Does carbamazepine-epoxide conjugate with glutathione? On an existing but almost forgotten possibility.

The anticonvulsant carbamazepine is widely used to treat affective disorders and behavioural disorders in non-epileptic children. We report an elevated plasma level of carbamazepine-10,11-epoxide in a cystinuric child after daily medication with 300 mg carbamazepine while the serum level of carbamazepine was in the therapeutic range. The concentrations of carbamazepine and its epoxide derivative were determined by HPLC. The formation of a glutathione conjugate of carbamazepine-10,11-epoxide is raised as a hypothesis.

Methylglyoxal in living organisms: chemistry, biochemistry, toxicology and biological implications.

Despite the growing interest towards methylglyoxal and glyoxalases their real role in metabolic network is still obscure. In the light of developments several reviews have been published in this field mainly dealing with only a narrow segment of this research area. In this article a trial is made to present a comprehensive overview of methylglyoxal research, extending discussion from chemistry to biological implications by reviewing some important characteristics of methylglyoxal metabolism and toxicity in a wide variety of species, and emphasizing the action of methylglyoxal on energy production, free radical generation and cell killing. Special attention is paid to the discussion of alpha-oxoaldehyde production in the environment as a potential risk factor and to the possible role of this a-dicarbonyl in diseases. Concerning the interaction of methylglyoxal with biological macromolecules (DNA, RNA, proteins) an earlier review (Kalapos, Toxicology Letters, 73, 1994, 3-24) means a supplementation to this paper, thus hoping the avoidance of unnecessary bombast. The paper arrives at the conclusion that since the early stage of evolution the function of methylglyoxalase pathway has been related to carbohydrate metabolism, but its significance has been changed over the thousands of years. Namely, at the beginning of evolution methylglyoxalase path was essential for the reductive citric acid cycle as an anaplerotic route, while in the extant metabolism it concerns with the detoxification of methylglyoxal and plays some regulatory role in triose-phosphate household. As there is a tight junction between methylglyoxal and carbohydrate metabolism its pathological role in the events of the development of diabetic complications emerges in a natural manner and further progress is hoped in this field. In contrast, significant advancement cannot be expected in relation to cancer research.

Possible physiological roles of acetone metabolism in humans.

Recently, the metabolic routes for acetone metabolism have been described. Therefore, acetone cannot further be regarded as a waste product of metabolism. However, its physiological role in biochemical machinery is not clear. Here, an integrative model for the role of acetone metabolism is presented that orders the events occurring in acetonemia in sequence: (i) acetone participates in pH regulation; and (ii) acetone degradation in the liver both contributes to glucostatic function of the liver and provides C3 fragments to peripheral tissues as additional fuel. The model raises a novel approach to the study of the physiological role of acetone metabolism.

A possible evolutionary role of formaldehyde.

Formaldehyde is a compound which is believed to have had a role in evolutionary processes. On the other hand, the (methyl)glyoxalase pathway is a route being present in all biological organisms whereas its function has not yet been recognized in the biochemical machinery. In this article it is raised that (methyl)glyoxalase path might have functioned as a bridge between formose and archaic reductive citric acid cycles in surface metabolists at the early stage of evolution. According to the theory, formaldehyde was essential for the mentioned system as a raw molecule. Based on thermodynamic calculations a simple way of regulation is also shown. The simplicity of the theory may be in a good agreement with and an explanation of why the (methyl)glyoxalase system is of ubiquitous nature.

On the promine/retine theory of cell division: now and then.

Although the glyoxalase system was discovered in 1913, its function in the biological network is still a subject of debate. An attractive theory on its role was described by Albert Szent-Györgyi in the 1960s. From a bird's eye view, the promine/retine concept of Szent-Györgyi seems to give a plausible role for this ubiquitous enzyme system, but on going into detail, it obviously suffers from several uncertainties which have not been discussed until now. Here, a critical overview of the theory is presented by taking the pros and cons into account. It looks as though more data object to the theory than give support to it; and the search for anticancer medicines stimulated by the theory has not resulted in a new way of treatment of tumors, either. Hence, it is feared that the theory suggested for the biological role of glyoxalase pathway cannot be accepted, as it is.

From mineral support to enzymatic catalysis--further assumptions for the evolutionary history of glyoxalase system.

In a previous paper we suggested that the methylglyoxalase pathway might have been an anaplerotic route for the archaic reductive citric acid cycle of surface metabolists at the early stage of evolution. The present paper tries to outline a possible way for the further development of enzyme catalysed glyoxalase path by separating the global process into several plausible evolutionary stages. A sequence of events is proposed which might have led to the emergence of energy rich bonds, especially to the formation of the thiol-ester bond. An explanation is given for the cofactor function of transition metals, as well. A proposition is also made for how nature may preserve molecular mechanisms using them for different purposes if the innovation has proved successful.

An evolutionary role of formaldehyde.

The evolution can be divided into three stages: chemical, prebiological and biological evolution. Most of the problems emerge when the development of cellular organization, the so-called prebiological evolution, is investigated. Here the possible evolutionary roles for formaldehyde as well as for the methylglyoxalase pathway are proposed. The theory, on the one hand, ascertaines a pathway serving as an anaplerotic route for the reductive citric acid cycle of surface metabolists and using formaldehyde as raw molecule. On the other hand, an explanation for the glyoxalase enigma is offered hoping that in this way a long lasting mystery of almost nine decades biochemical research can be solved.

[Acetone metabolism: biochemistry, toxicology and clinical implications]. / Aceton-anyagcsere: biokémia, toxikológia és klinikai vonatkozások.

In the article, the routes of acetone metabolism are summarized and the conditions in which acetonemia occurs, are taken into account. Acetonemiae are classified as to be of exogenous and endogenous origin, and the biochemical mechanisms of the development of different acetonemiae are analyzed. Referring to the medical significance of acetone and isopropanol it is suggested that the previous view on those should be revised. As a summary, a hypothesis is raised according to which the physiological role of acetone production would be, on the one hand, to regulate the pH buffering capacity of body fluids and, on the other hand, in ketotic states emerging in diseases, to provide additional fuel for the particularly sensitive tissues (e.g. brain).

Polyadenylated mRNA in Escherichia coli: modulation of poly(A) RNA levels by polynucleotide phosphorylase and ribonuclease II.

The effect of 3'-exoribonucleases on the polyadenylation of mRNA in Escherichia coli was studied by comparing the synthesis and levels of poly(A) RNA in wild-type E coli and mutant strains defective in the two major 3'-exoribonucleases: polynucleotide phosphorylase and ribonuclease II. Mutations which substantially reduced the activity of these 3'-exonucleases caused a 10-fold increase in pulse-labeling of total poly(A) RNA in intact cells. When the net rate of RNA synthesis was measured in permeabilized cells, the mutant with defective 3'-exonucleases showed 20- to 60-fold increased synthesis of total poly(A) RNA as well as of specific polyadenylated mRNAs, with less than two-fold changes in non-poly(A) RNA. Measurement of mRNA polyadenylation in permeable cells under conditions when 3'-exoribonucleases were inactive showed a 6-fold higher rate of poly(A) synthesis in the exonuclease-deficient mutant strain, suggesting a higher concentration of mRNA 3'-ends amenable to polyadenylation. Steady-state levels of poly(A) RNA, measured by the ability to serve as template for oligo(dT)-dependent complementary DNA synthesis, also increased more than 40-fold when the 3'-exonucleases were inactivated. Monitoring of the length of the poly(A) tracts by denaturing polyacrylamide gel electrophoresis showed chain lengths of up to 45 residues in the 3'-exonuclease-deficient mutant, whereas most of the poly(A) tracts in the parent strain were shorter than 12 residues. These results show that 3'-exonucleases reduce the level of polyadenylated mRNA in E coli not merely by causing its degradation but also by reducing its rate of synthesis, presumably by competing with poly(A) polymerase for the 3'-ends of mRNA.

Identification of ribosomal protein S1 as a poly(A) binding protein in Escherichia coli.

To elucidate the metabolic function of mRNA polyadenylation in Escherichia coli. we searched for a polyadenylate-binding protein as a potential mediator of the function of the poly(A) moiety. Using a nitrocellulose filter-binding assay and a Northwestern blot technique, a protein in the ribosomal supernatant fraction of E coli was identified and purified to homogeneity. N-terminal sequence analysis yielded a 25-residue sequence which corresponded to the 25 N-terminal amino acids of protein S1, one of the proteins of the E coli 30S ribosomal subunit. Poly(A) binding to S1 protein was inhibited by Mg2+ and Mn2+ and by ATP and stimulated 8-fold by 100 mM KCl. The binding of S1 to poly(A) occurred with an association constant of 3 x 10(6) M-1 and seemed to be only mildly cooperative. Competition studies of the binding of poly(A) and poly(C) to purified S1 protein were consistent with the presence of two polynucleotide binding sites, of which one binds poly(A) five times more strongly than poly(C), whereas the other binds poly(C) 50 times more strongly than poly(A). Poly(A) bound to 30S ribosomal subunits but not to 50S ribosomes. To study possible association of S1 with the poly(A) tracts of E coli mRNA in the process of translation, poly(A) RNA was isolated from polysomes by oligo(dT) cellulose chromatography and the poly(A) RNA with bound protein was eluted either directly or after digestion with RNase T1 and A. When subjected to Western blot analysis with antibody to S1, both poly(A) RNA and isolated poly(A) tracts revealed bound S1 protein. The implications of these results for the possible interaction of poly(A) tracts of mRNA and the translational machinery of E coli are discussed.

Glucose formation from methylglyoxal in hepatocytes from streptozotocin-induced diabetic mice: the effect of insulin.

Acetol and methylglyoxal are intermediates of the intrahepatic metabolism of acetone leading to pyruvate formation. In hepatocytes prepared from fasted streptozotocin-induced diabetic mice, net glucose production could be measured from methylglyoxal but not from acetone or acetol. Insulin increased glucose formation from methylglyoxal in a concentration-dependent manner, whereas it was ineffective when pyruvate was used as substrate. Drug oxidation, as evidenced by p-aminophenol formation from aniline, was enhanced by methylglyoxal, and insulin proved to be stimulatory in this case as well. It is concluded that insulin might be involved in the regulation of glucose formation from methylglyoxal, but its mode of action is not yet clear.

Increased oxidation and decreased conjugation of drugs in the liver caused by starvation. Altered metabolism of certain aromatic compounds and acetone.

Starvation causes several changes in the various processes of biotransformation. The focus of this review is on biotransformation of various aromatic and other compounds whose metabolism is catalyzed in phase I by isozymes belonging to the CYP2E1 gene subfamily, while in phase II phenol-UDPGT or conjugation with GSH play a dominant role. The other ways of conjugation are beyond the scope of this review. The reason why this aspect has been chosen is that the capacity of these reactions is profoundly altered by nutritional conditions. There is a balance between the two phases of biotransformation. Therefore, under standard circumstances in a well-fed state the intermediate formed in the course of phase I is converted to a conjugated compound rapidly, as a result of phase II. However, in starvation the pattern of drug metabolism is altered and the balance between the two phases is changed. This alteration of drug metabolism upon starvation is partly connected to the changes of cofactor supplies due to the metabolic state.

Net glucose production from acetone in isolated murine hepatocytes. The effect of different pretreatments of mice.

1. To evaluate the condition under which net glucose production from acetone, added as sole substrate, occurs different pretreatments of mice, in combination with starvation, were used; (i) acetone pretreatment (acetone is a known inducer of cytochrome P-450 isozymes involved in this pathway), (ii) fructose pretreatment (to induce NADPH+H+ generating enzymes) or (iii) their combination. 2. There was net glucose formation from acetone only in that case, when the cells were prepared from 48 hr fasted animals pretreated with both acetone and fructose. However, using 2-14C-acetone, incorporation of 14C-carbon into glucose could be detected in all the cases and, at the same time, acetone was without any effect on protein synthesis. 3. The addition of acetone increased gluconeogenesis from alanine in almost all the cases. The only exception from this general rule was that the case, when hepatocytes were prepared from acetone pretreated 48 hr starved mice where, instead of the elevation of glucose formation, a decrease of that was caused by acetone. 4. Acetone decreased 14C-carbon incorporation into glucose from 14C-(U)-alanine added at saturating concentration in hepatocytes prepared from starved mice. 5. Similarly to acetone there was no net glucose formation from acetone either when added alone, however, it enhanced gluconeogenesis from alanine at non-saturating concentrations of the amino acid. 6. Methylglyoxal proved gluconeogenic in all the cases. 7. It is concluded that net glucose formation from acetone as sole substrate occurs only under those conditions which are far from a physiological situation, however, when gluconeogenesis from another substrate takes place, acetone can contribute to net glucose formation in hepatocytes prepared from fasted mice.

Methylglyoxal and cell viability.

Methylglyoxal by depleting glutathione stores increased Trypan-blue uptake by the cells incubated in glucose, pyruvate and amino acids free medium. Only a transient fall of glutathione concentration without any effect on cell viability was caused by methylglyoxal when the medium was supplemented with above-mentioned compounds. The role of gamma-glutamyl-transpeptidase is discussed.

Methylglyoxal toxicity in mammals.

Although the interest is growing towards glyoxalases and methylglyoxal, their role in metabolism is still an enigma. In this paper, the effects of methylglyoxal in both in vivo and in vitro mammalian systems are reviewed and correlated with its interaction with macromolecules (nucleic acids, proteins). The theories on the role of methylglyoxal and glyoxalases are also discussed. Recently, data obtained have focused attention on the possible role of disturbed methylglyoxal metabolism in the development of diabetic complications and it is hoped that the contribution of methylglyoxal to pathological events can be ascertained in the near future.

[D-lactate, D-lactic acidosis. Biochemical review of D-lactic acid metabolism and its clinical implications]. / D-laktát, D-laktacidózis. A D-tejsav anyagcsere biokémiai áttekintése és klinikai vonatkozásai.

D-lactate is an intermediate of methylglyoxal metabolism and the article gives an overview of D-lactatate metabolism as well as discusses the possible pathological role of this lactate stereoisomer. The increase of D-lactate concentration in a given body fluid can be used as a diagnostic sign, especially in patients who underwent small bowel resection or bypass operation or who have infections or suffer from certain metabolic disorders. Determination of D-lactate concentration should be considered as a useful tool both in the diagnosis and in controlling of the management of infectious diseases, particularly in those when the smear was negative or when the patient has already received antibiotics. Spite of these, the clinical value of the measurement of D-lactate concentration cannot, however, be evaluated yet.

Identification of a second poly(A) polymerase in Escherichia coli.

A second poly(A) polymerase (PAP II) has been identified in Escherichia coli using a strain carrying a deletion of pcnB (the structural gene for PAP I; Cao and Sarkar, 1992b) and pnp-7 (a null mutation in the structural gene for polynucleotide phosphorylase). While PAP I has a M(r) of 53,000, PAP II is a smaller protein with a native M(r)-35,000. PAP II differs from PAP I in preferring poly(A) over tRNA primers and being more thermolabile. The presence of multiple poly(A) polymerases in E. coli raises interesting questions regarding the role of polyadenylation in mRNA synthesis and decay.

[Submolecular theory of cancer. In memoriam Albert Szent-Györgyi]. / A rák szubmolekuláris elméletéröl. In memoriam Szent-Györgyi Albert.

The author surveys the way leading to the electronic theory of cancer and analyses the role of methylglyoxal and glyoxalases. Based on the comparison of experimental data with the theory it can be concluded that (i) data being at disposal are not convincing enough either to verify or to confute the theory; (ii) compounds having function to influence the glyoxalase system can, however, be considered as tumour-selective anticancer agents.

Has reactive oxygen a role in methylglyoxal toxicity? A study on cultured rat hepatocytes.

The toxicity of methylglyoxal and its ability to generate reactive oxygen species were investigated in cultured rat hepatocytes. Under aerobic and anaerobic conditions methylglyoxal increased lactate dehydrogenase (LDH) release and trypan blue uptake in a concentration dependent manner. Those concentrations of methylglyoxal causing cell injury (1 mM <) also caused the release of reactive oxygen species as indicated by peroxidase-catalyzed luminol chemiluminescence. Release of reactive oxygen was detectable only under aerobic conditions, and only became significant when a large portion of the cells had already lost their viability. It is concluded that methylglyoxal injuries cultured rat hepatocytes and induces the generation of reactive oxygen species. The reactive oxygen species, however, are essentially not involved in methylglyoxal hepatotoxicity but are released by already severely injured cells.

Gluconeogenesis from methylglyoxal in isolated murine hepatocytes. Does an alternative pathway exist in which pyruvate is not an intermediate?

1. Gluconeogenesis from alanine can be prevented by the addition of monoiodo acetic acid (an inhibitor of glyceraldehyde 3-phosphate dehydrogenase), while glucose production from methylglyoxal is only partially inhibited by this compound. 2. It is supposed that methylglyoxal can enter gluconeogenic sequence not only at pyruvate.