The formation of oxalate from glycolate in rat and human liver

In this study, we attempted to elucidate the metabolic pathway and enzymes actually involved in oxalate formation from glycolate in rat and human liver. In rat liver, the formation of oxalate from glycolate appeared to take place predominantly via glyoxylate. The oxalate formation from glycolate observed with crude enzyme preparations was almost entirely accounted for by the sequential actions of glycolate oxidase and xanthine oxidase (XOD) or lactate dehydrogenase (LDH). Under the conditions used, no significant activity was attributable to glycolate dehydrogenase, an enzyme reported to catalyze the direct oxidation of glycolate to oxalate. Among the three enzymes known to catalyze the oxidation of glyoxylate to oxalate, glycolate oxidase and XOD showed much lower activities (a higher Km and lower Vmax) toward glyoxylate than those with the respective primary substrates. As to LDH, none of the LDH subunit-deficient patients examined showed profoundly lowered urinary oxalate excretion. Based on the results obtained, the presumed efficacies in vivo of individual enzymes, as catalysts of glyoxylate oxidation, and the in vivo conditions assumed to allow their catalysis of oxalate production are discussed.     

Isolation and characterization of glycolic acid oxidase from human liver.

Glycolic acid oxidase has been isolated from human liver and purified over 3000-fold to a specific activity of 123 U/mg protein by a 5-step procedure. The preparation gave a single protein band on polyacrylamide gel electrophoresis, required flavin mononucleotide for catalytic activity, had a pH optimum between 8.2-8.8 depending on the substrate, and had a molecular weight of 105 000. The enzyme has a broad specificity towards alpha-hydroxy acids. Glycolate (Km = 3.3 . 10(-4) M) was the most effective substrate. The enzyme was stable for several months when stored as an (NH4)2SO4 precipitate or in 15% glycerol. Since glycolate inhibits the oxidation of glyoxylate to oxalate by glycolic acid oxidase, it is suggested that glycolic acid oxidase contributes to the synthesis of oxalate in vivo when the glyoxylate concentration is high and the glycolate concentration is low.     

植物中草酸积累与光呼吸乙醇酸代谢的关系

对几种C3 和C4 植物中草酸含量及相应的乙醇酸氧化酶活性测定结果表明 :叶片光呼吸强度及其关键酶活性大小与草酸积累量没有相关性 ;植物根中均能积累草酸 ,但未测出乙醇酸氧化酶活性。烟草根、叶中的草酸含量在不同生长时期差异明显 ,且二者呈极显著正相关 (y =2 .5 6 5lnx 2 .137,r =0 .749,P <0 .0 0 1) ,说明根中草酸可能来自叶片。氧化乙醇酸的酶的活性与氧化乙醛酸的酶的活性呈极显著线性正相关 (y =0 .2 41x 0 .0 0 6 ,r=0 .96 7,P <0 .0 0 0 1) ,进一步证实是乙醇酸氧化酶催化了两种底物的反应。烟草在不同生长期叶片中草酸总含量变化与相应的乙醇酸氧化酶活性变化亦没有相关性 ;低磷胁迫可显著诱导烟草根叶中的草酸形成和分泌 ,但并未影响乙醇酸氧化酶活性 ,进一步证明草酸积累与该酶活性大小无关     

Glycolate and glyoxylate metabolism in HepG2 cells

Oxalate synthesis in human hepatocytes is not well defined despite the clinical significance of its overproduction in diseases such as the primary hyperoxalurias. To further define these steps, the metabolism to oxalate of the oxalate precursors glycolate and glyoxylate and the possible pathways involved were examined in HepG2 cells. These cells were found to contain oxalate, glyoxylate, and glycolate as intracellular metabolites and to excrete oxalate and glycolate into the medium. Glycolate was taken up more effectively by cells than glyoxylate, but glyoxylate was more efficiently converted to oxalate. Oxalate was formed from exogenous glycolate only when cells were exposed to high concentrations. Peroxisomes in HepG2 cells, in contrast to those in human hepatocytes, were not involved in glycolate metabolism. Incubations with purified lactate dehydrogenase suggested that this enzyme was responsible for the metabolism of glycolate to oxalate in HepG2 cells. The formation of ¹⁴C-labeled glycine from ¹⁴C-labeled glycolate was observed only when cell membranes were permeabilized with Triton X-100. These results imply that peroxisome permeability to glycolate is restricted in these cells. Mitochondria, which produce glyoxylate from hydroxyproline metabolism, contained both alanine:glyoxylate aminotransferase (AGT)2 and glyoxylate reductase activities, which can convert glyoxylate to glycine and glycolate, respectively. Expression of AGT2 mRNA in HepG2 cells was confirmed by RT-PCR. These results indicate that HepG2 cells will be useful in clarifying the nonperoxisomal metabolism associated with oxalate synthesis in human hepatocytes. liver; peroxisomes; hepatocytes; hyperoxaluria; alanine:glyoxylate aminotransferase; glyoxylate reductase     

Hydroxyproline metabolism in mouse models of primary hyperoxaluria

Primary hyperoxaluria type 1 (PH1) and type 2 (PH2) are rare genetic diseases that result from deficiencies in glyoxylate metabolism. The increased oxalate synthesis that occurs can lead to kidney stone formation, deposition of calcium oxalate in the kidney and other tissues, and renal failure. Hydroxyproline (Hyp) catabolism, which occurs mainly in the liver and kidney, is a prominent source of glyoxylate and could account for a significant portion of the oxalate produced in PH. To determine the sensitivity of mouse models of PH1 and PH2 to Hyp-derived oxalate, animals were fed diets containing 1% Hyp. Urinary excretions of glycolate and oxalate were used to monitor Hyp catabolism and the kidneys were examined to assess pathological changes. Both strains of knockout (KO) mice excreted more oxalate than wild-type (WT) animals with Hyp feeding. After 4 wk of Hyp feeding, all mice deficient in glyoxylate reductase/hydroxypyruvate reductase (GRHPR KO) developed severe nephrocalcinosis in contrast to animals deficient in alanine-glyoxylate aminotransferase (AGXT KO) where nephrocalcinosis was milder and with a lower frequency. Plasma cystatin C measurements over 4-wk Hyp feeding indicated no significant loss of renal function in WT and AGXT KO animals, and significant and severe loss of renal function in GRHPR KO animals after 2 and 4 wk, respectively. These data suggest that GRHPR activity may be vital in the kidney for limiting the conversion of Hyp-derived glyoxylate to oxalate. As Hyp catabolism may make a major contribution to the oxalate produced in PH patients, Hyp feeding in these mouse models should be useful in understanding the mechanisms associated with calcium oxalate deposition in the kidney.     

光呼吸代谢物乙醇酸、乙醛酸和草酸对烟草叶片硝酸还原的影响

乙醇酸、乙醛酸和草酸能明显促进烟草(Nicotiana rustica)叶片在黑暗中的硝酸还原,光呼吸抑制剂a-羟基吡啶甲烷磺酸能消除前二者的促进作用而不能完全消除草酸的作用。草酸+NAD~+能显著促进离体的硝酸还原。烟叶提取液加入草酸和NAD~+后生成NADH和CO_2认为活体内由乙醛酸氧化生成的草酸是经脱氢生成NADH供硝酸还原之用。未能证明在烟叶内存在乙醇酸脱氨酶,因此排除由乙醇酸直接脱氢以还原硝酸的可能。     

The inhibition of oxalate biosynthesis in isolated perfused rat liver by DL-phenyllactate and n-heptanoate

Glycolate oxidase was isolated and partially purified from human and rat liver. The enzyme preparation readily catalyzed the oxidation of glycolate, glyoxylate, lactate, hydroxyisocaproate and α-hydroxybutyrate. The oxidation of glycolate and glyoxylate by glycolate oxidase was completely inhibited by 0.02 m dl-phenyllactate or n-heptanoate. The oxidation of glyoxylate by lactic dehydrogenase or xanthine oxidase was not inhibited by 0.067 m dl-phenyllactate or n-heptanoate. The conversion of [U-¹⁴C] glyoxylate to [¹⁴C] oxalate by isolated perfused rat liver was completely inhibited by dl-phenyllactate and n-heptanoate confirming the major contribution of glycolate oxidase in oxalate synthesis. Since the inhibition of oxalate was 100%, lactic dehydrogenase and xanthine oxidase do not contribute to oxalate biosynthesis in isolated perfused rat liver. dl-Phenyllactate also inhibited [¹⁴C] oxalate synthesis from [1-¹⁴C] glycolate, [U-¹⁴C] ethylene glycol, [U-¹⁴C] glycine, [3-¹⁴C] serine, and [U-¹⁴C] ethanolamine in isolated perfused rat liver. Oxalate synthesis from ethylene glycol was inhibited by dl-phenyllactate in the intact male rat confirming the role of glycolate oxidase in oxalate synthesis in vivo and indicating the feasibility of regulating oxalate metabolism in primary hyperoxaluria, ethylene glycol poisoning, and kidney stone formation by enzyme inhibitors.     

荞麦与大豆叶片中草酸含量差异及其可能的原因

用1/5浓度Hoagland营养液培养荞麦和大豆幼苗l0 d后,荞麦叶片、根及其分泌物中的草酸含量均明显高于大豆,说明荞麦叶片的草酸形成能力强.荞麦叶片中存在少量的草酸氧化酶活性,而大豆中未检测到该酶活性,表明荞麦具有一定的降解草酸的能力.乙醇酸氧化酶(GO)催化乙醇酸氧化的活性两种植物之间虽差异不明显,但该酶催化乙醛酸氧化的活性荞麦显著高于大豆.荞麦GO对乙醛酸的Km值明显低于大豆GO,同时其乙醛酸含量也较高,因此其叶片中由乙醛酸形成草酸的速率应高于大豆.由此认为,由乙醛酸氧化生成草酸可能是植物草酸合成限速步骤之一,其反应速率高低可能导致不同种类植物叶片中草酸含量的差异.     

Relationship between hydroxypyruvate and the production of oxalate in vitro.

Chicken liver lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC1.1.1.27) catalyses the reversible reduction reaction of hydroxypyruvate to L-glycerate. It also catalyses the oxidation reaction of the hydrated form of glyoxylate to oxalate and the reduction of the non-hydrated form of glyoxylate to oxalate and the reduction of the non-hydrated form to glycolate. At pH 8, these latter two reactions are coupled. The coupled system equilibrium is attained when the NAD+/NADH ratio is greater than unity. Hydroxypyruvate binds to the enzyme at the same site as the pyruvate. When there are substances with greater affinity to this site in the reaction medium and their concentration is very high, hydroxypyruvate binds to the enzyme at the L-lactate site. In vitro and with purified preparation of lactate dehydrogenase, hydroxypyruvate stimulates the production of oxalate from glyoxylate-hydrated form and from NAD; the effect is due to the fact that hydroxypyruvate prevents the binding of non-hydrated form of glyoxylate to the lactate dehydrogenase in the pyruvate binding site. At pH 8, THE L-glycerate stimulates the production of glycolate from glyoxylate-non-hydrated form and NADH since hydroxypyruvate prevents the binding of glyoxylate-hydrated form to the enzyme     

荞麦与大豆叶片乙醇酸氧化酶的纯化和性质比较研究

分别从荞麦与大豆叶片中部分纯化了乙醇酸氧化酶 (GO ,EC1 .1 .3 .1 ) ,并研究其部分性质。结果显示荞麦与大豆叶片中GO的催化特性有明显差异 :大豆叶片中GO对乙醇酸Km值为 0 .3 1mmol/L ,对乙醛酸Km值为 1 .98mmol/L。外源草酸对GO氧化乙醇酸活性影响很小 ,但对其氧化乙醛酸活性抑制明显 ,5mmol/L草酸可抑制 44%。而荞麦叶片中GO性质有所不同 :GO对乙醇酸Km为 0 .46mmol/L ,对乙醛酸Km为 0 .85mmol/L。草酸对荞麦GO氧化乙醇酸活性影响也很小 ,对其氧化乙醛酸活性的抑制作用明显小于大豆 ,5mmol/L草酸只抑制 2 4%。上述研究结果表明 ,荞麦GO对乙醛酸的亲和力明显强于大豆 ,并且草酸对其GO氧化乙醛酸活性影响较小。因此相对于大豆而言 ,GO可能在荞麦叶片草酸合成中起重要作用。     

The synthesis of oxalate from hydroxypyruvate by isolated perfused rat liver the mechanism of hyperoxaluria in L-lyceric aciduria

Hydroxypyruvate and glycolate inhibited the oxidation of [U-¹⁴C]glyoxylate to [¹⁴C]oxalate in isolated perfused rat liver, but stimulated total oxalate and glycolate synthesis. [¹⁴C]Oxalate synthesis from [¹⁴C]glycine similarly inhibited by hydroxypyruvate, but conversion of [¹⁴C₁]glycolate to [⁴C]oxalate was increased three-fold. Pyruvate had no effect on the synthesis of [¹⁴C]oxalate or total oxalate. The inhibition studies suggest that hydroxypyruvate is a precursor of glycolate and oxalate and that the conversion of glycolate to oxalate does not involve free glyoxylate as an intermediate. [¹⁴C₃]Hydroxypyruvate, but not [¹⁴C₁]hydroxypyruvate, was oxidized to [¹⁴C]oxalate in isolated perfused rat liver. Isotope dilution studies indicate the major pathway involves the decarboxylation of hydroxypyruvate forming glycolaldehyde which is subsequently oxidized to oxalate via glycolate. The oxidation of serine to oxalate appears to proceed predominantly via hydroxypyruvate rather than glycine or ethanolamine. The hyperoxaluria of L-glyceric aciduria, primary hyperoxaluria type II, is induced by the oxidation of the hydroxypyruvate, which accumulates because of the deficiency of D-glyceric dehydrogenase, to oxalate.     

Primary hyperoxaluria type 1 in Japan

Glyoxylate is an immediate precursor of oxalate, but in its metabolism the conversion into glycine catalyzed by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT) appears to be the main route. When SPT/AGT is missing as in the case of primary hyperoxaluria type 1 (PH1) more glyoxylate is used for the oxalate production, resulting in calcium oxalate urolithiasis and finally systemic oxalosis. SPT/AGT is a unique enzyme of species-specific dual organelle localization; it is located largely in mitochondria in carnivores and entirely in peroxisomes in herbivores and man. For herbivores, the peroxisomal localization of SPT/AGT is indispensable to avoid massive production of oxalate, probably because liver peroxisomes are the main site of glyoxylate production from glycolate, and plants contain glycolate much more than animal tissues. Recently, we took charge of laboratory examination for 8 cases of primary hyperoxaluria in Japan, and felt that symptoms of some of the Japanese PH1 patients are apparently milder than those of Western patients. The reason of this is not clear, but from the above mentioned seemingly indispensable association of grass-eating with the peroxisomal localization of SPT/AGT it may be related, at least in part, to the food habit of Japanese, especially that of old generation, that they prefer boiled greens rather than frying or raw vegetables.     

Oxalate accumulation from citrate by Aspergillus niger. I. Biosynthesis of oxalate from its ultimate precursor.

Carbon-14 was incorporated from citrate-1,5-14C, glyoxylate-14C(U), or glyoxylate-1-14C into oxalate by cultures of Aspergillus niger pregrown on a medium with glucose as the sole source of carbon. Glyoxylate-14C(U) was superior to glyoxylate-1-14C and citrate-1,5-14C as a source of incorporation. By addition of a great amount of citrate the accumulation of oxalate was accelerated and its maximum yield increased. In a cell-free extract from mycelium forming oxalate from citrate the enzyme oxaloacetate hydrolase (EC3.7.1.1) was identified. Its in vitro activity per flask exceeded the rate of in vivo accumulation of oxalate. Glyoxylate oxidizing enzymes (glycolate oxidase, EC1.1.3.1; glyoxylate oxidase, EC1.2.3.5;NAD(P)-dependent glyoxylate dehydrogenase; glyoxylate dehydrogenase, CoA-oxalylating, EC1.2.1.7) could not be detected in cell-free extracts. It is concluded that in cultures accumulating oxalate from citrate after pregrowth on glucose, oxalate arises by hydrolytic cleavage of oxaloacetate but not by oxidation of glyoxylate.     

Active site and loop 4 movements within human glycolate oxidase: implications for substrate specificity and drug design

Human glycolate oxidase (GO) catalyzes the FMN-dependent oxidation of glycolate to glyoxylate and glyoxylate to oxalate, a key metabolite in kidney stone formation. We report herein the structures of recombinant GO complexed with sulfate, glyoxylate, and an inhibitor, 4-carboxy-5-dodecylsulfanyl-1,2,3-triazole (CDST), determined by X-ray crystallography. In contrast to most alpha-hydroxy acid oxidases including spinach glycolate oxidase, a loop region, known as loop 4, is completely visible when the GO active site contains a small ligand. The lack of electron density for this loop in the GO-CDST complex, which mimics a large substrate, suggests that a disordered to ordered transition may occur with the binding of substrates. The conformational flexibility of Trp110 appears to be responsible for enabling GO to react with alpha-hydroxy acids of various chain lengths. Moreover, the movement of Trp110 disrupts a hydrogen-bonding network between Trp110, Leu191, Tyr134, and Tyr208. This loss of interactions is the first indication that active site movements are directly linked to changes in the conformation of loop 4. The kinetic parameters for the oxidation of glycolate, glyoxylate, and 2-hydroxy octanoate indicate that the oxidation of glycolate to glyoxylate is the primary reaction catalyzed by GO, while the oxidation of glyoxylate to oxalate is most likely not relevant under normal conditions. However, drugs that exploit the unique structural features of GO may ultimately prove to be useful for decreasing glycolate and glyoxylate levels in primary hyperoxaluria type 1 patients who have the inability to convert peroxisomal glyoxylate to glycine.     

Identification and characterization of HAOX1, HAOX2, and HAOX3, three human peroxisomal 2-hydroxy acid oxidases

Computer-based approaches identified three distinct human 2-hydroxy acid oxidase genes, HAOX1, HAOX2, and HAOX3, that encode proteins with significant sequence similarity to plant glycolate oxidase, a prototypical 2-hydroxy acid oxidase. The products of these genes are targeted to peroxisomes and have 2-hydroxy acid oxidase activities. Each gene displays a distinct tissue-specific pattern of expression, and each enzyme exhibits distinct substrate preferences. HAOX1 is expressed primarily in liver and pancreas and is most active on the two-carbon substrate, glycolate, but is also active on 2-hydroxy fatty acids. HAOX2 is expressed predominantly in liver and kidney and displays highest activity toward 2-hydroxypalmitate. HAOX3 expression was detected only in pancreas, and this enzyme displayed a preference for the medium chain substrate 2-hydroxyoctanoate. These results indicate that all three human 2-hydroxy acid oxidases are involved in the oxidation of 2-hydroxy fatty acids and may also contribute to the general pathway of fatty acid alpha-oxidation. Primary hyperoxaluria type 1 (PH1) is caused by defects in peroxisomal alanine-glyoxylate aminotransferase, the enzyme that normally eliminates intraperoxisomal glyoxylate. The presence of HAOX1 in liver and kidney peroxisomes and the ability of HAOX1 to oxidize glyoxylate to oxalate implicate HAOX1 as a mediator of PH1 pathophysiology.     

Primary hyperoxalurias: Disorders of glyoxylate detoxification

Glyoxylate detoxification is an important function of human peroxisomes. Glyoxylate is a highly reactive molecule, generated in the intermediary metabolism of glycine, hydroxyproline and glycolate mainly. Glyoxylate accumulation in the cytosol is readily transformed by lactate dehydrogenase into oxalate, a dicarboxylic acid that cannot be metabolized by mammals and forms tissue-damaging calcium oxalate crystals. Alanine-glyoxylate aminotransferase, a peroxisomal enzyme in humans, converts glyoxylate into glycine, playing a central role in glyoxylate detoxification. Cytosolic and mitochondrial glyoxylate reductase also contributes to limit oxalate production from glyoxylate. Mitochondrial hydroxyoxoglutarate aldolase is an important enzyme of hydroxyproline metabolism. Genetic defect of any of these enzymes of glyoxylate metabolism results in primary hyperoxalurias, severe human diseases in which toxic levels of oxalate are produced by the liver, resulting in progressive renal damage. Significant advances in the pathophysiology of primary hyperoxalurias have led to better diagnosis and treatment of these patients, but current treatment relies mainly on organ transplantation. It is reasonable to expect that recent advances in the understanding of the molecular mechanisms of disease will result into better targeted therapeutic options in the future. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of peroxisomes in Health and Disease.     

Evidence for the presence in tobacco leaves of multiple enzymes for the oxidation of glycolate and glyoxylate

The enzymic oxidation of glycolate to glyoxylate and glyoxylate to oxalate by preparations purified from tobacco (Nicotiana tabacum var Havana Seed) leaves was studied. The K_m values for glycolate and glyoxylate were 0.26 and 1.0 millimolar, respectively. The ratio of glycolate to glyoxylate oxidation was 3 to 4 in crude extracts but decreased to 1.2 to 1.5 on purification by (NH₄)₂SO₄ fractionation and chromatography on agarose A-15 and hydroxylapatite. This level of glyoxylate oxidation activity was higher than that previously found for glycolate oxidase (EC 1.1.3.1). The ratio of the two activities was changed by reaction with the substrate analog 2-hydroxy-3-butynoate (HBA) which at all concentrations inhibited glyoxylate oxidation to a greater extent than glycolate oxidation. The ratio of the two activities could also be altered by changing the O₂ concentration. Glycolate oxidation increased 3.6-fold when the O₂ atmosphere was increased from 21 to 100%, whereas glyoxylate oxidation increased only 1.6-fold under the same conditions. These changes in ratio during purification, on inhibition by HBA, and under varying O₂ concentrations imply that tobacco leaves contain at least two enzymes capable of oxidizing glycolate and glyoxylate.     

A preliminary account of the properties of recombinant human Glyoxylate reductase (GRHPR), LDHA and LDHB with glyoxylate, and their potential roles in its metabolism

Human lactate dehydrogenase (LDH) is thought to contribute to the oxidation of glyoxylate to oxalate and thus to the pathogenesis of disorders of endogenous oxalate overproduction. Glyoxylate reductase (GRHPR) has a potentially protective role metabolising glyoxylate to the less reactive glycolate. In this paper, the kinetic parameters of recombinant human LDHA, LDHB and GR have been compared with respect to their affinity for glyoxylate and related substrates. The Km values and specificity constants (Kcat/K(M)) of purified recombinant human LDHA, LDHB and GRHPR were determined for the reduction of glyoxylate and hydroxypyruvate. K(M) values with glyoxylate were 29.3 mM for LDHA, 9.9 mM for LDHB and 1.0 mM for GRHPR. For the oxidation of glyoxylate, K(M) values were 0.18 mM and 0.26 mM for LDHA and LDHB respectively with NAD+ as cofactor. Overall, under the same reaction conditions, the specificity constants suggest there is a fine balance between the reduction and oxidation reactions of these substrates, suggesting that control is most likely dictated by the ambient concentrations of the respective intracellular cofactors. Neither LDHA nor LDHB utilised glycolate as substrate and NADPH was a poor cofactor with a relative activity less than 3% that of NADH. GRHPR had a higher affinity for NADPH than NADH (K(M) 0.011 mM vs. 2.42 mM). The potential roles of LDH isoforms and GRHPR in oxalate synthesis are discussed.     

Metabolism of Glycolate in Isolated Spinach Leaf Peroxisomes : KINETICS OF GLYOXYLATE, OXALATE, CARBON DIOXIDE, AND GLYCINE FORMATION

The flow of glyoxylate derived from glycolate into various metabolic routes in the peroxisomes during photorespiration was assessed. Isolated spinach leaf peroxisomes were fed [¹⁴C] glycolate in the absence or presence of exogenous glutamate, and the formation of radioactive glyoxylate, CO₂, glycine, oxalate, and formate was monitored at time intervals. In the absence of glutamate, 80% of the glycolate was consumed within 2 hours and concomitantly glyoxylate accumulated; CO₂, oxalate, and formate each accounted for less than 5% of the consumed glycolate. In the presence of equal concentration of glutamate, glycolate was metabolized at a similar rate, and glycine together with some glyoxylate accumulated; CO₂, oxalate, and formate each accounted for an even lesser percentage of the consumed glycolate. CO₂ and oxalate were not produced in significant amounts even in the absence of glutamate, unless glycolate had been consumed completely and glyoxylate had accumulated for a prolonged period. These in vitro findings are discussed in relation to the extent of CO₂ and oxalate generated in leaf peroxisomes during photorespiration.     

Relationship between hydroxypyruvate and the production of oxalate in vitro

Chicken liver lactate dehydrogenase (l-lactate: NAD⁺ oxidoreductase, EC 1.1.1.27) catalyses the reversible reduction reaction of hydroxypyruvate to l-glycerate. It also catalyses the oxidation reaction of the hydrated form of glyoxylate to oxalate and the reduction of the non-hydrated form to glycolate. At pH 8, these latter two reactions are coupled. The coupled system equilibrium is attained when the NAD⁺/NADH ratio is greater than unity.Hydroxypyruvate binds to the enzyme at the same site as the pyruvate. When there are substances with greater affinity to this site in the reaction medium and their concentration is very high, hydroxypyruvate binds to the enzyme at the l-lactate site. In vitro and with purified preparation of lactate dehydrogenase, hydroxypyruvate stimulates the production of oxalate from glyoxylate-hydrated form and from NAD; the effect is due to the fact that hydroxypyruvate prevents the binding of non-hydrated form of glyoxylate to the lactate dehydrogenase in the pyruvate binding site. At pH 8, the l-glycerate stimulates the production of glycolate from glyoxylate-non-hydrated form and NADH since hydroxypyruvate prevents the binding of glyoxylate-hydrated form to the enzyme.