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Oxalate Synthesis, Transport and the Hyperoxaluric Syndromes
0022-534 7/ /89 /1413-07 42$2.00 /0 Vol. 141, March
THE JOURNAL OF UROLOGY
Copyright © 1989 by The Williams & Wilkins Co.
Printed in U.S.A.
OXALATE SYNTHESIS, TRANSPORT AND THE HYPEROXALURIC SYNDROMES HIBBARD E. WILLIAMS
THEODORE R. W ANDZILAK
AND
From the University of California, Davis, School of Medicine. Davis, California
ABSTRACT
This article reviews the mechanisms involved in the synthesis, absorption, excretion and transport of oxalic acid, and the factors controlling these processes in man. The clinical syndromes associated with hyperoxaluria and recurrent calcium oxalate stone disease are reviewed, including new studies that raise the possibility of a generalized oxalate transport abnormality in some patients with renal stone disease. The important role of oxalate in the determination of calcium oxalate solubility in patients with calcium oxalate stone disease is emphasized and future directions for research in the prevention of recurrent calcium oxalate stone disease are discussed. (J. Ural., part 2, 141: 742-747, 1989) The subject of this presentation is oxalic acid and its role in renal stone disease. Oxalic acid is a strong organic acid with a pKa, of 1.27 and a pKa, of 3.8. It is the simplest dicarboxylic acid in nature, and at physiological pH it forms a number of salts; soluble salts with sodium and potassium, and an insoluble salt with calcium. It is this insolubility of the calcium oxalate salt that gives oxalic acid its clinical importance in man. To our knowledge, oxalic acid has no significant useful role in human metabolism. Approximately 75 per cent of all kidney stones are composed predominantly of calcium oxalate. This review will emphasize the important role that urinary oxalate has in the formation of calcium oxalate stones. Factors that control the amount of oxalate in the urine, causes of increased oxalate production and excretion, and possible directions for future research also will be discussed. URINARY OXALATE
Oxalate normally is excreted in urine at levels between 15 and 40 mg./24 hours. Various methods have confirmed this normal range, and the ion-chromatographic method and the enzymatic method for oxalate determination in urine appear to produce similar results. 1 A diurnal variation in urinary oxalate concentration and excretion exists with greatest values between 11 a.m. and 11 p.m.," as well as a seasonal variation with higher values in the summer months. i Approximately 10 to 20 per cent of excreted oxalate arises from absorbed dietary oxalate and the remainder of the oxalate in the urine arises as the result of endogenous metabolism from its 2 major precursors, glyoxylate and ascorbic acid.4 Oxalate is present in many common foods and it occurs in particularly high levels in cocoa, tea, rhubarb, spinach, parsley, pepper, peanuts and beets. The normal dietary intake of oxalate ranges between 80 and 100 mg. per day.'' Factors controlling the gastrointestinal absorption of ingested oxalate will be discussed. METABOLIC SYNTHESIS OF OXALATE
Oxalic acid represents an apparent useless end product of metabolism and in this way it resembles uric acid. Enzymes that metabolize oxalate to formic acid and carbon dioxide do not exist in man. Therefore, once oxalate is produced it must be excreted by the kidneys. No gastrointestinal route of oxalate excretion is known to exist. The 2 major precursors of oxalate are ascorbic acid and glyoxylic acid. Conversion of ascorbic acid to oxalic acid apparently occurs in the liver by a process
involving diketogulonic acid with the first 2 carbons of ascorbic acid being converted to oxalate.'; Little is known about the factors controlling this metabolic conversion but it is estimated that approximately 35 to 50 per cent of urinary oxalate comes from ascorbic acid.' Apparently the enzymes involved in the conversion of ascorbic acid to oxalate are saturable at low concentrations of ascorbic acid, since ingestion of fairly large doses of ascorbic acid does not appear to increase urinary oxalate. However, some studies have demonstrated that with high doses of ascorbic acid (greater that 5 gm. per day) some elevation of urinary oxalic acid can occur. 8 - 11 It should be emphasized that under certain conditions ascorbic acid can undergo apparent nonenzymatic oxidation to oxalate in urine, 12 making evaluation of reports of conversion of ascorbate to oxalate difficult. More studies need to be performed with present methods of oxalate determination that avoid any nonenzymatic conversion of ascorbate to oxalate in urine samples or in the assay procedure. Chalmers and associates have demonstrated an increased urinary oxalate and decreased ascorbate excretion in recurrent stone formers compared to controls after oral (2 gm.) but not intravenous (500 mg.) administration of ascorbic acid. 1 '1 The authors suggested that there was an increased conversion of oxalate from ascorbate in the gastrointestinal tract of recurrent stone formers. The other major precursor of oxalate is glyoxylate, which accounts for the synthesis of approximately 50 to 70 per cent of urinary oxalate (fig. 1).4 The major sources of glyoxylate in man include glycine, glycolic acid and serine. Glycine is converted to glyoxylate by D-amino acid oxidase. Glycolate is oxidized to glyoxylate by glycolic acid oxidase, and serine can be converted to glycine or to glycolic acid via ethanolamine and glycoaldehyde. An additional source of serine and glycolic acid is hydroxypyruvate. The oxidation of glyoxylate to oxalate can be catalyzed by 3 different enzymes: lactic dehydrogenase, xanthine oxidase and glycolic acid oxidase. Evidence to date supports the important role of lactic dehydrogenase in this oxidative reaction with only a limited if any role for glycolic acid oxidase or xanthine oxidase in this conversion_.4 Glyoxylate can be metabolized to glycine, a reaction controlled by the peroxisomal enzyme alanine, glyoxylate aminotransferase, a transaminase that is pyridoxine-dependent. Glyoxylate also can be reduced to glycolic acid by the enzyme glyoxylate reductase and it can be metabolized to a-hydroxy, /3-ketoadipate by a carboligase enzyme that is dependent on thiamine pyrophosphate and uses a-ketoglutarate.'
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OXALATE SYNTHESIS, TRANSPORT AND HYPEROXALURIC SYNDROMES
'i
Hydroxypyruvate
/
Serine
Glycolate
~t Ascorbic Acid
Glycine
Ca
OX
\' Ca
B6
ox FIG. 1. Metabolic pathways involved in oxalate synthesis RENAL CLEARANCE OF OXALATE
A number of studies during the last 20 years have documented the major mechanisms involved in the renal handling of oxalate. Studies on the renal clearance of oxalate using in vivo C-14 oxalate infusion have demonstrated an oxalate/creatinine or inulin clearance ratio greater than 1, 14 - 17 suggesting that tubular secretion is the predominant mechanism for the renal handling of oxalate. Osswald and Hautmann studied the renal handling of oxalate in 6 subjects by the rapid injection of C-14 oxalate into the renal artery and the subsequent collection of urine via a catheter inserted into the renal pelvis. 18 They demonstrated 2.3-fold higher excretion of oxalate compared to inulin, confirming that tubular secretion has a major role in oxalate excretion in man. A number of studies in experimental animals also have documented that tubular secretion has a major role in oxalate excretion in these species. Studies have been done in the rat, 19 dog,2° sheep 21 and chicken. 22 Weinman and associates, using free-flow micropuncture and intratubular microinjection techniques, examined the renal handling of oxalate in the rat kidney.'" These studies demonstrated that oxalate is secreted in the early part of the proximal convoluted tubule, undergoes bidirectional transport in the mid portions of the proximal convoluted tubule and is probably not transported further down the tubule. Tremaine and associates, using the Sperber in vivo chicken preparation, demonstrated saturable excretory transport of oxalate through the renal tubular cell. 22 The transport of oxalate was not affected by probenecid, suggesting a separate system from that of uric acid, but it was reduced by a-ketoglutaric acid, indicating a possible involvement of the dicarboxylate transporter. Studies in man have documented the renal clearance of oxalic acid to be approximately 170 ml. per minute, 14 and an oxalate half life of elimination of 92 plus or minus 8 minutes.'" By extrapolating these clearance figures, the plasma oxalate concentration has been estimated to be 7 to 15 ,ug./100 ml. in normal man. GASTROINTESTINAL HANDLING OF OXALATE
As noted, oxalate is a constituent of the normal diet and approximately 80 to 100 mg. per day are ingested. Most ingested oxalate appears to be bound by intraluminal calcium in the small intestine and it is excreted as insoluble calcium oxalate complexes (fig. 2). 21 It is this phenomenon that probably accounts for the fact that only 10 to 20 per cent of ingested oxalate is absorbed in normal individuals. The concentration of intraluminal calcium appears to have a major role in determining the amount of dietary oxalate absorbed, and an inverse relationship exists between the amount of calcium ingested and the amount of oxalate absorbed by the gastrointestinal tract. Oxalate absorption appears to occur along the course of the entire gastrointestinal tract. The studies of Prenen and associates, which demonstrated a peak of oxalate be-
FIG. 2. Gastrointestinal tract handling of oxalate
tween 2 and 4 hours after ingestion, suggest that the small bowel is a major site of oxalate absorption in normal individuals.24 The mechanisms of oxalate absorption by the gastrointestinal tract recently have been investigated in detail. Early studies suggested that gastrointestinal oxalate transport was a nonenergy-dependent nonsaturable passive process. 25 - 27 However, these experiments were all performed with a calcium-free buffer system, and it was later demonstrated that calcium is required to maintain the integrity of the conductive pathways across gastrointestinal epithelium. Freel28 and Hatch 28 and their associates, using the technique of isolated short-circuited segments of rat and rabbit colon, demonstrated a net transport of 0xalate and chloride. This transport was abolished by the metabolic inhibitor dinitrophenol and the anion transport inhibitor, 4-acetamido-4' -isothiocyanatostilbene-2,2' -disulfonic acid. These results suggested that oxalate transport is indeed an energy-dependent process and that oxalate and chloride share a common transport pathway. More recently, Knickelbein and associates demonstrated the presence of oxalate transport in brush border membrane vesicles of rabbit ileum. 30 Oxalate could be transported by exchange for either hydroxyl or chloride ions and it could be inhibited by the anion exchange inhibitor, 4,4' -diisothiocyanatostilbene-2,2' -disulfonic acid (DIDS). Among other organic acids only formate and oxaloacetate could stimulate oxalate and chloride uptake. These studies demonstrated further that the cellular absorption of oxalate is an active transport process. Evidence in man does not suggest secretion of oxalate by the gastrointestinal tract. These conclusions were based primarily on the studies of intravenous C-14 oxalate administration in which recovery of oxalate in urine during a 2-day period was demonstrated. 01 These studies do not rule out a bidirectional transport system for oxalate in the gastrointestinal tract. DISORDERS OF OXALATE METABOLISM AND EXCRETION
Before the abnormalities of oxalate synthesis and excretion that lead to hyperoxaluria and the potential role of oxalate in idiopathic calcium oxalate stone disease are reviewed, it is important to define hyperoxaluria. As noted previously, the normal 24-hour excretion of oxalate is between 15 and
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WILLIAMS AND W ANDZILAK
40 mg./24 hours. Upper limits of normal for urinary oxalate vary between 40 and 45 mg. depending upon the method used_. The normal ratio of urinary oxalate-to-urinary creatinine varies between 0.008 and 0.054. Using these figures, the incidence of hyperoxaluria in calcium oxalate stone formers varies from approximately 2 to 3 per cent to as high as 40 to 50 per cent. 32- 36 In our opinion most recent studies of calcium oxalate stone formers using accurate methods for urinary oxalate suggest that the true incidence of hyperoxaluria is between 10 and 20 per cent. We wish to emphasize at this point the important role urinary oxalate has in determining the state of saturation of the urine with respect to calcium oxalate. Studies by Robertson and associates have indicated that the oxalate concentration in urine is one of the most important determinants of calcium oxalate solubility." 7 Clinical evidence to support this important role for oxalate comes from studies that have demonstrated that urinary oxalate concentration has a greater effect on the formation of calcium oxalate crystalluria than comparable changes in calcium,°8 and that the incidence of stone episodes in patients with calcium oxalate stones is much more directly dependent upon urinary oxalate levels than on urinary calcium."'-, These studies imply that high normal oxalate levels may be an important determinant of calcium oxalate crystal formation and they emphasize the need to understand those factors involved in controlling the synthesis, excretion and transport of oxalic acid. DISORDERS OF OXALATE SYNTHESIS
The term primary hyperoxaluria refers to 2 genetically distinct syndromes, both associated with the same clinical picture; namely, the early onset of recurrent calcium oxalate stones and eventually the deposition of calcium oxalate crystals in various tissues, a condition referred to as oxalosis.4 In these conditions marked over production of oxalate has been documented in addition to increased production and excretion of glyoxylic and glycolic acids in type I primary hyperoxaluria, and increased production of L-glyceric acid in type II primary hyperoxaluria. Nearly 20 years ago Koch and associates demonstrated a deficiency of a cytosolic carboligase in a patient with primary hyperoxaluria type l." 9 This carboligase uses a-ketoglutarate and glyoxylate to produce a-hydroxy, ~-ketoadipate. It was postulated that a defect in this enzyme would lead to glyoxylate accumulation and increased synthesis of oxalate and glycolate, accounting for the increased excretion of these organic acids. Bourke and associates were unable to confirm a defect in this carboligase in hyperoxaluric patients.4° Recently, Danpure and associates demonstrated a deficiency in the activity of the peroxisomal enzyme, alanine:glyoxylate aminotransferase (AGT) in the liver of 6 patients with primary hyperoxaluria type 1.41 The activity of AGT appeared to be inversely correlated to urinary oxalate excretion. The patient with the mildest disease had the highest relative activity and the patient with the next mildest form had less AGT activity. Since primary hyperoxaluria type I appears to be a heterogeneous disease clinically,4 2 enzymologically41 and immunologically,4" it is possible that patients with mild elevations of urinary oxalate could have a mild deficiency of this enzyme. Danpure and associates 44 have suggested that the previously defined carboligase defect in patients with primary hyperoxaluria type I represents a methodological artifact, since the methods of preparation of cytosolic fractions by Koch and associates'i 9 could have led to release of mitochondrial carboligase activity. Presently, it is not possible to determine whether there are 2 separate enzyme defects to explain the type I syndrome or whether all patients with the type I syndrome have a deficiency of AGT. It is important to emphasize that some patients with primary hyperoxaluria type I appear to respond to large doses of pyridoxine by lowering somewhat the urinary excretion of oxalate.4 5
Other authors have demonstrated a response to low doses of pyridoxine in some patients 4 G or a variable response to high dose pyridoxine. 47 Most of the patients studied by Danpure and associates have been pyridoxine resistant. This variable response to pyridoxine in patients with primary hyperoxaluria type I documents clinical heterogeneity that has not been fully explained on a molecular basis. Determinations of hepatic enzymes involved in oxalate metabolism have not been performed in patients with calcium oxalate nephrolithiasis who are not hyperoxaluric or only modestly hyperoxaluric. It is certainly possible that mild defects in the enzymes that metabolize glyoxylate could lead to modest hyperoxaluria and recurrent calcium oxalate stone disease. Dietary studies tend to diminish somewhat the possible role endogenous oxalate production may have in idiopathic calcium oxalate stone disease. If increased endogenous oxalate production causes mild hyperoxaluria then the removal of dietary sources should have little or no effect on oxalate excretion. However, fasting has been shown to abolish the difference in the oxalate-to-creatinine ratio between controls and stone formers. 48 In addition, the concentration of oxalate in fasting urine did not differ between controls and stone formers. Therefore, the elevated levels of oxalate seen in some patients with calcium oxalate stone disease may not be the result of endogenous over production but clearly more work is needed to resolve this question. Other causes of over production of oxalate leading to hyperoxaluria include the ingestion of substances that can be converted to oxalate and pyridoxine deficiency.4 Ingestion of large amounts of ethylene glycol can lead to hyperoxaluria, since ethylene glycol can be converted to glycoaldehyde and to glycolate in the liver. The 2-carbon anesthetic agent methoxyflurane also can be converted to oxalate and some cases of hyperoxaluria following methoxyflurane anesthesia have been reported. 49 • 50 Pyridoxine deficiency in rats can certainly lead to hyperoxaluria but documented cases of pyridoxine deficiency and hyperoxaluria in man are limited. DISORDERS OF INTESTINAL OXALATE ABSORPTION
Enteric hyperoxaluria is a condition in which severe chronic bowel disease associated with fat malabsorption leads to the hyperabsorption of dietary oxalate, hyperoxaluria and recurrent calcium oxalate stones. A wide variety of chronic gastrointestinal disorders and surgical procedures of the gastrointestinal tract have been shown to produce this syndrome. 51 The mechanism for the hyperoxaluria in these patients is the hyperabsorption of oxalate. In the presence of severe fat malabsorption fatty acids accumulate in the lumen of the small bowel. These fatty acids avidly bind intraluminal calcium reducing the amount of calcium available for complexation with oxalate. This leaves more oxalate in a soluble form and hyperabsorption of oxalate ensues. In addition to this mechanism, other investigators have demonstrated that malabsorbed bile acids increase the permeability of the colon with regard to oxalate and increased colonic absorption of oxalate can occur in that situation. 52 Is it possible that increased absorption of dietary oxalate could explain the modest hyperoxaluria seen in some patients with idiopathic calcium oxalate nephrolithiasis? Schwille and associates showed that with fasting the amount of urinary oxalate in patients with idiopathic stone disease did not differ from that in controls but patients exhibited a greater degree of postprandial hyperoxaluria.4 8 Robertson and associates demonstrated that the addition of sodium oxalate to a basal diet led to a greater degree of urine saturation and formation of calcium oxalate crystals in patients with calcium oxalate stone disease than in controls. 38 Zarembski and Hodgkinson studied the effect that a high oxalate diet (given in the form of tea or rhubarb) had on increasing urinary oxalate, and they showed that stone formers had a 10.3 per cent increase versus 3.4 per
OXALATE SYNTHESIS, TRANSPORT AND HYPEROXALURIC SYNDROMES
cent in controls when expressed as a percentage of the increase in dietary oxalate.'·' These data suggest that an increase in intestinal absorption of oxalate occurs in some patients with idiopathic calcium oxalate stone disease. Hodgkinson hypothesized that the increase in urinary oxalate excretion is due to an increase in oxalate absorption from the intestine, secondary to the increased absorption of calcium.'' 4 Increased calcium absorption would lead to decreased intraluminal calcium and indirectly allow for an increase in the absorption of free oxalate. However, some patients with idiopathic stone disease have normocalciuria and, t.11erefore, increased intestinal absorption of oxalate could be independent of increased calcium absorption. No studies of ,;he intraluminal calcium concentration in the gastrointestinal tract of normocalciuric stone formers have been reported. ABNORMALITIES IN THE RENAL CLEARANCE OF OXALATE
The question of abnormalities in the renal clearance of oxalate as a cause of hyperoxaluria remains open. Previous studies on the renal clearance of oxalate have been hampered by the lack of reproducible and accurate methods for plasma oxalate quantitation and, therefore, they had to rely on C-14 oxalate infusion studies to determine plasma oxalate. Several studies on the renal clearance of oxalate have been reported. Williams and associates measured the renal clearance of oxalate in 6 normal subjects. 14 Using a constant infusion of C14 oxalic acid, the renal clearance was shown to be 169 ml. per minute, with a range of 101 to 217 ml. per minute. The oxalate/ creatinine clearance ratio in these subjects was between 1.33 and 2.09 with a mean of 1.64. It also was demonstrated that the renal clearance of oxalate in 2 patients with primary hyperoxaluria was not different from controls. Constable and associates, comparing 2 different methods for oxalate measurement, enzymatic and radioisotopic, determined the mean oxalate clearance in 5 normal subjects to be 168 ml. per minute per 1.73 m.", with a range of 105 to 227.rn Hodgkinson and associates determined the renal clearance of oxalate in 3 controls and 15 patients with recurring calciumcontaining renal stones, specific type unspecified. 0 ' The oxalate clearances in normal subjects ranged from 162 to 358, mean 249 ml. per minute, and they did not differ from patients (range 95 to 287, mean 201 ml. per minute). The oxalate/creatinine clearance ratio ranged from 1.42 to 2.6 (mean 1.95) in the controls, compared to 1.04 to 2.38 (mean 1.80) in the patients. On the other hand, Pinto and associates found an increase in oxalate clearance in a subpopulation of 8 recurrent oxalate stone formers (380 to 615 ml. per minute per 1.73 m.", mean 562) compared to 6 controls (170 to 250 ml. per minute per 1.73 m.", mean 203)." t; The recent availability of more accurate methods for the measurement of plasma oxalate should allow standard renal clearance studies to be done in patients with calcium oxalate nephrolithiasis to determine if increased renal clearance could explain modest hyperoxaluria. TRANSPORT OF OXALIC ACID
Recently, Laxmanan and associates reported specific binding of oxalate to homogenates of rat and human kidney and liver tissues.'· 7 Upon further subcellular fractionation, this binding was localized to the inner mitochondrial membrane. The binding was saturable, occurred with high affinity and showed ligand specificity. Strzelecki and Menon also demonstrated carrier mediated transport of oxalate by rat liver and kidney mitochondria.'·' This transport was phosphate-linked and mediated through the dicarboxylate transporter. Recently, our laboratory, using standard tissue culture techniques, has preliminary data demonstrating oxalate uptake in the kidney epithelial LLCPKl cell line" This transport was time and concentration
745
Baggio and associates reported an altered red blood cell transmembrane transport of oxalate in a group of 90 patients with idiopathic calcium oxalate nephrolithiasis, many of whom had moderate hyperoxaluria. 5 " The rate of oxalate exchange across the red cell membrane was significantly faster in stone forming patients than in controls, and it was shown to be an inherited characteristic. Oral treatment with the diuretics hydrochlorothiazide and amiloride restored the red cell oxalate exchange rate to nearly normal in all patients. The oxalate exchange rate in red blood cells also was inhibited in vitro by hydrochlorothiazide'rn and the anion transport inhibitor DIDS." 1 Red blood cells possess a specific anion transport protein called band 3 protein. "2 The proposed mechanism by which these drugs act is by covalently binding to band 3 (the anion transporter protein)."" Baggio and associates have also demonstrated that red blood cell ghosts from patients with idiopathic calcium oxalate nephrolithiasis exhibit increased phosphorylation of band 3 and band 2 (spectrin), which can be decreased by hydrochlorothiazide. 64 Since red blood cells are commonly used as models for the study of membrane function and transport systems, the authors were comfortable in suggesting that the results from red blood cells could be extrapolated to other types of cells, and they hypothesized that an inherited defect in the cellular transport of oxalate may be a common factor in idiopathic calcium oxalate stone disease. These studies have suggested the possibility that abnormally rapid oxalate transport seen in red blood cells could represent a generalized membrane abnormality. This abnormality could also be present in the gastrointestinal epithelial cells and renal epithelial cells, accounting for increased absorption and increased clearance of oxalate. These interesting hypotheses will require further study in patients with and without this red blood cell abnormality. FUTURE DIRECTIONS IN RESEARCH
The amount of oxalic acid in urine clearly has an important role in the formation of calcium oxalate stones. This is particularly true in patients with the aforementioned classical hyperoxaluric syndromes. The finding that urinary oxalate levels are modestly elevated in some patients with calcium oxalate stone disease and the evidence that supports a major role for oxalate in determining calcium oxalate crystalluria indicate that research efforts in the future should be directed toward developing methods for lowering urinary oxalate. If one could successfully and safely lower urinary oxalate to levels below the normal lower limit, it would be difficult to form a calcium oxalate stone regardless of the specific etiology of the stone disease. The concentration of oxalate in urine can be substantially lowered by high fluid intake but this requires a degree of patient compliance that is extremely difficult to achieve. Therefore, we believe that a search for safe and effective methods of lowering urinary oxalate is potentially useful for the prevention of calcium oxalate stone disease. To develop these approaches will require additional studies of oxalate synthesis, factors that control that synthesis and pharmacological agents that can safely inhibit the synthesis of oxalate. Although in normal individuals only a small amount of dietary oxalate is absorbed, dietary oxalate does have a significant effect on urinary oxalate in stone formers and, therefore, measures to block the absorption of dietary oxalate could be useful in the treatment of patients with stone disease. Finally, since oxalate is secreted by the proximal renal tubule, agents that can block oxalate secretion could effectively lower urinary oxalate. Whether this can be done safely without increasing plasma or tissue levels of oxalate remains to be determined. Nevertheless, the more we can learn about the molecular mechanisms involved in oxalate synthesis and transport the closer we should be to developing effective and safe methods for oxalate and calcium oxalate
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L Liedtke, R.R., Wilson, D. M., Moyer, T. P., Wandzilak, T. and Williams, H. E.: Analysis of an immobilized oxalate oxidase method in urine-problems solved and methods compared. Urol. Res., 16: 188, abstract A57, 1988. 2. Hargreave, T. B., Sali, A., Mackay, C. and Sullivan, M.: Diurnal variation in urinary oxalate. Brit. J. Urol., 49: 597, 1977. 3. Robertson, W. G., Peacock, M., Marshall, R. W., Speed, R. and Nordin, B. E. C.: Seasonal variations in the composition of urine in relation to calcium stone formation. Clin. Sci. Mol. Med., 49: 597, 1975. 4. Williams, H. E. and Smith, L. H., Jr.: Primary hyperoxaluria. In: The Metabolic Basis of Inherited Disease, 5th ed. Edited by J. B. Stanbury, J.B. Wyngaarden, D.S. Fredrickson, J. L Goldstein and M. S. Brown. New York: McGraw-Hill Book Co., chapt. 10, pp. 204-229, 1983. 5. Ney, D., Hofmann, A. F., Fischer, C. and Stubblefield, N.: The Low Oxalate Diet Book. San Diego: University of California, 1981. 6. Baker, E. M., Saari, J. C. and Tolbert, B. M.: Ascorbic acid metabolism in man. Amer. J. Clin. Nutr., 19: 371, 1966. 7. Rivers, J.M.: Safety of high-level vitamin C ingestion. Ann. N. Y. Acad Sci., 498: 445, 1987. 8. Lamden, M. P. and Chrystowski, G. A.: Urinary oxalate excretion by man following ascorbic acid ingestion. Proc. Soc. Exp. Biol. Med., 85: 190, 1954. 9. Takenouchi, K., Aso, K., Kawase, K., Ichikawa, H. and Shiomi, T.: On the metabolites of ascorbic acid, especially oxalic acid, eliminated in urine, following the administration of large amounts of ascorbic acid. J. Vitaminology, 12: 49, 1966. 10. Kallner, A.: Serum bile acids in man during vitamin C supplementation and restriction. Acta Med. Scand., 202: 283, 1977. 11. Schmidt, K. H., Hagmaier, V., Hornig, D. H., Vuilleumier, J. P. and Rutishauser, G.: Urinary oxalate excretion after large intakes of ascorbic acid in man. Amer. J. Clin. Nutr., 34: 305, 1981. 12. Chalmers, A.H., Cowley, D. M. and McWhinney, B. C.: Stability of ascorbate in urine: relevance to analyses for ascorbate and oxalate. Clin. Chem., 31: 1703, 1985. 13. Chalmers, A. H., Cowley, D. M. and Brown, J. M.: A possible etiological role for ascorbate in calculi formation. Clin. Chem., 32: 333, 1986. 14. Williams, H. E., Johnson, G. A. and Smith, L. H., Jr.: The renal clearance of oxalate in normal subjects and patients with primary hyperoxaluria. Clin. Sci., 41: 213, 1971. 15. Hodgkinson, A. and Wilkinson, R.: Plasma oxalate concentration and renal excretion of oxalate in man. Clin. Sci. Mol. Med., 46: 61, 1974. 16. Constable, A. R., Joekes, A. M., Kasidas, G. P., O'Regan, P. and Rose, Q, A.: Plasma level and renal clearance of oxalate in normal subjects and in patients with primary hyperoxaluria or chronic renal failure or both. Clin. Sci., 56: 299, 1979. 17. Prenen, J. A. C., Boer, P., Mees, E. J. D., Endeman, H.J., Spoor, S. M. and Oei, H. Y.: Renal clearance of [14C]oxalate: comparison of constant-infusion with single-injection techniques. Clin. Sci., 63: 47, 1982. 18. Osswald, H. and Hautmann, R.: Renal elimination kinetics and plasma half-life of oxalate in man. Urol. Int., 34: 440, 1979. 19. Weinman, E. J., Frankfurt, S. H., Ince, A. and Sansom, S.: Renal tubular transport of organic acids. Studies with oxalate and paraaminohippurate in the rat. J. Clin. Invest., 61: 801, 1978. 20. Cattell, W.R., Spencer, A.G., Taylor, G. W. and Watts, R. W. E.: The mechanism of the renal excretion of oxalate in the dog. Clin. Sci., 22: 43, 1962. 21. McIntosh, G. H. and Belling, G. B.: An isotopic study of oxalate excretion in sheep. Aust. J. Exp. Biol. Med. Sci., 53: 479, 1975. 22. Tremaine, L. M., Bird, J. E. and Quebbemann, A. J.: Renal tubular excretory transport of oxalate in the chicken. J. Pharm. Exp. Ther., 233: 7, 1985. 23. Earnest, D. L., Johnson, G., Williams, H. E. and Admirand, W. H.: Hyperoxaluria in patients with ileal resection: an abnormality in dietary oxalate absorption. Gastroenterology, 66: 1114, 1974. 24. Prenen, J. A. C., Boer, P. and Dorhout Mees, E. J.: Absorption kinetics of oxalate from oxalate-rich food in man. Amer. J. Clin. Nutr., 40: 1007, 1984. 25. Binder, H.J.: Intestinal oxalate absorption. Gastroenterology, 67: 441, 1974.
26. Caspary, W. F.: Intestinal oxalate absorption. I. Absorption in vitro. Res. Exp. Med., 171: 13, 1977. 27. Schwartz, S. E., Stauffer, J. Q., Burgess, L. W. and Cheney, M.: Oxalate uptake by everted sacs of rat colon. Regional differences and the effects of pH and ricinoleic acid. Biochim. Biophys. Acta, 596: 404, 1980. 28. Freel, R. W., Hatch, M., Earnest, D. L. and Goldner, A. M.: Oxalate transport across the isolated ·rat colon. A re-examination. Biochim. Biophys. Acta, 600: 838, 1980. 29. Hatch, M., Freel, R. W., Goldner, A. M. and Earnest, D. L.: Oxalate and chloride absorption by the rabbit colon: sensitivity to metabolic and anion transport inhibitors. Gut, 25: 232, 1984. 30. Knickelbein, R. G., Aronson, P. S. and Dobbins, J. W.: Oxalate transport by anion exchange across rabbit ileal brush border. J. Clin. Invest., 77: 170, 1986. 31. Elder, T. D. and Wyngaarden, J.B.: The biosynthesis and turnover of oxalate in normal and hyperoxaluric subjects. J. Clin. Invest., 39: 1337, 1960. 32. Bailey, R.R., Dann, E., Greenslade, N. F., Little, P. J., McRae, C. U. and Utley, W. L. F.: Urinary stones: a prospective study of 350 patients. New Zeal. Med. J., 79: 961, 1974. 33. Wallace, M. R., Mason, K. and Gray, J.: Urine oxalate imd calcium in idiopathic renal stone formers. New Zeal. Med. J., 94: 87, 1981. 34. Revusova, V., Zvara, V. and Gratzlova, J.: Urinary oxalatl\ excretion in patients with urolithiasis. Urol. Int., 26: 277, 1971. 35. Robertson, W. G. and Peacock, M.: The cause of idiopathic calcium stone disease: hypercalciuria or hyperoxaluria? Nephron, :26: 105, 1980. 36. Baggio, B., Gambaro, G., Favaro, S. and Borsatti, A.: Prevalence of hyperoxaluria in idiopathic calcium oxalate kidney stone disease. Nephron, 35: 11, 1983. 37. Robertson, W. G., Peacock, M. and Nordin, B. E. C.: Measurement of activity products in urine from stone-formers and normal subjects. In: Urolithiasis; Physical Aspects. Edited by B. Finlayson, L. L. Hench and L. H. Smith. Washington, D. C.: National Academy of Science, p. 79, 1972. 38. Robertson, W. G., Peacock, M. and Nordin, B. E. C.: Calcium oxalate crystalluria and urine saturation in recurrent renal stoneformers. Clin. Sci., 40: 365, 1971. 39. Koch, J., Stokstad, E. L., Williams, H. E. and Smith, L. H., Jr.: Deficiency of 2-oxo-glutarate: glyoxylate carboligase activity in primary hyperoxaluria. Proc. Natl. Acad. Sci., 57: 1123, 1967. 40. Bourke, E., Frindt, G., Flynn, P. and Schreiner, G. E.: Primary hyperoxaluria with normal alpha-ketoglutarate: glyoxylate carboligase activity. Treatment with isocarboxazid. Ann. Intern. Med., 76: 279, 1972. 41. Danpure, C. J., Jennings, P.R. and Watts, R. W. E.: Enzymological diagnosis of primary hyperoxaluria type 1 by measurement of hepatic alanine: glyoxylate aminotransferase activity. Lancet, 1: 289, 1987. 42. Watts, R. W. E., Veall, N., Purkiss, P., Mansell, M. A. and Haywood, E. F.: The effect ofpyridoxine on oxalate dynamics in three cases of primary hyperoxaluria (with glycollic aciduria). Clin. Sci., 69: 87, 1985. 43. Wise, P. J., Danpure, C. J. and Jennings, P. R.: Immunological heterogeneity of hepatic alanine: glyoxylate aminotransferase in primary hyperoxaluria type I. FEBS Letter, 222: 17, 1987. 44. Danpure, C. J., Purkiss, P., Jennings, P.R. and Watts, R. W. E.: Mitochondrial damage and the subcellular distribution of 2oxoglutarate: glyoxylate carboligase in normal human and rat liver and in the liver of a patient with primary hyperoxaluria type I. Clin. Sci., 70: 417, 1986. 45. Gibbs, D. A. and Watts, R. W. E.: The action of pyridoxine in primary hyperoxaluria. Clin. Sci., 38: 277, 1970. 46. Yendt, E. R. and Cohanim, M.: Response to a physiologic dose of pyridoxine in type I primary hyperoxaluria. New Engl. J. Med., 312: 953, 1985. 47. Gill, H. S. and Rose, G. A.: Mild metabolic hyperoxaluria and its response to pyridoxine. Urol. Int., 41: 393, 1986. 48. Schwille, P. 0., Hanisch, E. and Scholz, D.: Postprandial hyperoxaluria and intestinal oxalate absorption in idiopathic renal stone disease. J. Urol., 132: 650, 1984. 49. Frascino, J. A., Vanamee, P. and Rosen, P. P.: Renal oxalosis and azotemia after methoxyflurane anesthesia. New Engl. J. Med., 283: 676, 1970. 50. McIntyre, J. W. R., Russell, J. C. and Chambers, M.: Oxalemia
OXALATE SYNTHESIS, TRANSPORT AND HYPEROXALURIC SYNDRON:ES
51. 52.
53.
54.
55.
56.
57.
following methoxyflurane anesthesia in man. Anesth. Analg., 52: 946, 1973. Williams, H. E.: Oxalic acid and the hyperoxaluric syndromes. Kidney Int., 13: 410, 1978. Dobbins, J. W. and Binder, H.J.: Effect of bile salts and fatty acids on the colonic absorption of oxalate. Gastroenterology, 70: 1096, 1976. Zarembski, P. M. and Hodgkinson, A.: Some factors influencing the urinary excretion of oxalic acid in man. Clin. Chim. Acta, 25: 1, 1969. Hodgkinson, A.: Evidence of increased oxalate absorption in patients with calcium-containing renal stones. Clin. Sci. Mo!. Med., 54: 291, 1978. Hodgkinson, A., Wilkinson, R. and Nordin, B. E. C.: The concentration of oxalic acid in human blood. In: Urinary Calculi: Recent Advances in Aetiology, Stone Structure and Treatment. Edited by L. Cifuentes Delatte, A. Rapado and A. Hodgkinson. Basel: S. Karger, p. 18, 1973. Pinto, B., Crespi, G., Sole-Balcells, F. and Barcelo, P.: Patterns of oxalate metabolism in recurrent oxalate stone formers. Kidney Int., 5: 285, 1974. Laxmanan, S., Selvam, R., Mahle, C. J. and Menon, M.: Binding of oxalate to mitochondrial inner membranes of rat and human kidney. J. Urol., 135: 862, 1986.
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58. Strzelecki, T. and Menon, M.: The uptake of oxalate by rat liver and kidney mitochondria. J. Biol. Chem., 261: 12197, 1986. 59. Baggio, B., Gambaro, G., Marchini, F., Cicerello, E., Tenconi, R., Clementi, M. and Borsatti, A.: An inheritable anomaly of redcell oxalate transport in "primary" calcium nephrolithiasis correctable with diuretics. New Engl. J. Med., 314: 599, 1986. 60. Cousin, J. L. and Motais, R.: The role of carbonic anhydrase inhibitors on anion permeability into ox red blood cells. J. Physiol., 256: 61, 1976. 61. Baggio, B., Gambaro, G., Borsatti, A., Clari, G. and Moret, V.: Relation between band 3 red blood cell protein and transmembrane oxalate flux in stone formers. Lancet, 2: 223, 1984. 62. Cabantchik, Z. I., Knauf, P. A. and Rothstein, A.: The anion transport system of the red blood cell: the role of membrane protein evaluated by the use of probes. Biochim. Biophys. Acta, 515: 239, 1978. 63. Rothstein, A., Cabantchik, Z. I. and Knauf, P.: Mechanism of anion transport in red blood cells: role of membrane proteins. Fed. Proc., 35: 3, 1976. 64. Baggio, B., Clari, G., Marzaro, G., Gambaro, G., Borsatti, A. and Moret, V.: Altered red blood cell membrane protein phosphorylation in idiopathic calcium oxalate nephrolithiasis. IRCS Med. Sci., 14: 368, 1986.
THE JOURNAL OF UROLOGY
Copyright © 1989 by The Williams & Wilkins Co.
Printed in U.S.A.
OXALATE SYNTHESIS, TRANSPORT AND THE HYPEROXALURIC SYNDROMES HIBBARD E. WILLIAMS
THEODORE R. W ANDZILAK
AND
From the University of California, Davis, School of Medicine. Davis, California
ABSTRACT
This article reviews the mechanisms involved in the synthesis, absorption, excretion and transport of oxalic acid, and the factors controlling these processes in man. The clinical syndromes associated with hyperoxaluria and recurrent calcium oxalate stone disease are reviewed, including new studies that raise the possibility of a generalized oxalate transport abnormality in some patients with renal stone disease. The important role of oxalate in the determination of calcium oxalate solubility in patients with calcium oxalate stone disease is emphasized and future directions for research in the prevention of recurrent calcium oxalate stone disease are discussed. (J. Ural., part 2, 141: 742-747, 1989) The subject of this presentation is oxalic acid and its role in renal stone disease. Oxalic acid is a strong organic acid with a pKa, of 1.27 and a pKa, of 3.8. It is the simplest dicarboxylic acid in nature, and at physiological pH it forms a number of salts; soluble salts with sodium and potassium, and an insoluble salt with calcium. It is this insolubility of the calcium oxalate salt that gives oxalic acid its clinical importance in man. To our knowledge, oxalic acid has no significant useful role in human metabolism. Approximately 75 per cent of all kidney stones are composed predominantly of calcium oxalate. This review will emphasize the important role that urinary oxalate has in the formation of calcium oxalate stones. Factors that control the amount of oxalate in the urine, causes of increased oxalate production and excretion, and possible directions for future research also will be discussed. URINARY OXALATE
Oxalate normally is excreted in urine at levels between 15 and 40 mg./24 hours. Various methods have confirmed this normal range, and the ion-chromatographic method and the enzymatic method for oxalate determination in urine appear to produce similar results. 1 A diurnal variation in urinary oxalate concentration and excretion exists with greatest values between 11 a.m. and 11 p.m.," as well as a seasonal variation with higher values in the summer months. i Approximately 10 to 20 per cent of excreted oxalate arises from absorbed dietary oxalate and the remainder of the oxalate in the urine arises as the result of endogenous metabolism from its 2 major precursors, glyoxylate and ascorbic acid.4 Oxalate is present in many common foods and it occurs in particularly high levels in cocoa, tea, rhubarb, spinach, parsley, pepper, peanuts and beets. The normal dietary intake of oxalate ranges between 80 and 100 mg. per day.'' Factors controlling the gastrointestinal absorption of ingested oxalate will be discussed. METABOLIC SYNTHESIS OF OXALATE
Oxalic acid represents an apparent useless end product of metabolism and in this way it resembles uric acid. Enzymes that metabolize oxalate to formic acid and carbon dioxide do not exist in man. Therefore, once oxalate is produced it must be excreted by the kidneys. No gastrointestinal route of oxalate excretion is known to exist. The 2 major precursors of oxalate are ascorbic acid and glyoxylic acid. Conversion of ascorbic acid to oxalic acid apparently occurs in the liver by a process
involving diketogulonic acid with the first 2 carbons of ascorbic acid being converted to oxalate.'; Little is known about the factors controlling this metabolic conversion but it is estimated that approximately 35 to 50 per cent of urinary oxalate comes from ascorbic acid.' Apparently the enzymes involved in the conversion of ascorbic acid to oxalate are saturable at low concentrations of ascorbic acid, since ingestion of fairly large doses of ascorbic acid does not appear to increase urinary oxalate. However, some studies have demonstrated that with high doses of ascorbic acid (greater that 5 gm. per day) some elevation of urinary oxalic acid can occur. 8 - 11 It should be emphasized that under certain conditions ascorbic acid can undergo apparent nonenzymatic oxidation to oxalate in urine, 12 making evaluation of reports of conversion of ascorbate to oxalate difficult. More studies need to be performed with present methods of oxalate determination that avoid any nonenzymatic conversion of ascorbate to oxalate in urine samples or in the assay procedure. Chalmers and associates have demonstrated an increased urinary oxalate and decreased ascorbate excretion in recurrent stone formers compared to controls after oral (2 gm.) but not intravenous (500 mg.) administration of ascorbic acid. 1 '1 The authors suggested that there was an increased conversion of oxalate from ascorbate in the gastrointestinal tract of recurrent stone formers. The other major precursor of oxalate is glyoxylate, which accounts for the synthesis of approximately 50 to 70 per cent of urinary oxalate (fig. 1).4 The major sources of glyoxylate in man include glycine, glycolic acid and serine. Glycine is converted to glyoxylate by D-amino acid oxidase. Glycolate is oxidized to glyoxylate by glycolic acid oxidase, and serine can be converted to glycine or to glycolic acid via ethanolamine and glycoaldehyde. An additional source of serine and glycolic acid is hydroxypyruvate. The oxidation of glyoxylate to oxalate can be catalyzed by 3 different enzymes: lactic dehydrogenase, xanthine oxidase and glycolic acid oxidase. Evidence to date supports the important role of lactic dehydrogenase in this oxidative reaction with only a limited if any role for glycolic acid oxidase or xanthine oxidase in this conversion_.4 Glyoxylate can be metabolized to glycine, a reaction controlled by the peroxisomal enzyme alanine, glyoxylate aminotransferase, a transaminase that is pyridoxine-dependent. Glyoxylate also can be reduced to glycolic acid by the enzyme glyoxylate reductase and it can be metabolized to a-hydroxy, /3-ketoadipate by a carboligase enzyme that is dependent on thiamine pyrophosphate and uses a-ketoglutarate.'
742
743
OXALATE SYNTHESIS, TRANSPORT AND HYPEROXALURIC SYNDROMES
'i
Hydroxypyruvate
/
Serine
Glycolate
~t Ascorbic Acid
Glycine
Ca
OX
\' Ca
B6
ox FIG. 1. Metabolic pathways involved in oxalate synthesis RENAL CLEARANCE OF OXALATE
A number of studies during the last 20 years have documented the major mechanisms involved in the renal handling of oxalate. Studies on the renal clearance of oxalate using in vivo C-14 oxalate infusion have demonstrated an oxalate/creatinine or inulin clearance ratio greater than 1, 14 - 17 suggesting that tubular secretion is the predominant mechanism for the renal handling of oxalate. Osswald and Hautmann studied the renal handling of oxalate in 6 subjects by the rapid injection of C-14 oxalate into the renal artery and the subsequent collection of urine via a catheter inserted into the renal pelvis. 18 They demonstrated 2.3-fold higher excretion of oxalate compared to inulin, confirming that tubular secretion has a major role in oxalate excretion in man. A number of studies in experimental animals also have documented that tubular secretion has a major role in oxalate excretion in these species. Studies have been done in the rat, 19 dog,2° sheep 21 and chicken. 22 Weinman and associates, using free-flow micropuncture and intratubular microinjection techniques, examined the renal handling of oxalate in the rat kidney.'" These studies demonstrated that oxalate is secreted in the early part of the proximal convoluted tubule, undergoes bidirectional transport in the mid portions of the proximal convoluted tubule and is probably not transported further down the tubule. Tremaine and associates, using the Sperber in vivo chicken preparation, demonstrated saturable excretory transport of oxalate through the renal tubular cell. 22 The transport of oxalate was not affected by probenecid, suggesting a separate system from that of uric acid, but it was reduced by a-ketoglutaric acid, indicating a possible involvement of the dicarboxylate transporter. Studies in man have documented the renal clearance of oxalic acid to be approximately 170 ml. per minute, 14 and an oxalate half life of elimination of 92 plus or minus 8 minutes.'" By extrapolating these clearance figures, the plasma oxalate concentration has been estimated to be 7 to 15 ,ug./100 ml. in normal man. GASTROINTESTINAL HANDLING OF OXALATE
As noted, oxalate is a constituent of the normal diet and approximately 80 to 100 mg. per day are ingested. Most ingested oxalate appears to be bound by intraluminal calcium in the small intestine and it is excreted as insoluble calcium oxalate complexes (fig. 2). 21 It is this phenomenon that probably accounts for the fact that only 10 to 20 per cent of ingested oxalate is absorbed in normal individuals. The concentration of intraluminal calcium appears to have a major role in determining the amount of dietary oxalate absorbed, and an inverse relationship exists between the amount of calcium ingested and the amount of oxalate absorbed by the gastrointestinal tract. Oxalate absorption appears to occur along the course of the entire gastrointestinal tract. The studies of Prenen and associates, which demonstrated a peak of oxalate be-
FIG. 2. Gastrointestinal tract handling of oxalate
tween 2 and 4 hours after ingestion, suggest that the small bowel is a major site of oxalate absorption in normal individuals.24 The mechanisms of oxalate absorption by the gastrointestinal tract recently have been investigated in detail. Early studies suggested that gastrointestinal oxalate transport was a nonenergy-dependent nonsaturable passive process. 25 - 27 However, these experiments were all performed with a calcium-free buffer system, and it was later demonstrated that calcium is required to maintain the integrity of the conductive pathways across gastrointestinal epithelium. Freel28 and Hatch 28 and their associates, using the technique of isolated short-circuited segments of rat and rabbit colon, demonstrated a net transport of 0xalate and chloride. This transport was abolished by the metabolic inhibitor dinitrophenol and the anion transport inhibitor, 4-acetamido-4' -isothiocyanatostilbene-2,2' -disulfonic acid. These results suggested that oxalate transport is indeed an energy-dependent process and that oxalate and chloride share a common transport pathway. More recently, Knickelbein and associates demonstrated the presence of oxalate transport in brush border membrane vesicles of rabbit ileum. 30 Oxalate could be transported by exchange for either hydroxyl or chloride ions and it could be inhibited by the anion exchange inhibitor, 4,4' -diisothiocyanatostilbene-2,2' -disulfonic acid (DIDS). Among other organic acids only formate and oxaloacetate could stimulate oxalate and chloride uptake. These studies demonstrated further that the cellular absorption of oxalate is an active transport process. Evidence in man does not suggest secretion of oxalate by the gastrointestinal tract. These conclusions were based primarily on the studies of intravenous C-14 oxalate administration in which recovery of oxalate in urine during a 2-day period was demonstrated. 01 These studies do not rule out a bidirectional transport system for oxalate in the gastrointestinal tract. DISORDERS OF OXALATE METABOLISM AND EXCRETION
Before the abnormalities of oxalate synthesis and excretion that lead to hyperoxaluria and the potential role of oxalate in idiopathic calcium oxalate stone disease are reviewed, it is important to define hyperoxaluria. As noted previously, the normal 24-hour excretion of oxalate is between 15 and
744
WILLIAMS AND W ANDZILAK
40 mg./24 hours. Upper limits of normal for urinary oxalate vary between 40 and 45 mg. depending upon the method used_. The normal ratio of urinary oxalate-to-urinary creatinine varies between 0.008 and 0.054. Using these figures, the incidence of hyperoxaluria in calcium oxalate stone formers varies from approximately 2 to 3 per cent to as high as 40 to 50 per cent. 32- 36 In our opinion most recent studies of calcium oxalate stone formers using accurate methods for urinary oxalate suggest that the true incidence of hyperoxaluria is between 10 and 20 per cent. We wish to emphasize at this point the important role urinary oxalate has in determining the state of saturation of the urine with respect to calcium oxalate. Studies by Robertson and associates have indicated that the oxalate concentration in urine is one of the most important determinants of calcium oxalate solubility." 7 Clinical evidence to support this important role for oxalate comes from studies that have demonstrated that urinary oxalate concentration has a greater effect on the formation of calcium oxalate crystalluria than comparable changes in calcium,°8 and that the incidence of stone episodes in patients with calcium oxalate stones is much more directly dependent upon urinary oxalate levels than on urinary calcium."'-, These studies imply that high normal oxalate levels may be an important determinant of calcium oxalate crystal formation and they emphasize the need to understand those factors involved in controlling the synthesis, excretion and transport of oxalic acid. DISORDERS OF OXALATE SYNTHESIS
The term primary hyperoxaluria refers to 2 genetically distinct syndromes, both associated with the same clinical picture; namely, the early onset of recurrent calcium oxalate stones and eventually the deposition of calcium oxalate crystals in various tissues, a condition referred to as oxalosis.4 In these conditions marked over production of oxalate has been documented in addition to increased production and excretion of glyoxylic and glycolic acids in type I primary hyperoxaluria, and increased production of L-glyceric acid in type II primary hyperoxaluria. Nearly 20 years ago Koch and associates demonstrated a deficiency of a cytosolic carboligase in a patient with primary hyperoxaluria type l." 9 This carboligase uses a-ketoglutarate and glyoxylate to produce a-hydroxy, ~-ketoadipate. It was postulated that a defect in this enzyme would lead to glyoxylate accumulation and increased synthesis of oxalate and glycolate, accounting for the increased excretion of these organic acids. Bourke and associates were unable to confirm a defect in this carboligase in hyperoxaluric patients.4° Recently, Danpure and associates demonstrated a deficiency in the activity of the peroxisomal enzyme, alanine:glyoxylate aminotransferase (AGT) in the liver of 6 patients with primary hyperoxaluria type 1.41 The activity of AGT appeared to be inversely correlated to urinary oxalate excretion. The patient with the mildest disease had the highest relative activity and the patient with the next mildest form had less AGT activity. Since primary hyperoxaluria type I appears to be a heterogeneous disease clinically,4 2 enzymologically41 and immunologically,4" it is possible that patients with mild elevations of urinary oxalate could have a mild deficiency of this enzyme. Danpure and associates 44 have suggested that the previously defined carboligase defect in patients with primary hyperoxaluria type I represents a methodological artifact, since the methods of preparation of cytosolic fractions by Koch and associates'i 9 could have led to release of mitochondrial carboligase activity. Presently, it is not possible to determine whether there are 2 separate enzyme defects to explain the type I syndrome or whether all patients with the type I syndrome have a deficiency of AGT. It is important to emphasize that some patients with primary hyperoxaluria type I appear to respond to large doses of pyridoxine by lowering somewhat the urinary excretion of oxalate.4 5
Other authors have demonstrated a response to low doses of pyridoxine in some patients 4 G or a variable response to high dose pyridoxine. 47 Most of the patients studied by Danpure and associates have been pyridoxine resistant. This variable response to pyridoxine in patients with primary hyperoxaluria type I documents clinical heterogeneity that has not been fully explained on a molecular basis. Determinations of hepatic enzymes involved in oxalate metabolism have not been performed in patients with calcium oxalate nephrolithiasis who are not hyperoxaluric or only modestly hyperoxaluric. It is certainly possible that mild defects in the enzymes that metabolize glyoxylate could lead to modest hyperoxaluria and recurrent calcium oxalate stone disease. Dietary studies tend to diminish somewhat the possible role endogenous oxalate production may have in idiopathic calcium oxalate stone disease. If increased endogenous oxalate production causes mild hyperoxaluria then the removal of dietary sources should have little or no effect on oxalate excretion. However, fasting has been shown to abolish the difference in the oxalate-to-creatinine ratio between controls and stone formers. 48 In addition, the concentration of oxalate in fasting urine did not differ between controls and stone formers. Therefore, the elevated levels of oxalate seen in some patients with calcium oxalate stone disease may not be the result of endogenous over production but clearly more work is needed to resolve this question. Other causes of over production of oxalate leading to hyperoxaluria include the ingestion of substances that can be converted to oxalate and pyridoxine deficiency.4 Ingestion of large amounts of ethylene glycol can lead to hyperoxaluria, since ethylene glycol can be converted to glycoaldehyde and to glycolate in the liver. The 2-carbon anesthetic agent methoxyflurane also can be converted to oxalate and some cases of hyperoxaluria following methoxyflurane anesthesia have been reported. 49 • 50 Pyridoxine deficiency in rats can certainly lead to hyperoxaluria but documented cases of pyridoxine deficiency and hyperoxaluria in man are limited. DISORDERS OF INTESTINAL OXALATE ABSORPTION
Enteric hyperoxaluria is a condition in which severe chronic bowel disease associated with fat malabsorption leads to the hyperabsorption of dietary oxalate, hyperoxaluria and recurrent calcium oxalate stones. A wide variety of chronic gastrointestinal disorders and surgical procedures of the gastrointestinal tract have been shown to produce this syndrome. 51 The mechanism for the hyperoxaluria in these patients is the hyperabsorption of oxalate. In the presence of severe fat malabsorption fatty acids accumulate in the lumen of the small bowel. These fatty acids avidly bind intraluminal calcium reducing the amount of calcium available for complexation with oxalate. This leaves more oxalate in a soluble form and hyperabsorption of oxalate ensues. In addition to this mechanism, other investigators have demonstrated that malabsorbed bile acids increase the permeability of the colon with regard to oxalate and increased colonic absorption of oxalate can occur in that situation. 52 Is it possible that increased absorption of dietary oxalate could explain the modest hyperoxaluria seen in some patients with idiopathic calcium oxalate nephrolithiasis? Schwille and associates showed that with fasting the amount of urinary oxalate in patients with idiopathic stone disease did not differ from that in controls but patients exhibited a greater degree of postprandial hyperoxaluria.4 8 Robertson and associates demonstrated that the addition of sodium oxalate to a basal diet led to a greater degree of urine saturation and formation of calcium oxalate crystals in patients with calcium oxalate stone disease than in controls. 38 Zarembski and Hodgkinson studied the effect that a high oxalate diet (given in the form of tea or rhubarb) had on increasing urinary oxalate, and they showed that stone formers had a 10.3 per cent increase versus 3.4 per
OXALATE SYNTHESIS, TRANSPORT AND HYPEROXALURIC SYNDROMES
cent in controls when expressed as a percentage of the increase in dietary oxalate.'·' These data suggest that an increase in intestinal absorption of oxalate occurs in some patients with idiopathic calcium oxalate stone disease. Hodgkinson hypothesized that the increase in urinary oxalate excretion is due to an increase in oxalate absorption from the intestine, secondary to the increased absorption of calcium.'' 4 Increased calcium absorption would lead to decreased intraluminal calcium and indirectly allow for an increase in the absorption of free oxalate. However, some patients with idiopathic stone disease have normocalciuria and, t.11erefore, increased intestinal absorption of oxalate could be independent of increased calcium absorption. No studies of ,;he intraluminal calcium concentration in the gastrointestinal tract of normocalciuric stone formers have been reported. ABNORMALITIES IN THE RENAL CLEARANCE OF OXALATE
The question of abnormalities in the renal clearance of oxalate as a cause of hyperoxaluria remains open. Previous studies on the renal clearance of oxalate have been hampered by the lack of reproducible and accurate methods for plasma oxalate quantitation and, therefore, they had to rely on C-14 oxalate infusion studies to determine plasma oxalate. Several studies on the renal clearance of oxalate have been reported. Williams and associates measured the renal clearance of oxalate in 6 normal subjects. 14 Using a constant infusion of C14 oxalic acid, the renal clearance was shown to be 169 ml. per minute, with a range of 101 to 217 ml. per minute. The oxalate/ creatinine clearance ratio in these subjects was between 1.33 and 2.09 with a mean of 1.64. It also was demonstrated that the renal clearance of oxalate in 2 patients with primary hyperoxaluria was not different from controls. Constable and associates, comparing 2 different methods for oxalate measurement, enzymatic and radioisotopic, determined the mean oxalate clearance in 5 normal subjects to be 168 ml. per minute per 1.73 m.", with a range of 105 to 227.rn Hodgkinson and associates determined the renal clearance of oxalate in 3 controls and 15 patients with recurring calciumcontaining renal stones, specific type unspecified. 0 ' The oxalate clearances in normal subjects ranged from 162 to 358, mean 249 ml. per minute, and they did not differ from patients (range 95 to 287, mean 201 ml. per minute). The oxalate/creatinine clearance ratio ranged from 1.42 to 2.6 (mean 1.95) in the controls, compared to 1.04 to 2.38 (mean 1.80) in the patients. On the other hand, Pinto and associates found an increase in oxalate clearance in a subpopulation of 8 recurrent oxalate stone formers (380 to 615 ml. per minute per 1.73 m.", mean 562) compared to 6 controls (170 to 250 ml. per minute per 1.73 m.", mean 203)." t; The recent availability of more accurate methods for the measurement of plasma oxalate should allow standard renal clearance studies to be done in patients with calcium oxalate nephrolithiasis to determine if increased renal clearance could explain modest hyperoxaluria. TRANSPORT OF OXALIC ACID
Recently, Laxmanan and associates reported specific binding of oxalate to homogenates of rat and human kidney and liver tissues.'· 7 Upon further subcellular fractionation, this binding was localized to the inner mitochondrial membrane. The binding was saturable, occurred with high affinity and showed ligand specificity. Strzelecki and Menon also demonstrated carrier mediated transport of oxalate by rat liver and kidney mitochondria.'·' This transport was phosphate-linked and mediated through the dicarboxylate transporter. Recently, our laboratory, using standard tissue culture techniques, has preliminary data demonstrating oxalate uptake in the kidney epithelial LLCPKl cell line" This transport was time and concentration
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Baggio and associates reported an altered red blood cell transmembrane transport of oxalate in a group of 90 patients with idiopathic calcium oxalate nephrolithiasis, many of whom had moderate hyperoxaluria. 5 " The rate of oxalate exchange across the red cell membrane was significantly faster in stone forming patients than in controls, and it was shown to be an inherited characteristic. Oral treatment with the diuretics hydrochlorothiazide and amiloride restored the red cell oxalate exchange rate to nearly normal in all patients. The oxalate exchange rate in red blood cells also was inhibited in vitro by hydrochlorothiazide'rn and the anion transport inhibitor DIDS." 1 Red blood cells possess a specific anion transport protein called band 3 protein. "2 The proposed mechanism by which these drugs act is by covalently binding to band 3 (the anion transporter protein)."" Baggio and associates have also demonstrated that red blood cell ghosts from patients with idiopathic calcium oxalate nephrolithiasis exhibit increased phosphorylation of band 3 and band 2 (spectrin), which can be decreased by hydrochlorothiazide. 64 Since red blood cells are commonly used as models for the study of membrane function and transport systems, the authors were comfortable in suggesting that the results from red blood cells could be extrapolated to other types of cells, and they hypothesized that an inherited defect in the cellular transport of oxalate may be a common factor in idiopathic calcium oxalate stone disease. These studies have suggested the possibility that abnormally rapid oxalate transport seen in red blood cells could represent a generalized membrane abnormality. This abnormality could also be present in the gastrointestinal epithelial cells and renal epithelial cells, accounting for increased absorption and increased clearance of oxalate. These interesting hypotheses will require further study in patients with and without this red blood cell abnormality. FUTURE DIRECTIONS IN RESEARCH
The amount of oxalic acid in urine clearly has an important role in the formation of calcium oxalate stones. This is particularly true in patients with the aforementioned classical hyperoxaluric syndromes. The finding that urinary oxalate levels are modestly elevated in some patients with calcium oxalate stone disease and the evidence that supports a major role for oxalate in determining calcium oxalate crystalluria indicate that research efforts in the future should be directed toward developing methods for lowering urinary oxalate. If one could successfully and safely lower urinary oxalate to levels below the normal lower limit, it would be difficult to form a calcium oxalate stone regardless of the specific etiology of the stone disease. The concentration of oxalate in urine can be substantially lowered by high fluid intake but this requires a degree of patient compliance that is extremely difficult to achieve. Therefore, we believe that a search for safe and effective methods of lowering urinary oxalate is potentially useful for the prevention of calcium oxalate stone disease. To develop these approaches will require additional studies of oxalate synthesis, factors that control that synthesis and pharmacological agents that can safely inhibit the synthesis of oxalate. Although in normal individuals only a small amount of dietary oxalate is absorbed, dietary oxalate does have a significant effect on urinary oxalate in stone formers and, therefore, measures to block the absorption of dietary oxalate could be useful in the treatment of patients with stone disease. Finally, since oxalate is secreted by the proximal renal tubule, agents that can block oxalate secretion could effectively lower urinary oxalate. Whether this can be done safely without increasing plasma or tissue levels of oxalate remains to be determined. Nevertheless, the more we can learn about the molecular mechanisms involved in oxalate synthesis and transport the closer we should be to developing effective and safe methods for oxalate and calcium oxalate
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WILLIAMS AND WANDZILAK
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