147
Clinica Chimica Acta, 60 (1975) 147-155 @ Elsevier Scientific Publishing Company,
Amsterdam
-
Printed
in The Netherlands
CCA 6862
DETERMINATION OF HUMAN LIVER CYTOCHROME P-450 LEVEL ELECTRON PARAMAGNETIC RESONANCE OF LIVER BIOPSIES
LUC GABRIELLEa, FRANCOIS and CHARLES LAVERDANTb
LETERRIERa,
PIERRE
BY
CRISTAUb
=Division de Biophysique, Centre de Recherches du Service de Santk des Armies, 1 bis rue du Lieutenant Raoul Batany, 92140 Clamart (France) and bService de GastroentBrologie, HGpital d ‘Instruction des ArmPes Bkgin, 94160 Saint-Mande (France) (Received
September
30, 1974)
Summary Human liver cytochrome P-450 level has been determined by direct electron paramagnetic resonance spectroscopy at 77°K of liver specimens obtained by surgical biopsy. A preliminary experiment with rat livers was performed in order to calibrate the intensity of the cytochrome P-450 EPR signal as a function of the cytochrome concentration obtained by the classical spectrophotometric method. 44 subjects have been examined. We found a cytochrome P-450 value of 21.5 * 11.2 nmoles per gram of liver in normal subjects. Variations of this level in some pathological liver states are discussed.
Introduction The structure and functions of cytochrome P-450 have been extensively studied in various animal tissues since it was first described [1,2] It has been demonstrated that the role of this hemoprotein is fundamental in the metabolism of endogenous and xenobiotic compounds. However little is known about the variations of cytochrome P-450 level in humans either in healthy subjects or in various liver diseases [3--g]. Human cytochrome P-450 studies are obviously limited by the practical difficulties of obtaining adequate liver samples. Post-mortem hepatic fragments are not suitable because the cytochrome level decreases very quickly after death [7]. Alvares et al. [ 31 have examined liver specimens obtained during abdominal surgery. It is easier to obtain human hepatic tissue by needle or surgical biopsy, but the liver fragments are small (1 to 20 mg) and it is necessary to use micromethods. Such a micromethod has been recently proposed by Schoene et al. [7] .
148
They use a very sensitive absorption spectrophotometer. The present work gives the results obtained by electron spin resonance spectroscopy performed directly on the liver samples. Our method has been calibrated by preliminary measurements on rat liver, and was applied to the examination of 44 human liver biopsies. Material and Methods
(1) Rat liver tissue, mitochondria
and microsomes
preparation
Sprague-Dawley male rats weighing about 200 g were anesthetised with ether after 12 h of fasting. Two liver tissue samples (about 20 mg each) were cut with scissors and immediately introduced into 4-mm internal diameter quartz tubes, weighed and immersed in liquid nitrogen (77°K). The animals were then sacrificed and their livers were used for the preparation of mitochondria and microsomes by the classical differential ultracentrifugation method. Microsomes (105 000 X g 1 h, in 0.25 M sucrose) were washed with 0.05 M, pH 7.5 phosphate buffer containing 0.1 M KC1 and 10e3 M EDTA. Protein concentrations were measured by the method of Gomall et al. [lo]. (2) Pretreatment of animals In order to obtain large variations of cytochrome P-450 levels, the following drugs were administered to the rats: 80 mg/kg phenobarbital in saline intraperitoneally during 3 days, 25 mg/kg methylcholanthrene in corn oil intraperitoneally, two consecutive days, and 1 mg/kg tetrachloromethane (50 g/100 ml paraffin oil) by intubation 24 h before liver sampling [ll]. (3) Cytochrome P-450 measurements The classical spectrophotometric measurement [ 121 was performed on microsomes with a Cary 15 spectrophotometer. Electron paramagnetic resonance spectra were recorded in liquid nitrogen with a Varian E 3 spectrometer. Liver samples were studied without preparation in their quartz tubes. 0.3 ml of mitochondria, microsomes (about 20 mg/ml protein) or 105 000 X g supernatant were examined in the same type of quartz tubes and under the same conditions as liver samples. Before each series of measurements the base line was carefully recorded and the spectrometer sensitivity was checked with the Varian pitch No. 90445002. The amplitude of the low spin g = 2.27 line was considered as proportiond to the cytochrome P-450 concentration in the sample. This amplitude was eventually corrected for the variations of the spectrometer sensitivity and base line. It was then divided by the liver sample weight or by the microsomal or mitochondrial protein concentrations. The results were expressed as EPR arbitrary units in mm per gram of liver or per mg/ml protein. (4) Origin of the human liver samples Liver biopsies were performed under laparoscopy in the Gastroenterology Departement of the H8pital Begin (Professors Ch. Laverdant and P. Cristau). One part of the liver fragment was used for classical histological examination, the other part was placed in a glass tube and immediately transported to the
149
laboratory in a container filled with solid CO, . The liver samples were stored at -35°C and the EPR spectra recorded as soon as possible under the same conditions as the rat liver samples. 44 subjects have been examined. They have been classified under clinical, biological and histological criteria in the following groups: (a) normal livers, (b) schistosomiasis treated by nitrothiazole, (c) cirrhosis, (d) cytosteatosis (two subgroups: mild and severe) and (e) viral hepatitis. Results (1) EPR spectra of microsomes, mitochondria and whole rat livers The spectra recorded with microsomal suspensions (Fig. la) show the three classical low spin lines of cytochrome P-450 at g = 1.91, 2.27 and 2.42 [13,15]. Whole liver sample spectra are more complicated (Fig. lb). However, the g = 2.27 line is well resolved, and its amplitude can be measured without difficulty. The g = 2.42 line is also present, but its intensity is too low to allow precise determinations. Between g = 1.9 and g = 2.1, numerous lines are observable. They are due to mitochondria (Fig. lc). An intense g = 2.003 free radical signal is observed in some samples.
Fig. 1. Rat liver EPR spectra. a, microsomes; b, whole liver fragment (20 me); c, mitochondria. Experimental conditions: Varian E3 spectrometer; temperature 77’K: microwave power 100 mW; modulation amplitude 20 g; gain 104; scan speed 33 gauss/min.
150
High-spin signals of cytochrome P-450 are not observable at 77°K [13,14]. However, a low amplitude g = 6 line of unknown origin is sometimes recorded. No EPR signal was recorded in the 105 000 X g supernatant. (2) Calibration of the EPR measurements as a function of the spectrophotometric cytochrome P-450 determination In order to calibrate the EPR arbitrary units (y), we have plotted them as a function of the cytochrome P-450 concentrations (x) measured by spectrophotometry. These plots are shown in Figs 2 and 3 for microsomes and whole liver tissue samples, respectively. The following regression lines have been calculated by the least-squares method: microsomes: y = 4.93 x + 0.5 (r = 0.897), whole liver: y’ = 160 x’ + 2260 (r = 0.866). The regression coefficients r are highly significant (48 couples of values, p < 0.001). We have verified that the amplitude of the EPR cytochrome P-450 signal increased linearly as a function of the liver sample weight. Results obtained with various fragments of the same rat liver are shown in Fig. 4. Direct proportionality is observed between 0 and 40 mg. (3) EPR spectra of human liver samples A typical EPR spectrum of human liver is shown in Fig. 5. It is qualitatively identical with the rat liver spectrum. The g = 2.27 cytochrome P-450 line is clearly observable and measurable. The amplitude of this line does not decrease with time when the samples are stored at -35” C. Ten human liver fragments have been recorded immediately after the biopsy, then stored for 1 month, and again recorded; no difference was observable between both series of spectra.
P-450
(nmol /mg
protein)
Fig. 2. Plot of the EPR arbitrary units (mmlmg protein) versus cytochrome P-450 concentration of rat microsomes. The regression line is Y = 4.93 x + 0.5. Tetrachloromethane-treated rats (m); controls, first series (0); second series (0); third series (0); methylcholanthrene-treated rats (A); phenobarbital-treated rats (A).
151
I
I
/
I
I I 10
I
I
I
I 20
I
I
I
II
30
II
I
II
40
I
I
II
11 5o
11 P-450
11
“1
Ct-Roll’s
liver)
Fig. 3. Plot of the EPR arbitrary units (mm/g liver) versus cytochrome P-450 content of rat liver (nmolk liver). The equation of the regression line is Y = 160 x + 2260. Same symbols as on Fig. 2.
EPR
signal
liver
Fig. 4. Plot of the EPR signal intensity
weight [me]
versus rat liver sample weight.
152
Fig.
5.
Fig.
1.
Human
liver
EPR
spectrum.
The
sample
weight
was
5.2
mg.
Same
experimental
condition
as in
(4) Evaluation of the human liver cytochrome P-450 content The mean weight of the human liver fragments was 4.51 + 2.24 mg with a minimum of 1.4 and a maximum of 11.7 mg. The smallness of these samples did not allow us to perform parallel spectrophotometric determinations by the method of Schoene et al. [7], with a classical spectrophotometer. Thus, we TABLE
1
DETERMINATION
Normal
liver
OF
Subjects ving
Nunber
nmollg of
CYTOCHROME
recei-
case
Liver
nitrothia-
ZOk
liver Num-
nmol/g
ber of
liver
P-450
CONTENT
cirrhosis
Liver ~_._~
-~-Nun-
nmol/g
ber of
liver
IN
HUMAN
steatosis
Viral hepatitis .~__~__
Severe
case
Mild
Num-
nmol/g
Num-
nmol/g
her of
liver
ber of
liver
case
case
LIVER
Num-
nmol/g
ber of
liver
case
case
__.~ 2
16.1
4
53.5
38
0
5
16.7
14
19.7
58
9.6
7
0
19
19.8
1
0
21
15.6
22
37.8
9
15 26
11
12.6
16
22.9
60
22.8
33
16.4
28
9.5
41
12
32.0
20
43.8
62
0
49
10.8
34
24.3
50
0
18
17.6
56
14.0
51
39
36.2
54
53
61
61.5
55
19.6
5.8
23
21.4
53
14.6
24
48.2
64
14
26
9.1
32
20.9
36
7.0
42
26.7
43
37.5
48
20.2
52
10.6
63
20.8
65**
1
In = 11.0*
m = 31.5*
m = 16.37
u
o
= 18.1
0
@
Clinica Chimica Acta, 60 (1975) 147-155 @ Elsevier Scientific Publishing Company,
Amsterdam
-
Printed
in The Netherlands
CCA 6862
DETERMINATION OF HUMAN LIVER CYTOCHROME P-450 LEVEL ELECTRON PARAMAGNETIC RESONANCE OF LIVER BIOPSIES
LUC GABRIELLEa, FRANCOIS and CHARLES LAVERDANTb
LETERRIERa,
PIERRE
BY
CRISTAUb
=Division de Biophysique, Centre de Recherches du Service de Santk des Armies, 1 bis rue du Lieutenant Raoul Batany, 92140 Clamart (France) and bService de GastroentBrologie, HGpital d ‘Instruction des ArmPes Bkgin, 94160 Saint-Mande (France) (Received
September
30, 1974)
Summary Human liver cytochrome P-450 level has been determined by direct electron paramagnetic resonance spectroscopy at 77°K of liver specimens obtained by surgical biopsy. A preliminary experiment with rat livers was performed in order to calibrate the intensity of the cytochrome P-450 EPR signal as a function of the cytochrome concentration obtained by the classical spectrophotometric method. 44 subjects have been examined. We found a cytochrome P-450 value of 21.5 * 11.2 nmoles per gram of liver in normal subjects. Variations of this level in some pathological liver states are discussed.
Introduction The structure and functions of cytochrome P-450 have been extensively studied in various animal tissues since it was first described [1,2] It has been demonstrated that the role of this hemoprotein is fundamental in the metabolism of endogenous and xenobiotic compounds. However little is known about the variations of cytochrome P-450 level in humans either in healthy subjects or in various liver diseases [3--g]. Human cytochrome P-450 studies are obviously limited by the practical difficulties of obtaining adequate liver samples. Post-mortem hepatic fragments are not suitable because the cytochrome level decreases very quickly after death [7]. Alvares et al. [ 31 have examined liver specimens obtained during abdominal surgery. It is easier to obtain human hepatic tissue by needle or surgical biopsy, but the liver fragments are small (1 to 20 mg) and it is necessary to use micromethods. Such a micromethod has been recently proposed by Schoene et al. [7] .
148
They use a very sensitive absorption spectrophotometer. The present work gives the results obtained by electron spin resonance spectroscopy performed directly on the liver samples. Our method has been calibrated by preliminary measurements on rat liver, and was applied to the examination of 44 human liver biopsies. Material and Methods
(1) Rat liver tissue, mitochondria
and microsomes
preparation
Sprague-Dawley male rats weighing about 200 g were anesthetised with ether after 12 h of fasting. Two liver tissue samples (about 20 mg each) were cut with scissors and immediately introduced into 4-mm internal diameter quartz tubes, weighed and immersed in liquid nitrogen (77°K). The animals were then sacrificed and their livers were used for the preparation of mitochondria and microsomes by the classical differential ultracentrifugation method. Microsomes (105 000 X g 1 h, in 0.25 M sucrose) were washed with 0.05 M, pH 7.5 phosphate buffer containing 0.1 M KC1 and 10e3 M EDTA. Protein concentrations were measured by the method of Gomall et al. [lo]. (2) Pretreatment of animals In order to obtain large variations of cytochrome P-450 levels, the following drugs were administered to the rats: 80 mg/kg phenobarbital in saline intraperitoneally during 3 days, 25 mg/kg methylcholanthrene in corn oil intraperitoneally, two consecutive days, and 1 mg/kg tetrachloromethane (50 g/100 ml paraffin oil) by intubation 24 h before liver sampling [ll]. (3) Cytochrome P-450 measurements The classical spectrophotometric measurement [ 121 was performed on microsomes with a Cary 15 spectrophotometer. Electron paramagnetic resonance spectra were recorded in liquid nitrogen with a Varian E 3 spectrometer. Liver samples were studied without preparation in their quartz tubes. 0.3 ml of mitochondria, microsomes (about 20 mg/ml protein) or 105 000 X g supernatant were examined in the same type of quartz tubes and under the same conditions as liver samples. Before each series of measurements the base line was carefully recorded and the spectrometer sensitivity was checked with the Varian pitch No. 90445002. The amplitude of the low spin g = 2.27 line was considered as proportiond to the cytochrome P-450 concentration in the sample. This amplitude was eventually corrected for the variations of the spectrometer sensitivity and base line. It was then divided by the liver sample weight or by the microsomal or mitochondrial protein concentrations. The results were expressed as EPR arbitrary units in mm per gram of liver or per mg/ml protein. (4) Origin of the human liver samples Liver biopsies were performed under laparoscopy in the Gastroenterology Departement of the H8pital Begin (Professors Ch. Laverdant and P. Cristau). One part of the liver fragment was used for classical histological examination, the other part was placed in a glass tube and immediately transported to the
149
laboratory in a container filled with solid CO, . The liver samples were stored at -35°C and the EPR spectra recorded as soon as possible under the same conditions as the rat liver samples. 44 subjects have been examined. They have been classified under clinical, biological and histological criteria in the following groups: (a) normal livers, (b) schistosomiasis treated by nitrothiazole, (c) cirrhosis, (d) cytosteatosis (two subgroups: mild and severe) and (e) viral hepatitis. Results (1) EPR spectra of microsomes, mitochondria and whole rat livers The spectra recorded with microsomal suspensions (Fig. la) show the three classical low spin lines of cytochrome P-450 at g = 1.91, 2.27 and 2.42 [13,15]. Whole liver sample spectra are more complicated (Fig. lb). However, the g = 2.27 line is well resolved, and its amplitude can be measured without difficulty. The g = 2.42 line is also present, but its intensity is too low to allow precise determinations. Between g = 1.9 and g = 2.1, numerous lines are observable. They are due to mitochondria (Fig. lc). An intense g = 2.003 free radical signal is observed in some samples.
Fig. 1. Rat liver EPR spectra. a, microsomes; b, whole liver fragment (20 me); c, mitochondria. Experimental conditions: Varian E3 spectrometer; temperature 77’K: microwave power 100 mW; modulation amplitude 20 g; gain 104; scan speed 33 gauss/min.
150
High-spin signals of cytochrome P-450 are not observable at 77°K [13,14]. However, a low amplitude g = 6 line of unknown origin is sometimes recorded. No EPR signal was recorded in the 105 000 X g supernatant. (2) Calibration of the EPR measurements as a function of the spectrophotometric cytochrome P-450 determination In order to calibrate the EPR arbitrary units (y), we have plotted them as a function of the cytochrome P-450 concentrations (x) measured by spectrophotometry. These plots are shown in Figs 2 and 3 for microsomes and whole liver tissue samples, respectively. The following regression lines have been calculated by the least-squares method: microsomes: y = 4.93 x + 0.5 (r = 0.897), whole liver: y’ = 160 x’ + 2260 (r = 0.866). The regression coefficients r are highly significant (48 couples of values, p < 0.001). We have verified that the amplitude of the EPR cytochrome P-450 signal increased linearly as a function of the liver sample weight. Results obtained with various fragments of the same rat liver are shown in Fig. 4. Direct proportionality is observed between 0 and 40 mg. (3) EPR spectra of human liver samples A typical EPR spectrum of human liver is shown in Fig. 5. It is qualitatively identical with the rat liver spectrum. The g = 2.27 cytochrome P-450 line is clearly observable and measurable. The amplitude of this line does not decrease with time when the samples are stored at -35” C. Ten human liver fragments have been recorded immediately after the biopsy, then stored for 1 month, and again recorded; no difference was observable between both series of spectra.
P-450
(nmol /mg
protein)
Fig. 2. Plot of the EPR arbitrary units (mmlmg protein) versus cytochrome P-450 concentration of rat microsomes. The regression line is Y = 4.93 x + 0.5. Tetrachloromethane-treated rats (m); controls, first series (0); second series (0); third series (0); methylcholanthrene-treated rats (A); phenobarbital-treated rats (A).
151
I
I
/
I
I I 10
I
I
I
I 20
I
I
I
II
30
II
I
II
40
I
I
II
11 5o
11 P-450
11
“1
Ct-Roll’s
liver)
Fig. 3. Plot of the EPR arbitrary units (mm/g liver) versus cytochrome P-450 content of rat liver (nmolk liver). The equation of the regression line is Y = 160 x + 2260. Same symbols as on Fig. 2.
EPR
signal
liver
Fig. 4. Plot of the EPR signal intensity
weight [me]
versus rat liver sample weight.
152
Fig.
5.
Fig.
1.
Human
liver
EPR
spectrum.
The
sample
weight
was
5.2
mg.
Same
experimental
condition
as in
(4) Evaluation of the human liver cytochrome P-450 content The mean weight of the human liver fragments was 4.51 + 2.24 mg with a minimum of 1.4 and a maximum of 11.7 mg. The smallness of these samples did not allow us to perform parallel spectrophotometric determinations by the method of Schoene et al. [7], with a classical spectrophotometer. Thus, we TABLE
1
DETERMINATION
Normal
liver
OF
Subjects ving
Nunber
nmollg of
CYTOCHROME
recei-
case
Liver
nitrothia-
ZOk
liver Num-
nmol/g
ber of
liver
P-450
CONTENT
cirrhosis
Liver ~_._~
-~-Nun-
nmol/g
ber of
liver
IN
HUMAN
steatosis
Viral hepatitis .~__~__
Severe
case
Mild
Num-
nmol/g
Num-
nmol/g
her of
liver
ber of
liver
case
case
LIVER
Num-
nmol/g
ber of
liver
case
case
__.~ 2
16.1
4
53.5
38
0
5
16.7
14
19.7
58
9.6
7
0
19
19.8
1
0
21
15.6
22
37.8
9
15 26
11
12.6
16
22.9
60
22.8
33
16.4
28
9.5
41
12
32.0
20
43.8
62
0
49
10.8
34
24.3
50
0
18
17.6
56
14.0
51
39
36.2
54
53
61
61.5
55
19.6
5.8
23
21.4
53
14.6
24
48.2
64
14
26
9.1
32
20.9
36
7.0
42
26.7
43
37.5
48
20.2
52
10.6
63
20.8
65**
1
In = 11.0*
m = 31.5*
m = 16.37
u
o
= 18.1
0
@
