homocystinuria due to cystathionine synthase deficiency: the metabolism...

Journal of Clinical InvestigationVol. 44, No. 10, 1965

Homocystinuria Due to Cystathionine Synthase Deficiency:The Metabolism of L-Methionine *

LEONARDLASTER,t S. HARVEYMUDD,JAMESD. FINKELSTEIN, ANDFILADELFOIRREVERREWITH THETECHNICALASSISTANCEOF BRINSONCONERLY

(From the National Institute of Arthritis and Metabolic Diseases and the National Institute ofMental Health, National Institutes of Health, Bethesda, Md.)

During surveys of mentally retarded patientsfor evidence of metabolic errors, investigators inIreland (1) and in America (2) independently dis-covered that abnormal excretion of homocystinein the urine may accompany retardation. A syn-drome has since been defined (3-9) with the rela-tively constant clinical features of mental defi-ciency, fine, fair hair, dislocated ocular lenses,malar flush, peculiar gait, and genu valgum; andwith the variable features of pes cavus, long ex-tremities and digits, convulsions, thrombotic in-cidents that have been attributed to abnormalstickiness of the platelets (10), cardiovascular dis-orders, and fatty liver. Almost without exceptionthe patients have had elevated concentrations ofmethionine and homocystine in the blood plasmain addition to increased amounts of homocystinein the urine.

A mentally retarded child, C.T., with many ofthe clinical and all of the biochemical abnormalitiesof the disorder, has been shown to lack detectableactivity of cystathionine synthase I in her liver(12). This enzyme catalyzes a reaction involvedin methionine metabolism (reaction 4, Figure 1),and we have proposed that deficiency of cystathio-nine synthase activity is the fundamental defect inthe disease. The parents of C.T., who are free ofclinical stigmata of the disease and who do not ex-crete abnormal amounts of homocystine in the urine,

* Submitted for publication May 5, 1965; accepted July6, 1965.

t Address requests for reprints to Dr. Leonard Laster,Gastroenterology Unit, National Institute of Arthritisand Metabolic Diseases, Bethesda, Md. 20014.

1 Although we have referred to this enzyme as "cysta-thionine synthetase" in earlier publications, we are nowusing the designation "cystathionine synthase" to con-form to the presently recommended nomenclature (11).Cystathionine synthase is the trivial name for the en-zyme L-serine hydro-lyase (adding L-homocysteine),E.C.4.2.1.21.

have approximately half the mean control hepaticcystathionine synthase activity and thus appear tobe heterozygous for the abnormal gene (13). Apaternal cousin of C.T. excretes abnormal amountsof homocystine in the urine, but is neither men-tally retarded nor otherwise afflicted with clinicalmanifestations of the syndrome. Her hepaticcystathionine synthase activity is between that ofthe heterozygous parents and that of C.T. (13).Although her genetic status is not clear, the cousindemonstrates that cystathionine synthase defi-ciency severe enough to cause homocystinuriadoes not necessarily cause mental retardation.

The concentration of cystathionine in brain tis-sue from patients with the typical clinical featuresof cystathionine synthase deficiency has beenshown to be markedly lower than in brain tissuefrom control subjects (14, 15). This abnormalityis consistent with the concept that cystathioninesynthase deficiency is the fundamental defect, sincecurrent knowledge suggests that the body's endoge-nous cystathionine derives exclusively from the re-action catalyzed by the synthase.

In the present study some of the metabolic con-sequences of cystathionine synthase deficiency wereexplored. Feeding methionine to normal humansubjects increases urinary excretion of inorganicsulfate (16, 17). There is abundant evidenceshowing that the pathway represented by reactions1 to 6 in Figure 1 is a possible route in mammalsfor the conversion of the sulfur of methionine toinorganic sulfate (reviewed in references 18 and19). However, the evidence that this pathway isthe predominant one is far less convincing. It waspossible to test whether reaction 4 is an obligatorystep in the pathway by feeding L-methionine andL-cysteine to control subjects and to patients withcystathionine synthase deficiency and determiningtheir subsequent urinary excretion of inorganic

1708

METHIONINE METABOLISMIN CYSTATHIONINE SYNTHASEDEFICIENCY

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LASTER, MUDD, FINKELSTEIN, AND IRREVERRE

acid (23), and the supernatant solution was stored frozen.Amino acids of blood plasma and urine were determinedwith an automatic amino acid analyzer according toSpackman, Stein, and Moore (24). Urinary content oftotal sulfur, inorganic and ethereal sulfates, and neutralsulfur was determined according to Fiske's modificationof the method of Rosenheim and Drummond (25).

Results

Prolonged feeding of L-lnethionine or L-cysteine.The results of a typical study of a control subjectare shown in Figure 2. The dietary intake ofmethionine and cystine was constant during the27-day study. The first 3 days were control days;during days 4 to 6, when the subject received sup-plementary L-cysteine, his urinary excretion of in-organic sulfate rose promptly and remained ele-vated throughout this period. During days 10 to12 he received supplementary L-methionine, and

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L-CYSTEINE TO PATIENTS WITH CYSTATHIONINE SY-NTIASE

DEFICIENCY. The charts are explained in the legend toFigure 2. The following changes in the protocol had tobe made: since C.T. develolied anorexia on day 23, herdose of L-methionine was halved; 'M.A.G. developed ano-rexia and nausea and decreased her dietary intake ofmethionine on days 7 and S.

onlile ( Figutre 3, day-s 20 to 23 and 45 to 47, andFigure 4, days 7 and 8).

The relationship between the total intake ofmethionine and the urinary excretion of inorganic

ssulfate was analyzed lb administration of a rangeof doses of supplemientary i-methionine to thecontrol subjects (Figutre 5). This range bracketedthe doses given to the two patients with cvstathio-nine svnthase deficiency. The total intake of meth-ionime was calculated vbadding, the values for die-tary methionine and supplementary L-methionine.The ol)servedl values for urinary inorganic sulfatewere converted to values for urinary inorganicsulfate attributable to methionine intake by makinga small correction for estimated urinary inorganicsulfate attributable to cvst(e)ine intake. First, itwas assumed that during the coiltrol periods thetotal urinary inorganic sulfate was made up of in-organic sulfate attril)butable to dietary c-stine andto dietary methfiolnine in proportiol to the dietaryintake of each amino acid. On the basis of thisassumpltion a value was calculated for urinary sul-fate attributable to dietary cvstine intake duringthe control leriods. This value was subtracted

from the value for total urinary inorganic sulfateduring the first and second days of L-methioninesupplemenitation. The correction was small anddid not change the qcualitative considerations.

The urinary excretion of inorganic sulfate bythe control subjects increased in relationship tomethionine intake, an increment in dose producingan approximately eqluivalent increment in excre-tion. of inorganic sulfate. The values for the two

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patients are below the curves established for thecontrol subjects, regardless of whether the data areexpressed in terms of body weight, as shown inFigure 5, or in terms of body surface area, and re-gardless of whether the first or second day ofL-methionine supplementation is considered.Thus, each patient with cystathionine synthase de-ficiency fails to excrete as much inorganic sulfatein response to an increase in methionine intake asdo control subjects, and appears, therefore, to havea reduced capacity to catabolize L-methionine toinorganic sulfate.

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In contrast, the responses of the two patientswith cystathionine synthase deficiency to the sup-plementary administration of L-cysteine did notdiffer significantly from those of the control sub-jects (Figures 2 to 4). In both the patients andthe control subjects there was a rapid and sig-nificant increase in urinary excretion of inorganicsulfate after an increase in intake of L-cysteine.A graphic analysis of the dose-response relation-ship for L-cysteine, analogous to the one describedfor L-methionine, is presented in Figure 6. Thevalues for urinary excretion of inorganic sulfate

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FIG. 5. DOSE-RESPONSERELATIONSHIP BETWEENMETHIONINE INTAKE AND URINARY EXCRE-TION OF INORGANIC SULFATE. The data are from the studies in which supplementary L-methio-nine was given repeatedly for several days. Responses are presented for the first and seconddays of supplementation. Open -symbols represent day 1; black symbols represent day 2. Thedotted lines show the limits of the control range. The homocystinuric patients are indicatedby their initials on the Figure.

The subjects included, in addition to the normal volunteers, S.M., the grandmother of C.T.;V.S., a 33-year-old maternal aunt of C.T. who has no clinical evidence of cystathionine syn-thase deficiency; and D.A.G. and D.M.G., two children of M.A.G. who are both free of clinicalevidence of the disorder.

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FIG. 6. DOSE-RESPONSERELATIONSHIP BETWEENCYST(E)INE INTAKE ANDURINARY EXCRETIONOF INORGANIC SULFATE. The symbols in this Figure areidentical to those in Figure 5.

by C.T. and M.A.G. during both the first andsecond days of L-cysteine supplementation arewithin the control range. Thus, under the condi-tions of this study the ability of each of these pa-tients to increase urinary excretion of inorganicsulfate in response to a dose of L-cysteine is notimpaired.

One possible explanation for the reduced ca-pacity of the patients with cystathionine synthasedeficiency to increase urinary excretion of inor-ganic sulfate after an increase in the dose ofL-methionine is that their intestinal absorption ofL-methionine is impaired. To explore this possi-bility we determined the concentration of methio-nine and its sulfoxide in the plasma of C.T. beforeand after a period during which she received sup-plementary L-methionine. On day 14 (Figure 3)there were 8 jAmoles per 100 ml of methionine inher plasma and only a trace of methionine sulfox-ide, whereas on day 23, after she had received sup-plementary L-methionine for 3 days, the concen-tration of methionine in her plasma had increasedto 135 Mmoles per 100 ml, and there was 57 Mmoles

of methionine sulfoxide per 100 ml. Comparablevalues for a study of a control subject were methi-onine, 2, and methionine sulfoxide, 0, ,moles per 100ml of plasma before the administration of L-methi-onine; and methionine, 2, and methionine sulfox-ide, trace, Lmoles per 100 ml of plasma after 4days of L-methionine supplementation. Thesefindings show that the L-methionine had been ab-sorbed by C.T. and had accumulated in her bodyfluids.

The two patients with cystathionine synthase de-ficiency differed from the control subjects not onlyin the magnitude of their responses to an increasein the intake of L-methionine, but also in the pat-tern of their responses to the omission of the sup-plementary L-methionine. On each of the daysthat the L-methionine supplement was first dis-continued, the control subjects excreted less inor-ganic sulfate in the urine than they had excretedduring the last day of feeding the supplementaryL-methionine, whereas C.T. (days 45 to 47, Fig-ure 3) and M.A.G. (days 7 and 8, Figure 4) ex-creted more inorganic sulfate in the urine during

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the first day of L-methionine omission. Theseobservations suggest that there was a delayed, butdepressed, response to the administration of sup-plementary L-methionine in the patients with cys-tathionine synthase deficiency.

Response to a single dose of L-methionine orL-cysteine. Although assays of liver biopsy speci-mens have indicated that E.T. and L.T., the parentsof C.T., are heterozygous for cystathionine syn-thase deficiency (13), they neither excrete abnor-mal amounts of homocystine in the urine, norpresent clinical features of the syndrome associatedwith this deficiency. The response to a singledose of L-methionine was studied to learn whetherit would provide a practical clinical means ofdetecting heterozygous subjects such as theparents.

The impaired ability of the homozygous patient,C.T., to metabolize L-methionine to inorganic sul-fate is reflected not only in her response to thechronic administration of L-methionine, describedabove, but also in her response to a single dose ofL-methionine (day 52, Figure 3). Her urinaryexcretion of inorganic sulfate on the date shereceived the single dose rose only slightly and toa much lesser extent than that of the control sub-jects. Figure 7 is a graph of the relationship be-tween size of the single dose of L-methionine andthe increment in 24-hour excretion of urinary in-organic sulfate. The value for C.T. is far belowthe control range.

Despite their partial reductions in hepatic cysta-thionine synthase activity, E.T. and L.T. re-sponded to single doses of L-methionine in thesame manner as the control subjects (Figure 7).The results of repeat tests of the parents werealmost identical, and studies at two dosage levelsfailed to differentiate them from control subjects.The possibility was explored that measurement ofthe urinary excretion of inorganic sulfate duringthe early time periods after ingestion of L-methio-nine (0 to 5, 5 to 8, 8 to 16, and 16 to 24 hours)might prove to be a more sensitive test of a meta-bolic difference between the heterozygous parentsand the control subjects, but once again the re-sults for the two groups were indistinguishable.

During studies of three control subjects and ofE.T. and L.T. the rate of excretion of inorganicsulfate after a single dose of L-cysteine consistentlyreached a maximal value at an earlier time than

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FIG. 7. EFFECT OF A SINGLE DOSEOF L-METHIONINE ONURINARY EXCRETION OF INORGANIC SULFATE DURING THE

SUBSEQUENT24 HOURS. The increment in methionine in-take was the single dose of L-methionine. The incre-ment in urinary excretion of inorganic sulfate was the dif-ference between the excretion during the day of methi-onine administration and the immediately preceding daywhen no supplementary L-methionine had been given.Symbols not defined in this Figure are identical to thosein Figure 5.

it did after a single dose of L-methionine (Figure8). This observation is compatible with the pos-sibility that one of the reactions between methio-nine and cysteine (Figure 1) is rate limiting forthe over-all pathway from methionine to inorganicsulfate. That the delayed conversion of L-methio-nine to urinary inorganic sulfate is not attributableto delayed intestinal absorption of the amino acidis suggested by the finding that when a normalvolunteer was fed the same dose of L-methioninethat was used in the studies referred to in Figure8, the plasma concentration of methionine reacheda peak value between L and 1 hour. The valuesat 0, i, 1, 2, 3, 4, and 5 hours after ingestion ofthe methionine were 3.5, 45.6, 69.6, 64.8, 52.8,48.0, and 39.6 omoles of methionine per 100 mlof plasma.

The chemical forms in which C.T. excretessulfur in her urine during periods of L-methionineadministration. The preceding evidence indicat-ing that a patient with cystathionine synthase de-ficiency converts much less of his dietary methio-nine to inorganic sulfate than a control subject

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1714


does is in keeping with the formulation that thefundamental defect underlying homocystinuria isa block at reaction 4 (Figure 1). One might ex-pect, too, that a patient with this inborn errorwould excrete a rather large fraction of the sulfurof his dietary methionine as homocystine. This is

not the case. When C.T eats an average diet,with a daily methionine content of approximately7 to 14 mmoles, she usually excretes an amount ofhomocystine in her urine that is equivalent to onlyabout 0.4 to 0.5 mmole of sulfur. Because of thisdiscrepancy we studied the fate of the remaining

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FIG. 8. RATE OF URINARY EXCRETION OF INORGANIC SULFATE AFTER ORAL ADMINIS-TRATION OF A SINGLE DOSE OF L-METHIONINE OR L-CYSTEINE TO THREE CONTROLSUB-JECTS ANDTO THE PARENTSOF C.T. In each study the dose of L-cysteine equaled thatOf L-methionine; the values were: J.R.McC., 0.4; TM-., 0.7; and R.N., E.T., and L.T.,0.5 mmole per kg. The periods of the urine collections after the dose of an amino acidwere 0 to 5, 5 to 8, 8 to 16, and 16 to 24 hours. The values given in the Figure asper cent represent -the per cent of the single dIose of amino acid that was excreted asexcess inorganic sulfate during the 24 hours after ingestion of the amino acid. Theresults of repeat studies of the effects Of L-methionine administration to E.T. and L.T.are presented. *- *, L-cysteine; ---- and - - - - A, L-methionine.

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TABLE I

Sulfur-containing compounds in the urine after L-methionine administration

Chemical forms of urinary sulfurSupple-mentary Sulfate Neutral sulfur Bound

Study L-methi- methi-Subject day onine Inorganic Ethereal Amino acid* Unidentifiedt oninel

mmoles/day mmoles/ mmoles/day mmoles/ mmoles/day mmoles/day day day

15 None 5.7 0.3 1.32 1.216 None 5.7 0.3 1.16 2.019 None 4.9 0.5 1.47 0.2 0.25

C.T,21 23.5 6.4 0.8 0.51 4.222 23.5 9.5 0.8 0.63 3.0 0.6123 11.7 9.2 1.0 1.21 4.0 0.86

7 None 11.3 1.0 0.53 1.38 None 6.0 0.5 0.62 2.8 0.13

T.H.9 53.6 32.1 1.9 0.60 Negative value

10 53.6 44.1 0.5 0.86 0.4311 53.6 45.1 2.2 0.94 0.33 0.27

9 None 13.3 0.2 0.67 Negative value 0.18J.R.McC.

11 46.9 58.1 Negative value 0.89 Negative value12 46.9 58.1 Negative value 0.69 Negative value 0.16

* The value for sulfur as urinary amino acids was calculated from the peaks observed in column chromatograms, which were regarded as cor-responding to cysteic acid, taurine, methionine sulfoxide, cystine, cystathionine, methionine, mixed disulfide of homocysteine and cysteine, andglutathione.

t The value for neutral sulfur in unidentified chemical form was obtained by subtracting the value for sulfur in the form of amino acids (seeprevious footnote) from the value for the total amount of neutral sulfur in the urine.

$ Bound methionine represents methionine liberated by acid hydrolysis.

methionine to learn whether C.T. excretes in herurine metabolites of methionine or of homocysteinethat are usually absent from or present only intrace amounts in the urine of control subjects.

The sulfur-containing compounds in urine thatwe identified by ion exchange column chromatog-raphy are listed in a footnote to Table I. Theexcretion of these compounds was measured forselected days from the studies of C.T. and thecontrol subjects, T.H. and J.R.McC. Adminis-tration of supplementary L-methionine to C.T. inthe doses shown did not significantly increase herurinary excretion of homocystine or of the mixeddisulfide of homocysteine and cysteine. Othershave also noted that no more than a small fractionof a load of homocystine or methionine is excretedas homocystine by homocystinuric subjects (4, 6,9), but the explanation of this finding is not ap-parent. During L-methionine administration C.T.excreted an average of only 0.1 mmole of homo-cystine daily. After oral administration of L-methionine, C.T. and the two control subjectsincreased their urinary excretion of methionineand of methionine sulfoxide to approximately thesame extent. Methionine excretion rose fromabout 0.02 to an average of about 0.20 mmole per

day, and methionine sulfoxide excretion rose fromundetectable levels to an average of about 0.25mmole per day. Thus, the amount of these twocompounds excreted fails to balance the discrep-ancy between the intake of methionine sulfur andthe amount of urinary sulfate in C.T.

The urinary excretion of excess methionine bythe control subjects stopped within about 2 daysof discontinuation of L-methionine supplementa-tion, but C.T. continued to excrete increasedamounts of methionine and its sulfoxide in herurine for about 10 days after L-methionine supple-mentation was stopped on day 24 (Figure 3).

When the amounts of inorganic and etherealsulfates are subtracted from the total sulfur ofthe urine, the difference is termed neutral sulfur.This value includes sulfur as the compounds iden-tified with the amino acid analyzer and remainingsulfur in unidentified form. The latter is desig-nated unidentified neutral sulfur. The value forneutral sulfur is obtained by subtraction of onelarge number from another, and the precision ofthe determinations is such that at times negative,meaningless values may be obtained. Despite thisshortcoming of the method, the data in Table Ido suggest that C.T. excretes relatively large

1716


amounts of sulfur as one or more unidentifiedcompounds in the urine when she receives L-methionine, whereas the control subjects do not.Thus, the excretion of unidentified neutral sulfurby the control subjects was no higher during theperiods of L-methionine supplementation than dur-ing control periods, but C.T. excreted an addi-tional 2 to 3 mmoles of unidentified neutral sulfurwhen she was given supplementary L-methionine.

To explore the chemical form of the excessurinary unidentified neutral sulfur excreted byC.T. after she received L-methionine, we meas-ured the amount of urinary methionine present inbound form. Samples of urine were evaporatedto dryness; 20 ml of constant-boiling HCl wasadded for each 10 ml of urine originally present;and the solution was heated at 1100 C for 24hours, evaporated to dryness, brought to pH 2.2,and analyzed for its amino acid content. Methio-nine liberated by such acid hydrolysis representsbound methionine, and the amounts detected arelisted in the last column of Table I. WhenJ.R.McC. or T.H. received L-methionine, theurinary excretion of bound methionine did not risein one case and rose only 0.14 mmole in the other.When C.T. received a smaller dose of L-methio-nine, her excretion of bound methionine roseapproximately 0.5 mmole.

The urine collected during days 1, 9, and 23of C.T.'s study (Figure 3) was analyzed for thea-keto analogue of methionine, a-keto-y-methiobu-tyrate (26). No a-keto-y-methiobutyrate wasfound in the urine collected during day 1 (controlperiod) and day 9 (period of L-cysteine adminis-tration), and 0.16 mmole was detected in the urineexcreted on day 23. Thus, the a-keto-y-methio-butyrate does not account for a major portion ofthe unidentified neutral sulfur in the urine of C.T.

The amino acid chromatograms of the urinepassed by C.T. during the period when L-methio-nine was administered revealed several as yetunidentified peaks that were not present in thechromatograms of the urine collected during anyother periods.

Discussion

The nature of the enzymatic defect responsiblefor the clinical syndrome associated with homo-cystinuria appears to have been firmly established.

The direct evidence that cystathionine synthaseactivity is deficient (12, 13) has recently beensupplemented by demonstrations (14, 15) that theconcentration of cystathionine in brain tissue fromhomocystinuric patients is markedly-lower than itis in brain tissue from control subjects. Many ofthe other enzymes involved in the metabolism- ofthe sulfur amino acids [reactions 1 and 5, Figure1 (12, 13, 27) and reactions 7 and 9 (14)] areunchanged in this condition.. The evidence pre-sented in this paper of the normal capacity of suchpatients to convert the sulfur of L-cysteine to in-organic sulfate suggests that cystathionine syn-thase deficiency is not accompanied by a sig-nificant alteration in the activity of the enzymesbeyond reaction 5.

Earlier studies (17-19) have established thatthe conversion of methionine to inorganic sulfateby mammals can occur via the sequence of reac-tions 1 to 6, but the evidence that this is a majoror obligatory pathway is less conclusive. Theo-retically other known biochemical reactions canconstitute alternative pathways, and the relativecontributions of these possible pathways of methio-nine metabolism in normal man have not yet beenfully assessed. The results of the present studyindicate that patients whose deficiency of cysta-thionine synthase activity is such that they excreteabnormal amounts of homocystine in the urinehave a reduced capacity to form inorganic sulfatefrom orally administered L-methionine. Weinter-pret this finding as evidence that cystathionine syn-thesis is an obligatory reaction in the conversionof methionine to sulfate in man.

The finding also suggests that the deficiency ofcystathionine synthase activity detected in the liveris associated with a generalized reduction in thebody's capacity to convert homocysteine to cysta-thionine. Evidence from other studies (27, 28)indicates that two factors lead to this generalizedreduction: a) the liver is the dominant site ofcystathionine synthesis; and b) the enzyme defectis present in other tissues. Assays of rat andmonkey tissues demonstrated cystathionine syn-thase activity to be present in the liver, brain;and pancreas (27). There was activity in ratkidney but not in monkey kidney. The relativelyhigh concentration of cystathionine synthase activ-ity in the liver and the size of this organ combineto make it the major site of mammalian cysta-

1717


thionine synthesis. Thus, an isolated hepatic de-fect would probably result in a marked reductionin the body's capacity to form cystathionine.However, post-mortem brain tissue from a patientwith homocystinuria had a specific defect in cysta-thionine synthase (28), suggesting that the enzy-matic defect first discovered in hepatic tissue alsoinvolves other organs.

The two patients with cystathionine synthasedeficiency were able to convert some L-methionineto inorganic sulfate (Figures 3 to 5). This smalldegree of conversion could be due either to thepresence in these patients of residual cystathioninesynthase activity sufficient to achieve the observedconversion or to the existence of an alternativepathway for methionine metabolism. The datapresented in Figure 5 establish that the normalsubject can convert at least 0.85 mmole of methio-nine per kg per day to inorganic sulfate. There-fore, a patient with 12% of the mean controlhepatic cystathionine synthase activity, such asM.A.G. (13), should be able to convert at least0.1 mmole per kg per day. Thus, it seems reason-able to attribute the ability of the two patients toincrease urinary excretion of inorganic sulfateafter the administration of L-methionine to theirresidual cystathionine synthase activity.

The present studies reveal that patients withcystathionine synthase deficiency retain abnormalamounts of ingested methionine. During the stud-ies described in Table I, C.T. excreted in the urinean average of only 20%o of the sulfur contained inher L-methionine supplement, whereas T.H. andJ.R.McC. excreted 58 and 90%o, respectively.Furthermore, she accumulated abnormal amountsof methionine and methionine sulfoxide in theplasma and continued to excrete large quantitiesof these two compounds in the urine for an ab-normally long time after supplementary L-methio-nine had been discontinued. This evidence alsosuggests that during the period of L-methioninesupplementation C.T. was in abnormally positivesulfur balance, although this conclusion can beconfirmed only by complete balance determina-tions. The chemical form and the tissue in whichmethionine is stored and the cause of the pro-longed retention must be explained.

Although attention has been focused-mainly onthe urinary excretion of homocystine in cystathio-nine synthase deficiency, the data presented in

Table I indicate that homocystine is not neces-sarily the most abundant abnormal sulfur-contain-ing compound in the urine. During day 23 ofC.T.'s study she excreted 0.12 mmole of homo-cystine, 0.34 mmole of methionine, 0.48 mmole ofmethionine sulfoxide, 0.86 mmole of bound methi-onine, and 4.0 mmoles of unidentified neutral sul-fur. Although the amounts of methionine and itssulfoxide were not significantly greater than thevalues observed in studies of control subjects, theamounts of bound methionine and unidentifiedneutral sulfur were distinctly abnormal. Thesevalues emphasize the extent to which the amountof unidentified neutral sulfur exceeds that of thesulfur in known amino acids. Attempts to deter-mine the chemical nature of the unidentified neu-tral sulfur are now in progress.

Summary1. Patients with homocystinuria due to cysta-

thionine synthase deficiency have an impaired ca-pacity to convert the sulfur of orally administeredL-methionine, but not L-cysteine, to urinary inor-ganic sulfate. This observation suggests thatcystathionine formation is an obligatory step inhuman catabolism of methionine to inorganicsulfate.

2. The patients were able to convert a smallamount of L-methionine to inorganic sulfate. Thislimited catabolism may be due to their residualhepatic cystathionine synthase activity.

3. The urinary excretion of inorganic sulfateafter the administration of a single dose of L-methionine distinguished the homozygote for cysta-thionine synthase deficiency from control subjects.The responses of two heterozygous subjects werewithin the control range.

4. During the administration of L-methionineto a patient with cystathionine synthase deficiencythe urinary excretion of homocystine did not in-crease markedly. Abnormal amounts of methio-nine and methionine sulfoxide accumulated in theplasma, and these compounds were excreted inlarge quantities in the urine for an abnormallylong time after L-methionine supplementation hadbeen discontinued.

5. Homocystine is not necessarily the mostabundant abnormal sulfur-containing compound inthe urine of these patients. During L-methioninesupplementation excessive amounts of bound meth-

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ionine and of unidentified neutral sulfur weredetected in the urine. Their quantities exceededthat of the homocystine.

AcknowledgmentsWe are grateful to Dr. William C. Alford for per-

forming the analyses for the various forms of urinarysulfur during the early studies of C.T. and for helping usset up the assay methods in our laboratory. Mrs. Min-nie L. Woodson and Mr. William A. Edwards providedtechnical assistance during the study. Miss Jeanne M.Reid calculated and supervised the preparation and feed-ing of the metabolic diets. Dr. Robert K. Ockner con-tributed greatly to the clinical management of many ofthe subjects. That the subjects were as cooperative asthey were and their urine collections as acurate as theywere is attributable to the diligence of the nursing staffof ward 9 West of the Clinical Center. Dr. George L.Spaeth referred C.T. and M.A.G. to us for study.

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