Quantitation of amino sugar stereoisomer and muramic acid biomarkers by hydrophilic interaction liquid chromatography-mass spectrometry.

A rapid separation and quantitation of the stereoisomer amino sugars glucosamine, galactosamine, and mannosamine, along with muramic acid, is needed. These compounds, when their quantities are accurate, can be used to understand the origin and fate of natural organic matter (NOM) in the environment. These target molecules are biomarkers of fungi and bacteria and allow the deconvolution of microbial transformations and degradation of NOM in a wide variety of environmental matrices. Analytical methods applied to this suite of biomarkers are needed to understand carbon and nitrogen biogeochemistry with a changing global climate. Traditional separations of these analytes by gas chromatography require sample derivatization, as does reverse phase liquid chromatography. In contrast, ion chromatography can separate the analytes directly, but requires a separate analytical method to quantify muramic acid. In this work we present a direct analysis of all these molecules using hydrophilic liquid interaction chromatography. Solvent composition, buffer strength, pH, flow rate, and column temperature were optimized. The method can separate these four compounds and the biopolymeric precursor molecule N-acetylglucosamine in a single run in under 8 min with equivalent resolution to the best previously reported separations that did not require derivatization prior to analysis. Detection of the analytes was performed by both tandem and time-of-flight mass spectrometry. The method was assessed for its quantitative capabilities through i) peak area assignment, ii) check standards with ratios of the target analytes likely to be present in real samples, iii) an injection internal standard, and iv) quantitative analysis of real soil hydrolysates by external calibration and standard addition approaches. Across their expected analytical ranges the response for each analyte was highly linear with good accuracy (<25%) and precision (<15%) over three orders of magnitude. Detection limits of 20 µg L-1 were found for galactosamine and 5 µg L-1 for the remainder of the analytes, comparable to the majority of other methods reported in the literature. Overall, this new approach can directly and rapidly quantify amino sugars recovered in environmental hydrolysates.

An in vitro evaluation of the ability of ozone to kill a strain of Enterococcus faecalis.

AIM: To evaluate the potential of ozone as an antibacterial agent using Enterococcus faecalis as the test species. METHODOLOGY: Ozone was produced by a custom-made bench top generator and its solubility in water determined by ultraviolet (258 nm) spectrophotometric analysis of solutions through which ozone was sparged for various time-periods. The antibacterial efficacy of ozone was tested against both broth and biofilm cultures. Ozone was sparged for 30, 60, 120 and 240 s, through overnight broth cultures of a strain of E. faecalis (E78.2) and compared with those that were centrifuged, washed and resuspended in water. Enterococcus faecalis (E78.2) biofilms were grown on cellulose nitrate membrane filters for 48 h and suspended in water through which ozone gas was sparged with stirring for 60, 120 and 240 s in a standard fashion. In a separate test, biofilms were also exposed to gaseous ozone. Sodium hypochlorite (NaOCl) was used as a positive control. All experiments were repeated four times. RESULTS: There were significant (P < 0.05) reductions of bacteria in the unwashed (2 log(10) reductions) and washed (5 log(10) reductions) broth cultures following 240 s applications. Biofilms incubated for 240 s with ozonated water showed no significant reduction in cell viability attributable to ozone alone, whereas with NaOCl no viable cells were detected over the same time. Gaseous ozone applied for 300 s had no effect on these biofilms. CONCLUSIONS: Ozone had an antibacterial effect on planktonic E. faecalis cells and those suspended in fluid, but little effect when embedded in biofilms. Its antibacterial efficacy was not comparable with that of NaOCl under the test conditions used.

VLDL output by hepatocytes from obese Zucker rats is resistant to the inhibitory effect of insulin.

The effects of insulin (0-780 nM) on the secretion of very-low-density lipoprotein (VLDL) apolipoprotein B (apoB) and triacylglycerol (TAG) in hepatocytes from obese Zucker rats and from their lean littermates were studied over a total period of 48 h in chemically defined culture medium. Cells from the obese Zucker rats initially secreted more TAG than those from the lean animals. Cells from the former were resistant to the inhibitory effect of insulin on the secretion of TAG. These changes were accompanied by an increased rate of TAG synthesis. Although the hepatocytes from the obese animals initially secreted less apoB than those from the lean, apoB output from the former could not be suppressed by insulin. Prolonging the length of the culture period resulted in the acquisition of sensitivity to the inhibitory effect of insulin in the hepatocytes from the obese rats. This occurred more rapidly for the secretion of TAG than of apoB. Under these conditions, the initial difference in the rate of TAG synthesis in the hepatocytes from the obese and from the lean animals was also abolished.

Decreased sensitivity to the inhibitory effect of insulin on the secretion of very-low-density lipoprotein in cultured hepatocytes from fructose-fed rats.

Hepatocytes were prepared from rats fed a chow diet (control-fed) and from rats fed a similar diet in which the drinking water contained 10% (wt/vol) fructose (fructose-fed). Both types of hepatocyte preparations were cultured for < or = 48 hours in supplemented Waymouth's medium containing increasing concentrations of bovine insulin (0 to 780 nmol/L). During the first 24 hours of culture, hepatocytes from fructose-fed rats secreted more very-low-density lipoprotein (VLDL) triacylglycerol (TAG) than hepatocytes from control-fed rats. This difference persisted at all concentrations of insulin. There was no difference in the rate of secretion of apolipoprotein B (apo B). In both control-fed and fructose-fed animals, the inhibitory effect of insulin on the secretion of VLDL was greater on the second versus the first day of culture. Under these conditions, hepatocytes from fructose-fed groups were less sensitive to insulin inhibition as compared with those from the control-fed group. This was evidenced by the following: (1) the decreased inhibitory effect of insulin on the secretion of both total and newly synthesized VLDL TAG, (2) the attenuated inhibitory effect of insulin on the secretion of VLDL apo B, (3) the decreased potency of insulin in suppressing the secretion of VLDL TAG in TAG-depleted hepatocytes from fructose-fed as compared with control-fed animals, and (4) the larger proportion of newly synthesized TAG secreted as VLDL in hepatocytes from fructose-fed rats as compared with controls. This difference was exacerbated at higher concentrations of insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of chronic uraemia on the formation of glucose and urea plus ammonia from L-alanine, L-glutamine and L-serine in isolated rat hepatocytes.

The effects of chronic uraemia on glucose production and nitrogen release (urea plus ammonia formation) from alanine, glutamine or serine in isolated rat hepatocytes were studied. Uraemia increased the rate of formation of urea plus ammonia from all three amino acids by 38-93% when they were present at a final concentration of 10 mmol/l. At lower concentrations (2 mmol/l) the rate of nitrogen release was not significantly increased. Hepatocytes from normal rats whose food intake had been restricted to the level of that of uraemic rats did not show the increased rates of nitrogen release. The increased rates of nitrogen release with hepatocytes from uraemic rats were not accompanied by increased rates of glucose synthesis. Instead, accumulation of metabolic intermediates occurred: lactate and pyruvate (alanine or serine as substrates) and glutamate (glutamine as substrate). Livers of uraemic rats had increased activities of glutaminase (30%) and serine dehydratase (100%). Hepatocytes from normal rats treated with phlorhizin to increase the plasma glucagon/insulin ratio behaved in a similar manner to hepatocytes from uraemic rats. They had increased serine dehydratase activity, and increased rates of utilization of serine or glutamine. The possible implications of these findings for human uraemia are discussed.

Mitochondrial compartmentation of metabolic CO2 resulting from its site of origin in relation to urea synthesis.

In isolated hepatocytes the entry into urea of metabolic 14CO2 derived from [14C] formate is modified by the addition of dichloroacetate and hydroxypyruvate. An explanation is that this results from changes in the cytoplasmic/mitochondrial pH gradient. 14CO2 derived from [1-14C] alanine enters into urea more readily than 14CO2 arising from [1-14C]glutamate. It is proposed that the difference, which is more than 4-fold, is indicative of a preferred pathway for metabolic CO2 in liver mitochondria from pyruvate dehydrogenase to carbamoylphosphate synthetase than form oxoglutarate dehydrogenase. Acetazolamide inhibition of carbonic anhydrase is without effect on this observed incorporation into urea.

The effect of dichloroacetate and hydroxypyruvate on the entry of 14C from [1-14C]alanine into urea in rat hepatocytes.

Entry of metabolic 14CO2 into urea is shown to be decreased by dichloroacetate although the production of 14CO2 is stimulated 2-fold. Hydroxypyruvate, a product of dichloroacetate metabolism, increases the incorporation of metabolic 14CO2 into urea. It is proposed that these effects result from changes in the cytoplasmic-mitochondrial pH gradient.

The effect of cysteine oxidation on isolated hepatocytes.

Isolated hepatocytes incubated with 4mM-cysteine lose reduced glutathione, adenine nucleotides and intracellular enzymes, thus showing extensive membrane damage. The toxic effects of cysteine are enhanced by NH4Cl. Lactate, ethanol and unsaturated fatty acids afford significant protection against cysteine-induced cytoxicity. Addition of catalase to the incubation medium also protected against cysteine toxicity, indicating that H2O2 formed during the oxidation of cysteine is involved in the toxic effects observed. Under anaerobic conditions cysteine did not cause leakage of lactate dehydrogenase from cells, confirming that rapid autoxidation is an essential condition for development of the toxic effects of cysteine.

Equilibration of metabolic CO2 with preformed CO2 and bicarbonate. An unexpected finding.

Entry of metabolic 14CO2 into urea is shown to occur more readily than it equilibrates with the general pool of cellular plus extracellular bicarbonate plus CO2. Since the sites of CO2 production (pyruvate dehydrogenase and oxoglutarate dehydrogenase) and of fixation (carbamoylphosphate synthetase) are intramitochondrial, it is likely that the fixation of CO2 is also more rapid than its equilibration with the cytoplasmic pool of bicarbonate plus CO2. This observation may point to a more general problem concerning the interpretation of isotope data, when compartmentation or proximity of sites of production and utilisation of metabolites may result in the isotope following a preferred pathway.

The production of free radicals during the autoxidation of cysteine and their effect on isolated rat hepatocytes.

Autoxidizing cysteine has been shown to produce thiyl and hydroxyl radicals. Hydrogen peroxide increased the yield of both radicals which was inhibited by catalase but stimulated by copper/zinc superoxide dismutase. This effect is due to increased hydrogen peroxide production by copper/zinc superoxide dismutase as a result of superoxide dismutation. The production of superoxide radicals could not be detected probably because of its low reactivity, however, measurement of oxygen uptake and reduction of ferricytochrome c by autoxidizing cysteine clearly implicate the involvement of super oxide radicals. The production of hydroxyl radicals is postulated to proceed through a fenton reaction, however, this may not necessarily be metal ion controlled. Autoxidizing cysteine disrupts the integrity of hepatocytes causing release of glutathione, adenosine triphosphate and lactate dehydrogenase indicating that it is of little use as a therapeutic agent.

The metabolic fate of lactate in renal cortical tubules.

1. Isolated kidney cortex tubules prepared from fed rats and incubated with near-physiological concentrations of [(14)C]lactate decrease the specific radioactivity of the added lactate. This effect may be attributable to at least two mechanisms; formation of lactate from endogenous precursors, or entry of unlabelled carbon into the lactate pool as a result of substrate cycling, via phosphoenolpyruvate, pyruvate and oxaloacetate, together with equilibration of the oxaloacetate pool with malate and fumarate. Such substrate cycling could occur within a single cell, or between two populations of different cells, one glycolytic and the other gluconeogenic. These possibilities have been investigated by using metabolic inhibitors or alternative metabolic substrates. 2. Tubules from fed rats produced a fall in specific radioactivity of 14.4% when incubated for 40min with 2mm-lactate alone. A mathematical treatment of this result is presented, which allows the rate of fall in specific radioactivity to be expressed as the addition of unlabelled lactate to the pool. This corresponds to a rate of formation of unlabelled lactate of 121+/-22mumol/h per g dry wt., a rate close to that of gluconeogenesis. In tubules from fasting rats, there was no reduction of the specific radioactivity of lactate, indicating that fasting for 24h suppresses production of unlabelled-lactate carbon. 3. Addition of 2mm-fumarate resulted in a significantly greater decrease in the specific radioactivity of lactate, but aspartate (2mm), malate (2mm) and glucose (5mm) were without effect. Total inhibition of gluconeogenesis with 3-mercaptopicolinate did not prevent the fall in specific radioactivity of lactate observed in tubules from fed-rat kidney, thereby excluding significant activity of the substrate cycle pyruvate-->oxaloacetate-->phosphoenolpyruvate-->pyruvate. 4. The capacity of pyruvate kinase under the test conditions in tubules prepared from kidneys of fed or starved rats was at least ten times higher than the observed rate of production of lactate, so that failure to observe recycling of lactate in starved-rat tubules indicates suppression of pyruvate kinase activity. 5. The endogenous glycogen and glucose content of isolated renal cortex tubules is too low to account for the dilution of label of lactate. Endogenous concentrations of glycerol and amino acids were also very low. As for glycogen, the possibility that very rapid turnover of these metabolites, in fed rats but not in starved rats, may account for formation of unlabelled lactate cannot be excluded. 6. It is concluded that substrate cycling via phosphoenolpyruvate does not occur to any significant extent in either fed or starved-rat kidney. In fed rats recycling of lactate carbon does occur and the rate of this reaction is similar to the rate of gluconeogenesis at physiological concentrations of lactate. The present results favour participation of oxaloacetate decarboxylase rather than ;malic' enzyme in this cycle.

Utilization of energy-providing substrates in the isolated working rat heart.

1. An improved perfusion system for the isolated rat heart is described. It is based on the isolated working heart of Neely, Liebermeister, Battersby & Morgan (1967) (Am. J. Physiol. 212, 804-814) and allows the measurement of metabolic rates and cardiac performance at a near-physiological workload. The main improvements concern better oxygenation of the perfusion medium and greater versatility of the apparatus. Near-physiological performance (cardiac output and aortic pressure) was maintained for nearly 2 h as compared with 30 min or less in the preparations of earlier work. 2. The rates of energy release (O2 uptake and substrate utilization) were 40-100% higher than those obtained by previous investigators, who used hearts at subphysiological workloads. 3. Values are given for the rates of utilization of glucose, lactate, oleate, acetate and ketone bodies, for O2 consumption and for the relative contributions of various fuels to the energy supply of the heart. Glucose can be replaced to a large extent by lactate, oleate or acetate, but not by ketone bodies. 4. Apart from quantitative differences there were also major qualitative differences between the present and previous preparations. Thus insulin was not required for maximal rates of glucose consumption at near-physiological, in contrast with subphysiological, workloads when glucose was the sole added substrate. When glucose oxidation was suppressed by the addition of other oxidizable substrates (lactate, acetate or acetoacetate), insulin increased the contribution of glucose as fuel for cardiac energy production at high workload. 5. In view of the major effects of workload on cardiac metabolism, experimentation on hearts performing subphysiologically or unphysiologically is of limited value to the situation in vivo.

Loss of cell constituents from hepatocytes on centrifugation.

In studies of the metabolism of isolated hepatocytes, it is often necessary to measure the concentrations of cell constituents both in cells and medium. When hepatocytes are separated in the special tubes of Hems, Lund & Krebs (1975) (Biochem. J. 150, 47--50), they lose much glucose, urea and Na+, whereas there is no loss of K+, glutamate, aspartate and adenine nucleotides. Cell water is also lost, as measured by the distribution of 3H2O. This loss is mainly due to an exchange of cell water with the aqueous solution in the stems of the tubes through which the cells pass on centrifugation. In general, substances are lost only when the intracellular concentration is equal to, or lower than, the extracellular concentration. Probably solutes are lost because they travel with the water unidirectionally out of the cell. A loss of solute does not occur when the cells are centrifuged in conical tubes with a layer of silicone oil between the cell suspension and the deproteinizing layer. The reasons for the loss occurring in the special separation tubes are discussed.

Sources of ammonia for mammalian urea synthesis.

The initial rate of incorporation of [15N]alanine into the 6-amino group of the adenine nucleotides in rat hepatocytes was about one-eighteenth of the rate of incorporation into urea. Thus the purine nucleotide cycle cannot provide most of the ammonia needed in urea synthesis for the carbamoyl phosphate synthase reaction (EC 2.7.2.5). On the other hand, contrary to the view expressed by McGivan & Chappell [(1975) FEBS Lett. 52, 1--7], the experiments support the view that hepatic glutamate dehydrogenase can supply the required ammonia.

Maintenance of glutathione content is isolated hepatocyctes.

1. During the standard procedure for the preparation of rat hepatocytes, about half of the cellular GSH (reduced glutathione) is lost. 2. This loss is prevented by the addition of 0.1 mM-EGTA (but no EDTA) to the perfusion medium. 3. On incubation with and without EGTA, isolated hepatocytes prepared in the presence of EGTA lose GSH. This loss is prevented by near-physiological concentrations of methionine or homocysteine, but not of cysteine. 4. Cysteine, at concentrations above 0.2 mM, causes a loss of GSH probably by non-enzymic formation of a mixed disulphide. 5. Serine together with methionine or homocystein increases GSH above the value in cells from starved rats in vivo. This is taken to suggest that cystathionine may be a cysteine donor in the synthesis of gamma-glutamylcysteine, the precursor of GSH.

Reaction of formiminoglutamate with liver glutamate dehydrogenase.

1. Kinetic aspects of the reaction between crystalline bovine liver glutamate dehydrogenase and formiminoglutamate were investigated to establish the conditions under which the latter may interfere with the assay of glutamate by using glutamate dehydrogenase and to explain why formiminoglutamate accumulates in vivo after histidine loading, although it can react with glutamate dehydrogenase. The Km and Vmax. values were compared with those of the enzyme reacting with glutamate. At pH 7.4 Km for formiminoglutamate was much higher and Vmax. much lower than the values for glutamate. 2. The equilibrium constant at pH 7.0 was 0.017 micrometer with formiminoglutamate, i.e. about one two-hundredths that with glutamate. 3. In vivo the interaction between glutamate dehydrogenase and formiminoglutamate is minimal even when the concentration of the latter in the liver is greatly raised, as in cobalamine or folate deficiency after histidine loading. 4. At pH 9.3, i.e. under the conditions for the assay of glutamate by glutamate dehydrogenase, formiminoglutamate reacts readily with the enzyme.

The regulation of folate and methionine metabolism.

1. The isolated perfused rat liver and suspensions of isolated rat hepatocytes fail to form glucose from histidine, in contrast with the liver in vivo. Both rat liver preparations readily metabolize histidine. The main end product is N-formiminoglutamate. In this respect the liver preparations behave like the liver of cobalamin- or folate-deficient mammals. 2. Additions of L-methionine in physiological concentrations (or of ethionine [2-amino-4-(ethylthio)butyric acid]) promotes the degradation of formiminoglutamate, as is already known to be the case in cobalamin of folate deficiency. Added methionine also promotes glucose formation from histidine. 3. Addition of methionine accelerates the oxidation of formate to bicarbonate by hepatocytes. 4. A feature common to cobalamin-deficient liver and the isolated liver preparations is taken to be a low tissue methionine concentration, to be expected in cobalamin deficiency through a decreased synthesis of methionine and caused in liver preparations by a washing out of amino acids during the handling of the tissue. 5. The available evidence is in accordance with the assumption that methionine does not directly increase the catalytic capacity of formyltetrahydrofolate dehydrogenase; rather, that an increased methionine concentration raises the concentration of S-adenosylmethionine, thus leading to the inhibition of methylenetetrahydrofolate reductase activity [Kutzbach & Stokstad (1967) Biochim. Biophys. Acta 139, 217-220; Kutzbach & Stokstad (1971) Methods Enzymol. 18B, 793-798], that this inhibition causes an increase in the concentration of methylenetetrahydrofolate and the C1 tetrahydrofolate derivatives in equilibrium with methylenetetrahydrofolate, including 10-formyltetrahydrofolate; that the increased concentration of the latter accelerates the formyltetrahydrofolate dehydrogenase reaction, because the normal concentration of the substrate is far below the Km value of the enzyme for the substrate. 6. The findings are relevant to the understanding of the regulation of both folate and methionine metabolism. When the methionine concentration is low, C1 units are preserved by the decreased activity of formyltetrahydrofolate dehydrogenase and are utilized for the synthesis of methionine, purines and pyrimidines. On the other hand when the concentration of methionine, and hence adenosylmethionine, is high and there is a surplus of C1 units as a result of excess of dietary supply, formyltetrahydrofolate dehydrogenase disposes of the excess. When ample dietary supply causes an excess of methionine, which has to be disposed of by degradation, the increased activity of formyltetrahydrofolate dehydrogenase decreases the supply of methyltetrahydrofolate. Thus homocysteine, instead of being remethylated, enters the pathway of degradation via cystathionine. 7. The findings throw light on the biochemical abnormalities associated with cobalamin deficiency (megaloblastic anaemia), especially on the 'methylfolate-trap hypothesis'. This is discussed. 8...

Rapid separation of isolated hepatocytes or similar tissue fragments for analysis of cell constituents.

A simple device is described for the rapid centrifugal separation of isolated hepatocytes or similar tissue fragments from the suspending medium. Rapid separation is essential if meaningful information on the concentrations of cell constituents and their distribution between cell and suspension medium is to be obtained. The usefulness of the technique is illustrated by measurements of hepatocyte constituents (glutamate, aspartate, alanine, K+) which show large concentration gradients against the extracellular medium.