CO~ZYME Q DEPLETION IN RAT PLASMA AFTER PARTIAL HEPATECTOMY

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1 Vol. 39, No. 6, August 1996 Pages CO~ZYME Q DEPLETION IN RAT PLASMA AFTER PARTIAL HEPATECTOMY G. Formiggini, C. Castelluccio, M. Mer]o Pich, M. L. Genova, C. Bovina, M.Marchetti, G. Lenaz Department of Biochemistry "G. Moruzzi", University of Bologna, via Irnerio 48, Bologna, Italy Received June 12, 1996 SUMMARY. Coenzyme Q content was monitored in blood plasma and regenerating liver mitochondria of hepatectomized rats, using as controls either sham-operated or non-operated animals. Mitochondrial CoQ 9 content increased in sham-operated rats, whilst it was significantly lower in hepatectomized in comparison with non-operated animals at all considered times. On the other hand plasma CoQ9 levels dramatically decreased in hepatectomized animals, while strongly increased in sham-operated in comparison with non-operated rats. The quinone decrease in hepatectomized animals is likely to be due to the attainment of a rate-limiting step in CoQ biosynthesis. KEY WORDS: Coenzyme Q,liver regeneration,plasma,mitochondria, antioxidant. INTRODUCTION Liver regeneration after partial hepatectomy is accompanied by increased mitochondrial biogenesis, with enhanced replication of mitochondrial DNA and biosynthesis of mitochondrial enzymic complexes involved in oxidative phosphorylation (i). It therefore represents a unique system where to investigate the coordination of the molecular systems involved in the oxidative phosphorylation process. Previous studies on mitochondrial function during liver regeneration (1-4) did not consider the levels of Coenzyme Q (ubiquinone, CoQ), a lipophilic benzoquinone enbedded in the lipid milieu of the membrane (5) and representing the redox link between flavoprotein dehydrogenases and the cytochromes in the respiratory chain (6). The concentration of CoQ in the mitochondrial membrane is not kinetically saturating for NADH oxidation (7) so that changes in its concentration in the membrane are expected to determine changes of respiratory activity. Its behavior would therefore be crucial in conditions of high energy demand, such as regeneration of the liver ~6/061135~ /0 Cop~ght 1996 ~ Aca~m~ Press Austs~l~ All ~h~ ~ reproduction in a~ ~nn reserved.

Moreover CoQ in its reduced form is now widely recognized to lipoproteins (9): its level in a condition of stress as hepatectomy may therefore be crucial also to contribute to hepatocyte protection

2 Moreover CoQ in its reduced form is now widely recognized to lipoproteins (9): its level in a condition of stress as hepatectomy may therefore be crucial also to contribute to hepatocyte protection during regeneration. In this study we have investigated the CoQ levels in liver mitochondria and in blood plasma of rats after partial hepatectomy using as controls both non-operated and sham-operated animals. MATERIALS A/~D METHODS Male albino rats of the Wistar strain, weighing g, were maintained under controlled conditions for one week and then divided into three groups. One group was subjected to partial hepatectomy under diethyl ether anesthesia with removal of 70% of the tissue as described in (i0); another group was subjected to laparatomy and used as control; the third group was sacrificed without any previous treatment except anesthesia. All procedures were performed according to the ethical guidelines issued by the University of Bologna. The animals were killed by decapitation at intervals of 12, 36, and 84 hours after surgery; mitochondria were isolated from livers as described by Kun et al (ii), without digitonin treatment. CoQ was extracted from mitochondria (12) and from plasma (13) and the extracts were analyzed by reverse phase hplc (14) using a Waters Data Module M370-Model 721 Programmable system Controller equipped with a Lambda-Max Model 481 LC spectrophotometer. Under the conditions employed, CoQ was completely present in its oxidized form. Statistical analysis was performed using means ± S.E.M. and comparisons were made by the unpaired t test. Only probability values p<0.05 were accepted as statistically significant. RESULTS In accordance with the reported increase of mitochondrial protein synthesis (I), the yield of mitochondrial protein was increased in the hepatectomized rats at 12 hours with respect to the non-operated controls; it was however increased also in the sham-operated controls. In spite of some subsequent decrease, the levels remained higher than in the non-operated controls even at 84 hours. Accordingly, there was a dramatic increase of the rate of succinate oxidation in the homogenate after surgery shown). (not Mitochondrial CoQ9 significantly increased in the sham- operated rats at 12 hours (Table i), and the levels remained higher than in the non-operated controls at further times after 1136

3 Vol. 3g, No. 6, 1996 Table 1. CoQg content of liver mitochondria from non-operated, sham-operated and hepatectomized rats. Hours after sham-operation or hepatectomy Animals CoQ9 content (pmoles/mg protein) 0 Non-operated Sham-operated 2162 _+ 178 # Hepatectomized * 36 Sham-operated Hepatectomized " 84 Sham-operated # Hepatectomized * Data are means + S.E.M. from 6-8 animals #Significantly different from non-operated control. *Significantly different from corresponding sham-operated control. operation; on the other hand the CoQ9 levels in hepatectomized rats remained significantly lower than in sham-operated controls rats remained significantly lower than in sham-operated controls at all times after surgery. Similar changes occurred for COQ1o, with a constant CoQg/COQ~o ratio in all groups (not shown). The changes of CoQ content in the total homogenate were small (not shown), indicating that most variations were present only in the mitochondrial fraction. In plasma (Table 2) there was a dramatic decrease of CoQ 9 content in the hepatectomized animals with respect to the nonoperated controls, with a minimum at 36 hours; on the other hand, the levels in the sham-operated rats were almost double those in the non-operated controls at all times after surgery. DISCUSSION A survey of enzymatic activities showed an increase of Complex III and Complex IV activities in the regenerating livers in accordance with previous observations (I); CoQ, however, does not 1137

4 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL Table 2. CoQ9 content of blood plasma from non-operated, sham operated and hepatectomized rats. Hours after sham-operation or hepatectomy Animals CoQ9 content (pmoles/ml) 0 Non-operated Sham-operated 267 _+ 63 Hepatectomized * 36 Sham-operated # Hepatectomized #* 84 Sham-operated # Hepatectomized 85 _+ 14 #* Data are means +SE,M. from 5-8 animals. # Significantly different fron non-operated control. * Significantly different from corresponding sham-operated control. match the behavior of the protein components. In fact mitochondrial CoQ did not significantly increase in the regenerating livers, and actually was significantly lower than in the sham-operated controls at all times after operation. This means that its biosynthesis and/or incorporation in mitochondria was not significantly increased over the average rate of biosynthesis and assembly of all mitochondrial protein components. This occurs in spite of the increased CoQ content in the liver mitochondria of the sham-operated controls, which is presumably a consequence of the surgical oxidative stress (15). This suggests that, in the regenerating livers, the stimulation of the quinone biosynthesis due to the oxidative stress cannot cope with the overwhelming demand of newly synthetized quinone for the rapidly growing mitochondrial population. It has to be borne in mind that the amount of newly synthetized quinone per cell must be much higher in the regenerating liver than in the control because the former are actively synthetizing new 1138

5 mitochondrial materials. Thus, some step in CoQ biosynthesis must become rate-limiting when the overall process is turned on. There are extensive indications in the literature that CoQ biosynthesis may be limiting under pathological conditions (cf. 16), leading to decreased CoQ levels. A possible bottleneck in CoQ The increase of CoQ in plasma in the sham-operated animals reflects the hepatic increase; on the other hand, in rats whose livers are regenerating the plasma CoQ levels approach zero, suggesting its preferred incorporation into proliferating mitochond~ia, so that its delivery to the newly formed lipoproteins (where hematic CoQ is localized) (9,18) is impaired. The dramatically decreased CoQ levels in plasma under liver regeneration may have serious pathological consequences. Plasma CoQ has been shown to protect lipoproteins from oxidation (9) and oral CoQ supplementation increases its content in the LDL thereby increasing their resistance to oxidative stress (19); CoQ is one of the major antioxidants in human lipoproteins and has a key role in preventing the uptake of oxidized lipoproteins in the endothelia and hence of development of atherosclerotic lesions (20). It is therefore likely that conditions, such as liver regeneration, leading to increased demand for CoQ, may have a better course and be devoid of pathological complications by maintaining high CoQ levels in the body; exogenous CoQ administration enhances plasma levels (21) and possibly tissue levels as well (22). A CoQ supplementation prior to and/or after liver surgery or transplantation or during liver degenerative diseases could be suggested as a preventive tool against harmful complications. ACKNOWLEDGEMENTS This study was supported by grants from MURST, Rome. REFERENCES i. Nagino, M., Tanaka, M., Nishikimi, M., Nimura, Y., Kubota, I., Kanai,,M., Kato, I. and Ozawa, T. (1989) Cancer Reso 9, Kamiyama, Y., Ozawa, K. and Honjo, I. (1976) J. Biochem. 80,

6 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL 3. Ozawa, K., Yamada, T., Ukikusa, M., Ngala, K., Ida, T., Nakase, A. and Tobe T. (1981) J. Surg. Res. 31, Nishihira, T., Tanaka, J., Nishikawa, K., Jikko, A., Taki, Y., Morimoto, T., Koizumi, K., Kamiyama, Y., Ozawa, K. and Tobe T. (1986) Hepatology 6: Samori, B., Lenaz, G., Battino, M., Marconi, G. and Domini, I., (1992) J. Membr. Biol. 128, Lenaz, G. (1988) J. Membr. Biol. 104, Estornell, E., Fato, R., Castelluccio, C., Cavazzoni, M., Parenti Castelli, G. and Lenaz, G. (1992) FEBS Lett. 311, Beyer, R.E. (1990) Free Rad. Biol. Med. 8, Stocker, R., Bowry, V.W. and Frei, B. (1991) Proc. Natl. Acad. Sci. US 88, i0. Higgins, G.M. and Anderson R.M. (1931) Arch. Pathol. 7, ii. Kun, E., Kirsten, E. and Piper, W.N. (1979) Methods Enzymol. 55, Genova, M.L., Bovina, C., Formiggini, G., Ottani, V., Sassi, S. and Marchetti, M. (1994) Molec. Aspects Med. 15S, Grossi, G., Bargossi, A.M., Fiorella, P.L., Piazzi, S., Battino, M. and Bianchi, G.P. (1992) J. Chromatogr. 593, Valls, V., Castelluccio, C., Fato, R., Genova, M.L., Bovina, C., Saez, G., Marchetti, M., Parenti Castelli, G. and Lenaz, G. (1994) Biochem. Mol. Biol. Int. 33, Tsai, A.L., Kauten, R. and Palmer, G. (1985) Anal. Biochem. 151, Folkers, K., Vadhanavikit, S. and Mortensen, S.A. (1985) Proc. Natl. Acad. Sci. US 82, Gr~nler, J., Ericsson, J. and Dallner, G. (1994) Biochim. Biophys. Acta 1212, Karlsson, J., Liska, J.. Gunnes,S., Koul, B., Semb, B., ~str6m, H., Diamant, S. and Folkers, K. (1993) Clin. Investig. 71S, Mohr, D., Bowry, V.W. and Stocker, R. (1992) Biochim.Biophys. Acta 1126, Steinbrecher, U.P., Zhang, H. and Longheed, M. (1990) Free Rad. Biol. Med. 9, Okamoto, T., Matsuya, T., Fulunaga, Y., Kishi, T. and Yamagami, T. (1989) Int. J. vit. Nutr. Res. 59, Mortensen, S.A. (1993) Clin. Investig. 71S,

Determination of Coenzyme Q in Human Plasma

Physiol. Res. 44: 39-45, 1995 Determination of Coenzyme Q in Human Plasma P. KAPLAN, N. SEBESTIANOVA1, J. TURIAKOVA, I. K U CERA 1 Department o f Biochemistry, Faculty o f Medicine and Department o f Biochemistry,