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Biotin content of human arterial and venous tissue
Journal of A therosclerosis Research Elsevier Publishing Company, A m s t e r d a m - Printed in The N e t h e r l a n d s
B I O T I N CONTENT OF HUMAN A R T E R I A L AND VENOUS T I S S U E
j . E. K I R K AND R. S A N W A L D
Division of Gerontology, Washington University School of Medicine, St. Louis, Mo. (U.S.A.) (Received D e c e m b e r 14th, 1965)
INTRODUCTION
Biotin is a coenzyme involved in f a t t y acid synthesis and other significant intermediary metabolic reactions, but no studies have previously been reported on the biotin content of vascular tissue. Since lipid deposit in the arterial wall is a conspicuous aspect of atherosclerosis in human subjects it was considered of importance to determine the concentration of this cofactor in various types of human blood vessels. In the present investigation assays were made on samples of the ascending aorta, thoracic descending aorta, abdominal aorta, pulmonary artery, coronary artery, and inferior vena cava. In order to evaluate the correlation between atherosclerosis and tissue biotin 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 intimamedia layers of vascular specimens obtained fresh at autopsy. A total of 445 samples of human vascular tissue was included in the study. METHODS
After removal of the adventitia from the vascular samples, 10 % aqueous homogenates were made with a Kontes Dual type tissue grinder. For release of biotin from the protein-bound biotin compound, the homogenates were treated with trypsin 1. Two ml of the homogenate was pipetted into a 125 ml Erlenmeyer flask and 10.0 ml of a freshly prepared 0 . 1 % solution of recrystallized trypsin (Pentex, Inc., Kankakee, Ill.) was added. The volume was subsequently adjusted to 20.0 ml by addition of 8.0 ml redistilled water. The flask was covered with parafilm and incubated for 3 h in a water b a t h at a temperature of 50~ At the end of the incubation period the parafilm was removed and a special glass stopper inserted. The flask was then autoclaved for 5 min at a temperature of 121 ~ Following this denaturation of proteins, the hot sample was filtered through a W h a t m a n No. 42 filter paper. The filtrate was then centrifuged for 10 min at 25,000 r.p.m, and the clear supernatant used for analyses. j . Atheroscler. Res., 6 (1966) 440-446
BIOTIN CONTENT OF HUMAN ARTERIAL AND VENOUS TISSUE
O.D. 0.500
0.20C
/-
.,/
441
f
0.10(
0
o:,
o:~
o'.~
o'.~
BIOTIN (mjzg)
Fig. 1. Biotin standard curve for Lactobacillus plantarum.
For biotin measurement the microbiological Lactobacillus plantarum (ATTCNo. 8014) method was employed as described by DIFco 2. Only newly purchased test tubes carefully cleansed with redistilled water were used in that procedure. (Agar culture medium, inoculum broth, and biotin assay medium were obtained from Difco Labs., Inc., Detroit.) A biotin standard curve, ranging from 0.0 to 0.4 m/~g of biotin was prepared for each set of tests (Fig. 1). All vascular samples were analyzed in duplicate at two different tissue concentrations; 2.0 and 4.0 ml aliquots of the homogenate supernatants were used, corresponding to 20 and 40 mg flesh tissue, respectively. Duplicate blanks derived from redistilled water to which trypsin had been added were similarly assayed, requiring a total of 8 inoculated sample tubes for each microbiological biotin determination. The growth of Lactobacillus plantarum was measured turbidimetrically after 20 h incubation of the tubes in a water bath at 37~ the optical density readings wef-e made at 640 m/~ with a Beckman DU spectrophotometer. To check the efficiency of the total analytical procedure outlined in the present paper, separate experiments were conducted in which known quantities of biotin were added to original tissue homogenate samples; quantitative recovery was achieved by subsequent microbiological analyses. RESULTS
Normal samples The average biotin concentrations found for the intinla-media layers of various types of blood vessels are listed in Table I. The assays of different anatomical sections of the aorta indicate a tendency toward an increase in the biotin level from the ascending to the abdominal aorta. A comparison of values recorded for pulmonary artery and descending thoracic aortic specimens from the same subjects revealed approximately similar concentrations for these two blood vessels, the average biotin content of the pulmonary artery being 95.5 % (wet tissue; t, 1.63) and 105.2 % (tissue nitrogen; t, 1.40) of that observed j. Atheroscler. Res., 6 (1966) 440-446
442
J. E. KIRK, R. SANWALD
TABLE I BIOTIN
CONTENT
OF HUMAN
VASCULAR
Vascular samples
TISSUE
Number of samples
Ascending aorta, n o r m a l Descending thoracic aorta normal atherosclerotic, lipid atherosclerotic, fibrous P u l m o n a r y artery, n o r m a l Coronary a r t e r y normal atherosclerotic, lipid Vena cava inferior
16 152 75 16 89 19 13 42
Biotin (mltg/g) * wet tissue
tissue nitrogen
4.41 :k 0.14
111.5 4-
3.61
5.02 6.17 5.55 4.76
130.0 181.5 170.0 136.5
3.86 7.60 9.88 5.75
4~ 4~
0.14 0.25 0.48 0.17
4,97 • 0.41 7.56 :[: 0.81 3.30 4- 0,29
* Mean value 4- s t a n d a r d error of the mean.
TABLE II BIOTIN
CONTENT
Age group (years)
OF VASCULAR
SAMPLES
Number of samples
IN VARIOUS
AGE GROUPS
Biotin (mlzg/g) * wet tissue
tissue nitrogen
2.73 4.28 5.45 4.98 5.36 5.27 5.76 5.00
• 4• • 4• • ~:
0.25 0.56 0.37 0.31 0.32 0.29 0.30 0.43
64.2 94.5 120.1 128.4 143.0 139.3 155.2 139,2
4- 6.9 4- 13.9 i 8.7 4- 8.0 • 8.5 J: 7.4 4- 8.2 • 10.6
3.10 3.31 4.76 4.36 5.06 4.58 5,40 5~08
4• • • 44•
0.31 0.63 0.31 0.45 0.39 0.38 0.47
79.0 81.1 117.0 121.1 141.8 132.1 165.8 156.4
4- 8.4 4- 13.8 4- 8.7 4-4- 11.8 4- 14.0 4- 12.3 • 16.6
0.47 0.45 0.58 0.74 0.32 0.70
62.0 94.0 60.0 93.6 83.8 99.8 102,3 72.1
-[= + • • • 4-
Normal thoracic descending aorta O- 9 10-19 20 29 30-39 40 49 50-59 60-69 70-86
13 8 14 20 22 30 24 21
Normal pulmonary artery 0- 9 10-19 20--29 30-39 40-49 50-59 60--69 70-86
2 6 8 l0 16 16 17 14
Vena cava inferior 0- 9 10- 19 20-29 30--39 40--49 50--59 60-69 70-86
1 1 5 5 6 5 12 7
3.20 3.55 2.54 3.49 3.36 3.54 3.76 2.64
• 4• 444-
* Mean value 4- s t a n d a r d error of the mean.
J. Atheroscler. Res., 6 (1966) 440-446
11.1 11.2 13.4 17.6 6,9 17.8
4~: 44-
144.5 4- 10.90 231.0 ~ 25.60 87.2 4- 8.56
BIOTIN CONTENT OF HUMAN ARTERIAL AND VENOUS TISSUE
443
for the aorta. In contrast to this, distinctly lower concentrations were found for the inferior vena cava; when expressed on the basis of wet tissue weight and tissue nitrogen, the values for the vena cava were, respectively, 63.4 % (t, 5.05) and 69.20//o (t, 4.44) of the aortic biotin content. The mean biotin concentrations of the thoracic descending aorta, p u l m o n a r y artery, and inferior vena cava found for various age groups are presented in Table II. The rather large n u m b e r of samples included in the s t u d y made it possible to evaluate the relationship between age and tissue biotin content. Analysis of the d a t a showed a notable increase in the biotin levels of the thoracic aorta and the p u l m o n a r y artery from early childhood to adulthood (Figs. 2, 3) whereas no significant variation with age was recorded for the vena cava. The coefficients of correlation between age and biotin content of vascular tissues are presented in Table I I I .
o:
5
u~
e.
/
"'. ,/
4
L~ 3 Aorto ...... P u / m o n o r y
Artery
2 .2
E
Jo 2'0 3() 4o
5'0 4o ~o B'o 9'0
Age (years)
Fig. 2. Variation with age in biotin content of normal aortic and pulmonary artery tissue.
150
L C
JO0
.40rio
,E
"~ so
.......
Pulmonary Artery
m
E
~, 2o ~o ,~ ~o ~o -~ ~o ~o Age (years)
Fig. 3. Variation with age in biotin content of normal aortic and pulmonary artery tissue. j . Atheroscler. Res., 6 (1966) 440-446
j.E.
444
KIRK, R. SANWALD
TABLE III COEFFICIENTS
OF C O R R E L A T I O N B E T W E E N
Correlation between age and
N o r m a l thoracic descending a o r t a Atlxerosclerotic a o r t a lipid fibrous Ascending aorta, n o r m a l A b d o m i n a l aorta, n o r m a l Pulmonary artery Coronary artery, n o r m a l Atherosclerotic c o r o n a r y artery, lipid Inferior v e n a cava
AGE AND BIOTIN CONTENT OF VASCULAR TISSUE
Age group (years)
Number of samples
0-86 20-86
Wet tissue
Tissue nitrogen
r
t
r
t
152 131
+ 0.42 + 0.03
5.69 0.34
+ 0.47 + 0.32
6.55 3.84
20-86 20-86 0-86 0-86 20-86 0-86 20-86 0-86
75 16 16 23 20 89 81 19
+ -+ + -+ + +
0.06 0.06 0.03 0.0l 0.08 0.27 0.13 0.20
0.52 0.23 0.11 0.05 0.34 2.63 1.18 0.85
+ -+ + -+ + +
0.09 0.18 0.24 0.12 0.06 0.40 0.27 0.38
0.78 0.67 0.91 0.55 0.26 4.30 2.50 1.72
20-86 0-86
13 42
+ 0.18 + 0.05
0.60 0.32
+ 0.05 + 0.12
0.10 0.78
Atherosclerotic samples The results of analyses of atherosclerotic samples are listed in Table I. Comparison of biotin concentrations in normal tissue portions and in pathological areas of the vascular wall displayed significantly higher values in samples with lipid deposits, whereas no great changes were found for the fibrous plaques (Table IV). Because of the observed elevated biotin concentrations in lipid-atherosclerotic tissue sections it was considered of interest to compare values recorded for adult male and female subjects: it is an established fact that premenopausal women are less susceptible to atherosclerosis than men. As seen from Table V, in the present study approximately similar biotin levels were observed for both sexes.
TABLE IV B I O T I N C O N T E N T OF A T H E R O S C L E R O T I C T I S S U E P O R T I O N S E X P R E S S E D
IN PERCENTAGES
OF N O R M A L T I S S U E OF T H E S A M E A O R T I C S A M P L E S
Age group (years)
Number of samples
Wet tissue
Tissue nitrogen
%
t
%
t
23 50 73
127 117 120
3.50 4.02 5.26
136 131 133
4.35 6.02 7.48
15
104
0.42
121
2.68
Lipid changes 20-49 50-86 Total
Fibrous changes 20-86
J. Atheroscler. Res., 6 (1966) 440-446
OF CONTENT
BIOTIN CONTENT OF HUMAN ARTERIAL AND VENOUS TISSUE
445
TABLE V C O M P A R I S O N OF B I O T I N C O N C E N T R A T I O N S
Age group (years)
18-54 55-86 18-86
OF AORTIC S A M P L E S F R O M M A L E A N D F E M A L E S U B J E C T S
Normal tissue
A therosclerotic-lipid tissue
wet tissue
tissue nitrogen
%*
%*
90.0 93.5 91.5
t
1.98 0.95 1.78
98.0 91.2 95.0
wet tissue
t
d.f.**
0.27 1.02 1.27
71 56 129
%*
88.2 98.8 93.8
tissue nitrogen
t
%*
t
d.f.**
1.06 0.115 1.07
91.3 96.5 94.0
0,67 0.35 0.87
33 37 72
* Values for female subjects expressed in % of concentrations recorded for male individuals, ** Degrees of freedom.
DISCUSSION T h e m e a n b i o t i n v a l u e of 5.02 mbtg/g of h u m a n aortic tissue o b s e r v e d in t h e p r e s e n t i n v e s t i g a t i o n is a b o u t 7 % 0f t h a t recorded b y BAKER AND SOBOTKA3 for h u m a n b r a i n a n d 15 t i m e s higher t h a n t h e a v e r a g e biotin s e r u m c o n c e n t r a t i o n enc o u n t e r e d in n o r m a l subjects. Since b i o t i n is i n v o l v e d in lipogenesis, t h e higher v a l u e s f o u n d for a t h e r o s c l e r o t i c - l i p i d t h a n for n o r m a l a r t e r i a l tissue is an o b s e r v a t i o n ot definite interest. As p o i n t e d o u t b y BAKER AND SOBOTKA3, t h e possible r e l a t i o n s h i p of b i o t i n to t h e a b i I i t y of a n i m a l s to esterify a n d store f a t t y acids as well as cholesterol is a m a i o r topic of biological research. I t is g e n e r a l l y a s s u m e d t h a t a large p r o p o r t i o n of biotin is s u p p l i e d t h r o u g h t h e a c t i o n of i n t e s t i n a l b a c t e r i a ; for t h a t reason, v a r i a t i o n s w i t h age in i n t e s t i n a l flora deserve c o n s i d e r a t i o n in connection w i t h e v a l u a t i o n of o b s e r v e d changes with age in tissue c o n t e n t s of this cofactor.
ACKNOWLEDGEMENTS The i n v e s t i g a t i o n was s u p p o r t e d b y t h e St. Louis H e a r t Association a n d b y a g r a n t from t h e N a t i o n a l I n s t i t u t e s of H e a l t h , P u N i c H e a l t h Service (HE-00891).
SUMMARY D e t e r m i n a t i o n s were m a d e of t h e b i o t i n c o n c e n t r a t i o n in v a r i o u s t y p e s of h u m a n b l o o d vessels. T h e a n a l y s e s were p e r f o r m e d a f t e r l i b e r a t i o n of p r o t e i n - b o u n d b i o t i n t h r o u g h t r e a t m e n t of tissue h o m o g e n a t e s w i t h r e c r y s t a l l i z e d t r y p s i n ; t h e m e a s u r e m e n t s were m a d e microbiologically b y the Lactobacillus p l a n t a r u m procedure. A t o t a l of 445 v a s c u l a r s a m p l e s was included in t h e s t u d y . T h e m e a n biotin values o b s e r v e d j . Atheroscler. Res., 6 (1966) 440-446
446
/1. E. KIRK, R. SANWALD
for intima-media layers of normal tissue portions expressed as m/~g/g of wet tissue were: ascending aorta, 4.41; descending thoracic aorta, 5.02; abdominal aorta, 5.96; pulmonary artery, 4.76; coronary artery, 4.97; and inferior vena cava, 3.30. Analyses of atherosclerotic tissue portions consisting mainly of lipid deposits showed significantly higher biotin levels than in the normal tissue, whereas no great changes were recorded for fibrous atherosclerotic plaques.
REFERENCES 1 j . O. LAMPEN, G. P. BAHLER AND W . H. PETERSON, J. Nutr., 23 (1942) 11. '2 Difco Manual, Difco Labs., Inc., Detroit, Michican, 9th edition, 1962, p. 220. 3 H. BAKER AND H. SOBOTKA, Advanc. Clin. Chem., 5 (1962) 173.
Journal of Atherosclerosis Research
B I O T I N CONTENT OF HUMAN A R T E R I A L AND VENOUS T I S S U E
j . E. K I R K AND R. S A N W A L D
Division of Gerontology, Washington University School of Medicine, St. Louis, Mo. (U.S.A.) (Received D e c e m b e r 14th, 1965)
INTRODUCTION
Biotin is a coenzyme involved in f a t t y acid synthesis and other significant intermediary metabolic reactions, but no studies have previously been reported on the biotin content of vascular tissue. Since lipid deposit in the arterial wall is a conspicuous aspect of atherosclerosis in human subjects it was considered of importance to determine the concentration of this cofactor in various types of human blood vessels. In the present investigation assays were made on samples of the ascending aorta, thoracic descending aorta, abdominal aorta, pulmonary artery, coronary artery, and inferior vena cava. In order to evaluate the correlation between atherosclerosis and tissue biotin 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 intimamedia layers of vascular specimens obtained fresh at autopsy. A total of 445 samples of human vascular tissue was included in the study. METHODS
After removal of the adventitia from the vascular samples, 10 % aqueous homogenates were made with a Kontes Dual type tissue grinder. For release of biotin from the protein-bound biotin compound, the homogenates were treated with trypsin 1. Two ml of the homogenate was pipetted into a 125 ml Erlenmeyer flask and 10.0 ml of a freshly prepared 0 . 1 % solution of recrystallized trypsin (Pentex, Inc., Kankakee, Ill.) was added. The volume was subsequently adjusted to 20.0 ml by addition of 8.0 ml redistilled water. The flask was covered with parafilm and incubated for 3 h in a water b a t h at a temperature of 50~ At the end of the incubation period the parafilm was removed and a special glass stopper inserted. The flask was then autoclaved for 5 min at a temperature of 121 ~ Following this denaturation of proteins, the hot sample was filtered through a W h a t m a n No. 42 filter paper. The filtrate was then centrifuged for 10 min at 25,000 r.p.m, and the clear supernatant used for analyses. j . Atheroscler. Res., 6 (1966) 440-446
BIOTIN CONTENT OF HUMAN ARTERIAL AND VENOUS TISSUE
O.D. 0.500
0.20C
/-
.,/
441
f
0.10(
0
o:,
o:~
o'.~
o'.~
BIOTIN (mjzg)
Fig. 1. Biotin standard curve for Lactobacillus plantarum.
For biotin measurement the microbiological Lactobacillus plantarum (ATTCNo. 8014) method was employed as described by DIFco 2. Only newly purchased test tubes carefully cleansed with redistilled water were used in that procedure. (Agar culture medium, inoculum broth, and biotin assay medium were obtained from Difco Labs., Inc., Detroit.) A biotin standard curve, ranging from 0.0 to 0.4 m/~g of biotin was prepared for each set of tests (Fig. 1). All vascular samples were analyzed in duplicate at two different tissue concentrations; 2.0 and 4.0 ml aliquots of the homogenate supernatants were used, corresponding to 20 and 40 mg flesh tissue, respectively. Duplicate blanks derived from redistilled water to which trypsin had been added were similarly assayed, requiring a total of 8 inoculated sample tubes for each microbiological biotin determination. The growth of Lactobacillus plantarum was measured turbidimetrically after 20 h incubation of the tubes in a water bath at 37~ the optical density readings wef-e made at 640 m/~ with a Beckman DU spectrophotometer. To check the efficiency of the total analytical procedure outlined in the present paper, separate experiments were conducted in which known quantities of biotin were added to original tissue homogenate samples; quantitative recovery was achieved by subsequent microbiological analyses. RESULTS
Normal samples The average biotin concentrations found for the intinla-media layers of various types of blood vessels are listed in Table I. The assays of different anatomical sections of the aorta indicate a tendency toward an increase in the biotin level from the ascending to the abdominal aorta. A comparison of values recorded for pulmonary artery and descending thoracic aortic specimens from the same subjects revealed approximately similar concentrations for these two blood vessels, the average biotin content of the pulmonary artery being 95.5 % (wet tissue; t, 1.63) and 105.2 % (tissue nitrogen; t, 1.40) of that observed j. Atheroscler. Res., 6 (1966) 440-446
442
J. E. KIRK, R. SANWALD
TABLE I BIOTIN
CONTENT
OF HUMAN
VASCULAR
Vascular samples
TISSUE
Number of samples
Ascending aorta, n o r m a l Descending thoracic aorta normal atherosclerotic, lipid atherosclerotic, fibrous P u l m o n a r y artery, n o r m a l Coronary a r t e r y normal atherosclerotic, lipid Vena cava inferior
16 152 75 16 89 19 13 42
Biotin (mltg/g) * wet tissue
tissue nitrogen
4.41 :k 0.14
111.5 4-
3.61
5.02 6.17 5.55 4.76
130.0 181.5 170.0 136.5
3.86 7.60 9.88 5.75
4~ 4~
0.14 0.25 0.48 0.17
4,97 • 0.41 7.56 :[: 0.81 3.30 4- 0,29
* Mean value 4- s t a n d a r d error of the mean.
TABLE II BIOTIN
CONTENT
Age group (years)
OF VASCULAR
SAMPLES
Number of samples
IN VARIOUS
AGE GROUPS
Biotin (mlzg/g) * wet tissue
tissue nitrogen
2.73 4.28 5.45 4.98 5.36 5.27 5.76 5.00
• 4• • 4• • ~:
0.25 0.56 0.37 0.31 0.32 0.29 0.30 0.43
64.2 94.5 120.1 128.4 143.0 139.3 155.2 139,2
4- 6.9 4- 13.9 i 8.7 4- 8.0 • 8.5 J: 7.4 4- 8.2 • 10.6
3.10 3.31 4.76 4.36 5.06 4.58 5,40 5~08
4• • • 44•
0.31 0.63 0.31 0.45 0.39 0.38 0.47
79.0 81.1 117.0 121.1 141.8 132.1 165.8 156.4
4- 8.4 4- 13.8 4- 8.7 4-4- 11.8 4- 14.0 4- 12.3 • 16.6
0.47 0.45 0.58 0.74 0.32 0.70
62.0 94.0 60.0 93.6 83.8 99.8 102,3 72.1
-[= + • • • 4-
Normal thoracic descending aorta O- 9 10-19 20 29 30-39 40 49 50-59 60-69 70-86
13 8 14 20 22 30 24 21
Normal pulmonary artery 0- 9 10-19 20--29 30-39 40-49 50-59 60--69 70-86
2 6 8 l0 16 16 17 14
Vena cava inferior 0- 9 10- 19 20-29 30--39 40--49 50--59 60-69 70-86
1 1 5 5 6 5 12 7
3.20 3.55 2.54 3.49 3.36 3.54 3.76 2.64
• 4• 444-
* Mean value 4- s t a n d a r d error of the mean.
J. Atheroscler. Res., 6 (1966) 440-446
11.1 11.2 13.4 17.6 6,9 17.8
4~: 44-
144.5 4- 10.90 231.0 ~ 25.60 87.2 4- 8.56
BIOTIN CONTENT OF HUMAN ARTERIAL AND VENOUS TISSUE
443
for the aorta. In contrast to this, distinctly lower concentrations were found for the inferior vena cava; when expressed on the basis of wet tissue weight and tissue nitrogen, the values for the vena cava were, respectively, 63.4 % (t, 5.05) and 69.20//o (t, 4.44) of the aortic biotin content. The mean biotin concentrations of the thoracic descending aorta, p u l m o n a r y artery, and inferior vena cava found for various age groups are presented in Table II. The rather large n u m b e r of samples included in the s t u d y made it possible to evaluate the relationship between age and tissue biotin content. Analysis of the d a t a showed a notable increase in the biotin levels of the thoracic aorta and the p u l m o n a r y artery from early childhood to adulthood (Figs. 2, 3) whereas no significant variation with age was recorded for the vena cava. The coefficients of correlation between age and biotin content of vascular tissues are presented in Table I I I .
o:
5
u~
e.
/
"'. ,/
4
L~ 3 Aorto ...... P u / m o n o r y
Artery
2 .2
E
Jo 2'0 3() 4o
5'0 4o ~o B'o 9'0
Age (years)
Fig. 2. Variation with age in biotin content of normal aortic and pulmonary artery tissue.
150
L C
JO0
.40rio
,E
"~ so
.......
Pulmonary Artery
m
E
~, 2o ~o ,~ ~o ~o -~ ~o ~o Age (years)
Fig. 3. Variation with age in biotin content of normal aortic and pulmonary artery tissue. j . Atheroscler. Res., 6 (1966) 440-446
j.E.
444
KIRK, R. SANWALD
TABLE III COEFFICIENTS
OF C O R R E L A T I O N B E T W E E N
Correlation between age and
N o r m a l thoracic descending a o r t a Atlxerosclerotic a o r t a lipid fibrous Ascending aorta, n o r m a l A b d o m i n a l aorta, n o r m a l Pulmonary artery Coronary artery, n o r m a l Atherosclerotic c o r o n a r y artery, lipid Inferior v e n a cava
AGE AND BIOTIN CONTENT OF VASCULAR TISSUE
Age group (years)
Number of samples
0-86 20-86
Wet tissue
Tissue nitrogen
r
t
r
t
152 131
+ 0.42 + 0.03
5.69 0.34
+ 0.47 + 0.32
6.55 3.84
20-86 20-86 0-86 0-86 20-86 0-86 20-86 0-86
75 16 16 23 20 89 81 19
+ -+ + -+ + +
0.06 0.06 0.03 0.0l 0.08 0.27 0.13 0.20
0.52 0.23 0.11 0.05 0.34 2.63 1.18 0.85
+ -+ + -+ + +
0.09 0.18 0.24 0.12 0.06 0.40 0.27 0.38
0.78 0.67 0.91 0.55 0.26 4.30 2.50 1.72
20-86 0-86
13 42
+ 0.18 + 0.05
0.60 0.32
+ 0.05 + 0.12
0.10 0.78
Atherosclerotic samples The results of analyses of atherosclerotic samples are listed in Table I. Comparison of biotin concentrations in normal tissue portions and in pathological areas of the vascular wall displayed significantly higher values in samples with lipid deposits, whereas no great changes were found for the fibrous plaques (Table IV). Because of the observed elevated biotin concentrations in lipid-atherosclerotic tissue sections it was considered of interest to compare values recorded for adult male and female subjects: it is an established fact that premenopausal women are less susceptible to atherosclerosis than men. As seen from Table V, in the present study approximately similar biotin levels were observed for both sexes.
TABLE IV B I O T I N C O N T E N T OF A T H E R O S C L E R O T I C T I S S U E P O R T I O N S E X P R E S S E D
IN PERCENTAGES
OF N O R M A L T I S S U E OF T H E S A M E A O R T I C S A M P L E S
Age group (years)
Number of samples
Wet tissue
Tissue nitrogen
%
t
%
t
23 50 73
127 117 120
3.50 4.02 5.26
136 131 133
4.35 6.02 7.48
15
104
0.42
121
2.68
Lipid changes 20-49 50-86 Total
Fibrous changes 20-86
J. Atheroscler. Res., 6 (1966) 440-446
OF CONTENT
BIOTIN CONTENT OF HUMAN ARTERIAL AND VENOUS TISSUE
445
TABLE V C O M P A R I S O N OF B I O T I N C O N C E N T R A T I O N S
Age group (years)
18-54 55-86 18-86
OF AORTIC S A M P L E S F R O M M A L E A N D F E M A L E S U B J E C T S
Normal tissue
A therosclerotic-lipid tissue
wet tissue
tissue nitrogen
%*
%*
90.0 93.5 91.5
t
1.98 0.95 1.78
98.0 91.2 95.0
wet tissue
t
d.f.**
0.27 1.02 1.27
71 56 129
%*
88.2 98.8 93.8
tissue nitrogen
t
%*
t
d.f.**
1.06 0.115 1.07
91.3 96.5 94.0
0,67 0.35 0.87
33 37 72
* Values for female subjects expressed in % of concentrations recorded for male individuals, ** Degrees of freedom.
DISCUSSION T h e m e a n b i o t i n v a l u e of 5.02 mbtg/g of h u m a n aortic tissue o b s e r v e d in t h e p r e s e n t i n v e s t i g a t i o n is a b o u t 7 % 0f t h a t recorded b y BAKER AND SOBOTKA3 for h u m a n b r a i n a n d 15 t i m e s higher t h a n t h e a v e r a g e biotin s e r u m c o n c e n t r a t i o n enc o u n t e r e d in n o r m a l subjects. Since b i o t i n is i n v o l v e d in lipogenesis, t h e higher v a l u e s f o u n d for a t h e r o s c l e r o t i c - l i p i d t h a n for n o r m a l a r t e r i a l tissue is an o b s e r v a t i o n ot definite interest. As p o i n t e d o u t b y BAKER AND SOBOTKA3, t h e possible r e l a t i o n s h i p of b i o t i n to t h e a b i I i t y of a n i m a l s to esterify a n d store f a t t y acids as well as cholesterol is a m a i o r topic of biological research. I t is g e n e r a l l y a s s u m e d t h a t a large p r o p o r t i o n of biotin is s u p p l i e d t h r o u g h t h e a c t i o n of i n t e s t i n a l b a c t e r i a ; for t h a t reason, v a r i a t i o n s w i t h age in i n t e s t i n a l flora deserve c o n s i d e r a t i o n in connection w i t h e v a l u a t i o n of o b s e r v e d changes with age in tissue c o n t e n t s of this cofactor.
ACKNOWLEDGEMENTS The i n v e s t i g a t i o n was s u p p o r t e d b y t h e St. Louis H e a r t Association a n d b y a g r a n t from t h e N a t i o n a l I n s t i t u t e s of H e a l t h , P u N i c H e a l t h Service (HE-00891).
SUMMARY D e t e r m i n a t i o n s were m a d e of t h e b i o t i n c o n c e n t r a t i o n in v a r i o u s t y p e s of h u m a n b l o o d vessels. T h e a n a l y s e s were p e r f o r m e d a f t e r l i b e r a t i o n of p r o t e i n - b o u n d b i o t i n t h r o u g h t r e a t m e n t of tissue h o m o g e n a t e s w i t h r e c r y s t a l l i z e d t r y p s i n ; t h e m e a s u r e m e n t s were m a d e microbiologically b y the Lactobacillus p l a n t a r u m procedure. A t o t a l of 445 v a s c u l a r s a m p l e s was included in t h e s t u d y . T h e m e a n biotin values o b s e r v e d j . Atheroscler. Res., 6 (1966) 440-446
446
/1. E. KIRK, R. SANWALD
for intima-media layers of normal tissue portions expressed as m/~g/g of wet tissue were: ascending aorta, 4.41; descending thoracic aorta, 5.02; abdominal aorta, 5.96; pulmonary artery, 4.76; coronary artery, 4.97; and inferior vena cava, 3.30. Analyses of atherosclerotic tissue portions consisting mainly of lipid deposits showed significantly higher biotin levels than in the normal tissue, whereas no great changes were recorded for fibrous atherosclerotic plaques.
REFERENCES 1 j . O. LAMPEN, G. P. BAHLER AND W . H. PETERSON, J. Nutr., 23 (1942) 11. '2 Difco Manual, Difco Labs., Inc., Detroit, Michican, 9th edition, 1962, p. 220. 3 H. BAKER AND H. SOBOTKA, Advanc. Clin. Chem., 5 (1962) 173.
Journal of Atherosclerosis Research