Enzymatic reduction of delta-keto acids to the corresponding optically active hydroxy acids.
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Hydroxy Acids↗
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Enhancement of hydroxyl radical generation in the Fenton reaction by alpha-hydroxy acid.
The effect of various organic acids on hydroxyl radical (.OH) generation in the Fenton reaction were examined by the ESR spin trapping technique, where 5,5-dimethyl-1-pyroline-N-nitroxide (DMPO) and alpha-phenyl-tert-butyl nitrone (PBN) were used as the spin trapping reagents. alpha-Hydroxy acids such as lactic acid, glycolic acid and 2-hydroxy isobutyric acid were found to markedly enhance .OH generation in the reaction. In contrast, beta-hydroxy acid, alpha-keto acid, esters of alpha-hydroxy acids, aldehydes and other straight chain organic acids had no such enhancing activity. alpha-Amino acids had also no enhancing effect. The results suggest that the alpha-hydroxy acid moiety is prerequisite for the enhancement of .OH generation in the Fenton reaction. Superoxide dismutase did not inhibit the enhancing effect of alpha-hydroxy acids whereas catalase completely inhibited the .OH generation. Thus, alpha-hydroxy acids directly enhanced the .OH generation via the Fenton reaction but not the Haber-Weiss reaction. Possible role of lactic acid manipulating .OH generation is discussed in relation to the ischemia-reperfusion cell damage.
Catalase↗
The role of a beta barrel loop 4 extension in modulating the physical and functional properties of long-chain 2-hydroxy-acid oxidase isozymes.
Peroxisomal long-chain 2-hydroxy-acid oxidase, an FMN-dependent enzyme, catalyzes the oxidation of a variety of L-2-hydroxy acids into keto acids at the expense of oxygen. We recently reported the cloning and sequencing of its CDNA and the existence of a weakly expressed isozyme [Belmouden, A., Le, K. H. D., Lederer, F. & Garchon, H. J. (1993) Eur. J. Biochem. 214, 17-251. This isozyme, beta 2 differs from the major one in having a three-residue insertion, -VRK-, in loop 4 of the beta 8 alpha 8 barrel. In the crystal structures of homologous flavocytochrome beta 2, and glycolate oxidase, the corresponding region of loop 4 is disordered. We now report on the constitutive high-level expression of isozymes beta 1, and beta 2 in Escherichia coli under control of the lambda pL promoter, and on the influence of the E. coli genetic background and the growth medium on the expression level. We describe the properties of isozyme beta 2 and compare them with those of pure isoform beta 1. The visible spectra of the purified enzymes differ in the position of the near-ultraviolet band of the prosthetic group. pH titration studies indicate that the FMN ionizes at N3 at a lower pH than free flavin and that there is a small pKa difference between the isozymes. To our knowledge, the only other known case of a lowered pKa for the protein-bound flavin is that of glycolate oxidase. In the CD spectra of the FMN region, a marked difference between isozymes in the 270-300-nm region appears to be related to the pKa difference for the N3-H bond. Kinetic parameters for a number of substrates and inhibitors are indistinguishable within the limits of experimental error, with the exception of values for kcat for mandelate (the most active substrate), Km for hydroxyhippurate (a new substrate), Ki for cinnamate and oxalate, and Kd for sulfite. The differences are no larger than twofold. The foregoing comparison between isozymes beta 1 and beta 2 shows that the naturally engineered insertion in loop 4 exerts some influence on the flavin spectral properties and the active-site reactivity. Since the corresponding loop 4 regions in the three-dimensional structures of flavocytochrome 2 and glycolate oxidase are 1.5-2.0 nm removed from the flavin, it would appear either that loop 4 has a very different conformation in hydroxy-acid oxidase, or that it may interact with the active site due to mobility.
Absorption↗
Sensitive high-performance liquid chromatographic method for the determination of the lactone form and the lactone plus hydroxy-acid forms of the new camptothecin derivative DX-8951 in human plasma using fluorescence detection.
A sensitive quantitation of the lactone form and the lactone plus hydroxy-acid forms of DX-8951, a camptothecin derivative, in human plasma has been investigated by high-performance liquid chromatography (HPLC). This assay method consisted of two analytical procedures. In Procedure I, the lactone form was collected by the stepwise separation on a C18 cartridge. In Procedure II, the lactone plus hydroxy-acid forms were collected using another batch of the plasma sample by co-elution of the two forms from a C18 cartridge with acidic solution. The hydroxy-acid form of DX-8951 was quantitated from the difference of the lactone plus hydroxy-acid forms and the lactone form. Thereafter, these pre-treated samples were assayed by HPLC under the same HPLC conditions with a spectrofluorometer and a reverse-phase ODS column. The mobile phase was acetonitrile/0.05 M potassium dihydrogen phosphate (pH 3) (18:82, v/v) at a flow-rate of 1.0 ml/min. For the assay of the lactone form and the lactone plus hydroxy-acid forms of DX-8951 in plasma, analytical method were validated over the range 0.2-50 ng/ml.
Antineoplastic Agents, Phytogenic↗
A theory for the mechanism of action of the alpha-hydroxy acids applied to the skin.
A theory for the mechanism of action of alpha-hydroxy acids topically applied to the skin is proposed on the basis of an analysis with the experimental and clinical data. The alpha-hydroxy acids reduce the calcium ion concentration in the epidermis and remove calcium ions from the cell adhesions by chelation. This causes a loss of calcium ions from the cadherins of the desmosomes and adherens junctions, from the tight junctions, and possibly also from other divalent metallic cation-dependent cell adhesion molecules. The cell adhesions are thereby disrupted, resulting in desquamation. Desquamation is enhanced by cleavage of the endogenous stratum corneum chymotryptic enzyme on the cadherins, which are otherwise protected from proteolysis by conjugation with calcium ions. The decrease of calcium ion level so brought about in the epidermis also tends to promote cell growth and retard cell differentiation, giving rise to a younger-looking skin. This property of alpha-hydroxy acids suggests that caution should be taken with excessive and chronic use of these compounds and studies in this regard are warranted. Alpha-hydroxy acids may also possess anti-inflammatory capacities.
Cations, Divalent↗
Oxidation of cortisol to hydroxy acid metabolites by liver cytosol.
Cytosols (post-microsomal supernatants) prepared from rat, hamster and mouse livers oxidized cortisol to 11 beta, 17, 20-trihydroxy-3-oxo-pregn-4-en-21-oic acids. Mouse liver enzymes yielded over 90% 20 alpha-hydroxy epimer from cortisol, 21-dehydrocortisol (11 alpha, 17-dihydroxy-3,20-dioxo-pregn-4-en-21-aldehyde), and 20 alpha-isocortisol (11 alpha, 17, 20 alpha-trihydroxy-3-oxo-pregn-4-en-21-aldehyde). The 20 beta-epimer of isocortisol yielded both 20 alpha- and 20 beta-hydroxy acid. Rat and hamster liver cytosols converted, 21-dehydrocortisol and 20 alpha-isocortisol to both 20 alpha and 20 beta-hydroxy acids, with the former predominant. The hamster enzyme oxidized 20 beta-isocortisol mainly to the 20 beta-hydroxy acid. The results support our conclusion that both 17 alpha-hydroxy and 17-deoxy corticosteroids are oxidized to hydroxy acids by similar pathways and that isosteroids are obligatory intermediates.
Animals↗
Simultaneous determination of alpha and beta hydroxy acids in personal care products by capillary gas chromatography.
A simple and rapid procedure is described for the isolation, silylation, and simultaneous capillary gas chromatographic quantitation of alpha hydroxy acids and beta hydroxy acids in various personal care products. The sample is dissolved in acidified N,N-dimethylformamide to simultaneously acidify/extract the hydroxy acids; a portion is then trimethylsilyl derivatized with BSTFA and quantified by capillary gas chromatography (GC) using flame ionization detection.
Chromatography, Gas↗
Molecular cloning and nucleotide sequence of cDNA encoding rat kidney long-chain L-2-hydroxy acid oxidase. Expression of the catalytically active recombinant protein as a chimaera.
Long-chain L-alpha-hydroxy acid oxidase from rat kidney is a member of the family of FMN-dependent alpha-hydroxy-acid-oxidizing enzymes. With the knowledge of the recently determined amino acid sequence, the cDNA encoding the enzyme has now been cloned using the polymerase chain reaction. The 1648-bp cDNA contains an open reading frame coding for the 352 residues of the previously determined sequence, preceded by a methionine codon. In addition, several clones were found to present a nine-base insertion, predicting the existence of an isoform with a tripeptide VRK inserted between residues 188 and 189 of the mature protein. The presence of about 10% of this isoform in the oxidase purified from rat kidney was indeed identified by amino acid sequencing. A recombinant active enzyme was obtained as a protein fused to glutathione S-transferase using the bacterial expression plasmid pGEX-3X. Physico-chemical characterization indicated, for the fused enzyme, properties similar to those of the rat kidney protein. When the chimaera was submitted to factor Xa, proteolysis at the engineered cleavage point was poor. Separation of hydroxy acid oxidase from glutathione S-transferase could not be achieved with trypsin either. With both proteases, the initial cleavage point appeared to be in a peptide loop internal to the hydroxy acid oxidase sequence, close to or in the tripeptide insertion locus and not at the engineered factor-Xa-cleavage point. Comparative tryptic proteolysis of the rat kidney enzyme yielded a form cleaved in the same loop.
Alcohol Oxidoreductases↗
Alpha hydroxy acids: procedures for use in clinical practice.
Alpha hydroxy acids and alpha keto acids applied topically in lower concentrations reduce the thickness of hyperkeratotic stratum corneum by reducing corneocyte cohesion at lower levels of the stratum corneum. This property permits efficient clinical control of dry skin, ichthyosis, follicular hyperkeratosis, and other conditions characterized by retention of stratum corneum. Applied topically in higher concentrations, these acids cause epidermolysis. This property provides a new alternative for treating seborrheic keratoses, keratoses commonly known as "age spots," actinic keratoses, and verrucae vulgares; all of which lesions involve distinct epidermal hyperplasia as well as retention of stratum corneum. Facial wrinkles can be modified with topical alpha hydroxy acids, applied in higher concentrations as office procedures, and concomitant daily home application of lower concentrations.
Acne Vulgaris↗
Quantitation of multiple pathways for the metabolism of nephrotoxic cysteine conjugates using selective inhibitors of L-alpha-hydroxy acid oxidase (L-amino acid oxidase) and cysteine conjugate beta-lyase.
In this study, we have established the selectivity of inhibitors for rat kidney cysteine conjugate beta-lyase and L-alpha-hydroxy acid oxidase (L-amino acid oxidase) and have used these inhibitors to explore the relative roles of these two enzymes in the metabolism of nephrotoxic cysteine conjugates by rat kidney homogenate. In addition, we have investigated the relationship between structure and the metabolism of toxic cysteine conjugates by purified rat kidney L-alpha-hydroxy acid oxidase. With purified enzyme, S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (PCBDC) was about four times more active than S(1,2-dichlorovinyl)-L-cysteine (DCVC). Three alkyl conjugates were less active than DCVC. Purified L-alpha-hydroxy acid oxidase was not inhibited by the beta-lyase inhibitor aminooxyacetic acid but was inactivated by 2-hydroxy-3-butynoate. PCBDC metabolism in rat kidney homogenate was inhibited 74% by aminooxyacetic acid and 42% by 2-hydroxy-3-butynoate, whereas DCVC metabolism was inhibited 77% by aminooxyacetic acid and 28% by 2-hydroxy-3-butynoate. However, only aminooxyacetic acid inhibited the binding of 35S label from [35S]DCVC. Based on these results we have reached three conclusions. First, L-alpha-hydroxy acid oxidase plays a significant role in the metabolism of some cysteine conjugates. Second, metabolism of DCVC by L-alpha-hydroxy acid oxidase does not contribute directly to covalent binding. Third, as much as 65% of DCVC may be metabolized to its corresponding alpha-keto acid. The results are discussed with regard to the nephrotoxicity of cysteine conjugates.
Amino Acid Oxidoreductases↗
Ligand and metal-ion effects in metal-ion clusters used for chiral analysis of alpha-hydroxy acids by the kinetic method.
Chiral recognition of alpha-hydroxy acids has been achieved, and mixtures of enantiomers have been quantified in the gas phase, by using the kinetics of competitive unimolecular dissociation of singly-charged transition metal ion-bound trimeric complexes, [M(II)(A)(ref*)(2)-H](+) (M(II)=divalent transition metal ion; A=alpha-hydroxy acid; ref*=chiral reference ligand), to form the dimeric complexes [M(II)(A)(ref*)-H](+) and [M(II)(ref*)(2)-H](+). Chiral selectivity, the ratio of these two fragment ion abundances for the complex containing the analyte in one enantiomeric form expressed relative to that for the fragments of the corresponding complex containing the other enantiomer, ranges from 0.65 to 7.32. Chiral differentiation is highly dependent on the choice of chiral reference compound and central metal ion. The different coordination geometry of complexes resulting from the different d-orbital electronic configurations of these transition metal ions plays a role in chiral discrimination. Of all the transition metal ions examined chiral recognition is lowest for Cu(II), because of large distortion of the coordination complexes, and hence weak metal-ligand interactions and small stereochemical effects. It seems that two independent pi-cation interactions occur when N-acetyl-substituted aromatic amino acids used as the reference ligands and this accounts for improved chiral discrimination. If both metal-ligand and ligand-ligand interactions are optimized, large chiral selectivity is achieved. The sensitive nature of the methodology and the linear relationship between the logarithm of the fragment ion abundance ratio and the optical purity, which are intrinsic to the kinetic method, enable mixtures to be analyzed for small enantiomeric excess ( ee) by simply recording the ratios of fragment ion abundances in a tandem mass spectrum.
Calibration↗
Asymmetric synthesis of beta-hydroxy acid via stereoselective dirhodium(II)-catalyzed C-H insertion of alpha-alkoxydiazoketone.
A new methodology for the asymmetric synthesis of beta-hydroxy acid was developed. Dirhodium(II)-catalyzed C-H insertion of alpha-alkoxydiazoketone (3), which was prepared from primary alkyl halide (1) and readily available chiral alpha-hydroxy acid (2), gave stereoselectively 2,5-cis-disubstituted 3(2H)-furanone (4). The Baeyer-Villiger reaction of 4 followed by treatment with an acid afforded chiral beta-hydroxy acid (6) with high optical purity.
Alkylation↗
Enzymatic synthesis of alpha-butylglucoside lactate: a new alpha-hydroxy acid derivative.
An alpha-hydroxy acid derivative, alpha-butylglucoside lactate, was successfully prepared by enzymatic transesterification of alpha-butylglucoside with a lactate alkyl ester in a non-aqueous medium using immobilized lipase as biocatalyst. Ester synthesis in organic solvent was optimized. Solvent choice was made on the basis of substrate solubility and enzyme stability in the medium. A solvent-free reaction using butyllactate as lactate donor led to the highest yields. In the presence of 0.5M alphabutylglucoside and 100 g/L Novozym(R), a 67 % yield could be obtained within 40 h at 50 degrees C. However, the presence of butanol by-product limited the reaction to a maximum that could not be exceeded in closed systems. The elimination of the alcohol under reduced pressure resulted in the complete equilibrium shift of the transesterification reaction in favor of synthesis; below 15 mbars, more than 95% of 0.5M alpha-butylglucoside could be converted within 30 h. Moreover, simultaneous evaporation of water allowed hydrolysis of butyllactate to be eliminated. Consequently, a very high alpha-butylglucoside lactate concentration (170 g/) could be obtained in a single batch reaction. A single purification procedure, consisting of butyllactate extraction with hexane, enabled the product to be obtained at a purity above 95% (w/w). 1H and 13C NMR analysis later demonstrated that lactic acid was exclusively grafted onto the primary hydroxyl group of alphabutylglucoside.
Bioreactors↗
Determination of alpha hydroxy acids in fruits by capillary electrophoresis.
Alpha hydroxy acids, malic acid, citric acid, tartaric acid, glycolic acid and lactic acid, were analyzed simultaneously using capillary electrophoresis with direct UV detection at 200 nm. The separation was carried out with uncoated fused-silica (50 cm x 50 microns i.d.), pressure injection at 15 psi s and operated at -15 kV potential. The separation buffers were prepared with 180 mM Na2HPO4, 1 mM cetyltrimethylammonium bromide and 15% (v/v) methanol and adjusted to pH 7.2 by phosphoric acid. Validation was performed for citric acid and malic acid. The obtained parameters were adequate and the limits of detection were 2.5 and 5 micrograms ml-1 for citric acid and malic acid, respectively. AHAs from natural fruit juices (orange and grape) were determined and measured with this method.
Buffers↗
Stereoselective antibodies to free alpha-hydroxy acids.
This work describes antibodies exhibiting high stereoselectivity and class-specificity towards the enantiomers of free alpha-hydroxy acids. Since the antibodies interact primarily with the carboxyl-hydroxyl-hydrogen triad about the stereogenic center, they are useful for enantiomer analysis of a variety of structurally different alpha-hydroxy acids including aromatic and aliphatic compounds, e.g. lactic acid. The utility of such antibodies for enantiomer separation in chromatography was demonstrated. Comparative studies of these and previously described anti-alpha-amino acid antibodies revealed that both types of antibodies bind only to analytes that possess both the corresponding target structure and the correct configuration. Thus, substitution of an amino group for the alpha-hydroxyl group results in a complete loss of binding activity with the anti-alpha-hydroxy acid antibodies, while an alpha-amino group is essential for the interaction between analytes and anti-alpha-amino acid antibodies.
Animals↗
Observations on the elimination of water from 2-hydroxy acids in the metabolism of amino acids by Clostridium sporogenes.
Cell-free extracts of Clostridium sporogenes catalyse the water elimination from (2R)-phenyllactate in the presence of one of the energy-rich compounds acetyl-CoA, acetylphosphate or ATP and coenzyme A. Water is eliminated from (2R)-phenyllactoyl-CoA without any of the aforementioned additions. Cinnamoyl-CoA also acts catalytically. One molecule of cinnamoyl-CoA causes the elimination of water from more than 8 molecules phenyllactate. This is important from an energetic point of view since less than 2 mol ATP are formed per 2-3 mol metabolized amino acids. An activation of the hydroxy group of the alpha-hydroxy acid in form of a phosphate ester can also be excluded for energetic reasons.
Adenosine Triphosphate↗
Fatty acid metabolism in Serratia marcescens. II. The occurrence of hydroxy acids.
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Fatty Acids↗
The NAD-linked aromatic alpha-hydroxy acid dehydrogenase from Trypanosoma cruzi. A new member of the cytosolic malate dehydrogenases group without malate dehydrogenase activity.
Trypanosoma cruzi, the protozoan parasite causing Chagas disease, contains a novel aromatic alpha-hydroxy acid dehydrogenase. This enzyme is responsible, together with tyrosine aminotransferase, for the catabolism of aromatic amino acids, which leads to the excretion of aromatic lactate derivatives into the culture medium. The gene encoding the aromatic alpha-hydroxy acid dehydrogenase has been cloned through a combined approach using screening of an expression genomic library with antibodies, peptide sequencing and PCR amplification. Its sequence shows high similarity to the cytosolic malate dehydrogenases. However, the enzyme has no malate dehydrogenase activity. The gene seems to be present in a single copy per haploid genome and is differentially expressed throughout the parasite's life cycle, the highest levels being found in the insect forms of T. cruzi. The purified recombinant enzyme, expressed in Escherichia coli, was unable to reduce oxaloacetate and had kinetic constants similar to those of the natural aromatic alpha-hydroxy acid dehydrogenase. Sequence comparisons suggest that the aromatic alpha-hydroxy acid dehydrogenase derives from a cytosolic malate dehydrogenase no longer present in the parasite, made redundant by the presence of a glycosomal malate dehydrogenase as a member of a shuttle device involving the mitochondrial isoenzyme.
Alcohol Oxidoreductases↗