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 synthase (EC 4.2.1.24) in erythrocytes from patients with hereditary 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 as succinylacetone (4,6-dioxoheptanoic acid) by gas/liquid chromatography-mass spectrometry. Fresh urine samples contained succinylacetoacetate (3,5-dioxooctanedioic acid) as well as succinylacetone. The inhibition of porphobilinogen synthase explains the high excretion of 5-aminolevulinate observed in hereditary tyrosinemia. Succinylacetone and succinylacetoacetate presumably originate from maleylacetoacetate or fumarylacetoacetate, or both, and their accumulation indicates a block at the fumarylacetoacetase (EC 3.7.1.2) step in the degradation of tyrosine. We suggest that the severe liver and kidney damage in hereditary tyrosinemia may be due to the accumulation of these tyrosine metabolites and that the primary enzyme defect in hereditary tyrosinemia may be decreased activity of fumarylacetoacetase. In the inborn error of metabolism called hereditary tyrosinemia, the main clinical findings are liver failure, which develops into liver cirrhosis in early childhood, and multiple renal tubular defects with hypophosphatemic rickets (1, 2). The derangement in tyrosine metabolism (i.e., hypertyrosinemia and high urinary excretion of 4-hydroxyphenylpyruvate, 4-hydroxyphenyllactate, and to a lesser extent 4-hydroxyphenylacetate) is due to a low activity of the enzyme 4-hydroxyphenylpyruvate dioxygenase [4-hydroxyphenylpyruvate:oxygen oxidoreductase (hydroxylating, decarboxylating), EC 1.13.11.27] (3), which catalyzes the formation of homogentisate (III) (Fig. 1) from 4-hydroxyphenylpyruvate (II). The increased excretion of these phenolic metabolites of tyrosine does not explain the liver and kidney damage in hereditary tyrosinemia because a similar large excretion has been found also in patients without liver and kidney disease-e.g., in a 5-year-old boy with multiple congenital anomalies and with negligible activity of soluble tyrosine aminotransferase (4) and in at least three other patients who were mentally retarded (2, 5, 6). A large excretion of the same metabolites has also been found in hereditary fructose intolerance (7). In 1967 we reported on a patient who had symptoms similar to those characteristic of acute intermittent porphyria (8). An increased excretion of 5-aminolevulinate has since then been observed in all patients studied by us, even in those without these symptoms (3, 9), but so far it has not been possible to find a biochemical link between the altered tyrosine metabolism and the increased excretion of a porphyrin precursor. In this report we present evidence for an enzyme defect in tyrosine catabolism in hereditary tyrosinemia that explains the increased excretion of 5-aminolevulinate. We also present a hypothesis The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

suggesting that the metabolites that accumulate cause the liver and kidney damage in hereditary tyrosinemia. METHODS Patients. The diagnosis of hereditary tyrosinemia was based on the criteria given in the introduction. Some clinical data are given with the Results. Porphobilinogen Synthase in Erythrocytes. The enzyme activity was determined as described by Collier (10), in 3' ml. Porphobilinogen Synthase in Liver. Liver tissue obtained at autopsy 1 hr after death was put on dry ice. The assay was carried out as described by Gibson et al. (11) on a 100,000 X g supernatant of a 33% homogenate prepared in 0.1 M potassium phosphate buffer at pH 6.5. 4-Hydroxyphenylpyruvate Dioxygenase in Liver. The activity of 4-hydroxyphenylpyruvate dioxygenase was determined from the formation of labeled homogentisate from labeled 4-hydroxyphenylpyruvate (12, 13). Assay for Inhibition of Porphobilinogen Synthase. Erythrocytes from normal donors were washed, hemolyzed, and used as the source of porphobilinogen synthase. To each incubation was added enzyme from approximately 1.5 X 109 erythrocytes. The solution to be tested for inhibitory activity was added to the incubation mixture at the start of a 15-min preincubation period. The reaction was started by addition of 5-aminolevulinate. The assay procedure described above for porphobilinogen synthase in erythrocytes was then carried out in the absence of MnCl2 and dithiothreitol. Isolation of the Inhibitor of Porphobilinogen Synthase. The isolation procedure was carried out on fresh pooled urine samples from'patients suffering from hereditary tyrosinemia. Diethyl ether extracts were fractionated by chromatography on both anion and cation exchange resins. The fractions were then assayed by the procedure described above and those containing inhibitory activity were pooled. Thin-layer chromatography of the fraction thus obtained from the last ion exchange chromatography showed one segment with inhibitory activity. This fraction was eluted and subjected to gas/liquid chromatography-mass spectrometry. Mass Spectrometry. Electron impact mass spectra were recorded at 70 eV on an LKB 9000 gas chromatograph-mass spectrometer; ion-source temperature, 270°; acceleration voltage, 3.5 kV. Methoximes and methyl esters were prepared at room temperature with methoxylamine hydrochloride in pyridine followed by treatment with diazomethane. Organic Acids in Urine. Urines were examined for organic acids by gas/liquid chromatography-mass spectrometry as described (14). Preparation of Synthetic Succinylacetone. Succinylacetone *

4641

This paper was presented at the Swedish Medical Congress, 1976.

Proc. Natl. Acad. Sci. USA 74 (1977)

Medical Sciences: Lindblad et al.

4642

-I

CCH2 2

OH COO ~~oo

CH2

CH2

Table 2. Inhibition of porphobilinogen synthase from erythrocytes by serum and urine from patients with hereditary tyrosinemia

OH

OH

OH

Porphobilinogen synthase activity, % of control incubations

-

HC-NH3+ COO-

COO-

I1

Ill

111

1

0

Patient

CH2-C-CH2-COO-

H

-ON*OOC

COO-

H CH2-C-CH2-COO-

0

CH-COO-

0

-OOC-CHi-CH)-C-CH.-C-CE z *1 *z z 4.-COO-

-

-OOC-CH VIII

VIV 0

OOC-CH2

0

0

CH3-C-CH2-COO-

-CH2-C-CH2-C -CH3

lx VII arrows indicate the of The heavy FIG. 1. Degradation tyrosine. normal degradation of tyrosine. The broken arrows represent the proposed abnormal metabolism of compounds that accumulate due to a primary block shown by a solid line across the arrow. The block (broken line across the arrow) is suggested to be a secondary block.

obtained by hydrogenation of synthetic fumarylacetone (15) with 10% palladium on charcoal as catalyst.

was

RESULTS Porphobilinogen Synthase in Erythrocytes and Liver in Hereditary Tyrosinemia. Table 1 shows the activity of porphobilinogen synthase in erythrocytes from three patients with hereditary tyrosinemia and in a liver sample obtained at autopsy. The activity of the enzyme in erythrocytes from the patients was less than 5% of that in a reference group of 15 healthy subjects. In the liver tissue the activity was less than 1% of the reference value reported (16) but was still measurable. Inhibition of Porphobilinogen Synthase by Urine and Serum from Patients with Hereditary Tyrosinemia. When urine from patients with hereditary tyrosinemnia was added to an assay for porphobilinogen synthase containing enzyme from normal subjects, a marked inhibition of the enzyme activity was noted (Table 2). Less than 1 Al of urine from some patients caused 50% inhibition of the enzyme from 1.5 X 109 erythrocytes. This is more than 100 times the inhibitory activity of urine Table 1. Porphobilinogen synthase in liver and erythrocytes in hereditary tyrosinemia Patient SF autopsy liver Reference valuest (n = 5) MW HA KeJ Reference values (n = 15) *

MW

HA

lV

Porphobilinogen synthase, milliunits Per g of Hb* Per g of liver 0.05t 14-17

Assayed by the method of Collier (10).

t Liver tissue was obtained 1 hr after death. Reported by Perlroth et al. (16).

1.2 1.6 2.1

46(25-59)

JS KaJ EE KeJ MF

With serum (250 tl) 24 30-61 34 77 83 88 88

2,u

With urine 50 Al

22 20-70 22 83

85 65

2 2-5 2 18 15 18 5

Hemolyzed erythrocytes from normal donors were used as the source of porphobilinogen synthase. To each incubation was added enzyme from approximately 1.5 X 109 erythrocytes. The assay was carried out as described by Collier (10), but without dithiothreitol or MnCl2. Control and patient incubations contained the indicated amounts of serum or urine as the source of inhibitory activity.

from control subjects. Serum from patients, but not from control subjects, also inhibited porphobilinogen synthase but the inhibitory activity of serum was much less than that of urine. 4-Hydroxyphenylpyruvate, 4-hydroxyphenyllactate, and 4-hydroxyphenylacetate, which are metabolites of tyrosine occurring in increased amount in hereditary tyrosinemia, did not inhibit porphobilinogen synthase when tested in the concentrations present in urine from patients. Isolation from Urine of the Inhibitor of Porphobilinogen Synthase in Patients with Hereditary Tyrosinemia. The inhibitory activity in urine was followed during a series of chromatographic steps. In no step of the purification procedure was more than one peak containing inhibitory activity obtained. When the final fraction had been derivatized and was analyzed by gas/liquid chromatography-mass spectrometry, one major and two minor components were seen. Each peak contained only one component as judged from mass spectra taken from several parts of each peak. Also, all three peaks had identical mass spectra, suggesting that the final fraction from the purification procedure contained only one component. The three peaks in the gas chromatogram probably represented steric isomers of the derivative of the purified inhibitor. The most likely structure of the parent compound giving rise to the mass spectrum shown in Fig. 2 is succinylacetone (4,6dioxoheptanoic acid). The structure was, confirmed by comparing the mass spectrum of the inhibitor with the mass spectrum of the same derivative of authentic succinylacetone. Synthetic succinylacetone was a potent inhibitor of porphobilinogen synthase; 1 nmol (0.3 1M) caused about 50% inhibition of the enzyme from 1.5 X 109 erythrocytes. Organic Acids in Urine of Patients with Hereditary Tyrosinemia. Fresh urine samples from patients with hereditary tyrosinemia were analyzed by gas chromatography-mass spectrometry after derivatization (Fig. 3). The large peaks 6, 11, and 12 were due to phenolic acids derived from tyrosine. Peak 5 was identified as a derivative of succinylacetone and peak 13 had a mass spectrum that identified it as a derivative of succinylacetoacetate (3,5-dioxooctanedioic acid). The combined excretion of these two compounds was usually 1-2 mmol/day. Succinylacetone and succinylacetoacetate were regularly present in fresh urine although in somewhat varying proportion; in stored urine samples, no succinylacetoacetate and

Proc. Natl. Acad. Sci. USA 74 (1977)

Medical Sciences: Lindblad et al.

4643

YURINARY COMPOUND

80 z

LJ 60 CL

a:

60

0

80

100

120

ilW

160

200

180

220

20

280

260

30 o

m/e

LU

100 60 80-

z

72

LUJ

60

LLUa_

60

59

N ~~~~~~~~~~~~~~~N Mx230 OCH3

8t"-2_67 t M-32*87) 72 I~

0 10

87

311OCH3

402j0-

86

59CH3 -C-C2-C -CH2 - CH2 - COOCH3

60

M31

M;

-1

N

6M(131

4L~~~~~~~~li ~ ~ J 80

100

120

1N0

180 160 m/e

200

220

240

260

280

300

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 of screening of several hundred urine samples from healthy subjects and patients with diseases other than tyrosinemia, including severe liver disease, we have never observed the presence of these two compounds, although an excretion of about 20 ,umol/day would have been detected. 4-Hydroxyphenylpyruvate Dioxygenase in Hereditary Tyrosinemia. Table 3 shows determinations of 4-hydroxyphenylpyruvate dioxygenase activity in liver biopsies and the clinical severity of the disease in a number of patients. It appears that subjects with a low activity of 4-hydroxyphenylpyruvate dioxygenase have a more protracted form of the disease, whereas those with a high residual activity die young. DISCUSSION A puzzling aspect of hereditary tyrosinemia has been the occurrence of episodes of symptoms of the type that occur in acute hepatic porphyria and a high excretion of 5-aminolevulinic acid. The present finding that a potent inhibitor of porphobilinogen synthase is formed in this disease explains the excretion of 5-aminolevulinic acid. We have previously reported (17) an increased activity of 5-aminolevulinate synthase [succinylCoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37] in a hepatoma removed from a patient with tyrosinemia, and a similar observation was later made by Kang and Gerald (18). In the light of the present findings it appears that this may have been a secondary phenomenon due to release of the feedback control of the enzyme as appears to be the case in the porphyrias. The relationship between the clinical symptoms in acute intermittent porphyria, porphyria variegata, hereditary coproporphyria, and lead intoxication and the altered porphyrin metabolism has not been established (19). Patients with the above conditions excrete 5-aminolevulinic acid, but other porphyrin precursors are also present (20). 5Aminolevulinic acid administered to man (21) or animals (22) does not produce toxic symptoms, but in vitro experiments with this compound have demonstrated inhibition of brain Na+, K+-ATPase and Na+ transport in frog skin (19). A correlation between the excyetion of 5-aminolevulinic acid and porphobilinogen and the severity of attacks in acute intermittent porphyria (19, 20) suggests, however, a causal relationship. The availability of a specific inhibitor of porphobilicourse

synthase may help to elucidate the biological effect of 5-aminolevulinic acid because, possibly, administration of 5aminolevulinic acid may not be equivalent to an endogenous overproduction of the acid. The enzyme porphobilinogen synthase has been purified from several sources (20). It is inhibited by some metal ions (e.g., lead and copper) and by sulfhydryl reagents, and it has also been reported that the herbicide 3-amino-1,2,4-triazole inhibits it (23). Succinylacetone (III, cf. Fig. 1) is not known to occur as an intermediate in any metabolic pathway. Two structurally related compounds, maleylacetoacetate (IV) and fumarylacetoacetate (V), occur as intermediates in the normal degradation of tyrosine. If these compounds were reduced in vivo, succinylacetoacetate (VI) would be formed. The procedure used to isolate the inhibitor, which involved extraction at acidic pH and repeated chromatography, would most likely lead to decarboxylation of this #-keto acid to yield succinylacetone. When we examined a number of urine samples' from tyrosinemic patients without prior fractionation and by a technique that minimized destruction, we could establish the presence of a compound with the mass spectrum expected for succinylacetoacetate. Maleylacetoacetate and fumarylacetoacetate were not found, but they are known to react easily with SH groups (24) (e.g., in proteins) and therefore may not occur in urine. The accumulation of metabolites of maleylacetoacetate or fumarylacetoacetate would imply a low activity for one or both enzymes involved in the metabolism of these compounds-i.e., maleylacetoacetate isomerase (4-maleylacetoacetate cistrans-isomerase, EC 5.2.1.2) and fumarylacetoacetase (4fumarylacetoacetate fumarylhydrolase, EC 3.7.1.2). It is known that succinylacetoacetate is a substrate for fumarylacetoacetase (25) and one is therefore led to conclude that in the tyrosinemic patient it is the activity of fumarylacetoacetase that is decreased below normal. It should be pointed out that inhibition of the isomerase by the product, fumarylacetoacetate, might occur if it is not further degraded. In the corresponding bacterial enzyme system that metabolizes gentisate to maleylpyruvate and fumarylpyruvate, an adduct can be formed between fumarylpyruvate and glutathione that is not attacked by the hydrolase but is an inhibitor of the isomerase (26). Fumnarylacetoacetate forms a similar dead-end complex (24). If this adduct inhibits maleylacetoacetate isomerase, maleylacetoacetate could nogen

.4644

Proc. Nati. Acad. Sci. USA 74 (1977)

Medical Sciences: Lindblad et al.

z

0 0.

LUW 533

Cr

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 to succinylacetoacetate. Such severe liver and kidney damage as observed in hereditary tyrosinemia does not occur in the hepatic porphyric conditions and seems unrelated to the inhibition of porphobilinogen synthase now reported. In the light of the present findings one must consider the possibility that maleylacetoacetate or its immediate metabolites cause tissue damage. A renal tubular dysfunction similar to that in hereditary tyrosinemia (i.e., a Fanconi syndrome) occurs also in Wilson disease, cystinosis, and cadmium intoxication (27). The presumed toxic compounds in these diseases have in common that they react with SH groups. Maleate also forms adducts with SH-containing compounds (28), and maleylacetoacetate is expected to react in a similar way. As discussed above, fumarylacetoacetate forms a stable adduct with glutathione and it is therefore probable that it also can be toxic to the kidney. The liver damage in hereditary tyrosinemia causes liver cirrhosis in early childhood. (1, 2, 29). Patients with the chronic form of the disease also develop hepatocellular tumors (30). It is generally believed that compounds that form reactive metabolites that can combine with macromolecules cause cellular necrosis and even induce cancer (31). Tissues may be protected by the presence of SH-containing compounds (e.g., glutathione) (32). Paracetamol is a much-used model compound for studies of this type of tissue damage (32-35). It is nontoxic in low doses but will induce cellular necrosis in high doses. A toxic metabolite of paracetamol first binds to glutathione and, when the supply of glutathione is exhausted, it will bind to macromolecules and cause cellular necrosis. Diethylmaleate by itself does not cause liver necrosis, but it will aggravate the toxicity of paracetamol by glutathione consumption. Maleylacetoacetate, which is a reactive compound, may have direct toxic effects but, because it would react with glutathione, it may also make the liver more vulnerable to other toxic compounds. A high concentration of potentially toxic metabolites of tyrosine is expected to occur in liver and kidney because only these tissues contain 4-hydroxyphenylpyruvate dioxygenase (36) which catalyzes the first irreversible step in the degradation sequence leading to maleylacetoacetate. The data in Table 3 illustrate the fact that patients with a more benign form of the

disease have the lowest activity of 4-hydroxyphenylpyruvate dioxygenase in the liver. This is in accord with our idea that maleylacetoacetate or its subsequent metabolites are responsible for the tissue damage. If maleylacetoacetate or fumarylacetoacetate are causing the kidney and liver damage, one should attempt to increase their elimination. Treatment of patients with a diet restricted in phenylalanine and tyrosine has resulted in reversal of the renal tubular dysfunction but the effect on liver function has been uncertain, even when the diet has been started at an early age (37). A possible way to increase the elimination of toxic metabolites would be by administration of SH-containing compounds that form adducts with maleylacetoacetate and fumarylacetoacetate. Glutathione forms such adducts and preliminary data have shown that penicillamine also forms adducts in vitro. We have established an increased formation of succinylacetoacetate and succinylacetone in hereditary tyrosinemia and the mechanism behind the increased 5-aminolevulinate excretion. We have also presented a hypothesis suggesting that an accumulation of the metabolites following homogentisate is characteristic of hereditary tyrosinemia and causes the liver and kidney damage. The cause of the low activity of 4-hydroxyphenylpyruvate dioxygenase in hereditary tyrosinemia (3, 12) remains to be discussed. It seems less likely that these patients have two structural gene defects resulting in low activities of both 4-hydroxyphenylpyruvate dioxygenase and fumarylacetoacetase. The most obvious explanation would be that 4-hydroxyphenylpyruvate dioxygenase is inhibited by the newly found metabolites. A pure preparation of this enzyme (38, 39), however, was not inhibited by succinylacetone, and the enzyme, which contains approximately five free thiol groups, is not particularly sensitive to SH reagents (39). With the use of an antiserum against the enzyme, we showed the absence of immunoreactive protein in liver biopsies from two patients who lacked enzyme activity (3). These results speak against an enzyme inhibition. There is then the possibility of a partially or completely repressed synthesis of the enzyme or that an altered enzyme is very rapidly eliminated. One should also consider a defect of a regulatory gene, common for 4hydroxyphenylpyruvate dioxygenase and fumarylacetoacetase.

Medical Sciences: Lindblad et al. Table 3. Correlation of catalytic activity of 4hydroxyphenylpyruvate dioxygenase in liver biopsies and clinical findings in patients with hereditary tyrosinemia 4-Hydroxyphenylpyruvate dioxygenase Clinical % of Age at Patient assay munits/g normal* findings 32 Died, 6 wk 20 6 wk MW 2 mo 9 Died, 2 mo 26 RW 5 mo 20 Died, 5 mo 19 SF 3.8 Liver cell tumor removed, 3.5 10 mo FP age 10 mo Dietary therapy; present age, 11/2 yr 3.5 Dietary therapy; good con3.D 11/4 yr HA dition; present age, 4 yr 8.5 Only slight dietary restricRM 2 yr tion; polyuria; present age, 8 yr Hepatoma removed, age 16 150 16 yr MS yr; died at age 18 yr from hepatoma 4.2 18 yr Good clinical and social MF 51/2 yr ND condition; only slight dietary restriction; present age 20 yr. 0.8 14 yr ND Good clinical and social 12 yr KeJ condition; no dietary restriction; present age, 26 yr.

20yr Reference

340 (150-

0.7 100

(38180) n =6 n = 13 * Calculated by using activity of several other enzymes as references (12). ND, Not detectable. t From refs. 12 and 13.

valuest

630)

This type of gene defect has been suggested to occur in orotic aciduria, another inborn error with two enzyme defects (40). In summary, the present findings suggest that the critical enzyme defect may be located at the fumarylacetoacetase step and provide possible explanations for the characteristic features of hereditary tyrosinemia which have been difficult to explain as a consequence of partial or complete lack of 4-hydroxyphenylpyruvate dioxygenase. This work has been supported by a grant from the Swedish Medical Research Council, 13X-585. 1. Gentz, J., Jagenburg, R. & Zetterstrom, R. (1965) J. Pediat. 66, 670-696. 2. La Du, B. N. & Gjessing, L. R. (1972) in The Metabolic Basis of Inherited Disease, eds. Stanbury, J. B., Wyngaarden, J. B. & Fredrickson, D. S. (McGraw-Hill Book Co., New York), 3rd ed.,

pp. 296-307. 3. Lindblad, B., Lindstedt, G., Lindstedt, S. & Rundgren, M. (1972) in Organic Acidurias, eds. Stern, J. & Toothill, C. (Churchill

Livingstone, London), pp. 63-81. 4. Kennaway, N. G. & Buist, N. R. M. (1971) Pediatr. Res. 5, 287-297. 5. Wadman, S. K., Van Sprang, F. J., Maas, J. W. & Ketting, D. (1968) J. Ment. Defic. Res. 12,269-281. 6. Holston, J. L., Jr., Levy, H. L., Tomlin, G. A., Atkins, R. J., Patton,

Proc. Natl. Acad. Sci. USA 74 (1977)

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T. H. & Hosty, T. S. (1971) Pediatrics 48,393-400. 7. Lindemann, R., Gjessing, L. R., Merton, B., Loken, C. A. & Halvorsen, S. (1970) Acta Paediat. Scand. 59, 141-147. 8. Gentz, J., Lindblad, B., Lindstedt, S., Levy, L., Shasteen, W. & Zetterstr6m, R. (1967) Am. J. Dis. Child. 113,31-37. 9. Gentz, J., Johansson, S., Lindblad, B., Lindstedt, S. & Zetterstr6m, R. (1969) Clin. Chim. Acta 23,257-263. 10. Collier, H. B. (1971) Clhn. Biochem. 4,222-232. 11. Gibson, K. D., Neuberger, A. & Scott, J. J. (1955) Biochem. J. 61, 618-629. 12. Gentz, J. & Lindblad, B. (1972) Scand. J. Clin. Lab. Invest. 29, 115-126. 13. Lindblad, B. (1971) Clin. Chim. Acta 34, 113-121. 14. Bjorkman, L., McLean, C. & Steen, G. (1976) Clin. Chem. 22, 49-52. 15. Nilsson, M. (1964) Acta Chem. Scand. 18, 441-446. 16. Perlroth, M. G., Tschudy, D. P., Marver, H. S., Berard, C. W., Ziegel, R. F., Reichcil, M. & Collins, A. (1966) Am. J. Med. 41, 149-162. 17. Gentz, J., Heinrich, J., Lindblad, B., Lindstedt, S. & Zetterstrom, R. (1969) Acta Paediat. Scand. 58,393-396. 18. Kang, E. S. & Gerald, P. S. (1970) J. Pediat. 77,397-406. 19. Kramer, S., Becher, D. & Viljoen, D. (1973) S. Afr. Med. J. 47, 1735-1738. 20. Marver, H. S. & Schmid, R. (1972) in The Metabolic Basis of Inherited Disease, eds. Stanbury, J. B., Wyngaarden, J. B. & Fredrickson, D. S. (McGraw-Hill Book Co., New York), 3rd ed., pp. 1087-1140. 21. Jarrett, A., Rimington, C. & Willoughby, D. A. (1956) Lancet i, 125-127. 22. Kennedy, G. L., Arnold, D. W. & Calendra, J. C. (1976) Fd Cosmet. Toxicol. 14, 45-48. 23. Tschudy, D. P. & Collins, A. (1957) Science 126, 168. 24. Edwards, S. W. & Knox, W. E. (1956) J. Biol. Chem. 220, 7991. 25. Knox, W. E. & Edwards, S. W. (1955) J. Biol. Chem. 216, 489-498. 26. Lack, L. (1961) J. Biol. Chem. 236,2835-2840. 27. Schneider, J. A. & Seegmiller, J. E. (1972) in The Metabolic Basis of Inherited Disease, eds. Stanbury, J. B., Wyngaarden, J. B. & Fredrickson, D. S. (McGraw-Hill Book Co., New York), 3rd ed., pp. 1581-1604. 28. Morgan, E. J. & Friedmann, E. (1938) Biochem. J. 32, 733742. 29. Woolf, L. (1966) in Symposium on Tyrosinosis, ed. Giessing, L. R. (Universitetsforlaget, Oslo), pp. 82-91. 30. Weinberg, A. G., Mize, C. E. & Worthen, H. G. (1976) J. Pediatr. 88,434-438. 31. Gilette, J. R. (1974) Biochem. Pharmacol. 23,2785-2794. 32. Mitchell, J. R., Jollow, D. J., Potter, W. Z., Gilette, J. R. & Brodie, B. B. (1973) J. Pharmacol. Exp. Ther. 187,211-217. 33. Mitchell, J. R., Jollow, D. J., Potter, W. Z., Davis, D. C., Gilette, J. R. & Brodie, B. B. (1973) J. Pharmacol. Exp. Ther. 187, 185-194. 34. Jollow, D. J., Mitchell, J. R., Potter, W. Z., Davis, D. C., Gilette, J. R. & Brodie, B. B. (1973) J. Pharm. Exp. Ther. 187, 195202. 35. Potter, W. Z., Davis, D. C., Mitchell, J. R., Jollow, D. J., Gilette, J. R. & Brodie, B. B. (1973) J. Pharm. Exp. Ther. 187, 203210. 36. Fellman, J. H., Fujita, T. S. & Roth, E. S. (1972) Biochim. Blophys. Acta 284,90-100. 37. Bodegard, G., Gentz, J., Lindblad, B., Lindstedt, S. & Zetterstrom, R. (1969) Acta Paediat. Scand. 58,37-48. 38. Lindblad, B., Lindstedt, S., Olander, B. & Omfeldt, M. (1971) Acta Chem. Scand. 25,329-330. 39. Lindblad, B., Lindstedt, G., Lindstedt, S. & Rundgren, M. (1977) J. Biol. Chem. 252, 5073-5084. 40. Smith, L. H., Huguley, C. M. & Bain, J. A. (1972) in The Metabolic Basis of Inherited Disease, eds. Stanbury, J. B., Wyngaarden, J. B. & Fredrickson, D. S. (McGraw-Hill Book Co., New York), 3rd ed., pp. 1003-1029.

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-iam...
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