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Thiamine content of human arterial and venous tissue
Atherosclerosis Elsevier Publishing
Company,
Amsterdam
THIAMINE
CONTENT
T. KHEIM
AND J. E. KIRK
Division
of Gerontology,
(Received
December
OF
Washington
207
- Printed in The Netherlands
HUMAN
University
ARTERIAL
AND
School of Medicine,
VENOUS
St. Louis,
TISSUE
MO. 63139 (U.S.A.)
!29th, 1969)
SUMMARY
Quantitative determinations various types of human vascular
were made of the thiamine concentrations in tissue. The mean contents observed for intima-
media layers of normal tissue portions expressed as ,uugthiamine hydrochloride/g of wet tissue, were: descending thoracic aorta, 0.206; pulmonary artery, 0.228; coronary artery, 0.311; and inferior vena cava, 0.178. Analyses of atherosclerotic tissue portions consisting normal
mainly
tissue,
sclerotic
of lipid deposits
whereas
showed essentially
significantly
lower values
similar
thiamine
were recorded
levels as in
for fibrous
athero-
plaques.
Key words:
Thiamine
- Atherosclerotic
tissue - Human vascular tissue - No? ma1 tissue
- Venous tissue
INTRODUCTION
Thiamine
pyrophosphate
is one of the cofactors
required
in animal
tissues
for
oxidative decarboxylation of pyruvic acid; it also acts as a coenzyme in the transketolation reaction. Quantitative study of the thiamine content in human vasculartissue was therefore considered desirable. This investigation included assays on ‘samples of the thoracic descending aorta, pulmonary artery, coronary artery, and inferior vena cava. In order to evaluate the correlation between atherosclerosis and tissue thiamine concentration, 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 made from intima-media layers of vascular specimens obtained fresh at autopsy from persons who did not This research was supported
by U.S. Public Health Service Grant HE-00891. Atherosclerosis,
1970, 12: 207-210
208
T.
display signs of metabolic human vascular tissue.
or infectious
diseases.
The study
KHEIM, J. E. KIRK
included
194 samples
of
METHOD The employed procedure was based on the techniques described by SARETT AND CHELDELIN~. FITZGERALD AND HUGHES~ and CARVALHO DA SILVA et al.3. Although hydrolysis
with sulfuric
acid is assumed
to release both thiamine
and thiamine
pyro-
phosphate quantitatively from the tissue, the takadiastase-papain digestion was used also; in agreement with FITZGERALD AND HUGHE+’ the present
method authors
found
reliable
that
this combined
treatment
of vascular
tissue
consistently
gives
results. 1 g of tissue was homogenized with 10 ml redistilled water and the volume subsequently made up to 40 ml with sulfuric acid to make a final concentration of 0.1 N H&04. The acidified sample was steamed at 100°C for 30 min in an autoclave. After cooling, the pH was adjusted to 4.5 with a 2.5 M sodium acetate solution. 20 mg of takadiastase and papain then were ad&a and the sample was incubated under toluene overnight
at 37°C. The hydrolyzate-digest
while hot through
Whatman
was steamed
No. 1 filter paper. The volume
for 15 min and filtered of the filtrate
was made
up to 50 ml with redistilled water and the pH adjusted to 6.5 by adding a few drops of 10% NaOH. The sample was steamed again for 15 min and filtered. At this stage the sample
can be stored frozen until analyzed. For determination of the total amount of thiamine compounds hydrolyzatedigest, the microbiological Lactobacillus fermenti 36
present in the (ATCC 9388)
method1
and thiamine
was used;
this
bacterium
responds
to both
free thiamine
phosphates. Agar culture medium and inoculum broth were obtained from Difco Labs., Inc., Detroit and the thiamine assay medium from General Biochemicals, Chagrin Falls, Ohio. All vascular samples were assayed in triplicate at three different concentrations,
requiring
nine
inoculated
sample
tubes
for
each
microbiological
TABLE 1 THIAMINE
CONTENT
OF HUMAN
VASCULAR
Vascular samples
Descending thoracic aorta normal atherosclerotic, lipid atherosclerotic, fibrous Pulmonary artery, normal Coronary artery normal atherosclerotic, lipid Vena cava inferior
TISSUE
No. of samples
1970, 12: 207-210
(pguglg)” tissue nitrogen
0.206 f 0.005
10 33
0.194 -& 0.007 0.184 + 0.012 0.228 + 0.008
5.57 * 0.15
44
3 8 27
0.311 0.289 & 0.011 0.178 + 0.006
9.27 11.12 * 0.47 5.24 f 0.18
69
* Mean values + standard error of the mean. Atherosclerosis,
Thiamine-HCl wet tissue
5.76 f 0.21 5.32 & 0.38 6.82 & 0.26
THIAMINE
CONTENT
thiamine
OF HUMAN
determination,
ARTERIAL
AND VENOUS
Of the described
209
TISSUE
2 y0 hydrolyzate-digest
2.0,3.0,
and 4.0 ml
aliquots were employed, corresponding to 40, 60, and 80 mg wet tissue, respectively. A standard curve ranging from 0.000-0.025 ,ug of thiamine hydrochloride and a blank were run in triplicate 36 was determined the optical density
with each set of analyses.
The growth
of Lactobacillus
fermenti
turbidimetrically after 16-18 h incubation of the tubes at 37°C; readings were made at 546 rnp with a Beckman DU spectrophoto-
meter. The thiamine
values
are expressed
tissue and per g tissue nitrogen; tions
of nitrogen.
average
difference
RESULTS
AND
Normal
as ,ug of thiamine
the Kjeldahl
A high reproducibility between
triplicate
procedure
of the thiamine
analyses
hydrochloride
per g wet
was used for the determinaassays
was obtained,
the
being only 5.3%.
DISCUSSION
samples
The average
thiamine
concentrations
for intima-media
layers of various
types
of blood vessels are listed in Table 1. The mean value of 0.206 ,ug/g of wet tissue for 69 samples of the descending thoracic aorta is definitely higher than that reported for human
blood. A comparison
the same subjects
of values for pulmonary
did not reveal statistically
artery
significant
and aortic specimens
differences,
from
the average thia-
mine concentration of the pulmonary artery being 107.7% (wet tissue; t 0.83) and llS.Oo/o (tissue nitrogen; t 1.82) of that observed for the aorta. For the inferior vena cava, when expressed on the basis of wet tissue weight and tissue nitrogen content the values were, respectively, content. The coefficients vascular
84.9o/o (t 1.98) and 98.6o/o (t 0.18) of the aortic thiamine of correlation between age and thiamine concentrations of
tissues are listed in Table 2; as seen from these data, no significant
with age was found in thiamine
TABLE
2
COEFFICIENTS OF CORRELATION BETWEEN
Correlation
variation
levels.
between
age and
Descending thoracic aorta normal atherosclerotic, lipid Pulmonary artery, normal Vena cava inferior
Age
AGE
AND
No. of
group (yea-1
samples
O-84 20-84 18-84 17-72
69 44 33 27
Atherosclerotic samples The results of analyses
of atherosclerotic
THIAMINE
CONTENT
OF VASCULAR
Tissue
Wet tissue
TISSUE
nitrogen
Y
t
Y
t
+0.04 0.00 $0.12 -0.19
0.33 0.00 0.66 0.97
+0.15 +0.09 +0.15 -0.08
1.23 0.58 0.85 0.46
samples
are listedin Atherosclerosis,
Table 1. Compari1970,
12: 207-210
T. KHEIM,
210
son of thiamine the vascular great changes TABLE
concentrations
in normal
wall showed significantly were found for samples
tissue portions lower values
and in pathological
in fibrous
with lipid deposit
J. E. KIRK
plaques,
areas of
whereas
no
(Table 3).
3
THIAMINE CONTENT OF ATHEROSCLEROTIC CONTENT OF NORMALTISSUE OFTHE SAME
Age group (years)
No. of
TISSUE PORTIONS AORTIC SAMPLES
%
IN PERCENTAGES
OF
Tissue nitrogen
Wet tissue
samcles
EXPRESSED
t
%
t
Lipid changes 20-49 50-59 60-84 total
16 12 12 40
91.5 92.9 99.0 94.2
1.09 1.06 0.13 1.28
94.7 102.2 116.3 103.5
0.66 0.26 1.72 0.68
Fibrous changes 34-69
10
78.4
2.42
83.0
2.05
REFERENCES SARETT, H. P. AND V. H. CHELDELIN, The use of Lactobacillus fermenturn 36 for thiamine assay, J. biol. Chem.. 1944, 155: 153. FITZGERALD, E. E. AND E. B. HUGHES, The microbiological assay of aneurine: an improved method employing Lactobacillus fermenti 36, Analyst, 1949, 74: 340. CARVALHO DA SILVA, A., S. R. MONSAOAND R. CARLOTTA DE A~~~~~~,Tissulardist.ribution and biliaryexcretionofthiamineonnephrectomizedanimals, Actaphysiol.latino-amer., 1958, 8: 111.
Atherosclerosis,
1970, 12: 207-210
Company,
Amsterdam
THIAMINE
CONTENT
T. KHEIM
AND J. E. KIRK
Division
of Gerontology,
(Received
December
OF
Washington
207
- Printed in The Netherlands
HUMAN
University
ARTERIAL
AND
School of Medicine,
VENOUS
St. Louis,
TISSUE
MO. 63139 (U.S.A.)
!29th, 1969)
SUMMARY
Quantitative determinations various types of human vascular
were made of the thiamine concentrations in tissue. The mean contents observed for intima-
media layers of normal tissue portions expressed as ,uugthiamine hydrochloride/g of wet tissue, were: descending thoracic aorta, 0.206; pulmonary artery, 0.228; coronary artery, 0.311; and inferior vena cava, 0.178. Analyses of atherosclerotic tissue portions consisting normal
mainly
tissue,
sclerotic
of lipid deposits
whereas
showed essentially
significantly
lower values
similar
thiamine
were recorded
levels as in
for fibrous
athero-
plaques.
Key words:
Thiamine
- Atherosclerotic
tissue - Human vascular tissue - No? ma1 tissue
- Venous tissue
INTRODUCTION
Thiamine
pyrophosphate
is one of the cofactors
required
in animal
tissues
for
oxidative decarboxylation of pyruvic acid; it also acts as a coenzyme in the transketolation reaction. Quantitative study of the thiamine content in human vasculartissue was therefore considered desirable. This investigation included assays on ‘samples of the thoracic descending aorta, pulmonary artery, coronary artery, and inferior vena cava. In order to evaluate the correlation between atherosclerosis and tissue thiamine concentration, 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 made from intima-media layers of vascular specimens obtained fresh at autopsy from persons who did not This research was supported
by U.S. Public Health Service Grant HE-00891. Atherosclerosis,
1970, 12: 207-210
208
T.
display signs of metabolic human vascular tissue.
or infectious
diseases.
The study
KHEIM, J. E. KIRK
included
194 samples
of
METHOD The employed procedure was based on the techniques described by SARETT AND CHELDELIN~. FITZGERALD AND HUGHES~ and CARVALHO DA SILVA et al.3. Although hydrolysis
with sulfuric
acid is assumed
to release both thiamine
and thiamine
pyro-
phosphate quantitatively from the tissue, the takadiastase-papain digestion was used also; in agreement with FITZGERALD AND HUGHE+’ the present
method authors
found
reliable
that
this combined
treatment
of vascular
tissue
consistently
gives
results. 1 g of tissue was homogenized with 10 ml redistilled water and the volume subsequently made up to 40 ml with sulfuric acid to make a final concentration of 0.1 N H&04. The acidified sample was steamed at 100°C for 30 min in an autoclave. After cooling, the pH was adjusted to 4.5 with a 2.5 M sodium acetate solution. 20 mg of takadiastase and papain then were ad&a and the sample was incubated under toluene overnight
at 37°C. The hydrolyzate-digest
while hot through
Whatman
was steamed
No. 1 filter paper. The volume
for 15 min and filtered of the filtrate
was made
up to 50 ml with redistilled water and the pH adjusted to 6.5 by adding a few drops of 10% NaOH. The sample was steamed again for 15 min and filtered. At this stage the sample
can be stored frozen until analyzed. For determination of the total amount of thiamine compounds hydrolyzatedigest, the microbiological Lactobacillus fermenti 36
present in the (ATCC 9388)
method1
and thiamine
was used;
this
bacterium
responds
to both
free thiamine
phosphates. Agar culture medium and inoculum broth were obtained from Difco Labs., Inc., Detroit and the thiamine assay medium from General Biochemicals, Chagrin Falls, Ohio. All vascular samples were assayed in triplicate at three different concentrations,
requiring
nine
inoculated
sample
tubes
for
each
microbiological
TABLE 1 THIAMINE
CONTENT
OF HUMAN
VASCULAR
Vascular samples
Descending thoracic aorta normal atherosclerotic, lipid atherosclerotic, fibrous Pulmonary artery, normal Coronary artery normal atherosclerotic, lipid Vena cava inferior
TISSUE
No. of samples
1970, 12: 207-210
(pguglg)” tissue nitrogen
0.206 f 0.005
10 33
0.194 -& 0.007 0.184 + 0.012 0.228 + 0.008
5.57 * 0.15
44
3 8 27
0.311 0.289 & 0.011 0.178 + 0.006
9.27 11.12 * 0.47 5.24 f 0.18
69
* Mean values + standard error of the mean. Atherosclerosis,
Thiamine-HCl wet tissue
5.76 f 0.21 5.32 & 0.38 6.82 & 0.26
THIAMINE
CONTENT
thiamine
OF HUMAN
determination,
ARTERIAL
AND VENOUS
Of the described
209
TISSUE
2 y0 hydrolyzate-digest
2.0,3.0,
and 4.0 ml
aliquots were employed, corresponding to 40, 60, and 80 mg wet tissue, respectively. A standard curve ranging from 0.000-0.025 ,ug of thiamine hydrochloride and a blank were run in triplicate 36 was determined the optical density
with each set of analyses.
The growth
of Lactobacillus
fermenti
turbidimetrically after 16-18 h incubation of the tubes at 37°C; readings were made at 546 rnp with a Beckman DU spectrophoto-
meter. The thiamine
values
are expressed
tissue and per g tissue nitrogen; tions
of nitrogen.
average
difference
RESULTS
AND
Normal
as ,ug of thiamine
the Kjeldahl
A high reproducibility between
triplicate
procedure
of the thiamine
analyses
hydrochloride
per g wet
was used for the determinaassays
was obtained,
the
being only 5.3%.
DISCUSSION
samples
The average
thiamine
concentrations
for intima-media
layers of various
types
of blood vessels are listed in Table 1. The mean value of 0.206 ,ug/g of wet tissue for 69 samples of the descending thoracic aorta is definitely higher than that reported for human
blood. A comparison
the same subjects
of values for pulmonary
did not reveal statistically
artery
significant
and aortic specimens
differences,
from
the average thia-
mine concentration of the pulmonary artery being 107.7% (wet tissue; t 0.83) and llS.Oo/o (tissue nitrogen; t 1.82) of that observed for the aorta. For the inferior vena cava, when expressed on the basis of wet tissue weight and tissue nitrogen content the values were, respectively, content. The coefficients vascular
84.9o/o (t 1.98) and 98.6o/o (t 0.18) of the aortic thiamine of correlation between age and thiamine concentrations of
tissues are listed in Table 2; as seen from these data, no significant
with age was found in thiamine
TABLE
2
COEFFICIENTS OF CORRELATION BETWEEN
Correlation
variation
levels.
between
age and
Descending thoracic aorta normal atherosclerotic, lipid Pulmonary artery, normal Vena cava inferior
Age
AGE
AND
No. of
group (yea-1
samples
O-84 20-84 18-84 17-72
69 44 33 27
Atherosclerotic samples The results of analyses
of atherosclerotic
THIAMINE
CONTENT
OF VASCULAR
Tissue
Wet tissue
TISSUE
nitrogen
Y
t
Y
t
+0.04 0.00 $0.12 -0.19
0.33 0.00 0.66 0.97
+0.15 +0.09 +0.15 -0.08
1.23 0.58 0.85 0.46
samples
are listedin Atherosclerosis,
Table 1. Compari1970,
12: 207-210
T. KHEIM,
210
son of thiamine the vascular great changes TABLE
concentrations
in normal
wall showed significantly were found for samples
tissue portions lower values
and in pathological
in fibrous
with lipid deposit
J. E. KIRK
plaques,
areas of
whereas
no
(Table 3).
3
THIAMINE CONTENT OF ATHEROSCLEROTIC CONTENT OF NORMALTISSUE OFTHE SAME
Age group (years)
No. of
TISSUE PORTIONS AORTIC SAMPLES
%
IN PERCENTAGES
OF
Tissue nitrogen
Wet tissue
samcles
EXPRESSED
t
%
t
Lipid changes 20-49 50-59 60-84 total
16 12 12 40
91.5 92.9 99.0 94.2
1.09 1.06 0.13 1.28
94.7 102.2 116.3 103.5
0.66 0.26 1.72 0.68
Fibrous changes 34-69
10
78.4
2.42
83.0
2.05
REFERENCES SARETT, H. P. AND V. H. CHELDELIN, The use of Lactobacillus fermenturn 36 for thiamine assay, J. biol. Chem.. 1944, 155: 153. FITZGERALD, E. E. AND E. B. HUGHES, The microbiological assay of aneurine: an improved method employing Lactobacillus fermenti 36, Analyst, 1949, 74: 340. CARVALHO DA SILVA, A., S. R. MONSAOAND R. CARLOTTA DE A~~~~~~,Tissulardist.ribution and biliaryexcretionofthiamineonnephrectomizedanimals, Actaphysiol.latino-amer., 1958, 8: 111.
Atherosclerosis,
1970, 12: 207-210