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Microcalorimetric study of metabolic inhibition by humic acids in mitochondria from Oryctolagus cuniculus domestica liver cells
Chemosphere, Vol. 33, No. 1, pp. 99-105, 1996 Copyright 0 1996 Elscvier Science Ltd Printed in Oreat Britain. All rigbta reserved 0045-6535196 $15.00+0.00
PII: soo45-6535(96)00151.8
Microcalorimetric
study of metabolic inhibition by
humic acids in mitochondria from Oryctolagus cuniculus
Yi Liu*,
qimg Wang, Chang - li Xie , Song - sheng
Xiao-
(Department P.
domestica liver cells
of Chemistry,
Wuhan
University,
Wuhan
QU
430072,
R. China) Feng - jiao Deng,
(School
of Life Science,
Wuhan
University,
Yu Guo
P . R . China)
Wuhan 430072,
(Received in USA 12 January 1996; accepted 8 April 1996)
Abstract
ohe
from Oryctolagus
metabolic
cuniculus
thermogenesis
domestica
ing an LKB - 2277 bioactivity dP/dt P,= was estabolished
and Keshan
isolated
After isolation,
nutrients.
mitochondria
The thennokinetic
by usmaintain
equation:
= k,=P’ Pa.+
k,*t
for the activity recovery phase of metabolism.
ies , the order of metabolism tochondria
of mitochondria
liver tissue have been determined
monitor.
activity when stored in appropriate
curves
was inhibited diseases,
For these stud-
was found to be zero. The metabolic
by humic acid, Experimental
a potential
results indicate
the heat output and lag time of mitochondrial Elsevier Science Ltd
99
toxicant
activity of mi-
in Kashin - Beck
that humic acid will effect
metabolism,
Copyrkht 0 19%
00
INTRODUCTION Mitochondria are important organelle involved in ATP production. isolation, they remain active and release heat during metabolism.
After
They are of-
ten referred to as the “power house” of the cell. ATP may be produced (by oxidation of a variety of elementary substrates) within mitochondria present in virtually all plant and animal cells,
therefore the study of metabolism by mito-
chondria is very important. Some enviromental toxicants can cause structural damage and metabolic changes in mitochondria . Recent research has indicated that humic acid can extensively damage liver cells.
The main organelle effected are mitochondria,
which become cavitas (marrow - like) structures”‘. In this paper, the thermogenesis curves and metabolic heat ouput of mitochondria isolated from Oryctolagus cuniculus domestica liver tissue was studied using an LKB - 2277 bioactivity monitor. The thermogenesis curves of mitochondria were obtained by the ampoule method. The first samples were isolated in normal isolating medium (A) , while the second samples were isolated in a humic acid - containing medium (B)-. Our objective was to determin if humic acids inhibited mitochondrial metabolism, in Kashin - Beck and Keshan diseases.
and thus could be a causative agest
Preliminany results indicated that there
were some differences in the thermogenesis
curves.
It was found that the
metabolic heat output was greater than normal when the isolating medium contained humic acids,
and that the lag phase time was less than normal. These
fact may be significant for toxicant physiology.
INSRUMENTAND MATERIAIS Instrument An LKB - 2277 bioactivity moniter was used for all thermogenesis studies ; the
performance
previouly[2’31.
and
details
of
its
construction
has
been
described
101
Materials Oryctolagus cuniculus domestica were provided by the animal farm of the School of Life Science, The hunk
WuhanUniversity.
acid was produced by Shanghai Second Chemical Reagent Fac-
tory, Shanghai, China. Isolating medium A was sucmse 0. 25 mol. Tris-HCl
10 mmol.
L-‘;
pH=7.
L-l;
hunk
L-l;
L-‘;
L-‘;
5.
Isolating medium B was sucrose 0. 25 mol. Tris - HCl 10 mmol.
L-r; EDTA 1 mmol.
acid 0. 3 wt.%;
EDTA 1 nunol.
pH=7.
5.
EXF'ERIMENTALMETHODS Isolation of mitochondria Hepatic cell of mitochondria were isolated by first removing the liver from the Oryctolagus cunicul us domestica and washing it with steriled isolating medium (one part was treated with isolating medium A ; the other part was treated with isolating medium B) . A portion of the liver was then weighted (2.
Og) ,
homogenised and centrifuged at 900 x g for 1Omin; the clear supematant was centrifuged twice for 2Omin at 900 x g each time, after each step.
the sediment being discarded
Th e c 1ear liquid was then centrifuged twice at high speed
(12ooO x g) 1Omin each time, to deposit the mitochondria as sediment.
This
was resuspended in the isolating medium for measurememts . All the above operations were performed aseptically at 272 - 277K. Experimental determination The metabolic thennogenesis curves of mitochondria were recorded using sealed ampoules, medium,
one containing a reference solution such as the isolating ’
the other containing the sample ( suspension of mitochondria) . The
102 sample normally occupied position A in the monitor and the reference occupied position B . Each ampoule contained a 1mL sample (mitochondria from 0.
5g
liver) or reference and 2mL of air. Sample A (treated with isolating medium A) was examined in the first channel and sample B (treated with isolating medium B) in the second channel. The temperature of all experiments was 35. 00% and the amplifiers of the monitor were set at 1OOpW.
RESULTS AND DISCUSSION The thermogenesis curves for two samples are shown in Fig.
1. Curve a is
the tbermogenesis curve of mitochondria metabolism of sample A; curve b is the thermogenesis curve of sample B (humic acid treated mitochondria)
.
35 . 30 . 25. 5‘ t 20. iz E 15. 10. 50 900 120015001800210024002700300033003600390042004500480051005400 TIME(min) Fig.
1 Thermogenesis curves of mitochondrial metabolism at 35 9: curve a: sample A (without humic acids) ; curve b: sample B (with humic acids)
103
As previously reportedC3*‘I, analysis of the thermogenesis curves reveals four regions : lag phase ; activity recovery phase ; stationary phase ; decline phase.
The
metabolism.
activity
recovery
From Fig.
phase
is
characteristic
of
mitochondrial
1, we can see that the thermogenesis curves of the ac-
tivity recovery phase was linear. P,= PO+ k;t dP,/dt
(1)
= k,=
k;Ps
(2)
The metabolic rate constant, k, , can be calculated from equation ( 1) . From ewe determined the order of metabolism to be zero. The heat out-
quation (21, put ( AHr
) of recovery phase, and the maximum heat power are shown in
Table 1. km and the corelation coefficient r are shown in Table 2. Table 1
Lag time, AHr,
liver weight and maximum thermal power of
mitochondrial metabolism
Sample
A B
Lag time
Heat
Table 2 Sample
(J)
t (mm>
U-h
1500
0. 9054
1270
AHr/W
Liver weight
1. 3635
Max. Power
(J. g-‘>
W (g)
6-m
0. 5
1. 8108
28. 0
0. 5
2. 7270
34. 6
Values of the metabolic rate constant k, , r and ,G k,
(uW.
min-‘)
r
p
(min-‘)
A
0. 02640
0. 99979
2. 681 x 1o-3
B
0. 03410
0. 99984
3. 404x 1o-3
104
Specific rate of power increase p was: )l=dP/PdP. During the recovery phase,
the specific rate of power increasing was :
dP/PdP = kJ’.
p=
The specific rate p decreased gradually during the recovery phase. If we adopt the data of P at a fLu time distance, we can calculate the mean specific rate p as follow: p = (kJP, + kJP,+ = (l/P,+ =
k,,,/n
l/Pz+ i=
. . . . . . + kflJ
/n
. . . . . . + l/P,,)
kJn
Clf’pi
(3)
i=n The values of ji are shown in Table 2. From these data we can see that AHr , the maximum heat output, k, and ,ciof sample B are greater than those of sample A. Certainly, the lag phase time of the former is less. At first appearance the metabolic activity of mitochondria is increased in the isolating medium containing humic acid. The reason for this is that the humic acid has possibly damaged the structure of mitochondria, and thus influenced their metabolism.
Some researchers have indicated that humic
acid can inhibit ATP synthesis in mitochondxia[“,
so the energy met&o lism of
nutrients was for the most part released with the heat output. This results is significant for studies of the relationship between some local diseases,
Kaahin -
Beck and Keshan and the toxicant humic acids.
ACKNOWLEDGEMENT This project was supported by National Nature Science Foundation of China.
105
REFERENCES 1 T. -S. Yang, Yixue Yanjiu Tongxun, 19(2) (1990) 32-33 2 J. Suurkuuskand I. Wadso, Chem. Ser., 242(1982) US- 163 3 C. -L. Xie, H. -K. Tang, Z. -H. Song and S. -S. Qu, Thermochim. Acta,123(1988) 33 -41 4 X. -Q. Wang, C. -L. Xie and S. -S. Qu, Thermochimi. Acta, 176 (1991) 69-74 5 Y. -Z. Wang, Yixue Yanjiu Tongxun, 19(5) (1990) 5 -7
PII: soo45-6535(96)00151.8
Microcalorimetric
study of metabolic inhibition by
humic acids in mitochondria from Oryctolagus cuniculus
Yi Liu*,
qimg Wang, Chang - li Xie , Song - sheng
Xiao-
(Department P.
domestica liver cells
of Chemistry,
Wuhan
University,
Wuhan
QU
430072,
R. China) Feng - jiao Deng,
(School
of Life Science,
Wuhan
University,
Yu Guo
P . R . China)
Wuhan 430072,
(Received in USA 12 January 1996; accepted 8 April 1996)
Abstract
ohe
from Oryctolagus
metabolic
cuniculus
thermogenesis
domestica
ing an LKB - 2277 bioactivity dP/dt P,= was estabolished
and Keshan
isolated
After isolation,
nutrients.
mitochondria
The thennokinetic
by usmaintain
equation:
= k,=P’ Pa.+
k,*t
for the activity recovery phase of metabolism.
ies , the order of metabolism tochondria
of mitochondria
liver tissue have been determined
monitor.
activity when stored in appropriate
curves
was inhibited diseases,
For these stud-
was found to be zero. The metabolic
by humic acid, Experimental
a potential
results indicate
the heat output and lag time of mitochondrial Elsevier Science Ltd
99
toxicant
activity of mi-
in Kashin - Beck
that humic acid will effect
metabolism,
Copyrkht 0 19%
00
INTRODUCTION Mitochondria are important organelle involved in ATP production. isolation, they remain active and release heat during metabolism.
After
They are of-
ten referred to as the “power house” of the cell. ATP may be produced (by oxidation of a variety of elementary substrates) within mitochondria present in virtually all plant and animal cells,
therefore the study of metabolism by mito-
chondria is very important. Some enviromental toxicants can cause structural damage and metabolic changes in mitochondria . Recent research has indicated that humic acid can extensively damage liver cells.
The main organelle effected are mitochondria,
which become cavitas (marrow - like) structures”‘. In this paper, the thermogenesis curves and metabolic heat ouput of mitochondria isolated from Oryctolagus cuniculus domestica liver tissue was studied using an LKB - 2277 bioactivity monitor. The thermogenesis curves of mitochondria were obtained by the ampoule method. The first samples were isolated in normal isolating medium (A) , while the second samples were isolated in a humic acid - containing medium (B)-. Our objective was to determin if humic acids inhibited mitochondrial metabolism, in Kashin - Beck and Keshan diseases.
and thus could be a causative agest
Preliminany results indicated that there
were some differences in the thermogenesis
curves.
It was found that the
metabolic heat output was greater than normal when the isolating medium contained humic acids,
and that the lag phase time was less than normal. These
fact may be significant for toxicant physiology.
INSRUMENTAND MATERIAIS Instrument An LKB - 2277 bioactivity moniter was used for all thermogenesis studies ; the
performance
previouly[2’31.
and
details
of
its
construction
has
been
described
101
Materials Oryctolagus cuniculus domestica were provided by the animal farm of the School of Life Science, The hunk
WuhanUniversity.
acid was produced by Shanghai Second Chemical Reagent Fac-
tory, Shanghai, China. Isolating medium A was sucmse 0. 25 mol. Tris-HCl
10 mmol.
L-‘;
pH=7.
L-l;
hunk
L-l;
L-‘;
L-‘;
5.
Isolating medium B was sucrose 0. 25 mol. Tris - HCl 10 mmol.
L-r; EDTA 1 mmol.
acid 0. 3 wt.%;
EDTA 1 nunol.
pH=7.
5.
EXF'ERIMENTALMETHODS Isolation of mitochondria Hepatic cell of mitochondria were isolated by first removing the liver from the Oryctolagus cunicul us domestica and washing it with steriled isolating medium (one part was treated with isolating medium A ; the other part was treated with isolating medium B) . A portion of the liver was then weighted (2.
Og) ,
homogenised and centrifuged at 900 x g for 1Omin; the clear supematant was centrifuged twice for 2Omin at 900 x g each time, after each step.
the sediment being discarded
Th e c 1ear liquid was then centrifuged twice at high speed
(12ooO x g) 1Omin each time, to deposit the mitochondria as sediment.
This
was resuspended in the isolating medium for measurememts . All the above operations were performed aseptically at 272 - 277K. Experimental determination The metabolic thennogenesis curves of mitochondria were recorded using sealed ampoules, medium,
one containing a reference solution such as the isolating ’
the other containing the sample ( suspension of mitochondria) . The
102 sample normally occupied position A in the monitor and the reference occupied position B . Each ampoule contained a 1mL sample (mitochondria from 0.
5g
liver) or reference and 2mL of air. Sample A (treated with isolating medium A) was examined in the first channel and sample B (treated with isolating medium B) in the second channel. The temperature of all experiments was 35. 00% and the amplifiers of the monitor were set at 1OOpW.
RESULTS AND DISCUSSION The thermogenesis curves for two samples are shown in Fig.
1. Curve a is
the tbermogenesis curve of mitochondria metabolism of sample A; curve b is the thermogenesis curve of sample B (humic acid treated mitochondria)
.
35 . 30 . 25. 5‘ t 20. iz E 15. 10. 50 900 120015001800210024002700300033003600390042004500480051005400 TIME(min) Fig.
1 Thermogenesis curves of mitochondrial metabolism at 35 9: curve a: sample A (without humic acids) ; curve b: sample B (with humic acids)
103
As previously reportedC3*‘I, analysis of the thermogenesis curves reveals four regions : lag phase ; activity recovery phase ; stationary phase ; decline phase.
The
metabolism.
activity
recovery
From Fig.
phase
is
characteristic
of
mitochondrial
1, we can see that the thermogenesis curves of the ac-
tivity recovery phase was linear. P,= PO+ k;t dP,/dt
(1)
= k,=
k;Ps
(2)
The metabolic rate constant, k, , can be calculated from equation ( 1) . From ewe determined the order of metabolism to be zero. The heat out-
quation (21, put ( AHr
) of recovery phase, and the maximum heat power are shown in
Table 1. km and the corelation coefficient r are shown in Table 2. Table 1
Lag time, AHr,
liver weight and maximum thermal power of
mitochondrial metabolism
Sample
A B
Lag time
Heat
Table 2 Sample
(J)
t (mm>
U-h
1500
0. 9054
1270
AHr/W
Liver weight
1. 3635
Max. Power
(J. g-‘>
W (g)
6-m
0. 5
1. 8108
28. 0
0. 5
2. 7270
34. 6
Values of the metabolic rate constant k, , r and ,G k,
(uW.
min-‘)
r
p
(min-‘)
A
0. 02640
0. 99979
2. 681 x 1o-3
B
0. 03410
0. 99984
3. 404x 1o-3
104
Specific rate of power increase p was: )l=dP/PdP. During the recovery phase,
the specific rate of power increasing was :
dP/PdP = kJ’.
p=
The specific rate p decreased gradually during the recovery phase. If we adopt the data of P at a fLu time distance, we can calculate the mean specific rate p as follow: p = (kJP, + kJP,+ = (l/P,+ =
k,,,/n
l/Pz+ i=
. . . . . . + kflJ
/n
. . . . . . + l/P,,)
kJn
Clf’pi
(3)
i=n The values of ji are shown in Table 2. From these data we can see that AHr , the maximum heat output, k, and ,ciof sample B are greater than those of sample A. Certainly, the lag phase time of the former is less. At first appearance the metabolic activity of mitochondria is increased in the isolating medium containing humic acid. The reason for this is that the humic acid has possibly damaged the structure of mitochondria, and thus influenced their metabolism.
Some researchers have indicated that humic
acid can inhibit ATP synthesis in mitochondxia[“,
so the energy met&o lism of
nutrients was for the most part released with the heat output. This results is significant for studies of the relationship between some local diseases,
Kaahin -
Beck and Keshan and the toxicant humic acids.
ACKNOWLEDGEMENT This project was supported by National Nature Science Foundation of China.
105
REFERENCES 1 T. -S. Yang, Yixue Yanjiu Tongxun, 19(2) (1990) 32-33 2 J. Suurkuuskand I. Wadso, Chem. Ser., 242(1982) US- 163 3 C. -L. Xie, H. -K. Tang, Z. -H. Song and S. -S. Qu, Thermochim. Acta,123(1988) 33 -41 4 X. -Q. Wang, C. -L. Xie and S. -S. Qu, Thermochimi. Acta, 176 (1991) 69-74 5 Y. -Z. Wang, Yixue Yanjiu Tongxun, 19(5) (1990) 5 -7