scholarly journals Heterogeneity of the sn-glycerol 3-phosphate pool in isolated hepatocytes, demonstrated by the use of deuterated glycerols and ethanol

1984 ◽  
Vol 224 (3) ◽  
pp. 731-739 ◽  
Author(s):  
T Cronholm ◽  
T Curstedt
Keyword(s):  
Mass Spectrometry ◽  
Alcohol Dehydrogenase ◽  
Organic Acids ◽  
Ethanol Oxidation ◽  
Isolated Hepatocytes ◽  
Female Rats ◽  
Dihydroxyacetone Phosphate ◽  
Deuterium Excess ◽  
Relative Contribution ◽  
Glycerol Uptake

Hepatocytes were isolated from female rats and incubated with [1,1,3,3-2H4]glycerol or [2-2H]glycerol. The deuterium excess in phosphatidylcholines, sn-glycerol 3-phosphate and other organic acids was determined by g.l.c./mass spectrometry. The unlabelled fraction of the major phosphatidylcholines decreased exponentially, and the turnover was not changed by the presence of ethanol. The relative contribution of the two deuterated glycerols was about the same in the major phosphatidylcholine as in sn-glycerol 3-phosphate, indicating that formation by acylation of dihydroxyacetone phosphate is insignificant. [1,1,3,3-2H4]Glycerol had lost deuterium to a larger extent when it was incorporated in the phosphatidylcholine than when it was incorporated in sn-glycerol-3-phosphate, indicating that the phosphatidylcholines are formed from a separate pool of sn-glycerol 3-phosphate. Deuterium at C-2 was transferred between sn-glycerol 3-phosphate molecules to about 25%. Ethanol decreased the extent of deuterium transfer, the extent of glycerol uptake and the loss of deuterium at C-1 and C-3 in sn-glycerol 3-phosphate. The results indicate that the oxidation to dihydroxyacetone phosphate was inhibited by the NADH formed during ethanol oxidation. [2-2H]Glycerol also labelled an alcohol dehydrogenase substrate, malate and lactate, indicating oxidation of sn-glycerol 3-phosphate in the cytosol. The two acids appeared to be formed in reductions with different pools of NADH.

scholarly journals Effect of ethanol on the redox state of the coenzyme bound to alcohol dehydrogenase studied in isolated hepatocytes

1987 ◽  
Vol 248 (2) ◽  
pp. 567-572 ◽  
Author(s):  
T Cronholm
Keyword(s):  
Alcohol Dehydrogenase ◽  
Redox State ◽  
Ethanol Oxidation ◽  
Equilibrium Composition ◽  
Isolated Hepatocytes ◽  
Indicator System ◽  
Female Rats ◽  
Equilibrium System ◽  
Efficient Removal ◽  
Redox Indicator

Hepatocytes were isolated from fed female rats and incubated with a redox indicator system consisting of cyclohexanone and unlabelled or perdeuterated cyclohexanol. The concentrations and deuterium contents of these were measured by g.l.c. and g.l.c.-m.s. of oxime t-butyldimethylsilyl derivatives. The equilibrium composition represented the redox state of the coenzyme bound to alcohol dehydrogenase, since 4-methylpyrazole inhibited the interconversion. Reduction appeared to be catalysed to a small extent also by an NADPH-dependent aldehyde reductase. The NADH/NAD+ ratio on alcohol dehydrogenase was 3 orders of magnitude higher in the presence of ethanol than in its absence. This redox shift has the degree expected from reported kinetic constants. The shift was due both to a decreased rate of oxidation and to an increased rate of reduction in the indicator system. The results indicate that the redox effect of ethanol on the free NAD system is due to efficient removal of acetaldehyde from a near-equilibrium system consisting of ethanol, acetaldehyde and bound coenzymes, together with dissociation of NADH from the enzyme. The effect on the redox state of the bound coenzyme was less marked when the ethanol was deuterated at C-1, indicating an isotope effect. The 2H excess in the cyclohexanol formed was about 70% of that in the [1,1-2H2]ethanol. This dilution, which is caused by binding of free NADH to the enzyme, indicates that reoxidation of cytosolic NADH partly limits the rate of ethanol oxidation.

scholarly journals The reversibility of cytosolic dehydrogenase reactions in hepatocytes from starved and fed rats. Effect of fructose

1984 ◽  
Vol 222 (2) ◽  
pp. 437-446 ◽  
Author(s):  
C Vind ◽  
N Grunnet
Keyword(s):  
Lactate Dehydrogenase ◽  
Alcohol Dehydrogenase ◽  
Ethanol Oxidation ◽  
Maximal Activity ◽  
Isolated Hepatocytes ◽  
Single Compartment ◽  
Dehydrogenase Reaction ◽  
Order Of Magnitude ◽  
Rate Limiting

The metabolism of [2-3H]lactate was studied in isolated hepatocytes from fed and starved rats metabolizing ethanol and lactate in the absence and presence of fructose. The yields of 3H in ethanol, water, glucose and glycerol were determined. The rate of ethanol oxidation (3 mumol/min per g wet wt.) was the same for fed and starved rats with and without fructose. From the detritiation of labelled lactate and the labelling pattern of ethanol and glucose, we calculated the rate of reoxidation of NADH catalysed by lactate dehydrogenase, alcohol dehydrogenase and triosephosphate dehydrogenase. The calculated flux of reducing equivalents from NADH to pyruvate was of the same order of magnitude as previously found with [3H]ethanol or [3H]xylitol as the labelled substrate [Vind & Grunnet (1982) Biochim. Biophys. Acta 720, 295-302]. The results suggest that the cytoplasm can be regarded as a single compartment with respect to NAD(H). The rate of reduction of acetaldehyde and pyruvate was correlated with the concentration of these metabolites and NADH, and was highest in fed rats and during fructose metabolism. The rate of reoxidation of NADH catalysed by lactate dehydrogenase was only a few per cent of the maximal activity of the enzymes, but the rate of reoxidation of NADH catalysed by alcohol dehydrogenase was equal to or higher than the maximal activity as measured in vitro, suggesting that the dissociation of enzyme-bound NAD+ as well as NADH may be rate-limiting steps in the alcohol dehydrogenase reaction.

Mass spectrometry‐based quantification and spatial location of small organic acids exudate in plant roots under phosphorous deficiency and aluminium toxicity

2021 ◽  
Author(s):  
David Gomez‐Zepeda ◽  
Moises Frausto ◽  
Héctor‐Rogelio Nájera‐González ◽  
Luis Herrera‐Estrella ◽  
José‐Juan Ordaz‐Ortiz
Keyword(s):  
Mass Spectrometry ◽  
Organic Acids ◽  
Spatial Location ◽  
Aluminium Toxicity ◽  
Plant Roots

scholarly journals Effects of bicarbonate on intercompartmental reducing-equivalent translocation in isolated parenchymal cells from rat liver

1974 ◽  
Vol 140 (3) ◽  
pp. 355-361 ◽  
Author(s):  
Michael N. Berry ◽  
Harold V. Werner ◽  
Ernest Kun
Keyword(s):  
Malate Dehydrogenase ◽  
Inhibitory Action ◽  
Ethanol Oxidation ◽  
Specific Inhibitor ◽  
Isolated Liver Cells ◽  
Glycerol Uptake ◽  
Parenchymal Cells ◽  
Rate Limiting ◽  
Isolated Liver ◽  
Phosphate Medium

1. Incubation of isolated liver cells in a medium containing bicarbonate raises malate concentrations almost sixfold compared with values obtained in a bicarbonate-free phosphate medium. The malate concentration of about 0.3mm in bicarbonate medium is of the same order as the Km for malate dehydrogenase. 2. The utilization of ethanol, glyercol and sorbitol was increased by 20–35% in bicarbonate medium. 3. Fluoromalate, a specific inhibitor of malate dehydrogenase and the malate carrier, inhibited or ethanol oxidation by 23%, glycerol uptake by 20% and sorbitol uptake by 42% in bicarbonate medium, but had a much smaller inhibitory action in phosphate medium. In consequence fluoromalate almost abolished the stimulatory effects of bicarbonate on substrate utilization. 4. Difluoro-oxaloacetate, a specific inhibitor of aspartate aminotransferase, had about one-half the inhibitory activity of fluoromalate. The two inhibitors in combination were less effective than fluoromalate by itself. 5. It is concluded that bicarbonate stimulates the utilization of reduced substrates, which are oxidized in the cytoplasmic compartment of the liver cell, by increasing the activity of rate-limiting malate dehydrogenase-dependent intercompartmental hydrogen shuttles. Both malate–oxaloacetate and malate–aspartate systems are involved in these hydrogen-translocation processes.

scholarly journals National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines: Follow-Up Testing for Metabolic Disease Identified by Expanded Newborn Screening Using Tandem Mass Spectrometry; Executive Summary

2009 ◽  
Vol 55 (9) ◽  
pp. 1615-1626 ◽  
Author(s):  
Dennis J Dietzen ◽  
Piero Rinaldo ◽  
Ronald J Whitley ◽  
William J Rhead ◽  
W Harry Hannon ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Tandem Mass Spectrometry ◽  
Organic Acids ◽  
Newborn Screening ◽  
Tandem Mass ◽  
Confirmatory Testing ◽  
The Us ◽  
The Impact

Abstract Background: Almost all newborns in the US are screened at birth for multiple inborn errors of metabolism using tandem mass spectrometry. Screening tests are designed to be sufficiently sensitive so that cases are not missed. The NACB recognized a need for standard guidelines for laboratory confirmation of a positive newborn screen such that all babies would benefit from equal and optimal follow-up by confirmatory testing. Methods: A committee was formed to review available data pertaining to confirmatory testing. The committee evaluated previously published guidelines, published methodological and clinical studies, clinical case reports, and expert opinion to support optimal confirmatory testing. Grading was based on guidelines adopted from criteria derived from the US Preventive Services Task Force and on the strength of recommendations and the quality of the evidence. Three primary methods of analyte measurement were evaluated for confirmatory testing including measurement of amino acids, organic acids, and carnitine esters. The committee graded the evidence for diagnostic utility of each test for the screened conditions. Results: Ample data and experience were available to make strong recommendations for the practice of analyzing amino acids, organic acids, and acylcarnitines. Likewise, strong recommendations were made for the follow-up test menu for many disorders, particularly those with highest prevalence. Fewer data exist to determine the impact of newborn screening on patient outcomes in all but a few disorders. The guidelines also provide an assessment of developing technology that will fuel a refinement of current practice and ultimate expansion of the diseases detectable by tandem mass spectrometry. Conclusions: Guidelines are provided for optimal follow-up testing for positive newborn screens using tandem mass spectrometry. The committee regards these tests as reliable and currently optimal for follow-up testing. .

INTERACTION OF ETHANOL OXIDATION WITH GLUCURONIDATION IN ISOLATED HEPATOCYTES

Abstracts ◽  
1978 ◽  
pp. 461
Author(s):  
P. Moldéus ◽  
B. Andersson ◽  
A. Norling ◽  
M. Berggren
Keyword(s):  
Ethanol Oxidation ◽  
Isolated Hepatocytes
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