- Email: [email protected]
Determination of the activities of the enzyme complexes of the electron transport chain in human fibroblasts
•
/!
ELSEVIER
Clinica Chimica Acta 253 (1996) 79-90
Determination of the activities of the enzyme complexes of the electron transport chain in human fibroblasts S t e p h a n Krfihenbiihl *a, Theres Sch~ifer b, Ulrich W i e s m a n n b aDivision of Clinical Pharmacology and Toxicology, Department of Internal Medicine. University Hospital CH-8091 Z'itrich, Switzerland bDivision of Pediatric Metabolism, Children Hospital University of Berne, Berne, Switzerland Received 18 October 1995; revision received l l March 1996; accepted 4 April 1996
Abstract
In order to improve the determination of the activities of the enzyme complexes of the electron transport chain (ETC) in fibroblasts, we characterized the isolation of mitochondria and measured enzyme activities in mitochondrial preparations from fibroblasts of control subjects and patients with suspected mitochondrial cytopathy. The isolation procedure yielded 54% of the citrate synthase activity in fibroblasts, with a 6-fold enrichment in this mitochondrial marker enzyme. The activities of the complexes of the ETC were linear with time and with the mitochondrial protein concentration used. The coefficients of variation for the enzyme activities determined were in the range of 10% in mitochondria from identical fibroblast cultures and between 30 and 70% in mitochondria from different fibroblast cultures of the same or of different patients. Decreased activities of one or more enzyme complexes (defined as an activity below the 95% confidence limit of control values) were found in 15 of 22 patients investigated. When compared with activities obtained in liver or skeletal muscle obtained at autopsy, the results were identical in three but different in two patients. The studies show that the activities of the enzyme complexes of the ETC can be determined reliably and reproducibly in mitochondria isolated from fibroblasts and that the results obtained are potentially useful for the diagnosis of mitochondrial cytopathies in patients with suggestive symptoms and signs.
Keywords: Electron transport chain; Fibroblast; Mitochondria; Enzyme complexes
*Corresponding author, Tel: +41 1 2552068; fax: +41 12554411. 0098-8981/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S0009-8981(96)06338-3
80
S. Kr?thenb~hl et al. I Clinica Chimica Acta 253 (1996) 79-90
1. Introduction
A reduced activity of enzyme complexes of the mitochondrial electron transport chain (ETC) can be associated with symptoms and signs of mitochondrial cytopathies such as lactacidemia, myopathy, cardiomyopathy, encephalopathy and hepatopathy I-1-3]. Diagnoses of mitochondrial cytopathies are principally based on clinical presentation, biochemical analysis of the enzyme complexes of the ETC and on the demonstration of mutations in the mitochondrial genome I-1-3]. Reduced activities of the enzyme complexes of the ETC in patients with mitochondrial cytopathy have been demonstrated in biopsies from the organs clinically involved but also in other tissues or cells, including cultured fibroblasts and lymphocytes 1,3-5]. In comparison to tissue biopsies, fibroblasts have the advantages that they can be obtained easily from skin biopsies and can be cultured over long periods of time. Usually, permeabilized or lysed, but unfractionated, fibroblasts are used for the measurement of the activity of the ETC or of specific enzyme activities [5,6]. However, for the enzyme complexes of the ETC, in particular complex I, this may be unsuitable, since, in unfractionated fibroblasts, their activities have to be determined against a variably high background activity, resulting either from unspecific enzymatic or non-enzymatic reactions. Pilot experiments with unfractionated fibroblasts have shown that in particular the determination of the rotenone-sensitive activity of complex I is unreliable with a poor reproducibility. Therefore, we worked out a simple procedure for the isolation of mitochondria from fibroblasts with subsequent determination of the enzyme activities in the mitochondrial fraction. The results show that the activities of the enzyme complexes of the ETC can be determined reliably in mitochondria isolated from cultured fibroblasts and that this procedure can be useful for the diagnosis of mitochondrial cytopathies. 2. Materials and methods
2.1. Materials The chemicals used were obtained from the identical suppliers and in the same quality as described in the publications describing the enzyme assays cited below. Decylubiquinone and 1,10-phenanthroline were obtained from Sigma (Buchs, Switzerland). The enzyme assays were performed at 30°C on an Uvikon 930 spectrophotometer.
2.2. Cultivation of fibroblasts and &olation of mitochondria Skin biopsies for the cultivation of fibroblasts were obtained from 17 healthy control subjects and from 22 patients with clinical suspicion of a
S. KrT~henb~hl et al. I Clinica Chimica Acta 253 (1996) 79-9(I
81
mitochondrial disorder. Fibroblasts were cultured under standard conditions in M E M containing 200 mmol/1 uridine and 10% fetal bovine serum as described previously 1,5,1.They were harvested with a rubber policeman, washed twice with saline and once with phosphate-buffered saline. From the resulting fibroblast pellet, mitochondria were isolated at 5°C according to Darley-Usmar et al. with some modifications 1,7]. Briefly, the fibroblast pellet was frozen at - 8 0 ° C for at least 15 min. After thawing, the pellet was suspended in 2 ml isolation buffer (0.25 mol/1 sucrose, 1 mmol/1 EGTA, 10 mmol/1 Hepes, pH 7.4, and 0.5% bovine serum albumin) and centrifuged for 2 min at 500 x g. The supernatant was discarded and the pellet was suspended in 3 ml isolation buffer and homogenized using a tight fitting Potter-Elvejhelm homogenizer (fibroblast homogenate). After centrifugation (10 min at 1500 x g), the supernatant was kept on ice and the pellet was rehomogenized arid centrifuged as described above. The two supernatants were combined and centrifuged at 10 000 x g for 10 min. The resulting mitochondrial pellet was washed twice with albumin-free isolation buffer and stored at - 8 0 ° C until used. For the enzyme assays and the protein determination, the pellet was suspended in 1 ml HeO (mitochondrial suspension). The protein contents of the fibroblast homogenate and the mitochondrial suspension were determined by the Lowry method with bovine serum albumin as a standard [8]. For the calculation of the fibroblast protein content, the albumin concentration of the isolation buffer was subtracted from the total protein concentration of the fibroblast homogenate.
2.3. Enzyme assays The activity of complex I (NADH:ubiquinone oxidoreductase) was determined according to Veitch et al. [9,1 with the modifications described previously 1,10,1. Decylubiquinone was used as an electron acceptor at a final concentration of 75 mmol/l. Since pilot experiments have shown that both rotenone (final concentration 5 mmol/l) or 1,10-phenanthroline (final concentration 400 mmol/1) 1,11-1could be used with identical results for the inhibition of complex I activity, either rotenone or 1,10-phenanthroline were used. The activities of complex II (succinate:ubiquinone oxidoreductase) and complex III (ubiquinol:ferricytochrome c oxidoreductase) were determined as described previously 1,5,10,12]. The activity of complex IV (ferrocytochrome c:oxygen oxidoreductase or cytochrome c oxidase) was determined according to Wharton and Tzogaloff I-13,1. The activity of citrate synthase was determined according to Srere [14,1 and the activity of catalase according to Aebi 1,15,1. The activity of arylsulfatase A was measured spectrophotometrically according to Baum et al. 1,16]. The activity of succinate dehydrogenase was measured spectrophotometrically according to Hoppel and Cooper 1,17].
82
S. Kr~thenb~hl et al. I Clinica Chimica Acta 253 (1996) 79-90
H u m a n tissues (skeletal muscle or liver) were homogenized in 20 mmol/l potassium phosphate buffer p H 7.2 (50 mg tissue per 1 ml p h o s p h a t e buffer). This homogenate was used as the enzyme source for the determination of the activities of the mitochondrial enzymes which were assayed as described above. All assays were determined in duplicate or in triplicate and the average of the individual determinations is given.
2.4. Stat&tics D a t a are presented as mean ___S.D. (Table 1 and text), mean + S.D. and coefficient of variation (Table 2) or mean + 95% confidence interval (Fig. 2).
3. Results The aim of the current studies was to establish and validate simple spectrophotometric assays for the measurement of the activities of the enzyme complexes of the E T C in h u m a n fibroblasts. Since in particular complex I is difficult to measure in sonicated but unfractionated fibroblasts, we choose to isolate mitochondria from the fibroblasts and to measure the activities of the enzyme complexes of the E T C in isolated mitochondria. The mitochondrial preparation is characterized in Table 1. Approximately 10% of the fibroblast protein was recovered in the mitochondrial pellet. In comparison, the recovery of citrate synthase, the mitochondrial marker enzyme used, was 54%, resulting in a 6.1 ___2.0-fold enrichment. In pilot experiments, the recovery of succinate dehydrogenase, an enzyme located in the inner mitochondrial membrane, was in the same range as
Table 1 Characterization of mitochondria isolated from cultured human fibroblasts. Mitochondria were isolated as described in Methods. Values represent the mean __+S.D. of mitochondrial preparations of fibroblasts from seven different control subjects. The recovery is given in parentheses (in % of the fibroblast homogenate)
Protein (rag) Citrate synthase (mU) Arylsulfatase A (mU) Catalase (mU)
Fibroblast homogenate
Mitochondrial pellet
3.90 + 1.38 43.5 __+20.2 28.0 __+26.0 17.6 + 10.9
0.37 + 0.17 (9.5 + 3.2) 22.7+ 11.6(54.3 __+14.1) 4.2 ___+2.6 (20.9 + 13.3) 7.2 __ 2.0 (47.7 + 14.7)
S. Kr?thenb~hl et al. I Clinica Chimica A cta 253 (1996) 79-90
COMPLEX I
COMPLEX II
1.2
O
0
A3
0.9 0.6
> 1
16A
83
0.3
I
I
I
I
40
80
120
160
0.0
COMPLEX III
I
I
I
60
90
120
COMPLEX IV
2.0
12-
I
30
1.5 o o o
i
4
o
r,
/
1.0
o
0.5 El
i , 10 20 ~g protein/assay
~
30
0.0
I
0
2
I
I
I
4 6 8 gg protein/assay
I
10
Fig. 1. Protein dependency of the activities of the enzyme complexes of the electron transport chain in mitochondria from fibroblasts of control subjects. Mitochondria were isolated and enzyme activities were determined as described in Methods. The determinations were carried out in duplicate or triplicate with the average of the individual determinations presented. O, total activities; F-l, the blank (obtained in the presence of a specific inhibitor as described in Methods); A, net activity (obtained by subtraction of the blank from total activity).
obtained for citrate synthase. Therefore, only citrate synthase activity was determined as a mitochondrial m a r k e r enzyme in the final studies. The enrichment of arylsulfatase A, a lysosomal marker, was 2.1 _ 0.9, and of catalase, a peroxisomal marker, 5.2 _ 1.6. Using this preparation, time and protein dependencies of the assays for citrate synthase and the enzyme complexes of the ETC were characterized. With the protein concentrations used in Fig. 1, the reactions were linear during at least 2 (complex III and complex IV) or 3 min (complex I, complex II and citrate synthase). The protein dependencies are shown in Fig. 1. Linear ranges (in/~g protein per assay) were found between 20 and 150 /~g for complex I, 10 and 120 /~g for complex II, 4 and 30 #g for complex III and 1 and 10 #g for complex IV. Accordingly, the a m o u n t of protein per assay routinely used in the subsequent determinations was 100 #g for complex I and II, 25 #g for complex III and 10/~g for complex IV.
84
S. Kr~ihenb~hl et al. I Clinica Chimica Acta 253 (1996) 79-90
Importantly, the inhibition of NADH oxidation by rotenone or 1,10phenanthroline was 50% or more at all protein concentrations used, indicating that by the isolation of mitochondria, a significant portion of the inhibitor-insensitive NADH oxidation in unfractionated fibroblasts could be eliminated. Similarly, at the protein concentrations usually used, the inhibitor-sensitive activities of complexes II, III and IV were also clearly above 50%. For citrate synthase, the linear range was between 2 and 35/~g protein per assay (not shown in Fig. 1). As a next step, the variability of the enzyme assays was investigated in mitochondrial preparations from fibroblasts of the same or of different control subjects (Table 2). The coefficients of variation of the activities of the enzyme complexes in different mitochondrial preparations from identical fibroblast cultures were in the range of 10% or lower and did not depend on the way the activity was expressed (per mg mitochondrial protein or per activity of citrate synthase or complex IV). In comparison, the variability of the same assays in mitochondrial preparations from different fibroblast cultures originating from the same control subject (multiple skin biopsies obtained at different time points) were higher, with coefficients of variation ranging from 39 to 59%. When expressed per citrate synthase or per complex IV, the variability did not improve significantly. Similarly, the coefficients of variation of the assays in mitochondria from fibroblast cultures of different control subjects were in the range of 40% and did not improve significantly when expressed per citrate synthase or per complex IV activity. Mitochondria of fibroblasts cultured from skin biopsies of 22 patients whose symptoms and signs were suggestive of a mitochondrial cytopathy (e.g. lactacidemia, myopathy, cardiomyopathy, encephalopathy or hepatopathy) were isolated and the activities of citrate synthase and of the complexes of the ETC determined. As illustrated in Fig. 2, when expressed per mg protein, the number of patients having reduced activities (defined as an activity below the 95% confidence interval of the mean obtained in ten healthy control subjects) were one for complex I, eight for complex II, six for complex III and none for complex IV. When expressed per activity of citrate synthase, two additional patients had a reduced activity of complex I, six out of the eight patients mentioned above had a reduced activity of complex II, six out of six patients had a reduced activity of complex III and two patients had a reduced activity of complex IV. Seven patients had an isolated reduction of the activity of complex II and three of complex III, respectively. Two patients had reduced activities of complexes I and III and one patient of complexes II and IV. One patient had reduced activities of complexes I, II and III and another patient of complexes I, III and IV. Citrate synthase activity was normal (within the
S. Kr?~henb#hl et al. I Clinica Chimica Acta 253 (1996) 79-90
8 ~ •
_
o ~ ¢-.Io
oe °' , . - . ,,,~- ,(::5
+1 +1
~= +1 +1
t ' ~ I'~-
.~
~
•"~
~•
_
+~
~
-~
,,,.4 ~ ,r,,"~ o
,.-,~',.,4
+1 +1
£"4 I "~.-
~
"'"~'~"
I'~
~'.s'._
" ~ e ~ ~___~ ~
~ +1+1+1
~ +1+1+1
= +1+1+1
•- - -
,~
e~
qD
£
II
e~
~2
~
~ ~
~,~
~
+1+1+1
~ +1+1 +1 ~ +1 +1+1
~----,,
o.,~-
_
+1+1+1
~ +1+1+1
~"~ +1+1+1
°~
,,~
e.,-
e.,
8 0o
0
"-d
o<
.o
o
~='-"
..0
~=~, .~
e.,
~ +~
~ +~
~
o
•~ 8
~ .o .~ :~E~8
- 2 ,.-'~'-'~-=°
E E
.~ E ~ E
.-=
e.,
£ .£ N.~
~ ~
=,.,
.:-
e-,
s.~ ~'~
E
~
N
85
S. Kr~henb~hl et al. I Clinica Chiraica Acta 253 (1996) 79-90
86
ACTIVITY PER MITOCHONDRIAL PROTEIN
"3
100
10
80
S
500
400
•
.I.-95%CL
6
200
• t-
95% CL
I
mean
120
300
!
E
4
mean
>
2o
.
-~- 95% CL I
0
COMP
I
"If" 95%CL
! 80
95% CL
100 -
|
I
mean
i 40 - -~.. 95% CL
|
L. 95% CL
0
t
I
I . -*-- 9"5Y. CL 2 mean
!
200
!
160
0
0
C O M P III
COMPII
C O M P IV
ACTIVITY PER CITRATE SYNTHASE A
|
2.0
0.5
35
14
30
12
25
10
20
8
15
6
II
t-
>, M
0.4
1.5
J o
•
0.3
•
0.2
•
1.0
-o-- 9S%CL
E
v
0.5
> ,< 0.0
10
"i'" 95% CL 0.1 | mean "1"" 95% C L 0.0
COMPI
I
mean
i - ~-- 95% CL
COMP II
. . . . 95*/* C L
~ 5 0
4
mean
/.I..95%CL "
COMP Ill
e
:-- 95%CL 2
"1"- mean • IF- 9 5 % C L
0 C O M P IV
Fig. 2. Activities of the enzyme complexes (COMP) of the electron transport chain in mitochondria isolated from fibroblasts cultivated from 22 patients with clinicallysuspected mitochondrial cytopathy. Activities are expressed per mg mitochondrial protein (upper panel) or per citrate synthase activity (lower panel). Mitochondria were isolated and enzyme activities determined as described in Methods• Mean and 95% confidence limits (CL) of previously determined control values (obtained from ten differentcontrol subjects) are indicated. A total of 15 patients had a decreased activity of one or more enzyme complexes.
95% confidence interval of control subjects) for all patients with reduced activities of enzyme complexes. F o r five patients, the activities of the enzyme complexes of the E T C were also determined in skeletal muscle a n d / o r liver (tissue samples obtained at autopsy). In three patients, the results were identical (reduced activities of complex III in two and normal activities in one patient). One
S. Kr~henb~hl et al. I Clinica Chimica Acta 253 (1996) 79-90
87
patient had a reduced activity of complex II in fibroblasts which was normal in skeletal muscle. Another patient had reduced activities of complexes I and III in skeletal muscle but normal activities in fibroblasts.
4. Discussion
The current studies show that mitochondria can be isolated quickly and in a good yield from isolated fibroblasts and that the activities of the enzyme complexes of the ETC can be determined reliably in these mitochondria. As shown in Table 1, approximately 50% of the activity of citrate synthase was recovered in the mitochondrial preparation, suggesting that the isolation procedure used yields a representative sample of the mitochondria in fibroblasts. The ratio of the marker enzymes citrate synthase (mitochondria), arylsulfatase A (lysosomes) and catalase (peroxisomes) was 1:0.64:0.40 in the fibroblast homogenate and 1:0.18:0.32 in mitochondria, showing that, in comparison to mitochondria, both lysosomes and, to a lesser extent also peroxisomes, were depleted during the isolation procedure. Using the specific activities (activity per mg protein of the respective organelle) of catalase 1-18] or arylsulfatase A 1-19] in rat liver, a crude estimate for the contamination with these organelles can be obtained. It can be estimated that 1 mg protein of the mitochondrial preparation contained approximately 3 #g of peroxisomal and 25 /~g of lysosomal protein. Thus, the current procedure results in the isolation of a sufficient amount of mitochondrial protein from fibroblasts and the isolated mitochondria have an acceptable degree of purity. The activity of the enzyme complexes of the ETC could be determined reliably with the assays used but the variability among mitochondria obtained from fibroblasts originating from different biopsies of the same subject or from different subjects was considerable. Since the variability of the activities in different mitochondrial preparations from identical fibroblast cultures was in the range of 10% or less, the major part of the variability must result from the origin of the fibroblasts but not from the enzyme assays or from the isolation of the mitochondria. Expression of the enzyme activities per citrate synthase or per complex IV activity did not result in a decrease in the variability, again suggesting that the major part of the variability observed originated from the mitochondria themselves but not from the isolation procedure or from the enzyme assays. As shown in Fig. 1, the activities of the enzyme complexes of the ETC could be determined reliably with an acceptable protein dependency and good inhibition by the respective specific inhibitors. In particular for complex I, the inhibition was 50% or more and was well reproducible for
88
S. Kr~henb~hl et al. I Clinica Chiraica Acta 253 (1996) 79-90
both inhibitors used, rotenone and 1,10-phenanthroline. These results are clearly better than those obtained in unfractionated fibroblasts, demonstrating the value of the isolation procedure. The assays described were used to determine the activities of the enzyme complexes of the ETC in mitochondrial preparations from fibroblasts grown from 22 patients with symptoms and signs of mitochondrial cytopathies. As shown in Fig. 2 and described in the Results section, a total of 15 patients had a reduced activity (lower than the 95% confidence interval of control fibroblasts) of one or more enzyme complexes of the ETC. In some patients, the isolation procedure and the enzyme assays were repeated in other fibroblast cultures and the results were identical. In comparison with the same enzyme activities determined in tissues, an identical result was obtained in 3 and a different result in 2 patients. While the divergence between the low enzyme activities in tissues and normal activities in mitochondria from fibroblasts in one patient may be explained by the delay between death and autopsy, differences between fibroblasts and tissues are also compatible with the well known tissue-specific expression of the deficiencies of the enzyme complexes of the ETC 1-20,21]. These differences between the enzyme activities in fibroblasts and tissues suggest that the activities obtained in fibroblasts are complementary rather than confirmatory to the activities obtained in tissues and have to be interpreted together with the clinical picture and, if available, with molecular studies of the mitochondrial genome. Interestingly, as shown in Fig. 2, increased activities of enzyme complexes of the ETC were also found in mitochondria isolated from fibroblasts of some patients. Since increased activities were mostly found in mitochondrial preparations with a decrease in the activity of one or more other enzyme complexes, this may reflect increased expression and/or decreased degradation of unaffected enzyme complexes, a finding also observed in hepatic mitochondria from rats with liver cirrhosis 1-22]. In conclusion, we have developed and characterized a simple and rapid isolation procedure for mitochondria from fibroblasts which allows a reliable and reproducible determination of the activities of the enzyme complexes of the ETC in fibroblasts from patients with clinically suspected mitochondrial cytopathies. However, due to tissue-specific expression of the defects, the results obtained in fibroblasts should be interpreted with caution and should be confirmed at the molecular level.
Acknowledgement The work was supported by a grant from the Swiss National Foundation to SK (SNF 32-37280.93).
S. KrZthenbVthlet al. I Clinica Chimica Acta 253 (1996) 79-90
89
References [1] Holt IJ, Harding AE, Cooper JM et al. Mitochondrial myopathies: clinical and biochemical features of 30 patients with major deletions of muscle mitochondrial DNA. Ann Neurol 1989;26:699-708. [2] Zeviani M, Antozzi C. Defects of mitochondrial DNA. Brain Pathol 1992;2:121-132. [3] Rustin P, Chretien D, Bourgeron T et al. Biochemical and molecular investigations in respiratory chain deficiencies. Clin Chim Acta 1994;228:35-51. [4] Scholte HR, Busch HFM, Luyt-Houwen EM, Vaandrager-Verduin MHM, Przyrembel H, Arts WFM. Defects in oxidative phosphorylation. Biochemical investigations in skeletal muscle and expression of the lesion in other cells. J Inher Metab Dis 1987;10(Suppl. 0:81-97. [5] Kriihenbiihl S, Talos C, Wiesmann U, Hoppel CU Development and evaluation of a spectrophotometric assay for complex III in isolated mitochondria, tissues and fibroblasts from rats and humans. Clin Chim Acta 1994;230:177-187. [6] Wanders RJA, Ruiter JPN, Wijburg FA. Studies on mitochondrial oxidative phosphorylation in permeabilized human skin fibroblasts: application to mitochondrial encephalomyopathies. Biochim Biophys Acta 1993;1181:219-222. [7] In: Darley-Usmar VM, Rickwood D, Wilson MT, eds. Mitochondria, a practical approach. Oxford, Washington: IRL Press, 1987; pp. 4-6. [8] Lowry OH, Rosebrough N J, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-275. [9] Veitch K, Hombroeckx A, Caucheteux D, Pouleur H, Hue L. Global ischemia induces a biphasic response of the mitochondrial respiratory chain. Biochem J 1992;281:709- 715. [10] Kr/ihenbiihl S, Talos C, Reichen J. Mechanisms of impaired hepatic fatty acid metabolism in rats with long-term bile duct ligation. Hepatology 1994;19:12721281. [I1] Finel M, Majander A. Studies on the proton-translocating NADH:ubiquinone oxidoreductases of mitochondria and Escherichia coli using the inhibitor 1,10phenanthroline. FEBS Lett 1994;339:142-146. [12] Kriihenbiihl S, Chang M, Brass EP, Hoppel C. Decreased activities of ubiquinol:ferricytochrome c oxidoreductase (complex III) and ferrocytochrome c:oxygen oxidoreductase (complex IV) in liver mitochondria from rats with hydroxycobolamin[c-lactam]-induced methylmalonic aciduria. J Biol Chem 1991; 266: 20998- 21003. [13] Wharton DC, Tzagoloff A. Cytochrome oxidase from beef heart mitochondria. Methods Enzymol 1967;10:245-250. [14] Srere P. Citrate synthase. Methods Enzymol 1969;13:3-11. [15] ,~.bi H. Catalase. In: Bergmeyer HU, ed. Methods of enzymatic analysis. 3rd ed. Weinheim, Germany: Verlag Chemie GmbH 1983;273-286. [16] Baum H, Dodgson KS, Spencer B. The assay of arylsulfatases A and B in human urine. Clin Chim Acta 1959;4:453-455. [17] Hoppel CL, Cooper C. The action of digitonin on rat liver mitochondria. The effects on enzyme content. Biochem J 1968;107:367-375. [18] V61kl A, Fahimi HD. Isolation and characterization of peroxisomes from the liver of normal untreated rats. Eur J Biochem 1985;149:257-265. [19] Dufour JF, Gehr P, Reichen J. Hepatic accumulation of lysosomes and defective transcytotic vesicular pathways in cirrhotic rat liver. Hepatology 1992;16:9971006.
90
S. Kr~henb~hl et al. I Clinica Chimica Acta 253 (1996) 79-90
1-201 Suomalainen A, Majander A, Haltia M et al. Multiple deletions of mitochondrial DNA in several tissues of a patient with severe retarded depression and familial progressive external ophthalmoplegia. J Clin Invest 1992;90:61-66. [21] Shoji Y, Sato W, Hayasaka K, Takada G. Tissue distribution of mutant mitochondrial DNA in mitochondrial myopathy, encephalopathy, lactic acidosis and strokelike episodes (MELAS). J Inher Metab Dis 1993;16:27-30. [22] Kr~ihenbiihl S, Reichen J, Zimmermann A, Gehr P, Stucki J. Mitochondrial structure and function in CC14-induced cirrhosis in the rat. Hepatology 1990;12:526-532.
/!
ELSEVIER
Clinica Chimica Acta 253 (1996) 79-90
Determination of the activities of the enzyme complexes of the electron transport chain in human fibroblasts S t e p h a n Krfihenbiihl *a, Theres Sch~ifer b, Ulrich W i e s m a n n b aDivision of Clinical Pharmacology and Toxicology, Department of Internal Medicine. University Hospital CH-8091 Z'itrich, Switzerland bDivision of Pediatric Metabolism, Children Hospital University of Berne, Berne, Switzerland Received 18 October 1995; revision received l l March 1996; accepted 4 April 1996
Abstract
In order to improve the determination of the activities of the enzyme complexes of the electron transport chain (ETC) in fibroblasts, we characterized the isolation of mitochondria and measured enzyme activities in mitochondrial preparations from fibroblasts of control subjects and patients with suspected mitochondrial cytopathy. The isolation procedure yielded 54% of the citrate synthase activity in fibroblasts, with a 6-fold enrichment in this mitochondrial marker enzyme. The activities of the complexes of the ETC were linear with time and with the mitochondrial protein concentration used. The coefficients of variation for the enzyme activities determined were in the range of 10% in mitochondria from identical fibroblast cultures and between 30 and 70% in mitochondria from different fibroblast cultures of the same or of different patients. Decreased activities of one or more enzyme complexes (defined as an activity below the 95% confidence limit of control values) were found in 15 of 22 patients investigated. When compared with activities obtained in liver or skeletal muscle obtained at autopsy, the results were identical in three but different in two patients. The studies show that the activities of the enzyme complexes of the ETC can be determined reliably and reproducibly in mitochondria isolated from fibroblasts and that the results obtained are potentially useful for the diagnosis of mitochondrial cytopathies in patients with suggestive symptoms and signs.
Keywords: Electron transport chain; Fibroblast; Mitochondria; Enzyme complexes
*Corresponding author, Tel: +41 1 2552068; fax: +41 12554411. 0098-8981/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S0009-8981(96)06338-3
80
S. Kr?thenb~hl et al. I Clinica Chimica Acta 253 (1996) 79-90
1. Introduction
A reduced activity of enzyme complexes of the mitochondrial electron transport chain (ETC) can be associated with symptoms and signs of mitochondrial cytopathies such as lactacidemia, myopathy, cardiomyopathy, encephalopathy and hepatopathy I-1-3]. Diagnoses of mitochondrial cytopathies are principally based on clinical presentation, biochemical analysis of the enzyme complexes of the ETC and on the demonstration of mutations in the mitochondrial genome I-1-3]. Reduced activities of the enzyme complexes of the ETC in patients with mitochondrial cytopathy have been demonstrated in biopsies from the organs clinically involved but also in other tissues or cells, including cultured fibroblasts and lymphocytes 1,3-5]. In comparison to tissue biopsies, fibroblasts have the advantages that they can be obtained easily from skin biopsies and can be cultured over long periods of time. Usually, permeabilized or lysed, but unfractionated, fibroblasts are used for the measurement of the activity of the ETC or of specific enzyme activities [5,6]. However, for the enzyme complexes of the ETC, in particular complex I, this may be unsuitable, since, in unfractionated fibroblasts, their activities have to be determined against a variably high background activity, resulting either from unspecific enzymatic or non-enzymatic reactions. Pilot experiments with unfractionated fibroblasts have shown that in particular the determination of the rotenone-sensitive activity of complex I is unreliable with a poor reproducibility. Therefore, we worked out a simple procedure for the isolation of mitochondria from fibroblasts with subsequent determination of the enzyme activities in the mitochondrial fraction. The results show that the activities of the enzyme complexes of the ETC can be determined reliably in mitochondria isolated from cultured fibroblasts and that this procedure can be useful for the diagnosis of mitochondrial cytopathies. 2. Materials and methods
2.1. Materials The chemicals used were obtained from the identical suppliers and in the same quality as described in the publications describing the enzyme assays cited below. Decylubiquinone and 1,10-phenanthroline were obtained from Sigma (Buchs, Switzerland). The enzyme assays were performed at 30°C on an Uvikon 930 spectrophotometer.
2.2. Cultivation of fibroblasts and &olation of mitochondria Skin biopsies for the cultivation of fibroblasts were obtained from 17 healthy control subjects and from 22 patients with clinical suspicion of a
S. KrT~henb~hl et al. I Clinica Chimica Acta 253 (1996) 79-9(I
81
mitochondrial disorder. Fibroblasts were cultured under standard conditions in M E M containing 200 mmol/1 uridine and 10% fetal bovine serum as described previously 1,5,1.They were harvested with a rubber policeman, washed twice with saline and once with phosphate-buffered saline. From the resulting fibroblast pellet, mitochondria were isolated at 5°C according to Darley-Usmar et al. with some modifications 1,7]. Briefly, the fibroblast pellet was frozen at - 8 0 ° C for at least 15 min. After thawing, the pellet was suspended in 2 ml isolation buffer (0.25 mol/1 sucrose, 1 mmol/1 EGTA, 10 mmol/1 Hepes, pH 7.4, and 0.5% bovine serum albumin) and centrifuged for 2 min at 500 x g. The supernatant was discarded and the pellet was suspended in 3 ml isolation buffer and homogenized using a tight fitting Potter-Elvejhelm homogenizer (fibroblast homogenate). After centrifugation (10 min at 1500 x g), the supernatant was kept on ice and the pellet was rehomogenized arid centrifuged as described above. The two supernatants were combined and centrifuged at 10 000 x g for 10 min. The resulting mitochondrial pellet was washed twice with albumin-free isolation buffer and stored at - 8 0 ° C until used. For the enzyme assays and the protein determination, the pellet was suspended in 1 ml HeO (mitochondrial suspension). The protein contents of the fibroblast homogenate and the mitochondrial suspension were determined by the Lowry method with bovine serum albumin as a standard [8]. For the calculation of the fibroblast protein content, the albumin concentration of the isolation buffer was subtracted from the total protein concentration of the fibroblast homogenate.
2.3. Enzyme assays The activity of complex I (NADH:ubiquinone oxidoreductase) was determined according to Veitch et al. [9,1 with the modifications described previously 1,10,1. Decylubiquinone was used as an electron acceptor at a final concentration of 75 mmol/l. Since pilot experiments have shown that both rotenone (final concentration 5 mmol/l) or 1,10-phenanthroline (final concentration 400 mmol/1) 1,11-1could be used with identical results for the inhibition of complex I activity, either rotenone or 1,10-phenanthroline were used. The activities of complex II (succinate:ubiquinone oxidoreductase) and complex III (ubiquinol:ferricytochrome c oxidoreductase) were determined as described previously 1,5,10,12]. The activity of complex IV (ferrocytochrome c:oxygen oxidoreductase or cytochrome c oxidase) was determined according to Wharton and Tzogaloff I-13,1. The activity of citrate synthase was determined according to Srere [14,1 and the activity of catalase according to Aebi 1,15,1. The activity of arylsulfatase A was measured spectrophotometrically according to Baum et al. 1,16]. The activity of succinate dehydrogenase was measured spectrophotometrically according to Hoppel and Cooper 1,17].
82
S. Kr~thenb~hl et al. I Clinica Chimica Acta 253 (1996) 79-90
H u m a n tissues (skeletal muscle or liver) were homogenized in 20 mmol/l potassium phosphate buffer p H 7.2 (50 mg tissue per 1 ml p h o s p h a t e buffer). This homogenate was used as the enzyme source for the determination of the activities of the mitochondrial enzymes which were assayed as described above. All assays were determined in duplicate or in triplicate and the average of the individual determinations is given.
2.4. Stat&tics D a t a are presented as mean ___S.D. (Table 1 and text), mean + S.D. and coefficient of variation (Table 2) or mean + 95% confidence interval (Fig. 2).
3. Results The aim of the current studies was to establish and validate simple spectrophotometric assays for the measurement of the activities of the enzyme complexes of the E T C in h u m a n fibroblasts. Since in particular complex I is difficult to measure in sonicated but unfractionated fibroblasts, we choose to isolate mitochondria from the fibroblasts and to measure the activities of the enzyme complexes of the E T C in isolated mitochondria. The mitochondrial preparation is characterized in Table 1. Approximately 10% of the fibroblast protein was recovered in the mitochondrial pellet. In comparison, the recovery of citrate synthase, the mitochondrial marker enzyme used, was 54%, resulting in a 6.1 ___2.0-fold enrichment. In pilot experiments, the recovery of succinate dehydrogenase, an enzyme located in the inner mitochondrial membrane, was in the same range as
Table 1 Characterization of mitochondria isolated from cultured human fibroblasts. Mitochondria were isolated as described in Methods. Values represent the mean __+S.D. of mitochondrial preparations of fibroblasts from seven different control subjects. The recovery is given in parentheses (in % of the fibroblast homogenate)
Protein (rag) Citrate synthase (mU) Arylsulfatase A (mU) Catalase (mU)
Fibroblast homogenate
Mitochondrial pellet
3.90 + 1.38 43.5 __+20.2 28.0 __+26.0 17.6 + 10.9
0.37 + 0.17 (9.5 + 3.2) 22.7+ 11.6(54.3 __+14.1) 4.2 ___+2.6 (20.9 + 13.3) 7.2 __ 2.0 (47.7 + 14.7)
S. Kr?thenb~hl et al. I Clinica Chimica A cta 253 (1996) 79-90
COMPLEX I
COMPLEX II
1.2
O
0
A3
0.9 0.6
> 1
16A
83
0.3
I
I
I
I
40
80
120
160
0.0
COMPLEX III
I
I
I
60
90
120
COMPLEX IV
2.0
12-
I
30
1.5 o o o
i
4
o
r,
/
1.0
o
0.5 El
i , 10 20 ~g protein/assay
~
30
0.0
I
0
2
I
I
I
4 6 8 gg protein/assay
I
10
Fig. 1. Protein dependency of the activities of the enzyme complexes of the electron transport chain in mitochondria from fibroblasts of control subjects. Mitochondria were isolated and enzyme activities were determined as described in Methods. The determinations were carried out in duplicate or triplicate with the average of the individual determinations presented. O, total activities; F-l, the blank (obtained in the presence of a specific inhibitor as described in Methods); A, net activity (obtained by subtraction of the blank from total activity).
obtained for citrate synthase. Therefore, only citrate synthase activity was determined as a mitochondrial m a r k e r enzyme in the final studies. The enrichment of arylsulfatase A, a lysosomal marker, was 2.1 _ 0.9, and of catalase, a peroxisomal marker, 5.2 _ 1.6. Using this preparation, time and protein dependencies of the assays for citrate synthase and the enzyme complexes of the ETC were characterized. With the protein concentrations used in Fig. 1, the reactions were linear during at least 2 (complex III and complex IV) or 3 min (complex I, complex II and citrate synthase). The protein dependencies are shown in Fig. 1. Linear ranges (in/~g protein per assay) were found between 20 and 150 /~g for complex I, 10 and 120 /~g for complex II, 4 and 30 #g for complex III and 1 and 10 #g for complex IV. Accordingly, the a m o u n t of protein per assay routinely used in the subsequent determinations was 100 #g for complex I and II, 25 #g for complex III and 10/~g for complex IV.
84
S. Kr~ihenb~hl et al. I Clinica Chimica Acta 253 (1996) 79-90
Importantly, the inhibition of NADH oxidation by rotenone or 1,10phenanthroline was 50% or more at all protein concentrations used, indicating that by the isolation of mitochondria, a significant portion of the inhibitor-insensitive NADH oxidation in unfractionated fibroblasts could be eliminated. Similarly, at the protein concentrations usually used, the inhibitor-sensitive activities of complexes II, III and IV were also clearly above 50%. For citrate synthase, the linear range was between 2 and 35/~g protein per assay (not shown in Fig. 1). As a next step, the variability of the enzyme assays was investigated in mitochondrial preparations from fibroblasts of the same or of different control subjects (Table 2). The coefficients of variation of the activities of the enzyme complexes in different mitochondrial preparations from identical fibroblast cultures were in the range of 10% or lower and did not depend on the way the activity was expressed (per mg mitochondrial protein or per activity of citrate synthase or complex IV). In comparison, the variability of the same assays in mitochondrial preparations from different fibroblast cultures originating from the same control subject (multiple skin biopsies obtained at different time points) were higher, with coefficients of variation ranging from 39 to 59%. When expressed per citrate synthase or per complex IV, the variability did not improve significantly. Similarly, the coefficients of variation of the assays in mitochondria from fibroblast cultures of different control subjects were in the range of 40% and did not improve significantly when expressed per citrate synthase or per complex IV activity. Mitochondria of fibroblasts cultured from skin biopsies of 22 patients whose symptoms and signs were suggestive of a mitochondrial cytopathy (e.g. lactacidemia, myopathy, cardiomyopathy, encephalopathy or hepatopathy) were isolated and the activities of citrate synthase and of the complexes of the ETC determined. As illustrated in Fig. 2, when expressed per mg protein, the number of patients having reduced activities (defined as an activity below the 95% confidence interval of the mean obtained in ten healthy control subjects) were one for complex I, eight for complex II, six for complex III and none for complex IV. When expressed per activity of citrate synthase, two additional patients had a reduced activity of complex I, six out of the eight patients mentioned above had a reduced activity of complex II, six out of six patients had a reduced activity of complex III and two patients had a reduced activity of complex IV. Seven patients had an isolated reduction of the activity of complex II and three of complex III, respectively. Two patients had reduced activities of complexes I and III and one patient of complexes II and IV. One patient had reduced activities of complexes I, II and III and another patient of complexes I, III and IV. Citrate synthase activity was normal (within the
S. Kr?~henb#hl et al. I Clinica Chimica Acta 253 (1996) 79-90
8 ~ •
_
o ~ ¢-.Io
oe °' , . - . ,,,~- ,(::5
+1 +1
~= +1 +1
t ' ~ I'~-
.~
~
•"~
~•
_
+~
~
-~
,,,.4 ~ ,r,,"~ o
,.-,~',.,4
+1 +1
£"4 I "~.-
~
"'"~'~"
I'~
~'.s'._
" ~ e ~ ~___~ ~
~ +1+1+1
~ +1+1+1
= +1+1+1
•- - -
,~
e~
qD
£
II
e~
~2
~
~ ~
~,~
~
+1+1+1
~ +1+1 +1 ~ +1 +1+1
~----,,
o.,~-
_
+1+1+1
~ +1+1+1
~"~ +1+1+1
°~
,,~
e.,-
e.,
8 0o
0
"-d
o<
.o
o
~='-"
..0
~=~, .~
e.,
~ +~
~ +~
~
o
•~ 8
~ .o .~ :~E~8
- 2 ,.-'~'-'~-=°
E E
.~ E ~ E
.-=
e.,
£ .£ N.~
~ ~
=,.,
.:-
e-,
s.~ ~'~
E
~
N
85
S. Kr~henb~hl et al. I Clinica Chiraica Acta 253 (1996) 79-90
86
ACTIVITY PER MITOCHONDRIAL PROTEIN
"3
100
10
80
S
500
400
•
.I.-95%CL
6
200
• t-
95% CL
I
mean
120
300
!
E
4
mean
>
2o
.
-~- 95% CL I
0
COMP
I
"If" 95%CL
! 80
95% CL
100 -
|
I
mean
i 40 - -~.. 95% CL
|
L. 95% CL
0
t
I
I . -*-- 9"5Y. CL 2 mean
!
200
!
160
0
0
C O M P III
COMPII
C O M P IV
ACTIVITY PER CITRATE SYNTHASE A
|
2.0
0.5
35
14
30
12
25
10
20
8
15
6
II
t-
>, M
0.4
1.5
J o
•
0.3
•
0.2
•
1.0
-o-- 9S%CL
E
v
0.5
> ,< 0.0
10
"i'" 95% CL 0.1 | mean "1"" 95% C L 0.0
COMPI
I
mean
i - ~-- 95% CL
COMP II
. . . . 95*/* C L
~ 5 0
4
mean
/.I..95%CL "
COMP Ill
e
:-- 95%CL 2
"1"- mean • IF- 9 5 % C L
0 C O M P IV
Fig. 2. Activities of the enzyme complexes (COMP) of the electron transport chain in mitochondria isolated from fibroblasts cultivated from 22 patients with clinicallysuspected mitochondrial cytopathy. Activities are expressed per mg mitochondrial protein (upper panel) or per citrate synthase activity (lower panel). Mitochondria were isolated and enzyme activities determined as described in Methods• Mean and 95% confidence limits (CL) of previously determined control values (obtained from ten differentcontrol subjects) are indicated. A total of 15 patients had a decreased activity of one or more enzyme complexes.
95% confidence interval of control subjects) for all patients with reduced activities of enzyme complexes. F o r five patients, the activities of the enzyme complexes of the E T C were also determined in skeletal muscle a n d / o r liver (tissue samples obtained at autopsy). In three patients, the results were identical (reduced activities of complex III in two and normal activities in one patient). One
S. Kr~henb~hl et al. I Clinica Chimica Acta 253 (1996) 79-90
87
patient had a reduced activity of complex II in fibroblasts which was normal in skeletal muscle. Another patient had reduced activities of complexes I and III in skeletal muscle but normal activities in fibroblasts.
4. Discussion
The current studies show that mitochondria can be isolated quickly and in a good yield from isolated fibroblasts and that the activities of the enzyme complexes of the ETC can be determined reliably in these mitochondria. As shown in Table 1, approximately 50% of the activity of citrate synthase was recovered in the mitochondrial preparation, suggesting that the isolation procedure used yields a representative sample of the mitochondria in fibroblasts. The ratio of the marker enzymes citrate synthase (mitochondria), arylsulfatase A (lysosomes) and catalase (peroxisomes) was 1:0.64:0.40 in the fibroblast homogenate and 1:0.18:0.32 in mitochondria, showing that, in comparison to mitochondria, both lysosomes and, to a lesser extent also peroxisomes, were depleted during the isolation procedure. Using the specific activities (activity per mg protein of the respective organelle) of catalase 1-18] or arylsulfatase A 1-19] in rat liver, a crude estimate for the contamination with these organelles can be obtained. It can be estimated that 1 mg protein of the mitochondrial preparation contained approximately 3 #g of peroxisomal and 25 /~g of lysosomal protein. Thus, the current procedure results in the isolation of a sufficient amount of mitochondrial protein from fibroblasts and the isolated mitochondria have an acceptable degree of purity. The activity of the enzyme complexes of the ETC could be determined reliably with the assays used but the variability among mitochondria obtained from fibroblasts originating from different biopsies of the same subject or from different subjects was considerable. Since the variability of the activities in different mitochondrial preparations from identical fibroblast cultures was in the range of 10% or less, the major part of the variability must result from the origin of the fibroblasts but not from the enzyme assays or from the isolation of the mitochondria. Expression of the enzyme activities per citrate synthase or per complex IV activity did not result in a decrease in the variability, again suggesting that the major part of the variability observed originated from the mitochondria themselves but not from the isolation procedure or from the enzyme assays. As shown in Fig. 1, the activities of the enzyme complexes of the ETC could be determined reliably with an acceptable protein dependency and good inhibition by the respective specific inhibitors. In particular for complex I, the inhibition was 50% or more and was well reproducible for
88
S. Kr~henb~hl et al. I Clinica Chiraica Acta 253 (1996) 79-90
both inhibitors used, rotenone and 1,10-phenanthroline. These results are clearly better than those obtained in unfractionated fibroblasts, demonstrating the value of the isolation procedure. The assays described were used to determine the activities of the enzyme complexes of the ETC in mitochondrial preparations from fibroblasts grown from 22 patients with symptoms and signs of mitochondrial cytopathies. As shown in Fig. 2 and described in the Results section, a total of 15 patients had a reduced activity (lower than the 95% confidence interval of control fibroblasts) of one or more enzyme complexes of the ETC. In some patients, the isolation procedure and the enzyme assays were repeated in other fibroblast cultures and the results were identical. In comparison with the same enzyme activities determined in tissues, an identical result was obtained in 3 and a different result in 2 patients. While the divergence between the low enzyme activities in tissues and normal activities in mitochondria from fibroblasts in one patient may be explained by the delay between death and autopsy, differences between fibroblasts and tissues are also compatible with the well known tissue-specific expression of the deficiencies of the enzyme complexes of the ETC 1-20,21]. These differences between the enzyme activities in fibroblasts and tissues suggest that the activities obtained in fibroblasts are complementary rather than confirmatory to the activities obtained in tissues and have to be interpreted together with the clinical picture and, if available, with molecular studies of the mitochondrial genome. Interestingly, as shown in Fig. 2, increased activities of enzyme complexes of the ETC were also found in mitochondria isolated from fibroblasts of some patients. Since increased activities were mostly found in mitochondrial preparations with a decrease in the activity of one or more other enzyme complexes, this may reflect increased expression and/or decreased degradation of unaffected enzyme complexes, a finding also observed in hepatic mitochondria from rats with liver cirrhosis 1-22]. In conclusion, we have developed and characterized a simple and rapid isolation procedure for mitochondria from fibroblasts which allows a reliable and reproducible determination of the activities of the enzyme complexes of the ETC in fibroblasts from patients with clinically suspected mitochondrial cytopathies. However, due to tissue-specific expression of the defects, the results obtained in fibroblasts should be interpreted with caution and should be confirmed at the molecular level.
Acknowledgement The work was supported by a grant from the Swiss National Foundation to SK (SNF 32-37280.93).
S. KrZthenbVthlet al. I Clinica Chimica Acta 253 (1996) 79-90
89
References [1] Holt IJ, Harding AE, Cooper JM et al. Mitochondrial myopathies: clinical and biochemical features of 30 patients with major deletions of muscle mitochondrial DNA. Ann Neurol 1989;26:699-708. [2] Zeviani M, Antozzi C. Defects of mitochondrial DNA. Brain Pathol 1992;2:121-132. [3] Rustin P, Chretien D, Bourgeron T et al. Biochemical and molecular investigations in respiratory chain deficiencies. Clin Chim Acta 1994;228:35-51. [4] Scholte HR, Busch HFM, Luyt-Houwen EM, Vaandrager-Verduin MHM, Przyrembel H, Arts WFM. Defects in oxidative phosphorylation. Biochemical investigations in skeletal muscle and expression of the lesion in other cells. J Inher Metab Dis 1987;10(Suppl. 0:81-97. [5] Kriihenbiihl S, Talos C, Wiesmann U, Hoppel CU Development and evaluation of a spectrophotometric assay for complex III in isolated mitochondria, tissues and fibroblasts from rats and humans. Clin Chim Acta 1994;230:177-187. [6] Wanders RJA, Ruiter JPN, Wijburg FA. Studies on mitochondrial oxidative phosphorylation in permeabilized human skin fibroblasts: application to mitochondrial encephalomyopathies. Biochim Biophys Acta 1993;1181:219-222. [7] In: Darley-Usmar VM, Rickwood D, Wilson MT, eds. Mitochondria, a practical approach. Oxford, Washington: IRL Press, 1987; pp. 4-6. [8] Lowry OH, Rosebrough N J, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-275. [9] Veitch K, Hombroeckx A, Caucheteux D, Pouleur H, Hue L. Global ischemia induces a biphasic response of the mitochondrial respiratory chain. Biochem J 1992;281:709- 715. [10] Kr/ihenbiihl S, Talos C, Reichen J. Mechanisms of impaired hepatic fatty acid metabolism in rats with long-term bile duct ligation. Hepatology 1994;19:12721281. [I1] Finel M, Majander A. Studies on the proton-translocating NADH:ubiquinone oxidoreductases of mitochondria and Escherichia coli using the inhibitor 1,10phenanthroline. FEBS Lett 1994;339:142-146. [12] Kriihenbiihl S, Chang M, Brass EP, Hoppel C. Decreased activities of ubiquinol:ferricytochrome c oxidoreductase (complex III) and ferrocytochrome c:oxygen oxidoreductase (complex IV) in liver mitochondria from rats with hydroxycobolamin[c-lactam]-induced methylmalonic aciduria. J Biol Chem 1991; 266: 20998- 21003. [13] Wharton DC, Tzagoloff A. Cytochrome oxidase from beef heart mitochondria. Methods Enzymol 1967;10:245-250. [14] Srere P. Citrate synthase. Methods Enzymol 1969;13:3-11. [15] ,~.bi H. Catalase. In: Bergmeyer HU, ed. Methods of enzymatic analysis. 3rd ed. Weinheim, Germany: Verlag Chemie GmbH 1983;273-286. [16] Baum H, Dodgson KS, Spencer B. The assay of arylsulfatases A and B in human urine. Clin Chim Acta 1959;4:453-455. [17] Hoppel CL, Cooper C. The action of digitonin on rat liver mitochondria. The effects on enzyme content. Biochem J 1968;107:367-375. [18] V61kl A, Fahimi HD. Isolation and characterization of peroxisomes from the liver of normal untreated rats. Eur J Biochem 1985;149:257-265. [19] Dufour JF, Gehr P, Reichen J. Hepatic accumulation of lysosomes and defective transcytotic vesicular pathways in cirrhotic rat liver. Hepatology 1992;16:9971006.
90
S. Kr~henb~hl et al. I Clinica Chimica Acta 253 (1996) 79-90
1-201 Suomalainen A, Majander A, Haltia M et al. Multiple deletions of mitochondrial DNA in several tissues of a patient with severe retarded depression and familial progressive external ophthalmoplegia. J Clin Invest 1992;90:61-66. [21] Shoji Y, Sato W, Hayasaka K, Takada G. Tissue distribution of mutant mitochondrial DNA in mitochondrial myopathy, encephalopathy, lactic acidosis and strokelike episodes (MELAS). J Inher Metab Dis 1993;16:27-30. [22] Kr~ihenbiihl S, Reichen J, Zimmermann A, Gehr P, Stucki J. Mitochondrial structure and function in CC14-induced cirrhosis in the rat. Hepatology 1990;12:526-532.