Resistance training increases skeletal muscle oxidative capacity and net intramuscular triglyceride breakdown in type I and II fibres of sedentary males.

Recent in vitro and in vivo experimental observations suggest that improvements in insulin sensitivity following endurance training are mechanistically linked to increases in muscle oxidative capacity, intramuscular triglyceride (IMTG) utilization during endurance exercise and increases in the content of the lipid droplet-associated perilipin 2 (PLIN2) and perilipin 5 (PLIN5). This study investigated the hypothesis that similar adaptations may also underlie the resistance training (RT)-induced improvements in insulin sensitivity. Thirteen sedentary men (20 ± 1 years old; body mass index 24.8 ± 0.8 kg m(-2)) performed 6 weeks of whole-body RT (three times per week), and changes in peak O2 uptake (in millilitres per minute per kilogram) and insulin sensitivity were assessed. Muscle biopsies (n = 8) were obtained before and after 60 min steady-state cycling at ~65% peak O2 uptake. Immunofluorescence microscopy was used to assess changes in oxidative capacity (measured as cytochrome c oxidase protein content), IMTG and PLIN2 and PLIN5 protein content. Resistance training increased peak O2 uptake (by 8 ± 3%), COX protein content (by 46 ± 13 and 61 ± 13% in type I and II fibres, respectively) and the Matsuda insulin sensitivity index (by 47 ± 6%; all P < 0.05). In type I fibres, IMTG (by 52 ± 11%; P < 0.05) and PLIN2 content (by 107 ± 19%; P < 0.05) were increased and PLIN5 content tended to increase (by 54 ± 22%; P = 0.054) post-training. In type II fibres, PLIN2 content increased (by 57 ± 20%; P < 0.05) and IMTG (by 46 ± 17%; P = 0.1) and PLIN5 content (by 44 ± 24%; P = 0.054) tended to increase post-training. Breakdown of IMTG during moderate-intensity exercise was greater in both type I and type II fibres (by 43 ± 5 and 37 ± 5%, respectively; P < 0.05) post-RT. The results confirm the hypothesis that RT enhances muscle oxidative capacity and increases IMTG breakdown and the content of PLIN2 and PLIN5 in both type I and type II fibres during endurance-type exercise.

Sprint interval and traditional endurance training increase net intramuscular triglyceride breakdown and expression of perilipin 2 and 5.

Intramuscular triglyceride (IMTG) utilization is enhanced by endurance training (ET) and is linked to improved insulin sensitivity. This study first investigated the hypothesis that ET-induced increases in net IMTG breakdown and insulin sensitivity are related to increased expression of perilipin 2 (PLIN2) and perilipin 5 (PLIN5). Second, we hypothesized that sprint interval training (SIT) also promotes increases in IMTG utilization and insulin sensitivity. Sixteen sedentary males performed 6 weeks of either SIT (4-6, 30 s Wingate tests per session, 3 days week(-1)) or ET (40-60 min moderate-intensity cycling, 5 days week(-1)). Training increased resting IMTG content (SIT 1.7-fold, ET 2.4-fold; P < 0.05), concomitant with parallel increases in PLIN2 (SIT 2.3-fold, ET 2.8-fold; P < 0.01) and PLIN5 expression (SIT 2.2-fold, ET 3.1-fold; P < 0.01). Pre-training, 60 min cycling at ∼65% pre-training decreased IMTG content in type I fibres (SIT 17 ± 10%, ET 15 ± 12%; P < 0.05). Following training, a significantly greater breakdown of IMTG in type I fibres occurred during exercise (SIT 27 ± 13%, ET 43 ± 6%; P < 0.05), with preferential breakdown of PLIN2- and particularly PLIN5-associated lipid droplets. Training increased the Matsuda insulin sensitivity index (SIT 56 ± 15%, ET 29 ± 12%; main effect P < 0.05). No training × group interactions were observed for any variables. In conclusion, SIT and ET both increase net IMTG breakdown during exercise and increase in PLIN2 and PLIN5 protein expression. The data are consistent with the hypothesis that increases in PLIN2 and PLIN5 are related to the mechanisms that promote increased IMTG utilization during exercise and improve insulin sensitivity following 6 weeks of SIT and ET.

Preferential utilization of perilipin 2-associated intramuscular triglycerides during 1 h of moderate-intensity endurance-type exercise.

The lipid droplet (LD)-associated protein perilipin 2 (PLIN2) appears to colocalize with LDs in human skeletal muscle fibres, although the function of PLIN2 in the regulation of intramuscular triglyceride (IMTG) metabolism is currently unknown. Here we investigated the hypothesis that the presence of PLIN2 in skeletal muscle LDs is related to IMTG utilisation during exercise. We therefore measured exercise-induced changes in IMTG and PLIN2 distribution and changes in their colocalization. Muscle biopsies from the vastus lateralis were obtained from seven lean, untrained men (22 ± 2 years old, body mass index 24.2 ± 0.9 kg m(-2) and peak oxygen uptake 3.35 ± 0.13 l min(-1)) before and after 1 h of moderate-intensity cycling at ~65% peak oxygen uptake. Cryosections were stained for perilipin 2, IMTG and myosin heavy chain type I and viewed using wide-field and confocal fluorescence microscopy. Exercise induced a 50 ± 7% decrease in IMTG content in type I fibres only (P < 0.05), but no change in PLIN2 content. Colocalization analysis showed that the fraction of PLIN2 associated with IMTG was 0.67 ± 0.03 before exercise, which was reduced to 0.51 ± 0.01 postexercise (P < 0.05). Further analysis revealed that the number of PLIN2-associated LDs was reduced by 31 ± 10% after exercise (P < 0.05), whereas the number of PLIN2-null LDs was unchanged. No such changes were seen in type II fibres. In conclusion, this study shows that PLIN2 content in skeletal muscle is unchanged in response to a single bout of endurance exercise. Furthermore, the PLIN2 and IMTG association is reduced postexercise, apparently due to preferential utilization of PLIN2-associated LDs. These results confirm the hypothesis that the PLIN2 association with IMTG is related to the utilization of IMTG as a fuel during exercise.

Inhibition of bovine plasma semicarbazide-sensitive amine oxidase by caffeine.

Semicarbazide-sensitive amine oxidase (SSAO) is a copper-containing enzyme that catalyzes the oxidative deamination of endogenous and exogenous primary amines. SSAO exists in mammals both as a plasma-soluble and as a membrane-bound form, and its active site is able to come into contact with numerous xenobiotic, amine-containing compounds. The kinetic studies performed in this work showed that caffeine inhibition of bovine serum amine oxidase was noncompetitive when benzylamine was used as substrate and mixed when the substrate used was methylamine. Since caffeine contains an imidazole ring, it cannot be excluded that it might bind to an inhibitory imidazoline-binding site on SSAO.

Exogenous amino acids stimulate net muscle protein synthesis in the elderly.

We have investigated the response of amino acid transport and protein synthesis in healthy elderly individuals (age 71+/-2 yr) to the stimulatory effect of increased amino acid availability. Muscle protein synthesis and breakdown, and amino acid transport were measured in the postabsorptive state and during the intravenous infusion of an amino acid mixture. Muscle-free amino acid kinetics were calculated by means of a three compartment model using data obtained by femoral arterio-venous catheterization and muscle biopsies from the vastus lateralis during the infusion of stable isotope tracers of amino acids. In addition, muscle protein fractional synthetic rate (FSR) was measured. Peripheral amino acid infusion significantly increased amino acid delivery to the leg, amino acid transport, and muscle protein synthesis when measured either with the three compartment model (P < 0.05) or with the traditional precursor-product approach (FSR increased from 0. 0474+/-0.0054 to 0.0940+/-0.0143%/h, P < 0.05). Because protein breakdown did not change during amino acid infusion, a positive net balance of amino acids across the muscle was achieved. We conclude that, although muscle mass is decreased in the elderly, muscle protein anabolism can nonetheless be stimulated by increased amino acid availability. We thus hypothesize that muscle mass could be better maintained with an increased intake of protein or amino acids.

Sodium bicarbonate enhances membrane-bound and soluble human semicarbazide-sensitive amine oxidase activity in vitro.

Semicarbazide-sensitive amine oxidase (SSAO) is a multifunctional enzyme with different biological roles that depend on the tissue where it is expressed. Because SSAO activity is altered in several pathological conditions, we were interested in studying the possible regulation of the human enzyme activity. It has been previously reported that SSAO activity is increased in the presence of Dulbecco's modified Eagle medium (DMEM) in vitro. The aim of the present work was to investigate the effects of the different constituents of DMEM on human SSAO activity. We found that sodium bicarbonate was the only component able to mimic the enhancement of both human aorta and plasma SSAO activity in vitro, suggesting a possible physiological role of bicarbonate as an intrinsic modulator of the human enzyme. Failure to take this activating effect into account could also result in inaccuracies in the reported tissue activities of this enzyme.

Inhibition of amine oxidases by the histamine-1 receptor antagonist hydroxyzine.

The effects of the drug hydroxyzine on the activities of the rat liver monoamine oxidases (EC 1.4.3.6; MAO) and the membrane-bound and soluble forms of bovine semicarbazide-sensitive amine oxidase (EC 1.4.3.6; SSAO) were studied. Hydroxyzine was found to be a competitive inhibitor of MAO-B (Ki - 38 microM), whereas it had a low potency towards MAO-A (IC50 > 630 microM). Although it was a relatively potent competitive inhibitor of bovine plasma SSAO (Ki approximately 1.5 microM), it was a weak inhibitor of the membrane-bound form of the enzyme from bovine lung (IC50 approximately 1 mM). These findings extend our knowledge of the drug binding capabilities of the amine oxidases and suggest that these interactions may contribute to the complex actions of this drug.

Activation of glutamate dehydrogenase by L-leucine.

The activation of glutamate dehydrogenase (L-glutamate: NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3) by L-leucine has been studied. Apparently homogeneous preparations from ox liver and brain were found to respond similarly. Commercially obtained preparations of the enzyme, which had suffered limited proteolysis during the purification procedure, were shown to behave similarly to preparations which had not suffered such proteolysis when the effects of L-leucine on the oxidative deamination reaction were studied using either NAD+ or NADP+ as the coenzyme. There was also no significant difference in the responses when the reductive reaction was determined with NADPH or with 40 microM NADH. At higher concentrations of NADH (160 microM) the unproteolysed preparations were activated by L-leucine to a considerably greater extent than those which had suffered limited proteolysis. These results accord with the greater sensitivity of the former preparations to inhibition by high concentrations of NADH and the relief of such inhibition by L-leucine. This amino acid was also found to relieve the inhibition of the enzyme by GTP, resulting in an apparent increase in the activation observed in the presence of this nucleotide. In contrast, under the conditions used in this work, the apparent degree of activation by L-leucine was found to be decreased in the presence of the activators ATP or ADP. The presence of high concentrations of NADH (200 microM) potentiated the high substrate inhibition by 2-oxoglutarate, and L-leucine significantly reduced this effect. The effects of L-leucine on the activity of glutamate dehydrogenase thus appear to be composed of a direct effect on the activity of the enzyme together with a relief of high substrate inhibition. The effects of GTP and 2-oxoglutarate in potentiating inhibition by NADH can account for their effects in enhancing the apparent activation by L-leucine. The marked differences in the responses of proteolysed and unproteolysed preparations of the enzyme result from the effects of proteolysis in decreasing the sensitivity to high concentrations of NADH.

Modifications in the allosteric properties of phosphofructokinase in rat erythrocytes and reticulocytes cross-linked with dimethyl suberimidate and 3,3'-dithiobispropionimidate.

The kinetic behaviour of phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) has been studied in situ, by using rat erythrocytes and reticulocytes treated with dimethyl suberimidate and 3,3'-dithiobispropionimidate as cross-linking reagents and with digitonin as the delipidating agent. Comparison of the ATP and fructose-6-P saturation curves of phosphofructokinase in dimethyl suberimidate-permeabilized cells with those obtained in haemolysates showed the enzyme to have reduced allosteric properties under in situ conditions, although it still responded to cyclic AMP (300 microM) added as allosteric effector. Non-sigmoidal fructose-6-P saturation curves were also observed using 3,3'-dithiobispropionimidate-permeabilized erythrocytes, either in the absence or in the presence of cyclic AMP. A hyperbolic behaviour was shown after cross-linking reversal of 3,3'-dithiobispropionimidate-permeabilized erythrocytes by treatment with dithiothreitol. Specific activity values of phosphofructokinase were always lower in permeabilized cells than in haemolysates. A significant inhibition of phosphofructokinase specific activity, without any effect on its allosteric behaviour, is exerted by reaction of dimethyl suberimidate or 3,3'-dithiobispropionimidate with erythrocyte lysates in the presence of an inhibitory concentration of ATP. These results suggest that penetration of the cross-linking reagent and its subsequent reaction with intracellular phosphofructokinase will have a direct effect upon the results obtained using this in situ approach.U

Fasciculin inhibition of acetylcholinesterase is prevented by chemical modification of the enzyme at a peripheral site.

Fasciculin 2 (FAS) is a 61 amino acid peptide present in Dendroaspis angusticeps snake venom, with a selective and potent inhibitory activity towards acetylcholinesterase (AChE). The specific interaction of FAS with peripheral sites present in Electrophorus electricus AChE (Ki = 0.04 nM FAS) was investigated by chemical modification with N,N-dimethyl-2-phenylaziridinium (DPA) in the presence of active or peripheral anionic site protective agents. An enzyme was obtained that compared to the native AChE is 10(6)-times less sensitive to FAS, is fully inhibited by edrophonium and tacrine, and is 25-170-times less sensitive to several peripheral site ligands. Characterization of catalytic functions showed that Km for acetylthiocholine was 4-fold lower in the DPA-modified enzyme, whereas Km for phenylacetate remained the same. Values for Kcat determined with both substrates were unchanged. Diminished catalytic efficiency reflects that hydrolysis and/or supply of cationic substrates to the active site was affected by DPA reaction at a peripheral site. Previous data implicate Trp-279 (Torpedo AChE sequence numbering) as the residue actually involved in DPA modification. Our results strongly support FAS binding to an AChE peripheral site which partially overlaps the site of other peripheral site ligands including acetylthiocholine.

Purification and properties of low-Km aldehyde reductase from ox brain.

A low-Km aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2), which may be identical with aldose reductase (alditol:NADP+ 1-oxidoreductase, EC 1.1.1.21), has been purified from ox brain to homogeneity. It was shown to be a monomer with Mr values of 31 000 and 35 100 being obtained by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, respectively. The enzyme catalyses the NADPH-dependent reduction of a number of aromatic and sugar aldehydes. The activity of the enzyme with 133 microM NADH was about one-third of that with 120 microM NADPH. Activity with both these coenzymes was optimum at pH 6.2 and was inhibited by increasing the ionic strength with KCl, NaCl or NaNO3. In contrast, the activity was stimulated by sodium phosphate. The activity with NADH as the coenzyme was more sensitive to stimulation by phosphate and to inhibition by increasing ionic strength than that determined with NADPH.

Activation of human lung semicarbazide sensitive amine oxidase by a low molecular weight component present in human plasma.

Semicarbazide-sensitive amine oxidase (SSAO) encodes a wide family of enzymes named E.C.1.4.3.6 [amine:oxygen oxidoreductase (deaminating) (copper containing)] that metabolises primary aliphatic and aromatic amines. It is present in almost all vascularised and nonvascularised mammalian tissues, and it is also present in soluble form in plasma. SSAO appears to show different functions depending on the tissue where it is expressed. Here we describe, for the first time, the activation of the SSAO from human lung by human plasma. The extent of activation was greater when the human plasma came from diabetic and heart infarcted patients. A kinetic mechanism of such effect is proposed. The activation was lost after the plasma was dialysed, indicating a low molecular weight component (MW <3800 Da) to be responsible. The activator component is heat stable and resistant to proteolysis by chymotrypsin and trypsin and also resistant to perchloric acid treatment. However, treatment with 35% formic acid, completely abolished activation, suggesting involvement of lipid material. The possibility of that lysophosphatidylcholine (LPC), an amphiphilic phospholipid derived from the phosphatidylcholine, the major component in plasma accumulated in pathological conditions, was studied. LPC was shown to behave as a "competitive activator" of human lung SSAO at concentrations below its critical micellar concentration (CMC value=50 microM). Thus LPC may be a component of the SSAO activatory material present in human plasma.

Purification of liver glutamate dehydrogenase by affinity precipitation and studies on its denaturation.

In the presence of glutaric acid, N2,N2'-adipodihydrazido-bis(N6-carbonylmethyl-NAD+)(bis-NAD+ ) forms cross-links between molecules of glutamate dehydrogenase, resulting in precipitation. The dependence of this process on bis-NAD+ and enzyme concentration has been investigated. This procedure has been shown to be effective in the purification of glutamate dehydrogenase from rat and ox liver, and a procedure is presented in which this affinity precipitation procedure is used instead of the affinity chromatography used in an earlier method (McCarthy, A.D., Walker, J.M. and Tipton, K.F. (1980) Biochem. J. 191, 605-611). The ox liver enzyme prepared in this way had not suffered the limited proteolysis that occurs during the preparation of the enzyme by other commonly used procedures. After the purified enzyme had been denatured by treatment with urea, guanidine hydrochloride, or low pH, no recovery of activity could be demonstrated following dilution or, in the last case, dialysis.

Survival after high blood alcohol levels. Association with first-order elimination kinetics.

A patient survived acute alcohol intoxication with an unprecedented blood alcohol level of 1,500 mg/dL. Treatment included peritoneal dialysis and intravenously administered fructose. Alcohol elimination followed first-order kinetics, in which the elimination rate is proportional to concentration, rather than zero order in which the metabolic rate is independent of concentration. The report provides further evidence for first-order elimination kinetics with high blood alcohol levels and for an adaptive increase in alcohol metabolism with long-term alcohol consumption.

Eukaryotic aldehyde dehydrogenase (ALDH) genes: human polymorphisms, and recommended nomenclature based on divergent evolution and chromosomal mapping.

As currently being performed with an increasing number of superfamilies, a standardized gene nomenclature system is proposed here, based on divergent evolution, using multiple alignment analysis of all 86 eukaryotic aldehyde dehydrogenase (ALDH) amino-acid sequences known at this time. The ALDHs represent a superfamily of NAD(P)(+)-dependent enzymes having similar primary structures that oxidize a wide spectrum of endogenous and exogenous aliphatic and aromatic aldehydes. To date, a total of 54 animal, 15 plant, 14 yeast, and three fungal ALDH genes or cDNAs have been sequenced. These ALDHs can be divided into a total of 18 families (comprising 37 subfamilies), and all nonhuman ALDH genes are named here after the established human ALDH genes, when possible. An ALDH protein from one gene family is defined as having approximately < or = 40% amino-acid identity to that from another family. Two members of the same subfamily exhibit approximately > or = 60% amino-acid identity and are expected to be located at the same subchromosomal site. For naming each gene, it is proposed that the root symbol 'ALDH' denoting 'aldehyde dehydrogenase' be followed by an Arabic number representing the family and, when needed, a letter designating the subfamily and an Arabic number denoting the individual gene within the subfamily; all letters are capitalized in all mammals except mouse and fruit fly, e.g. 'human ALDH3A1 (mouse, Drosophila Aldh3a1).' It is suggested that the Human Gene Nomenclature Guidelines (http://++www.gene.ucl.ac.uk/nomenclature/guidelines.h tml) be used for all species other than mouse and Drosophila. Following these guidelines, the gene is italicized, whereas the corresponding cDNA, mRNA, protein or enzyme activity is written with upper-case letters and without italics, e.g. 'human, mouse or Drosophila ALDH3A1 cDNA, mRNA, or activity'. If an orthologous gene between species cannot be identified with certainty, sequential naming of these genes will be carried out in chronological order as they are reported to us. In addition, 20 human ALDH variant alleles that have been reported to date are listed herein and are recommended to be given numbers (or a number plus a capital letter) following an asterisk (e.g. 'ALDH3A2*2, ALDH2*4C'). It is anticipated that this eukaryotic ALDH gene nomenclature system will be extended to include bacterial genes within the next 2 years and that this nomenclature system will require updating on a regular basis; an ALDH Web site has been established for this purpose (http://++www.uchsc.edu/sp./sp./alcdbase/a ldhcov.html) and will serve as a medium for interaction amongst colleagues in this field.

The UDP glycosyltransferase gene superfamily: recommended nomenclature update based on evolutionary divergence.

This review represents an update of the nomenclature system for the UDP glucuronosyltransferase gene superfamily, which is based on divergent evolution. Since the previous review in 1991, sequences of many related UDP glycosyltransferases from lower organisms have appeared in the database, which expand our database considerably. At latest count, in animals, yeast, plants and bacteria there are 110 distinct cDNAs/genes whose protein products all contain a characteristic 'signature sequence' and, thus, are regarded as members of the same superfamily. Comparison of a relatedness tree of proteins leads to the definition of 33 families. It should be emphasized that at least six cloned UDP-GlcNAc N-acetylglucosaminyltransferases are not sufficiently homologous to be included as members of this superfamily and may represent an example of convergent evolution. For naming each gene, it is recommended that the root symbol UGT for human (Ugt for mouse and Drosophila), denoting 'UDP glycosyltransferase,' be followed by an Arabic number representing the family, a letter designating the subfamily, and an Arabic numeral denoting the individual gene within the family or subfamily, e.g. 'human UGT2B4' and 'mouse Ugt2b5'. We recommend the name 'UDP glycosyltransferase' because many of the proteins do not preferentially use UDP glucuronic acid, or their nucleotide sugar preference is unknown. Whereas the gene is italicized, the corresponding cDNA, transcript, protein and enzyme activity should be written with upper-case letters and without italics, e.g. 'human or mouse UGT1A1.' The UGT1 gene (spanning > 500 kb) contains at least 12 promoters/first exons, which can be spliced and joined with common exons 2 through 5, leading to different N-terminal halves but identical C-terminal halves of the gene products; in this scheme each first exon is regarded as a distinct gene (e.g. UGT1A1, UGT1A2, ... UGT1A12). When an orthologous gene between species cannot be identified with certainty, as occurs in the UGT2B subfamily, sequential naming of the genes is being carried out chronologically as they become characterized. We suggest that the Human Gene Nomenclature Guidelines (http://www.gene.acl.ac.uk/nomenclature/guidelines.html++ +) be used for all species other than the mouse and Drosophila. Thirty published human UGT1A1 mutant alleles responsible for clinical hyperbilirubinemias are listed herein, and given numbers following an asterisk (e.g. UGT1A1*30) consistent with the Human Gene Nomenclature Guidelines. It is anticipated that this UGT gene nomenclature system will require updating on a regular basis.

What is it that l-deprenyl (selegiline) might do?

There have been many claims that l-deprenyl may have distinct properties in slowing and perhaps even in reversing the progression of Parkinson's disease and other neurodegenerative conditions. This article will consider the paucity of evidence that such is the case in humans and the more detailed results from studies with experimental animals indicating that deprenyl may indeed express such a property. The conflicting data on its mechanism of action are considered and the concept that it may function to enhance neuronal fitness is advanced as an alternative to the neuroprotection and neurorescue hypotheses. Possible lines of experimental development that would help resolve some of the many unanswered questions regarding l-deprenyl function are outlined.

Effect of orthotopic liver transplantation and chemical denervation of the liver on the activities of hepatic monoamine oxidase and catechol-O-methyltransferase.

The denervation of some tissue is associated with a fall in the activities of monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT). Here we report on the effect of orthotopic liver transplantation and chemical denervation of the liver on the enzymes. Liver transplantation was performed on Lewis rats (n = 7). Denervation (n = 8) was by intraportal injection of 6-hydroxydopamine (75 mg/kg). A control group (n = 8) was also included. The norepinephrine content of the transplanted and denervated livers was reduced by greater than 99% (P < 0.001) and 95% (P < 0.001), respectively. The activity of hepatic COMT (substrate: catechol [5 mM] was not affected by transplantation or denervation. The activity of MAO with 0.1 mM 5-hydroxytryptamine (5-HT) (substrate for MAO-A) and with 0.01 mM 2-phenylethylamine (substrate for MAO-B) were not affected by denervation. In the transplanted liver, the activity of MAO with 5-HT and 2-phenylethylamine was increased by 26% (P < 0.05) and by 53% (P < 0.001), respectively. The ratios of the activities of the A to B forms of MAO (approximately 70% A to 30% B) was not affected by either procedure. Enzyme sensitivity for MAO inhibitors clorgyline and deprenyl were not significantly altered by transplantation. The concentration of plasma norepinephrine in the transplantation group was significantly lower than either the control (P < 0.001) or denervation groups (P < 0.05). We conclude from our results that the metabolism of circulating catecholamines by the liver is unlikely to be impaired after liver transplantation.

Deuterium isotope effect of phenelzine on the inhibition of rat liver mitochondrial monoamine oxidase activity.

Phenelzine is a suicide monoamine oxidase (MAO) inhibitor with antidepressant properties. The present study compares the inhibition of rat liver mitochondrial MAO by phenelzine and 1,1-dideuterated phenelzine and the metabolism of these drugs by that enzyme. Phenylacetaldehyde, which was measured by a high performance liquid chromatographic procedure, was found to be the major metabolite of phenelzine after incubation with MAO. The time-courses of aldehyde formation were non-linear due to the time-dependent inhibition of MAO. The reaction rate was reduced substantially when the hydrogen atom in the 1-carbon position was replaced by deuterium. The VH/VD value was 3.1, indicating a primary isotope effect. Such a substitution of deuterium in the phenelzine molecule did not affect significantly the initial reversible inhibition of MAO, which was determined by comparison of their Ki values. The irreversible inhibition, as estimated from IC50 values, however, was potentiated substantially by deuteration. These results support the notion that the irreversible inhibition of MAO activity by phenelzine proceeds via a phenylethyldiazene intermediate, which reacts with the enzyme to form a covalent adduct. An alternative pathway involving hydrogen abstraction from carbon-1 of phenelzine or via rearrangement of the diazine on the enzyme surface could occur to form a phenylethylidene hydrazine intermediate which would subsequently be hydrolyzed to phenylacetaldehyde. The reduction in the rate of phenylethylidene hydrazine formation due to the isotope effect could lead to the accumulation of phenylethyldiazene intermediate and thus potentiate the inhibition of MAO activity.

The effects of assay temperature on the complex kinetics of acetaldehyde oxidation by aldehyde dehydrogenase from human erythrocytes.

Several studies have shown preparations of the cytosolic aldehyde dehydrogenase (EC 1.2.1.3) from sheep and human liver and from human erythrocytes to exhibit complex kinetic behaviour in which the dependence of the initial velocity on the concentration of acetaldehyde gives rise to downwardly curving double-reciprocal plots. This behaviour has often been analysed in terms of a sharp discontinuity in the double-reciprocal plots and its possible implications for the oxidation of acetaldehyde and other pharmacologically important aldehydes has been a subject of speculation. In the present work, it is shown that the purified, apparently homogeneous, enzyme from human erythrocytes exhibits such complex kinetic behaviour when initial rates are determined at 25 degrees, although the double-reciprocal plots describe a smooth curve with no sharp discontinuity. However, when the assays were performed at 37 degrees there was no significant deviation from Michaelis-Menten kinetics over a wide range of acetaldehyde concentrations (0.2-30 mM). At higher concentrations of acetaldehyde inhibition occurred which was competitive with respect to NAD+. These results, which indicate that the complex kinetic behaviour of aldehyde dehydrogenase is not important at physiological temperature, are interpreted in terms of the mechanisms that have been advanced to explain the phenomena.