Importance of homocysteine-induced abnormalities of proteoglycan ...

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Jan 15, 1970 - Address for reprint requests: Dr. Kilmer S. McCully, Department ..... HARRIS ED JR, SJOERDSMA A: Collagen profile in various clinical ...
Importance of Homocysteine-lnduced Abnormalities of Proteoglycan Structure in Arteriosclerosis Kilmer S. McCully, MD

HOMOCYSTEINEMIA was found to be the key factor leading to arterial damage antI accelerated arteriosclerosis in two individuals, each with a different enzymatic disorder of sulfur amino acid metabolism.' In one of these individuals, cystathionine synthetase was deficient, and homocysteinemia resulted from a block in the conversion of homocysteine to cystathionine In addition to accelerated arteriosclerosis and mental retardation, individuals with cystathionine synthetase deficiency are affected also by widespread connective tissue abnormalities, including ectopia lentis, genu valgum, pectus carinatum, arachnodactyly, osteoporosis and inguinal hernia.3'4 In the second individual with accelerated arteriosclerosis, reduced 5N methyltetrahy-drofolate homocysteine methyl transferase activity led to the homocysteinemia, presumably because methylation of homocysteine to methionine occurred at a reduced rate.5 The nature of the metabolic and macromolecular abnormalities produced by homocysteinemia, which lead to arterial damage and connective tissue abnormalities in these syndromes, has not been explained adequately at a molecular level. In this report, observations are described concerning the effect of homocysteine on the structure of proteoglyeans synthesized by cultured human skin cells. Cells cultured from two cystathionine synthetasedeficient individuals, from two different families, were found to synthesize abnormal proteoglyeans that are granular, aggregated and flocculent. This abnormality resulted in marked distortion and destruction of the normal fibrillar structure and was also produced in the proteoglycan synthesized by normal cultured cells by adding homocysteine to the culture medium. Analysis of the cultured cell monolayer protein showed that only traces of hydroxyproline are synthesized by normal cells and that the amino acid composition of these cells resembles that of connective tissue proteoglycan. The results are interpreted to indicate that elevated concentrations of endogenous or exogenous homo.

From the Department of Pathology, Harvard Medical School, and the James Homer Wright Pathology Laboratories, Massachusetts General Hospital, Boston. Supported by Grant-in-Aid 924 from the Greater Boston Chapter of the Massachusetts Heart Association and by Grant CA11119 from the US Public Health Service. Accepted for publication Jan 15, 1970. Address for reprint requests: Dr. Kilmer S. McCully, Department of Pathology, Massachusetts General Hospital, Boston, Mass 02114. 181

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cysteine produce pathologic changes in the arteries and other connective tissues by altering the state of aggregation and normal fibrillar structure of proteoglycan molecules. The key role of the macromolecular abnormality produced by homocysteine in the pathogenesis of arteriosclerosis is discussed. This interpretation suggested therapeutic measures for the prevention of arteriosclerotic lesions. Methods Cultures of Human Skin Cells

Fragments of dermis were obtained from a skin biopsy of a 9-year-old, mentally retarded female with documented cystathionine synthetase deficiency and homocystinuria. A serially cultivated cell line, 49 HC, was obtained as an outgrowth of cells from the dermis fragments. The child is the niece of a previously studied patient with presumed cystathionine synthetase deficiency.1'6 A second cell line, 21 HC, obtained from a 27-year-old-male with homocystinuria, and 2 normal cell lines, 26 JL and 120 JL, obtained from a three-year-old female and a 20-year-old male were cultivated in the same way. After the cell lines were established, vials containing cell suspensions were stored in liquid nitrogen. After growth from frozen vials, the 4 cell lines were each passaged 1-3 times prior to use. Cell lines were serially cultivated at 37°C in a 5% CO2 humidified atmosphere in Falcon petri dishes containing Eagle's minimal essential medium F-li or F-15 with 10% fetal calf serum (Gibco). The lines were passaged every 1-3 weeks by trypsinization and growth in fresh medium. Cultures were refed routinely with fresh medium twice weekly. Homocysteine, ascorbic acid, 14C proline and other compounds were dissolved in medium, filtered, and stored at 40C before they were added to the various experimental cultures. To visualize cellular and extracellular material, the monolayer was rinsed with phosphate buffered saline, fixed for 10 min in 10% buffered formalin, pH 7.0, rinsed' with distilled water, and stained with Giemsa or toluidine blue. To demonstrate birefringence, fragments of monolayer were scraped into a small amount of phosphate buffered saline, mounted under a coverslip, and examined under polarized light. Chemical Analysis of Cell Monolayers

For labeling of proline and hydroxyproline, confluent monolayers, refed for an additional 2 weeks with F-15 medium containing 1 mM pyruvate, were incubated for 4 days in F-11 medium containing 0.1 g mol/ml L-proline, 5.30 x 105 cpm/t mol, with or without added ascorbic acid, 50 it g/ml. The monolayers were prepared for analysis by removing the medium, rinsing twice .with cold (0-4°C) phosphate buffered saline (PBS), pH 7.0, and scraping the entire monolayer into cold PBS. The suspension was homogenized thoroughly in a glass hand homogenizer, and protein was determined on aliquots of the homogenate by the Lowry method.7 Comparison of the results of protein determination of trypsinized, washed cells with the results for scraped monolayers led to the conclusion that a large proportion of the protein determined in the homogenates of the scraped monolayer was either extracellular or lost during trypsinization. Aliquots of monolayer homogenates were dialysed against cold phosphate buffered saline and hydrolyzed in 6N HCL at 110°C for 22 hr. Amino acids were separated and estimated according to the method of Moore et al,8 using a 60 cm Aminex A-6 (Bio Rad) column at 500C, pH 3.25 and pH 4.25 or by gradient elution, pH 2.875 to pH 5.0. Radioactivity was estimated in the Packard liquid scintillation counter using Bray's counting solution.

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Results

Cells from the patients with cystathionine synthetase deficiency, 49 HC and 21 HC, consistently produced moderate amounts of extracellular and intracellular material which formed aggregates and granules of varying size, frequently aligned along cellular processes and extracellular appearing fibrils but also scattered between the cells (Fig 1). The material was light red when stained with Giemsa but metachromatic red-violet when stained with toluidine blue. Reduced numbers of fibrils of normal morphology were present either in confluent areas or in sparsely populated areas. The abnormal material was produced throughout the growth cycle. When homocysteine or homocysteine thiolactone was added to the culture medium, larger amounts of the abnormal substance formed. In addition, comparison with control cultures revealed that the total number of cells in the monolayer was decreased after 1 week's growth when dl-homocysteine 1 ,umol/ml was added to the medium. The addition of dl-homocysteine, in concentrations varying from 0.2 to 2.0 jimol/ml, to the medium did not, however, alter the cloning efficiency (26%) of the cystathionine synthetase deficient cells. In cultures with homocysteine in the medium, some of the cells were found to contain small, uniform sized cytoplasmic granules, less than 1 [t in diameter, frequently distributed in a short linear array at the margin of the cytoplasm, within the cytoplasm, and extending along extracellular appearing fibrils (Fig 2). Cells from normal patients formed large numbers of long extracellular and intracellular fibrils of varying length and diameter without evidence of granular aggregated material (Fig 3). Addition of homocysteine or homocysteine thiolactone to the medium, however, resulted in the formation of moderate amounts of light red-staining granular flocculent substance and small amounts of fine granular material associated with fibrils (Fig 4). Examination of the confluent monolayer of normal cells, by polarized light, revealed numerous, long, moderately birefringent fibrils (Fig 5). However, only small numbers of short, irregular, clumped birefringent fibrils were observed in the confluent monolayer of cystathionine synthetase deficient cells (Fig 6). In order to determine the amount of hydroxyproline synthesized within the confluent cell monolayer, normal cells were cultivated according to the method of Schafer et all9 and exposed to medium containing 14C proline for 4 days. The proteins of the monolayer were hydrolyzed, and the resulting amino acids were chromatographed to determine the amount of 14C present in proline, hydroxyproline, and the other amino acids. As demonstrated by the data in Table 1, essen-

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Table 1. Incorporation of 1'C Proline in Human Skin Monolayer Cultures Recovery of Conversion of 14C in aliquot 14C as proline 14C proline to of monolayer l4C recovered '4C recovered as and 14C hydroxyhydrolyzate as proline hydroxyproline hydroxyproline proline Samples* (cpm) (cpm) (cpm) (90 (%) 1.69 X 104 1.78 X 104 0.039 X 104 97 2.3 A1 2.75 X 104 2.73 X 104 .060 X 104 101 2.2 A2 2.34 X 104 2.41 X 104 .058 X 104 105 2.3 A3T 1.71 X 104 1.74 X 104 .040 X 104 104 2.2 A3D 2.42 X 104 2.24 X 104 .061 X 104 95 B, 2.7 2.10 X 104 2.11 X 104 .058 X 104 103 2.7 B2 * Samples B, and B2 contained ascorbic acid, 50 ,Ag/ml, during the entire period of culture. Sample A3D homogenate was dialyzed against PBS before hydrolysis.

tially all of the 14C in the hydrolyzate was recovered as proline and hydroxyproline. A very small fraction (2.2-2.3%) of the total 14C was recovered as hydroxyproline, and a slight (2.7%) increase in the recovery of 14C hydroxyproline was observed when the medium also contained ascorbic acid, 50 itg/ml. No ninhydrin-positive material was eluted at the position expected fot hydroxyproline. However, since bound proline in peptide linkage is the precursor of hydroxyproline,'0 the amount of hydroxyproline synthesized in the monolayer can be estimated, assuming the specific activity of the recovered 14C hydroxyproline to be equal to that determined for the recovered 14C proline. For sample B2 the result of the calculation is 0.018 imol hydroxyproline/mg total protein of monolayer, approximately one-third of the amount found by Schafer et al,9 using a different method to determine hydroxyproline. The composition of the proteins of the confluent monolayer of normal human skin cells, 26 JL, was determined by column chromatography of the hydrolyzates of the samples summarized in Table 1. A representative analysis is presented in Table 2, which sets forth the amino acid composition of the A2 monolayer. No significant differences were found in the composition data for samples dialysed before hydrolysis or for samples from ascorbic acid supplemented cultures. Analysis demonstrated that monolayer proteins are very rich in acidic and basic amino acids, aspartic acid, glutamic acid, lysine, histidine and arginine. Abundant threonine, serine and phenylalanine were present, and only small amounts of cystine and methionine were found. The content of hydroxyproline, estimated from the '4C proline incorporation experiment in Table 1, is very low. No ninhydrin-positive material was eluted in the positions expected for hydroxylysine, desmosine or isodesmosine."

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Table 2. Amino Acid Composition of Monolayer Culture of Human Skin Cells Amino g/100 g protein acid

aspartic acid threonine serine proline (hydroxyproline)* glutamic acid glycine alanine valine cystine methionine isoleucine leucine tyrosine phenylalanine lysine histidine arginine TOTAL

9.5 6.7 5.7 6.9 (0.15) 16.5 6.8 4.6 4.5 1.7 0.5 4.8 8.6 2.9 4.9 7.7 3.4 7.7 103.6

The composition data are referred to total protein, determined by the Lowry method.7 * Hydroxyproline content was calculated from recovered 14C hydroxyproline, assuming the specific activity is equal to that of recovered 14C proline.

Discussion and Conclusions

The demonstration that cultured cystathionine synthetase deficient cells 12 elaborate a morphologically abnormal intracellular and extracellular proteoglycan matrix is a finding with importance both for arteriosclerosis and for the other connective tissue abnormalities associated with homocystinuria. A prominent feature of arterial changes found in cystathionine synthetase deficiency is the accumulation of metachromatic material in the media of the aorta and arteries,'3 presumably consisting of abnormal granular fragmented material elaborated by vessel wall cells similar to that observed in cultured cells (Fig 1). The altered solubility of the glycoprotein and proteoglycan fractions isolated from the aorta of a patient with homocystinuria 1 is probably the result of an altered state of aggregation and molecular conformation of proteoglycans synthesized by aortic cells. The ectopia lentis commonly observed in the syndrome was found to result from replacement of the normal fibrillar structure of the zonular fibers by irregular, granular material.'5 This morphologic change is very similar to the replacement of fibrils by granular fragmented material observed in the cultured cells (Fig 1). The findings of increased amounts of

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soluble collagen and an increase in the amount of noncross-linked collagen in two individuals with homocystinuria,16 as well as some features of the various connective tissue abnormalities in the syndrome, are presumably due to partial inhibition of normal collagen crosslinking by abnormal proteoglycans elaborated by the cystathionine synthetase deficient cells of connective tissues. Investigation of the amino acid composition and 14C hydroxyproline synthesis from '4C proline in the cultured monolayer was undertaken to define the possible molecular basis for the morphologic abnormalities observed in the cultured cells. The very low hydroxyproline content found in the monolayer (Table 1) excludes the possibility that collagen fibrils were present in significant numbers. Therefore, the abnormalities of fibrils of cultured cells resulting from excess concentrations of endogenous (Fig 1) or exogenous (Fig 4) homocysteine are not due to abnormalities in collagen synthesis or collagen cross-linking within the cultured monolayer. The amino acid composition of the cultured monolayer (Table 2) is very different from that of purified human skin collagen 17 or purified mammalian elastin 18 but is very similar to that reported for the noncollagenous protein fraction isolated from various connective tissues, such as aorta, bone, cartilage, skin or ligament.19-2' Although the analysis in Table 2 reflects to a considerable degree the amino acid content of the abundant intracellular and extracellular fibrils observed morphologically, amino acids from proteins of other cellular constituents are also present in the monolayer hydrolyzate. In a study of extracellular fibrils produced by fetal rat fibroblasts in culture, Daniel et al22 found that the isolated fibrils contained appreciable amounts of hydroxyproline. However, the cells used in their study were isolated by trypsinization of embryonic skin, and the human skin cells used in the present study were obtained as spontaneous outgrowths from skin biopsy fragments. The present findings of trace amounts of hydroxyproline (Table 1) and the similarity of the amino acid composition of the cell monolayer (Table 2) to connective tissue proteoglycans suggest that very few cells obtained as spontaneous outgrowths from dermis fragments are capable of forming collagen under the conditions of culture, that most of the cells are probably derived from poorly differentiated mesenchymal cells or from endothelial cells, and that the cultured cells synthesize significant quantities of proteoglycans. The proteoglycan fractions from connective tissue contain appreciable amounts of polysaccharide and hexosamine, depending on the tissue and method of preparation. Cultured human skin cell monolayers, similar to those used in the present study, also contain

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appreciable amounts of sulfated and nonsulfated glycosaminoglycans.9 The nature of the structural changes at a molecular level which are responsible for the aggregation and fragmentation of extracellular and intracellular fibrillar material of the cultured cells, either produced by increased exogenous homocysteine in the medium (Fig 4) or by increased endogenous homocysteine as a result of cystathionine synthetase deficiency (Fig 1), cannot be defined with certainty from the data presented. It is unlikely that the observed changes are due to an effect on the primary polypeptide structure of the proteoglycans, since homocysteine is not a peptide-bound constituent of proteins and since both methionine and cystine, found in small amounts in the cultured monolayer (Table 2), are both present in adequate concentrations in the culture medium. The glycosaminoglycans of human skin cultures do, however, contain appreciable amounts of esterified sulfate.9'm If increased homocysteine concentration within the cell produced a change in the number or distribution of esterified sulfate groups on the carbohydrate side chains of the proteoglycan molecules synthesized by the cell, a drastic change in the conformation of the macromolecule at physiologic pH might be expected to result from the consequent alteration in the ratio of charge on polypeptide core to charge on surrounding carbohydrate envelope. Purification of the proteoglycans of cultured cells and further studies of the function of homocysteine as an intermediate in sulfate esterification are needed to confirm this preliminary formulation of the molecular basis for the homocysteine effect on proteoglycan structure. The finding that the addition of homocysteine to the culture medium of normal cells produced proteoglycan abnormalities (Fig 4) similar to those found in cystathionine synthetase-deficient cell cultures (Fig 1) suggests that elevated concentrations of exogenous homocysteine are capable of producing arteriosclerotic and connective tissue changes in vivo in normal individuals without enzyme deficiencies. In addition, any type of metabolic abnormality that elevates or depresses concentrations of endogenous homocysteine within tissue cells may predispose to or protect from, respectively, the pathologic changes in vessels and connective tissues associated with homocysteinemia 1,13 because of the consequent increased or decreased production of abnormal proteoglycans. For example, the well known protection against arteriosclerosis found in severe chronic liver disease, such as cirrhosis, can be explained by the presumed low endogenous homocysteine concentration which results from the failure of methionine demethylation and the excretion of excess methionine sulfur as methyl mercaptan.24

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The homocysteine effect on cellular proteoglycan synthesis is considered to be a key factor in the initiation of arteriosclerotic lesions because several lines of evidence from experimental and pathologic literature show that changes in sulfate esterified proteoglycans are an essential feature of the evolution of the arteriosclerotic process. Virchow first observed the accumulation of metachromatic mucoid substance in early arteriosclerotic lesions,25 and, in a recent study, the mucoid change was found to occur at an earlier age than lipid deposition in the evolution of coronary arteriosclerosis.26 Increased amounts of metachromatic substance and a very active uptake of 35S sulfate are found in cellular areas of arteriosclerotic lesions at all stages of development.27 Serum ,8-lipoprotein, which is associated with a predisposition to arteriosclerosis when present in elevated concentration, is known to bind to proteoglycans isolated from aorta.28 Sulfated proteoglycans of abnormal macromolecular configuration resulting from homocysteinemia may interact with 0-lipoprotein from serum to stimulate further synthesis of proteoglycans by vascular cells. If this interpretation of the pathogenesis of arteriosclerosis is correct, a promising approach to the prevention of progressive arteriosclerotic lesions would be restriction of dietary methionine, administration of choline and pyridoxine,2930 or other similar measures to reduce concentrations of endogenous and exogenous homocysteine in vascular cells. Summary Cells cultured from the skin of 2 individuals with cystathionine synthetase deficiency have been discovered to synthesize abnormal proteoglycans that are granular, aggregated and flocculent. This abnormality resulted in marked distortion of the normal fibrillar structure and has been produced in cultured normal cells by adding homocysteine to the culture medium. The amino acid composition of normal cell cultures has been found to resemble that of connective tissue proteoglycan; only traces of the hydroxylation of proline were found to occur in the cultured cell protein. The results are interpreted to indicate that elevated concentrations of endogenous or exogenous homocysteine produce pathologic changes in arteries and other connective tissues by altering the state of aggregation and the normal fibrillar structure of proteoglycan molecules. A possible molecular basis for the homocysteine-induced alterations in the structural conformation of proteoglycan is discussed. Interpretation of the findings suggested therapeutic measures for the prevention of arteriosclerotic lesions.

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References 1.

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Vascular pathology of homocysteinemia: Implications for the pathogenesis of arteriosclerosis. Amer J Path 56:111, 1969. MUDD SH, FINKLESTEIN JD, IRREVERE F, LASTER L: Homocystinuria: An enzymatic defect. Science 143:1443, 1964. CARSON NAJ, CUSWORTH DC, DENT CE, FIELD CMB, NEILL DW, WESTALL RD: Homocystinuria, a new inborn error of metabolism associated with mental deficiency. Arch Dis Child 38:425, 1963. SCHIMKE RN, McKusICK VA, HUANG T, POLLACK AD: Homocystinuria. Studies of 20 families with}38 affected members. JAMA 193:711, 1965. MUDD SH, LEVY HL, ABELES RH: A derangement in B12 metabolism leading to homocystinuria, cystathioninuria and methyl malonic aciduria. Biochem Biophys Res Comm 35:121, 1969. Case Records of the Massachusetts General Hospital. Marked cerebral symptoms following a limp of three months' duration. Case 19471. New Eng J Med 209:1063, 1933. LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265, 1951. MOORE S, SPACKMAN DH, STEIN WH: Chromatography of amino acids on sulfonated polystyrene resins. Anal Chem 30:1185, 1958. SCHAFER IA, SILVERMAN L, SULLIVAN JC, ROBERTSON W VAN B: Ascorbic acid deficiency in cultured human fibroblasts. J Cell Biol 34:83, 1967. PROCKOP DJ, JUVA K: Synthesis of hydroxyproline in vitro by the hydroxylation of proline in a precursor of collagen. Proc Nat Acad Sci 53:661, 1965. THOMAS J, ELSDEN DF, PARTRIDGE SM: Partial structure of two major degradation products from the cross-linkages in elastin. Nature (London) 200:651, 1963. UHLENDORF BW, MUDD SH: Cystathionine synthase in tissue culture

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derived from human skin: Enzyme defect in homocystinuria: Clinical and

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pathological review of ten cases. Science 160:1007, 1968. GIBSON JB, CARSON NAJ, NEILL DW: Pathological findings in homocystinuria. I Clin Path 17:427, 1964. CARSON NAJ, DENT CE, FIELD CMB, GAULL GE: Homocystinuria. J Pediat 66:565, 1965. HENKIND P, ASHTON N: Ocular pathology in homocystinuria. Trans Ophthal Soc UK 85:21, 1965. HARRIS ED JR, SJOERDSMA A: Collagen profile in various clinical conditions.

Lancet 2:707, 1966. 17. FLEISCHMAJER R, FISHMAN L: Amino acid composition of human dermal collagen. Nature (London) 205:264, 1965. 18. GOTTE L, STERN P, ELSDEN DF, PARTRIDGE SM: Composition of elastin from three bovine tissues. Biochem J 87:344, 1963. 19. PARTRIDGE SM, DAvIS HF, ADAIR GS: The constitution of the chondroitin sulphate-protein complex in cartilage. Biochem 1 79:15, 1961. 20. CLEARY EG, SANDBERG LB, JACKSON DS: The changes in chemical composition during development of the bovine nuchal ligament. J Cell Biol 33:469, 1967. 21. EASTOE JE, EASTOE B: The organic constituents of mammalin compact bone. Biochem J 57:453, 1954.

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22. DANIEL MR, DINGLE JT, Lucy JA: Cobalt tolerance and mucopolysaccharide production in rat dermal fibroblasts in culture. Exp Cell Res 24: 88, 1961. 23. GROSSFELD H, MEYER K, GOODMAN G, LINKER A: Mucopolysaccharides produced in tissue culture. J Biophys Biochem Cytol 3:391, 1957. 24. CHALLENGER F, WALSHE JM: Methyl mercaptan in relation to foetor hepaticus. Biochem J 59:372, 1955. 25. VIRCHow R: Gesammelte Abhandlungen zur Wissenschaftlichen, Medicin. Frankfurt-am-Main, 1856, p 458. 26. OsBoRN CR: The Incubation Period of Coronary Thrombosis. Butterworths, London, 1963, p 18. 27. CURRAN RC, CRANE WAJ: Mucopolysaccharides in the atheromatous aorta. J Path Bact 84:405, 1962. 28. GERO S, GERGELEY J, DEVENYI T, JAXAB L, SZEKELY J, VIRAG S: Role of intimal mucoid substances in the pathogenesis of atherosclerosis. I. Complex formation in vitro between mucopolysaccharides from atherosclerotic aortic intimas and plasma ,-lipoprotein and fibrinogen. J Athero Res 1: 67, 1961. 29. PERRY TL, HANSEN S, LOVE DL, CRAWFORD LE, TISCHLER B: Treatment of homocystinuria with a low methionine diet, supplemental cystine and a methyl donor. Lancet 474, 1968. 30. BARBER GW, SPAETH GL: Pyridoxine therapy in homocystinuria (abst). Lancet 1:337, 1967. The advice, encouragement, and samples of the human cell lines used in this study from Dr. J. W. Littlefield and the assistance and advice of Dr. V. E. Shih in performing the amino acid chromatography are gratefully acknowledged. The excellent technical assistance of R. Corrigan and M. Goldmann is also acknowledged.

Legends for Figures Fig 1. (upper) Cystathionine synthetase-deficient cells, 49 HC, cultured in F-11 medium for 7 days, produced granular material between cells and aligned along cell processes and fibrils. Giemsa. X 150. Fig 2. (lower) Cystathionine synthetase-deficient cells, 49 HC, cultured for 7 days with dI-homocysteine, 1.0 atmol/ml, in F-li medium, produced abundant granular material and intracytoplasmic granules aligned along cell processes and at margin of cytoplasm. Giemsa. X 600.

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Fig 3. (upper) Normal cells, 26 JL, cultured for 7 days in F-11 medium, produced fine fibrillar material varying in diameter located between cells and within cell cytoplasm. Giemsa. X 600. Fig 4. (lower) Normal cells, 26 JL, cultured for 7 days in F-11 medium with dl-homocysteine, 1.0 gmol/ml, produced granular material aligned along cell processes, between cells and within cell cytoplasm. Giemsa. X 600.

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Fig 5. (upper) Birefringent fibrils within monolayer of normal cells, 26 JL, cultured for 2 weeks in F-11 medium, are numerous, long, and parallel. X 10. Fig 6. (lower) Birefringent fibrils within monolayer of cystathionine synthetase-deficient cells, 49 HC, cultured for 2 weeks in F-11 medium are short, irregular, clumped, and fewer than in normal cell monolayer. X 10.

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American Journal of Pathology