Study On The Metabolism Of Mandelic Acid In Vitro

In the present study, the enzyme types for the stereoselectivity metabolism of mandelic acid were firstly studied by invitro metabolism models. The enzymes that could possibly catalyze the stereoselectivity metabolism of mandelic acid were speculated. The recombinant human and rat L-2-hydroxy acid oxidase isozyme B were cloned and expressed successfully. The difference in enzyme kinetics of mandelic acid catalyzed by the two enzymes was compared and the enzyme kinetics characteristics of different mandelic acid analogues were further studied.Objective To study the enzyme types for the stereoselectivity metabolism of mandelic acid and speculate the enzymes that catalyze this reaction. To clone and express human and rat L-2-hydroxy acid oxidase isozyme B, compare the difference in enzyme kinetics of mandelic acid catalyzed by the two enzymes. To study the effect of the substrates’ structures on enzyme kinetics characteristics of the two enzymes.Methods1) The S9fractions of different tissues and mitochondrion and cytosol of rat liver were prepared by gradient centrifugation. After incubation with L-mandelic acid, the metabolism amounts of L-mandelic acid were investigated. The effect of specific inhibitors of different enzymes on the metabolism amounts of L-mandelic acid were investigated and then verified by commercial enzymes.2) The human HAOX2gene was amplified from human liver cDNA, and was cloned into pMAL-c2p vector as pMALc2p-hHao2recombinant plasmid. The rat LCHAO gene was amplified from rat kidney cDNA, and was cloned into pET-28a(+) vector as pET28a-rHao2recombinant plasmid. Then the recombinant plasmids were transformed into E. coli strain Rosetta(DE3). The expression of recombinant protein was induced by IPTG, and purified by affinity column. It was confirmed by SDS-PAGE and western blot assay. The kinetic parameters of classic substrates were determined by measuring the change of DCIP in A605to identify the enzyme activities.3) The recombinant human and rat L-2-hydroxy acid oxidase isozyme B were incubated with mandelic acid and analogues. The metabolism amounts of different substrates were investigated. Series concentrations of active compounds were incubated with the recombinant human and rat L-2-hydroxy acid oxidase isozyme B. The enzyme kinetic parameters for each substrate were calculated, regarding the formation rate of metabolites as metabolic rate.Results1) L-mandelic acid could be metabolized in rat liver S9, kidney S9, liver mitochondrion and cytosol protein, but couldn’t be metabolized in lung and intestine S9. The metabolism of L-mandelic acid in each fraction could be inhibited by the inhibitors of malic dehydrogenase, oxaloacetate and L-malate and the inhibitors of L-2-hydroxy acid oxidase, sodium diethyldithiocarbamate, hydroxylamine hydrochloride and trans-cinnamate. However, L-mandelic acid couldn’t be metabolized in commercial human malic dehydrogenase.2) The pMALc2p-hHao2, pET28a-rHao2(β,) and pET28a-rHao2(β2) recombinant plasmids were successfully constructed and the human HAOX2and rat LCHAO isozymes (β1,β2were successfully induced and expressed. SDS-PAGE analysis showed that the purity of human HAOX2was more than85%and there was only a single band of rat LCHAO after purification. They were both identified as corresponding target protein by western blotting. The Km value of2-hydroxyoctanoic acid was (0.69±0.07) mmol·L-1for human HAOX2. The Km values of2-hydroxyoctanoic acid were (25.1±1.9) and (24.6±1.2) μmol·L-1for the rat LCHAO isozymes β1and β2respectively. The Km values of L-2-hydroxyisocaproic acid were (24.6±2.3) and (22.4±1.6) μmol·L-1respectively.3) The human and rat L-2-hydroxy acid oxidase isozyme B could catalyze the metabolism of L-mandelic acid, L-2-chloromandelic acid and L-3-phenyllactic acid, but couldn’t catalyze the metabolism of L-methyl mandelate. The Km values of L-mandelic acid were (264.6±7.5),(47.2±3.1) and (43.3±2.6)μmol·L-1for the human HAOX2and rat LCHAO isozymes β1, β2, respectively. The Km values of L-2-chloromandelic acid were (47.6±2.2),(20.7±0.7) and (20.5±1.7)μmol·L-1, respectively. The Km values of L-3-phenyllactic acid were (29.9±1.1),(88.9±5.7) and (127.8±7.8)μmol·L-1, respectively.Conclusion The microsmal enzyme is not involved in the stereoselectivity metabolism of mandelic acid, and L-2-hydroxy acid oxidase may catalyze the stereoselectivity metabolism of L-mandelic acid in rats. Human HAOX2and rat LCHAO isozymes β1, β2were successfully cloned and expressed with high purity and good enzyme activities. The enzyme kinetic profiles of these enzymes are different for L-mandelic acid and analogues. The order of catalytic efficiency for human HAOX2is L-3-phenyllactic acid> L-2-chloromandelic> L-mandelic acid. The order of catalytic efficiency for rat LCHAO, however, is reverse. It may be of relevance to the differences in active amino acid residues and loop4in human and rat L-2-hydroxy acid oxidase isozyme B crystal structures.