A Review of the Clinical Presentation and Laboratory Findings in Two - - PDF document

SLIDE 1

A Review of the Clinical Presentation and Laboratory Findings in Two Uncommon Hereditary Disorders of Sulfur Amino Acid Metabolism, 13-Mercaptolactate Cysteine Disulfideuria and Sulfite Oxidase Deficiency

• J. C. CRAWHALL

Division of Clinical Biochemistry, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, P.Q. Canada Two hereditary disorders of sulfur amino acid metabolism, 13-mer- captolactate-cysteine disulfideuria and sulfite oxidase deficiency, were described twenty years ago. Other examples

• f these disorders

have been limited to about 5 of each in the world literature since then. Reasons for the apparent rarity of these conditions are discussed and the analytical procedures to identify them are reviewed. The detection of the first depends on the positive result of a cyanide-nitroprusside test followed by positive identification of the specific mixed disulfide. The enzyme mercaptopyruvate sulfur transferase has been shown to be deficient. In the second dis-

• rder
• f sulfite oxidase deficiency, the clinical presentation with pro-

gressive dystonia and dislocated lenses in an infant should suggest further laboratory investigations for this disorder which would not be detected by conventional laboratory screening procedures. Lab-

• ratory

diagnosis can be obtained by use of the Merckoquant sulfite test on a fresh urine sample. Quantitative thiosulfate and taurine measurements can also be made. Positive identification of the specific amino acid S-sulfo-L-cysteine should also be made. The enzyme sulfite oxidase is missing from such organs as liver, kidney and brain. This latter condition may also be associated with xanthi-

• nuria. For this combined disorder of sulfite oxidase and xanthine
• xidase,

a deficiency of a molybdenum-containing cofactor has been demonstrated.

KEY WORDS: ~-mercaptolactate-cysteine disulfideuria, sulfite oxidase deficiency, xanthine oxidase deficiency, bio- chemical genetic screening, sulfur amino acid metabolism, cysteine. su~l-Mercaptolactate-cysteine disulfideuria and sulfite

• xidase deficiency were two hereditary disorders of

fur amino acid metabolism described in the United States in the mid 1960's. It is now twenty years later and only about five other cases of each have been found in the Western world. In the disorder ~-mercapto- lactate-cysteine disulfideuria, no other case has been reported in North America out of a total population of about 250 million people. Only one other case of sulfite 0xidase deficiency has been reported in North America. These are apparently very rare disorders, and in this report I will review the accumulated information on these patients up to the present time, including their clinical presentation and the laboratory procedures available for diagnosis. The relevant structural formulae and metabolic re- lationships are shown in Figure 1. It can be seen that cysteine plays a central role in the production of many interesting intermediates and important end products Correspondence: Dr. J. C. Crawhall, Division of Clinical Biochemistry, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec H3A 1A1. Manuscript submitted June 12, 1984; revised manuscript received September 19, 1984, accepted for publication October 9,1984. whose significance will be discussed in the course of this paper. Clinical presentation of ~-mercaptolactate- cysteine disulfideuria The first patient to be detected with this disorder was an adult male found as the result of a metabolic screen- ing program established by Efron and Bixby at the Wrentham State School for the Mentally Retarded in

• Massachusetts. The urines of five patients were found

to be positive by the cyanide-nitroprusside test. Of the- se, four were shown to be due to cystinuria, and in the fifth, the presence of a hitherto unknown sulfur- containing amino acid was observed (1). The unknown spot was identified by the method of two-way sepa- ration by high voltage electrophoresis followed by pa- per chromatography developed by Efron (2). This sub- stance was eventually isolated and characterised as ~-mercaptolactate-cysteine disulfide with confirmation

• f the structure by mass spectrometry (3, 4). Synthesis
• f this compound was carried out by Hope and Walti (5)

and the same authors synthesised the various isomers and identified the appropriate isomer excreted in this patient's urine (6). The presence of traces of this un- usual mixed disulfide can sometimes be found in normal human urine (7). It has also been shown that administration of cysteine to normal human subjects can give rise to the presence of ~-mercaptolactate- cysteine disulfide in the urine (8). The next subjects with this abnormal metabolite in the urine were found as the result of a screening pro- gram of healthy school children in Zurich which identi- fied two female siblings, age 11 and 13. They were extensively investigated (9). The fourth subject to be found was a 20-year-old male with mild mental re- tardation and bilateral congenital dislocation of the lenses who was found as the result of a screening program of 3000 mentally handicapped patients in Aberdeen, Scotland (10). Since then, only one other female patient has been reported. Her associated dis- eases were ulcerative colitis and autoimmune hemo- lytic anemia but these were probably not related to her metabolic abnormality. She was mentally normal (11). The first patient was studied to identify the specific enzyme defect which was shown to be a deficiency of mercaptopyruvate sulfurtransferase (E.C.2.8.1.2) ac- tivity in erythrocytes (12). This enzyme defect was also confirmed in the fifth patient (11). This latter report included an extensive study of several metabolites in the urines of the propositus and many members of the CLINICAL BIOCHEMISTRY, VOLUME 18, JUNE 1985 139

SLIDE 2

CRAWHALL ~..~" H S- Hydrosulfide

~

SCN- Thiocyanate S2022-Thiosulfite Mercaptopyruvate Sulfur Transferase (M ST) (Y Mercaptopyruvate CH2--C--COOH

I

II SH

#Mercaptolaetate

CH2--CHCOOH

I I

SH OH + Cysteine

Mercaptolactate- cysteine disulfide Thiosulfate (8203) -2 Cysteine m Cysteamine

CH2--CHCOOH ~H2QH2--NH2 SH ~NH 2 SH

Cysteine sulfinic acid Hypotaurine

CH2--CHCOOH CH2--CH2--NH 2

I ~ I

SO2H NH S02H ~, Sulfinylpyruvic acid ~ S03H CH 2- C--COOH ~ I II Cysteic acid

SO2H 0 CH2~ CHCOOH

1 ' '

SO3H NH 2 + Cysteine

Sulfite

~ HO2SSCH2CHCOOH

(S03) 2- I~IH 2 Sulfite oxidase S-Sulfocysteine

Figure 1 -- The metabolism of cysteine showing the derivation of ~-mercapto- lactate, sulfite and S-sulfocysteine.

Sulfate (S04) 2-

• family. These studies allowed a tentative classification
• f

the disorder as being hereditary autosomal recessive. Of the five patients, two have been reported to be mentally retarded and three have been found to be mentally normal (Table 1). It is not clear that the met- abolic defect is related to the mental retardation and it is just possible that these people were found because of specifically directed screening programs within mental

• institutions. At present no treatment for the condition

is known and possibly would not be indicated. How- ever, the rarity of the condition at present precludes any generalisations concerning the significance of the phenotype. Laboratory criteria for establisment of the diagnosis of mercaptolactate-eysteine disulfideuria The first finding is generally that the urine is posi- tive by the cyanide-nitroprusside

• test. Other conditions

giving this result would be cystinuria, some hetero- zygotes for cystinuria and homocystinuria. These latter possibilities can be eliminated on the basis of con- ventional chromatography. The abnormal substance should then be identified by a separate technique. This could be the high voltage electrophoresis followed by paper chromatography described by Efron (2). A method for analysis by gas chromatography has re- cently been proposed (13). However, in this technique the mixed disulfide first has to be reduced on a thiopropyl-Sepharose column, and two further chromatographic steps are required before the mer- captolactate is finally converted to its chromatographic derivative by extractive alkylation. Final confirmation

• f the structure has been made in two of the patients by

mass spectrometry (4, 9). The presence of the appropri- ate enzyme defect can be determined by measurement

• f beta-mercaptopyruvate sulfurtransferase activity in

erythrocytes as previously described (11, 12). Clinical presentation of sulfite oxidase deficiency This disorder (Table 2) was first described in a 30-month-old boy with extreme brain damage, mental retardation and dislocated lenses. The first biochemical abnormality to be detected in the urine was the excre- tion of an unusual amino acid S-sulfo-L-cysteine (14). Up to that time S-sulfo-L-cysteine had only been found as a urinary excretion product in the mammalian species called the blotched Kenya genet (15). Other findings in the patient's urine were a lack of inorganic sulfate, an abnormally increased quantity of sulfite and abnormal quantities of thiosulfate and taurine. A defi- ciency of the enzyme sulfite oxidase (Sulfite:oxygen

• xidoreductase E.C. 1.8.3.1) was confirmed by these

authors in tissue obtained at autopsy in liver, kidney and brain of this patient (16). Since that time only one

• ther patient with this condition has been described

(17). However, in 1978, another patient was described who had the same clinical findings but in addition had hypouricaemia and xanthinuria (18). Investigation of this patient showed that she not only had sulfite ox- 140 CLINICAL BIOCHEMISTRY, VOLUME 18, JUNE 1985

SLIDE 3

TABLE 1 Patients Described with ~-Mercaptolactate-cysteine Disulfi- deuria Sex Age Clinical findings Reference M 47 Mental retardation 1 F 11 Normal 9 F 12 Normal 9 M 20 Mild mental retardation: Bilateral congenital dislocation

• f the lenses

10 F 32 Mentally normal, associated findings--ulcerative colitis, autoimmune hemolytic anemia • 11 TABLE 2 Patients Described with Sulfite Oxidase Deficiency Sex Age Clinical findings Reference M 30 months Brain damage, dystonia, mental retardation, bilateral dislocated lenses 14 M 4.5 yr Bilateral choreo-athetoid-- spastic hemiparesis, dislocated lenses 17 Sulfite Oxidase Deficiency Associated with Xanthine Oxidase Deficiency F 3 days Severe central nervous dysfunction, dislocated lenses 21 F 5 months Severe central nervous dysfunction, dislocated lenses 21 M 5 days Severe central nervous dysfunction, ophthalmological examination unremarkable 21 M 8 weeks Severe central nervous

• dysfunction. No comment was

made concerning the lenses 21 F 5 years Severe central nervous dysfunction from birth. Dislocated lenses 21 idase deficiency but also xanthine oxidase deficiency. It is known that the trace element molybdenum is essen- tial in man for the proper functioning of these enzymes as well as aldehyde oxidase (E.C. 1.2.3.1). Studies on nonmammalian systems have shown that the molyb- doenzymes require not only the metal molybdenum but also an associated cofactor. The exact structure of this cofactor is not known, but it is known that it contains a reduced form of an unusual pterin (19) and is believed to contain sulfur in the molecule (20). A review of the clinical history and biochemical findings of these patients has recently been published (21). Laboratory criteria for the establishment of the diagnosis Urinary sulfite is most readily detected in fresh urine by use of the appropriate test stick (22) (Merckoquant sulfite test, E. Merck, Darmstadt, F.R. Germany) avail- able in Canada through British Drug Houses. Urinary sulfite is an unstable compound and the test has to be carried out within a few hours even with refrigeration 0fthe urine. We have tested the sensitivity of these test strips on serial aqueous solutions of sodium sulfite and did not achieve the sensitivity claimed by the manu-

• facturers. A very pale color change was said to occur at

0.08 mmol/L with a more obvious change to a pink color at 0.32 mmol/L. Our limit of sensitivity was about 0.40 mmol/L. The accumulated values of Wadman et al. (21) for 5 patients was in the range of 1.1 to 4.2 mmol/g creatinine so these would still be readily detectable by the test strips. A mucolytic agent, 2-mercaptoethane sulfonate, has also been shown to give a positive test with the Merckoquant sulfite strips (23). These authors also showed that this drug caused a positive cyanide- nitroprusside test in the urine which could lead to cenfusion with other disorders of sulfur amino acid

• metabolism. On the other hand, thiosulfate, taurine

and S-sulfo-cysteine are more stable compounds. The latter can be detected by two-dimensional electro- phoresis and chromatographic separation (24). S-sulfo- cysteine can also be identified by automatic amino acid analysis (14, 15). Recently a method has been described for the determination of S-sulfo-cysteine in the urine by high performance liquid chromatography. Detection was by fluorometry after pre-column conversion to the dansyl derivative (25). Thiosulfate may be measured by the spectrophotometric method of Sorbo (26) or as modified by Shih et al. (27) or by Sorbo and Ohman (28). Antenatal detection of combined xanthine and sulfite oxidase deficiences The mother of the first propositus described with this condition had two further pregnancies. Amniotic fluid was obtained at 17 weeks of the pregnancies. S-Sulfo- cysteine concentration was measured in the fluid and sulfite oxidase activity was measured in the cellular

• preparation. During the first of the two subsequent

pregnancies the S-sulfocysteine concentration was less than the detectable limit (2 ~Lmol/L) and the sulfite

• xidase activity of the amniotic cellular preparation

was comparable with normal control values. A normal male child was born. In the second pregnancy the S- sulfocysteine concentration was grossly elevated (32 ~mol/L) in the amniotic fluid and sulfite oxidase activ- ity was missing from the amniotic cell preparation. A therapeutic abortion was performed. The karyotype of the fetus was 47 XY trisomy 21 (29). Conclusion ~-Mercaptolactate-cysteine disulfideuria may be a metabolic disorder with no adverse clinical effects even though studies have shown that the enzyme mercapto- pyruvate sulfurtranferase is missing in these patients and that it is an autosomal recessive hereditary defect

• pattern. The apparent extreme rarity of this condition

could be explained by several hypotheses. Modern neonatal genetic screening programs are usually based

• n blood spot analyses which would not detect this

disorder, and urine is only analysed if there is some specific indication and the Brand cyanide-nitroprusside test would be required to reveal this abnormality. Further chromatographic analysis would then be re- quired to separate this condition from homozygotes and heterozygotes for cystinuria. Finally, there is a possi- bility that the mixed disulfide does not appear in the urine until after the neonatal period when screening would most frequently be done. The youngest patient described with this condition was 11 years of age so it is not certain that the condition could be detected by CLINICAL BIOCHEMISTRY, VOLUME 18, JUNE 1985 141

SLIDE 4

CRAWHALL screening in the neonatal period. Sulfite oxidase defi- ciency is unlikely to be missed clinically because of the association with severe neurological -impairment and dislocated lenses in early childhood. However, the bio- chemical tests to confirm the diagnosis are not readily

• available. The identification of sulfite in the urine is

easily overlooked because of its ready oxidation, but if a case were suspected the appropriate test sticks could be quickly obtained through the Merck Diagnostic Company or its appropriate agency in North America. The exact metabolic significance of these enzyme deficiencies is difficult to investigate as no animal models exist. It can be seen from Figure 1 that a defi- ciency of mercaptopyruvate sulfurtransferase would prevent the formation of sulfide ion, thiocyanate and thiosuffite, but this probably has no effect on metabo- lism which is clinically significant. The enzyme defect also leads to an accumulation of mercaptolactate and its derivatives, which creates the biochemically de- tectable abnormality which may be benign. In the case

• f sulfite oxidase deficiency the defect occurs at the last

stage of the degradative pathway leading to an accu- mulation of sulfite, thiosulfate and S-sulfocysteine. Whether earlier metabolites accumulate is not known, but the accepted probability would be that it is the accumulation of sulfite which is toxic and which seems to exert its principal effect on the central nervous

• system. The possible etiology of the dislocated ocular

lens in this disease has been discussed (14). Although no direct relationships have been identified it is in- teresting that this phenomenon is also seen in another disorder of sulfur amino acid metabolism, homocystinuria. References

• 1. Ampola MG, Efron ML, Bixby EM, Meshorer E. Mental

deficiency and a new amino aciduria. Am J Dis Child 1969; 117: 66-70.

• 2. Efron ML. Two-way separation of amino acids and other

ninhydrin-reacting substances by high voltage electro- phoresis followed by paper chromatography. Biochem J 1959; 72: 691-4.

• 3. Crawhall JC, Parker R, Sneddon W, Young EP, Ampola

MG, Efron ML, Bixby EM. ~-Mercaptolactate-cysteine disulfide: Analog of cysteine in the urine of a mentally retarded patient. Science 1968; 160: 419-20.

• 4. Crawhall JC, Parker R, Sneddon W, Young EP. ~-Mer-

captolactate-cysteine disulfide in the urine of a mentally retarded patient. Am J Dis Child 1969; 117: 71-82.

• 5. Hope DB, Walti M. Synthesis of the alpha-hydroxy

analogues of S-benzylcysteine and cysteine. J Chem Soc, Section C 1970; 2475-8.

• 6. Walti M, Hope DB. Synthesis of the isomers of the mono-

and dihydroxy analogues of cystine and comparison with metabolites excreted in the urine. J Chem Soc, Section C 1971; 2326-8.

• 7. Ubuka T, Kobayashi K, Yao K, et al. S-(2-Hydroxy-2-

carboxymethylthio)-cysteine and S-(carboxymethylthio)- cysteine in human urine. Biochim Biophys Acta 1968;

158: 493-5.

• 8. Crawhall JC, Bir~ K, Purkiss P, Stanbury JB. Sulfur

amino acids as precursors of ~-mercaptolactate-cysteine disulfide in human subjects. Biochem Med 1971; 5: 109-15.

• 9. Niederwieser A, Gilberti P, Baerlocher K. ~-Mercapto-

lactate cysteine disulfideuria in two normal sisters. Iso- lation and characterisation of ~-mercaptolactate-cysteine 142

• disulfide. Clin Chim Acta 1973; 43: 405-16.
• 10. Law EA, Fowler B. ~-Mercaptolactate-cysteine

disul- fideuria in a mentally retarded Scottish male. J Mental Defic Res 1976; 20: 99-104.

• 11. Hannestad U, Martennson J, Sjodahl R, Sorbo B. 3-Mer-

captolactate-cysteine disulfideuria. Biochemical studies

• n affected and unaffected members of a family. Biochem

Med 1981; 26: 106-14.

• 12. Shih VE, Carney MM, Fitzgerald L, et aI. ~-Mercap-

tolactate pyruvate sulfurtransferase deficiency, the en- zyme defect in ~-mercaptolactate-cysteine disulfideuria. Pediatr Res 1977; 11: 464.

• 13. Hannestad U, Sorbo B. Determination of 3-mercapto-

lactate, mercaptoacetate and N-acetylcysteine in urine by gas chromatography. Clin Chim Acta 1979; 95: 189-200.

• 14. Irreverre F, Mudd SH, Heizer WD, Laster L. Sulfite oxi-

dase deficiency: Studies of a patient with mental retar- dation, dislocated ocular lenses and abnormal urinary excretion of S-sulfo-L-cysteine, sulfite and thiosulfate. Biochem Med 1967; 1: 187-217.

• 15. Crawhall JC, Segal S. Sulfocysteine in the urine of

the blotched Kenya genet. Nature (London) 1965; 208: 1320-2.

• 16. Mudd SH, Irreverre F, Laster L. Sulfite oxidase deficiency

in man: Demonstration of the enzyme defect. Science 1967; 156: 1599-602.

• 17. Shih VE, Abroms IF, Johnson JL, et al. Sulfite oxidase
• deficiency. Biochemical and clinical investigations of a

hereditary disorder in sulfur metabolism. N Engl J Med 1977; 297: 1022-8.

• 18. Duran M, Beemer FA, van der Heiden C, et al. Combined

deficiency of xanthine oxidase and sulfite oxidase: A defect of molybdenum metabolism or transport. J Inher Metab Dis 1978; 1: 175-8.

• 19. Johnson JL, Hainline BE, Rajagopalan KV. Char-

acterisation of the molybdenum cofactor of sulfite oxidase, xanthine oxidase and nitrate reductase. J Biol Chem 1980; 255: 1783-6.

• 20. Johnson JL, Rajagopalan K. The structural and metabolic

relationship between the molybdenum cofactor and uro-

• thione. Proc Natl Acad Sci USA 1982; 79: 6856-60.
• 21. Wadman SK et al. Absence of hepatic molybdenum

cofactor: An inborn error of metabolism leading to a combined deficiency of sulfite oxidase and xanthine de-

• hydrogenase. J Inher Metab Dis 1983; 6: Suppl. 1, 78-83.
• 22. Wadman SK, Cats BP, de Bree PK. Sulfite oxidase defi-

ciency and the detection of urinary sulfite. Eur J Pediatr 1983; 141: 62-3.

• 23. Duran M, Aarsen G, Fokkens RH, et al. 2-Mercapto-

ethanesulfonate-cysteine disulfide excretion following the administration of 2-mercaptoethanesulfonate--a pit- fall in the diagnosis of sulfite oxidase deficiency. Clin Chim Acta 1981; 111: 47-53.

• 24. Shih VE. Laboratory techniques for the detection of hered-

itary metabolic disorders. Pp. 11-77. Cleveland: CRC Press, 1973.

• 25. Kagedal B, Kallberg M, Sorbo B. Determination of S-

sulfo-cysteine in urine by high-performance liquid chro-

• matography. J Chromatog 1983; 276: 418-22.
• 26. Sorbo B. A colorimetric method for the determination of
• thiosulfate. Biochim Biophys Acta 1957; 23: 412-6.
• 27. Shih VE, Carney MM, Mandell R. A simple screening test

for sulfite oxidase deficiency: Detection of urinary thio- sulfate by a modification of Sorbo's method. Clin Chim Acta 1979; 95: 143-5.

• 28. Sorbo B, Ohman S. Determination of

thiosulfate in urine. Scand J Clin Lab Invest 1978; 38: 521-7.

• 29. Ogier H, Wadman SK, Johnson JL, et al. Antenatal

diagnosis of combined xanthine and sulfite oxidase

• deficiencies. Lancet 1983; 2: 1363-4.

CLINICAL BIOCHEMISTRY, VOLUME 18, JUNE 1985