Growth Disorders and Homocysteine Metabolism*

KILMER S. McCULLY, M.D.

Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114

ABSTRACT Inherited disorders of homocysteine metabolism produce accelerated growth and arteriosclerosis with myointimal hyperplasia. The growth of cell cultures from cystathionine synthetase deficient inidividuals with homocystinuria is characterized by abnormal contact inhibition and production of an aggregated proteoglycan matrix which binds excess sulfate. Homocysteic acid, a precursor of sulfate ester, increases the growth rate of normal guinea pigs. Synthesis of homocysteic acid from homocysteine thiolactone is more rapid in the livers of young animals than adults, and hypophysectomy results in a pattern of homo cysteine thiolactone metabolism resembling that in liver of adult animals. Homocysteine thiolactone metabolism differs in guinea pig, an herbivorous species, and in rat, an omnivorous species. Sulfate binding by cultured human cells is slightly increased when homocysteic acid is present in the culture medium. These observations suggest a relationship between homocysteic acid and somatomedin, a serum polypeptide which mediates the action of growth hormone. The growth disorders associated with homocystinuria, including arteriosclerosis and accelerated growth, are believed to result from increased conversion of methionine to homocysteine thiolactone and homocysteic acid.

Introduction mal hyperplasia and arteriosclerosis are Homocystine excretion was discovered found in arteries and arterioles of individ in mentally retarded children2’6 with ac uals with homocystinuria caused by each celerated growth, dislocated lenses, osteo of these enzyme deficiencies.12 The growth porosis, pectus deformities, hernias, light of cells cultured from the skin of individ colored hair and cardiovascular disease.17 uals with cystathionine synthetase defi Homocystinuria is associated with an in ciency is characterized by abnormal contact herited deficiency of one of three enzymes, inhibition and producton of an aggregated cystathionine synthetase,18 N5 methyltetra- proteoglycan matrix which binds more sul hydrofolate methyl transferase,19 and meth- fate than the fibrillar matrix of normal ylenetetrahydrofolate reductase.20 Myointi- cells.15 The sulfur of homocysteine thiolac tone is a precursor of phosphoadenosine * Supported by grants from the National Insti phosphosulfate and proteoglycan sulfate tutes of Health, AM-15978, CDA-HL-18747 and the American Heart Association, 73-655. ester in these cell cultures.14 Somatomedin,5 147 1 4 8 MCCULLY a serum polypeptide which increases sul styrene resin columns (Aminex A-6),f fate binding by cartilage fragments, is be using stepwise elution by citrate bufferf at lieved to mediate the physiological effects pH 3.25, 4.25 and 7.0 at 50°. Aliquots of of growth hormone.23 Because of these ob the fractions were counted in Bray’s solu servations, a relationship between homo tion,1 and the position of eluted radioactive cysteine metabolism and the physiological peaks was compared to that of authentic action of growth hormone was suggested.14 amino acids. The radioactive peaks were Homocysteic acid, the sulfonic acid de pooled, desalted on a 0.9 X 10 cm Dowex rivative of homocysteine, is a precursor of AG 50 X 8, 200-450 meshf column, eluted phosphoadenosine phosphosulfate and pro with 1.4 M NH4OH and lyophylized. The teoglycan sulfate ester.14 In order to inves residue was dissolved in H20 and chromat tigate the relationship between homocys ographed on silica gel TLC-TGF plates,£ teine metabolism and the physiological ac using butanolacetic acid—H20 (4:1:1 by tion of growth hormone, the conversion of volume), for comparison with authentic homocysteine thiolactone to homocysteic compounds. acid was compared in young, adult and Cells cultured from normal human skin hypophysectomized animals. The growth biopsies were maintained in Eagle’s min promoting activity of homocysteic acid was imal essential medium with 10 percent fetal determined by weight acquisition in nor calf serum, according to standard tech mal and scorbutic guinea pigs, and the niques. Equal numbers of cells were pas eifect of homocysteic acid on sulfate bind saged into media containing S5S04, 1 /xCi ing was studied in human cell cultures. per ml, 0.8 mM, and homocysteic acid and refed twice weekly. The cultures became Materials and Methods confluent after seven days, and half the cultures were incubated an additional ten The conversion of homocysteine thiolac days. The media were removed, and the tone to free amino acid derivatives was monolayer was rinsed with cold phosphate studied by injecting 5.0 ¿u.Ci of carboxyl-14C buffered saline, scraped into saline and 1-homocysteine thiolactone hydrochloride* homogenized. Radioactivity of the homog- with 7.5 mg dl-homocysteine thiolactone enate was determined, using Bray’s solu hydrochloride in 5 ml of normal saline tion, and protein was determined, using the intraperitoneally in guinea pigs or rats. Lowry method.11 After one hour the livers were removed, Three groups of six guinea pigs weighing rinsed with buffer, chilled, diced with 320 to 390 g were injected subcutaneously scissors and homogenized in a Waring every day for 22 days with 30 mg per kg blender for 20 to 30 seconds with two vol of neutralized homocysteic acid or an equi- umes of cold sucrose, KHC03, KC1 buffer8 molar quantity of saline. Initial mean containing 15 mg per 1 of thiodiglycol and weights were 347 to 357 g. The animals 0.5 g per 1 of ethylene diamine tetraacetic were fed chow* or an ascorbic acid defi acid (EDTA). The extract was decanted, cient diet.f Average growth rates, calcu deproteinized by adding excess sulfosali- lated from biweekly weights, were linear cylic acid and centrifuged at 10,000 g for during the experimental period. P values 15 minutes. Amino acids were separated by were calculated, using the student t test, standard techniques of amino acid chroma tography on 0.9 X 60 cm sulfonated poly | Malinckrodt. * Calbiochem. * Agway. f Bio-Rad. f Nutritional Biochemicals, Inc. GROWTH AND HOMOCYSTEINE f * ! 149

R esu lts T A B L E I A The conversion of homocysteine thiolac Metabolism of Homocysteine Thiolactone in Liver Percentage of 1I+C Recovered by Chromatography tone to free amino acids of liver was deter mined in young and adult guinea pigs, in Guinea Guinea Hypox P ig - P ig R a t R a t R a t rats and in hypophysectomized rats. 14C- Amino A cid (545 g ) (125 g ) (400 g ) (120 g ) (1 2 0 g ) carboxyl 1-homocysteine thiolactone was in jected intraperitoneally; after one hour, the Homocysteic 6.8 13.8 10.2 24.4 18.0 acid livers were homogenized, deproteinized Homocysteine 7.8 13.6 13.8 21.4 12.0 sulfinic acid and chromatographed on sulfonated poly Homocysteine 0.5 <0.1 20.3 16.3 23.0 styrene resin. The percentage of recovered Homocystine <0.1 <0.1 5.9 <1.0 7.0 Homocysteine 1.5 0.03 18.4 <1.0 6.7 14C is listed in table I for each free amino thiolactone Unidentified 78.1 68.6 27.8 34.6 31.2 acid recovered by chromatography. homocysteine derivatives (10) Cystathionine 5.4 3.3 3.1 <1.0 <1.0 The conversion of homocysteine thiolac Adenosyl <1.0 <1.0 <1.0 3.2 <1.0 tone to homocysteic acid and homocysteine homocysteine sulfinic acid in liver was greater in young, rapidly growing guinea pigs and rats than in adult animals. Only traces of unmetab- than in rat liver. Conversion of homocys olized homocysteine thiolactone were found teine thiolactone to multiple unidentified in young animals, whereas a large amount homocysteine derivatives was greater in was recovered from adult rat liver and a guinea pig than in rat liver. The sulfur of small amount from adult guinea pig liver. these unidentified derivatives is oxidized, Conversion of homocysteine thiolactone to since they all yielded homocysteic acid cystathionine was greater in adult than in when desalted, isolated, and chromato young animals. Decreased conversion of graphed on thin layers of silica gel. homocysteine thiolactone to homocysteic Three groups of six guinea pigs were acid and homocysteine sulfinic acid was injected subcutaneously once daily with found in hypophysectomized rat liver, com neutralized homocysteic acid or an equi- pared to the conversion found in liver of a molar quantity of saline. Weights were re young rat of the same size. corded twice weekly, and the final mean Considerable differences in homocysteine weights were compared (table II). The thiolactone metabolism were found in growth rates of the groups fed chow were guinea pigs compared to rats (table I). linear, as illustrated in figure 1. Larger amounts of homocysteine occurred Homocysteic acid increased the growth in rat liver, but only traces of homocysteine rate of normal young guinea pigs when were recovered from guinea pig liver. Ho given subcutaneously for 22 days in a dose mocystine was recovered from adult rat of 30 mg per kg per day (table II and liver and hypophysectomized rat liver but figure 1). A higher dose of 80 mg per kg not from adult guinea pig liver or from per day resulted in the same growth rate the liver of young animals of either species. as the lower dose, but the minimum dose The conversion of homocysteine thiolactone necessary to produce the effect was not to homocysteic acid and homocysteine sul determined. The guinea pigs given an as finic acid was greater in young rat liver corbic acid deficient diet and parenteral than young guinea pig liver, and the same homocysteic acid all failed to gain weight relationship was found in adult animals. and died of scurvy. Adenosyl homocysteine was recovered only The binding of 35S0 4 to human cell cul from young rat liver, and conversion to tures was increased slightly when homocys cystathionine was greater in guinea pig teic acid, 0.01 to 1.0 mM, was present in 1 50 MCCULLY

TABLE I I

Effect of Homocysteic Acid on Growth

Dose (s.c.) mg p e r D u r a t i o n Final Weight (g) Growth Rate Com pound k g p e r d a y (D a y s ) D i e t S u r v i v o r s M ean ± SEM p g per day

_ _ 22 Chow 5/6 418 ± 25 3.2 Homocysteic acid 30 22 Chow 5/6 523 ± 19 <0.01 7.5 Homocysteic acid 30 22 Ascorbic acid 0/6 300 0.0 deficient the culture medium during growth to con teic acid, resembles the metabolism in adult fluence at seven days (figure 2). Parallel liver more than the metabolism in young cultures were maintained in medium con rat liver. Growth hormone and other pitu taining homocysteic acid for an additional itary hormones are not secreted by hypo ten days after confluence was reached, and physectomized rats, resulting in a reduced the binding of 35S0 4 was slightly increased growth rate. These experiments suggest in all cultures (figure 2). that pituitary hormones, including growth hormone, act upon the liver to increase the D iscussion conversion of methionine to homocysteic The results show that parenteral homo acid, a substance which promotes growth. cysteic acid promotes increased growth of Methionine is metabolized to homocys normal guinea pigs, as determined by teine thiolactone24 and cystathionine in weight acquisition. The conversion of ho liver. The greater conversion of homocys mocysteine thiolactone to homocysteic acid teine thiolactone to cystathionine in the is greater in young, growing rats and liver of older animals, compared to young guinea pigs than in older animals, correlat animals, suggests that the trans-sulfuration ing with more rapid utilization of methi pathway for conversion of methionine to onine and other nutrients for growth in cystathionine and cysteine metabolites com young animals. The metabolism of homo petes with the pathway for conversion of cysteine thiolactone in hypophysectomized methionine to homocysteic acid, which is rat liver, including conversion to homocys- greater in young animals than in older an imals. The failure to recover cystathionine

DAYS

Figure 1. Effect of homocysteic acid on growth F i g u r e 2. Effect of homocysteic acid on KS0 4 of normal and Scorbutic guinea pigs. binding by cultured human cells. GROWTH AND HOMOCYSTEINE 151 and the greater conversion of homocysteine mined by direct analysis and chemical syn thiolactone to homocysteic acid in hypo- thesis. physectomized rat liver than in adult rat Individuals with homocystinuria have ac liver suggest that failure of normal secre celerated skeletal growth, and homocysteic tion of pituitary hormones, including acid has been isolated from their urine.21 growth hormone, produces abnormalities in The results of the present study suggest metabolism of methionine to homocysteic that the accelerated skeletal growth of acid or in transport of homocysteic acid to these individuals is produced by increased the tissues. synthesis of homocysteic acid from methi Although oxidation of homocysteine thio onine. The myointimal hyperplasia charac lactone to homocysteic acid is diminished teristic of the fibrous arteriosclerotic plaques in scurvy,14 administration of homocysteic found in individuals with homocystinuria acid to scorbutic guinea pigs failed to pro is attributable to the growth promoting ef mote weight gain or to prevent death from fect of homocysteine derivatives,12’13 since scurvy. This result suggests that ascorbic homocysteine thiolactone and homocysteic acid is required for vital processes other acid produce arteriosclerosis in rabbits.13’16 than oxidation of homocysteine thiolactone The lathyritic properties of the connective to homocysteic acid. tissues in homocystinuria have been attrib Slightly increased sulfate binding in nor uted to the reversible formation of tetra- mal skin cell cultures is produced by homo hydrothiazine derivatives of the aldehyde cysteic acid in the culture medium during precursors of collagen cross-links from ho growth to confluence and survival after mocysteine.9’10 confluence. The fetal calf serum in the cul Carnivorous species such as rats, cats ture medium in these experiments contains and dogs are generally resistant to athero somatomedin,23 and the increased sulfation genic diets, whereas herbivorous species observed may have resulted from augmen such as rabbits, primates and horses are tation of the effect of somatomedin by ho susceptible to atherogenic diets. Differ mocysteic acid. Increased sulfation by ho ences in metabolism of homocysteine thio mocysteic acid was also found in chick lactone may determine whether a species embryo chondrocyte cultures incubated is carnivorous or herbivorous and, hence, with serum from hypopituitary patients.22 resistant or susceptible to experimental ar Although homocysteic acid does not have teriosclerosis. Important differences were somatomedin activity in vitro, the serum found in the metabolism of homocysteine from hypophysectomized animals with a thiolactone between rat, which is carnivo rous, and guinea pig, which is herbivorous. growth response to homocysteic acid has Similar metabolic studies in other carniv normal somatomedin activity.3 Also homo- orous and herbivorous species are needed cystine has been reported to have somato to confirm these observations. The associa medin activity in cartilage fragments.4 tion of arteriosclerosis with homocystinuria These observations suggest that homocys in man7’12 suggests that the metabolism of teine derivatives, including homocysteic methionine in man may resemble that in acid, are required for synthesis of somato herbivorous species more than carnivorous medin by the metabolic action of cells and species. Prevention of human arteriosclero tissues. Although highly purified somato sis would be expected to result from reduc medin from plasma has been found to be tion in dietary consumption of animal pro a polypeptide,25 the chemical structure of tein and an increase in vegetable protein the active molecule has not been deter consumption, since animal protein contains 1 52 MCCULLY more methionine than vegetable protein.13 ment with the Folin phenol reagent. J. Biol. The growth disorders associated with al Chem. 193:265-275, 1951. 12. M c C u l l y , K. S.: Vascular pathology of homo- tered homocysteine metabolism, including cysteinemia: Implications for the pathogenesis arteriosclerosis and accelerated skeletal of arteriosclerosis. Amer. J. Pathol. 56:111- growth, have been interpreted to result 128, 1969. 13. M c C u l l y , K. S. a n d R a g s d a l e , B. D.: Pro-, from increased conversion of methionine to duction of arteriosclerosis by homocysteinemia. homocysteine thiolactone and homocysteic Amer. J. Pathol. 62:1-11, 1970. 14. M c C u l l y , K. S.: Homocysteine metabolism acid. Further studies are needed to eluci in scurvy, growth and arteriosclerosis. Nature date the biochemical and physiological 232:391-392, 1971. processes by which homocysteic acid pro 15. M c C u l l y , K. S.: Macromolecular basis for homocysteine-induced changes in proteoglycan motes growth of animals and contributes structure in growth and arteriosclerosis. Amer. to the genesis of growth disorders in man. J. Pathol. 66:83-95, 1972. 16. M c C u l l y , K. S. a n d W i l s o n , R. B.: Homo cysteine theory of arteriosclerosis. Unpub Acknowledgments lished, 1974. 17. M c K u s i c k , V. A., H a l l , J. G., a n d C h a r , F.: Thanks are extended to A. J. Wertheimer and The clinical and genetic characteristics of M. Goldmann for technical assistance. homocystinuria. Inherited Disorders of Sul phur Metabolism. Carson, N. A. J. and Raine, D. N., eds., Churchill Livingstone, Edinburgh R eferences and London, pp. 179-203, 1971. 18. M u d d , S. H., F i n k e l s t e i n , J. D., Ir r e v e r e , 1. Bray, G. A.: A simple, efficient liquid scintil F., a n d L a s t e r , L.: Homocystinuria: an enzy lator for counting aqueous solutions in a liquid matic defect. Science 143:1443—1445, 1964. scintillation counter. Anal. Biochem. 2:278- 19. M u d d , S. H., L e v y , H. L., a n d A b e l e s , R. H.: 285, 1960. A derangement in B12 metabolism leading to 2. C a r s o n , N. A. J. a n d N e i l l , D. W.: Meta homocystinuria, cystathioninuria, and methyl bolic abnormalities detected in a survey of malonic aciduria. Biochem. Biophys. Res. mentally backward individuals in Northern Comm. 35:121-126, 1969. Ireland. Arch. Dis. Child. 37:505-513, 1962. 20. M u d d , S. H., U h l e n d o r f , B. W., F r e e m a n , 3. C l o p a t h , P., IIi n t z , R. L., a n d M c C u l l y , J. M., F i n k e l s t e i n , J. D., a n d Shih, V. E.: K. S.: Growth promoting activity of oxidized Homocystinuria associated with decreased homocystine compounds. Unpublished, 1974. methylenetetrahydrofolate reductase activity. 4. D e h n e l , J. M. a n d F r a n c i s , M. J. O.: So Biochem. Biophys. Res. Comm. 46:905-912, matomedin (sulfation factor)-like activity of 1972. homocystine. Clin. Sci. 43:903—906, 1972. 21. O h m o r i , S., K o d a m a , H., Ik e g a m a , T., 5. D a u g h a d a y , W. H., H a l l , K., R a b e n , M. S., M i z u r h a r a , S., O u r a , T., Is s h i k i , G., a n d S a l m o n , W . D., V a n d e n B r a n d e , J. L., a n d U e m u r a , I.: Unusual sulfur-containing amino V a n W y k , J. J.: Somatomedin: proposed des acids in the urine of homocystinuric patients. ignation for sulfation factor. Nature 235:107, III. Homocysteic acid, homocysteine sulfinic 1972. acid, S- (carboxymethylthio) homocysteine, 6. G e r r i t s e n , T., V a u g h n , J. 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