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DIRECTIONS AND PROSPECTS FOR FUTURE RESEARCH ON GOUT J. EDWIN SEEGMILLER Knowledge of the causes and mechanism of development of gouty arthritis has been greatly expanded within the past 15 years (12). Nevertheless many gaps still exist. T h e molecular basis of the ability of the crystal of monosodium urate (3,4), but not that of cystine (5,6), to produce a disruption of the lysosomal membrane needs more detailed investigation. Additional information is still needed on the precise role of uric acid and its salt in the genesis of renal dysfunction in gout. T h e presence of monosodium urate crystals in the renal parenchyma of at least some of the patients with gout (7) raises the possibility that part of the pathologic process leading to renal dysfunction involves an acute intermittent inflammatory response to monosodium urate crystals similar to that observed in peripheral joints. Such an acute gouty nephropathy as part of the pathology of gout has not heretofore been recognized either c h i cally or pathologically (1,Z). I n order to assess this possibility clinicians should be alert to the possibility of acute gouty nephropathy in their patients with known gout. Pain or tenderness in the renal area in the absence of infection or renal calculi should prompt a urine analysis. T h e presence of transient proteinuria or a sterile pyruria, perhaps with systemic symptoms of malaise, J. Edwin Seegmiller, M.D.: Professor of Medicine, Department of Medicine, University of California, San Diego, La Jolla, California 92093. Address reprint requests to Dr. Seegmiller.

fever, and leukocytosis, might constitute evidence suggesting the presence of such a process. T h e actual method to be employed in demonstrating conclusively the presence or absence of an acute gouty inflammation involving the kidney presents a more difficult problem. A needle biopsy of the kidney with demonstration, in frozen sections or alcohol-fixed tissues, of urate crystals undergoing phagocytosis by infiltrating accumulations of neutrophils would present conclusive evidence of such a n acute gouty nephropathy. However the biopsy procedure presents difficulties. Because the crystals are characteristically located in the medullary portions of the kidney and are confined to discrete areas, one cannot be certain that the biopsy, even if it is sufficiently deep to provide medullary tissue, includes the involved area. T h e extent to which the suspected acute inflammatory process might present problems of excessive bleeding following the biopsy must also be carefully evaluated. Other noninvasive approaches are also needed. A method for detecting the presence of monosodium urate crystals within the renal parenchyma would be of extraordinary value. A society with technology that is now capable of detecting, by physical means, flights of insects in tremendous expanses of atmosphere (8) should be capable of developing a system for the detection of small solid crystals in soft tissues. T h e possibility that ultrasound might be used to detect such crystals has been explored through this author’s col-

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laboration with Dr. Glen Wade of the Department Electrical Engineering and Computer Science at Santa Barbara. H e has developed by means of an ultrasonic laser beam an interference pattern holography (9) that depicts the entire inner organ structure of small tropical fish i n three-dimensional display. Unfortunately monosodium urate crystals injected into the fish could not be visualized with the ultrasonic wavelength that he was using. T h e possibility of modifications of his procedure to permit detection of monosodium urate crystals within soft tissues would be of tremendous clinical value in detecting not only gouty nephropathy but also other crystaldeposition diseases such as chondrocalcinosis (10) as well as deposition or presence of crystals in muscles as a cause of myalgia (1 1). Another problem deserving more detailed investigation in the kidney is the mechanism of uric acid transport. T h e details of the transport process are now being investigated intensively i n bacterial as well as i n mammalian cells and a number of biochemical mechanisms and accompaniments have been identified (12). Natural human mutations resulting in a deficient renal reabsorption of uric acid in the kidney tubules should provide important “experiments of nature” for gaining insight into the transport mechanism for uric acid (13-15). At the present time we know of only two biochemical reactions that uric acid undergoes other than the reaction with uricase, which of course is missing in human tissue. One of these is the peroxidative destruction of uric acid observed i n leukocytes (16,17); tlie other has so far been described only i n beef erythrocytes (18,19). Hatfield has purified the enzyme (20) and found that it converts uric acid to a ribonucleotide by reaction with phosphoribosylpyrophosphate. Moreover the purified enzyme reacts much more rapidly with uracil than with uric acid, and ribose phosphate is added to the 3 position of the pyrimidine ring rather than to the 9 position of the purine ring. Becker has explored the possibility that cultured human cells may be able to carry out the reaction but so far he has been unable to demonstrate this reaction in human fibroblasts. Whether or not this reaction is involved in the tubular transport mechanism remains to be demonstrated. Another approach would be to look for a uric acid-binding protein in human kidney tissue. T h e possible role of uric acid and its salt in the development of cardiovascular disease also de-

SEEGMILLER

serves investigation. A correlation of hyperuricemia with development of myocardial infarction was found in prospective studies of the members of the Framingliam community in Massachusetts (21), although it. was not confirmed in a similar prospective study of Tecumseh, Michigan (22). An investigation of diseases developing i n hyperuricemic patients at the Kaiser Hospital in San Francisco (23) also failed to confirm this study. However, considering the importance of cardiovascular disease, a study is needed of the possible role of monosodium urate crystals in the development of cardiovascular lesions. For this study coronary artery samples obtained from hyperuricemic patients at autopsy should be examined in polarized light with polarizing filters for the presence of monosodium urate crystals i n frozen sections or in alcohol-fixed tissues after paraffin removal but before staining, and correlations should be made with serum urate concentration before death. Another area deserving attention in the future is identification of additional chemical disorders that are associated with hyperuricemia. Such studies can probably best be done in institutions where chemical data and diagnoses are available through the computer. T h e fact that around 2.50/, of the normal population exceeds the upper limit of normal (two standard deviations, by definition) assures the presence of hyperuricemia in a portion of patients with a wide range of diseases. I n order for an association of hyperuricemia with disease states to be significant rather than incidental, association must be significantly higher than 2.5 to 3y0. Obtaining a sufficient number of patients for evaluation may therefore be a difficult problem, particularly for some of the more rare disorders. A summary of presently available correlations of hyperuricemia and hypouricemia with a large number of other diseases has been assembled by Newcombe (24). A complicating factor of course is the liyperuricemic effect of salicylates and other drugs in common use, such as antihypertensive drugs, which must also be considered. A possible association of hyperuricemia with retinitis pigmentosum has been observed i n several members of a family (25). Hyperuricemia in association with polycystic kidney disease and an etiologic role for uric acid i n tlie development of polycystic kidney have been deduced without additional supporting data (26). Hyperuricemia with Osgood-Slatter’s disease has also been reported informally to the present author by a number of physicians (25). Another correlation to be considered is

FUTURE RESEARCH

the association of gout with a macular degeneration. An acquaintance of tlie author has seen at least 12 patients with this combination of disorders in the course of his clinical practice. This type of association holds forth the possibility that a n understanding of the abnormality in purine metabolism in some of these associated disorders might eventually lead to the primary genetic defect that may underlie many diseases that now are but poorly understood at a biochemical and genetic level. Development of diabetes mellitus in patients with a preexisting hyperuricemia has been reported (19,27). Whether tlie hyperuricemia represents but one side of tlie metabolic coin that includes diabetes and cardiovascular disease on the reverse side, or whether the hyperuricemia is of etiologic importance in tlie generation of diabetes has not been fully assessed. I n previous investigations in Denmark no evidence of an association of diabetes and gout was found (28). Nevertheless alloxan, a chemical agent able to induce diabetes in experimental animals, is formed from uric acid as a product of uric acid peroxidation in a n acid environment like that found within leukocytes (29,30); its possible role in regard to development of diabetes deserves more detailed investigation in human cells and hyperuricemic patients. T h e mechanism by which a number of environmental and physiologic factors act to increase serum urate concentration also deserves special attention. T h e mechanism responsible for hyperuricemia as a late effect of lead poisoning is poorly understood. Likewise the mechanism by which obesity produces hyperuricemia deserves more detailed investigation, particularly because weight reduction produces a substantial decrease in serum urate in the obese hyperuricemic individual. T h e mechanism of action of estrogenic hormones in maintaining the serum urate of women around 1 mg% lower than that of men until after the menopause also deserves more intensive investigation. T h e remarkably large load of uric acid excreted by the pregnant woman provides a teleologic reason for this extra safety factor for preventing hyperuricemia in women. T h e possible role of tlie large load of uric acid in the genesis of toxemia of pregnancy presents an interesting area for further investigation. We are now aware of a number of clinical hereditary disorders associated with overproduction of uric acid and presumably with a primary defect

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somewhere in tlie regulation of the rate of purine biosynthesis. I n addition to those disorders described in Kelley and Becker’s presentations (pp 673-680, pp 687-693), several others have been described in association with a n excessive rate of purine synthesis. These include a child with autistic behavior (31) and an encephalopathy with self-mutilation in a female (32). Ataxia, weakness, deafness, and renal insufficiency with varying degrees of disability were found in 5 of 22 members of a kindred (33). Gout with benign symmetrical lipomatosis (Launois-Bensaude adenolipomatosis) associated with a n excessive rate of purine synthesis has been described in a 30-year-old woman with oligomenorrliea, muscle cramps, pes caws deformity, and extensor plantar reflexes (34). An idiopathic tremor of the right arm was found to be associated with overproduction of uric acid in a 15-year-old boy with a history of renal calculi from age 2 (25). Frequent uncontrollable seizures in a hyperuricemic child were ultimately controlled with allopurinol therapy (35). These disorders each hold promise not only of greatly increasing our knowledge of genetic defects leading to gouty arthritis but also of giving insight into the biochemical causes of neurologic diseases. T h e neurologic disorder that has been most thoroughly studied, however, is the Lescli-Nyhan syndrome (37,38). Although the genetic and biochemical primary defect has been identified, the mechanism by which the neurologic dysfunction is produced remains an enigma. Because i t is not abnormally high in concentration in the cerebrospinal fluid of affected children, uric acid probably does not mediate the neurologic dysfunction (39). Furthermore allopurinol has no effect on the neurologic problem even when begun shortly after birth (40). T h e difficulty probably resides in a functional disturbance in the neuron produced by tlie enzyme deficiency in tlie brain cells, particularly the basal ganglia, where the enzyme is normally most abundant (39). T h e identification at birth of a child with a gross deficiency of HPRT enzyme led to an evaluation of one new therapeutic approach. T h e rationale used was a bit beyond tlie solid base of knowledge, but clinical research must frequently extend itself in this manner in a n effort to meet clinical problems as they arise. Because adenine is known to correct a megaloblastic anemia that develops in some of these children (41,42), it was reasoned that a n adenine supplement in tlie diet might prevent the development

SEEGMILLER

of the neurologic dysfunction in this particular child. It is known that adenine by itself can be nephrotoxic in high doses because xantliine oxidase in the body converts it to the very sparingly soluble product, 2,8-dioxyadenine, which is then deposited in the kidneys (43).This process leads to severe renal dysfunction and death in experimental animals. Nevertheless it was felt that as large a dose as possible had to be given in order to provide the very best opportunity for it to act on the central nervous system (44). T h e dose of adenine was therefore gradually increased in the baby to the point at which the infant had a transient elevation of BUN for just 1 day, accompanied by an increase in the ratio of absorption in the urinary sediments at 305 mp, the absorption for 2,8-deoxyadenine, as compared to 292 mp, the absorption maximum for uric acid. When the drug was stopped at this point, the BUN promptly returned to normal. T h e n full doses of allopurinol were given because this drug blocks xanthine oxidase and thereby prevents to a considerable extent the formation of the toxic product 2,8-dioxyadenine. Despite continuation of both medications, the patient nevertheless developed neurologic symptoms at the expected time; thus it was realized that the adenine had been completely ineffective. Knowledge of the biochemical and molecular basis of dominantly inherited diseases is very meager. Further investigation of gout promises increased knowledge in a n area that includes many more common diseases. Defects in membrane structure or receptor proteins of various types are reasonable possibilities. A fascinating new defect in purine metabolism has been discovered by Giblett et al, who identified a gross deficiency of adenosine deaminase in 2 patients with combined immunodeficiency disease (45). Since that time over 14 families have been found with this unusual combination (46).T h e defect is transmitted by an autosomal recessive gene and the present author has succeeded in identifying a heterozygote fetus in utero through determination of adenosine deaminase activities in the amniotic fluid (47). T h e enzyme defect itself holds forth the possibility of providing useful new insights into the role of this enzyme and of purine metabolism in the development of the immune response. More recently Gilbett and others have described a child with immunodeficiency from impaired T-cell

function associated with a gross deficiency of purine nucleoside phosphorylase (48). One interesting new prospect for the future is the possibility of developing human cell lines deficient in specific enzymes and thus removing the necessity of first awaiting their identification in affected patients (49,50). From this type of approach substantial progress can be made in characterizing the metabolic effects of enzyme defects, such as their effect on the rate of purine synthesis. Such characterization would provide a more rational approach to the identification of these same enzyme defects in patient populations. T h u s the prospects for gaining further insight into the mechanism by which a genetic abnormality in metabolism leads to a pathologic state such as gouty arthritis hold great promise, not only of expanding our knowledge of human metabolic diseases but also of providing new and rational approaches for interrupting the sequence of events that produces the pathology.

REFERENCES 1. Seegmiller JE: Diseases of purine and pyrimidine me-

tabolism, Duncan’s Diseases of Metabolism. Seventh edition. Edited by PK Bondy, LE Rosenberg. Philadelphia, W. B. Saunders, 1974, pp 655-774 2. Wyngaarden JB, Kelley WN: Gout, T h e Metabolic Basis of Inherited Disease. Third edition. Edited by JB Stanbury, JB Wyngaarden, DS Fredrickson. New York, McGraw-Hill Book Company, 1972, pp 889-968 3. Weissmann G, Rita GA: Molecular basis of gouty inflammation: interaction of monosodium urate crystals with lysosomes and liposomes. Nature [New Biol] 240: 167-172, 1972 4. Mandel N: Gout: crystal structure of monosodium urate. Science (in press) 5. Seegmiller JE: Cystinosis, Lysosomes and Storage Diseases. First edition. Edited by H G Hers, F Van Hoof. New York, Academic Press, 1973, p p 485-518 6. Seegmiller JE: Acute gout-a study in depth. Hosp Prac 6:9-13, 1971 (editorial) 7. Seegmiller JE, Frazier PD: Biochemical considerations of the renal damage of gout. Ann Rheum Dis 25: 668-672, 1966 8. Richter JH, Jensen DR, Noonkester VR, et al: Remote radar sensing: atmospheric structure and insects. Science 180:1176-1178, 1973 9. Landry J, Powers J, Wade G: Ultrasonic imaging of internal structure by Bragg diffraction. Appl Phys Letters 15:186-188, 1969 10. McCarty DJ: Crystal deposition joint disease. Annu Rev Med 25:279-288, 1974

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11. Chalmers RA, Watts RWE, Pallis C, et al: Crystalline deposits in striped muscle in xanthinuria. Nature 221: 170-171, 1969 12. Boos W: Bacterial transport. Annu Rev Biochem 43: 123-146, 1974 13. Praetorius E, Kirk JE: Hypouricemia with evidence for tubular elimination of uric acid. J Lab Clin bled 35: 865, 1950 14. Greene ML, Marcus R, Aurbach GD, et al: Hypouricemia due to isolated renal tubular defect. Dalmatian dog mutation in man. Am J Med 53:361-367, 1972 15. Simkin PA, Skeath MD, Healey LA: Suppression of uric acid secretion in a patient with renal hypouricemia. Adv Exp Biol Med 41B:723-728, 1974. 16. Howell RR, Seegmiller JE: Uricolysis in human leukocytes. Nature 196:482-483, 1962 17. Goldfinger SE, Howell RR, Seegmiller JE: Suppression of metabolic accompaniments of phagocytosis by colchicine. Arthritis Rheum 8: 1112-1 122, 1965 18. Benedict SR: Studies in uric acid metabolism. I. On the uric acid in ox and chicken blood. J Biol Chem 20:633, 1915 19. Davis AR, Newton EB, Benedict SR: T h e combined uric acid in beef blood. J Biol Chem 54:595, 1922 20. Hatfield D, Wyngaarden JB: 3-Ribosylpurines. I. Synthesis of 3-ribosyluric acid, 5'-phosphate and 3-ribosylxanthine 5'-phosphate by a pyrimidine ribonucleotide pyrophosphorylase of beef erythrocytes. J Biol Chem 239:2580-2586, 1964 21. Hall AP: Correlations among hyperuricemia, hypercholesterolemia, coronary disease and hypertension. Arthritis Rheum 8:846-852, 1965 22. Mikkelsen WM, Dodge HJ, Valkenburg H: T h e distribution of serum uric acid values in a population unselected as to gout or hyperuricemia, Tecumseh, Michigan, 1959-60. Am J Med 39:242-251, 1965 23. Fessel WJ, Siegelaub AB, Johnson ES: Correlates and consequences of asymptomatic hyperuricemia. Arch Intern Med 13244-54, 1973 24. Newcombe DS: Inherited Biochemical Disorders and Uric Acid Metabolism. First edition. Baltimore, University Park Press, 1975 25. Seegmiller JE: Unpublished observations 26. Newcombe DS: Gouty arthritis and polycystic kidney disease. Ann Intern Med 79:605, 1973 27. Herman JB, Medalie JH, Goldbourt U: Diabetes and uric acid-a relationship investigated by the epidemiological method. Adv Exp Biol Med 41B:483484, 1974 28. Hauge M, Harvald B: Heredity in gout and hyperuricemia. Acta Med Scand 152:247-257, 1955 29. Soberon G, Cohen PP: Peroxidative formation of alloxan from uric acid by leukocytes. Arch Biochem Biophys 103:331-337, 1963

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30. Canellakis ES, Tuttle AL, Cohen PP: Comparative study of end-products of uric acid oxidation by peroxidases. J Biol Chem 213:397-404, 1955 31. Nyhan WL, James JA, Teberg A, et al: A new disorder of purine metabolism with behavioral manifestations. J Pediatr 74:20-27, 1969 32. Hooft C, Van Nevel C, Schaepdryver AFD: Hyperuricosuric encephalopathy without hyperuricemia. Arch Dis Child 43:734-737, 1968 33. Kosenberg AL, Bergstrom L, Troost BT, et al: Hyperuricemia and neurologic deficits: a family study. N Engl J Med 282:992-997, 1970 34. Greene ML, Glueck CJ, Fujimoto WY, et al: Benign symmetric lipomatosis (Launois-Bensaude adenolipomatosis) with gout and hyperlipoproteinemia. Am J Med 48:239-245, 1970 35. Coleman hf: Reversal of organic brain syndrome with seizures and hyperuricosuria subsequent to allopurinol therapy. Trans Am Neurol Assoc 96:113-117, 1971 36. Bazelon M, Stevens H, Davis M, et al: Mental retardation, self-mutilation and hyperuricemia in females. Trans Am Neurol Assoc 93:187-188, 1968 37. Lesch M, Nyhan WL: A familial disorder of uric acid metabolism and central nervous system function. Am J Med 36:561-570, 1964 38. Seegmiller JE, Rosenbloom FM, Kelley WN: Enzyme defect associated with a sex-linked human neurological disorder and excessive purine synthesis. Science 155: 1682-1684, 1967 39. Rosenbloom FM, Kelley WN, Miller J, et al: Inherited disorder of purine metabolism: correlation between central nervous system dysfunction and biochemical defects. JAMA 202: 175-177, 1967 40. Marks JF, Baum J, Keele DK, et al: Lesch-Nyhan syndrome treated from the early neonatal period. Pediatrics 42:357-359, 1968 41. Van der Zee SP, Lommen EJP, Trijbels JMF, et al: T h e influence of adenine on the clinical features and purine metabolism in the Lesch-Nyhan syndrome. Acta Paediatr Scand 59:259-264, 1970 42. Lommen EJP, Vogels GD, Van der Zee SP, et al: Concentrations of purine nucleotides in erythrocytes of patients with the Lesch-Nyhan syndrome before and during oral administration of adenine. Acta Paediatr Scand 60:642-646, 1971 43. Bendich A, Brown GB, Phillips FS, et al: Direct oxidation of adenine in vivo. J Biol Chem 183:267, 1950 44. Schulman JD, Greene ML, Fujimoto WY, et al: Adenine therapy for Lesch-Nyhan syndrome. Pediatr Res 5:77-82, 1971 45. Giblett ER, Anderson JE, Cohen F, et al: Adenosine deaminase deficiency in two patients with severely impaired cellular immunity. Lancet 2: 1067-1069, 1972

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46. Chen S-H, Scott CR, Swedberg KR: Heterogeneity for adenosine deaminase deficiency: expression of the enzyme i n cultured skin fibroblasts and amniotic fluid cells. Am J H u m Genet 27:46-52 ,1975 47. Snyder F, Seegmiller JE: Unpublished observations 48. Giblett ER, Ammann AJ, Wara DW, et al: Nucleoside phosphorylase deficiency i n a child with severely defective T-cell immunity an d normal B-cell immunity. Lancet 2:lOlO-1013, 1975

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49. Nuki G, Lever J , Seegmiller JE: Biochemical characteristic\ of 8-azaguanine resistant human lymphoblast mutants selected in uitro. Adv Exp N e d Biol 41A:255267, 1974 50. Lever JE, Nuki G, Seegmiller JE: Expression of purine overproduction in a series of 8-azaguanine resistant diploid human lymphoblast lines. Proc Natl Acad Sci USA 71 :2679-2683,1974