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Table 1. Oxidation of isomers of a-pherrylethanolamine by mitochondria1 monoamine oxidase Enzyme activity was measured by measuring O2 uptake at pH 7.4 and at 30°C. Substrate concentrations were 3 mM. O2 uptake (nmol/h per mg of protein) in the presence of: /I-Phenylethanolamine isomer L(+)
N o inhibitor 36.5 75 48
Clorgyline (100nM) Deprenil (1 PM) 35.5 17 36 60
was similarly used to bIock the B enzyme, a similar decrease in the rate of oxidation of the L(+)-isomer was observed, with a lesser effect on the other optical isomer. This suggests that monoamine oxidase A acts only on D(-)-B-phenylethanolamine, whereas the B enzyme will oxidize both D(-)- and L(+)-forms. This apparent optical specificity is particularly noteworthy because the D(-)-isomer has the same configuration as the naturally occurring laevorotatory form of noradrenaline, itself a substrate for monoamine oxidase. It is not clear from previous reports whether other phenylethanolamines such as adrenaline, noradrenaline and their 0-methyl derivatives show the same behaviour, since most studies of the optical specificity of monoamine oxidase were carried out before the multiple nature of the enzyme was established. There are reports that (-)noradrenaline is oxidized more readily than is the d-form by monoamine oxidase from several different tissues (Blaschko et a/., 1937; Pratesi & Blaschko, 1959; Giachetti & Shore, 1966). These findings agree with those of the present study, where the absolute specificity of monoamine oxidase became apparent only when the B enzyme was partly inhibited. The present findings do not reveal why /?-phenylethanolamine does not conform to the specificity criteria outlined earlier, i.e. why it acts as a substrate at all. In delineating the specificities of monoamine oxidases A and B, Houslay & Tipton (1974) pointed out the anomalous behaviour of 5-hydroxytryptamine. It now appears that a-phenylethanolamine also fails to fit into the general pattern that they described. Blaschko, H. Richter, D. & Schlossman, H. (1938) Biochem. J. 31,2187-2196 Giachetti, A. &Shore, P. A. (1966) Life Sci. 5, 1373-1378 Hawkins, J. ( I 952) Biochem. J. 50,577-58 1 Houslay, M. D. & Tipton, K. F. (1974) Biochem. J. 139,645-652 Pratesi, P. & Blaschko, H. (1959) Br. J . Pharrnacol. 14,256-260 Williams, C . H. (1974) Biochem. Phnrmacal. 23,615-628
A Sensitive Quantitative Assay Method for Dolichols, Cholesterol and Ubiquinone using High-pressure Liquid Chromatography 1. ANTHONY TAVARES, NEIL J . JOHNSON and FRANK W. HEMMING
Department of Biochemistry, University Hospital and Medical School, Nottingham NG7 2UH, U.K.
There is currently no satisfactory sensitive assay for polyisoprenoid alcohols. In the past these compounds have been assayed either by weighing purified samples or by spot size on t.1.c. (e.g. Butterworth & Hemming, 1968). Donnahey & Hemming (1975) described an assay procedure based on high-pressure liquid chromatography in which a refractiveindex detector was used. I n this procedure individual ficaprenols could be separated VOl.
BIOCHEMICAL SOCIETY TRANSACTIONS
completely from isoprenologues and assayed individually in the 2OOpg-5 mg range. Individual dolichols from pig liver were incompletely separated from their isoprenologues, but could be assayed as a mixture at about the same order of sensitivity. The use of a variable-wavelength U.V. monitor at a wavelength (210nm) approaching the absorption peak for isolated double bonds affords a much more sensitive assay procedure for these compounds. Results of applying this procedure are described below. In addition the use of the method for the assay of ubiquinone and cholesterol in the same extracts of unsaponifiable lipid is described. High-pressure liquid chromatography was performed on Waters Associates equipment comprising a solvent delivery system, model 6000A. an injection system, model U6K, and a reverse-phase column (300mmx 3.9mm, initially 5120 theoretical plates according to Waters recommended test procedure) of p-Bondapak C18/Porasil. The eluting solvent was monitored in a Pye-Unicam variable-wavelength u.v.-detector, model LC3. A natural mixture of pig liver dolichols (dolichols-17, -18, -19, -20 and -21) were successfully separated into the various components in an eluting solvent of methanol/ propan-2-ol/hexane/water(30 :65 :5 :1, by vol.) at 1ml/min. The U.V. detector was used at low wavelength (210nm) for detection of isolated double bonds. Dolichols-17, -18, -19, -20 and -21 had retention times of 8.8,9.8, 1I .O, 12.2 and 13.6min (to 1.3min) with bandwidths of O.8,l .l, 1.4,1.5 and 1.6min respectively. The mixture contained 2.5,19.8, 43.5, 28.6 and 5.5 % of these isoprenologues respectively. A calibration curve of total peak height against quantity (ng) injected, was linear in the 50-500ng range. The lowest amount of accurately measurable dolichols was 50 ng, whereas the minimum detectable amount was long. The coefficient of variation on five repeated injections was 1.9 % at the 500ng level. Yeast dolichols (-1 3, -14, -15, -16, -1 7) were also separated into the variouscomponents (respectively 3.0, 14.1, 43.5, 34.5 and 4.8% of the mixture) by using the above system. Dolichol-13, -14, -15, -16 and -17 had retention times of 5.8, 6.4, 7.0, 7.7 and 8.8min (to 1.3min) and bandwidths of 0.45,0.6,0.8,0.8 and 0.8min. With all of these dolichols and a column of above 5000 theoretical plates, it is possible to achieve complete separation of each isoprenologue. The improvement in this respect over the system described by Donnahey & Hemming (1975) may be due in part to use of a different solvent and to use of a more sensitive detector which allows smaller quantities to be injected on to the column. Cholesterol was eluted with a mixture of propan-2-01 and methanol (1 :9, v/v) at 1ml/min and monitored at 210nm on the U.V. detector. It gave a single peak with a retention time of 7.0min (to 0.9min) and a bandwidth of 1.3min. A calibration curve of peak height against quantity injected was linear in the 20-2000ng range. The minimum level of detection was approx. long and the lowest level of accurately measurable cholesterol was 20ng. The coefficient of variation on five repeat injections was 1.8 % at the 1 pg level. Ubiquinone-9 was eluted with a mobile phase of propan-2-ol/methanol (3 :7, v/v) at 1.4ml/min and assayed by using the U.V. detector at 275 nm. Ubiquinone-9 was eluted as a single peak with a retention time of 7.8min (to 0.8min; ubiquinone-10 retention time 9.9min) and a bandwidth of 1.4min. A straight line was obtained on plotting peak height against quantity injected, in the 10-1000ng range. The minimum amount detectable was 2.5ng. The lowest amount of accurately measurable ubiquinone-9 was long. The coefficient of variation on five repeat injections was 3.9% at the 15ng level. With these chromatographic systems the dolichols, cholesterol and ubiquinone-9 of rat liver were assayed. The unsaponifiable lipid of the liver was recovered (e.g. Burgos et al., 1963) dissolved in propan-2-01 at a suitable dilution and a sample (2-1Op1) was injected directly on to the column for assay. Dolichols-16, -17, -18, -19, -20 and -21 were present at an overall concentration of 21.5pg/g wet wt. of liver, the percentage composition of the mixture being 1.0, 16.2, 56.8, 23.9 and 2.0 respectively. Cholesterol was shown to be present at 1.97mg/g wet wt. of liver and ubiquinone-9 at a concentration of 74pg/g wet wt. of liver. 1977
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Prior partial purification of these compounds on alumina was shown to offer no advantage to the method. There was no evidence of contamination interfering with the assay. Losses of the compounds during the chromatography were shown to be negligible. The procedure is simpler and more sensitive than the spectrophotometric assay for ubiquinone (Crane et al., 1959) and is probably more reliable in that the compound is separated from contaminants and also that different isoprenologues can be assayed separately. The cholesterol determination is also a little more sensitive and simpler than the usual colorimetric methods (e.g. Rodnight, 1957) and involves separation from at least someinterfering materials. However, the concentrationofcholesterol in most tissues is sufficiently high for the colorimetric assays to suffice in most instances. In this situation the main advantage of high-pressure liquid chromatography is its convenience and rapidity, especially if other assays are being carried out by the method. The above values indicate that the procedure is capable of assaying these lipids from very small samples of tissue or cell fractions. It also appears to be useful for measuring concentrations of dolichol phosphate by a modified extraction and chromatographic met hod. The work was supported by a Project Grant from the Medical Research Council. Burgos, J., Hemming, F. W., Pennock, J. F. & Morton, R. A. (1963) Biochem. J . 88,470-482 Butterworth, P. H. W. & Hemming, F. W. (1968) Arch. Biochem. Biophys. 128,503-508 Crane, F. L., Lester, R. L., Widmer, C. & Hatefi, Y. (1959) Biochim. Biophys. Actu 32, 73-82 Donnahey, P. L. & Hemming, F. W. (1975) Biochem. SOC.Trans. 3,775-776 Rodnight, R. (1957) J. Neurochem. 1,207-216