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Coenzyme a content of human arterial and venous tissue
JOURNAL OF ATHEROSCLEROSIS RESEARCH
497
COENZYME A C O N T E N T OF HUMAN A R T E R I A L AND VENOUS T I S S U E
R. S A N W A L D AND J. E. K I R K
Division of Gerontology, Washington University School of Medicine, St. Louis, Mo. (U.S.A.) (Received September 21, 1964)
INTRODUCTION
Coenzyme A is an essential factor involved in lipid synthesis and other intermediary metabolic reactions, but no studies have previously been reported on the coenzyme A content of human vascular tissue. Investigations by PAOLETTI et al.1, ~ have shown t h a t animal species which are most susceptible to atherosclerosis have the highest coenzyme A concentrations in the arterial tissue. It was further demonstrated b y in vitro experiments that animal aortas with the greatest coenzyme A levels exhibited the highest rate of lipid synthesis from 14C-labelled acetate. Since lipid deposit in the arterial wall is a conspicuous aspect of atherosclerosis in h u m a n subjects it was considered advisable to determine the concentration of coenzyme A in various types of h u m a n blood vessels. In the investigation reported in the present paper, coenzyme A determinations were made on a total of 257 samples of h u m a n vascular tissue, including mainly specimens of the thoracic descending aorta, pulmonary artery, and coronary artery. For comparative purposes, a limited number of analyses were also performed on the ascending aorta, abdominal aorta, and inferior vena cava. In order to evaluate the correlation between atherosclerosis and tissue coenzyme A concentration, the measurements were made separately on normal and atherosclerotic tissue portions and distinction was made between lipid and fibrous atherosclerotic plaques. All analyses were performed on homogenates from intima-media layers of vascular specimens obtained fresh at autopsy. METHODS
The assay of coenzyme A in human vascular samples is more complicated t h a n the determination of this compound in animal tissue specimens because h u m a n arterial samples usually are not available immediately after death. I t is an established fact t h a t an appreciable portion of coenzyme A present in tissues is split quickly after death b y autolysis with release of pantothenic acid; for that reason, the rather simple acetylation procedure cannot be applied to human vascular samples. Quantij . Atheroscler. Res., 5 (1965) 497-503
498
R. SANWALD, J. E. KIRK
tative measurements of coenzyme A in h u m a n arterial specimens m u s t therefore be made on the basis of microbiological pantothenic acid analyses. Although t h a t procedure is rather time-consuming it has the a d v a n t a g e of being very sensitive and therefore permits reliable assay of the pantothenic acid content of tissues like vascular specimens which have a comparatively low coenzyme A content. I t has been reported b y NOVELLI et al.~ that nearly all pantothenic acid in tissues is present as a component of coenzyme A. To express measured pantothenic acid values as units of coenzyme A, recorded #g of pantothenic acid are multiplied b y the factor of 1.67. The procedure employed in the present experiments for enzymic release of pantothenic acid from coenzyme A is a modification of the technique outlined b y NOVELLI4. Aqueous homogenates of vascular samples were prepared with the use of a Kontes Dual type tissue grinder. Quantitative liberation of pantothenic acid from tissue coenzyme A was achieved b y t r e a t m e n t of the homogenates with intestinal alkaline phosphatase and with peptidase obtained from an acetone powder of hog kidney extract. Since it is of great importance to reduce the blank values in pantothenic acid assay tests, only resin-treated kidney acetone powder was used (Pentex Inc., Kankakee, Ili.). The activity of the employed alkaline phosphatase preparation was assayed b y the procedure of SCHMIDT AND THANNHAUSER5; 42 phosphatase units were used per g r a m of fresh vascular tissue. The efficiency of the hog kidney peptidase-like enzyme was determined as described b y NOVELLI4 b y testing its ability to release pantothenic acid from coenzyme A (Sigma Chemical Co., St. Louis) in the presence of an excess of alkaline phosphatase; 35 mg of hog kidney powder/g arterial tissue was found appropriate. The enzymic t r e a t m e n t of the vascular homogenates was accomplished b y incubating the samples in 0.135 M Tris buffer, p H 8.2, at 37°C for 6 h in a shaking water bath. A phosphatase and hog kidney powder blank in which the tissue homogenate was substituted with a similar volume of redistilled water, was run with each set of samples. At the end of the incubation period, the tissue samples and blanks were diluted 1 : 10 with redistilled water, boiled for 3 rain to stop the enzyme reaction, and then filtered through W h a t m a n No. 2 filter paper. The filtrates were subsequently centrifuged for 5 min at 25,000 r.p.m. For pantothenic acid determination in the resulting supernatants, the microbiological Lactobacillus plantarum (ATTC No. 8014) m e t h o d was employed as described b y SKEGGS AND WRIGHT6 and b y THE ASSOCIATION OF OFFICIAL AGRICULTURAL CHEMISTS7. Only newly purchased test tubes, carefully cleansed with redistilled water were used in t h a t procedure. Agar culture medium, inoculum broth, and p a n t o t h e n a t e assay medium were obtained from Difco Labs. Inc., Detroit. A pantothenic acid standard curve was prepared with each set of determinations (Fig. 1). All vascular samples and blanks were analysed in triplicate at two different concentrations, requiring a total of six inoculated sample tubes for each microbiological pantothenic acid determination. The growth of Lactobacillus plantarum was measured turbidimetrically after 20 hours' incubation of the tubes at 37°C; the optical density readings were made at 640 m/z with a B e c k m a n D U spectrophotometer. In order to ]. Atheroscler. Res., 5 (1965) 497-503
COENZYME A CONTENT OF HUMAN ARTERIAL AND VENOUS TISSUE
499
check the efficiency of the t o t a l analytical procedure outlined in the present paper, quantities of a freshly p r e p a r e d standard coenzyme A (Sigma Chemical Co., St. Louis) solution were added to original tissue homogenates; the coenzyme A content of the commercial preparation was verified through assay with the fl-hydroxyacyl dehy0. D.
iiilt
/
o3o0 • 0.200--
/
/
*
0 I00
I
OOt
I
I
0.03
I
I
I
~
I
0,05 0.07 Pontothenicocicl (H9)
Fig. 1. P a n t o t h e n i c a c i d s t a n d a r d c u r v e for Lactobacillus plantarum.
drogenase m e t h o d s. I t was ascertained t h a t after enzymic t r e a t m e n t of the samples with alkaline phosphatase and hog kidney extract, subsequent microbiological measurement of the released pantothenic acid revealed q u a n t i t a t i v e recovery of the added coenzyme A. RESULTS
Normal samples The average coenzyme A concentrations found for the i n t i m a - m e d i a layers of various types of blood vessels are listed in Table I. The mean value of 5.50 U/g of tissue observed for 100 samples of the descending thoracic a o r t a is definitely higher t h a n t h a t reported b y KAPLAN AND LIPMANN9 for h u m a n red blood cells (3-4 U/g). The assays of different a n a t o m i c a l sections of the aorta indicate a tendency toward an increase in the coenzyme A level from the ascending to the abdominal aorta. A comparison of values recorded for pulmonary artery and aortic specimens from the same subjects revealed distinctly higher concentrations in the p u l m o n a r y artery, the average coenzyme A content of that blood vessel being 129 % (wet tissue; t, 3.38) a n d 137 % (tissue nitrogen; t, 4.12) of the value observed for the aorta. Even higher coenzyme A levels were recorded for the coronary a r t e r y and inferior vena cava. The mean coenzyme A concentrations of the thoracic descending aorta found for various age groups are presented in Table II. The rather large n u m b e r of samples J. Atheroscler. Res., 4 (1965) 4 9 7 - 5 0 3
R. SANWALD, J. E. K I R K
500 TABLE I COENZYME
A CONTENT
OF HUMAN VASCULAR TISSUE
Coenzyme A (U/g) *
Number
Vascular samples
of
wet tissue
tissue nitrogen
8
4.37 4- 0.28
106 4-
100 29 10
5.50 -t- 0.29 4.78 4- 0.45 2.79 ± 0.67
139 4- 7.4 141 4- 13.3 83 4- 19.7
17 7 33
7.28 4- 0.71 5.89 4- 0.75 7.03 4- 0.49
186 4- 17.6 182 4- 21.2 197 4- 13.1
25 14 14
8.15 4- 0.61 7.30 4- 0.74 7.60 4- 0.66
229 4- 18.1 223 4- 21.5 196 4- 15.3
samples
Ascending aorta, normal Descending thoracic aorta Normal A t h e r o s c l e r o t i c , lipid A t h e r o s c l e r o t i c , fibrous Abdominal aorta Normal A t h e r o s c l e r o t i c , lipid Pulmonary artery, normal Coronary artery Normal A t h e r o s c l e r o t i c , lipid V e n a c a v a inferior
7.0
* M e a n v a l u e s 4- s t a n d a r d error of t h e m e a n .
TABLE II COENZYME
A CONTENT
OF NORMAL THORACIC DESCENDING
Age group (years)
Number of of samples
Coenzyme A (U/g) wet tissue
tissue nitrogen
0- 9 10-19 20-29 30-39 40-49 50-59 60-69 70-79 80-85
10 1 15 17 17 24 9 5 2
7.74 6.44 4.72 4.35 5.64 6.10 4.62 5.42 5.35
166 127 110 108 147 161 126 161 147
4- 1.32 4-t4444-
0.61 0.53 0.68 0.64 1.05 1.26
AORTA IN VARIOUS
AGE GROUPS
4- 32.0 444444-
13.5 13.3 16.3 15.8 31.5 38.6
~6
Age (years) Fig. 2. V a r i a t i o n w i t h age in c o e n z y m e A c o n t e n t of n o r m a l a o r t i c t i s s u e (N = 100).
j . Atheroscler. Res., 5 (1965) 4 9 7 - 5 0 3
COENZYME A CONTENT OF HUMAN ARTERIAL AND VENOUS TISSUE
501
included in the s t u d y made it possible to evaluate in detail the relationship between age and tissue coenzyme A content. Analysis of the data showed a tendency for the coenzyme A level to decrease from early childhood to the end of the thirties, whereas no significant variation in aortic coenzyme A values was observed for age groups above 40 years (Table III, Fig. 2). The coefficients of correlation between age and TABLE III COEFFICIENTS
OF
CORRELATION
BETWEEN
Age group (years)
Age/normal aorta
Age/atherosclerotic a o r t a , lipid Age/pulmonary artery Age/normal coronary artery
AGE
AND
COENZYME
A CONTENT
OF
ARTERIAL
Number of samples
Wet tissue
Tissue nitrogen
r
t
r
t
0-85 0-39 40-85 18-85
100 43 57 90
- - 0.09 --0.43 --0.05 + 0.10
0.90 3.07 0.37 1.03
+ 0.07 --0.35 0.00 + 0.20
0.69 2.40 0.00 1.91
18-85 0-85 18-85
29 33 31
--0.16 --0.11 --0.04
0.85 0.57 0.22
+ 0.04 0.00 + 0.02
0.21 0.00 0.11
0-85 18-85
25 23
+ 0.19 + 0.20
0.92 0.93
+ 0.20 + 0.28
0.97 1.34
TISSUE
TABLE IV COENZYME CONTENT
A
CONTENT
OF NORMAL
Age group (years)
OF
ATHEROSCLEROTIC
TISSUE
OF THE
SAME
TISSUE
AORTIC
PORTIONS
EXPRESSED
IN
PERCENTAGES
OF
SAMPLES
Number of samples
Wet tissue
Tissue nitrogen
%
t
%
t
12 17 29
96.5 93.0 94.5
0.27 0.84 0.79
109.0 98.9 102.6
0.56 O. 12 0.32
54.5
4.50
64.5
3.50
L i p i d changes 18-49 50-85
Total
Fibrous changes 18-85
10
coenzyme A content of the p u l m o n a r y artery and coronary artery (Table III) were not statistically significant, neither when calculated for both children and adults (0~85 years) nor for adults only (18-85 years). Atherosclerotic samples
The results of analyses of atherosclerotic tissue samples are listed in Table I. j . Atheroscler. Res., 5 (1965) 497-503
502
R. SANWALD,
j. E. KIRK
Comparison of coenzyme A concentrations in normal aortic tissue portions and in pathological areas of the vascular wall consisting mainly of lipid deposits showed essentially similar values (Table IV). In contrast to this, m a r k e d l y lower coenzyme A concentrations were found in fibrous atherosclerotic tissue portions of the aorta. DISCUSSION
The mean coenzyme A value of 5.50 U/g of h u m a n aortic tissue observed in the present investigation is distinctly lower t h a n the average concentrations reported b y PAOLETTI et al.1, ~ for animal aortas. The differences between h u m a n and animal coenzyme A values are presented in Table V, which demonstrates t h a t the h u m a n coenzyme A content is only one-third to one-half of the concentrations recorded for chicken, pigeon, rat, hamster, guinea pig, and rabbit aortas. On the basis of the conclusions b y PAOLETTI et al. 1,2 about a relationship between arterial coenzyme A levels and susceptibility to atherosclerosis, our findings might suggest t h a t the h u m a n aorta is less susceptible to lipid deposits t h a n aortas of animals reported in the table.
TABLE V C O E N Z Y M E A C O N C E N T R A T I O N S OF A O R T I C T I S S U E I N V A R I O U S S P E C I E S *
Species
Coenzyme A (U/g wet tissue)
Human Pigeon Rat Hamster Guinea pig Rabbit Chicken
5.5 9.3 9.5 10.1 12.1 17.3 18.0
* Animal values reported by Kilt et al. 2
B u t because the human species displays a m u c h higher longevity t h a n the studied animals, a comparative evaluation of the relationship between the coenzyme A content of arteries in humans and animals and the tendency to atherogenesis does not seem possible at the present stage of knowledge. ACKNOWLEDGEMENTS The authors are indebted to Dr. Lyle Morris, Difco Labs. Inc., Detroit, for valuable instruction about the microbiological pantothenic acid procedure. The investigation was supported b y St. Louis H e a r t Association and b y a grant from the National Institutes of Health, Public H e a l t h Service (PHS-891). J. Atheroscler. Res., S (1965) 497-503
COENZYME A C O N T E N T OF H U M A N A R T E R I A L AND VENOUS T I S S U E
503
SUMMARY
D e t e r m i n a t i o n s were m a d e of the c o e n z y m e A c o n c e n t r a t i o n i n various t y p e s of h u m a n b l o o d vessels. T h e c o e n z y m e A analyses were p e r f o r m e d b y assay of p a n t o t h e n i c acid l i b e r a t e d t h r o u g h t r e a t m e n t of tissue samples w i t h purified i n t e s t i n a l alkaline p h o s p h a t a s e a n d a c e t o n e powder of hog k i d n e y e x t r a c t . T h e q u a n t i t a t i v e l y released p a n t o t h e n i c a c i d was m e a s u r e d microbiologically b y t h e Lactobacillus p l a n t a r u m pl ocedure. A t o t a l of 257 v a s c u l a r samples were i n c l u d e d in the study. The m e a n c o e n z y m e A c o n c e n t r a t i o n s o b s e r v e d for i n t i m a - m e d i a layers of n o r m a l tissue p o r t i o n s expressed as U / g of wet tissue, were: ascending aorta, 4.37; d e s c e n d i n g thoracic a o r t a , 5.50; a b d o m i n a l a o r t a , 7.28; p u l m o n a r y artery, 7.03; c o r o n a r y a r t e l y , 8.15; a n d inferior v e n a cava, 7.60. A n a l y s e s of atherosclerotic tissue p o r t i o n s consisting m a i n l y of lipid deposits showed e s s e n t i a l l y similar coenzyme A levels as in the n o r m a l tissue, whereas m a r k e d l y lower v a l u e s wele recorded for fibrous atherosclerotic plaques. REFERENCES 1 2 3 4 5 8 7
R. PAOLETTI,L. TESSARIAND R. VERTUA, Ric. Sci., 29 (1959) 2382. j. j. KIM, R. PAOLETTIAND R. VERTUA,Atompraxis, 6 (1960) 55. G. D. NOVELLI, N. O. KAPLANAND F. LIPMANN, J. Biol. Chem., 177 (1949) 97. G. D. NOVELLI, Methods Biochem. Anal., 2 (1955) 182. G. SCHMIDTAND S. J. THANNHAUSER,J. Biol. Chem., 149 (1943) 369. H. R. SKEC.C,SAND L. D. WRIGHT, J. Biol. Chem., 156 (1944) 21. Official Method of Analysis of the Association of Official Agricultural Chemists, 9th edit., 1960. 8 G . 1V[ICHALAND H.-U. BERC,MEYER, Biochim. Biophys. Acta, 67 (1963) 599. 9 N. O. K~,PLANAND F. LIPMANN, .[. Biol. Chem., 174 (1948) 37. j . Atheroscler. Res., 5 (1965) 497-503
497
COENZYME A C O N T E N T OF HUMAN A R T E R I A L AND VENOUS T I S S U E
R. S A N W A L D AND J. E. K I R K
Division of Gerontology, Washington University School of Medicine, St. Louis, Mo. (U.S.A.) (Received September 21, 1964)
INTRODUCTION
Coenzyme A is an essential factor involved in lipid synthesis and other intermediary metabolic reactions, but no studies have previously been reported on the coenzyme A content of human vascular tissue. Investigations by PAOLETTI et al.1, ~ have shown t h a t animal species which are most susceptible to atherosclerosis have the highest coenzyme A concentrations in the arterial tissue. It was further demonstrated b y in vitro experiments that animal aortas with the greatest coenzyme A levels exhibited the highest rate of lipid synthesis from 14C-labelled acetate. Since lipid deposit in the arterial wall is a conspicuous aspect of atherosclerosis in h u m a n subjects it was considered advisable to determine the concentration of coenzyme A in various types of h u m a n blood vessels. In the investigation reported in the present paper, coenzyme A determinations were made on a total of 257 samples of h u m a n vascular tissue, including mainly specimens of the thoracic descending aorta, pulmonary artery, and coronary artery. For comparative purposes, a limited number of analyses were also performed on the ascending aorta, abdominal aorta, and inferior vena cava. In order to evaluate the correlation between atherosclerosis and tissue coenzyme A concentration, the measurements were made separately on normal and atherosclerotic tissue portions and distinction was made between lipid and fibrous atherosclerotic plaques. All analyses were performed on homogenates from intima-media layers of vascular specimens obtained fresh at autopsy. METHODS
The assay of coenzyme A in human vascular samples is more complicated t h a n the determination of this compound in animal tissue specimens because h u m a n arterial samples usually are not available immediately after death. I t is an established fact t h a t an appreciable portion of coenzyme A present in tissues is split quickly after death b y autolysis with release of pantothenic acid; for that reason, the rather simple acetylation procedure cannot be applied to human vascular samples. Quantij . Atheroscler. Res., 5 (1965) 497-503
498
R. SANWALD, J. E. KIRK
tative measurements of coenzyme A in h u m a n arterial specimens m u s t therefore be made on the basis of microbiological pantothenic acid analyses. Although t h a t procedure is rather time-consuming it has the a d v a n t a g e of being very sensitive and therefore permits reliable assay of the pantothenic acid content of tissues like vascular specimens which have a comparatively low coenzyme A content. I t has been reported b y NOVELLI et al.~ that nearly all pantothenic acid in tissues is present as a component of coenzyme A. To express measured pantothenic acid values as units of coenzyme A, recorded #g of pantothenic acid are multiplied b y the factor of 1.67. The procedure employed in the present experiments for enzymic release of pantothenic acid from coenzyme A is a modification of the technique outlined b y NOVELLI4. Aqueous homogenates of vascular samples were prepared with the use of a Kontes Dual type tissue grinder. Quantitative liberation of pantothenic acid from tissue coenzyme A was achieved b y t r e a t m e n t of the homogenates with intestinal alkaline phosphatase and with peptidase obtained from an acetone powder of hog kidney extract. Since it is of great importance to reduce the blank values in pantothenic acid assay tests, only resin-treated kidney acetone powder was used (Pentex Inc., Kankakee, Ili.). The activity of the employed alkaline phosphatase preparation was assayed b y the procedure of SCHMIDT AND THANNHAUSER5; 42 phosphatase units were used per g r a m of fresh vascular tissue. The efficiency of the hog kidney peptidase-like enzyme was determined as described b y NOVELLI4 b y testing its ability to release pantothenic acid from coenzyme A (Sigma Chemical Co., St. Louis) in the presence of an excess of alkaline phosphatase; 35 mg of hog kidney powder/g arterial tissue was found appropriate. The enzymic t r e a t m e n t of the vascular homogenates was accomplished b y incubating the samples in 0.135 M Tris buffer, p H 8.2, at 37°C for 6 h in a shaking water bath. A phosphatase and hog kidney powder blank in which the tissue homogenate was substituted with a similar volume of redistilled water, was run with each set of samples. At the end of the incubation period, the tissue samples and blanks were diluted 1 : 10 with redistilled water, boiled for 3 rain to stop the enzyme reaction, and then filtered through W h a t m a n No. 2 filter paper. The filtrates were subsequently centrifuged for 5 min at 25,000 r.p.m. For pantothenic acid determination in the resulting supernatants, the microbiological Lactobacillus plantarum (ATTC No. 8014) m e t h o d was employed as described b y SKEGGS AND WRIGHT6 and b y THE ASSOCIATION OF OFFICIAL AGRICULTURAL CHEMISTS7. Only newly purchased test tubes, carefully cleansed with redistilled water were used in t h a t procedure. Agar culture medium, inoculum broth, and p a n t o t h e n a t e assay medium were obtained from Difco Labs. Inc., Detroit. A pantothenic acid standard curve was prepared with each set of determinations (Fig. 1). All vascular samples and blanks were analysed in triplicate at two different concentrations, requiring a total of six inoculated sample tubes for each microbiological pantothenic acid determination. The growth of Lactobacillus plantarum was measured turbidimetrically after 20 hours' incubation of the tubes at 37°C; the optical density readings were made at 640 m/z with a B e c k m a n D U spectrophotometer. In order to ]. Atheroscler. Res., 5 (1965) 497-503
COENZYME A CONTENT OF HUMAN ARTERIAL AND VENOUS TISSUE
499
check the efficiency of the t o t a l analytical procedure outlined in the present paper, quantities of a freshly p r e p a r e d standard coenzyme A (Sigma Chemical Co., St. Louis) solution were added to original tissue homogenates; the coenzyme A content of the commercial preparation was verified through assay with the fl-hydroxyacyl dehy0. D.
iiilt
/
o3o0 • 0.200--
/
/
*
0 I00
I
OOt
I
I
0.03
I
I
I
~
I
0,05 0.07 Pontothenicocicl (H9)
Fig. 1. P a n t o t h e n i c a c i d s t a n d a r d c u r v e for Lactobacillus plantarum.
drogenase m e t h o d s. I t was ascertained t h a t after enzymic t r e a t m e n t of the samples with alkaline phosphatase and hog kidney extract, subsequent microbiological measurement of the released pantothenic acid revealed q u a n t i t a t i v e recovery of the added coenzyme A. RESULTS
Normal samples The average coenzyme A concentrations found for the i n t i m a - m e d i a layers of various types of blood vessels are listed in Table I. The mean value of 5.50 U/g of tissue observed for 100 samples of the descending thoracic a o r t a is definitely higher t h a n t h a t reported b y KAPLAN AND LIPMANN9 for h u m a n red blood cells (3-4 U/g). The assays of different a n a t o m i c a l sections of the aorta indicate a tendency toward an increase in the coenzyme A level from the ascending to the abdominal aorta. A comparison of values recorded for pulmonary artery and aortic specimens from the same subjects revealed distinctly higher concentrations in the p u l m o n a r y artery, the average coenzyme A content of that blood vessel being 129 % (wet tissue; t, 3.38) a n d 137 % (tissue nitrogen; t, 4.12) of the value observed for the aorta. Even higher coenzyme A levels were recorded for the coronary a r t e r y and inferior vena cava. The mean coenzyme A concentrations of the thoracic descending aorta found for various age groups are presented in Table II. The rather large n u m b e r of samples J. Atheroscler. Res., 4 (1965) 4 9 7 - 5 0 3
R. SANWALD, J. E. K I R K
500 TABLE I COENZYME
A CONTENT
OF HUMAN VASCULAR TISSUE
Coenzyme A (U/g) *
Number
Vascular samples
of
wet tissue
tissue nitrogen
8
4.37 4- 0.28
106 4-
100 29 10
5.50 -t- 0.29 4.78 4- 0.45 2.79 ± 0.67
139 4- 7.4 141 4- 13.3 83 4- 19.7
17 7 33
7.28 4- 0.71 5.89 4- 0.75 7.03 4- 0.49
186 4- 17.6 182 4- 21.2 197 4- 13.1
25 14 14
8.15 4- 0.61 7.30 4- 0.74 7.60 4- 0.66
229 4- 18.1 223 4- 21.5 196 4- 15.3
samples
Ascending aorta, normal Descending thoracic aorta Normal A t h e r o s c l e r o t i c , lipid A t h e r o s c l e r o t i c , fibrous Abdominal aorta Normal A t h e r o s c l e r o t i c , lipid Pulmonary artery, normal Coronary artery Normal A t h e r o s c l e r o t i c , lipid V e n a c a v a inferior
7.0
* M e a n v a l u e s 4- s t a n d a r d error of t h e m e a n .
TABLE II COENZYME
A CONTENT
OF NORMAL THORACIC DESCENDING
Age group (years)
Number of of samples
Coenzyme A (U/g) wet tissue
tissue nitrogen
0- 9 10-19 20-29 30-39 40-49 50-59 60-69 70-79 80-85
10 1 15 17 17 24 9 5 2
7.74 6.44 4.72 4.35 5.64 6.10 4.62 5.42 5.35
166 127 110 108 147 161 126 161 147
4- 1.32 4-t4444-
0.61 0.53 0.68 0.64 1.05 1.26
AORTA IN VARIOUS
AGE GROUPS
4- 32.0 444444-
13.5 13.3 16.3 15.8 31.5 38.6
~6
Age (years) Fig. 2. V a r i a t i o n w i t h age in c o e n z y m e A c o n t e n t of n o r m a l a o r t i c t i s s u e (N = 100).
j . Atheroscler. Res., 5 (1965) 4 9 7 - 5 0 3
COENZYME A CONTENT OF HUMAN ARTERIAL AND VENOUS TISSUE
501
included in the s t u d y made it possible to evaluate in detail the relationship between age and tissue coenzyme A content. Analysis of the data showed a tendency for the coenzyme A level to decrease from early childhood to the end of the thirties, whereas no significant variation in aortic coenzyme A values was observed for age groups above 40 years (Table III, Fig. 2). The coefficients of correlation between age and TABLE III COEFFICIENTS
OF
CORRELATION
BETWEEN
Age group (years)
Age/normal aorta
Age/atherosclerotic a o r t a , lipid Age/pulmonary artery Age/normal coronary artery
AGE
AND
COENZYME
A CONTENT
OF
ARTERIAL
Number of samples
Wet tissue
Tissue nitrogen
r
t
r
t
0-85 0-39 40-85 18-85
100 43 57 90
- - 0.09 --0.43 --0.05 + 0.10
0.90 3.07 0.37 1.03
+ 0.07 --0.35 0.00 + 0.20
0.69 2.40 0.00 1.91
18-85 0-85 18-85
29 33 31
--0.16 --0.11 --0.04
0.85 0.57 0.22
+ 0.04 0.00 + 0.02
0.21 0.00 0.11
0-85 18-85
25 23
+ 0.19 + 0.20
0.92 0.93
+ 0.20 + 0.28
0.97 1.34
TISSUE
TABLE IV COENZYME CONTENT
A
CONTENT
OF NORMAL
Age group (years)
OF
ATHEROSCLEROTIC
TISSUE
OF THE
SAME
TISSUE
AORTIC
PORTIONS
EXPRESSED
IN
PERCENTAGES
OF
SAMPLES
Number of samples
Wet tissue
Tissue nitrogen
%
t
%
t
12 17 29
96.5 93.0 94.5
0.27 0.84 0.79
109.0 98.9 102.6
0.56 O. 12 0.32
54.5
4.50
64.5
3.50
L i p i d changes 18-49 50-85
Total
Fibrous changes 18-85
10
coenzyme A content of the p u l m o n a r y artery and coronary artery (Table III) were not statistically significant, neither when calculated for both children and adults (0~85 years) nor for adults only (18-85 years). Atherosclerotic samples
The results of analyses of atherosclerotic tissue samples are listed in Table I. j . Atheroscler. Res., 5 (1965) 497-503
502
R. SANWALD,
j. E. KIRK
Comparison of coenzyme A concentrations in normal aortic tissue portions and in pathological areas of the vascular wall consisting mainly of lipid deposits showed essentially similar values (Table IV). In contrast to this, m a r k e d l y lower coenzyme A concentrations were found in fibrous atherosclerotic tissue portions of the aorta. DISCUSSION
The mean coenzyme A value of 5.50 U/g of h u m a n aortic tissue observed in the present investigation is distinctly lower t h a n the average concentrations reported b y PAOLETTI et al.1, ~ for animal aortas. The differences between h u m a n and animal coenzyme A values are presented in Table V, which demonstrates t h a t the h u m a n coenzyme A content is only one-third to one-half of the concentrations recorded for chicken, pigeon, rat, hamster, guinea pig, and rabbit aortas. On the basis of the conclusions b y PAOLETTI et al. 1,2 about a relationship between arterial coenzyme A levels and susceptibility to atherosclerosis, our findings might suggest t h a t the h u m a n aorta is less susceptible to lipid deposits t h a n aortas of animals reported in the table.
TABLE V C O E N Z Y M E A C O N C E N T R A T I O N S OF A O R T I C T I S S U E I N V A R I O U S S P E C I E S *
Species
Coenzyme A (U/g wet tissue)
Human Pigeon Rat Hamster Guinea pig Rabbit Chicken
5.5 9.3 9.5 10.1 12.1 17.3 18.0
* Animal values reported by Kilt et al. 2
B u t because the human species displays a m u c h higher longevity t h a n the studied animals, a comparative evaluation of the relationship between the coenzyme A content of arteries in humans and animals and the tendency to atherogenesis does not seem possible at the present stage of knowledge. ACKNOWLEDGEMENTS The authors are indebted to Dr. Lyle Morris, Difco Labs. Inc., Detroit, for valuable instruction about the microbiological pantothenic acid procedure. The investigation was supported b y St. Louis H e a r t Association and b y a grant from the National Institutes of Health, Public H e a l t h Service (PHS-891). J. Atheroscler. Res., S (1965) 497-503
COENZYME A C O N T E N T OF H U M A N A R T E R I A L AND VENOUS T I S S U E
503
SUMMARY
D e t e r m i n a t i o n s were m a d e of the c o e n z y m e A c o n c e n t r a t i o n i n various t y p e s of h u m a n b l o o d vessels. T h e c o e n z y m e A analyses were p e r f o r m e d b y assay of p a n t o t h e n i c acid l i b e r a t e d t h r o u g h t r e a t m e n t of tissue samples w i t h purified i n t e s t i n a l alkaline p h o s p h a t a s e a n d a c e t o n e powder of hog k i d n e y e x t r a c t . T h e q u a n t i t a t i v e l y released p a n t o t h e n i c a c i d was m e a s u r e d microbiologically b y t h e Lactobacillus p l a n t a r u m pl ocedure. A t o t a l of 257 v a s c u l a r samples were i n c l u d e d in the study. The m e a n c o e n z y m e A c o n c e n t r a t i o n s o b s e r v e d for i n t i m a - m e d i a layers of n o r m a l tissue p o r t i o n s expressed as U / g of wet tissue, were: ascending aorta, 4.37; d e s c e n d i n g thoracic a o r t a , 5.50; a b d o m i n a l a o r t a , 7.28; p u l m o n a r y artery, 7.03; c o r o n a r y a r t e l y , 8.15; a n d inferior v e n a cava, 7.60. A n a l y s e s of atherosclerotic tissue p o r t i o n s consisting m a i n l y of lipid deposits showed e s s e n t i a l l y similar coenzyme A levels as in the n o r m a l tissue, whereas m a r k e d l y lower v a l u e s wele recorded for fibrous atherosclerotic plaques. REFERENCES 1 2 3 4 5 8 7
R. PAOLETTI,L. TESSARIAND R. VERTUA, Ric. Sci., 29 (1959) 2382. j. j. KIM, R. PAOLETTIAND R. VERTUA,Atompraxis, 6 (1960) 55. G. D. NOVELLI, N. O. KAPLANAND F. LIPMANN, J. Biol. Chem., 177 (1949) 97. G. D. NOVELLI, Methods Biochem. Anal., 2 (1955) 182. G. SCHMIDTAND S. J. THANNHAUSER,J. Biol. Chem., 149 (1943) 369. H. R. SKEC.C,SAND L. D. WRIGHT, J. Biol. Chem., 156 (1944) 21. Official Method of Analysis of the Association of Official Agricultural Chemists, 9th edit., 1960. 8 G . 1V[ICHALAND H.-U. BERC,MEYER, Biochim. Biophys. Acta, 67 (1963) 599. 9 N. O. K~,PLANAND F. LIPMANN, .[. Biol. Chem., 174 (1948) 37. j . Atheroscler. Res., 5 (1965) 497-503