A Collagen Defect in Homocystinuria

ANDREwH. KAkNGand ROBERTL. TRELSTAD

From the Developmental Biology Laboratory, Departments of Medicine andPathology, Massachusetts General Hospital and Harvard Medical School,Boston, Massachusetts 02114, and the Departments of Medicine andBiochemistry, Veterans Administration Hospital, and The University ofTennessee Medical School, Memphis, Tennessee 38104

ABSTRACT The biochemical mechanism accountingfor the connective tissue abnormalities in homocystin-uria was explored by examining the effects of variousamino acids known to accumulate in the plasma ofpatients with this disease on cross-link formation incollagen. Neutral salt solutions of purified, rat skin col-lagen, rich in cross-link precursor aldehydes, werepolymerized to native type fibrils by incubating at 370Cin the presence of homocysteine, homocystine, or methi-onine. After the polymerization was completed, eachsample was examined for the formation of covalentintermolecular cross-links, assessed indirectly by solu-bility tests and directly by measuring the cross-linkcompounds after reduction with tritiated sodium boro-hydride and hydrolysis.

Collagen solutions containing homocysteine (0.01 M-0.1 M) failed to form insoluble fibrils. Furthermore,much less of the reducible cross-links, A"'7 dehydro-hydroxylysinonorleucine, A67 dehydrohydroxylysinohy-droxynorleucine, and histidino-dehydrohydroxymerodes-mosine were formed in the preparations containinghomocysteine as compared with the control and thesamples containing methionine or homocystine. The con-tent of the precursor aldehydes, a-aminoadipic-8-semi-aldehyde (allysine) and the aldol condensation product,was also markedly diminished in tropocollagen incu-bated with homocysteine. It is concluded that homocys-teine interferes with the formation of intermolecular

A portion of this work was presented at the joint meetingof the American Society for Clinical Investigation, AtlanticCity, N. J. April 1972. This is publication no. 610 ofRobert W. Lovett Group for Study of Diseases CausingDeformities.

Dr. Kang is a Medical Investigator of Veterans Adminis-tration.

Dr. Trelstad is a Fellow of the Helen Hay WhitneyFoundation.

Received for publication 5 Februarv 1973 and in revisedform 15 Mayt 1973.

cross-links that help stabilize the collagen macromolecu-lar network via its reversible binding to the aldehydicfunctional groups.

Analysis of the collagen cross-links in skin biopsysamples obtained from three patients with documentedlhomocystinuria showed that the cross-links were sig-nificantly decreased as compared with the age-matchedcontrols, supporting the tentative conclusions reachedfrom the in vitro model studies. In addition, the solu-bilitv of dermal collagen in non-denaturing solvents wassignificantly increased in the two patients examined,reflecting a functional defect in collagen cross-linking.Although the concentration of homocysteine used inthis study to demonstrate these effects in vitro is clearlyhigher than that which is observed in homocystinuric'splasma, the data do suggest a possible pathogeneticmechanism of connective tissue defect in homocystinuria.

INTRODUCTION

Homnocystinuria is a metabolic disease inherited as anautosomal recessive, characterized biochemically byhomocysteinemia, homocystinemia, hypermethioninemia,and homocystinuria, the last a variety of "overflow"amino aciduria due to the accumulation of the aminoacid in the blood (1). The basic defect has been shownto be the deficient activity of the enzyme, cystathioninesynthetase in these patients (2), which catalyzes thesynthesis of cystathionine from homocysteine and serinein the methionine pathway. Deficient activity of the en-zvme then results in the accumulation of homocysteineand other metabolites including methionine. Homocys-tine that the affected individual excretes is presumablyderived from oxidation of homocysteine.

The disease is characterized clinically by widespreaddeformities and malfunctions of connective tissue includ-ing joint laxity, kyphoscoliosis, pigeon breast, genuvalgum, severe osteoporosis. ectopia lentis, and vascular

The Journal of Clinical Investigation Volume 52 October 197.3- 2571-25782 25. -1I

disease in which dilatation and thrombosis of medium-sized arteries and veins occur frequently. Despite thedelineation of the enzymatic defect responsible for thealtered amino acid metabolism the pathogenetic mecha-nisms by which these metabolic defects lead to the con-nective tissue malformations are poorly understood. How-ever, in view of similarities in some of the clinicalmanifestations of connective tissue diseases in thesepatients to experimental osteolathyrism (3), it wassuggested by McKusick (1) that a collagen cross-link-ing defect might be similar to that shown in lathyrism(4, 5). The structural similarity of homocysteine to D-penicillamine led to the speculation that, like the latter,homocysteine might also interfere with the collagen(and elastin) cross-linking by binding to the precursoraldehydes (1, 6-8). Good evidence is available for sucha mechanism of action by D-penicillamine (9-11). How-ever, no experimental evidence for such a mechanismof action by homocysteine has been obtained to ourknowledge. Harris and Sjoerdsma (6) did report thatin two out of four patients with homocystinuria, solu-bility of dermal collagen was increased over controlpatients, suggesting a defect in collagen cross-linking.They did not, however, measure the cross-link contentdirectly.

Evidence to date indicates that collagen cross-linkinginvolves oxidative deamination of certain lysyl residuesto form allysine (a-aminoadipic-8-semialdehyde) thatthen reacts via Schiff base formation with an e-aminogroup of a lysyl or hydroxylysyl residue located on anadjacent molecule to form the reducible intermolecularcross-links, A67 dehydrolysinonorleucine or A6'7 dehydro-hydroxylysinonorleucine (12-16). In a separate path-way, two residues of the aldehyde, allysine, may con-dense in an aldol condensation to form the "intra"-molecular cross-link (17-21). The aldol product, whichcontains a reactive aldehyde, further participates in theformation of a higher complex cross-link, tentativelydesignated "post-histidine compound" (16, 22) and re-cently identified as histidino-hydroxymerodesmosine(23, 24). In addition, another Schiff base A6'7 dehydro-hydroxylysinohydroxynorleucine has been identified asa cross-link in certain collagens including human (25),but its biosynthetic origin has not yet been established.In all of these reactions, the integrity of the aldehydeappears essential, since its reduction by borohydrideresults in complete loss of the ability to form the cross-links by the reduced collagen (16). Similarly, bindingof the functional group by any agent such as D-penicil-lamine also interferes with cross-linking (10).

In the present paper, we report the results of our

studies on the effect of homocysteine and other relatedmetabolites on the collagen cross-linking and the resultsof examination of the dermal collagen of patients with

homocystinuria for direct evidence of cross-link im-pairment and alteration in solubility.

METHODSPreparation of collagen. Acetic acid-extracted collagen,

rich in aldehydic precursors of collagen cross-links (17,18, 21), was prepared from the dorsal skin of normal,young (150-200 g) male Sprague-Dawley rats (CD strain,Charles River Breeding Laboratories, Wilmington, Mass.).Lathyritic, cold neutral salt-extracted collagen was ob-tained from another group of animals that had been fed aPurina rat chow containing 2 g/kg of p-aminopropionitrilefumarate (Aldrich Chemical Co., Inc., Milwaukee, Wisc.)for 2 wk. The skins were cleaned, ground in a mechanicalmeat grinder in the cold, washed with cold distilled water,and extracted with 0.05 M Tris, pH 7.4 containing 1 MNaCl, overnight at 4°. The residue from the normal ani-mals was then extracted further with 0.5 M acetic acid.The collagen present in the 1 M NaCl extract of the lathy-ritic tissue and the 0.5 M acetic acid extract of the normaltissue was purified by the method described elsewhere (21).Purified collagen was stored in a lyophilized state in adesiccator over P2Os in the cold.

In vitro fibril formation. Native-type fibrils were formedfrom the soluble collagen by a modification (16) of themethod described by Gross and Kirk (26). Briefly, purifiedsoluble collagen was solubilized in cold 0.1 M acetic acid(1 mg/ml) and dialyzed vs. 0.05 M Tris, pH 7.4 containing0.16 M NaCl in the cold. Any insoluble material wasremoved by centrifugation in a Spinco ultracentrifuge(Spinco Div., Beckman Instruments, Palo Alto, Calif.) at105,000 g for 2 h. To aliquots of the neutral saline solutionof collagen, varying amounts of the amino acids, homo-cysteine (free base), homocystine, or methionine wereadded. In all experiments, homocysteine was freshly pre-pared from homocysteinethiolactone immediately prior touse by 2 N NaOH treatment (27). The reaction vesselswere immediately sealed under nitrogen and incubated ina water bath maintained at 370 to form the native-typefibrils (26). The course of fibril formation was followedby measuring the opacity in a Klett colorimeter (KlettManufacturing Co., Inc., New York). The gelation usuallyreached a maximum within hours. In some experimentsthe gels were cooled to 40 at the end of a 24 h incubationperiod. The opacity of the solution was used as an indexof the degree of collagen aggregation.

Reduction with tritiated sodium borohydride. In order toassay the cross-link content and other related compounds,collagen samples were reduced with calibrated NaB3H4(200 mCi/mM, New England Nuclear Corp., Boston,Mass.) using 100-fold molar excess as previously described(16, 28). The reaction was carried out for 30 min at room

temperature. Excess reagent was destroyed by adjustingthe pH of the reaction mixture to 4.0 with 1 M acetic acid,and the salts were removed by exhaustive dialysis against0.1 Macetic acid.

Solubility of thermally precipitated collagen reduced withNaBYH4. The collagen gels reduced with NaB3H4 anddialyzed against 0.1 M acetic acid as described above were

centrifuged at 40,000 g for 1 h at 40, and the precipitateand the supernates were analyzed for hydroxyproline on an

amino acid analyzer.Clinical materials. 6-mm punch biopsies were obtained

from two patients (K. S. and K. J.) with documentedhomocystinuria through the cooperation of Dr. Ellen S.Kang of the Children's Hospital Medical Center, and from

2572 A. H. Kang and R. L. Trelstad

another patient (T. K.) by Dr. Richard Erbe of Massa-chusetts General Hospital, Harvard Medical School, Bos-ton, Mass. 10 age-matched control samples were obtainedfrom normal volunteers and surgical patients at the Chil-dren's Hospital Medical Center, Massachusetts GeneralHospital or the Veterans Administration Hospital, Mem-phis, Tennessee.

Samples of dermis were frozen in liquid nitrogen andpulverized into powder using a stainless steel cup andpestle that had been immersed in liquid nitrogen. Afterthawing, the samples were washed with cold distilled waterand suspended in 0.05 M Tris, pH 7.4 containing 0.16 MNaC1, and reduced with NaB3H4 as described above.

Aliquots of the powderized samples from two of thepatients (K. S. and K. J.) were also examined for solu-bility of collagen in nondenaturing solvents. Each samplewas extracted in the cold with 50 vol (volume to wet weightratio) of 1.0 M NaCI, 0.05 M Tris, pH 7.4 for 3 dayswith two changes and 0.5 M acetic acid for 6 days withtwo changes. The insoluble residue was separated from thesupernate by centrifugation at 40,000 g at 40 for 30 min.The amount of extracted collagen and insoluble collagenwas determined by hydroxyproline assay (29). Two controlsamples were similarly studied.

Amino acid analysis and scintillation spectrometry. Eachsample was equally divided and hydrolyzed separately inconstant boiling HCl in tubes sealed under an atmosphereof nitrogen and in 2 N NaOH for 24 h at 1080. Analyseswere performed on a Beckman 121 or Jeolco automaticamino acid analyzer equipped with a stream-split device asdescribed previously (16). The acid hydrolyzates, used fordetection and quantitation of the reduced intermolecularcross-links (hydroxylysinonorleucine, hydroxylysinohydroxy-norleucine, and histidino-hydroxymerodesmosine), were ana-lyzed using a modification of the buffer system describedby Hamilton (30). The alkaline hydrolyzates, used forquantitation of the precursor aldehyde (hydroxynorleucine)and the intramolecular cross-link (aldol condensationproduct) were analyzed using the gradient of Burns, Curtis,and Kaeser (31). A portion of the effluent was continuouslymonitored for ninhydrin reactivity, and the remainingportion was collected in fractions of 1 ml. Radioactivityof the individual fractions was determined in a liquidscintillation counter using Aquasol (New England Nuclear).

Electron microscopy. The ultrastructure of thermally pre-cipitated collagen samples was monitored by electron mi-croscopy. Small aliquots of each sample containing fibrilswere applied to grids coated with a colloidion film, stainedwith 2% uranyl acetate for 15 min and examined in anRCA EMU3 G electron microscope. In addition, aliquotsof each sample were fixed in glutaraldehyde, embedded inEpon (Fisher Scientific Co., Pittsburgh, Pa.) and examinedafter sectioning in the electron microscope.

RESULTS

Neutral salt solutions (0.05 M Tris-0.16 M NaCl, pH7.4) of collagen were incubated at 370 in the presenceof varying concentrations of homocysteine, homocystine,and methionine along with a control and a solution oflathyritic collagen to compare their capacity to forminsoluble native-type fibril aggregates. The results areshown in Fig. 1. Collagen solutions containing variousamounts of homocysteine or methionine up to the con-centration of 0.1 M, and 0.05 M in the case of homo-

500O

400t

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0 9nrn

1

--- 410-2M

-0----O----O5xl0O2M

'9,* CONTROL Gs* 10-1M METHIONINE '- __ ---o010l M

A 5x1(02M HOMOCYSTINEo HOMOCYSTEINE* LATHYRITIC

2 3-

HOURS DA2 3 4

HOURS

FIGURE 1 Thermal gelation of purified lathyritic and acid-extracted normal collagen and the effects of various aminoacids. Collagen solutions (1 mg/ml) in 0.05 M Tris-0.16M NaCl, pH 7.4 were warmed to 370 and the rate ofthermal aggregation was measured by the change in opacityin. a Klett calorimeter. Test solutions contained variousconcentrations of homocysteine (10-' M, 5 X 10-' M, or

10-'M), methionine (10-1 M) or homocystine (5 X 10-' M).After incubation at 370 for 24 h, the tubes were cooled to4° in a water bath and the changes in opacity were recorded.

cystine, gel rapidly at 37° and reach a maximum opacitywithin 1 h of incubation as does a solution of lathyriticcollagen. Concentrations of homocystine greater than0.05 M were not tested because of its insolubility.There are no significant differences in the maximalopacity attained by the various samples. After incuba-tion at 37° for 24 h, each sample was examined forreversibility of fibril formation upon cooling to 40 asdescribed by Gross (32). The fibrils from lathyriticcollagen, containing low content of the cross-link pre-cursor aldehydes, are rapidly redissolved under theseconditions reflecting its inability to form intermolecularcrosslinks (5). However, the acid-extracted normalcollagen does not show significant redissolution, indi-cating formation of stable intermolecular cross-linksduring the incubation period. The presence of 0.1 Mhomocysteine in the normal collagen solution almostcompletely prevents the insolubilization of the fibrils,suggesting interference by the amino acid with cross-link formation. At lower concentrations, partial inhibi-tion is noted. That this inhibition of insolubilizationis specific for homocysteine is shown by the fact thatboth homocystine and methionine have no effect.

Since it is well established that the in vitro collagencross-link formation requires the stereospecificity of thenative-type fibrils, it was important to determinewhether homocysteine interfered with the native-typefibril aggregation, as it had been previously shown thatcross-linking failed to occur in any aggregates other

Collagen Defect in Homocystinuria 2573

e-uurI

00-

n

K(

TABLE ISolubility of Collagen Fibrils Thermally Polymerized in the

Presence of Homocysteine, Homocystine, or Methionineand Subsequently Reduced with NaB3H4

Collagen solublein 0.1 M

Collagen sample acetic acid

- Acetic acid-extracted normal collagen 12Lathyritic collagen 89Acetic acid-extracted normal collagen

+0.1 Mhomocysteine 85+0.05 Mhomocysteine 40+0.01 Mhomocysteine 19+0.05 Mhomocystine 14+0.1 Mmethionine 13

than the native-type 640 A banded fibrils (11). Analiquot of the collagen aggregated in the presence of0.1 M homocysteine, therefore, was examined byelectron microscopy. All of the fibrils display thetypical band pattern with 640 A periodicity as do thecontrol samples. Thus, the effect of homocysteine wasnot mediated through nonspecific interference withnative-type fibril formation.

These observations were further substantiated by thequantitative analysis of the various cross-link com-pounds and solubility of the thermally precipitated gelsafter 24 h incubation and reduction with NaB'H4. Re-duction with NaB'H4 introduces one atom per mole oflion-exchangeable tritium into the aldehydes of allysineand the aldol condensation product and into the Schiffbase forms of hydroxylysinonorleucine, hydroxylysino-hydroxynorleucine, and histidino-hydroxymerodesmo-sine (16, 22-24) thus allowing quantitation of thesecompounds by radioactivity assay and at the same

time stabilizing them to subsequent analytic procedure.The solubility data, summarized in Table I, clearlyshow that homocysteine prevents insolubilization ofthe collagen fibrils even after subsequent borohydridereduction, implying that the stable cross-links are notformed. Lathyritic collagen, lacking the necessary func-tional (aldehyde) groups, is unable to form insolublefibrils. These results are confirmed by the direct assayof the cross-link compounds as presented in Table II.The amounts of the intermolecular cross-links gener-ated, hydroxylysinonorleucine, hydroxylysinohydroxy-norleucine, and histidino-hydroxymerodesmosine aremarkedly diminished by 0.1 M homocysteine.

In order to investigate the mechanism by whichhomocysteine prevents the formation of the intermolecu-lar cross-links, the effect of the amino acid on theintegrity of the precursor aldehyde functional groupson the tropocollagen was investigated. The hypothesisto be tested was whether the amino acid might interactwith the aldehydes in a manner analogous to that de-scribed for D-penicillamine (10) and as has been sug-gested for homocysteine (1, 7, 8). Acid-extracted nor-mal collagen, solubilized in 0.05 M Tris, pH 7.4, con-taining 0.16 M NaCl was incubated with homocysteineat room temperature along with appropriate controlsamples and was directly reduced with NaB3H4. Thereduced samples were then analyzed for the contentof e-hydroxynorleucine (reduction product of allysine)and the reduced aldol condensate after hydrolysis in 2N NaOH. The results are presented in Table III. Thesedata indicate that homocysteine in concentrations of0.01-0.1 M interacts in some manner with the aldehydegroups of allysine and the aldol residues. Apparently,this interaction is reversible, since the collagen samplesincubated with 0.1 M homocysteine at 370 for 24 h arecapable of forming insoluble gels after exhaustive di-

TABLE I IContent of the Cross-links Generated in the Collagen Fibrils during Incubation

with Homocysteine, Homocystine, and Methionine*

Hydroxylysino- Hydroxylysino- Histidino-Collagen sample hydroxynorleucine norleucine hydroxymerodesmosine

Acetic acid-extracted normal collagen 20 X 10' 55 X 10' 80 X 104Lathyritic collagen 3 X 10' 9 X 10' 12 X 104Acetic acid-extracted normal collagen

+0.1 M homocysteine 1 X 10' 12 X) 104 10 X 10'+0.05 M homocysteine 10 X 10' 27 X 104 42 X 10'+0.01 Mhomocysteine 15 X 104 40 X 101 69 X 10'+0.05 Mhomocystine 19 X 104 57 X 104 82 X 101+0.1 M methionine 22 X 10' 52 X 104 78 X 104

* Measured after reduction with NaB'H4 and expressed as total cpm per 10 mgof collagen. The amounts ofcollagen were calculated from amino acid analysis assuming 92 residues of hydroxyproline per 1,000residues of amino acids. The data obtained from 6 N HCOhydrolvzates of the NaB3H4 reduced samples.

2574 A. H. Kang and R. L. Trelstad

TABLE IIIEffects of Homocysteine, Homocystine, and Mlethionine on the

Cross-link Precursor Aldehydes*

Collagen samples e-hydroxynorleucine Reduced aldol

Acid-extracted normalcollagen 62 X 104 70 X 104

Lathyritic collagen 9 X 10. 10 X to;

Acid-extracted nornalcollagen

+0.1 M llioocXsteiile 2 X 10' 2 X 104+0.05 MI liomocysteine 30( X 10'( 32 X I W

+0.1)1 N Iomocysteiln' 51 X 1()' 59 ) 10'+0.05 M 11omocsstilne 65 X l0' 72 X 10'

-0.1 M methionine 59 X t0' 69 X 10'

* Data obtained from 2 N NaOH hydrolyzates of the NaB3H4 reducedcollagen. Expressed as total cpm per 10 mg of protein. The amounts ofprotein were calculated from amino acid analysis assuming 28 residues oflysine per 1,000 total residues, since hydroxyproline is partly destroyedduring hydrolysis.

alysis against 0.05 M Tris-0.16 M NaCI, pH 7.4, asshown in Fig. 2.

In view of these in vitro experiments indicating"lathyrogenic" effects of homocysteine on collagen, weconsidered the possibility that similar effects might beobserved in the tissues of patients with homocystinuria.Accordingly, dermal collagen of skin biopsies from pa-tients with homocystinuria and age-matched controlswas examined for the content of e-hydroxynorleucineand the cross-link compounds after reduction withNaB0H4. The results are tabulated in Table IV. Thecontent of both the precursor aldehyde, e-hydroxynor-leucine, and the cross-link compounds, hydroxylysino-norleucine, hydroxylysinohydroxynorleucine, and histi-dino-hydroxymerodesmosine are significantly decreased.These findings are consistent with the above in vitrodata suggesting collagen aldehyde-homocysteine inter-action. Decreased content of these compounds are alsoobserved in the sodium borohydride-reduced dermaltissues of human patients who had been treated withD-penicillamine (Kang, A. H., unpublished data).

<100200o

1

CONTROL01

o 1O-1M HOMOCYSTEINEo LATHYRITIC

0 3 4

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FIGURE 2 Reversibility of homocysteine effect on thermalaggregation of collagen by dialysis. After 24 h incubation at370, the collagen solution containing 10' M homocysteinewas dialyzed vs. the same buffer for 48 h at 40 with sev-eral changes. Lathyritic and control samples were kept at40 without dialysis. Note that following dialysis, the testsolution becomes insoluble upon incubation at 37°.

In addition, the solubility of dermal collagen in non-denaturing solvents (1 M NaCl and 0.5 M acetic acid)of two patients examined (K. S. and K. J.) is signifi-cantly increased as compared with the controls (TableV). These results are consistent with the increasedsolubility of dermal collagen and increased a: P ratio inhomocystinuria previously reported by Harris andSjoerdsma (6). It is of interest to note that the twoyounger patients (K. S. and K. J.) who both showgreater diminution in the cross-link content (Table IV)are vitamin Bo unresponsive patients. Patient T. K., whoshows a lesser decrease, is a vitamin Be responsivepatient and was on therapy at the time the biopsy wasobtained. However, the small number of samples doesnot allow any definitive conclusion in this regard.

TABLE IVContent of e-Hydroxynorleucine and Cross-link Compounds of Dermal Collagen in Homocystinuria*

e-Hydroxy- Hydroxylysino- Hydroxylysino- Histidino-Patient Age norleucine hydroxynorleucine norleucine hydroxymerodesmosine

K. S. 12 1.8 X 10' 3.0 X 10 12.3 X 103 10.9 X 103K. J. 9 1.0 X 10: 2.0 X 103 8.9 X 103 7.4 X 103T. K. 27 4.0 X 10? 7.5 X 10' 21.3 X 103 18.0 X 103Control 9-12 5.5 X 10: 20.0 X 10 26.9 X 10i 30.2 X 10Control 20-30 9.0 X 103 19.1 X 10: 32.0 X 10' 39.4 X 103

* Measured after reduction with NaB3H4 and expressed as cpm per micromole of hydroxyproline.The data for hydroxyly sinohy droxynorleucine, hydroxylysinonorleucine, and histidino-hydroxymero-desmosine were obtained from 6 N HCI hydrolyzates and e-hydroxynorleucine from 2 N NaOHhydrolyzates.

Collagen Defect in Homocystinuria 2575

TABLE VSolubility of dermal collagen* in Two Subjects

with Homocystinuria

Patient Age Extracted

K. S. 12 7.8K. J. 9 10.1Controls (2) 11 2.4, 2.9

* Combined 1.0 MNaCl and 0.5 M acetic acid extracts.

DISCUSSIONThe unusual mechanical stability of collagen fibrils andthe collagenous tissues is largely dependent on the for-mation of covalent intermolecular cross-links. The criti-cal importance of these cross-links is best exem-plified by the dramatic functional failure of connec-tive tissues observed in experimental lathyrism (3, 4), astate that can be induced by administration of lathyro-gens such as P-aminopropionitrile and in which the af-fected animals display a variety of connective tissue ab-normalities including marked skeletal deformities suchas kyphoscoliosis and curvature of long bones and vas-cular abnormalities among others. Recent investigationsfrom several laboratories (33-35) have established thatP-aminopropionitrile acts by inhibiting the lysyl oxidaseinvolved in the biosynthesis of allysine (and presumablyhydroxylysine-derived aldehyde as well), the cardinal ini-tial reaction in a series of steps leading to cross-linkformation. Thus, lathyritic collagen is deficient in thecross-link precursor, allysine, and is unable to form stableintermolecular cross-links.

Recent studies have revealed another class of lathyro-genic compounds, best understood in the case of D-penicil-lamine, which exerts its effect at a different step in thebiosynthesis of collagen cross-links (9, 10). D-penicilla-mine does not interfere with the allysine formation butrather acts by binding to the aldehydic functional groups,preventing further reaction to form the cross-links (9,10).

The remarkable similarities in some of the clinicalmanifestations of lathyrism to those observed in homo-cystinuria and the structural similarity of D-penicillamineto homocysteine have led some authors (1, 6-8) to sug-gest the possibility that homocysteine might produce de-fects in collagen cross-linking in homocystinurics in amanner analogous to D-penicillamine and stimulated usto undertake the present investigation. We have shownthat only homocysteine but not homocystine and methio-nine, also increased in the plasma of the patients, inter-feres with stable intermolecular cross-link formation byinteraction with aldehydic groups; they are thus pre-vented from participating in the cross-linking reactions

as measured by the in vitro gelation method, by thereversible solubility of the "aged" and borohydridereduced gels, and by the direct assay of the precursoraldehydes and the cross-links generated during in vitroincubation. Although the concentrations of homocysteineused in our in vitro studies, 0.01 M-0.1 M, are clearlyhigher than the levels observed in the patient's plasma,the increased solubility of collagen in non-denaturingsolvents and the decreased content of the collagen cross-link precursor aldehyde and the cross-link compoundsobserved in the skin biopsies from the patients suggeststrongly that our interpretation for the mechanism ofreaction is correct.

The higher concentration of homocysteine requiredin our in vitro experiments should not necessarily in-validate the proposed mechanism, since a similar dis-parity between in vitro and in vivo studies with D-peni-cillamine also has been observed. It has been reportedthat a D-penicillamine concentration of 0.4 mg/ml is re-quired for any discernible effect on allysine of elastinin vitro (36). In our laboratory, using an identical invitro system as described in this paper, the minimumconcentration of D-penicillamine required for a definitelydiscernible effect on allysine of collagen is 0.5 mg/ml(Kang, A. H., and C. Franzblau, unpublished data).On the other hand, human patients treated with D-peni-cillamine at a dose of 2 g/day have been reported toshow an increased solubility and a decreased content ofP-components of collagen (37). Although the plasmaconcentration of D-penicillamine in these patients werenot reported, it would seem reasonable to suggest thatthe plasma concentration must be considerably lowerthan 0.5 mg/ml required for an in vitro effect. A plausibleexplanation of this disparity would be that, since totalcollagen turnover is very slow, a prolonged exposure ofcollagen to smaller concentrations of D-penicillamine orhomocysteine as in the patient may produce a morecumulative effect than that which can be reproduced inthe relatively acute experimental conditions in vitro.This possibility is further supported by our preliminaryobservation that the solubility of collagen of experi-mental rats that had been administered homocysteinein their diet for several weeks is significantly increaseddespite the fact that the plasma homocysteine levelachieved in these animals is lower than that commonlyobserved in human patients (Kang, A. H. unpublisheddata).

It should be noted that the apparently decreasedamount of measurable e-hydroxynorleucine after directsodium borohydride reduction of native collagenous tis-sues does not necessarily indicate a true diminution in thecontent of allysine per se but that it may be a result ofbinding of the aldehydic group by other compounds.Thus, analysis for e-hydroxynorleucine after sodium

2576 A. H. Kang and R. L. Trelstad

borohydride reduction of dermal collagenous tissue fromhuman patients and the laboratory animals such aschicks that had been administered D-penicillamine showslow content of the compound (Kang, A. H. unpublisheddata). However, the measurable content of e-hydroxy-norleucine is high if collagen present in such tissues isfirst extracted and purified since the D-penicillaminebinding to collagen is reversible and it is removed duringthe purification procedure (9-11). Unfortunately, dueto the limited amount of patient material available it wasnot possible for us to analyze for the allysine contentof homocystinuric collagen after extraction and purifi-cation. However, the diminution of measurable e-hy-droxynorleucine in the patient samples reduced directlywith borohydride as compared with the controls (TableIV) does suggest an interaction between the aldehydeand homocysteine in vivo. Considerations of chemicalstructures of the known collagen lysyl oxidase inhibitors,such as P-aminopropionitrile on the one hand and theaminothiol lathyrogens that act via direct interactionwith collagen aldehyde such as D-penicillamine on theother hand would also support such a mechanism ofaction by homocysteine.

The solubility of collagen of homocystinurics in non-denaturing solvents obtained in the present study is in-creased as compared to controls. Harris and Sjoerdsma(6) previously reported also increased solubility of col-lagen and increased a: , ratio in solubilized collagen,implying a defect in the "intra"-molecular cross-linkor the aldol cross-link formation. However, it was previ-ously shown that in the native type fibrils such as invivo tissues, the aldol cross-link exists only as a partof an intermolecular cross-link, the post-histidine com-pound (recently shown to be histidino-hydroxymerodes-mosine) and not as a separatae entity (11, 16, 23, 24). Inthis sense, the "intra"-molecular cross-link is only a re-sult of extraction and solubilization of collagen and re-flects the intermolecular cross-link, histidino-hydroxy-merodesmosine of which it is a structural component.Thus, the diminished P-content (or the aldol conden-sate) in the solubilized, homocystinuric collagen is con-

sistent with the diminished content of histidino-hydroxy-merodesmosine in the native-type tissue observed in thepresent investigation (Table IV).

Although the precise relationship between the solu-bility of collagen in non-denaturing solvents and theSchiff base type of cross-links has not been clearlyestablished, there is a reasonable amount of evidencethat implicates cross-linking as one of the factors in in-fluencing the collagen solubility. This includes the in-creased solubility in lathyrism vis-a-vi§ the deficientcross-linking as recently reviewed by Bornstein (34),the increased solubility observed in experimental animalsas well as humans after D-penicillamine administration

0/1 +

NORMALCOLLAGEN

HS-C2 S- CH

,CH2 CH /CH2H2N-CH J NH-CH

\ COOH 'C00H

HOMOCYSTEINE COLLAGEN-HOMOCYSTEINECOMPLEX

FIGURE 3 A mechanism proposed for the mode by whichhomocysteine acts to interfere with collagen cross-linking.

with defective cross-linking (9, 10, 37), and the in vitrostudies of Tanzer (38) relating the cross-linking andthe solubility.

The relatively easy removal of homocysteine fromcollagen upon dialysis (Fig. 2) implies that the inter-acting forces are not strong. At the present time, theprecise chemical nature of this interaction is not known.By analogy with the mechanism proposed for D-penicil-lamine, a possible mechanism is postulated in Fig. 3.It is known that compounds with suitably proximatethiol and amino groups will readily form a complex withaldehydes (39). Various cations, pH and the natureof the media as well as the nature of the ring substituentswill shift the equilibrium of the reaction (40). Underappropriate conditions, the ring structure postulated forthis interaction can dissociate to regenerate normalcollagen with reactive aldehydes and the amino acid.

The reversibility of the homocysteine effect on thecollagen molecule in the in vitro experiments also sug-gests that such might be the case as well in vivo. If thepatients could be maintained at a very low or negligiblelevel of homocysteinemia by therapeutic means such aswith vitamin Be and dietary therapy, one would expectthe severity of the collagen defect to diminish. A longi-tudinal study involving newly diagnosed vitamin B. re-sponsive patients before and after therapy would behighly informative.

ACKNOWLEDGMENTSThe authors are grateful to Dr. J. Gross for his supportduring the investigation, and to Miss Karen Lawley, Mrs.Joanne Hazard, and Mrs. Margaret Cirtain for expert tech-nical assistance. This work was supported by grants fromthe U. S. Public Health Service (AM 3564, AM 16506)and a Medical Investigatorship of the Veterans Adminis-tration. Hydroxylysinohydroxynorleucine was a generousgift of Dr. Gerald Mechanic.

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2578 A. H. Kang and R. L. Trelstad