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Pseudocholinesterase in obese and hyperlipemic subjects
CLWICA
CHIMICA
ACTA
PSEUDOCHOLINESTERASE
IS
IN OBESE
AND HYPERLIPEMIC
SUBJECTS
SUMMARY
It was found that serum pseudocholi~esterase increases not only in obese subjects but also in hyperlipemic patients with normal body weight. A good statistical correlation was found between serum pseudocholinesterase on one hand, and both serum cholesterol and the logarithm of serum triglycerides concentration, on the other. It cannot be stated whether increased pseudocholinesterase activity should be correlated with a possible role of the enzyme in the metabolism of lipids or with an unspecific and rather general stimulation of protein synthesis in the liver of obese and 11~7perlipemicsubjects.
Decrease of serum cholinesterase (pseudocholinesterase) in severe hepatic disease and malnutrition is a well known fact mentioned in various monographs1-3. It was attributed to an impairment of the proteosynthetic function of the liver. The increase of the enzyme reported in obese subjects4, diabetes mellitus5~6, hyperthyroidism’, nephrotic syndromes, psoriasisQ and essential hypertensionlO, is however rather difficult to explain since the physiological role of pseudocholinesterase (PCE) has not yet been established with certainty. According to Clitherow and ~011.11 the principal biological function of PCE should be to hydrolyse the acyl-choline esters and, especially, butyryl-choline that might arise as by-products during the catabolism of free fatty acids and lipogenesis in the liver. Starting from these considerationsll and from those of Augustinsson12 suggesting a linkage between PCE and the metabolism of fats, and also from several incidental observations, PCE was investigated in correlation with serum lipids. MATERIAL
AND
METHODS
Serum cholesterol (Zlatkis et n2.), serum triglycerides (Van Handel and Zilversmit) and PCE activityI were measured photocolorimetrically using a spectrophotometer Jobin-Yvon, in the following groups of subjects. * Director:
Prof.
St. Haragus. C&z. Chim.
AC&L,
22
(1968)
1 jr-155
=52
CUCUIAMJ
et
al.
A. The co&r01 groufi included 76 healthy subjects, 34 males 42 females aged IS-84 years (mean 3s years). Subjects exceeding the ideal weight by more than IO "/:, as well as those with a serum cholesterol level exceeding zoo mgY; were excluded from the control group. It should be mentioned that mean values for serum cholesterol of the Rumanian population were found to be about 170 mg% (ref. 14). R. The obese norrnolipemic group included 37 subjects, 9 males 2S females aged 15573 years (mean 39 years), exceeding the desirable weight by zo-So’): (mean +30(:X) but with a serum cholesterol under 200 rngfa. No severe glandular dyscrasia was observed; IO patients were found to have a diastolic blood pressure over IOO mm Hg, but none of them presented clinical signs of organic vascular disease. C. TJze obese hy~erlipemic ,pozq5 included 46 subjects, 26 males, 20 females, aged 16-72 years (mean 43 years) exceeding the desirable weight by 2oS5 yb (mean +34 y/o)and having a serum cholesterol level over 200 mg yo. Of this group, 22 patients were found to have increased blood pressure, 3 patients presented a mild diabetes mellitus and II had overt atherosclerotic changes. D. TJae hyperlipernic groq5 witlz normal body weight was composed
of 66 subjects,
3g males, 27 females, aged 19-77 years (mean 46 years). Of these, 23 patients had a diastolic blood pressure over IOO mm Hg; in 5 of them a mild diabetes was detected and 16 patients had clinical signs of atherosclerosis. In four patients a carbohydrateinduced hypertriglyceridemia could be found on dietetical grounds; three cases were identified as familial hypercholesterolemia. Though groups C and D were rather heterogeneous, all subjects had increased plasma lipoprotein levels. Also, the mechanisms leading to hyperlipemia remained ill-defined in most subjects with hyperlipemia. Subjects with evident hyperthyroidism or myxedema were not included in the statistical calculations. Also excluded were cases of recent myocardial infarction where a transient decrease of PCE is known to occurls. Cases with liver disease, heart failure, nephritis or nephrotic syndrome were not taken into consideration. The correlation of PCE and serum lipids was studied by means of correlation and regression analysis. In order to reduce the skewness of the distribution of triglycerides and facilitate calculations, the triglyceride values were converted into the corresponding
logarithms
and then reconverted,
as indicated
by Carlson*fl.
RESULTS
As shown in Table I, PCE was found to be increased not only in obese subjects but also in hyperlipemic subjects with normal body weight. Highest values were obtained in four cases with carbohydrate-induced hypertriglyceridemia. There was no significant difference in the PCE level of hyperlipemic patients with increased blood pressure and normotensive hyperlipemic subjects. Also, PCE was not significantly different in subjects with clinical atherosclerosis when compared with hyperlipemic subjects not manifestly affected by atherosclerosis. lower mean level of PCE was found As previously reported’7 a significantly in the 14 control normolipemic subjects over 60 years (222 & S.8). This decrease with age was, however, less evident in the IS hyperlipemic subjects from groups C and D above 60 years (329 A1 15). There was good statistical correlation between PCE and serum cholesterol Clin. Chim. Ada,
~2 (1968) 151-155
PSEUDOCHOLIXESTERASE TABLE
I
SERUM WITH AND
I53
IN OBESE SUBJECTS
CHOLESTEROL
NORMAL
HYPERLIPEMIC
Standard
;z
AND
BODY
Statistical
(A),
SUBJECTS
WITH
OBESE
LEVELS
NORMOLIPEMIC
NORMAL
BODY
IN
CONTROL
SUBJECTS
WEIGHT
(B),
NORMOLIPEMIC OBESE
SUBJECTS
HYPERLIPEMIC
(c)
(D)
error of the mean in brackets.
I67 I75 234 253
76 37 46 66
B C D
PSEUDOCHOLINESTER-4SE
WEIGHT
(i (:I (i (=
2.4)* 2.6) 6) 6.2)
24-l 308 355 33s
(2 (k (+ (&
4.3) 8.6) 7.4) 6.8)
significance
A US. B < 0.001 A US. c < 0.001 A vs. D < 0.001
B vs. C < 0.01 B U.S.D < 0.05 C US. D < 0.02
The remarkably low standard error for serum cholesterol according to their cholesterol levels.
*
(r = 0.574) ; satisfactory
correlation
is due to the selection
(Y = 0.56) was found also between
of the subjects
PCE and the
logarithm of serum triglycerides that were measured in 85 cases. For the same level of serum lipids (see Figs. I, z), there was a greater increase of PCE in obese subjects than in subjects with normal body weight. A discrepancy
Y
550 500
.
1
450 t
15’
i/
100 150
200
250
300
350
400
450
I
500
Fig. I. Correlation between serum cholesterol and pseudocholinesterase. Abscissa : serum cholesterol (mg %) ; ordinate : pseudocholinesterase activity (~moles/ml/h). l Subjects with normal body weight; 0 obese subjects; A subjects with hyperthyroidism; o subjects with hypothyroidism. Y = 0.574; regression equations: y = 0.45x + 212.40; x = 0.73 _Y- 14.38.
between PCE and serum cholesterol was noted in thyroid disease: high levels of PCE were associated with lower cholesterol values in five cases of hyperthyroidism. On the contrary, hypercholesterolemia was not accompanied by increased PCE in three cases of hypothyroidism. Cli+z. Chim. Acta,
22
(1968)
~51-155
‘50.
70 8090
x)0
125
150
200
250
300
400
500
600
L. Correlation between serum triglycerides and pseudocholinest~rase. Abscissa: serum triglycerides (mgq;) logarithmic scale; ordinate: pseudocholinesterase activity (~~moles~rnl~i~). l Subjects with normal body weight; o obese subjects. Y = 0.56; regression equations: y = 211x - 13r; x = 0.0015 y -+ 1.69, where x = logarithm of the triglyceride concentration.
Fig.
Our findings seem to support the hypothesis of Clitherow et aZ.ll about an active role of PCE in the metabolism of lipids. Increased availability and/or flux of unesterified fatty acids to the liver or increased lipogenesis from carbohydrates leads to an increased production of butyryl-coenzyme A, so that butyryl-choline production might also increase. This might be a stimulus for the synthesis or for the activation of PCE whose hydrolytic action on but~~ryl-choline allows the products of hydrolysis to return to their respective metabolic pathways. Hyperlipemia and high levels of PCE are therefore frequently associated as different effects of a common cause. Increased mobilisation of lipids is known to occur in hyperthyroidismr* where a high level of PCE was also noted?. However, high levels of serum cholesterol are uncol~ln~on in this condition, probably as a result of increased elimination of cholesterol and oxidation of lipids. Increased PCE activity should be therefore correlated rather with increased influx of unesteritied fatty acids and their uptake by the liver than with actual level of lipoproteins. It is worth mentioning that PCE is lower in newborn
infantslg
that have also
low levels of serum cholesterol, phospholipids and triglyceridesZo but high concenof trations of free-fatty acids 21. It seems that mechanisms leading to maturation lipogenesis in the liver and biosynthesis of PCE are somehow correlated. However, the increase of PCE as an expression of an unspecific and rather general stimulation of protein synthesis in the liver of obese and hyperlipemic subjects cannot be excluded. Accelerated lipogenesis in the liver implies also increased synthesis of the protein component of lipoproteins. It was found that inhibition of protein synthesis by puronlycin impairs the production of lipoproteins in rats, so that triglycerides accumulate in the livcrze. Also, perfusion of the isolated rat liver with a medium rich in fatty acid stimulated the synthesis of the protein moiety of lipoprotein+. C&z. Chim.
Acta,
22 (1968)
151-155
PSEUDOCHOLINESTERASE
IN OBESE
I55
SUBJECTS
Clinical investigations showed that a high level of ~-lipoproteins is accompanied by an increase of the B-protein and B-glycoprotein component of plasma24~26, and protein-bound hexoses increase not only in atherosclerotic but also in obese subjectP. The higher concentration of the inhibitors of fibrinolysis in atherosclerotic patients with high levels of /LlipoproteinP could also be related with increased protein synthesis in the liver, since serum lipids were not found to inhibit fibrinolysis by themseIveszR. REFERENCES I R. ABDERHALDEN, Klinische Enqfmologie, G. Thieme, Stuttgart, 1958. 2 B. RICHTERICH, Enz~mopathologie,Springer, Berlin--GSttingen-Heidelberg, 1958. 3 H. XV. GOEDDE, A. DOENICKE AND Ii.ALTLAND, Psezrdocholinestevase?z ~PIa~r~~kogenet~k, Bio-
cke~z~e,I~l~~~k~, Springer,Berlin-I-leidelberge~~York, 1967. 4 W. T. C. BERRY, P. J. COWIN AND D. R. D-AWES, &it. J. Xl&., 8 (1954) 79. 5 W. ANTOPOL, L. TIJCHMAN AND SCHIFRIN, Proc. Sot. Exptl.Biol. Med., 36 (1937) 46. 6 RI.FABER, Acta Med. &and., 114 (1943) 72. 7 J. c. THOMPSON AND 1\/1. ~HITTAKER, j. Clin.PUthOl.,18 (1965) 811. 8 N. STEFENELLI, K&n. Wochschr., 39 (1961) 1019. 9 L. HELMECZY AND E. NAGY, Acta Med. Acad. Sci. Hwag, 5 (x954) 109. IO R. RHEINFRANK AND H. I. WHETSTONE, ~~~t~oyd Hos$.BulL., 31 (1958) 32. or J. W. CLITHEROW, &I.MITCHARI~ AND N. J. HARPEK, h’ature, 199 (1963) 1000. I I I<. B. XUGUSTINSSON, in G. B. KOELLE (Ed.),Handbarch dev enperime~~tellen Pharnzakotogie, Vol. 15, Springer, Berlin-Giittingen-Heide!berg, 1963, p. 89. 13 J. DE LA HUERGA, C. YESIKICK AND H. POPPER, Am. j. Clin.Pathcd, 22 (1952) 1126. 14 A. MOGA AND ST. HARAGUS, Ateroscleroza, Ed. Academiei, Bucuresti, 1963. Tg R. HEINECKER AND I. MEYER, K&z. Wochschv., 35 (1957) 340. 16 L. A. CARLSON, Acta Med. Stand., 167 (1960) 377. A@?$. ~Z~~~ZU~Gmet., 23 (1959) 239. 17 W'. KALLOW AND D. B. GUM, 18 C. RICH, E. L. BERMAN AXD I. L. SCHWARTS, J. Clilz. Inwst., 36 (1959) 275. 19 H. LEHMXZJN, J. COOK AND E. Rsax, Pvoc. Ros. Sm. Med., 50 (1957) 147. 20 S. BRODP AND L. .4.CARLSON, Clin.Chim. Acta, 7 (1962) 694. 21 81. NOVAK, V. MELICHAROV, P. HAHN AND 0. KELDOVSKI, J. Lipid Res., 6 (1965) 91. 22 D. S. ROBINSON AND .4.SEAKISS, Biochzm. Biofihys.Acta, 62 (1962) 163. rj A. L. JONES, p\'. B. RUDER&IAN AND 31.G. HERREKA, J. Lipid Res., 8 (19G7) 429. 24 4. MOGA AND M. CUCUIAX, Rev. Roum. iWe’d. Inteme, I (1964) 403. 25 M. I:I?GEI ASD M. RIHARI-\TXRG.A,Chin.Clzirn. Acta, 16 (1967) 371. 26 ‘r. A. POPHSCL:, 3%. CUCUI.-\W, I. ORHA AxD E. POPESCU, St. Ct~cf. E~l~OC~~~#z., 16 (1965) 169. 27 A. RIoca, I. BACIXJ, P. PITEA AND %I. CUCUIANU, Sfudii Cercet. AWed. Irztevn., II (1960) 97, 28 31. CUCTIINU, Thromb. D&h. Haenaovrhh., 16 (1966)087. clilz.
Chim. .4cta,
22
(1968) 151-155
CHIMICA
ACTA
PSEUDOCHOLINESTERASE
IS
IN OBESE
AND HYPERLIPEMIC
SUBJECTS
SUMMARY
It was found that serum pseudocholi~esterase increases not only in obese subjects but also in hyperlipemic patients with normal body weight. A good statistical correlation was found between serum pseudocholinesterase on one hand, and both serum cholesterol and the logarithm of serum triglycerides concentration, on the other. It cannot be stated whether increased pseudocholinesterase activity should be correlated with a possible role of the enzyme in the metabolism of lipids or with an unspecific and rather general stimulation of protein synthesis in the liver of obese and 11~7perlipemicsubjects.
Decrease of serum cholinesterase (pseudocholinesterase) in severe hepatic disease and malnutrition is a well known fact mentioned in various monographs1-3. It was attributed to an impairment of the proteosynthetic function of the liver. The increase of the enzyme reported in obese subjects4, diabetes mellitus5~6, hyperthyroidism’, nephrotic syndromes, psoriasisQ and essential hypertensionlO, is however rather difficult to explain since the physiological role of pseudocholinesterase (PCE) has not yet been established with certainty. According to Clitherow and ~011.11 the principal biological function of PCE should be to hydrolyse the acyl-choline esters and, especially, butyryl-choline that might arise as by-products during the catabolism of free fatty acids and lipogenesis in the liver. Starting from these considerationsll and from those of Augustinsson12 suggesting a linkage between PCE and the metabolism of fats, and also from several incidental observations, PCE was investigated in correlation with serum lipids. MATERIAL
AND
METHODS
Serum cholesterol (Zlatkis et n2.), serum triglycerides (Van Handel and Zilversmit) and PCE activityI were measured photocolorimetrically using a spectrophotometer Jobin-Yvon, in the following groups of subjects. * Director:
Prof.
St. Haragus. C&z. Chim.
AC&L,
22
(1968)
1 jr-155
=52
CUCUIAMJ
et
al.
A. The co&r01 groufi included 76 healthy subjects, 34 males 42 females aged IS-84 years (mean 3s years). Subjects exceeding the ideal weight by more than IO "/:, as well as those with a serum cholesterol level exceeding zoo mgY; were excluded from the control group. It should be mentioned that mean values for serum cholesterol of the Rumanian population were found to be about 170 mg% (ref. 14). R. The obese norrnolipemic group included 37 subjects, 9 males 2S females aged 15573 years (mean 39 years), exceeding the desirable weight by zo-So’): (mean +30(:X) but with a serum cholesterol under 200 rngfa. No severe glandular dyscrasia was observed; IO patients were found to have a diastolic blood pressure over IOO mm Hg, but none of them presented clinical signs of organic vascular disease. C. TJze obese hy~erlipemic ,pozq5 included 46 subjects, 26 males, 20 females, aged 16-72 years (mean 43 years) exceeding the desirable weight by 2oS5 yb (mean +34 y/o)and having a serum cholesterol level over 200 mg yo. Of this group, 22 patients were found to have increased blood pressure, 3 patients presented a mild diabetes mellitus and II had overt atherosclerotic changes. D. TJae hyperlipernic groq5 witlz normal body weight was composed
of 66 subjects,
3g males, 27 females, aged 19-77 years (mean 46 years). Of these, 23 patients had a diastolic blood pressure over IOO mm Hg; in 5 of them a mild diabetes was detected and 16 patients had clinical signs of atherosclerosis. In four patients a carbohydrateinduced hypertriglyceridemia could be found on dietetical grounds; three cases were identified as familial hypercholesterolemia. Though groups C and D were rather heterogeneous, all subjects had increased plasma lipoprotein levels. Also, the mechanisms leading to hyperlipemia remained ill-defined in most subjects with hyperlipemia. Subjects with evident hyperthyroidism or myxedema were not included in the statistical calculations. Also excluded were cases of recent myocardial infarction where a transient decrease of PCE is known to occurls. Cases with liver disease, heart failure, nephritis or nephrotic syndrome were not taken into consideration. The correlation of PCE and serum lipids was studied by means of correlation and regression analysis. In order to reduce the skewness of the distribution of triglycerides and facilitate calculations, the triglyceride values were converted into the corresponding
logarithms
and then reconverted,
as indicated
by Carlson*fl.
RESULTS
As shown in Table I, PCE was found to be increased not only in obese subjects but also in hyperlipemic subjects with normal body weight. Highest values were obtained in four cases with carbohydrate-induced hypertriglyceridemia. There was no significant difference in the PCE level of hyperlipemic patients with increased blood pressure and normotensive hyperlipemic subjects. Also, PCE was not significantly different in subjects with clinical atherosclerosis when compared with hyperlipemic subjects not manifestly affected by atherosclerosis. lower mean level of PCE was found As previously reported’7 a significantly in the 14 control normolipemic subjects over 60 years (222 & S.8). This decrease with age was, however, less evident in the IS hyperlipemic subjects from groups C and D above 60 years (329 A1 15). There was good statistical correlation between PCE and serum cholesterol Clin. Chim. Ada,
~2 (1968) 151-155
PSEUDOCHOLIXESTERASE TABLE
I
SERUM WITH AND
I53
IN OBESE SUBJECTS
CHOLESTEROL
NORMAL
HYPERLIPEMIC
Standard
;z
AND
BODY
Statistical
(A),
SUBJECTS
WITH
OBESE
LEVELS
NORMOLIPEMIC
NORMAL
BODY
IN
CONTROL
SUBJECTS
WEIGHT
(B),
NORMOLIPEMIC OBESE
SUBJECTS
HYPERLIPEMIC
(c)
(D)
error of the mean in brackets.
I67 I75 234 253
76 37 46 66
B C D
PSEUDOCHOLINESTER-4SE
WEIGHT
(i (:I (i (=
2.4)* 2.6) 6) 6.2)
24-l 308 355 33s
(2 (k (+ (&
4.3) 8.6) 7.4) 6.8)
significance
A US. B < 0.001 A US. c < 0.001 A vs. D < 0.001
B vs. C < 0.01 B U.S.D < 0.05 C US. D < 0.02
The remarkably low standard error for serum cholesterol according to their cholesterol levels.
*
(r = 0.574) ; satisfactory
correlation
is due to the selection
(Y = 0.56) was found also between
of the subjects
PCE and the
logarithm of serum triglycerides that were measured in 85 cases. For the same level of serum lipids (see Figs. I, z), there was a greater increase of PCE in obese subjects than in subjects with normal body weight. A discrepancy
Y
550 500
.
1
450 t
15’
i/
100 150
200
250
300
350
400
450
I
500
Fig. I. Correlation between serum cholesterol and pseudocholinesterase. Abscissa : serum cholesterol (mg %) ; ordinate : pseudocholinesterase activity (~moles/ml/h). l Subjects with normal body weight; 0 obese subjects; A subjects with hyperthyroidism; o subjects with hypothyroidism. Y = 0.574; regression equations: y = 0.45x + 212.40; x = 0.73 _Y- 14.38.
between PCE and serum cholesterol was noted in thyroid disease: high levels of PCE were associated with lower cholesterol values in five cases of hyperthyroidism. On the contrary, hypercholesterolemia was not accompanied by increased PCE in three cases of hypothyroidism. Cli+z. Chim. Acta,
22
(1968)
~51-155
‘50.
70 8090
x)0
125
150
200
250
300
400
500
600
L. Correlation between serum triglycerides and pseudocholinest~rase. Abscissa: serum triglycerides (mgq;) logarithmic scale; ordinate: pseudocholinesterase activity (~~moles~rnl~i~). l Subjects with normal body weight; o obese subjects. Y = 0.56; regression equations: y = 211x - 13r; x = 0.0015 y -+ 1.69, where x = logarithm of the triglyceride concentration.
Fig.
Our findings seem to support the hypothesis of Clitherow et aZ.ll about an active role of PCE in the metabolism of lipids. Increased availability and/or flux of unesterified fatty acids to the liver or increased lipogenesis from carbohydrates leads to an increased production of butyryl-coenzyme A, so that butyryl-choline production might also increase. This might be a stimulus for the synthesis or for the activation of PCE whose hydrolytic action on but~~ryl-choline allows the products of hydrolysis to return to their respective metabolic pathways. Hyperlipemia and high levels of PCE are therefore frequently associated as different effects of a common cause. Increased mobilisation of lipids is known to occur in hyperthyroidismr* where a high level of PCE was also noted?. However, high levels of serum cholesterol are uncol~ln~on in this condition, probably as a result of increased elimination of cholesterol and oxidation of lipids. Increased PCE activity should be therefore correlated rather with increased influx of unesteritied fatty acids and their uptake by the liver than with actual level of lipoproteins. It is worth mentioning that PCE is lower in newborn
infantslg
that have also
low levels of serum cholesterol, phospholipids and triglyceridesZo but high concenof trations of free-fatty acids 21. It seems that mechanisms leading to maturation lipogenesis in the liver and biosynthesis of PCE are somehow correlated. However, the increase of PCE as an expression of an unspecific and rather general stimulation of protein synthesis in the liver of obese and hyperlipemic subjects cannot be excluded. Accelerated lipogenesis in the liver implies also increased synthesis of the protein component of lipoproteins. It was found that inhibition of protein synthesis by puronlycin impairs the production of lipoproteins in rats, so that triglycerides accumulate in the livcrze. Also, perfusion of the isolated rat liver with a medium rich in fatty acid stimulated the synthesis of the protein moiety of lipoprotein+. C&z. Chim.
Acta,
22 (1968)
151-155
PSEUDOCHOLINESTERASE
IN OBESE
I55
SUBJECTS
Clinical investigations showed that a high level of ~-lipoproteins is accompanied by an increase of the B-protein and B-glycoprotein component of plasma24~26, and protein-bound hexoses increase not only in atherosclerotic but also in obese subjectP. The higher concentration of the inhibitors of fibrinolysis in atherosclerotic patients with high levels of /LlipoproteinP could also be related with increased protein synthesis in the liver, since serum lipids were not found to inhibit fibrinolysis by themseIveszR. REFERENCES I R. ABDERHALDEN, Klinische Enqfmologie, G. Thieme, Stuttgart, 1958. 2 B. RICHTERICH, Enz~mopathologie,Springer, Berlin--GSttingen-Heidelberg, 1958. 3 H. XV. GOEDDE, A. DOENICKE AND Ii.ALTLAND, Psezrdocholinestevase?z ~PIa~r~~kogenet~k, Bio-
cke~z~e,I~l~~~k~, Springer,Berlin-I-leidelberge~~York, 1967. 4 W. T. C. BERRY, P. J. COWIN AND D. R. D-AWES, &it. J. Xl&., 8 (1954) 79. 5 W. ANTOPOL, L. TIJCHMAN AND SCHIFRIN, Proc. Sot. Exptl.Biol. Med., 36 (1937) 46. 6 RI.FABER, Acta Med. &and., 114 (1943) 72. 7 J. c. THOMPSON AND 1\/1. ~HITTAKER, j. Clin.PUthOl.,18 (1965) 811. 8 N. STEFENELLI, K&n. Wochschr., 39 (1961) 1019. 9 L. HELMECZY AND E. NAGY, Acta Med. Acad. Sci. Hwag, 5 (x954) 109. IO R. RHEINFRANK AND H. I. WHETSTONE, ~~~t~oyd Hos$.BulL., 31 (1958) 32. or J. W. CLITHEROW, &I.MITCHARI~ AND N. J. HARPEK, h’ature, 199 (1963) 1000. I I I<. B. XUGUSTINSSON, in G. B. KOELLE (Ed.),Handbarch dev enperime~~tellen Pharnzakotogie, Vol. 15, Springer, Berlin-Giittingen-Heide!berg, 1963, p. 89. 13 J. DE LA HUERGA, C. YESIKICK AND H. POPPER, Am. j. Clin.Pathcd, 22 (1952) 1126. 14 A. MOGA AND ST. HARAGUS, Ateroscleroza, Ed. Academiei, Bucuresti, 1963. Tg R. HEINECKER AND I. MEYER, K&z. Wochschv., 35 (1957) 340. 16 L. A. CARLSON, Acta Med. Stand., 167 (1960) 377. A@?$. ~Z~~~ZU~Gmet., 23 (1959) 239. 17 W'. KALLOW AND D. B. GUM, 18 C. RICH, E. L. BERMAN AXD I. L. SCHWARTS, J. Clilz. Inwst., 36 (1959) 275. 19 H. LEHMXZJN, J. COOK AND E. Rsax, Pvoc. Ros. Sm. Med., 50 (1957) 147. 20 S. BRODP AND L. .4.CARLSON, Clin.Chim. Acta, 7 (1962) 694. 21 81. NOVAK, V. MELICHAROV, P. HAHN AND 0. KELDOVSKI, J. Lipid Res., 6 (1965) 91. 22 D. S. ROBINSON AND .4.SEAKISS, Biochzm. Biofihys.Acta, 62 (1962) 163. rj A. L. JONES, p\'. B. RUDER&IAN AND 31.G. HERREKA, J. Lipid Res., 8 (19G7) 429. 24 4. MOGA AND M. CUCUIAX, Rev. Roum. iWe’d. Inteme, I (1964) 403. 25 M. I:I?GEI ASD M. RIHARI-\TXRG.A,Chin.Clzirn. Acta, 16 (1967) 371. 26 ‘r. A. POPHSCL:, 3%. CUCUI.-\W, I. ORHA AxD E. POPESCU, St. Ct~cf. E~l~OC~~~#z., 16 (1965) 169. 27 A. RIoca, I. BACIXJ, P. PITEA AND %I. CUCUIANU, Sfudii Cercet. AWed. Irztevn., II (1960) 97, 28 31. CUCTIINU, Thromb. D&h. Haenaovrhh., 16 (1966)087. clilz.
Chim. .4cta,
22
(1968) 151-155