GLYOXYLATE SYNTHESIS, AND ITS MODULATION AND INFLUENCE ON OXALATE SYNTHESIS

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THE JOURKAL OF UROLOGY Copyright 0 1998 by AMERICAN UROLOGICAL ASSOCIATION, IN(,.

Vol. 160, 1617-1624, November 1998 Printed in LISA.

Review Article GLYOXYLATE SYNTHESIS, AND ITS MODULATION AND INFLUENCE ON OXALATE SYNTHESIS ROSS P. HOLMES

AND

DEAN G. ASSIMOS

From the Department of Urology, Wake Forest University School of Medicine, Winston-Salem,North Carolina

ABSTRACT

Purpose: We define the major pathways of hepatic oxalate synthesis in h u m a n s , examine the association with other metabolic pathways a n d identify ways that oxalate synthesis m a y be modified. In addition, w e suggest w h a t is required for f u r t h e r progress in this area. Materials and Methods: We consolidated relevant data primarily from recently published literature, considered new pharmacological approaches to decrease oxalate synthesis, a n d formulated an overview of the regulation and modification of oxalate synthesis pathways. Results: Experiments with animals, including h u m a n s , animal cells and in vitro preparations of cellular components, support the existence of a major metabolic p a t h w a y linking the amino acids serine, glycine and alanine. Oxalate synthesis is a minor, secondary reaction of a cascade of reactions termed the glyoxylate pathway, which has a prominent role in gluconeogenesis and ureagenesis. The enzymatic steps and effectors which regulate glyoxylate and oxalate synthesis are not well characterized. Pharmacological approaches can reduce oxalate synthesis by diminishing the glyoxylate pool and possibly modifying enzymatic reactions leading t o glyoxylate synthesis. Conclusions: The individual steps associated w i t h glgoxglate and oxalate synthesis can be identified. The glyoxylate p a t h w a i h a s a significant fu&tional role in intermediary liver metabolism but the w a y it is regulated is uncertain. Oxalate synthesis can be modified by drugs, indicating that primary a n d idiopathic hyperoxaluria m a y respond t o pharmacological intervention. KEYWORDS:oxalates; glyoxylates; hyperoxaluria, primary

The majority of renal calculi (about 70%) are composed predominantly of calcium oxalate.1 Despite the obvious importance of oxalate in stone formation, many aspects of its biochemistry, physiology and function have been poorly characterized, including its synthesis which occurs primarily in the liver. There is neither a complete picture of the pathways associated with oxalate synthesis available, nor a n understanding of the regulation of these pathways and the flux of metabolites through them. Oxalate synthesis has been poorly characterized for various reasons, including the lack of suitable models of human hepatic metabolism, limited characterization of some key enzymes in the process, complex interactions between subcellular organelles and the cytoplasm, and difficulties in measuring some metabolites. Many of these issues were recognized in previous reviews of the topic, whereas others stem from more recent developments in our knowledge of metabolism.z--”A better understanding of OXalate synthesis could lead to the development of more effective pharmacological treatment for primary and idiopathic hyperoxaluria. Oxalate synthesis should be viewed only as a small part in a metabolic complex involving the central glyoxylate pathway that we have proposed. We outline this complex, and define the relationships with intermediary metabolism and synthesis of glucose, urea and other cellular components. We show that the daily flux of carbon through this pathway is indicative of its functional significance. The regulation of oxalate synthesis is likely closely associated with how these fluxes are regulated. Finally, the possibility

that oxalate affects the rate of motion of the central glyoxylate pathway by controlling carbon fluxes is also considered. PATHWAYS

The evidence for the liver as the principal site of oxalate synthesis is derived primarily from observations that the conversion of 14C-glycolate to 14C-oxalate is reduced by more than 80% in hepatectomized rats: which also indicates that a small amount of oxalate may be synthesized in nonhepatic tissues. The synthesis of oxalate is almost entirely dependent on that of glyoxylate. We have proposed that a series of reactions in the liver are linked in a sequence termed the glyoxylate pathway. This pathway links glyoxylate synthesis with the metabolism of serine, alanine and glycine, which are 3 prominent amino acids that the liver uses to synthesize glucose and dispose of nitrogen as urea. There are also apparent links with the pentose phosphate pathway, and a pathway that metabolizes glucose to glucuronic acid and xylulose. The interaction of these pathways and the molecules in the glyoxylate pathway that link them are shown in figure 1. Glyoxylate pathway. The reactions linking components in the proposed glyoxylate pathway are depicted in figure 2. The enzymic reactions converting serine to hydroxypyruvate (serine:pyruvate aminotransferase activity of a1inine:glyoxylate aminotransferase), glycolaldehyde to glycolate (aldehyde dehydrogenase), glycolate to glyoxylate (glycolate oxidase) and glyoxylate to glycine (a1anine:glyoxylate aminotransfer-

1617

GLYOXYLATE AND OXALATE SYNTHESIS

1619

enzyme has not yet been purified and more knowledge of its no enzyme activity could be detected using the human cDNA properties, activity and subcellular localization in liver, par- in several expression systems but no activity was achieved ticularly human liver, is required. With isolated rat hepato- with the rat cDNA either, suggesting that the coding secytes they showed a high conversion of hydroxypyruvate to quence of this gene may have unusual characteristics. The activity of this pathway is apparently stimulated by oxalate, about 2.5% of the hydroxypyruvate added t o the medium.16 Liao and Richardson observed an even higher fructose catabolism, which may be related to the enhanced conversion of hydroxypyruvate to oxalate in perfused rat oxalate synthesis observed with the metabolism of fructose liver, that is 19%of the radioactivity recovered in the liver.14 compared t o glucose. The differential effect of these sugars As the bulk of the glycolaldehyde formed (greater than 99%) on oxalate synthesis has been observed in humans and rat would normally be converted to glycine after sequential re- hepatocytes.17.25.26A key feature of fructose metabolism is actions with aldehyde dehydrogenase, glycolate oxidase and that the concentration of D-glycerate i n c r e a s e ~ 2 ~as, ~ ~ a1inine:glyoxylate aminotransferase, these conversion rates D-glyceraldehyde is reduced. The relationship of this into oxalate are high, suggesting that under experimental con- crease in D-glycerate, if any, to the increased synthesis of ditions substantial nonenzymatic breakdown of hydroxy- oxalate is not known. Xylulose pathway. The existence of a pathway t o oxalate pyruvate to oxalate 0ccurred.1~ The conversion of glycolaldehyde to glycolate is catalyzed through D-xylulose was originally discovered when it was by aldehyde dehydrogenase. Most attention has focused on recognized that xylitol infusions were fatal in some individthe activity of this enzyme with acetaldehyde as a substrate uals with oxalosis as a pathological feature.4 An oral dose of due to its importance in ethanol metabolism. Several liver 20 gm. xylitol has been shown to produce a transient 53% enzymes can catalyze this reaction and extensive studies of increase in urinary oxalate excretion in humans 2 hours after The reactions feeding sugars from the xylulose various isozymes have established that, particularly at low ingesti~n.~g levels of acetaldehyde, the reaction is catalyzed by the mito- pathway into the glyoxylate pathway are shown in figure 4. chondrial form of aldehyde dehydrogenase.18 Although the This pathway apparently functions in part to produce glucureactivity of these enzymes to glycolaldehyde has not been ronic acid, which is required for conjugation reactions.30 The extensively studied, when examined it was only slightly less main carbon source for the pathway is glucose. The key step that relates xylulose metabolism and oxalate than that of acetaldehyde. 19 The activity of mitochondrialaldehyde dehydrogenase in human liver has been reported as synthesis is the phosphorylation of D-xylulose, which occurs 0.65 Fmol. per minute per gm. tissue with acetaldehyde as a by xylulokinase to xylulose 5-phosphate or fructokinase to substrate.20 This level is a little less than the activity of 1 xylulose 1-phosphate. Xylulose 5-phosphate is a component bmol. per minute per gm. reported for glycolate oxidase in of the pentose phosphate pathway. The activities of the enhuman liver.21 If the activity of mitochondrial-aldehyde de- zymes in liver tissue are similar but as xylulokinase has a K, hydrogenase is similar with glycolaldehyde as a substrate, it for D-xylulose which is 10-fold lower than that of fructokiwill permit a sufficient synthesis of glycolate to account for nase, formation of xylulose 5-phosphate is apparently preThe relative amounts of these phosphorylated the speculated fluxes through the pathway that we describe. ferred.31~3~ The conversion of glycolate to glyoxylate is catalyzed by the xylulose sugars synthesized under a variety of metabolic flavin containing peroxisomal enzyme glycolate oxidase. This conditions warrant investigation. Oxalate pathway. The key reaction for our report is the enzyme is also referred to as short chain a-hydroxy acid oxidase, although alternative substrates t o glycolate which oxidation of glyoxylate to oxalate (fig. 5). The 2 enzymes that may be oxidized to any significant extent by it in the liver can catalyze this reaction are lactate dehydrogenase and have not been identified. Hamilton has proposed that thiol glycolate oxidase. Experiments that we recently performed glyoxylate adducts may be good substrates for the enzyme.22 with isolated peroxisomes and purified lactate dehydrogeUnfortunately, most of these studies were conducted with nase indicated that the latter most likely catalyzes the bulk renal long chain a-hydroxy acid oxidase. Despite the poten- of this reaction in vivo, as the concentrations of glycolate and tial importance in gluconeogenesis and oxalate synthesis, lactate in hepatocytes will inhibit the glycolate oxidase cathuman liver glycolate oxidase has not been well character- alyzed reaction.7 Glycolate oxidase has a 10-fold higher afized, and the amino acid and genomic sequences have not been determined. A key reaction in the glyoxylate pathway is the conversion of glyoxylate to glycine coupled with the conversion of alanine to pyruvate catalyzed by the pyridoxal phosphate dependent enzyme a1anine:glyoxylate aminotransferase. The enzyme is predominantly in peroxisomes in humans, and has XDH a central role in limiting oxalate synthesis through a high D-glucose L-xylulose xylitol affinity for glyoxylate. The enzyme removes greater than 99% of the glyoxylate, permitting only a small fraction to be oxidized to oxalate. This key function is illustrated by the pronounced hyperoxaluria that ensues when the enzyme is D-glucuronate / deficient in peroxisomes of individuals with primary hyperD-xylulose oxahria type I. Swine pathway. Serine is an important ghconeogenic amino acid in the liver, and the pathway that illustrates its linkage with gluconeogenic and glycolytic pathways is shown xylulose 5-phosphate in figure 3 with the biosynthetic pathway. An important xylulose I-phosphate distinction is that humans unlike most other animals appear to lack functional serine dehydratase activity for the direct conversion of serine to pyr~1vate.23There are still some questions regarding the existence of serine in human liver as PENTOSE PATHWAY glycolaldehyde screening a human complementary deoxyribonucleic acid (CDNA) library with a probe for rat liver serine dehydratase FIG. 4. Reactions of xylulose pathway. X D H , xylitol dehydrogerevealed that a messenper ribonucleic acid is expressed wlth nase. ZDH, L-iditol dehydrogenase. XK, xylulose kinase. PFK, phosohofructokinase. FBPA. fructose-bisDhosDhate - . aldolase. more than 80% homolo& to the rat message.24 Surprisingly, .

1620

hydroxyproline

GLYOXYLATEANDOXALATESYNTHESIS

would suggest that this is a contribution of about 1%. The principal evidence for degradation of phenylalanine, tyrosine and tryptophan to oxalate comes from the intraperitoneal injection of isotopes into rats that had fasted.3*,ss The degradation was calculated to contribute less than 3% of the urinary oxalate in the rat under these conditions. However, pathways have not been identified for the catabolism of these amino acids that could yield oxalate, and so these estimates must be treated with caution.

glycolate

NAD+y

SUBCELLULAR COMPARTMENTATION O F ENZYMES

1 4-OH-2-ketoglutarate

% glyoxylate TW

AGT

A potentially important feature of the glyoxylate pathway is that the enzymes are localized in different subcellular alanine [ pyrwate f compartments. The clinical significance is evident in primary hyperoxaluria type I when targeting of a1inine:glyoxylate aminotransferase, normally in peroxisomes, to mitochondria NADH makes it unable t o complete the task of converting glyoxylate to glycine.5 In the glyoxylate pathway there is a n interplay oxalate among peroxisomes, mitochondria and cytoplasm. Alanine: FIG. 5. Reactions associated with oxalate synthesis. LDH, lactate dehydrogenase; HKA, 4-hydroxy-2-ketoglutaratealdolase. NADPi, glyoxylate aminotransferase and glycolate oxidase are localoxidized form of nicotinamide-adenine dinucleotide phosphate. ized in peroxisomes. Aldehyde dehydrogenase, the serine hyNADPH,reduced form of nicotinamide-adenine dinucleotide phos- droxymethyltransferase converting serine to glycine, and much phate. H,O, hydrogen peroxide. NAD+, oxidized form of nicotinamide- of the hydroxypyruvate decarboxylase are localized in mitoadenine dinucleotide. O,,molecular oxygen. NADH,reduced form of chondria. The serine hydroxymethyltransferase converting nicotinamide-adeninedinucleotide. glycine to serine, some of the hydroxypyruvate decarboxylase, and the enzymes lactate dehydrogenase and D-glycerate dehydrogenase, which are closely associated with the glyoxyfinity for glycolate over glyoxylate,33 which also argues late pathway, are present in the cytoplasm. Transport of against a role for it as the concentration of glycolate is more metabolites in and out of organelles becomes an important than 10 times that of glyoxylate in guinea pig liver.34 Workstep, and more knowledge of these transport processes is ing with rat liver preparations Sharma and Schwille have required to characterize them and determine if they are rate similarly concluded that lactate dehydrogenase rather than limiting. glycolate oxidase is responsible for converting glyoxylate to oxalate.35 ENZYME DEFICIENCIES IN PRIMARY HYPEROXALURIA Glyoxylate may be converted back to glycolate in a reaction Primary hyperoxaluria type I . A deficiency in peroxisomal that can be catalyzed by lactate dehydrogenase or D-glycerate dehydrogenase using the reduced form of nicotinamide-adenine a1anine:glyoxylate aminotransferase results in primary hydinucleotide or the reduced form of nicotinamide-adenine dinu- peroxaluria type I: which is characterized by high excretions cleotide phosphate, respectively, as cofactors. Both reactions of oxalate and glycolate, although glycolate excretion may be will consume energy, and with the reconversion of glycolate to normal in some individuals. The glyoxylate pathway (fig. 2) glyoxylate the net reaction will be thermogenic because of the will be blocked in 2 places and gram amounts of intermediformation and resultant breakdown of hydrogen peroxide. Con- ates will not flow through it daily. Glycolaldehyde produced sidering the relative importance of these activities, at first it via the xylulose pathway will lead to the synthesis of glyoxywould seem that the higher activity of lactate dehydrogenase in late, and the conversion to glycolate and oxalate. The catabhuman liver (200versus 40 units per gm.) is offset by the higher olism of hydroxyproline and aromatic amino acids may also K, value for glyoxylate (5 versus 0.4mM.). When the concen- produce some glyoxylate as discussed previously. As individtrations of the reduced form of nicotinamide-adenine dinucle- uals with primary hyperoxaluria type I excrete a combined otide and the reduced form of nicotinamide-adenine dinu- total of 200 to 650 mg. oxalate and glycolate daily,40 this total cleotide phosphate in the cytoplasm where both enzymes are may represent the amount of glycolaldehyde that is synthelocalized are considered as well as their respective affinities sized daily by the xylulose pathway. However, this amount for the enzymes, it is apparent that D-glycerate dehydroge- may be an overestimate a s hydroxyproline degradation may nase and its glyoxylate reductase activity have the more also add to the glyoxylate pool. prominent role. However, it is not clear whether this converIn about 30% of individuals with primary hyperoxaluria sion occurs to any appreciable extent in normal liver but it type I subnormal a1anine:glyoxylate aminotransferase activcould have a prominent role in maintaining an elevated gly- ity is present in liver but mistargeted to mitochondria. The colate excretion in primary hyperoxaluria type I. apparent inability of this mitochondrial a1anine:glyoxylate Several minor reactions may also produce glyoxylate, aminotransferase to convert appreciable amounts of glyoxywhich include the catabolism of hydroxyproline and some of late to glycine suggests that mitochondria do not take up the aromatic amino acids. In hydroxyproline catabolism glyoxylate, despite the presumed high cytoplasmic glyoxylate 4-hydroxy-2-ketoglutarateis cleaved by the mitochondrial concentration in these individuals. aldolase to pyruvate and enzyme 4-hydroxy-2-ketoglutarate Primary hyperoxaluria type II. Hyperoxaluria in this disglyoxylate. The enzyme is present in liver and kidney tis- order is due t o a deficiency in D-glycerate dehydrogenase sue,36 suggesting that these are major sites of hydroxyproline activity but the exact mechanism by which oxalate synthesis breakdown. A rough estimate of the contribution of hy- is stimulated is not certain. The hallmark of this disease is an droxyproline breakdown to the glyoxylate pool is given by the enhanced urinary excretion of L-glycerate as well as oxalate. urinary excretion of hydroxyproline in individuals with hy- One result of the deficiency in D-glycerate dehydrogenase is droxyprolinemia, reportedly ranging from 285 to 550 mg. in that a significant increase in the concentration of hydroxy24 h0urs.3~If there is a complete blockage of hydroxyproline pyruvate should occur as the pathway to D-glycerate is breakdown in these individuals, we estimate that 161 to 311 blocked. The increase may produce an increased flux through mg. glyoxylate could be generated in 24 hours by this route. the glyoxylate pathway and result in an increase in the Estimates derived for the turnover of the glyoxylate pool synthesis of oxalate. The increased concentration of hydroxyglycine

GLYOXYLATE AND OXALATE SYNTHESIS

pyruvate is also believed to cause a n increased conversion of the hydroxypyruvate to L-glycerate by lactate dehydrogenase (fig. 5 ) . The absence of D-glycerate dehydrogenase and its glyoxylate reductase activity could conceivably decrease the amount of glyoxylate converted to glycolate, but more information on the occurrence of this reaction is required before any conclusions can be drawn. However, an increase in the hydroxypyruvate concentration in liver tissue of patients with primary hyperoxaluria type I1 has not been shown, which stems from the low level of hydroxypyruvate in liver tissue, rarity of the disorder and unavailability of tissues from affected individuals for analysis. Other types o f hyperoxaluria with hepatic origin. Van Acker et a1 identified 2 individuals with consistent hyperoxaluria and occasionally elevated excretion of glycolate.41 A liver biopsy indicated normal levels of a1anine:glyoxylate aminotransferase with the expected kinetic properties and peroxisoma1 localization. Analyses of the urinary excretions of family members suggested that the hyperoxaluria was inherited. However. the molecular basis remains unknown.

FLUXES THROUGH THE PATHWAY

1621

REGULATION OF THE GLYOXYLATE PATHWAY AND OXALATE SYNTHESIS

Regulation in these pathways could potentially involve the modulation of enzyme activity or changes in the concentration of enzymes. A role for the latter in these pathways has not been firmly established, although it is clear that changes in the metabolic state of the liver can change the levels of these enzymes. For instance we observed decreases in hepatic a1anine:glyoxylate aminotransferase and glycolate oxidase in guinea pigs with constant hyperglucagonemia, but oxalate synthesis increased as these levels decreased.34 In rats Sharma and Schwille observed a decrease in glycolate oxidase following clofibrate treatment concomitant with increased oxalate synthesis.35 They observed a n increase in lactate dehydrogenase and suggested that it may be a n important factor. However, changes in other factors which may modify these reactions should be determined before firm conclusions are made. Serine and glycine are major entry points of carbon into the glyoxylate pathway, with plasma the predominant source of each. Fluxes through the glyoxylate pathway could be modulated by the uptake of these amino acids into the hepatocyte .~~ as this is a rate limiting step in their ~ a t a b o l i s mGlucagon stimulates this process and resultant enhanced fluxes through the glyoxylate pathway may make a contribution to the increased oxalate synthesis observed with glucagon treatment in vivo and in v i t r 0 . 3 ~Changes ,~~ in the levels of alanine, lactate and pyruvate also appear t o have a role in the increased oxalate synthesis by modifying the activities of glycolate oxidase and a1anine:glyoxylate aminotransferase.9 Regulation of pathway fluxes could also occur through changes in the extrusion rate of glycolate from the liver cell, which will decrease substrate for glycolate oxidase. Such a change could explain the responses noted in patients receiving a xylitol infusion.48 Oxalate excretion increased 3-fold while glycolate excretion increased 60-fold. We observed evidence for such a mechanism in examining the response of individuals to diets containing either 0.6 or 1.8 gm. protein per kg. daily. There were only minor changes in oxalate excretion but glycolate excretion was twice as high on the high compared to low protein diet.45 This excretory response may be due t o the action of the monocarboxylic acid transporter, which functions to transport monocarboxylic acids in and out of the ~e11.~9 Overall our knowledge of the regulation of the biochemical pathways associated with oxalate synthesis is limited. However, this information is critical t o understanding how these pathways interact in liver metabolism and determining strategies t o modulate oxalate synthesis. A particularly important step may be the branch point with hydroxypyruvate where D-glycerate dehydrogenase and hydroxypyruvate decarboxylase compete for the substrate.

There are several indications that in the liver gram amounts of carbon flow through the glyoxylate and associated pathways daily. This evidence stems from calculations of whole body fluxes of pathway metabolites, knowledge of enzyme and substrate concentrations in liver tissue, and urinary excretions in individuals with inherited defects in the steps of these pathways. The amount of glucose synthesized daily by the liver is approximately 90 gm.42 Whole body glycine synthesis has been estimated t o be 44 gm. daily in humans,43 and a substantial amount of this synthesis will occur in the liver. However, the calculation of these rates with 15N-glycine may have underestimated liver glycine synthesis due to the compartmentation of glycine in different pools, cytoplasm and mitochondria, within the liver. Other estimates are higher at about 1 gm./kg. daily.9 The activity of a1anine:glyoxylate aminotransferase in the glyoxylate pathway is likely t o make a major contribution t o liver glycine synthesis. Some glycine may also be produced by serine hydroxymethyltransferase catalyzed conversion of serine t o glycine. The level of glycolate oxidase activity and intracellular concentration of glycolate provide a good guide to fluxes through the glyoxylate pathway. The concentration of glycolate in guinea pig liver is around 200 wM., close to the K,,, of the enzyme.34 Allowing for some inhibition of glycolate oxidase activity due to high concentration of lactate in liver,7 and assuming that the enzyme normally functions at a quarter of the measured activity, which is reported to be 1Km. per minute per gm. for human liver,21 the calculated oxidation of glycolate daily in a 1.5 kg. liver would be about 40 gm., resulting in the synthesis of a nearly equivalent amount of glyoxylate. A similar analysis can be performed for aldehyde dehydrogenase and glycolaldehyde oxidation to glycolate. Although glycolaldehyde concentrations in liver are not known, they may exceed the K,,, value of 200 nM. recently determined for human liver mitochondria1 aldehyde dehydrogen a ~ e . The ~ * activity of this enzyme in human liver is reported to be 0.24 units per gm. tissue.20 Operating at maximal capacity this enzyme would generate approximately 40 fl. glycolate daily. Thus, even if these estimates were off by 50% it is clear that gram amounts of carbon flow through the glyoxylate pathway daily. Furthermore, daily oxalate synthesis, which contributes only a portion of the mean 30 mg. excreted daily in urine25 represents less than 0.1% use of the gboxylate produced from glycolate.

FUNCTIONAL ROLES FOR THE GLYOXYLATE PATHWAY AND OXALATE SYNTHESIS

The glyoxylate pathway makes an important contribution to 2 essential metabolic functions of the liver, which are glucose and urea synthesis. The major compounds supporting fluxes through the pathway and these syntheses are serine and glycine as they are taken up from the circulation. With each cycle 1 mol. glycine will be consumed as 2 mol. glycine are required to form 1mol. serine. This metabolism of glycine will also generate 1 mol. each of carbon dioxide, the reduced form of nicotinamide-adenine dinucleotide and ammonium ion. The carbon dioxide and ammonium ion can be used to synthesize urea, and the reduced form of nicotinamideadenine dinucleotide will become an energy source. With the branch point in the glyoxylate pathway at hydroxypyruvate the cell can determine, based on its requirements, to funnel the carbon to glycine for urea and energy production (an

1622

GLYOXYLATE AND OXALATE SYNTHESIS

Ates) increased oxalate excretion, suggesting that there is a additional mol. of the reduced form of nicotinamide-adenine dinucleotide is produced with the oxidation of glycolaldehyde iosage effect.25 These results also indicate that amounts of to glycolate) or divert it to D-glycerate with subsequent forma- Fructose that increase oxalate synthesis are not likely to be tion of intermediates of the gluconeogenic pathway. Apart consumed in a normal diet. from an abnormal production of oxalate liver function apPHARMACOLOGICAL APPROACHES TO DECREASE OXALATE pears relatively normal in individuals with primary hyperSYNTHESIS oxaluria type I who have a dysfunctional glyoxylate pathway, although metabolism may be a little less efficient. Effective drug therapies that decrease oxalate synthesis An important question is whether oxalate itself has a func- would be valuable tools in the treatment of primary hypertional role in the body or is just an accident of nature due to oxaluria and possibly intractable cases of idiopathic calcium a lack of specificity of lactate dehydrogenase for its natural oxalate nephrolithiasis. A number of different treatments for substrate lactate. The 3 functional roles proposed for oxalate primary hyperoxaluria have been tested. Of these varied in humans are regulation of hepatic metabolism, mainte- approaches moderate results have been obtained only with nance of a population of intestinal oxalate degrading organ- long-term therapy with orthophosphate and pyridoxine.62 isms and facilitating renal sodium chloride reabsorption. However, pyridoxine therapy may benefit only 30% of paRegulation of metabolism could occur directly through the tients with primary hyperoxaluria type I, and be related to inhibition by oxalate of several enzymic reactions. Enzyme the mis-targeting of a1anine:glyoxylate aminotransferase to activities reported to be inhibited by low concentrations of mitochondria, which was the consensus at the Primary Hyoxalate (10 to 50 pmol.) include pyruvate c a r b o ~ y l a s e , ~ ~~~~ peroxaluria Workshop in Torino, Italy, March 1997. The lactate dehydrogenase, monophosphoglyceromutase and mechanism of action of pyridoxine in these individuals is not pyruvate kinase.52-54 Through the inhibition of pyruvate car- clear. Pyridoxal 5-phosphate can modulate the expression of boxylase oxalate was proposed as an inhibitor of gluconeo- a number of enzymes, which may be a factor.63 It is possible genesis. The regulation of metabolism could also be indirect that the expression of the mitochondrial-a1anine:glyoxylate through the activity of oxalyl thioesters, which appear to aminotransferase or g1utamate:glyoxylate aminotransferase have a potent biological activity.55 These esters are synthe- (alanine aminotransferase) is enhanced. sized from glyoxylate and the relationship with free oxalate Treatments that inhibit aldehyde dehydrogenase could also in the liver is unknown. Evidence for the intestinal secretion be considered due to the position of this enzyme in the pathway of oxalate has mainly been derived from studies of chronic described in figure 1. A good response to carbamide, 1 such renal failure.56 The occurrence in normal individuals and inhibitor, was reported by Solomons et a1 in 1individual- but relationship to maintaining populations of oxalate degrading other studies have failed to confirm this finding. Another pobacteria in the gut remain speculative, Finally, it has been tential inhibitor of this enzyme, disulfiram, has also been inefobserved that oxalate and formate facilitate sodium chloride fective.5 However, as described previously multiple isozymes of reabsorption in the proximal tubule.57 The physiological ram- aldehyde dehydrogenase exist and it is apparent that disulifications of these potentially important in vitro observations firam cannot directly inhibit mitochondrial-aldehyde dehydrohave not yet been determined. Thus, the available data sug- genase, the enzyme most likely associated with oxalate synthegest that a biological role for oxalate is possible but clear-cut sis.18 In a new pharmacological approach Tu et al used an evidence is lacking. oligonucleotidethat was successful in blocking the transcription of mitochondrial-aldehyde dehydrogenaset5 suggesting that this form of therapy warrants consideration. Such antisense DIETARY NUTRIENTS INFLUENCING OXALATE SYNTHESIS technology, pharmacological approaches that use knowledge of A common and increasingly popular topic in kidney stone gene sequences to block transcription or translation, could also research is dietary modification in patients to reduce the rate be useful in inhibiting other enzymes in the glyoxylate pathof stone formation. Dietary amino acids and sugars provide way, such as glycolate oxidase. the bulk of the carbon for glyoxylate and oxalate synthesis. In A pharmacological approach is best directed at the terminormal individuals increasing protein intake from 0.6 to 1.8 nal steps in oxalate synthesis shown in figure 2. Lactate gm./kg. daily increased glycolate excretion 70% but oxalate dehydrogenase may not be a good target as it performs other excretion was increased only The increase in glyco- important functions in energy metabolism. Thus, decreasing late excretion would suggest that dietary amino acids in- the glyoxylate pool and availability of substrate for the reaccrease fluxes through the glyoxylate pathway. The minor tion becomes the most logical choice. Adverse consequences effect on oxalate excretion could suggest that oxalate synthe- associated with the depletion of the glyoxylate pool are possis is tightly regulated. However, these and all other exper- sible but individuals with primary hyperoxaluria type I and iments to date examining the influence of dietary protein on an impaired glyoxylate pathway have not been reported to urinary oxalate excretion need to be reinterpreted in view of have an abnormal metabolism apart from elevations in glyour finding that dietary oxalate contributes more to urinary colate, glyoxylate and oxalate. Furthermore, glyoxylate is not oxalate excretion than previously recognized.58.59 Without known to be required for any biosynthetic reactions, and control of dietary oxalate and factors affecting intestinal serine and glycine can be metabolized through other pathabsorption such experiments are difficult to interpret. Con- ways. The glyoxylate pool could be reduced, either by desidering the effect that we observed on glycolate excretion creasing the activity of glycolate oxidase or taking advantage until the issue is clarified it is probably prudent to assume of the high reactivity of the aldehyde group on glyoxylate. that large intakes of protein may increase oxalate excretion. This latter approach is exemplified in the use of the drug The 2 main dietary sugars that have the potential to in- Procysteine* t o raise intracellular cysteine levels. Cysteine fluence oxalate synthesis are fructose and xylitol. Large will form a stable adduct with glyoxylate, thiazolidine-2,4amounts of xylitol are not consumed and use in parenteral dicarboxylate.66 We have recently determined that this drug nutrition has been largely discontinued? indicating that it decreases urinary oxalate excretion in normal men and beneed not be considered. Although Dills has noted that the use gun a phase I1 clinical trial t o test the effects on individuals of xylitol as a bulk sweetener is increasing,60 the ingestion of with primary hyperoxaluria type 1.67 Although serious adamounts that will influence oxalate synthesis is unlikely. verse reactions have not been reported with use in a number Large amounts of fructose may be ingested as a component of of clinical trials, the compound is likely not to be specific for sucrose or corn derived sweeteners. However, an oral dose of glyoxylate and may interact with the reactive aldehyde 75 gm.fructose had no effect on oxalate excretion.61 In contrast, a rapid intravenous infusion (about 35 gm. in 15 min* Transcend Therapeutics Inc., Cambridge, Massachusetts.

GLYOXYLATE AND OXALATE SYNTHESIS

groups of other compounds, suggesting that some caution with use is warranted. Inhibition of glycolate oxidase activity is also a prime target for therapeutic intervention, if it is the primary source of glyoxylate in the liver as argued previously. Potent inhibitors of glycolate oxidase have been developed by Rooney et a1 who reported that, while 1 of these drugs was able to prevent oxalate synthesis accompanying ethylene glycol ingestion in the rat, it did not affect urinary oxalate excretion in rats not taking ethylene glyco1.6S However, any effect of the drug may have been masked by contributions from dietary oxalate and the breakdown of ascorbic acid to oxalate which will occur as the rat urine alkalizes on standing. Further investigation of these drugs is warranted. CONCLUSIONS

Most of the data reviewed that are related to oxalate synthesis and reactions in the glyoxylate pathway have been derived from animal studies and cell culture experiments. Substantial differences exist between humans and rodents in the subcellular localization of a1anine:glyoxylate aminotransferase and presence of serine dehydratase activity, which will result in different pathways for the metabolism of serine, glycine and other components of the glyoxylate pathway. Limited information is available to confirm that the reactions described in figures 2 to 5 occur in human liver. Equally deficient is our knowledge of the fluxes of metabolites through the pathways in humans or other animals. More knowledge is required about the properties of such enzymes as hydroxypyruvate decarboxylase, D-glycerate dehydrogenase and hydroxymethyltransferases in human hepatocytes before we can have a complete understanding of the metabolic pathways associated with glyoxylate and oxalate synthesis, and quantify fluxes through them. The available data indicate that oxalate synthesis results from the oxidation of only a minor portion (0.1%) of the hepatic glyoxylate pool and that this reaction is catalyzed by lactate dehydrogenase. It is proposed that glyoxylate is a component of a major hepatic metabolic pathway that makes large contributions to ureagenesis and gluconeogenesis. To date the existence and function of this pathway have not been fully appreciated. A greater understanding of the factors that influence the individual steps in the pathway is required to comprehend better how this whole metabolic complex is arranged. Pharmacological approaches that deplete the glyoxylate pool to limit oxalate synthesis, by directly reacting with glyoxylate in the case of cysteine or decreasing synthesis by inhibiting glycolate oxidase, should be beneficial in the treatment of primary hyperoxaluria and possibly intractable cases of idiopathic calcium oxalate nephrolithiasis. REFERENCES

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