On the Enzymic Defects in Hereditary Tyrosinemia*

Proc. NatL. Acad. Scd. USA Vol. No. 74, No. 10, pp. 46414645, October 1977 Medical Sciences

On the enzymic defects in hereditary tyrosinemia* (5-iaminolevulinate/4,6-dioxoheptanoic acid, 3,5-dioxooctanedioic acid/fumarylacetoacetase / inborn error of metabolism/ porphobilinogen synthase) BENGT LINDBLAD, SVEN LINDSTEDT, AND GORAN STEEN Department of Clinical Chemistry, University of Gothenburg, Sahigren's Hospital, S-413 45 Gothenburg, Sweden Communicated by Jan G. Waldenstrom, August 1,1977

ABSTRACT The activity of the enzyme porphobilinogen suggesting that the metabolites that accumulate cause the liver synthase (EC 4.2.1.24) in erythrocytes from patients with he- and kidney damage in hereditary tyrosinemia. reditary tyrosinemia was less than 5% of that in a control group and the activity in liver tissue was less than 1% of the reported normal activity. Urine from patients with hereditary tyrosinemia contained an inhibitor that was isolated and identified METHODS as succinylacetone (4,6-dioxoheptanoic acid) by gas/liquid chromatography-mass spectrometry. Fresh urine samples Patients. The diagnosis of hereditary tyrosinemia was based contained succinylacetoacetate (3,5-dioxooctanedioic acid) as on the criteria given in the introduction. Some clinical data are well as succinylacetone. The inhibition of porphobilinogen given with the Results. synthase explains the high excretion of 5-aminolevulinate ob- Porphobilinogen Synthase in Erythrocytes. The enzyme served in hereditary tyrosinemia. Succinylacetone and succin- activity was determined as described by Collier (10), in 3' ylacetoacetate presumably originate from maleylacetoacetate or fumarylacetoacetate, or both, and their accumulation indi- ml. cates a block at the fumarylacetoacetase (EC 3.7.1.2) step in the Porphobilinogen Synthase in Liver. Liver tissue obtained degradation of tyrosine. We suggest that the severe liver and at autopsy 1 hr after death was put on dry ice. The assay was kidney damage in hereditary tyrosinemia may be due to the carried out as described by Gibson et al. (11) on a 100,000 X g accumulation of these tyrosine metabolites and that the primary supernatant of a 33% homogenate prepared in 0.1 M potassium enzyme defect in hereditary tyrosinemia may be decreased phosphate buffer at pH 6.5. activity of fumarylacetoacetase. 4-Hydroxyphenylpyruvate Dioxygenase in Liver. The ac- In the inborn error of metabolism called hereditary tyrosin- tivity of 4-hydroxyphenylpyruvate dioxygenase was deter- emia, the main clinical findings are liver failure, which develops mined from the formation of labeled homogentisate from la- into liver cirrhosis in early childhood, and multiple renal tubular beled 4-hydroxyphenylpyruvate (12, 13). defects with hypophosphatemic rickets (1, 2). The derangement Assay for Inhibition of Porphobilinogen Synthase. in tyrosine metabolism (i.e., hypertyrosinemia and high urinary Erythrocytes from normal donors were washed, hemolyzed, excretion of 4-hydroxyphenylpyruvate, 4-hydroxyphenyllac- and used as the source of porphobilinogen synthase. To each tate, and to a lesser extent 4-hydroxyphenylacetate) is due to incubation was added enzyme from approximately 1.5 X 109 a low activity of the enzyme 4-hydroxyphenylpyruvate diox- erythrocytes. The solution to be tested for inhibitory activity ygenase [4-hydroxyphenylpyruvate:oxygen oxidoreductase was added to the incubation mixture at the start of a 15-min (hydroxylating, decarboxylating), EC 1.13.11.27] (3), which preincubation period. The reaction was started by addition of catalyzes the formation of homogentisate (III) (Fig. 1) from 5-aminolevulinate. The assay procedure described above for 4-hydroxyphenylpyruvate (II). porphobilinogen synthase in erythrocytes was then carried out The increased excretion of these phenolic metabolites of in the absence of MnCl2 and dithiothreitol. tyrosine does not explain the liver and kidney damage in he- Isolation of the Inhibitor of Porphobilinogen Synthase. reditary tyrosinemia because a similar large excretion has been The isolation procedure was carried out on fresh pooled urine found also in patients without liver and kidney disease-e.g., samples from'patients suffering from hereditary tyrosinemia. in a 5-year-old boy with multiple congenital anomalies and with Diethyl ether extracts were fractionated by chromatography negligible activity of soluble tyrosine aminotransferase (4) and on both anion and cation exchange resins. The fractions were in at least three other patients who were mentally retarded (2, then assayed by the procedure described above and those 5, 6). A large excretion of the same metabolites has also been containing inhibitory activity were pooled. Thin-layer chro- found in hereditary fructose intolerance (7). matography of the fraction thus obtained from the last ion ex- In 1967 we reported on a patient who had symptoms similar change chromatography showed one segment with inhibitory to those characteristic of acute intermittent porphyria (8). An activity. This fraction was eluted and subjected to gas/liquid increased excretion of 5-aminolevulinate has since then been chromatography-mass spectrometry. observed in all patients studied by us, even in those without Mass Spectrometry. Electron impact mass spectra were these symptoms (3, 9), but so far it has not been possible to find recorded at 70 eV on an LKB 9000 gas chromatograph-mass a biochemical link between the altered tyrosine metabolism and spectrometer; ion-source temperature, 270°; acceleration the increased excretion of a porphyrin precursor. In this report voltage, 3.5 kV. Methoximes and methyl esters were prepared we present evidence for an enzyme defect in tyrosine catabo- at room temperature with methoxylamine hydrochloride in lism in hereditary tyrosinemia that explains the increased ex- pyridine followed by treatment with diazomethane. cretion of 5-aminolevulinate. We also present a hypothesis Organic Acids in Urine. Urines were examined for organic acids by gas/liquid chromatography-mass spectrometry as The costs of publication of this article were defrayed in part by the described (14). payment of page charges. This article must therefore be hereby marked Preparation of Synthetic Succinylacetone. Succinylacetone "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. * This paper was presented at the Swedish Medical Congress, 1976. 4641 Downloaded by guest on September 30, 2021 4642 Medical Sciences: Lindblad et al. Proc. Natl. Acad. Sci. USA 74 (1977)

OH OH OH Table 2. Inhibition of porphobilinogen synthase from erythrocytes by serum and urine from patients with hereditary tyrosinemia -I CCH2 2 CH2 CH2 OH COO~~oo - Porphobilinogen synthase HC-NH3+ activity, % of control

COO- COO- incubations I1 Ill With 1 111 0 serum With urine (250 2,u 50 CH2-C-CH2-COO- Patient tl) Al H COO- -ON*OOC H CH2-C-CH2-COO- MW 24 22 2 lV HA 30-61 20-70 2-5 JS 34 22 2 KaJ 77 83 18 EE 83 15 0 0 CH-COO- KeJ 88 85 18 -OOC-CHi-CH)-C-CH.-C-CE- z *1 *z - z 4.-COO- -OOC-CH MF 88 65 5 VIII VIV Hemolyzed erythrocytes from normal donors were used as the source of porphobilinogen synthase. To each incubation was added 0 0 0 enzyme from approximately 1.5 X 109 erythrocytes. The assay was OOC-CH2 -CH2-C-CH2-C -CH3 CH3-C-CH2-COO- carried out as described by Collier (10), but without dithiothreitol or lx MnCl2. Control and patient incubations contained the indicated VII amounts of serum or urine as the source of inhibitory activity. FIG. 1. Degradation of tyrosine. The heavy arrows indicate the normal degradation of tyrosine. The broken arrows represent the proposed abnormal metabolism of compounds that accumulate due from control subjects. Serum from patients, but not from control to a primary block shown by a solid line across the arrow. The block subjects, also inhibited porphobilinogen synthase but the in- (broken line across the arrow) is suggested to be a secondary block. hibitory activity of serum was much less than that of urine. 4-Hydroxyphenylpyruvate, 4-hydroxyphenyllactate, and was obtained by hydrogenation of synthetic fumarylacetone 4-hydroxyphenylacetate, which are metabolites of tyrosine (15) with 10% palladium on charcoal as catalyst. occurring in increased amount in hereditary tyrosinemia, did not inhibit porphobilinogen synthase when tested in the con- RESULTS centrations present in urine from patients. Isolation from Urine of the Inhibitor of Porphobilinogen Porphobilinogen Synthase in Erythrocytes and Liver in Synthase in Patients with Hereditary Tyrosinemia. The in- Hereditary Tyrosinemia. Table 1 shows the activity of por- hibitory activity in urine was followed during a series of chro- phobilinogen synthase in erythrocytes from three patients with matographic steps. In no step of the purification procedure was hereditary tyrosinemia and in a liver sample obtained at au- more than one peak containing inhibitory activity obtained. topsy. The activity of the enzyme in erythrocytes from the When the final fraction had been derivatized and was analyzed patients was less than 5% of that in a reference group of 15 by gas/liquid chromatography-mass spectrometry, one major healthy subjects. In the liver tissue the activity was less than 1% and two minor components were seen. Each peak contained of the reference value reported (16) but was still measurable. only one component as judged from mass spectra taken from Inhibition of Porphobilinogen Synthase by Urine and several parts of each peak. Also, all three peaks had identical Serum from Patients with Hereditary Tyrosinemia. When mass spectra, suggesting that the final fraction from the puri- urine from patients with hereditary tyrosinemnia was added to fication procedure contained only one component. The three an assay for porphobilinogen synthase containing enzyme from peaks in the gas chromatogram probably represented steric normal subjects, a marked inhibition of the enzyme activity was isomers of the derivative of the purified inhibitor. noted (Table 2). Less than 1 Al of urine from some patients The most likely structure of the parent compound giving rise caused 50% inhibition of the enzyme from 1.5 X 109 erythro- to the mass spectrum shown in Fig. 2 is succinylacetone (4,6- cytes. This is more than 100 times the inhibitory activity of urine dioxoheptanoic acid). The structure was, confirmed by com- paring the mass spectrum of the inhibitor with the mass spectrum of the same derivative of authentic succinylacetone. Synthetic succinylacetone was a potent inhibitor of porpho- Table 1. Porphobilinogen synthase in liver and erythrocytes bilinogen synthase; 1 nmol (0.3 1M) caused about 50% inhibi- in hereditary tyrosinemia tion of the enzyme from 1.5 X 109 erythrocytes. Porphobilinogen synthase, milliunits Organic Acids in Urine of Patients with Hereditary Tyrosinemia. Fresh urine samples from patients with heredi- Patient Per g of liver Per g of Hb* tary tyrosinemia were analyzed by gas chromatography-mass SF autopsy liver 0.05t spectrometry after derivatization (Fig. 3). The large peaks 6, Reference valuest (n = 5) 14-17 11, and 12 were due to phenolic acids derived from tyrosine. MW 1.2 Peak 5 was identified as a derivative of succinylacetone and HA 1.6 peak 13 had a mass spectrum that identified it as a derivative KeJ 2.1 of succinylacetoacetate (3,5-dioxooctanedioic acid). The Reference values (n = 15) 46(25-59) combined excretion of these two compounds was usually 1-2 were * Assayed by the method of Collier (10). mmol/day. Succinylacetone and succinylacetoacetate t Liver tissue was obtained 1 hr after death. regularly present in fresh urine although in somewhat varying Reported by Perlroth et al. (16). proportion; in stored urine samples, no succinylacetoacetate and Downloaded by guest on September 30, 2021 Medical Sciences: Lindblad et al. Proc. Natl. Acad. Sci. USA 74 (1977) 4643

80 YURINARY COMPOUND z LJ 60

a: 0 60 80 100 120 ilW 160 180 200 220 20 260 280 30o m/e LU 100 60 72 86 87 59 80- 59CH3 -C-C2-C -CH2 - CH2 - COOCH3 z LUJ 60 ~~~~~~~~~~~~~~~NN LLU 60 311OCH3 OCH3 Mx230 a_ 40- 8t"-2_67t M-32*87) M31 M; 72 2j0- I~ 6M(131 -1 N 0 4L~~~~~~~~li~ ~ J 10 60 80 100 120 1N0 160 180 200 220 240 260 280 300 m/e FIG. 2. Mass spectra of the methyl ester and methoxime of the isolated urinary inhibitor (Upper) and of synthetic succinylacetone (Lower). M+ is the molecular ion.

only small amounts of succinylacetone were found. In the nogen synthase may help to elucidate the biological effect of course of screening of several hundred urine samples from 5-aminolevulinic acid because, possibly, administration of 5- healthy subjects and patients with diseases other than tyrosi- aminolevulinic acid may not be equivalent to an endogenous nemia, including severe liver disease, we have never observed overproduction of the acid. The enzyme porphobilinogen the presence of these two compounds, although an excretion synthase has been purified from several sources (20). It is in- of about 20 ,umol/day would have been detected. hibited by some metal ions (e.g., lead and copper) and by 4-Hydroxyphenylpyruvate Dioxygenase in Hereditary sulfhydryl reagents, and it has also been reported that the Tyrosinemia. Table 3 shows determinations of 4-hydroxy- herbicide 3-amino-1,2,4-triazole inhibits it (23). phenylpyruvate dioxygenase activity in liver biopsies and the Succinylacetone (III, cf. Fig. 1) is not known to occur as an clinical severity of the disease in a number of patients. It appears intermediate in any metabolic pathway. Two structurally re- that subjects with a low activity of 4-hydroxyphenylpyruvate lated compounds, maleylacetoacetate (IV) and fumarylace- dioxygenase have a more protracted form of the disease, toacetate (V), occur as intermediates in the normal degradation whereas those with a high residual activity die young. of tyrosine. If these compounds were reduced in vivo, succinylacetoacetate (VI) would be formed. The procedure used to DISCUSSION isolate the inhibitor, which involved extraction at acidic pH and A puzzling aspect of hereditary tyrosinemia has been the oc- repeated chromatography, would most likely lead to decar- currence of episodes of symptoms of the type that occur in acute boxylation of this #-keto acid to yield succinylacetone. When hepatic porphyria and a high excretion of 5-aminolevulinic we examined a number of urine samples' from tyrosinemic acid. The present finding that a potent inhibitor of porphobil- patients without prior fractionation and by a technique that inogen synthase is formed in this disease explains the excretion minimized destruction, we could establish the presence of a of 5-aminolevulinic acid. We have previously reported (17) an compound with the mass spectrum expected for succinylace- increased activity of 5-aminolevulinate synthase [succinyl- toacetate. Maleylacetoacetate and fumarylacetoacetate were CoA:glycine C-succinyltransferase (decarboxylating), EC not found, but they are known to react easily with SH groups 2.3.1.37] in a hepatoma removed from a patient with tyrosi- (24) (e.g., in proteins) and therefore may not occur in urine. nemia, and a similar observation was later made by Kang and The accumulation of metabolites of maleylacetoacetate or Gerald (18). In the light of the present findings it appears that fumarylacetoacetate would imply a low activity for one or both this may have been a secondary phenomenon due to release of enzymes involved in the metabolism of these compounds-i.e., the feedback control of the enzyme as appears to be the case maleylacetoacetate isomerase (4-maleylacetoacetate cis- in the porphyrias. The relationship between the clinical trans-isomerase, EC 5.2.1.2) and fumarylacetoacetase (4- symptoms in acute intermittent porphyria, porphyria variegata, fumarylacetoacetate fumarylhydrolase, EC 3.7.1.2). It is known hereditary coproporphyria, and lead intoxication and the al- that succinylacetoacetate is a substrate for fumarylacetoacetase tered porphyrin metabolism has not been established (19). (25) and one is therefore led to conclude that in the tyrosinemic Patients with the above conditions excrete 5-aminolevulinic patient it is the activity of fumarylacetoacetase that is decreased acid, but other porphyrin precursors are also present (20). 5- below normal. It should be pointed out that inhibition of the Aminolevulinic acid administered to man (21) or animals (22) isomerase by the product, fumarylacetoacetate, might occur does not produce toxic symptoms, but in vitro experiments with if it is not further degraded. In the corresponding bacterial this compound have demonstrated inhibition of brain Na+, enzyme system that metabolizes gentisate to maleylpyruvate K+-ATPase and Na+ transport in frog skin (19). and fumarylpyruvate, an adduct can be formed between fu- A correlation between the excyetion of 5-aminolevulinic acid marylpyruvate and glutathione that is not attacked by the hy- and porphobilinogen and the severity of attacks in acute in- drolase but is an inhibitor of the isomerase (26). Fumnarylace- termittent porphyria (19, 20) suggests, however, a causal rela- toacetate forms a similar dead-end complex (24). If this adduct tionship. The availability of a specific inhibitor of porphobili- inhibits maleylacetoacetate isomerase, maleylacetoacetate could Downloaded by guest on September 30, 2021 .4644 Medical Sciences: Lindblad et al. Proc. Nati. Acad. Sci. USA 74 (1977)

z 0 0. WLU Cr 533 0 Uj 0~~~~~~~~~1 2

250 230 210 190 170 150 130 110 90 OVEN TEMPERATURE *C FIG. 3. Gas chromatogram of organic acids in urine from an infant with hereditary tyrosinemia. The methoxime trimethylsilyl derivatives were separated on a 3% OV-17 packed column, with temperature programming at a rate of 40/min. The peaks have been assigned the following identities: 1, urea + phosphoric acid; 2, succinic acid; 3, internal standard (2-methyl-3-hydroxybenzoic acid); 4, 2-oxoglutaric acid; 5, succinylacetone (4,6-dioxoheptanoic acid); 6, 4-hydroxyphenylacetic acid; 7, aconitic acid; 8, citric acid, 9, isocitric acid; 10, dihydroxyphenylpropionic acid; 11, 4-hydroxyphenyllactic acid; 12, 4-hydroxyphenylpyruvic acid; 13, succinylacetoacetate (3,5-dioxooctanedioic acid).

-accumulate together with fumarylacetoacetate and be reduced disease have the lowest activity of 4-hydroxyphenylpyruvate to succinylacetoacetate. dioxygenase in the liver. This is in accord with our idea that Such severe liver and kidney damage as observed in hered- maleylacetoacetate or its subsequent metabolites are responsible itary tyrosinemia does not occur in the hepatic porphyric for the tissue damage. conditions and seems unrelated to the inhibition of porphobil- If maleylacetoacetate or fumarylacetoacetate are causing inogen synthase now reported. In the light of the present the kidney and liver damage, one should attempt to increase findings one must consider the possibility that maleylaceto- their elimination. Treatment of patients with a diet restricted acetate or its immediate metabolites cause tissue damage. A in phenylalanine and tyrosine has resulted in reversal of the renal tubular dysfunction similar to that in hereditary tyrosi- renal tubular dysfunction but the effect on liver function has nemia (i.e., a Fanconi syndrome) occurs also in Wilson disease, been uncertain, even when the diet has been started at an early cystinosis, and cadmium intoxication (27). The presumed toxic age (37). A possible way to increase the elimination of toxic compounds in these diseases have in common that they react metabolites would be by administration of SH-containing with SH groups. Maleate also forms adducts with SH-containing compounds that form adducts with maleylacetoacetate and compounds (28), and maleylacetoacetate is expected to react fumarylacetoacetate. Glutathione forms such adducts and in a similar way. As discussed above, fumarylacetoacetate forms preliminary data have shown that penicillamine also forms a stable adduct with glutathione and it is therefore probable that adducts in vitro. it also can be toxic to the kidney. We have established an increased formation of succinyl- The liver damage in hereditary tyrosinemia causes liver acetoacetate and succinylacetone in hereditary tyrosinemia and cirrhosis in early childhood. (1, 2, 29). Patients with the chronic the mechanism behind the increased 5-aminolevulinate ex- form of the disease also develop hepatocellular tumors (30). It cretion. We have also presented a hypothesis suggesting that is generally believed that compounds that form reactive me- an accumulation of the metabolites following homogentisate tabolites that can combine with macromolecules cause cellular is characteristic of hereditary tyrosinemia and causes the liver necrosis and even induce cancer (31). Tissues may be protected and kidney damage. The cause of the low activity of 4-hy- by the presence of SH-containing compounds (e.g., glutathione) droxyphenylpyruvate dioxygenase in hereditary tyrosinemia (32). Paracetamol is a much-used model compound for studies (3, 12) remains to be discussed. It seems less likely that these of this type of tissue damage (32-35). It is nontoxic in low doses patients have two structural gene defects resulting in low ac- but will induce cellular necrosis in high doses. A toxic metabolite tivities of both 4-hydroxyphenylpyruvate dioxygenase and of paracetamol first binds to glutathione and, when the supply fumarylacetoacetase. The most obvious explanation would be of glutathione is exhausted, it will bind to macromolecules and that 4-hydroxyphenylpyruvate dioxygenase is inhibited by the cause cellular necrosis. Diethylmaleate by itself does not cause newly found metabolites. A pure preparation of this enzyme liver necrosis, but it will aggravate the toxicity of paracetamol (38, 39), however, was not inhibited by succinylacetone, and by glutathione consumption. Maleylacetoacetate, which is a the enzyme, which contains approximately five free thiol reactive compound, may have direct toxic effects but, because groups, is not particularly sensitive to SH reagents (39). With it would react with glutathione, it may also make the liver more the use of an antiserum against the enzyme, we showed the vulnerable to other toxic compounds. absence of immunoreactive protein in liver biopsies from two A high concentration of potentially toxic metabolites of ty- patients who lacked enzyme activity (3). These results speak rosine is expected to occur in liver and kidney because only against an enzyme inhibition. There is then the possibility of these tissues contain 4-hydroxyphenylpyruvate dioxygenase a partially or completely repressed synthesis of the enzyme or (36) which catalyzes the first irreversible step in the degradation that an altered enzyme is very rapidly eliminated. One should sequence leading to maleylacetoacetate. The data in Table 3 also consider a defect of a regulatory gene, common for 4- illustrate the fact that patients with a more benign form of the hydroxyphenylpyruvate dioxygenase and fumarylacetoacetase. Downloaded by guest on September 30, 2021 Medical Sciences: Lindblad et al. Proc. Natl. Acad. Sci. USA 74 (1977) 4645

Table 3. Correlation of catalytic activity of 4- T. H. & Hosty, T. S. (1971) Pediatrics 48,393-400. hydroxyphenylpyruvate dioxygenase in liver biopsies and clinical 7. Lindemann, R., Gjessing, L. R., Merton, B., Loken, C. A. & findings in patients with hereditary tyrosinemia Halvorsen, S. (1970) Acta Paediat. Scand. 59, 141-147. 8. Gentz, J., Lindblad, B., Lindstedt, S., Levy, L., Shasteen, W. & 4-Hydroxyphenyl- Zetterstr6m, R. (1967) Am. J. Dis. Child. 113,31-37. pyruvate 9. Gentz, J., Johansson, S., Lindblad, B., Lindstedt, S. & Zetterstr6m, dioxygenase R. (1969) Clin. Chim. Acta 23,257-263. Age at % of Clinical 10. Collier, H. B. (1971) Clhn. Biochem. 4,222-232. 11. Gibson, K. D., Neuberger, A. & Scott, J. J. (1955) Biochem. J. 61, Patient assay munits/g normal* findings 618-629. MW 6 wk 20 32 Died, 6 wk 12. Gentz, J. & Lindblad, B. (1972) Scand. J. Clin. Lab. Invest. 29, RW 2 mo 26 9 Died, 2 mo 115-126. SF 5 mo 19 20 Died, 5 mo 13. Lindblad, B. (1971) Clin. Chim. Acta 34, 113-121. FP 10 mo 3.5 3.8 Liver cell tumor removed, 14. Bjorkman, L., McLean, C. & Steen, G. (1976) Clin. Chem. 22, age 10 mo 49-52. Dietary therapy; present 15. Nilsson, M. (1964) Acta Chem. Scand. 18, 441-446. 16. Perlroth, M. G., Tschudy, D. P., Marver, H. S., Berard, C. W., age, 11/2 yr Ziegel, R. F., Reichcil, M. & Collins, A. (1966) Am. J. Med. 41, HA 11/4 yr 3.D 3.5 Dietary therapy; good con- 149-162. dition; present age, 4 yr 17. Gentz, J., Heinrich, J., Lindblad, B., Lindstedt, S. & Zetterstrom, RM 2 yr - 8.5 Only slight dietary restric- R. (1969) Acta Paediat. Scand. 58,393-396. tion; polyuria; present 18. Kang, E. S. & Gerald, P. S. (1970) J. Pediat. 77,397-406. age, 8 yr 19. Kramer, S., Becher, D. & Viljoen, D. (1973) S. Afr. Med. J. 47, MS 16 yr 150 Hepatoma removed, age 16 1735-1738. yr; died at age 18 yr from 20. Marver, H. S. & Schmid, R. (1972) in The Metabolic Basis of hepatoma Inherited Disease, eds. Stanbury, J. B., Wyngaarden, J. B. & 18 yr 4.2 Fredrickson, D. S. (McGraw-Hill Book Co., New York), 3rd ed., MF 51/2 yr ND Good clinical and social pp. 1087-1140. condition; only slight di- 21. Jarrett, A., Rimington, C. & Willoughby, D. A. (1956) Lancet etary restriction; present i, 125-127. age 20 yr. 22. Kennedy, G. L., Arnold, D. W. & Calendra, J. C. (1976) Fd 14 yr 0.8 Cosmet. Toxicol. 14, 45-48. KeJ 12 yr ND - Good clinical and social 23. Tschudy, D. P. & Collins, A. (1957) Science 126, 168. condition; no dietary re- 24. Edwards, S. W. & Knox, W. E. (1956) J. Biol. Chem. 220, 79- striction; present age, 26 91. 25. Knox, W. E. & Edwards, S. W. (1955) J. Biol. Chem. 216, yr. 489-498. 20yr - 0.7 26. Lack, L. (1961) J. Biol. Chem. 236,2835-2840. Reference 340 100 27. Schneider, J. A. & Seegmiller, J. E. (1972) in The Metabolic Basis valuest (150- (38- of Inherited Disease, eds. Stanbury, J. B., Wyngaarden, J. B. & 630) 180) Fredrickson, D. S. (McGraw-Hill Book Co., New York), 3rd ed., n =6 n = 13 pp. 1581-1604. * Calculated by using activity of several other enzymes as references 28. Morgan, E. J. & Friedmann, E. (1938) Biochem. J. 32, 733- (12). ND, Not detectable. t From refs. 12 and 13. 742. 29. Woolf, L. (1966) in Symposium on Tyrosinosis, ed. Giessing, L. R. (Universitetsforlaget, Oslo), pp. 82-91. This type of gene defect has been suggested to occur in orotic 30. Weinberg, A. G., Mize, C. E. & Worthen, H. G. (1976) J. Pediatr. aciduria, another inborn error with two enzyme defects (40). 88,434-438. In summary, the present findings suggest that the critical en- 31. Gilette, J. R. (1974) Biochem. Pharmacol. 23,2785-2794. zyme defect may be located at the fumarylacetoacetase step 32. Mitchell, J. R., Jollow, D. J., Potter, W. Z., Gilette, J. R. & Brodie, and provide possible explanations for the characteristic features B. B. (1973) J. Pharmacol. Exp. Ther. 187,211-217. which have been difficult to explain 33. Mitchell, J. R., Jollow, D. J., Potter, W. Z., Davis, D. C., Gilette, of hereditary tyrosinemia J. R. & Brodie, B. B. (1973) J. Pharmacol. Exp. Ther. 187, as a consequence of partial or complete lack of 4-hydroxy- 185-194. phenylpyruvate dioxygenase. 34. Jollow, D. J., Mitchell, J. R., Potter, W. Z., Davis, D. C., Gilette, This work has been supported by a grant from the Swedish Medical J. R. & Brodie, B. B. (1973) J. Pharm. Exp. Ther. 187, 195- Research Council, 13X-585. 202. 35. Potter, W. Z., Davis, D. C., Mitchell, J. R., Jollow, D. J., Gilette, 1. Gentz, J., Jagenburg, R. & Zetterstrom, R. (1965) J. Pediat. 66, J. R. & Brodie, B. B. (1973) J. Pharm. Exp. Ther. 187, 203- 670-696. 210. 2. La Du, B. N. & Gjessing, L. R. (1972) in The Metabolic Basis of 36. Fellman, J. H., Fujita, T. S. & Roth, E. S. (1972) Biochim. Blophys. Inherited Disease, eds. Stanbury, J. B., Wyngaarden, J. B. & Acta 284,90-100. Fredrickson, D. S. (McGraw-Hill Book Co., New York), 3rd ed., 37. Bodegard, G., Gentz, J., Lindblad, B., Lindstedt, S. & Zetterstrom, pp. 296-307. R. (1969) Acta Paediat. Scand. 58,37-48. 3. Lindblad, B., Lindstedt, G., Lindstedt, S. & Rundgren, M. (1972) 38. Lindblad, B., Lindstedt, S., Olander, B. & Omfeldt, M. (1971) in Organic Acidurias, eds. Stern, J. & Toothill, C. (Churchill Acta Chem. Scand. 25,329-330. Livingstone, London), pp. 63-81. 39. Lindblad, B., Lindstedt, G., Lindstedt, S. & Rundgren, M. (1977) 4. Kennaway, N. G. & Buist, N. R. M. (1971) Pediatr. Res. 5, J. Biol. Chem. 252, 5073-5084. 287-297. 40. Smith, L. H., Huguley, C. M. & Bain, J. A. (1972) in The Meta- 5. Wadman, S. K., Van Sprang, F. J., Maas, J. W. & Ketting, D. bolic Basis ofInherited Disease, eds. Stanbury, J. B., Wyngaar- (1968) J. Ment. Defic. Res. 12,269-281. den, J. B. & Fredrickson, D. S. (McGraw-Hill Book Co., New 6. Holston, J. L., Jr., Levy, H. L., Tomlin, G. A., Atkins, R. J., Patton, York), 3rd ed., pp. 1003-1029. Downloaded by guest on September 30, 2021