. The decline is not seen when Ca2+ is absent or when the respiratory chain is inhibited by rotenone. The decline in glycolysis is reversible, is not due to substrate exhaustion, and is the cause, rather than the effect, of lowered synaptosomal ATP/ADP ratios. The failure to maintain glycolysis when the terminal oxidase of the respiratory chain is inhibited may have relevance to the sensitivity of the brain to anoxic damage.