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Homocysteine content of plasma proteins in ischemic heart disease
Atherosclerosis, 69 (1988) 109-113 Elsevier Scientific Publishers Ireland,
109 Ltd.
ATH 04056
Homocysteine content of plasma proteins in ischemic heart disease Andrzej J. Olszewski and Wiktor B. Szostak National Food and Nutrition Institute, 02-903 Warsaw (Poland) (Received 21 November, 1986) (Revised, received 17 July, 1987) (Accepted 27 July, 1987)
Summary
It has been shown previously that accumulation of homocysteine produces atheromatous changes. The present study was done on 26 male survivors of myocardial infarction 2-3 months after the acute phase and 26 healthy males of the same age (30-60 years). The concentrations of homocysteine, its derivatives and other amino acids were determined in acid hydrolyzate of plasma and in deproteinized plasma. The plasma proteins of survivors of myocardial infarction were found to contain a high concentration of homocysteine. The average value was 958 k 84 pmol/l of plasma, which was about 25 times the quantity found in the control group. Large differences were also found in a-amino adipic acid and cystathionine concentrations. These substances were found in significantly higher concentrations in the plasma of the survivors compared to controls. The high positive correlation between homocysteine and a-amino adipic acid level (r = 0.83; P < 0.001) suggests a common source of these 2 compounds in the analyzed samples. The levels of the other 15 measured amino acids were not significantly different in the 2 groups. The results support the homocysteine theory and suggest a method for more exact diagnosis of atherosclerosis.
Key words:
a-Amino adipic acid; steine mixed disulfide
Atherosclerosis;
Introduction The accumulation of homocysteine and its derivatives produces atheromatous changes in the arterial wall [l-4]. These observations formed the basis of the homocysteine theory of atherosclerosis
Correspondence Nutrition Institute, land.
0021-9150/88/$03.50
to: A.J. Powiixka
Olszewski, National Food 61/63, PL-02-903 Warsaw,
and Po-
Homocysteine;
Scientific
Publishers
Ireland,
plasma;
Homocysteine-cy-
[5]. Some suggested pathophysiological mechanisms are: (1) Disintegration of the elastica interna by the binding of homocysteine to the allysine residue of tropoelastin and inhibition of elastin polymerization [5]. (2) Hyperplasia of arterial smooth muscle cells and synthesis of extracellular connective tissue macromolecules caused by homocysteic acid which has a growth hormone-like activity [5,6]. (3) Degradation of vascular glycocalyx and basement membrane by
deposition 0 1988 Elsevier
Human
Ltd.
of highly sulfated proteoglycosamino-
110 glycans within the arterial wall [5]. (4) Activation of Hageman factor and stimulation of platelet thromboxane TXB, [5,7,8]. Evidence for elevated concentrations of free plasma homocysteine was sought in previous studies of patients with ischemic heart and arteriosclerotic cerebrovascular diseases [9-121. Very low concentrations of homocystine and homocysteine-cysteine mixed disulfide were found to be slightly increased after doses of oral methionine in patients with atherosclerosis [9-111. This was, however, not confirmed by other investigators [12]. Small amounts of plasma homocysteine, found in healthy subjects [13] as well as in patients with coronary artery disease [14], are mainly present in protein-bound form. Protein-bound homocysteine appeared to be slightly but statistically significantly elevated in patients in comparison with normals. Until now, however, in such studies only homocysteine bound by disulfide linkage was determined. The aim of the present study was to measure the total amount of homocysteine bound to plasma proteins in survivors of myocardial infarction compared to unaffected controls. Total proteinbound homocysteine of the plasma was determined after acid hydrolysis. In contrast to previous studies, very large quantities of proteinbound homocysteine were found.
lyzed plasma and in deproteinized plasma by automated amino acid analysis (Beckman Model 119 CL with 126 Data System integrator). Proteins were precipitated with sulfosalicylic acid added to plasma in solid form to obtain a concentration of 5% (w/v)_ The hydrolysis of whole plasma was accomplished by heating samples in vacua in 4 N HCl (final concentration) at 110°C for 5 h. Then the samples were lyophilized, diluted in 0.2 N lithium citrate buffer (pH 2.2) and passed through Millipore filters. Concentration of homocysteine, homocysteine-cysteine mixed disulfide, cu-amino adipic acid, cystathionine, and 15 other amino acids (Asp, Thr, Glu, Pro, Abu, Val, Ile, Leu, Tyr, Phe, Gly, Ala, His, Arg) were determined. The analyzer operating parameters used in this study are shown in Table 1. A longer running time of the first buffer was used, and the second buffer had slightly lower pH value than recommended in the original Beckman physiological fluid analysis method. This modification produced better resolution of the homocysteine-cysteine mixed disulfide between Leu and Tyr. The chromatogram showing the elution profile of the determined compounds in acid hydrolyzed
TABLE
1
OPERATING YSIS
PARAMETERS
FOR AMINO
ACID
ANAL-
Materials and methods The study was done in 26 male survivors of myocardial infarction 2-3 months after the acute phase, and 26 healthy males aged 30-60 years. The basic criterion qualifying the individuals for the study was ECG abnormality estimated by the Minnesota code. The mean body mass index (weight/ height2) was 26.2 * 2.2 in the group of survivors and 24.2 $- 2.1 in the control group. Plasma samples were obtained after an overnight fast. In 21 cases the triglyceride level in survivors plasma was above 2.1 mmol/l and/or total cholesterol level was above 260 mg/dl, measured by the Boehringer-Mannheim enzymatic test. In all control cases plasma triglycerides were below 2.1 mmol/l and total cholesterol was below 260 mg/dl. Amino acids were determined in whole, hydro-
6x460 mm w-3P 220 mm pH 2.83 0.20 N Li+ pH 3.65 0.20 N Li’ pH 3.75 1.00 N Li + 0.30 N LiOH + 0.25 g EDTA (acid Regeneration reagent form)/ 44 ml/h Buffer flow rate 22 ml/h Ninhydrin flow rate 40°C,650C Column temperatures 70,162,260 min Buffer change times Temperature change time 40 min 100 ~1, 10 X final plasma dilution Sample size 6 in/h Chart speed 6 in/h Recorder chart speed 0.2 A Recorder range 2.0 A Integrator range Summation of 440 nm and 570 nm Upper pen trace signals 440 nm signal only Lower pen trace
Column Resin type Resin bed height Buffers (lithium citrate)
111 plasma of myocardial infarction survivors is shown in Fig. 1. The limit of detection for homocystine, homocysteine-cysteine mixed disulfide, cystathionine and ol-amino adipic acid in lo-fold diluted plasma hydrolyzate was approx. 0.5 pmol/l. Standard solutions of homocystine, homocysteine-cysteine mixed disulfide, and Pierce’s physiological amino acid calibration standard were submitted to all steps of the analytical procedure in parallel with plasma samples to minimize the error of determination. Recovery of homocysteine from the standard solution averaged 96 f 6% (n = 12). The mixed disulfide was produced from homocysteine (Serva) and cysteine (Pierce) by aeration of the solution of these amino acids at pH 11.5. The concentration of this disulfide standard was found to increase stoichiometrically, corresponding to a decrease in cysteine concentration. Total homocysteine was calculated in pmol/l as twice the concentration of homocystine plus the concentration of homocysteine-cysteine mixed disulfide. Plasma protein-bound homocysteine was calculated from the difference of whole plasma homocysteine and deproteinized plasma homocysteine. However, the concentrations of homocystine and homocysteine-cysteine mixed disulfide found in deproteinized plasma did not exceed several pmoles per liter, and, in the described method, were in the range of the analytical error. There-
fore, nearly total plasma homocysteine was bound to protein. The results are shown as mean f SEM. The statistical significance of differences between the examined groups was estimated by Student’s t-test. Results Fig. 2 presents the differences in the levels of plasma amino acids which were found to be altered in the whole plasma in patients after myocardial infarction, compared with the control group. In all samples containing both homocysteine and cysteine, homocystine and homocysteine-cysteine mixed disulfide are always found. Therefore homocystine as well as mixed disulfide were determined in the samples. The amounts of homocystine and homocysteine-cysteine mixed disulfide in the plasma of the control group averaged only 17.5 + 6 pmol/l for the former and several pmoles for the latter. The levels of these compounds were much higher in the survivors of infarction and averaged 116 k 8 pmol/l and 725 f 71 pmol/l, respectively. The total amount of homocysteine found in the survivors was 958 + 84 nmol/l, and the corresponding value for the control group was only 38 * 11 pmol/l. The concentration of cystathionine was 366 k
112 30 pmol/l in the patient group and 159 + 11 pmol/l in the control group. The level of a-amino adipic acid was also higher in the survivors (366 + 50 pmol/l), compared to the control group (78 k 19 pmol/l). The relations between total homocysteine and these 2 amino acids are illustrated in Fig. 3. Each of the levels of 15 other amino acids measured was similar in both investigated groups. In deproteinized plasma samples homocystine and homocysteine-cysteine mixed disulfide were found in very low levels (several pmol/l) and there were only traces of cY-amino adipic acid. So, the amounts of these substances in hydrolyzed plasma were derived mainly from plasma proteins. Discussion
Wne+Stine
p*MOl
MI& hano. TOtOl cy?x!Mmnt CystMa-cyshonocystmc !ClM dlwlfdc p. ODOl
Pmm
Fig. 2. The acid hydrolyzed derivatives,
infarction
and
a-amino
(a) compared
plasma
adipic
acid
to controls
p-oDO!
d-omno omplc OCld
p.oDn
levels of homocysteine, its in survivors of myocardial (0).
r
-’ I-AMINO ADl_flC ACID
,,k,s”
Fig. 3. Relation between a-amino and total homocysteine in acid myocardial infarction survivors.
.~-mm
MO
CYSTATHIONINE
w?ol/I
adipic acid, cystathionine hydrolyzate of plasma of
High levels of homocystine and homocysteinecysteine mixed disulfide were found in the survivors of myocardial infarction. In contrast, only insignificant amounts were found in healthy subjects. The total amount of homocysteine, calculated as the sum of mixed disulfide and twice homocystine, in the patient group was about 25 times higher than in the control group. Such large differences were found in every patient compared to the controls. Almost all homocysteine found in hydrolyzed plasma came from protein in which it might have been bound by tetrahydrothiazine, peptide, Schiff base or disulfide bonds. Our results are in accordance with the homocysteine theory of atherosclerosis [5]. It is suggested, that this method of homocysteine determination may be used as an indicator of atherogenesis. Other investigators have found only small quantities of homocysteine, either free after deproteinization of plasma [9-121 or released by 2-mercaptoethanol or dithioerythritol from disulfide binding with protein [13,14]. Our results agree with the finding of only trace quantities of free homocystine and mixed disulfide in deproteinized plasma. Cystathionine, the product of homocysteine catabolism, was also increased in the patient group. A high correlation between homocysteine and We cystathionine was found (r = 0.93; P < 0.001). suggest that cystathionine concentration, like that
113 of homocysteine may be used as an index of atheromatosis. cr-Amino adipic acid concentration was also markedly higher in survivors and highly correlated (r = 0.83; P < with homocysteine concentration 0.001). In all probability, the increased amount of cy-amino adipic acid is derived from allysine (CXamino adipic 6-semialdehyde) of elastin during plasma hydrolysis. This may suggest that elastin residues are present in the plasma of atherosclerotic patients in evidently higher amounts than in healthy subjects. There is also a possibility that cu-amino adipic acid is attached to the amino group of homocysteine bound to plasma protein. The present method of analysis employs complete hydrolysis of plasma protein which releases homocysteine from all types of protein binding. The results of our investigation not only confirm the homocysteine theory but may provide a method for more exact diagnosis, treatment and prevention of atherosclerosis in the future. References 1 Gibson, J.B., Carson, N.A.J. and Neill, D.W., Pathological findings in homocysteinuria, J. Clin. Pathol., 17 (1964) 427. 2 Harker, L.A., Ross, R., Slichter, S.J. and Scott, C.R., Homocysteine-induced arteriosclerosis. Role of endothelial cell injury and platelet response to its genesis, J. Clin. Invest., 58 (1976) 731. 3 Kanwar, Y.S., Manaligod, J.R. and Wong, W.K., Morphologic studies in a patient with homocystinuria due to 5,10methylenetetrahydrofolate reductase deficiency, Pediatr. Res., 10 (1976) 598.
4 McCully, K.S., Vascular pathology of homocysteinemia. Implications for the pathogenesis of arteriosclerosis, Am. J. Pathol., 56 (1969) 111. 5 McCully, K.S., Homocysteine theory of arteriosclerosis. Development and current status. In: Gotto, Jr. A.M., and Paoletti, R. (Eds.), Atherosclerosis Reviews, vol. 11, Raven Press, New York, 1983, pp. 157. 6 Clopath, P., Smith, V.C. and McCully, K.S., Growth promotion by homocysteic acid, Science, 192 (1976) 372. 7 Graeber, J.E., Slott, J.H., Ulane, R.E., Schulman, J.D. and Stuart, M.J., Effect of homocysteine and homocystine on platelet and vascular arachidonic acid metabolism, Pediatr. Res., 16 (1982) 490. 8 Ratnoff, O.D., Activation of Hageman factor by l-homocysteine, Science, 162 (1968) 1007. 9 Boers, G.H.J., Smals, A.G.H., Trijbels, F.J.M., Fowler, B., Bakkeren, J.A.J.M., Schoonderwalt, M.C., Kleijer, W.J. and Kloppenborg, P.W.C., Heterozygosity for homocystinuria in premature peripheral and cerebral occlusive arterial disease, N. Engl. J. Med., 313 (1985) 709. 10 Brattstrom, L., Hardebo, J. and Hultberg, B., Moderate homocysteinemia-a possible risk factor for arteriosclerotic cerebrovascular disease, Stroke, 15 (1984) 1012. 11 Murphy-Chontorian, D.R., Wexman, M.P., Grieco, A.J., Heininger, J.A., Glassman, E., Gaul, G.E., Ng, S.K.C., Feit, F., Wexman, K. and Fox, C.A., Methionine intolerance: a possible risk factor for coronary artery disease, J. Am. COB. Cardiol., 6 (1985) 725. 12 Wilcken, D., Reddy, S. and Gupta, V., Homocysteinemia, ischemic heart disease and the carrier state for homocystinuria, Metabolism, 32 (1983) 363. 13 Refsum, H., Helland, S. and Ueland, P.M., Radioenzymic determination of homocysteine in plasma and urine, Clin. Chem., 31 (1985) 624. 14 Kang, S., Wong, P.W.K., Cook, H.Y., Norusis, M. and Messer, J.V., Protein-bound homocysteine. A possible risk factor for coronary artery disease, J. Clin. Invest., 77 (1986) 1482.
109 Ltd.
ATH 04056
Homocysteine content of plasma proteins in ischemic heart disease Andrzej J. Olszewski and Wiktor B. Szostak National Food and Nutrition Institute, 02-903 Warsaw (Poland) (Received 21 November, 1986) (Revised, received 17 July, 1987) (Accepted 27 July, 1987)
Summary
It has been shown previously that accumulation of homocysteine produces atheromatous changes. The present study was done on 26 male survivors of myocardial infarction 2-3 months after the acute phase and 26 healthy males of the same age (30-60 years). The concentrations of homocysteine, its derivatives and other amino acids were determined in acid hydrolyzate of plasma and in deproteinized plasma. The plasma proteins of survivors of myocardial infarction were found to contain a high concentration of homocysteine. The average value was 958 k 84 pmol/l of plasma, which was about 25 times the quantity found in the control group. Large differences were also found in a-amino adipic acid and cystathionine concentrations. These substances were found in significantly higher concentrations in the plasma of the survivors compared to controls. The high positive correlation between homocysteine and a-amino adipic acid level (r = 0.83; P < 0.001) suggests a common source of these 2 compounds in the analyzed samples. The levels of the other 15 measured amino acids were not significantly different in the 2 groups. The results support the homocysteine theory and suggest a method for more exact diagnosis of atherosclerosis.
Key words:
a-Amino adipic acid; steine mixed disulfide
Atherosclerosis;
Introduction The accumulation of homocysteine and its derivatives produces atheromatous changes in the arterial wall [l-4]. These observations formed the basis of the homocysteine theory of atherosclerosis
Correspondence Nutrition Institute, land.
0021-9150/88/$03.50
to: A.J. Powiixka
Olszewski, National Food 61/63, PL-02-903 Warsaw,
and Po-
Homocysteine;
Scientific
Publishers
Ireland,
plasma;
Homocysteine-cy-
[5]. Some suggested pathophysiological mechanisms are: (1) Disintegration of the elastica interna by the binding of homocysteine to the allysine residue of tropoelastin and inhibition of elastin polymerization [5]. (2) Hyperplasia of arterial smooth muscle cells and synthesis of extracellular connective tissue macromolecules caused by homocysteic acid which has a growth hormone-like activity [5,6]. (3) Degradation of vascular glycocalyx and basement membrane by
deposition 0 1988 Elsevier
Human
Ltd.
of highly sulfated proteoglycosamino-
110 glycans within the arterial wall [5]. (4) Activation of Hageman factor and stimulation of platelet thromboxane TXB, [5,7,8]. Evidence for elevated concentrations of free plasma homocysteine was sought in previous studies of patients with ischemic heart and arteriosclerotic cerebrovascular diseases [9-121. Very low concentrations of homocystine and homocysteine-cysteine mixed disulfide were found to be slightly increased after doses of oral methionine in patients with atherosclerosis [9-111. This was, however, not confirmed by other investigators [12]. Small amounts of plasma homocysteine, found in healthy subjects [13] as well as in patients with coronary artery disease [14], are mainly present in protein-bound form. Protein-bound homocysteine appeared to be slightly but statistically significantly elevated in patients in comparison with normals. Until now, however, in such studies only homocysteine bound by disulfide linkage was determined. The aim of the present study was to measure the total amount of homocysteine bound to plasma proteins in survivors of myocardial infarction compared to unaffected controls. Total proteinbound homocysteine of the plasma was determined after acid hydrolysis. In contrast to previous studies, very large quantities of proteinbound homocysteine were found.
lyzed plasma and in deproteinized plasma by automated amino acid analysis (Beckman Model 119 CL with 126 Data System integrator). Proteins were precipitated with sulfosalicylic acid added to plasma in solid form to obtain a concentration of 5% (w/v)_ The hydrolysis of whole plasma was accomplished by heating samples in vacua in 4 N HCl (final concentration) at 110°C for 5 h. Then the samples were lyophilized, diluted in 0.2 N lithium citrate buffer (pH 2.2) and passed through Millipore filters. Concentration of homocysteine, homocysteine-cysteine mixed disulfide, cu-amino adipic acid, cystathionine, and 15 other amino acids (Asp, Thr, Glu, Pro, Abu, Val, Ile, Leu, Tyr, Phe, Gly, Ala, His, Arg) were determined. The analyzer operating parameters used in this study are shown in Table 1. A longer running time of the first buffer was used, and the second buffer had slightly lower pH value than recommended in the original Beckman physiological fluid analysis method. This modification produced better resolution of the homocysteine-cysteine mixed disulfide between Leu and Tyr. The chromatogram showing the elution profile of the determined compounds in acid hydrolyzed
TABLE
1
OPERATING YSIS
PARAMETERS
FOR AMINO
ACID
ANAL-
Materials and methods The study was done in 26 male survivors of myocardial infarction 2-3 months after the acute phase, and 26 healthy males aged 30-60 years. The basic criterion qualifying the individuals for the study was ECG abnormality estimated by the Minnesota code. The mean body mass index (weight/ height2) was 26.2 * 2.2 in the group of survivors and 24.2 $- 2.1 in the control group. Plasma samples were obtained after an overnight fast. In 21 cases the triglyceride level in survivors plasma was above 2.1 mmol/l and/or total cholesterol level was above 260 mg/dl, measured by the Boehringer-Mannheim enzymatic test. In all control cases plasma triglycerides were below 2.1 mmol/l and total cholesterol was below 260 mg/dl. Amino acids were determined in whole, hydro-
6x460 mm w-3P 220 mm pH 2.83 0.20 N Li+ pH 3.65 0.20 N Li’ pH 3.75 1.00 N Li + 0.30 N LiOH + 0.25 g EDTA (acid Regeneration reagent form)/ 44 ml/h Buffer flow rate 22 ml/h Ninhydrin flow rate 40°C,650C Column temperatures 70,162,260 min Buffer change times Temperature change time 40 min 100 ~1, 10 X final plasma dilution Sample size 6 in/h Chart speed 6 in/h Recorder chart speed 0.2 A Recorder range 2.0 A Integrator range Summation of 440 nm and 570 nm Upper pen trace signals 440 nm signal only Lower pen trace
Column Resin type Resin bed height Buffers (lithium citrate)
111 plasma of myocardial infarction survivors is shown in Fig. 1. The limit of detection for homocystine, homocysteine-cysteine mixed disulfide, cystathionine and ol-amino adipic acid in lo-fold diluted plasma hydrolyzate was approx. 0.5 pmol/l. Standard solutions of homocystine, homocysteine-cysteine mixed disulfide, and Pierce’s physiological amino acid calibration standard were submitted to all steps of the analytical procedure in parallel with plasma samples to minimize the error of determination. Recovery of homocysteine from the standard solution averaged 96 f 6% (n = 12). The mixed disulfide was produced from homocysteine (Serva) and cysteine (Pierce) by aeration of the solution of these amino acids at pH 11.5. The concentration of this disulfide standard was found to increase stoichiometrically, corresponding to a decrease in cysteine concentration. Total homocysteine was calculated in pmol/l as twice the concentration of homocystine plus the concentration of homocysteine-cysteine mixed disulfide. Plasma protein-bound homocysteine was calculated from the difference of whole plasma homocysteine and deproteinized plasma homocysteine. However, the concentrations of homocystine and homocysteine-cysteine mixed disulfide found in deproteinized plasma did not exceed several pmoles per liter, and, in the described method, were in the range of the analytical error. There-
fore, nearly total plasma homocysteine was bound to protein. The results are shown as mean f SEM. The statistical significance of differences between the examined groups was estimated by Student’s t-test. Results Fig. 2 presents the differences in the levels of plasma amino acids which were found to be altered in the whole plasma in patients after myocardial infarction, compared with the control group. In all samples containing both homocysteine and cysteine, homocystine and homocysteine-cysteine mixed disulfide are always found. Therefore homocystine as well as mixed disulfide were determined in the samples. The amounts of homocystine and homocysteine-cysteine mixed disulfide in the plasma of the control group averaged only 17.5 + 6 pmol/l for the former and several pmoles for the latter. The levels of these compounds were much higher in the survivors of infarction and averaged 116 k 8 pmol/l and 725 f 71 pmol/l, respectively. The total amount of homocysteine found in the survivors was 958 + 84 nmol/l, and the corresponding value for the control group was only 38 * 11 pmol/l. The concentration of cystathionine was 366 k
112 30 pmol/l in the patient group and 159 + 11 pmol/l in the control group. The level of a-amino adipic acid was also higher in the survivors (366 + 50 pmol/l), compared to the control group (78 k 19 pmol/l). The relations between total homocysteine and these 2 amino acids are illustrated in Fig. 3. Each of the levels of 15 other amino acids measured was similar in both investigated groups. In deproteinized plasma samples homocystine and homocysteine-cysteine mixed disulfide were found in very low levels (several pmol/l) and there were only traces of cY-amino adipic acid. So, the amounts of these substances in hydrolyzed plasma were derived mainly from plasma proteins. Discussion
Wne+Stine
p*MOl
MI& hano. TOtOl cy?x!Mmnt CystMa-cyshonocystmc !ClM dlwlfdc p. ODOl
Pmm
Fig. 2. The acid hydrolyzed derivatives,
infarction
and
a-amino
(a) compared
plasma
adipic
acid
to controls
p-oDO!
d-omno omplc OCld
p.oDn
levels of homocysteine, its in survivors of myocardial (0).
r
-’ I-AMINO ADl_flC ACID
,,k,s”
Fig. 3. Relation between a-amino and total homocysteine in acid myocardial infarction survivors.
.~-mm
MO
CYSTATHIONINE
w?ol/I
adipic acid, cystathionine hydrolyzate of plasma of
High levels of homocystine and homocysteinecysteine mixed disulfide were found in the survivors of myocardial infarction. In contrast, only insignificant amounts were found in healthy subjects. The total amount of homocysteine, calculated as the sum of mixed disulfide and twice homocystine, in the patient group was about 25 times higher than in the control group. Such large differences were found in every patient compared to the controls. Almost all homocysteine found in hydrolyzed plasma came from protein in which it might have been bound by tetrahydrothiazine, peptide, Schiff base or disulfide bonds. Our results are in accordance with the homocysteine theory of atherosclerosis [5]. It is suggested, that this method of homocysteine determination may be used as an indicator of atherogenesis. Other investigators have found only small quantities of homocysteine, either free after deproteinization of plasma [9-121 or released by 2-mercaptoethanol or dithioerythritol from disulfide binding with protein [13,14]. Our results agree with the finding of only trace quantities of free homocystine and mixed disulfide in deproteinized plasma. Cystathionine, the product of homocysteine catabolism, was also increased in the patient group. A high correlation between homocysteine and We cystathionine was found (r = 0.93; P < 0.001). suggest that cystathionine concentration, like that
113 of homocysteine may be used as an index of atheromatosis. cr-Amino adipic acid concentration was also markedly higher in survivors and highly correlated (r = 0.83; P < with homocysteine concentration 0.001). In all probability, the increased amount of cy-amino adipic acid is derived from allysine (CXamino adipic 6-semialdehyde) of elastin during plasma hydrolysis. This may suggest that elastin residues are present in the plasma of atherosclerotic patients in evidently higher amounts than in healthy subjects. There is also a possibility that cu-amino adipic acid is attached to the amino group of homocysteine bound to plasma protein. The present method of analysis employs complete hydrolysis of plasma protein which releases homocysteine from all types of protein binding. The results of our investigation not only confirm the homocysteine theory but may provide a method for more exact diagnosis, treatment and prevention of atherosclerosis in the future. References 1 Gibson, J.B., Carson, N.A.J. and Neill, D.W., Pathological findings in homocysteinuria, J. Clin. Pathol., 17 (1964) 427. 2 Harker, L.A., Ross, R., Slichter, S.J. and Scott, C.R., Homocysteine-induced arteriosclerosis. Role of endothelial cell injury and platelet response to its genesis, J. Clin. Invest., 58 (1976) 731. 3 Kanwar, Y.S., Manaligod, J.R. and Wong, W.K., Morphologic studies in a patient with homocystinuria due to 5,10methylenetetrahydrofolate reductase deficiency, Pediatr. Res., 10 (1976) 598.
4 McCully, K.S., Vascular pathology of homocysteinemia. Implications for the pathogenesis of arteriosclerosis, Am. J. Pathol., 56 (1969) 111. 5 McCully, K.S., Homocysteine theory of arteriosclerosis. Development and current status. In: Gotto, Jr. A.M., and Paoletti, R. (Eds.), Atherosclerosis Reviews, vol. 11, Raven Press, New York, 1983, pp. 157. 6 Clopath, P., Smith, V.C. and McCully, K.S., Growth promotion by homocysteic acid, Science, 192 (1976) 372. 7 Graeber, J.E., Slott, J.H., Ulane, R.E., Schulman, J.D. and Stuart, M.J., Effect of homocysteine and homocystine on platelet and vascular arachidonic acid metabolism, Pediatr. Res., 16 (1982) 490. 8 Ratnoff, O.D., Activation of Hageman factor by l-homocysteine, Science, 162 (1968) 1007. 9 Boers, G.H.J., Smals, A.G.H., Trijbels, F.J.M., Fowler, B., Bakkeren, J.A.J.M., Schoonderwalt, M.C., Kleijer, W.J. and Kloppenborg, P.W.C., Heterozygosity for homocystinuria in premature peripheral and cerebral occlusive arterial disease, N. Engl. J. Med., 313 (1985) 709. 10 Brattstrom, L., Hardebo, J. and Hultberg, B., Moderate homocysteinemia-a possible risk factor for arteriosclerotic cerebrovascular disease, Stroke, 15 (1984) 1012. 11 Murphy-Chontorian, D.R., Wexman, M.P., Grieco, A.J., Heininger, J.A., Glassman, E., Gaul, G.E., Ng, S.K.C., Feit, F., Wexman, K. and Fox, C.A., Methionine intolerance: a possible risk factor for coronary artery disease, J. Am. COB. Cardiol., 6 (1985) 725. 12 Wilcken, D., Reddy, S. and Gupta, V., Homocysteinemia, ischemic heart disease and the carrier state for homocystinuria, Metabolism, 32 (1983) 363. 13 Refsum, H., Helland, S. and Ueland, P.M., Radioenzymic determination of homocysteine in plasma and urine, Clin. Chem., 31 (1985) 624. 14 Kang, S., Wong, P.W.K., Cook, H.Y., Norusis, M. and Messer, J.V., Protein-bound homocysteine. A possible risk factor for coronary artery disease, J. Clin. Invest., 77 (1986) 1482.