Activities of mitochondrial oxidative phosphorylation enzymes in cultured amniocytes

Clinica Chimica Acta 298 (2000) 157–173 www.elsevier.com / locate / clinchim

Activities of mitochondrial oxidative phosphorylation enzymes in cultured amniocytes S.K.R. Chowdhury a b , Z. Drahota a , D. Floryk a b , P. Calda c , ˇ ˇ a b ,* J. Houstek a

´ ´ 1083, 142 20 Prague 4, Institute of Physiology, Academy of Sciences of the Czech Republic, Vıdenska Czech Republic b Department of Paediatrics, 1 st Faculty of Medicine, Charles University, Ke Karlovu 2, 120 00 Prague 2, Czech Republic c ´ ˇ ´ 18, Department of Obstetrics and Gynaecology, 1 st Faculty of Medicine, Charles University, Apolinarska 128 51 Prague 2, Czech Republic Received 4 January 2000; received in revised form 17 April 2000; accepted 20 April 2000

Abstract Amniocytes represent a population of foetal cells that can be used for prenatal diagnosis in families with suspected mitochondrial oxidative phosphorylation (OXPHOS) defects. In this paper, we present a complex protocol for evaluation of the function of mitochondrial OXPHOS enzymes in cultured amniocytes using three independent and complementary methods: (a) spectrophotometry as a tool for determination of the capacities of mitochondrial respiratory-chain enzymes (NADH ubiquinone oxidoreductase, succinate- and glycerophosphate cytochrome c reductase, cytochrome c oxidase and citrate synthase); (b) polarography as a tool for the evaluation of mitochondrial OXPHOS enzyme functions in situ using digitonin-permeabilised amniocytes (rotenone-sensitive oxidation of pyruvate 1 malate, antimycin A-sensitive oxidation of succinate, KCN-sensitive oxidation of cytochrome c, ADP-activated substrate oxidation) and (c) cytofluorometric determination of tetramethyl rhodamine methyl ester (TMRM) fluorescence in digitonin-permeabilised amniocytes as a sensitive way to determine the mitochondrial membrane potential under steady-state conditions (state 4 with succinate). These protocols are presented

Abbreviations: COX, cytochrome c oxidase; CS, citrate synthase; NQR, rotenone-sensitive NADH ubiquinone reductase; SCCR, succinate cytochrome c reductase; GCCR, glycerophosphate cytochrome c reductase; OXPHOS, oxidative phosphorylation; LM, lauryl maltoside; DCm , mitochondrial membrane potential; TMRM, tetramethyl rhodamine methyl ester; TMPD, N,N,N9,N9-tetramethyl-p-phenylenediamine *Corresponding author. Fax: 1420-2-475-2149. ˇˇ E-mail address: [email protected] (J. Houstek) 0009-8981 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 00 )00300-4

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together with reference control values using 9–22 independent cultures of amniocytes.  2000 Elsevier Science B.V. All rights reserved. Keywords: Prenatal diagnosis; Amniocytes; Oxidative phosphorylation; Respiratory-chain enzymes; Mitochondrial membrane potential; TMRM cytofluorometry

1. Introduction The number of patients with confirmed mitochondrial disorders is steadily increasing. Clinical symptoms of mitochondrial diseases caused by various defects in oxidative phosphorylation (OXPHOS) enzymes are highly heterogeneous, with the predominant affect being on tissues and organs with high energetic demands, such as muscle and brain (encephalomyopathies). Mitochondrial OXPHOS diseases often result in severe or fatal metabolic disorders for which no causal therapy is available and, thus, genetic counselling and prenatal diagnostics are of particular importance. Hitherto, most mitochondrial diseases were identified as mtDNA point mutations that are mainly maternally inherited [1]. However, their transmission is often unpredictable, because of uncertain segregation of heteroplasmic mtDNA defects [2]. Deletions and duplications of mtDNA are, in most cases, sporadic and, thus, also inaccessible to prenatal diagnostics. The prenatal diagnosis of mitochondrial defects caused by mutations in nuclear OXPHOS genes seems to present fewer difficulties. Several nuclear mutations of mitochondrial Complexes I, II, IV and V have been described recently that affect either subunits of these complexes [3–6] or specific assembly proteins, such as SURF or SCO2 (surfeit 1 or cytochrome oxidase deficient yeast homolog 2) proteins [7–9]; others might be characterised in the near future. The high incidence of OXPHOS disorders, particularly deficiencies of Complexes I and IV, and the cases of unknown DNA defects, led to the need for biochemical investigations in prenatal diagnostics. Cultured amniocytes have been repeatedly used for prenatal diagnosis of mitochondrial diseases but systematic analysis of OXPHOS enzymes is missing as, often, only the activity of the enzyme with a suspected defect and citrate synthase were measured [10–16]. Spectrophotometric evaluation of mitochondrial enzyme activities cannot reflect all changes in mitochondrial functions induced by genetic defects. Recent findings by Villani and Attardi [17] indicate that mitochondrial respiratory-chain enzyme activities determined in permeabilised cells by oxygen consumption measurements show a much lower ratio of COX to other respiratory chain complexes than when determined in the presence of detergent by spectrophotometric methods [17]. Evidently, under in situ conditions, COX

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activity is down-regulated. Hence, the measurements of maximum activities of mitochondrial respiratory-chain enzymes should be completed using polarographic analysis, which better reflects the situation in mitochondria in situ. Moreover, by using polarographic measurements in combination with cytofluorometric detection of mitochondrial membrane potential, DCm , the extent of mitochondrial coupling and the efficacy of ATP synthesis may also be evaluated. Therefore, we attempted to widen the scope of analysis of mitochondrial enzyme activities in amniocytes using the combination of spectrophotometric, polarographic and cytofluorometric methods. In this paper, we present optimum conditions for the determination of maximum activities of various mitochondrial OXPHOS enzymes and their reference values in fresh and frozen amniocytes (NADH ubiquinone oxidoreductase, succinate cytochrome c reductase, glycerophosphate cytochrome c reductase, cytochrome c oxidase and citrate synthase), conditions for the permeabilisation of amniocytes for polarographic measurements of mitochondrial respiratory enzyme activities in situ and for FACS measurements of DCm in digitonin-permeabilised amniocytes using tetramethyl rhodamine methyl ester (TMRM).

2. Materials and methods

2.1. Cell cultures and homogenate Foetal cells in amniotic fluid obtained by amniocentesis consist mainly of fibroblasts with a few epithelial cells, which are usually less viable under the culture conditions used. Therefore, the preparation of a sufficient quantity of cells for enzyme analysis (three–four passages) results in a rather homogeneous population of foetal fibroblasts with a minimum amount of epithelial cells. These cells are referred to as amniocytes, while the cultures of skin fibroblasts from healthy young individuals are referred to as fibroblasts. Amniotic fluid was used after informed consent was obtained from patients with negative results of prenatal diagnosis for suspected chromosomal aberrations. Amniocytes were cultured at 378C and 5% CO 2 in air in Amniomax medium (Gibco BRL, Paisley, UK) that was diluted using GPM-3 medium (SEVAC, Prague, Czech Republic) and supplemented with 10% foetal calf serum (Sigma, St. Louis, MO, USA). Cells were grown to approximately 90% confluence and harvested using 0.05% trypsin and 0.02% EDTA. Detached cells were diluted with ice-cold cultured medium, sedimented by centrifugation and washed twice using phosphate-buffered saline (PBS). Skin fibroblasts were cultured at 378C and 5% CO 2 in air in DMEM medium (SEVAC, Prague, Czech Republic) that was supplemented with 10% foetal calf

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serum and these were harvested in the same manner as the amniocytes (see above). Hearts from adult (90-day-old) male Wistar rats were homogenised in STE medium (0.25 mol / l sucrose, 10 mmol / l Tris–HCl, 1 mmol / l EDTA, pH 7.4) at 08C in a Teflon-glass homogenizer (50 mg of heart / 1 ml of STE buffer). The protein content was measured according to the method of Bradford [18] using bovine serum albumin as a standard. Cell samples were sonicated for 20 s prior to protein determination.

2.2. Spectrophotometric measurements Cytochrome c oxidase (COX) was measured at 308C by following the rate of oxidation of reduced cytochrome c at 550 nm [19]. Cytochrome c was reduced by sodium dithionite and excess dithionite was removed by filtration through a Sephadex G-25 column. The efficacy of reduction and the autooxidation rate were controlled. COX activity measurements in cultured cells were performed using 30 mmol / l cytochrome c, 20 mmol / l phosphate buffer, 0.1 mg of protein from cultured cells or tissue homogenate and 8 mg of lauryl maltoside / mg protein (0.16% LM), unless stated otherwise. The rotenone-sensitive NADH ubiquinone oxidoreductase (NQR) activity of Complex I was measured at 308C by following the reduction of NADH at 340 nm using decylubiquinone as an acceptor of electrons [20]. Obtained values were corrected for activity measured in the presence of 5 mmol / l rotenone. Enzyme activity was determined in 25 mmol / l potassium phosphate (pH 7.4), 2.5 mmol / l MgCl 2 , 2 mmol / l KCN, 5 mmol / l antimycin A, 50 mmol / l decylubiquinone, 0.25% bovine serum albumin (fatty acid free), 0.05 mg protein from amniocytes and 0.1 mmol / l NADH. Citrate synthase (CS) was determined according to Srere [21] at 308C in a medium containing 150 mmol / l Tris–HCl, pH 8.2, 8 mg lauryl maltoside / mg protein, 0.1 mmol / l dithionitrobenzoic acid and 0.2 mg protein from amniocytes. The reaction was started by the addition of 5 mmol / l acetyl CoA and changes of absorbance at 412 nm were measured for 1 min. This value was subtracted from the rate obtained after the subsequent addition of 0.5 mmol / l oxalacetic acid. Succinate cytochrome c reductase (SCCR) and glycerophosphate cytochrome c reductase (GCCR) were measured according to the method of Rustin et al. [20] in medium containing 10 mmol / l potassium phosphate (pH 7.8), 2 mmol / l EDTA, 0.01% bovine serum albumin (fatty acid free), 0.2 mmol / l ATP, 1 mmol / l KCN, 5 mmol / l rotenone, and 10 mmol / l succinate or 20 mmol / l glycerophosphate, respectively. Cells (0.2 mg protein) were incubated in the medium for 2 min at 308C, then the reaction was started by the addition of 40 mmol / l oxidised cytochrome c and measured for 5 min.

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2.3. Polarographic measurement Oxygen consumption by amniocytes was determined at 308C using the OROBOROS oxygraph (Innsbruck, Austria). For each measurement, 0.5–1.5 mg protein / ml of cultured amniocytes were used. Freshly harvested amniocytes were resuspended in KCl medium (80 mmol / l KCl, 10 mmol / l Tris–HCl, 3 mmol / l MgCl 2 , 1 mmol / l EDTA, 5 mmol / l potassium phosphate, pH 7.2) and cells were permeabilised by digitonin using 0.03 mg / mg of cell protein. Various respiratory substrates and inhibitors were used as indicated. Oxygen consumption was expressed as pmol oxygen / s / mg protein.

2.4. Cytofluorometric analysis Mitochondrial membrane potential (DCm ) measurements were performed on the FACSort flow cytometer (Becton Dickinson, San Jose, CA, USA) according to the method of Floryk and Houstek [22]. Amniocytes were resuspended in KCl medium containing 10 mmol / l succinate at a protein concentration of 1 mg / ml, and digitonin (Fluka, Buchs, Switzerland) was added at indicated concentrations (0.01–1.0 mg / mg of protein). Permeabilised amniocytes were resuspended in the KCl medium at 0.2 mg of protein / ml and incubated with 20 nmol / l TMRM (Molecular probes, OR, USA). The permeability of the plasma membrane was tested using 1 mg / ml propidium iodide. A minimum of 5000 cells were used for each FACS measurement. Data were acquired in log scale using CellQuest (Becton Dickinson) and were analysed using WinMDI 2.7 software (Trotter, J., TSRI, La Jolla, CA, USA). Arithmetic mean values of the fluorescence signal (in arbitrary units) were determined for each sample for subsequent graphic representation.

3. Results

3.1. Optimal conditions for determinations of COX activity COX activity depends on various factors (e.g. ionic composition of the medium, type and concentration of detergent) that appear to be specific even for particular cell populations [19,23–29]. In addition, the permeabilisation of cells by detergent for in situ measurements of mitochondrial enzyme activities requires different concentrations for different cell populations [26]. For measurement of maximum COX activity in mammalian tissues, 40–80 mmol / l potassium phosphate was recommended, whereas in cultured cells, lower optimal concentrations of phosphate (20–30 mmol / l) were reported [19,23–29]. As shown in Fig. 1, the maximum activity of COX in heart muscle

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Fig. 1. Effect of potassium phosphate (A) and potassium chloride (B) on COX activity in amniocytes, fibroblasts and rat heart homogenate. Spectrophotometric measurements of COX activity were performed at indicated concentrations of potassium phosphate or potassium chloride in the presence of 0.16% LM.

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homogenate was obtained at 60 mmol / l potassium phosphate or KCl. In cultured amniocytes, the maximum COX activity was found at lower concentrations (20 mmol / l) of potassium phosphate and KCl (Fig. 1). Similarly, in fibroblasts, the maximum COX activity was found at 20 mmol / l potassium phosphate and KCl (Fig. 1). When we measured COX activities in cultured amniocytes at different concentrations of LM, to obtain optimum proportions between cell protein and detergent, we found the maximum activity at 0.16% (w / v) LM, which corresponds to a detergent–protein ratio of 8 mg of LM per mg of protein. Further increases of the LM concentration (up to 0.6%) did not change the enzyme activity (Fig. 2). Measurements of COX activity under optimal conditions in 22 different amniocytes cultures are presented in Table1. The mean6S.D. of COX activity was 45.6613.3 nmol / min / mg protein, corresponding to a range of activities of 24.9–74.9. It is also evident from Table 1 that storage of amniocytes at 2 708C for 2–3 weeks decreased the COX activity by 30–50%.

3.2. Determination of succinate cytochrome c reductase ( SCCR), glycerophosphate cytochrome c reductase ( GCCR), rotenone-sensitive NADH ubiquinone oxidoreductase ( NQR) and citrate synthase ( CS) activity Maximum activities of SCCR and GCCR (Table 1) were obtained in fresh cells that had been sonicated for 3 3 10 s. The activity of SCCR, determined in

Fig. 2. Activation of COX in cultured amniocytes by LM. Spectrophotometric measurements of COX activity were performed in the presence of 20 mmol / l potassium phosphate at indicated concentrations of LM.

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Enzymes

Enzyme activity (nmol/min/mg protein)

Ratio to CS

Range

Ratio to COX

Range

Activity (%) remaining

Mean6S.D.

Range

Mean6S.D.

NQR (n512)

7.662.5

4.1–12.3

0.0760.010

0.06–0.08

0.1460.02

0.12–0.18

80–90

SCCR (n59)

9.662.1

7.7–13.4

0.1160.030

0.08–0.14

0.1960.03

0.16–0.19

80–90

GCCR (n59)

4.761.0

3.4–6.0

0.0560.001

0.04–0.07

0.0960.01

0.07–0.11

80–90

COX (n522)

45.6613.3

24.9–66.5

0.6560.130

0.43–0.84

2

2

50–70

CS (n513)

83.3615.8

66.1–117.4

2

2

1.5760.36

1.23–2.31

95–100

after storage at 2708C Mean6S.D.

a SCCR, succinate cytochrome c reductase; GCCR, glycerophosphate cytochrome c reductase; NQR, rotenone-sensitive NADH ubiquinone oxidoreductase; COX, cytochrome c oxidase; CS, citrate synthase; n, number of measurements.

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Table 1 Spectrophotometric measurements of respiratory-chain enzymes in cultured amniocytes a

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nine cultures of amniocytes, was 9.662.1 nmol / min / mg protein, varying in the range between 7.7 and 13.4. The activity of GCCR, determined in nine cultures of amniocytes, was 4.761.0 nmol / min / mg protein, varying from 3.4 to 6.0. Storage of cells at 2 708C decreased activities of both enzymes by 10–20% (Table 1). NQR activity, determined in 12 cultures of amniocytes, was 7.662.5 nmol / min / mg protein, varying between 4.1 and 12.3. Storage of cells at 2 70 8C decreased the enzyme activity by 10–20% (Table 1). The activity of CS, determined in 13 cultures of amniocytes, was 83.3615.8, varying between 66.1 and 117.4. The activity of this matrix enzyme was not changed during storage at 2 708C (Table 1). The ratios between individual enzyme activities, which often represent a better criterion for evaluation of mitochondrial enzyme defects [20], are summarised in Table 1. Ratios between COX and other respiratory-chain enzymes are helpful for evaluation of specific enzyme defects, whereas ratios between respiratory-chain enzymes and CS indicate the proportions between the inner mitochondrial membrane and matrix compartments.

3.3. Oxygen consumption in permeabilised amniocytes Fig. 3 shows representative measurements of the respiratory rates of permeabilised amniocytes in the presence of various substrates. In order to assess the free access of substrates and other ligands to mitochondria, measurements were performed in the presence of low concentrations of detergent (0.03 mg digitonin / mg protein), which had been found optimal in experiments with fibroblasts [30]. Under these conditions, amniocytes exert a maximum rate of substrate respiration at state 3 and show good respiratory control. Average values of oxygen consumption measurements from five amniocyte cultures are summarised in Table 2. Complex I function is determined as rotenone-sensitive respiration in the presence of NADH-dependent substrates. The activities of Complexes II and III are evaluated as antimycin A-sensitive respiration in the presence of flavoprotein-dependent substrates. Under the conditions used, respiration with pyruvate and malate was 95% sensitive to rotenone and that supported by succinate or glycerophosphate was 95% sensitive to antimycin A. COX activity measured in the presence of ascorbate and TMPD was 80% sensitive to KCN. The rate of endogenous respiration of permeabilised amniocytes (Table 2) was stimulated 1.4-fold by pyruvate 1 malate, and 3.2-fold by the subsequent addition of ADP, indicating the coupled state of mitochondria in permeabilised amniocytes. This respiration was also almost completely inhibited by rotenone. ADP-stimulated respiration of amniocytes with succinate in the presence of rotenone was four-times higher than the basal respiration rate and was

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Fig. 3. Oxygen consumption by digitonin-permeabilised amniocytes. Oxygen consumption was measured using 1.3 mg protein from amniocytes / ml. Subsequent additions of digitonin (0.03 mg / mg protein), pyruvate (5 mmol / l)1malate (1.5 mmol / l), ADP (1.5 mmol / l), rotenone (2 mg / ml), succinate (10 mmol / l), antimycin A (0.2 mg / ml), ascorbate (10 mmol / l), TMPD (0.2 mmol / l), cytochrome c (30 mmol / l) and KCN (1 mmol / l) are indicated.

completely inhibited by antimycin A. After the addition of ascorbate and TMPD, COX activity may also be determined (Fig. 3). Oxidation of both NADH- and flavoprotein-dependent substrates, as well as oxidation of ascorbate and TMPD, was not increased by the addition of cytochrome c, which indicated that the outer membrane of mitochondria in cells permeabilised by digitonin remained Table 2 Oxygen consumption of permeabilised amniocyte cultures (n55) in the presence of various substrates Oxygen consumption (pmol / s / mg protein)

Basal respiration Pyruvate1malate Pyruvate1malate1ADP Succinate1ADP Ascorbate1TMPD1ADP Cytochrome c1ascorbate 1TMPD1ADP Respiratory control

Ratio to ascorbate1 TMPD1ADP Mean6S.D.

Range

Mean6S.D.

Range

20.2569,37 45.9368.72 63.12618.04 62.68622.00 93.92649.72 92.51646.88

10.50–31.60 32.0–61.40 32.40–89.50 38.30–89.20 37.1–163.10 38.00–154.00

0.2260.05 0.6260.25 0.7960.24 0.7560.20 2 1.0060.05

0.15–0.31 0.26–0.88 0.41–1.11 0.54–1.17 2 0.93–1.03

3.2362.10

1.50–7.50

2

2

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Fig. 4. Influence of digitonin on the cytofluorometric analysis of DCm in cultured amniocytes. Cells resuspended in KCl medium were stained with 20 nmol / l TMRM (dotplots and histogram on the left) to assess DCm , and with 1 mg / ml of propidium iodide (dotplots on the right) to test the permeability of the plasma membrane. The ratio of digitonin to protein (mg / mg) was (A) 0.01, (B) 0.08 and (C) 1. The abscissa represents SSC-height (side scatter height, dotplots) or the intensity of fluorescence (histogram), the ordinate intensity of fluorescence (dotplots) or the relative cell number (histogram).

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intact. However, in frozen amniocytes, the oxidation of succinate was highly activated by the addition of cytochrome c (not shown).

3.4. Cytofluorometric measurement Analysis of DCm in cultured cells with TMRM also requires selective permeabilisation of the plasma membrane in order to facilitate free access of the probe to mitochondria and to prevent accumulation of the probe in cytosol [22]. Fig. 4 shows the influence of digitonin on TMRM fluorescence of cultured amniocytes incubated in a KCl medium with succinate but without ADP (state 4 conditions). At a very low digitonin concentration (0.01 mg digitonin / mg protein), two discrete populations of cells were observed by FACS analysis, differing substantially in the TMRM fluorescence intensity and indicating that digitonin did not permeabilise all cells. Only the population of cells with a high TMRM signal was stained well with the nuclear dye propidium iodide (Fig. 4A). A further increase in the digitonin concentration gradually increased the number of cells with a high TMRM signal and, at the optimal concentration of digitonin (0.05 mg digitonin / mg protein), all cells were sufficiently permeabilised without affecting mitochondria (Fig. 4B). The damage of mitochondria and the occurrence of cells with low TMRM fluorescence became apparent at digitonin concentrations above 0.3 mg / mg protein (Fig. 4C).

Fig. 5. Optimum digitonin concentration for TMRM cytofluorometry in cultured amniocytes. The cytofluorometric analysis was performed as in Fig. 4 in amniocytes treated with the indicated concentrations of digitonin. The relationship between the digitonin–protein ratio and the percentage of cells with a high TMRM signal (FL-2 height from the interval 300–10,000) is shown.

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Fig. 5 shows the relationship between the digitonin concentration used and percentage of cells with a high TMRM signal. Within the concentration range of 0.02–0.2 mg digitonin / mg protein, more than 90% of amniocytes displayed a high TMRM signal (cells with the rank of fluorescence 300–10,000). In comparison with other cell types previously analysed [22], the cultured amniocytes behave similarly to fibroblasts. As shown in Fig. 6, the high intensity TMRM signal determined in amniocytes under optimal conditions was fully collapsed by the uncouplers

Fig. 6. Effect of inhibitors on TMRM fluorescence. Cytofluorometry of amniocytes stained with 20 nmol / l TMRM was performed at 0.1 mg digitonin / mg protein. The inhibitors FCCP (1 mmol / l), antimycin A (10 mmol / l), KCN (1 mmol / l) and valinomycin (20 nmol / l) were used as indicated.

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FCCP and valinomycin and it was also completely abolished by inhibitors of the respiratory chain, KCN and antimycin A. Complete FACS analysis of DCm could be performed with , 0.5 mg protein from cultured amniocytes.

4. Discussion Amniocytes represent a foetal cell population that can easily be propagated in cell culture and used for prenatal diagnosis of mitochondrial defects in suspected cases. Prenatal diagnosis and the use of biochemical methods to assess the activities of OXPHOS enzymes in cultured amniocytes would not yield satisfactory results if the defect had tissue-specific expression and was absent in skin fibroblasts, or if the mtDNA mutation was heteroplasmic and the patient’s cells harbouring the mutation could be cloned out during cell culture [31]. In other cases, in particular when the DNA mutation affects nuclear genes, the biochemical approach is of key importance. In this paper, we describe the use of three independent methods (spectrophotometry, polarography and cytofluorometry) for evaluation of the specific activities of respiratory-chain enzymes and the function of the OXPHOS system in cultured amniocytes. Spectrophotometric measurements of the activity of various mitochondrial enzymes provide data showing the capacity of a particular enzyme. In the case of mitochondrial OXPHOS defects, simultaneous measurement of various membrane-bound and matrix-soluble enzymes is recommended as, due to large individual variation, the ratios of enzyme activities are often a better indicator of a particular enzymatic defect than is the specific activity of the enzyme itself [20,32]. Polarographic detection of oxygen consumption in amniocytes permeabilised by digitonin enables the evaluation of the function of mitochondrial enzymes in intact mitochondria under conditions that are closer to an in vivo situation. Cytofluorometry of permeabilised amniocytes represents a simple and rapid procedure for the detection of DCm , the key parameter of mitochondrial energy conversion, which gives general information about the function and coupling of the mitochondrial OXPHOS system. Based on our experimental data, the described protocol for assay of COX allows the determination of the maximum capacity of mitochondrial COX and excludes potential interference of COX down-regulation by various endogenous factors. These are eliminated by changes in the ion composition of the incubation medium and by the presence of detergent. Besides COX activity, we describe reference values obtained from nine–thirteen different cultures of amniocytes for NQR, SCCR, GCCR and for the matrix enzyme CS (Table 1).

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From these data, ratios between various membrane enzymes and between matrix and membrane enzymes may also be calculated. Comparison with previous studies on cultured fibroblasts shows that these ratios in amniocytes are similar to ratios found in human skin fibroblasts [20]. Recent findings on human cells [17] have shown that maximum values of COX activity obtained in the presence of detergent do not characterise the activity of this enzyme in intact cells. In accord with these data, we found, in permeabilised amniocytes, a different ratio of COX and Complex II when the enzyme activity was tested as oxygen consumption or measured spectrophotometrically. The COX activity was four-times higher than the SCCR activity when spectrophotometric detection was used (Table 1), but evaluation of polarographic measurements of oxygen consumption showed almost identical activity of COX and succinate oxidase (Table 2 and Fig. 3). These findings indicate that, in intact cells, COX has no reserve capacity and that even small defects of the enzyme may have an important impact on cell energy metabolism in vivo. As indicated in Table 2, in permeabilised amniocytes, both succinate and pyruvate 1 malate oxidation is activated by ADP, which indicates tight coupling of OXPHOS and well-preserved mitochondrial production of ATP. This could be further confirmed by cytofluorometric detection of DCm . Analysis of the mitochondrial membrane potential was successfully applied in our recent studies on the characterisation of a COX defect in maternally inherited mitochondrial encephalomyopathy [33]. These studies clearly showed that DCm represents a sufficiently sensitive method for the determination in cultured cells of OXPHOS disorders due to decreased activity of DCm -forming processes. Moreover, TMRM cytofluorometry was also able to detect hyperpolarisation of DCm resulting from the low capacity of ATP synthase to utilise DmH 1 due to a selective deficiency of the enzyme [10]. According to our protocol, a complete evaluation of mitochondrial respiratory-chain enzymes by spectrophotometry, polarography and FACS analysis (performed in duplicate) requires about 4.5 mg of protein from cultured amniocytes. Approximately two–three-times more can be obtained within 3 weeks of amniocyte culture, starting with 10 ml of amniotic fluid. Thus, the analysis of mitochondrial OXPHOS enzymes can be completed 3 weeks after amniocentesis performed between the 16th and 18th week of pregnancy. This allows sufficient time to opt for an abortion before the 22nd week of gestation. We conclude from our data that the combination of these three independent and complementary techniques can significantly improve the characterisation of mitochondrial enzymes in cultured amniocytes, which is essential for the evaluation of putative OXPHOS defects in the prenatal diagnosis of mitochondrial diseases.

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Acknowledgements The expert technical assistance of V. Fialova´ is gratefully acknowledged. This work was supported by grants from the Ministry of Health (4035-3), the Ministry of Education (ME 226) and Grant Agency of the Czech Republic (303 / 00 / 1658).

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