In vitro and ex vivo 13C-NMR spectroscopy studies of pyruvate recycling in brain

Pyruvate recycling is a well established pathway in the liver, but in the brain, the cellular localization of pyruvate recycling remains controversial and its physiological significance is unknown. In cultured cortical astrocytes, pyruvate formed from [U-13C]glutamate was shown to re-enter the TCA cycle after conversion to acetyl-CoA, as demonstrated by the labelling patterns in aspartate C-2 and C-3, lactate C-2, and glutamate C-4, which provides evidence for pyruvate recycling in astrocytes. This finding is in agreement with previous studies of astrocytic cultures, in which pyruvate recycling has been described from [U-13C]glutamine, in the presence of glutamate, and from [U-13C]aspartate. Pyruvate recycling in brain was studied in fasted rats receiving either an intraperitoneal or a subcutaneous injection of [1,2-13C]acetate followed by decapitation 30 min later. Extracts of cortical tissue were analysed with 13C-NMR spectroscopy and total amounts of amino acids quantified by HPLC. Plasma extracts were analysed with 1H- and 13C-NMR spectroscopy, and showed a significantly larger amount of [1, 2-13C]acetate in the intraperitoneal group compared to the subcutaneous group. Furthermore, a small amount of label was detected in glucose in both groups. In the subcutaneously injected rats, [4-13C]glutamate and [2-13C]GABA were less enriched than plasma glucose, which might have been the precursor. In the intraperitoneally injected rats, however, pyruvate formation from [1, 2-13C]acetate, and re-entry of this pyruvate into the TCA cycle was demonstrated by the presence of greater 13C enrichment in [4-13C]glutamate and [4-13C]glutamine compared to the subcutaneous group, probably resulting from the significantly higher [1, 2-13C]acetate concentration in brain and plasma.     

Biochemical effects of gadolinium chloride in rats liver and kidney studied by ^1H NMR metabolomics

The biochemical effects of gadolinium chloride were studied using high-resolution IH nuclear magnetic resonance (NMR) spec-troscopy to investigate the biochemical composition of tissue (liver and kidney) aqueous extracts obtained from control and gadolinium chlo-ride (GdCl3) (10 and 50 mg/kg body weight, intraperitoneal injection, i.p.) treated rats. Tissue samples were collected at 48, 96 and 168 h p.d. after exposure to GdCl3, and extracted using methanol/chloroform solvent system. 1H NMR spectra of tissue extracts were analyzed by pat-tern recognition using principal components analysis. The liver damages caused by GdCl3 were characterized by increased succinate and de-creased glycogen level and elevated lactate, alanine and betaine concentration in liver. Furthermore, the increase of creatine and lactate, and decrease of glutamate, alanine, phosphocholine, glycophosphocholine (GPC), betaine, myo-inositoi and trimethylamine N-oxide (TMAO)levels in kidney illustrated kidney disturbance induced by GdCl3.     

Conservation surgery for supraglottic carcinoma. Oncological and functional results

Listeria innocua NCTC 11289 was grown aerobically in continuous culture in defined media at 30 degrees C. Both acetate and lactate were produced, the proportion of acetate decreased with increasing dilution rate. Enzymatic analysis showed lactate dehydrogenase was activated 10-fold by fructose-1, 6-bisphosphate. The presence of phosphate acetyltransferase and acetate kinase but not pyruvate oxidase was detected, suggesting the sequential action of phosphate acetyltransferase and acetate kinase to produce acetate from acetyl CoA via acetylphosphate.     

Cultural factors influencing the utilization or production of acetate by Butyrivibrio fibrisolvens

Utilization of acetate by four strains of Butyrivibrio fibrisolvens was influenced by the composition of their growth medium. Growth-limiting glucose concentrations, low availability of CO2 and the presence of sodium lactate all stimulated acetate uptake by three strains. The type strain, D1, utilized acetate if the concentration of acetate added to the medium was at least 15 mumol ml-1. In batch culture, all strains produced acetate before entering a phase of acetate uptake. Continuous-culture studies with one strain showed that acetate uptake was dependent upon growth rate. The amount of n-butyrate produced in batch or continuous culture was closely linked to the amount of acetate taken up.     

Microencapsulation of human insulin DEAE-dextran complex and the complex in liposomes by the emulsion non-solvent addition method

The blood levels of ethanol, acetaldehyde, acetate, methanol, acetone, lactate, pyruvate, and glucose were measured in 23 male alcohol-dependent patients on days 2 to 6 after hospitalization and in 22 healthy male blood donors. Correlations between the biochemical parameters and 17 symptoms of the alcohol-withdrawal syndrome (AWS) were calculated. Abnormally high levels of ethanol, methanol, acetate, and acetone as well as hypoglycaemia were found on day 2, but lactacidaemia and pyruvataemia were pronounced throughout the observation period. AWS severity correlated positively with the acetone content on day 2 and with the acetate content on days 2 to 6. Negative correlations were found between ethanol levels and craving for alcohol, methanol levels and craving for alcohol, and between psychopathologic disorders and the total AWS severity score. The results suggest that concentrations of blood ethanol, methanol, acetate, and acetone exceeding their normal endogenous levels can be used only as indicators of recent heavy drinking. Linear discriminant analysis using the levels of the nine parameters studied enabled the correct classification of 91% to 96% of alcoholic patients during 1 week of abstinence and 100% of control subjects. The most informative parameters in the discrimination between alcoholics and controls were lactate, pyruvate, the lactate/pyruvate ratio, acetate, and acetone.     

Isoenzyme spectra of lactate dehydrogenase in tissues of pigs at different stages of embryonic development

Dynamics of the activity and correlation of isoenzyme spectra of lactate dehydrogenase in extracts of the liver, hip muscle and blood serum of the 30-60- and 108-day pig embryos was studied by polyacrilamide gel diskelectrophoresis. A high specificity of isoenzymes sets in all the tissues under study is established.     

Net flux of glucose, lactate, volatile fatty acids, and nitrogen metabolites across the portal-drained viscera and liver of pregnant ewes

Our objective for this study was to determine the pattern of nutrient flux across the portal-drained viscera (PDV) and liver in ewes with varying numbers of fetuses. Catheters were placed in the hepatic portal vein, a branch of the hepatic vein, a mesenteric vein, and the abdominal aorta of ewes. Blood flow and net nutrient release across the PDV and liver were determined before exposure to rams. Ewes were then mated, which resulted in two ewes not pregnant and in six ewes with single and 11 ewes with twin lambs. Additional measurements were taken 103, 82, 61, 39, 19, and 6 d before parturition. Net PDV glucose release did not differ from zero (-.4 +/- 8.4 mmol/h; P = .58). In ewes with singles, premating net hepatic glucose release was 34.4 +/- 2.4 mmol/h, and 19 d before parturition it was 46.2 +/- 3.8 mmol/h. In ewes with twins, premating net hepatic glucose release was 36.8 +/- 2.7 mmol/h, and 19 d before parturition it was 47.4 +/- 2.8 mmol/h. Net PDV lactate release did not differ with litter size (P = .58) or days from parturition (P = .14; 9.7 +/- 4.6 mmol/h). Net lactate uptake by the liver increased in pregnant ewes as the pregnancy progressed (P < .001). The hepatic extraction ratio for lactate increased in late pregnancy (P = .02). Net PDV and hepatic release of acetate and propionate were not different with litter size or days from parturition. Hepatic extraction ratios of VFA did not differ with litter size or day from parturition. The patterns of change in hepatic metabolite fluxes are similar to the patterns of change in gravid uterus metabolite uptake. Hepatic lactate uptake seems to be regulated during pregnancy.     

Antioxidant treatment during liver resection for alleviation of ischemia-reperfusion injury

BACKGROUND/AIMS: Many experimental studies on ischemia-reperfusion injury in animals suggest a preventive effect of antioxidants, but the clinical significance of these findings is still unclear. The aim of our study was to evaluate the effect of antioxidant treatment with vitamins on liver function parameters during liver resection. METHODOLOGY: Our prospective randomized study comprised 58 patients undergoing major liver surgery, including the Pringle maneuver. In the treatment group 32 patients received a multivitamin infusion (Omnibionta) which included 10 mg of alpha-tocopherol acetate, 2 mg of DL-alpha-tocopherol and 1 g of ascorbate. The control group consisted of 26 patients. Various parameters associated with liver function, such as transaminases, lactate, ammonia, bilirubin, cholinesterase and clotting parameters were measured preoperatively, at the beginning of liver ischemia, 15, 30 and 60 minutes after reperfusion onset and every 12 hours after the operation. RESULTS: The Mann-Whitney-Wilcoxon-Test showed statistically significant differences in the postischemic changes between the treatment group and the control group for the Quick test (prothrombin time): p = 0.01. The transaminases were also markedly better in the treatment group (splitting-up slightly more delayed than with the Quick test). A smaller effect was seen with cholinesterase. Lactate, however, increased intraoperatively with a strong correlation to the duration of ischemia and returned quickly to baseline values without any remarkable influence of the antioxidant treatment. CONCLUSION: In our study, antioxidant treatment with a multivitamin infusion showed a positive effect on postischemic liver function parameters.     

Effect of aeromonas salmonicida (furunculosis) infection on the incorporation of [1-14C]acetate into lipids of trout liver

[1-(14)C]acetate is incorporated into lipids more rapidly by liver from Aeromonas salmonicida-infected trout than by normal trout liver, both in vitro and in vivo. The increase was, in general, about twofold to less than threefold.     

Use of rat and human liver slices for the detection of steroid hormone-induced DNA-adducts in vitro by means of the (32)P-postlabeling technique

Precision cut liver slices from humans and rats were used to investigate the covalent binding of xenobiotics to the DNA by means of the (32)P-postlabeling technique. Human liver slices were incubated with the structurally related steroid hormones chlormadinone acetate (5 mu g/ml), cyproterone acetate (0.01-5 mu g/ml), megestrol acetate (5 mu g/ml), and the positive control 2-aminofluorene (0.01-5 mu g/ml), which is known for its marked ability to form DNA-adducts in vivo. Rat liver slices were incubated with cyproterone acetate in concentrations of 0.1, 1, and 5 mu g/ml. The functional viability and metabolic activity of the slices were shown to be sufficiently maintained during the incubation time by measurement of the intracellular K(+)-content and the metabolic turnover of the model substrate 7-ethoxycoumarin, respectively. All three test substances and the control induced DNA-adducts in human liver slices, however, with a different adduct pattern. While the total DNA-adduct levels obtained with cyproterone acetate and megestrol acetate were in the same order of magnitude (on average 1000 DNA-adducts/10(9) nucleotides after incubation with 5 mu g /ml), the relative adduct labeling calculated for chlormadinone acetate was about 400. Following in vitro incubation of rat liver slices with cyproterone acetate, the relative adduct labeling values increased proportionally with increasing concentrations and added linearily to in vivo generated DNA-adducts. At the level of liver slices, different DNA-adduct patterns were induced by cyproterone acetate in rat and man. In contrast to the finding of others, using rat hepatocytes, the relative adduct labeling values of cyproterone acetate and megestrol acetate were in the same order of magnitude after incubation with human liver slices. The present study indicates that liver slices are a useful tool to investigate the in vitro DNA-adduct inducing potential of xenobiotics.     

Intracellular redox state and control of gluconeogenesis in perfused chicken liver

The role of the cellular redox state in the control of gluconeogenesis was studied in hemoglobin-free perfused chicken liver, by fluorimetric measurement of the redox states of intracellular pyridine nucleotides. The aminotransferase inhibitor, aminooxyacetate, completely inhibited gluconeogenesis from lactate in the perfused rat liver and to a small extent in the perfused chicken liver. In chicken liver, the highest rate of glucose production was seen with lactate, followed by fructose, pyruvate, and glycerol. When compared at 5 mM, the rate of glucose production from pyruvate was only 10% of that from lactate. Glucose production from a pyruvate/lactate mixture decreased with increasing proportions of pyruvate, together with redox changes of pyridine nucleotides to a more oxidized state. Increased reduction of pyridine nucleotides upon infusion of ethanol was associated with an increased glucose production from pyruvate, and the increase was abolished during octanoate infusion. This abolishment was accompanied by an increase in the acetoacetate to beta-hydroxybutyrate ratio with an oxidation of pyridine nucleotides. The octanoate-inhibited gluconeogenesis occurred at the higher lactate concentration (10 mM) with a transient oxidation of pyridine nucleotides. No significant inhibition was observed at 1 mM lactate, although an instant reduction of pyridine nucleotides was taking place. The rate of beta-hydroxybutyrate generation during octanoate infusion was 2.2 times higher at 1 mM than at 10 mM lactate. The inhibitory effect of octanoate on glyconeogenesis was completely relieved by the addition of NH4Cl. The results demonstrate that the regeneration of NADH in the cytosol is limited in chicken liver, and that gluconeogenesis is regulated, in part, by alteration in the redox states of mitochondria and cytosol.     

Bilateral pulmonary hilar lymphadenopathy. An unusual manifestation of metastatic renal cell carcinoma

The significance of glucagon for the alterations in carbohydrate and fat metabolism during swimming has been evaluated. Fed, male rats were used. Blood was drawn by cardiac puncture for glucose analysis and either rabbit-antiglucagonserum (A-rats) or normal rabbitserum (N-rats) injected. Twenty-nine rats were then forced to swim (S-rats) with a tail weight for 60 min, while 16 rats were resting controls (C-rats). Subsequently blood was drawn and samples of liver and muscle tissue collected. In SN-rats glucagon concentrations increased from 152 +/- 18 (S.E.) pg/ml (CN-rats) to 332 +/- 61 (P less than 0.05), while liver glycogen decreased (P less than 0.001) and blood glucose increased (P less than 0.05). In SA-rats, however, the changes in liver glycogen and blood glucose were halved indicating that increased glucagon secretion enhances hepatic glycogen depletion during prolonged exercise. NEFA rose in SA-rats (P less than 0.005) as well as in SN-rats (P less than 0.05). Glycerol concentrations, however, only increased in SA-rats (P less than 0.05) indicating a shift towards lipid combustion in antibody treated rats. Muscle glycogen and plasma insulin diminished and blood lactate increased uniformly in exercised rats.     

On the mechanism of hypocholesterolemic action of amphotericin B in rat

The transient hypocholesterolemia phenomenon observed in rats, injected i.v. with amphotericin B (1.5 mg/kg/day) for three weeks, was investigated by in vitro [14C] acetate incorporation into the liver cholesterol. The incorporation of acetate in liver slices of treated animals increased more than two-fold (p less than 0.02) when compared with controls.When amphotericin B was added in vitro to liver slices of control rats, the incorporation of acetate into cholesterol decreased to about 65% of that observed with the corresponding controls (p less than 0.01). The results are consistent with the hypothesis that initially amphotericin B causes inhibition of de novo cholesterol synthesis resulting in hypocholesterolemia which disappears on continued treatment possibly due to compensatory increased rate of cholesterol biosynthesis.     

Hyperpolarization of the liver cell membrane by palmitate as affected by glucose and lactate: implications for control of feeding

Since the membrane potential of liver cells being in contact with vagal afferents has been proposed to represent a major signal in metabolic control of food intake, we investigated the effect of palmitate, glucose and lactate on the membrane potential of hepatocytes with microelectrodes using superfused mouse liver slices. The mice used for the experiments were fed a fat-enriched diet (18% fat). Palmitate (0.5 mM) hyperpolarized the membrane of hepatocytes by 3-4 mV, and this hyperpolarization was not affected by 5-10 mM glucose and 0.5-1 mM lactate. Glucose alone did not influence the potential, even when mice fed a high carbohydrate diet were employed. At lactate concentrations > or = 2 mM the palmitate induced hyperpolarization was eliminated and 5 mM lactate or pyruvate alone hyperpolarized the liver cell membrane. Similar to the palmitate induced hyperpolarization, the lactate induced hyperpolarization was prevented by the K-channel blocker TEA, suggesting that activation of K channels is involved in the hyperpolarization. The results show that physiological concentrations of glucose and lactate do not affect the hyperpolarization of the liver cell membrane due to fatty acid oxidation. The implications of these findings with regard to control of food intake by fatty acid oxidation and lactate metabolism are discussed. The observations are consistent with a signal function of the hepatic membrane potential in physiological control of food intake by fatty acid oxidation. Hepatic lactate metabolism at supraphysiological lactate concentrations may also produce a satiety signal coded by the hepatic membrane potential.     

1H NMR study of metabolic alterations in the small intestine of rats infected with Hymenolepis diminuta

1H NMR spectra of the duodenum, jejunum and ileum tissues of the small intestine of a rat showed metabolic gradients. 2. The concentrations of metabolites in these gut regions were altered by the presence of the tapeworm Hymenolepis diminuta. 3. In the infected duodenum there was significantly less glycogen, glucose and phosphocreatine/creatine, but significantly more lactate than in the corresponding controls. 4. Infected jejunum contained significantly less betaine but significantly more succinate, alanine and lactate. 5. Infected ileum had significantly less glycogen and taurine but significantly more alanine and lactate.     

Further studies on the bioaffinity chromatography of NAD(+)-dependent dehydrogenases using the locking-on effect

Previous studies have capitalized on ordered kinetic mechanisms in the design of biospecific affinity chromatographic methods for highly efficient purifications and mechanistic studies of enzymes. The most direct tactic has been the use of immobilised analogues of the following, usually enzyme-specific substrates, e.g., lactate/pyruvate in the case of lactate dehydrogenase for which NAD+ is the leading substrate. Such immobilised specific substrates are, however, often difficult or impossible to synthesise. The locking-on strategy reverses the tactic by using the more accessible immobilised leading substrate, immobilised NAD+, as adsorbent with soluble analogues of the enzyme-specific ligands (e.g., lactate in the case of lactate dehydrogenase) providing a substantial reinforcement of biospecific adsorption sufficient to effect adsorptive selection of an enzyme from a group of enzymes such as the NAD(+)-specific enzymes. The value of this approach is demonstrated using model studies with lactate dehydrogenase (LDH, EC 1.1.1.27), alcohol dehydrogenase (ADH, EC 1.1.1.1), glutamate dehydrogenase (GDH, EC 1.4.1.3) and malate dehydrogenase (MDH, EC 1.1.1.37). Purification of bovine liver GDH in high yield from crude extracts is described using the tactic.     

不同钙源对MICP固化氰化尾渣的影响

为探究固化氰化尾渣最佳参数,实现最优试验方案,采用微生物诱导碳酸钙沉淀（MICP）技术,通过设计4种不同钙源,对比研究尿素与氯化钙、乙酸钙、硝酸钙和乳酸钙的4种胶结液,进行固化氰化尾渣,讨论了在不同钙源添加下MICP固化氰化尾渣的效果,分析了固化体无侧限抗压强度、重金属浸出和重金属形态、SEM和XRD表征等方法的可行性。物理力学指标表明,乙酸钙和氯化钙作为钙源时,固化氰化尾渣的效果优于硝酸钙和乳酸钙,结合SEM、FT-IR和XRD结果,确定乙酸钙为最优钙源。该研究可以为MICP技术在固化氰化尾渣工程应用提供科学依据和参考。     

Comparison of lactate and glucose metabolism in cultured neocortical neurons and astrocytes using 13C-NMR spectroscopy

In cerebral cortical neurons, synthesis of the tricarboxylic acid (TCA) cycle-derived amino acids, glutamate and aspartate as well as the neurotransmitter of these neurons, gamma-aminobutyrate (GABA), was studied incubating the cells in media containing 0.5 mM [U-13C]glucose in the absence or presence of glutamine (0.5 mM). Lyophilized cell extracts were analyzed by 13C nuclear magnetic resonance (NMR) spectroscopy and HPLC. The present findings were compared to results previously obtained using 1.0 mM [U-13C]lactate as the labeled substrate for the neurons. Regardless of the amino acids studied, incubation periods of 1 and 4 h resulted in identical amounts of 13C incorporated. Furthermore, the metabolism of lactate was studied under analogous conditions in cultured cerebral cortical astrocytes. The incorporation of 13C from lactate into glutamate was much lower in the astrocytes than in the neurons. In cerebral cortical neurons the total amount of 13C in GABA, glutamate and aspartate was independent of the labeled substrate. The enrichment in glutamate and aspartate was, however, higher in neurons incubated with lactate. Thus, lactate appears to be equivalent to glucose with regard to its access to the TCA cycle and subsequent labeling of glutamate, aspartate and GABA. It should be noted, however, that incubation with lactate in place of glucose led to lower cellular contents of glutamate and aspartate. The presence of glutamine affected the metabolism of glucose and lactate differently, suggesting that the metabolism of these substrates may be compartmentalized.     

Retinyl substituted-benzyl ethers. Inhibition of mammary carcinogenesis by retinyl 3,4,5-trimethoxybenzyl ether (RTMBE)

Recently, we reported that retinyl 2-propynyl ether (RPE) inhibits MNU-induced mammary cancer in rats and is less toxic than RME and retinyl acetate. The preparation and biological investigations of retinyl ethers have now been extended to retinyl substituted-benzyl ethers, some of which bind to cellular retinol-binding protein. In long-term (160-180 days) experiments, retinyl 3,4,5-trimethoxybenzyl ether (RTMBE) has been shown to be active against MNU-induced mammary cancer in Sprague-Dawley rats. In effectiveness, RTMBE is comparable, at least, to retinyl acetate; but, unlike retinyl acetate, RTMBE is comparatively non-toxic to rats and mice, is not converted enzymatically to retinol, and does not cause significant increases in retinyl palmitate concentrations in the liver. RTMBE reaches high concentrations in mammary tissue. Two of the four RTMBE congeners that were evaluated in 90 day studies were moderately effective in inhibiting mammary carcinogenesis.     

1H-nuclear magnetic resonance studies of human synovial fluid in arthritic disease states as an aid to confirming metabolic activity in the synovial cavity

1. A 1H-n.m.r. method was used to measure concentrations of valine, alanine, lactate, acetate, hyaluronan and lipids in synovial fluid obtained, during the normal course of examination from the knee joints of patients attending rheumatology and orthopaedic clinics. Fluid was available from 16 patients with osteoarthritis, 18 patients with rheumatoid arthritis, four patients with meniscal tear and one patient each with systemic lupus erythematosis, mono-arthritis, synovitis and loose bodies. Four normal specimens were obtained for comparison. 2. Valine, alanine and acetate levels all showed a normal Gaussian distribution, reflecting the distributions within the serum of the sample population. 3. Lactate concentrations divided into two distinct patterns. At concentrations below 2.5 mmol/l the lactate levels showed a Gaussian distribution, reflecting the distribution in normal serum. The normal synovial fluid specimens belong to this distribution. Above 2.5-3.0 mmol/l, lactate levels were asymmetric in distribution with a long tail at higher concentrations. These high levels of lactate can be explained by the generation of lactate through anaerobic metabolism within the synovial cavity. This metabolic process is triggered by a general inflammatory condition such as in rheumatoid arthritis. 4. The distribution of n.m.r.-observable lipid concentrations in rheumatoid arthritis and osteoarthritis each shows a normal distribution and the mean concentration is significantly higher in rheumatoid arthritis. 5. An increased n.m.r.-observable hyaluronan concentration is associated with an inflammatory situation. 6. It is concluded that raised levels of lactate and n.m.r.-observable hyaluronan and lipids are useful markers to aid the clinical distinction between rheumatoid arthritis and osteoarthritis.(ABSTRACT TRUNCATED AT 250 WORDS)