Acta pharmacol. et tonicol. 1975, 36, 1-12.

From the Royal Dental College, Department of Biochemistry, Vennelyst Boulevard, DK-8000 Arhus C, Denmark

The Effect of Chlorhexidine on some Biochemical Parameters of Rat Liver Mitochondria

BY

Flemming Christensen, Henry S. Bleeg and Jsrn Erik Jensen (Received May 13, 1974; Accepted August 7, 1974)

Abstract: Chlorhexidine (CHX) binds to isolated rat liver mitochondria with a saturation of binding at 900 nmol per mg protein. The binding is completed within 20 sec. and the binding does not render the mitochondria capable of sedimentation at 600 X g. A slight solubilization of mitochondrial proteins is obtained with 100 pM CHX, whereas higher concentrations of CHX (250 yM) result in an increase in the amount of protein sedimenting at 15,000 X g. Low concentrations of CHX (25 yM) have an uncouplinglike effect on the mito- chondria] electron transport system. Inhibition of respiration is observed with 100 pM CHX. The activity of ATP-ase is slightly stimulated with 15 pM CHX and inhibited 5 0 % with 100 pM CHX. Membrane-bound mitochondrial enzymes, e. g. succinate: cytochrome c reductase, proline oxidase and kynurenine hydr- oxylase are inhibited by CHX at 70 pM, 200 pM and 1 mM CHX respectively. Malate dehydrogenase (a mitochondrial matrix enzyme) is solubilized by CHX. At 25 pM CHX 87 % of the activity is liberated from the mitochondrial matrix. The soluble enzyme is not inhibited by higher concentrations of CHX. The results are tentatively explained as being due to effects of binding of CHX to the mitochondrial membranes. Chlorhexidine is considered to be an amphi- pathic substance being capable of penetrating the non-polar regions of phospho- lipid bilayers and at the same time being able to bind as a bication to phosphate groups in the phospholipids.

Key-words: Chlorhexidine - mitochondria - binding - oxygen consumption - enzymes - kynurenine

succinate:cytochrome c reductase - proline oxidase - hydroxylase - malate dehydrogenase.

In a previous paper it was shown that chlorhexidine (NFN) (1,l-hexa- methylene bis (5-(4-chlorophenyl) biguanide)) exerts profound effects on the membranes of rat liver microsomes (CHRISTENSEN & JENSEN 1974). It was found that chlorhexidine binds to and precipitates the microsomes, denatures cytochrome P-450 and influences the activity of glucose-6-phos- phatase and p-nitroanisole-0-demethylase. It was suggested that these effects

2 FLEMMING CHRISTENSEN er al.

of chlorhexidine could be attributed, at least partly, to a non-specific ca- tionic detergent effect on the lipoprotein membranes of the microsomes. Other workers (HUGO & LONGWORTH 1964 & 1966; RYE & WISEMAN 1964 & 1965) have shown that the plasma membrane of some bacteria is also affected by chlorhexidine. It is thus possible that this compound affects rather generally the lipoprotein membranes of living organisms. We therefore decided to examine this possibility further and in the present paper we present evidence for such an effect of chlorhexidine on the lipoprotein membranes of rat liver mitochondria.

Materials and methods

Animals. White male rats of an inbred strain L from our animal stock were used throughout the experiments. The animals had free access to water and a laboratory food source (Altromin).

Preparation of mitochondria. Two animals were used in each experiment. They were sacrificed by decapitation, the livers removed as quickly as possible and then homogenized, with cooling, in 4 vol. of ice-cold 0.25 M sucrose in 3.4 mM tris-HC1 buffer (pH 7.4) containing 1 mM EGTA in a motordriven Teflon glass homogenizer. All succeeding tissue manipulations were performed at 0-4". The preparation of mito- chondria from this homogenate followed the method of CHAPPELL & HANSFORD (1972). The final mitochondria1 pellet was diluted with the same buffer solution as used for homogenization to give a total volume of 5-10 ml equivalent to 2 g liver (around 100 mg mitochondrial protein) per millilitre. Immediately following preparation the mitochondrial suspension exhibited respiratory control ratios of 2.7-3.0 (cf. results).

Binding of chlorhexidine to mitochondria. Mitochondria equivalent to 0.3 mg mito- chondrial protein were incubated for 30 min. at 22" in a mixture containing 250 pmol sucrose, 5 pmol tris-HCI, 0-600 pg of unlabelled chlorhexidine-2HCI and 0.7 pg (0.05 pci) of 14C-labelled chlorhexidine digluconate in a total volume of 1.0 ml. Fol- lowing incubation the mixture was centrifuged at 15,000 X g for 5 min. and the radioactivity of the supernatant (0.25 ml) was determined as described in the fol- lowing section. For each concentration of chlorhexidine a control sample prepared as described above but without mitochondria was run in parallel with the test sample. From the results obtained by measuring the radioactivity of these controls it was ap- parent that a fraction of the chlorhexidine was bound to the glass of the test tube especially when the concentration of the drug was lower than about 50 pM. The results given in fig. 1 are corrected for this glass-binding.

In order to study the kinetics of the binding of chlorhexidine 0.25 ml of a suspen- sion of mitochondria in the preparation buffer equivalent to 10 mg protein was added to 0.25 ml of tris-HCI buffer 0.02 M pH 7.4 in 0.25 M sucrose. At zero-time 0.5 ml of a solution containing 400 pg unlabelled chlorhexidine-2 HC1 and 1.4 pg chlor- hexidine digluconate labelled with l4C (0.1 pci) in the same buffer was added and the mixture thoroughly mixed. At various times after the addition of chlorhexidine the suspensions were filtered through Millipore@ membrane filters (HAWP) with a diameter of 13 mm and pore dimensions of 0.45 pm. The radioactivity of the

CHLORHEXIDlNE AND LIVER MITOCHONDRIA 3

material deposited on the filter was determined as described in the following section. Controls without mitochondria were run in parallel with the test samples and were used to correct for unspecific binding to the filters. The radioactivity of the labelled chlorhexidine added to the mitochondria was also determined.

Measurement of radioactivity. As a rule 250 pl of the radioactive solution was used and added to 6.75 ml scintillator fluid. This consisted of 80 g naphthalene, 5 g PPO and 0.05 g POPOP in a mixture of 333 g dioxane, 333 g xylene and 333 g ethanol. The radioactive material was placed in counting vials (5.5 X 2.5 cm) and measured in an IDL liquid scintillation counter. The radioactivity of the material deposited on the Millipore filter was determined as follows. The filters were placed in counting vials and to each of these were added 0.25 ml methanol and 0.25 ml concentrated ammonia followed by 6.75 ml scintillator fluid. After some time the filters were macerated and the radioactivity of the solution was determined.

Deterniinatiori of the e f fec t o f chlorhexidine on mitochondrial proteins. In order to examine if chlorhexidine precipitates mitochondria1 proteins 0.25 ml of the orig- inal mitochondrial suspension diluted 1 to 4 with 0.25 M sucrose (4.5 mg protein) was added to 0.25 ml 0.02 M tris-HC1 buffer pH 7.4 in 0.25 M sucrose. To this mixture was added 0.5 ml chlorhexidine-2HCl (0-600pg) in the same buffer and the mixture was left at room temperature for 30 min. and was then centrifuged at 600 x g for 15 min. The protein content of the resulting pellet was determined while the supernatant was further centrifuged for 5 min. at 15,000 X g. The protein con- centration of the supernatant from the last centrifugation was determined.

Determinution of protein. The protein content of suspensions of mitochondria was determined by the biuret method as described by LAYNE (1957).

Protein of other solutions was determined by the method of LOWRY et al. (1951) using bovine serum albumin as a standard. The results were corrected for interference by chlorhexidine with the LOWRY assay.

Determination of Mg2+-stimulated ATP-ase activity. This was performed as described by FLEISCHER & FLEISCHER (1967). The amount of inorganic phosphate liberated from ATP during the reaction was determined by a modification (BAGINSICI & ZAK 1960) of the method of FISKE & SUFIBAROW (1925). As chlorhexidine gives some colour by this reaction controls using zero time incubation but otherwise treated as the test samples were run in parallel with these at all the concentrations of chlorhexidine used.

Determination of the e f fec t of chlorhexidine on malate dehydrogenase. This was performed by a modification of the method described by DUPOURQUE & KUN (1969).

Mitochondria (10 PI, 0.3 mg protein), 10 ml incubation medium (0.225 M sucrose, 10 mM-KH,PO,/K,HPO,, 5 mM-MgCI,, 20 mM-KCl, 20 mM triethanolamine, pH 7.4) and chlorhexidine digluconate in water (10 pl) was preincubated at 25" for 15 min. A 3 ml portion of this mixture was placed in the sample cuvette in a Shimadzu MPS- 50L recording spectrophotometer while the reference cuvette contained 3 ml incubation medium. The cuvettes were thermostatically kept at 25". Three microlitres of oxalo- acetate (0.6 pmol) was added and the reaction initiated by the addition of 3 p1 NADH (0.3 pmol). The decrease in absorbance with time at 340 nm was recorded. The activity was determined from the slope of the graph during the first 10 sec. of the reaction. The amount of NADH oxidized was calculated using a molar extinction coefficient

4 FLEMMING CHRISTENSEN et al.

of 6.22 X loe cm-lM-l at 340 nm for NADH (HORECKER & KORNBERG 1948). Dis- integration of the mitochondria was performed by sonication for two min. with an MSE 150 Watts Ultrasonic Disintegrator. In order to determine the release of enzyme activity from the mitochondria on incubation with chlorhexidine, 10 p1 of mitochondria (0.3 mg protein) was incubated for 15 min. in 10 ml incubation medium with chlor- hexidine (25 pM) and centrifuged at 17,300 X g for 30 min. in a Sorvall RC 2 centri- fuge. The supernatant was aspirated and the pellet resuspended in 10 ml incubation medium with chlorhexidine (25 pM). The activities of the supernatant and the pellet were determined according to the method described above.

Determination of the effect of chlorhexidine on siiccinate: cytochrome c redcictase. This was performed by a modification of the method described by TISDALE (1967). The mitochondria were disintegrated by sonication for two rnin. with an MSE

150 Watts Ultrasonic Disintergrator. Mitochondria (60 pl, 3.1 mg protein), 5 ml in- cubation medium (0.25 M sucrose, 10 mM-KH,PO,/K,HPO,, 5 mM-MgCI,, 20 mM- KCI, 1 mM-NaN,, 20 mM triethanolamine, pH 7.4) and chlorhexidine digluconate in water (5 pl) were preincubated at 25" for 15 min. A 3 ml portion of the mixture was placed in the sample cuvette in a Shimadzu MPS-SOL recording spectrophotometer, while 3 ml incubation medium was placed in the reference cuvette. The cuvettes were thermostatically kept at 25". Disodium succinate (20 pl, 40 pmol) was added to the sample cuvette and the reaction initiated by the addition of oxidized cytochrome c (10 p1, 3 mg). The increase in absorbance with time at 550 nrn was recorded. The activity was determined from the slope of the graph during the first 10 sec. of the reaction. The amount of cytochrome c reduced was calculated using a molar ex- tinction coefficient of 21.0 X 109 cm-1M-1 for reduced cytochrome c (MASSEY 1959).

Mitochondria1 respiration was measured polarographically with a Beckman model 777 oxygen analyzer at 30". The total volume of the reaction mixture was 4.00 ml and consisted of "incubation buffer" (as described for the malate dehydrogenase assay) plus disodium succinate 2.5 mM and 4.8 mg mitochondrial protein. For examination of respiratory control ADP was added at a final concentration of 0.25 mM.

Kynurenine hydroxylase. The activity was estimated polarographically at 30" from the rate of kynurenine stimulated oxygen uptake in the presence of an NADPH generating system ( H A Y A I ~ 1962). The reaction mixture (4.00 ml) consisted of tris- HCI buffer 25 mM pH 8.0, sucrose 0.30 M, glucose-6-phosphate 1.0 mM, NADP 0.4 mM, glucose-6-phosphate dehydrogenase 48 pg, mitochondrial protein 2.4 mg and L-kynurenine sulphate 0.3 mM.

Proline oxidase was measured polarographically at 30" (JOHNSON & STRECKER 1962). The reaction mixture (4.00 ml) consisted of tris-HC1 buffer 25 mM pH 7.4, sucrose 0.3 M, mitochondrial protein 4 mg and L-proline 6 mM.

Chemicals. Unlabelled chlorhexidine dihydrochloride and chlorhexidine digluconate were obtained from ICI, Macclesfield, England. Chlorhexidine digluconate labelled with I4C was generously supplied by ICI, Macclesfield, England. L-kynurenine sulphate and L-proline were obtained from Calbiochem, Los Angeles, USA.

Glucose-6-phosphatase, glucose-6-phosphate dehydrogenase, nicotinamide-adenine- dinucleotide phosphate, adenosine-5'-diphosphate, adenosine-5'-triphosphate, disodium succinate and cytochrome c were obtained from Boehringer, Mannheim, GFR.

All other reagents were of analytical grade.

CHLORHEXIDINE AND LIVER MITOCHONDRIA 5

Chlorhexidine, phi

Fig. 1. The binding of chlorhexidine to rat liver mitochondria as determined from experiments with 14C-labelled chlorhexidine. The abscissa gives the total concentration of chlorhexidine in the reaction mixture. For further details see Materials and

Methods.

Results

Binding of chlorhexidine to rat liver mitochondria. This is shown in fig. 1, from which it is apparent that the amount of chlorhexidine bound to mitochondria increased nearly linearly with the total concentration up to 200 pM. At higher concentrations the percentage bound decreased. At about 800 pM the mitochondria were apparently saturated with chlorhexidine.

The binding of chlorhexidine to mitochondria was terminated even at 20 sec. following the addition of the compound to the mitochondrial sus- pension and it did not change when the time of incubation was gradually extended to 10 min.

Effect of chlorhexidine on mitochondrial proteins. Addition of chlor- hexidine to a suspension of mitochondria did not result in any visible precipitation of material nor did the amount of protein sedimented at 600 x g change significantly. However, when the concentration of chlor- hexidine was gradually raised to 100 pM, increasing amounts of proteins were apparently solubilized (fig. 2) as judged from the amount of protein remaining in solution following centrifugation at 15,000 X g for 5 min. When the concentration of chlorhexidine was raised to over 100 pM de- creasing amounts of protein remained in solution and at concentrations higher than 250 pM the supernatant contained even less protein than the con-

6 FLEMMING CHRISTENSEN et al.

I I I I I I

400 800 Chlorhexidine, pM

Fig. 2. The effect of chlorhexidine on soluble mitochondrial protein. The concentration of protein remaining in solution following centrifugation at 15,000 X g for 5 min. is shown related to the total concentration of chlorhexidine during the incubation. For

details see Materials and Methods.

trols without chlorhexidine. This might be an expression of a weak precipi- tating action of chlorhexidine on the soluble proteins.

Effect of chlorhexidine on mitochondrial respiration. Chlorhexidine has a very pronounced effect on the function of the mitochondria1 electron trans- port chain which is seen in fig. 3a. At very low concentrations i. e. 10-25 pM chlorhexidine (10-25 nmol per mg protein) an increased oxygen consump- tion is observed. This increase of 2.5 fold in the respiratory rate is of the same magnitude as the respiratory control ratio and also of the same magni- tude as the respiratory rate which was obtained by the uncoupler 2.4 dini- trophenol. Mitochondria which were stored on ice overnight showed no further respiratory control nor could their respiration be stimulated with chlorhexidine. Very low concentrations of chlorhexidine exhibited a lag of about 1 min. before the maximal effect was obtained. At higher concentra- tions, that were inhibitory (fig. 3a) to succinate oxidation, the effect was immediate.

The effect o f chlorhexidine on proline oxidase. Proline oxidase is a cyto- chrome c linked part of the mitochondrial electron transport system sensitive to cyanide (JOHNSON & STRECKER 1962). We therefore decided to compare the effect of chlorhexidine on this activity with the effect on succinate oxidation. In this case only an inhibition of proline oxidase could be ob- served, but the chlorhexidine concentrations necessary for 100 yo inhibition were about the same as for inhibition of succinate oxidation (fig. 3a).

The effect of chlorhexidine on mitochondrial ATP-use. When the con-

CHLORHEXIDINE AND LIVER MITOCHONDRIA 7

1" -0- MlTOCHONDRUl RESFIRATKlN I

2, MITOCHONDW ATP-ore )

C. MllocHoNDWAL

KYNURENINE HYDRQXYLASE

I I I 1 I

200 400 PM PM

Fig. 3. The abscissae show total concentrations of chlorhexidine while the ordinates show activities relative to control values obtained without chlorhexidine.

Fig. 3a. The effect of chlorhexidine on mitochondrial respiration and mitochondrial pro- line oxidase. Respiration with succinate and no chlorhexidine was 15.0 nmol 0, per min. per mg protein. The proline oxidation amounted to 4.7 nmol 0, per min. per mg protein.

Fig. 3b. Effect of chlorhexidine on mitochondria1 Mg*+-stimulated ATP-ase activity. Mitochondria equivalent to 0.9 mg protein was incubated with ATP (5 pmol), MgC1, (2.5 pmol) in a total volume of 1.5 ml at 30" and pH 7.4. Incubation time 5 min.

Activity without chlorhexidine 45.8 nmol per min. per mg protein.

Fig. 3c. Effect of chlorhexidine on mitochondria1 kynurenine hydroxylase. The activ- ity without chlorhexidine was 1.68 nmol 0, per min. per mg protein.

Fig. 3d. The effect of chlorhexidine on malate dehydrogenase and succinate: cyto- chrome c reductase. The graphs marked by 0-0 and 0-0 show the effect on malate dehydrogenase in whole (wm) and sonicated (sm) mitochondria respectively. The activity without chlorhexidine with whole mitochondria was 168 nmol NADH

8 FLEMMING CHRISTENSEN et a/.

centration of chlorhexidine was raised to 15 pM a slight stimulation of mitochondrial ATP-ase was observed (fig. 3b), but this was followed by an increasing depression of the activity when the concentration was further augmented. At concentrations exceeding 250 pM the depression was maxi- mal and the activity of mitochondrial ATP-ase reduced to one-half to one- third of its value without chlorhexidine.

The effect of chlorhexidine on kynurenine hydroxylase. As an enzyme normally attributed to the outer membrane of the mitochondria (BORST 1969) ,it can be seen from fig. 3c. that this activity too is sensitive to in- hibition by chlorhexidine. It is less sensitive than proline oxidase since a concentration of 1 mM chlorhexidine is necessary for 100 yo inhibition.

Effect of chlorhexidine on malate dehydrogenase. This is shown in fig. 3d. Controls with whole mitochondria oxidized 168 nmol NADH per min. per mg protein. Addition of chlorhexidine to whole mitochondria stimulated the activity, reaching a maximal value of 180 % at 25 pM chlorhexidine. At concentrations higher than 100 pM chlorhexidine the activity was stabilized at a level about 160 % of the control activity. Sonicated mitochondria oxidized 314 nmol NADH per min. per mg protein. Addition of chlor- hexidine (0-100 pM) to sonicated mitochondria stimulated the activity only slightly reaching a maximal value of 114 yo at 25 pM chlorhexidine. Chlor- hexidine in amounts exceeding 100 pM had no effect on the activity. Chlorhexidme (25 pM) released 87 Yo of the activity from the mitochondria and 13 % was left in the pellet.

Effect o f chlorhexidine on succinate: cytochrome c reductase. This is shown in fig. 3d. Sonicated mitochondria reduced 5.5 nmol cytochrome c per min. per mg protein. Addition of chlorhexidine depressed the reductase system and at a concentration of 67 pM chlorhexidine, the inhibition was total.

Discussion

The present investigation has shown that chlorhexidine binds to rat liver mitochondria. The maximal binding obtained was 900 nmol per milligram of mitochondrial protein which may be compared to a maximal binding of 500 nmol chlorhexidine per milligram protein observed with rat liver

oxidized per min. per mg protein. The activity without chlorhexidine with sonicated mitochondria was 314 nmol NADH oxidized per min. per mg protein. The graph represented by W-W shows the effect of chlorhexidine on succinate: cytochrome c reductase in sonicated mitochondria. The activity without chlorhexidine was 5.5 nmol

cytochrome c reduced per min. per mg protein. For details consult Materials and Methods.

CHLORHEXIDINE AND LIVER MITOCHONDRIA 9

microsomes (CHRISTENSEN & JENSEN 1974). The results indicate that the binding is of the same order of magnitude for the two kinds of particles but it is difficult to arrive at any definite conclusions from such data. No precipitation of mitochondria could be observed in the presence of chlor- hexidine even at the highest concentrations examined (about 1000 pM). This is in sharp contrast to the effect of chlorhexidine on liver microsomes which are practically totally precipitated by such concentrations (CHRISTEN- SEN & JENSEN 1974). The basis for the binding of chlorhexidine to liver mitochondria may be its cationic amphipathic nature supported by its af- finity for phosphate groups in the same way as proposed for microsomes (CHRISTENSEN & JENSEN 1974).

Chlorhexidine has apparently only a weak solubilizing effect on mito- chondrial proteins. The effect was only observable at rather low concentra- tions (< 200 KM) of the drug while at higher concentrations less protein was detectable in a soluble form perhaps due to a slight protein precipi- tating action of the compound at the higher concentrations. The solubilizing effect of chlorhexidine on mitochondrial proteins is very much less pro- nounced than that seen with many other detergents as for example with deoxy cholate.

The effect of chlorhexidine on succinate stimulated mitochondrial oxygen uptake was pronounced at low concentrations around 10 pM where a stimu- lation up to 2.5 times the control values was encountered. It has not been determined whether this stimulation is accompanied by an uncoupling of the ATP-generation from oxidation. However, only a very weak stimulation of mitochondrial ATP-ase activity was observed at the low concentrations of chlorhexidine enhancing the oxygen uptake. This would have been ex- pected since uncoupling is generally accompanied by increased activity of mitochondrial ATP-ase (HEMKER 1962), but the reason for the lack of stimulation of the ATP-ase in the present investigation remains undeter- mined. The increase in mitochondrial oxygen uptake under the influence of chlorhexidine is of the same order of magnitude as seen with classical uncouplers such as 2.4-dinitrophenol (HEMKER 1962). The uncoupling ef- fect of chlorhexidine may be related to its amphipathic nature as it is re- ported that other detergents can uncouple oxidative phosphorylation in liver mitochondria (LEHNINGER 1955). The inhibitory effect of chlorhexidine on mitochondrial oxygen consumption at higher concentrations (100 pM) may be related to an effect of the drug on the mitochondrial membrane caused by a disturbance of the membrane structure or of one or more of the enzymes of the respiratory chain. It seems appropriate here to mention that certain biguanides (alkyl or arylalkyl) depress mitochondrial oxygen uptake by inhibiting oxidative phosphorylation (SLATER 1967). Although we did not test this possibility directly in the case of chlorhexidine it seems

10 FLEMMING CHRISTENSEN et al.

rather unlikely because of the apparent uncoupling action of the compound observed at low concentrations. The depression of mitochondrial ATP-ase observed at the higher concentrations of chlorhexidine may be ascribed to a membrane effect of the same kind as proposed as the basis of the de- pression of mitochondrial oxygen uptake. It should be noted that a de- pression of bacterial ATP-ase in the presence of chlorhexidine has recently been described (HAROLD et al. 1969).

The effect of chlorhexidine on various enzyme activities of mitochondria was examined in order to test the hypothesis that the drug affects rather generally the membrane-bound enzymes of these particles. As representa- tives of the inner membrane enzymes chosen were succinate:cytochrome c reductase and proline oxidase respectively while kynurenine hydroxylase was chosen as a representative of the outer membrane and malate de- hydrogenase as a matrix-bound enzyme (BORST 1969). The membrane- bound enzymes (inchding the mitochondrial ATP-ase) were all depressed by chlorhexidine so that virtually no activity was observable at the highest concentrations of the drug studied. It seems reasonable to suggest that chlorhexidine affects these enzymes by changing their conformation in the membrane so as to render them catalytic inactive. In contrast to the mem- brane-bound enzymes the matrix-bound malate dehydrogenase was unique in being stimulated by chlorhexidine. It seems, however, to be a result of a solubilizing effect on the enzyme since an equally large stimulation of the enzyme was obtained by sonication of the mitochondria. The enzyme itself was practically uninfluenced by chlorhexidine as the activity of the sonicated mitochondria did not respond to increasing concentrations of the drug.

From the results described in this paper it can be concluded that chlor- hexidine binds to mitochondria and exerts concentration-dependent effects on various enzyme systems of these particles. The basis of the effects is the binding of chlorhexidine which presumably induces permeability changes in the mitochondrial membranes and results in conformational changes in the membrane-bound enzymes thereby rendering them more or less active. Some enzymes may even be totally displaced from the membranes and eventually solubilized. The effect of chlorhexidine at the highest concentra- tions studied may also be ascribed to a protein precipitating effect although this is presumably of minor importance. All the effects described may be related to the cationic amphipathic nature of chlorhexidine as previously suggested for its effects on the liver microsomal membranes.

A c k n o w l e d g e m e n t s

Andersen and Chr. Hoffmann for skilful technical assistance. The authors wish to thank Birte Esmann, Bente Poulsen, Hans Krogh

CHLORHEXIDINE AND LIVER MITOCHONDRIA 11

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