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Ubiquinone analyses in fish tissues and in some marine invertebrates
Comp. Biochem. Physiol.. Vol. 63~. pp. 395 to 397
"'
0305-0491/79/0107-0395502.00/0
~) Pergamon Press Lid 1979. Printed in Great Britain
UBIQUINONE ANALYSES IN FISH TISSUES AND IN SOME MARINE INVERTEBRATES T. FARBU and G. LAMBERTSEN Institute of Vitamin Research, Directorate of Fisheries, P.O. Box 187, N-5001 Bergen, Norway
{Received 29 November 1978) Abstract--l. Ubiquinone contents were determined in species of marine invertebrates, and in heart, red and white muscle and liver of three species of fish. 2. Three different methods of determination were compared, based on spectrophotometry, reduction and a reaction with the dimethoxy groups of ubiquinone. 3. Using ubiquinone homologues 6-10 prepared from beef heart and commercially available microorganisms {SCP) as standards, ubiquinone 10 was found in all samples. In addition were found minor amounts of Q-9 in samples of saithe heart and red muscle. 4. Less than 10 mg/kg wet wt of ubiquinone was found in the samples of marine invertebrates and in white muscle and liver of the fish samples, with one exception: 40 mg/kg in a sample of mackerel liver. 5. Higher contents of ubiquinone were found in fish heart and red muscle tissues, ranging from 24 to 116 mg/kg wet wt. The ubiquinone contents were comparable in the two tissues. 6. A test on cellular fragments of red muscle tissue of saithe showed that the ubiquinone was concentrated in the mitochondria fraction.
INTRODUCTION
on the ubiquinone concentration, 1-10 g of the freeze-dried samples were saponified for 15min in methanol (10ml/g sample) using 55% aqueous KOH (w/w, 1 ml/g) and with some grains of pyrogallol added as antioxidant. To the cooled mixture was added 50ml distilled water and the unsaponifiable matter was extracted three times with 50 ml of diethyl ether. The combined extracts were washed with 3 x 40 ml distilled water and evaporated under reduced pressure on a water bath (temp. <60°C), dissolved in a few millilitres of hexane and transferred to a glass column (180ram length, 7ram dia.) filled with 10g Silica gel (Woelm). The column was eluted with an increasing ratio of ethyl ether in hexane (0-15%) and 10ml fractious were collected and monitored by thin-layer chromatography (DC-Alufolien, Merck) using hexan/ethyl ether 80:20 as eluent and leucomethyleneblue as detector reagent. Leucomethylene was prepared from 1% aqueous methylene blue chloride, 1-3 g zink dust and 2 ml conc. H2SO,t, shaken and filtered through glass wool (Crane & Barr, 1971). A few of the extracts had to be purified by preparative TLC after the column chromatographic step. (Glass plates with silica gel G (Merck), eluant and detector as for qualitative TLC.)
A s active factors in the respiratory chain, the ubiquinones are of interest in comparative biochemistry. Methods of determination of ubiquinones have been described in detail for different tissues (Diplock et al., 1966, Crane & Barr, 1971). The ubiquinones and other biological quinones are often found in mixtures at low levels of concentration, and reliable identification of the ubiquinones is therefore an integral part of an analytical method. The presence in the tissues of fat in greatly varying concentrations may reduce the fidelity of a determination. We have studied three methods of determination of ubiquinone, based on different principles. These methods were used to determine the contents of ubiquinone in some marine invertebrates, as well as in light and red muscle tissue, heart and liver of some fish species. Further, the isoprenoid chain length of the ubiquinones has been verified using a standard set prepared from commercially available microorganisms (SCP).
Qualitative determination MATERIALS AND METHODS Live specimens of horse mussel (Modiolus mod~olus), blue mussel (Mytilus edulis), a crab (Cancer paourus), a sea urchin (Echinus acutus), and a starfish (Asteria rubens) were obtained from the Biological Station, Espegrend, University of Bergen, Norway. Mackerel (Scomber scombrus), saithe (Pollachius virens) and fillet of halibut (HippooIossus hippoglossus) were obtained in a fresh state at the Bergen fish market. Tissues were prepared from the samples upon arrival, followed by freeze-drying (Hetosicc Freeze, Type CD 13-2) and homogenization. The freeze-dried, samples were stored in closed vials in a freezer ( - 20°C) until analyo sis.
Methods of separation The samples were saponified mainly according to a method described by Mervyn & Morton (1959). Depending
The ubiquinone homologues were separated by thinlayer partition chromatography (Crane & Barr, 1971) using 10% squalane in hexane to impregnate the silica gel (Merck). The glass plates were developed with acetone: water (95:5), saturated with squalane. Ubiquinone homoIogues were prepared from commercially available SCP, obtained by the courtesy of the IS KIover-Mollenes Felleskontor, Troodheim, Norway (Fig. 1).
Quantitative determination Method A. Spectrophotometric assay using geometrical curve correction. Ubiquinone has a maxhnum extinction in ethanol at 275 run. Using the general 3-poiut correction formula given by Mulder & Keunig (1961) the following correction factors were calculated E ~ m corrected = 3.899-E27 s - 2.127"E2~a - 1.772'E2ss
395
T. FARBUand G. LAMBERTSEN
396
0
Q-IO
0
Q-9
0
Q-8 Q-7
"7.
0 0
Q-6 MIX.
0
0 0 0 0
Development Fig. 1. TLC-separation of ubliquinone homologues. A silica gel glass plate was impregnated with squalane and developed in acetone: water (95:5) saturated with squalane. Q-10 was prepared from beef heart, Q-9 from Candida tropicalis (B.P.), Q-8 from Pseudomonas sp. (I.C.1.), Q-7 from Candida utilis (B.P.) and 1~-6 from bakers yeast.
Method B. Spectrophotometry before and after reduction. Ubiquinone was reduced by a few crystals of NaBH,, in ethanol. The difference between the extinction of the oxidized and the reduced form at 275 nm was used for quantitative determination according to the formula given by Crane & Barr (1971). The spectrophotometric measurements in method A and B were performed in 1 cm quarts cuvettes on a Zeiss PM QII instrument. Method C. Colourimetry. The methoxygroups of ubiquinone develops a blue colour in alkaline solution with ethylcyanoacetate (Cravens test). 1 ml ethylcyanoacetate was added to 2ml of the sample extract in ethanol, followed by 1 ml 0.8N Triton B. The blue colour was measured after 10 min on a Gilford spectrophotometer at 630nm (Redalieu & Folkers, 1971). The amount of ubiquinone 10 was determined by preparing a standard curve from pure ubiquinone (Correlation coefficient r > 0.998). RESULTS AND DISCUSSION
Freeze-drying of the samples was practical because of the high water content in many of the marine invertebrates. Methanol was used instead of ethanol in the saponification step to avoid artifacts of ubiquinone (Linn et al., 1960). Overall recovery of added ubiquinone 10 to a beef heart preparation was 87.7 _ 11.2~o(S.D, n = 8). Based on a pure solution of ubiquinone 10 (Sigma), method A gave a recovery of 102.3~ + 0.6, method B 94.2~ ___ 3.1 and method C 106.3~o __+ 3.1 (S.D., n = 6). The three methods gave comparable results on a purified extract from beef hearts. Method A, spectrophotometry with geometrical curve correction should be used only when the calculated correction is below 30%. This method failed on some samples high in fat. Method B, spectrophototnetry before and after reduction, gave approx 8% lower results compared with methods A and C. This effect disappeared in biological material with low ubiquinone concentrations. Method B gave unreliable results on samples of liver due to interfering substances. Some of these problems could be corrected by an additional preparative TLC separation (see Methods). Method C, colourimetry, could be used in all cases. This method has a low detection limit, but demands a good preseparation to get results comparable with methods A and B.
Results are given in Table 1. Pennock t1966) found small amounts of ubiquinone in marine invertebrates. Among these were the following values: Patella t:ulgata 4 pg/g wet wt, Asteria rubens 3.5 pg/g, Nephrops norvegicus 2.2pg/g. Table 1 shows corresponding values. All three methods gave comparable results within narrow limits on the invertebrate samples. Thin layer partition chromatography showed only one spot for ubiquinone 10 in all invertebrate samples. A sea anemone (Bolocera tuediael was negative for ubiquinone, probably due to interferevce from a high content of wax esters in this sample. The values found for ubiquinone in mackerel and saithe hearts were l l6pg/g and 61pg/g respectively. These values were comparable to those found for beef and sheep heart, 77 and 199pg/g respectively. Diplock & Haselwood (1967) have found corresponding values. Small amounts of ubiquinone 9 could be detected in the saithe heart and red muscle samples. Ubiquinone 9 was detectable only when applying large amounts of ubiquinone to the TLC and was estimated to be a maximum of 5~,~ of the total ubiquinone content. Whistance et aL 11971) found bolh ubiquinone 9 and 10 in animal hearts, and also ubiquinone 8 could be detected in some cases. Values for ubiquinone in the red muscle varied from 24.2/~g/g (mackerel) to 82.5/~g/g isaithe) and in white muscle from 1.8/~g/g (saithe) to 3.6/~g/g/mackerel). Method B could not be used for the red muscle samples of mackerel. The ubiquinone content in fish red muscle was 7 45 times higher than that found in white muscle. The red muscle of saithe had a ubiquinone content even higher than that found in the heart of this fish species. Red muscle cells have numerous mitochondria and store more energy rich materials as glycogen and lipid than the white muscle fibres (Patterson & Goldspink, 1972). It is therefore not surprising to find a high concentration of ubiquinone in red muscle tissue. Cytochrome oxidase was found to have about 8 times higher activity in red muscle than in the white muscle of carp (Johnston et aL, 1977). It seems probable that red muscle is active during slow movements and obtains its energy from oxidative phosphorylation. whereas the white muscle is active during vigorous movements and gets its energy from anaerobic glycolysis (Johnston & Goidspink, 1973). A preliminary tissue fractionation of saithe red muscle tissue showed that ubiquinone was enriched to a higher degree in the mitochondria fraction than the marker enzyme glutamate dehydrogenase GDH (E.C. 1.4.1.2). 40~o of the total ubiquinone content was found in the mitochondria fraction. This is almost the same value that Jayerman & Ramasarma t1963! found for rat liver mitochondria, 40-60~. The ubiquinone content in fish livers will probably be influenced by such factors as season, sex and nutritional status. Mackerel liver samples from autumn and spring had greatly different ubiquinone values (4.76/~g/g and 40.0/~g/g). Diplock & Haselwood (1967) found 22.0/~g/g in mackerel (Scomber scrombus) liver, 106.2 #g/g in saithe (Pollachius t,irens) liver and 62.1 pg/g in cod (Gadus morhua) liver, whereas Penhock et al., 1962, reported 8.6 #g/g in cod (Gadus morhua) liver. According to the values presented, as well as from
397
Ubliquinone analyses in fish tissues and in some marine invertebrates Table 1. Analysis by three methods of the ubiquinone contents in some marine invertebrates and fish species. Method A--Spectrophotometry with geometrical curve correction. Method B--Spectrophotometry before and after reduction. Method C--Colorimetry after Craven. Average dry wt (g/kg)
Samples (No. of analyses) Invertebrates: Horse mussel (2) Modiolus modiolus Blue mussel (2) Mytilus edulis Crab (2) Cancer pagurus Sea urchin (1) Echinus acutus Star fish (2) Asteria rubens Fish species: Mackerel Scomber scombrus heart (6) red muscle (6) white muscle (4) liver, autumn (4) liver, spring (2) Saitbe Pollachius virens heart (3) red muscle (4) white muscle (4) liver (2) Halibut Hippoglossus hippogloossus red muscle (2) white muscle (2)
A
Analytical method B (mg/kg dry wt)
C
Average of A+ B+ C (mg/kg wet wt)
204
7.9
7.5
7.3
1.54
238
16.9
16.9
16.8
4.01
256
15.4
15.5
16.0
4.00
144
38.3
38.8
38.1
' 5.35
258
10.0
9.4
10.9
2.61
244 535 468 265 294
482 45.4 ----
448 19.7 --~" 18.0 118
500 45.2 7.7 17.9 154.0
116 24.2* 3.60 4.76 40.0
180 227 219 738
352 375 8.8 --
318 342 --t --
347 373 8.0 12.9
61.0 82.5 1.84 9.52
278 230
160 9.6
162 --~"
162 8.0
44.9 2.02
* This value is based on method A and C. ? Insufficient material for analysis.
literature reports, ubiquinone contents may vary greatly in tissues of marine organisms. It seems necessary, therefore, to check both identification and quantity of ubiquinone by two independent methods. The methods compared in the present report identify ubiquinone by its absorption curve in u.v.-light, by its redox properties, and by the presence of methoxy groups. REFERENCES CRANE F. L. & BARR R. (1971) Determination of ubiquinones. Meth. Eazym. 18C, 137-169. DIPLOCK A. T., GREENI., BUNYANl. & METTALED. (1966) The analysis of u-tocopherol and ubiquinone in rat liver. Br. J. Nutr. 20, 95-I01. DlPLOCK A. T. & HASELWOOD,G. A. D. (1967) The ubiquinone content of animal tissues. Biochem. J. 104, 1004-1010. J^'~RM^N I. & R^~tASARrO,T. (1963) Intracellular distribution of coenzyrne Q in rat liver. Archs Biochem. Biophys. 103, 258-266. JOHnS'tON I. A. & GOLI)SPINK G. (1973) A study of glycogen and lactate in the myotomal muscles and liver of the coalfish (Gadus virens L.) during sustained swimming. J. mar. biol. Ass. U.K. 53, 17-26.
JOHNSTON I. A., DAVISON W. & GOLDSPINK G. (1977) Energy metabolism of carp swimming muscles. 2. Comp. Physiol. 114, 203-216. L ~ B. O., TRENI~ER N. R., ARISON B. H., WESTON R. G, Sh'UNK C. H. & FOLr~RS K. (1960) Coenzyme Q. XlI. Ethoxy homologs of coenzyme QI0. Artifact of isolation. 2. Am. chem. Soc. 82, 1647-1652. MERVYN L. & MORTON R. A. (1959) Unsaponifiable fraction of lipid from normal and diseased human kidney. Biochem. 2. 72, 106. MULDER F. I. & KEUNm K. J. (1961) Spectrophotometric assay of ~-tocopberol. Recueil 80, 1029-1039. PATTERSON S. & GOLDSPlNK G. (1972) The fine structure of red and white myotomal muscle in a marine teleost, the coalfish (Gadus virens). Z. Zellforsch. mikrosk. Anat. 133, 463-474. I~NNOCK J. F. (1966) Occurrence of vitamine K and related quinones. Vitams Hot. 24, 307-330. PENNOCKJ. F., MORTONR. A., LAWSOND. E. M. & LAIDMAN D. L. (1969) Quinones and related compounds in ~ h tissues. 8iochem. J. 84, 637-640. REDALIEUE. & FOLKEILSK. (1971) Assay of coenzyme QI0 in blood. Meth. Enzym. 18C, 179-181. WmSTANC~ G. R., FmLD F. E. & THRELFALLD. R. (1971) Observations on the biosyntheses of ubiquinones by animals. Eur. J. Biochem. 18, 46-52.
"'
0305-0491/79/0107-0395502.00/0
~) Pergamon Press Lid 1979. Printed in Great Britain
UBIQUINONE ANALYSES IN FISH TISSUES AND IN SOME MARINE INVERTEBRATES T. FARBU and G. LAMBERTSEN Institute of Vitamin Research, Directorate of Fisheries, P.O. Box 187, N-5001 Bergen, Norway
{Received 29 November 1978) Abstract--l. Ubiquinone contents were determined in species of marine invertebrates, and in heart, red and white muscle and liver of three species of fish. 2. Three different methods of determination were compared, based on spectrophotometry, reduction and a reaction with the dimethoxy groups of ubiquinone. 3. Using ubiquinone homologues 6-10 prepared from beef heart and commercially available microorganisms {SCP) as standards, ubiquinone 10 was found in all samples. In addition were found minor amounts of Q-9 in samples of saithe heart and red muscle. 4. Less than 10 mg/kg wet wt of ubiquinone was found in the samples of marine invertebrates and in white muscle and liver of the fish samples, with one exception: 40 mg/kg in a sample of mackerel liver. 5. Higher contents of ubiquinone were found in fish heart and red muscle tissues, ranging from 24 to 116 mg/kg wet wt. The ubiquinone contents were comparable in the two tissues. 6. A test on cellular fragments of red muscle tissue of saithe showed that the ubiquinone was concentrated in the mitochondria fraction.
INTRODUCTION
on the ubiquinone concentration, 1-10 g of the freeze-dried samples were saponified for 15min in methanol (10ml/g sample) using 55% aqueous KOH (w/w, 1 ml/g) and with some grains of pyrogallol added as antioxidant. To the cooled mixture was added 50ml distilled water and the unsaponifiable matter was extracted three times with 50 ml of diethyl ether. The combined extracts were washed with 3 x 40 ml distilled water and evaporated under reduced pressure on a water bath (temp. <60°C), dissolved in a few millilitres of hexane and transferred to a glass column (180ram length, 7ram dia.) filled with 10g Silica gel (Woelm). The column was eluted with an increasing ratio of ethyl ether in hexane (0-15%) and 10ml fractious were collected and monitored by thin-layer chromatography (DC-Alufolien, Merck) using hexan/ethyl ether 80:20 as eluent and leucomethyleneblue as detector reagent. Leucomethylene was prepared from 1% aqueous methylene blue chloride, 1-3 g zink dust and 2 ml conc. H2SO,t, shaken and filtered through glass wool (Crane & Barr, 1971). A few of the extracts had to be purified by preparative TLC after the column chromatographic step. (Glass plates with silica gel G (Merck), eluant and detector as for qualitative TLC.)
A s active factors in the respiratory chain, the ubiquinones are of interest in comparative biochemistry. Methods of determination of ubiquinones have been described in detail for different tissues (Diplock et al., 1966, Crane & Barr, 1971). The ubiquinones and other biological quinones are often found in mixtures at low levels of concentration, and reliable identification of the ubiquinones is therefore an integral part of an analytical method. The presence in the tissues of fat in greatly varying concentrations may reduce the fidelity of a determination. We have studied three methods of determination of ubiquinone, based on different principles. These methods were used to determine the contents of ubiquinone in some marine invertebrates, as well as in light and red muscle tissue, heart and liver of some fish species. Further, the isoprenoid chain length of the ubiquinones has been verified using a standard set prepared from commercially available microorganisms (SCP).
Qualitative determination MATERIALS AND METHODS Live specimens of horse mussel (Modiolus mod~olus), blue mussel (Mytilus edulis), a crab (Cancer paourus), a sea urchin (Echinus acutus), and a starfish (Asteria rubens) were obtained from the Biological Station, Espegrend, University of Bergen, Norway. Mackerel (Scomber scombrus), saithe (Pollachius virens) and fillet of halibut (HippooIossus hippoglossus) were obtained in a fresh state at the Bergen fish market. Tissues were prepared from the samples upon arrival, followed by freeze-drying (Hetosicc Freeze, Type CD 13-2) and homogenization. The freeze-dried, samples were stored in closed vials in a freezer ( - 20°C) until analyo sis.
Methods of separation The samples were saponified mainly according to a method described by Mervyn & Morton (1959). Depending
The ubiquinone homologues were separated by thinlayer partition chromatography (Crane & Barr, 1971) using 10% squalane in hexane to impregnate the silica gel (Merck). The glass plates were developed with acetone: water (95:5), saturated with squalane. Ubiquinone homoIogues were prepared from commercially available SCP, obtained by the courtesy of the IS KIover-Mollenes Felleskontor, Troodheim, Norway (Fig. 1).
Quantitative determination Method A. Spectrophotometric assay using geometrical curve correction. Ubiquinone has a maxhnum extinction in ethanol at 275 run. Using the general 3-poiut correction formula given by Mulder & Keunig (1961) the following correction factors were calculated E ~ m corrected = 3.899-E27 s - 2.127"E2~a - 1.772'E2ss
395
T. FARBUand G. LAMBERTSEN
396
0
Q-IO
0
Q-9
0
Q-8 Q-7
"7.
0 0
Q-6 MIX.
0
0 0 0 0
Development Fig. 1. TLC-separation of ubliquinone homologues. A silica gel glass plate was impregnated with squalane and developed in acetone: water (95:5) saturated with squalane. Q-10 was prepared from beef heart, Q-9 from Candida tropicalis (B.P.), Q-8 from Pseudomonas sp. (I.C.1.), Q-7 from Candida utilis (B.P.) and 1~-6 from bakers yeast.
Method B. Spectrophotometry before and after reduction. Ubiquinone was reduced by a few crystals of NaBH,, in ethanol. The difference between the extinction of the oxidized and the reduced form at 275 nm was used for quantitative determination according to the formula given by Crane & Barr (1971). The spectrophotometric measurements in method A and B were performed in 1 cm quarts cuvettes on a Zeiss PM QII instrument. Method C. Colourimetry. The methoxygroups of ubiquinone develops a blue colour in alkaline solution with ethylcyanoacetate (Cravens test). 1 ml ethylcyanoacetate was added to 2ml of the sample extract in ethanol, followed by 1 ml 0.8N Triton B. The blue colour was measured after 10 min on a Gilford spectrophotometer at 630nm (Redalieu & Folkers, 1971). The amount of ubiquinone 10 was determined by preparing a standard curve from pure ubiquinone (Correlation coefficient r > 0.998). RESULTS AND DISCUSSION
Freeze-drying of the samples was practical because of the high water content in many of the marine invertebrates. Methanol was used instead of ethanol in the saponification step to avoid artifacts of ubiquinone (Linn et al., 1960). Overall recovery of added ubiquinone 10 to a beef heart preparation was 87.7 _ 11.2~o(S.D, n = 8). Based on a pure solution of ubiquinone 10 (Sigma), method A gave a recovery of 102.3~ + 0.6, method B 94.2~ ___ 3.1 and method C 106.3~o __+ 3.1 (S.D., n = 6). The three methods gave comparable results on a purified extract from beef hearts. Method A, spectrophotometry with geometrical curve correction should be used only when the calculated correction is below 30%. This method failed on some samples high in fat. Method B, spectrophototnetry before and after reduction, gave approx 8% lower results compared with methods A and C. This effect disappeared in biological material with low ubiquinone concentrations. Method B gave unreliable results on samples of liver due to interfering substances. Some of these problems could be corrected by an additional preparative TLC separation (see Methods). Method C, colourimetry, could be used in all cases. This method has a low detection limit, but demands a good preseparation to get results comparable with methods A and B.
Results are given in Table 1. Pennock t1966) found small amounts of ubiquinone in marine invertebrates. Among these were the following values: Patella t:ulgata 4 pg/g wet wt, Asteria rubens 3.5 pg/g, Nephrops norvegicus 2.2pg/g. Table 1 shows corresponding values. All three methods gave comparable results within narrow limits on the invertebrate samples. Thin layer partition chromatography showed only one spot for ubiquinone 10 in all invertebrate samples. A sea anemone (Bolocera tuediael was negative for ubiquinone, probably due to interferevce from a high content of wax esters in this sample. The values found for ubiquinone in mackerel and saithe hearts were l l6pg/g and 61pg/g respectively. These values were comparable to those found for beef and sheep heart, 77 and 199pg/g respectively. Diplock & Haselwood (1967) have found corresponding values. Small amounts of ubiquinone 9 could be detected in the saithe heart and red muscle samples. Ubiquinone 9 was detectable only when applying large amounts of ubiquinone to the TLC and was estimated to be a maximum of 5~,~ of the total ubiquinone content. Whistance et aL 11971) found bolh ubiquinone 9 and 10 in animal hearts, and also ubiquinone 8 could be detected in some cases. Values for ubiquinone in the red muscle varied from 24.2/~g/g (mackerel) to 82.5/~g/g isaithe) and in white muscle from 1.8/~g/g (saithe) to 3.6/~g/g/mackerel). Method B could not be used for the red muscle samples of mackerel. The ubiquinone content in fish red muscle was 7 45 times higher than that found in white muscle. The red muscle of saithe had a ubiquinone content even higher than that found in the heart of this fish species. Red muscle cells have numerous mitochondria and store more energy rich materials as glycogen and lipid than the white muscle fibres (Patterson & Goldspink, 1972). It is therefore not surprising to find a high concentration of ubiquinone in red muscle tissue. Cytochrome oxidase was found to have about 8 times higher activity in red muscle than in the white muscle of carp (Johnston et aL, 1977). It seems probable that red muscle is active during slow movements and obtains its energy from oxidative phosphorylation. whereas the white muscle is active during vigorous movements and gets its energy from anaerobic glycolysis (Johnston & Goidspink, 1973). A preliminary tissue fractionation of saithe red muscle tissue showed that ubiquinone was enriched to a higher degree in the mitochondria fraction than the marker enzyme glutamate dehydrogenase GDH (E.C. 1.4.1.2). 40~o of the total ubiquinone content was found in the mitochondria fraction. This is almost the same value that Jayerman & Ramasarma t1963! found for rat liver mitochondria, 40-60~. The ubiquinone content in fish livers will probably be influenced by such factors as season, sex and nutritional status. Mackerel liver samples from autumn and spring had greatly different ubiquinone values (4.76/~g/g and 40.0/~g/g). Diplock & Haselwood (1967) found 22.0/~g/g in mackerel (Scomber scrombus) liver, 106.2 #g/g in saithe (Pollachius t,irens) liver and 62.1 pg/g in cod (Gadus morhua) liver, whereas Penhock et al., 1962, reported 8.6 #g/g in cod (Gadus morhua) liver. According to the values presented, as well as from
397
Ubliquinone analyses in fish tissues and in some marine invertebrates Table 1. Analysis by three methods of the ubiquinone contents in some marine invertebrates and fish species. Method A--Spectrophotometry with geometrical curve correction. Method B--Spectrophotometry before and after reduction. Method C--Colorimetry after Craven. Average dry wt (g/kg)
Samples (No. of analyses) Invertebrates: Horse mussel (2) Modiolus modiolus Blue mussel (2) Mytilus edulis Crab (2) Cancer pagurus Sea urchin (1) Echinus acutus Star fish (2) Asteria rubens Fish species: Mackerel Scomber scombrus heart (6) red muscle (6) white muscle (4) liver, autumn (4) liver, spring (2) Saitbe Pollachius virens heart (3) red muscle (4) white muscle (4) liver (2) Halibut Hippoglossus hippogloossus red muscle (2) white muscle (2)
A
Analytical method B (mg/kg dry wt)
C
Average of A+ B+ C (mg/kg wet wt)
204
7.9
7.5
7.3
1.54
238
16.9
16.9
16.8
4.01
256
15.4
15.5
16.0
4.00
144
38.3
38.8
38.1
' 5.35
258
10.0
9.4
10.9
2.61
244 535 468 265 294
482 45.4 ----
448 19.7 --~" 18.0 118
500 45.2 7.7 17.9 154.0
116 24.2* 3.60 4.76 40.0
180 227 219 738
352 375 8.8 --
318 342 --t --
347 373 8.0 12.9
61.0 82.5 1.84 9.52
278 230
160 9.6
162 --~"
162 8.0
44.9 2.02
* This value is based on method A and C. ? Insufficient material for analysis.
literature reports, ubiquinone contents may vary greatly in tissues of marine organisms. It seems necessary, therefore, to check both identification and quantity of ubiquinone by two independent methods. The methods compared in the present report identify ubiquinone by its absorption curve in u.v.-light, by its redox properties, and by the presence of methoxy groups. REFERENCES CRANE F. L. & BARR R. (1971) Determination of ubiquinones. Meth. Eazym. 18C, 137-169. DIPLOCK A. T., GREENI., BUNYANl. & METTALED. (1966) The analysis of u-tocopherol and ubiquinone in rat liver. Br. J. Nutr. 20, 95-I01. DlPLOCK A. T. & HASELWOOD,G. A. D. (1967) The ubiquinone content of animal tissues. Biochem. J. 104, 1004-1010. J^'~RM^N I. & R^~tASARrO,T. (1963) Intracellular distribution of coenzyrne Q in rat liver. Archs Biochem. Biophys. 103, 258-266. JOHnS'tON I. A. & GOLI)SPINK G. (1973) A study of glycogen and lactate in the myotomal muscles and liver of the coalfish (Gadus virens L.) during sustained swimming. J. mar. biol. Ass. U.K. 53, 17-26.
JOHNSTON I. A., DAVISON W. & GOLDSPINK G. (1977) Energy metabolism of carp swimming muscles. 2. Comp. Physiol. 114, 203-216. L ~ B. O., TRENI~ER N. R., ARISON B. H., WESTON R. G, Sh'UNK C. H. & FOLr~RS K. (1960) Coenzyme Q. XlI. Ethoxy homologs of coenzyme QI0. Artifact of isolation. 2. Am. chem. Soc. 82, 1647-1652. MERVYN L. & MORTON R. A. (1959) Unsaponifiable fraction of lipid from normal and diseased human kidney. Biochem. 2. 72, 106. MULDER F. I. & KEUNm K. J. (1961) Spectrophotometric assay of ~-tocopberol. Recueil 80, 1029-1039. PATTERSON S. & GOLDSPlNK G. (1972) The fine structure of red and white myotomal muscle in a marine teleost, the coalfish (Gadus virens). Z. Zellforsch. mikrosk. Anat. 133, 463-474. I~NNOCK J. F. (1966) Occurrence of vitamine K and related quinones. Vitams Hot. 24, 307-330. PENNOCKJ. F., MORTONR. A., LAWSOND. E. M. & LAIDMAN D. L. (1969) Quinones and related compounds in ~ h tissues. 8iochem. J. 84, 637-640. REDALIEUE. & FOLKEILSK. (1971) Assay of coenzyme QI0 in blood. Meth. Enzym. 18C, 179-181. WmSTANC~ G. R., FmLD F. E. & THRELFALLD. R. (1971) Observations on the biosyntheses of ubiquinones by animals. Eur. J. Biochem. 18, 46-52.