Adult controls and patients with methylmalonic aciduria or propionic acidaemia were given either 100 or 400 mg of. L-carnitine/kg body weight orally or 40 mg/kg ...
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616th MEETING. LONDON object of reducing the accumulation of acyl-CoA intermediates by removing acyl groups as acylcarnitine conjugates. We have studied the changes in urinary (acyl) carnitine excretion before, during and after such treatment. Adult controls and patients with methylmalonic aciduria or propionic acidaemia were given either 100 or 400 mg of L-carnitine/kg body weight orally or 40 mg/kg intravenously. The excretion of free and acylcarnitines and other organic metabolites was monitored using H-n.m.r. spectroscopy (Iles et al., 1985). This technique allows the simultaneous measurement of all urinary metabolites in the millimolar range that contain at least one C-H bond regardless of molecular charge. Spectroscopy was carried out using either a Bruker AM 360 MHz or WH 400 MHz spectrometer. Samples of urine (0.5 ml) were mixed with 0.05 ml of D,O and 0.02 ml of 3-(trimethylsilyl)-2,2,3,3-tetradeuteropropionate (TSPd,; 500mmol/l) to act as a chemical shift reference standard. They were then run at room temperature using either a single-pulse technique (100-400 45" pulses; pulse recycle time 2 s or 5 s ) or the Hahn (9Ooz-18O0z) spinecho pulse sequence (z = 60ms). The water signal was suppressed by pre-saturation. Each spectrum took 5-10 min to accumulate. The appearance of acylcarnitine (propionyl) in the urine in the patients was rapid, less than an hour, whether carnitine was administered orally or intravenously, suggesting that intramitochondrial carnitine acyltransferase competes well for the propionyl-CoA compared with other pathways. Little reduction in the rate of disposal of propionate via the other major routes (3-hydroxypropionate and propionylglycine) occurred in the patient with propionic acidaemia at the 100 mg/kg dose. Administration of a further 400 mg of
'
carnitine/kg to a patient with methylmalonic aciduria on a daily lOOmg/kg regimen, resulted in a much greater excretion of propionylcarnitine (Fig. 1). In addition, acetylcarnitine excretion occurred (Fig. 1) and these changes were accompanied by a rapid fall in alternative routes of propionate disposal (mainly as methylmalonate). This indicates in the latter case that propionylcarnitine is now a major contributor to propionate excretion and that intracellular propionyl-CoA has been significantly lowered. Over 90% of the intravenous carnitine dose was recovered from the patients' urine as free and esterified carnitine, indicating that little, if any, is retained or degraded. However, only 10% of the oral dose appears in the urine, which indicates losses of carnitine in the gut. There was, however, no evidence in the n.m.r. spectra of urinary excretion of trimethyl derivatives arising from carnitine degradation (Bremer, 1983).
Bremer, J. (1983) Physiol. Rev. 63, 1420-1480 Chalmers, R. A. & Lawson, A. M. (1982) Organic Acids in Man; The Analyticul Chemistry, Biochemisiry and Diagnosis of the Organic Acidurias, Chapman and Hall Ltd., London Chalmers, R. A,, Roe, C. R., Stacey, T. E. & Hoppel. C. L. (1984) Paediatr. Res. 18, 1325 1328 Iles. R. A,. Hind, A. J. & Chalmers, R . A. (1985) Clin. Chem. 31, 1795.- 1801
Roe, C. R., Hoppel, C. L.. Stacey, T. E., Chalmers, R. A., Tracey, B. M . & Millington, D. S. (1983) Arch. Dis. Child. 58. 916920 Rosenberg, L. E. (1983) in The Meiubolic Basis of Inherited Disease (Stanbury, J. B., Wyngaarden, J. B., Frederickson, D. S., Goldstein, J. L. & Brown, M. s.,eds.), pp. 474-497, McGraw-Hill Book Co., New York and London
The mechanism of inhibition of mitochondrial fatty acid oxidation by mercaptoacetate: inhibition of acetoacetyl-CoA, 2-methylacetoacetyl-CoA and 3-oxoacyl-CoA thiolases R. KEITH VEITCH,* H. STANLEY A. SHERRATT,* ANTHONY G. CAUSEY,? KIM BARTLETTt and BRUCE MIDDLETON$ *Department qf Pharmacological Sciences and ?Department of Child Health and Clinical Biochemistry, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4 H H , U . K . , and $Department of Biochemistry, Queens Medical Centre, University of Nottingham, Nottingham NG7 2UH, U.K.
Mercaptoacetyl-CoA, formed in vitro when coupled mitochondria are incubated with mercaptoacetate (thiolacetic acid), or in vivo after intraperitoneal administration of mercaptoacetate, inhibits P-oxidation (Bauche et al., 1981, 1982). Inhibition of palmitoyl-carnitine was demonstrated in the presence of rotenone and 10 mM-oxaloacetate, when this was followed spectrophotometrically at 425-475 nm by reduction of ferricyanide as final electron acceptor at the level of cytochrome c by the method of Osmundsen & Bremer (1977). It was considered that the site of inhibition was palmitoyl-CoA dehydrogenase (Bauche et al., 1981). However, although with these conditions ferricyanide is reduced by electrons originating solely from acyl-CoA dehydrogenase activity, the P-oxidation sequence is proceeding and electrons originating from the activity of 3-hydroxyacylCoA dehydrogenase are transferred to oxaloacetate by malate dehydrogenase in the matrix (Osmundsen & Bremer, 1977). It cannot therefore be concluded that the site of inhibition of j-oxidation by mercaptoacetyl-CoA is necessarily palmitoyl-CoA dehydrogenase. Vol. 14
Fasted male albino rats (200g) were given mercaptoacetate (40pmo1/100g body wt.) or 0 . 1 4 ~ - N a C I intraperitoneally and after 3 h liver mitochondrial fractions were prepared in 0.3 M-mannitol/lO rnM-Hepes/o. 1 mM-EGTA, pH 7.2. Mitochondria1 oxidations were measured polarographically (Turnbull et al., 1984). It was confirmed that the State-3 rate of oxidation of succinate was uninhibited, but that the oxidation of palmitoyl-carnitine in the presence of either 1 mwmalate or 1 mwmalonate was inhibited by about 77% or by 50% respectively (Table 1). The mitochondrial fractions were extracted with 0.8 M-KCI/IOmMphosphate/100 ~ M - F A D / O . ~(v/v) % Triton X-100, pH 7.4, and the extract was centrifuged and washed with peroxidefree ether and used for assays of acyl-CoA dehydrogenase activities (Furata et al., 198I), using phenazine methosulphate instead of phenazine methosulphate. The other enzymes of P-oxidation were assayed essentially according to Holland et al. (1973) and Middleton & Bartlett (1983) in extracts of mitochondria made using 50 m~-phosphate/0.2% (v/v) Triton X-lOO/l .O mM-dithiothreitol, pH 7.4. The activities of butyryl-CoA, octanoyl-CoA and palmitoyl-CoA dehydrogenases, citrate synthase and enoyl-CoA hydratase were similar in both the control and mercaptoacetate-treated groups (Table 1). However, 3-oxoacyl-CoA, acetoacetyl-CoA and 2-methylacetoacetyl-CoA thiolases were inhibited by about 75% and 3-hydroxybutyryl-CoA dehydrogenase by about 33% (Table 1). These inhibitions were apparently irreversible and persisted in very dilute mitochondrial extracts. The results gave no evidence for inhibition of acyl-CoA dehydrogenase activity, although
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BIOCHEMICAL SOCIETY TRANSACTIONS Table 1 . Substrate oxidation by, and enzyme activities in, liver mitochondria1,fractionsfrom rats treated with mercaptoacetate or 0.14 M-NaCl Results are means S.E.M. The oxidations were measured in State-3 at 30°C and are expressed as ng-atom O/min per mg of protein. Enzyme activities were measured at 25°C and are expressed as pmol/min per mg of protein. Thiolase activities activities were measured in the presence of 10 mM-KCI. Significance of differences from controls: * P < 0.05; **P < 0.01. Acyl-CoA substrate Oxidations 10mM-Succinate 20pmol of palmitoylcarnitine plus 1 mMmalate 20 pmol of palmitoylcarnitine plus 5 mMmalonate Enzyme activities Citrate synthase Acyl-CoA dehydrogenase Acyl-CoA dehydrogenase Acyl-CoA dehydrogenase Enoyl-CoA hydratase 3-H ydroxybutyryl-CoA dehydrogenase Acetoacetyl-CoA thiolase 3-Oxohexyl-CoA thiolase 2-Methylacetoacetyl-CoA thiolase
-
Mercaptoacetate-treated (n = 4)
Inhibition
153 & 9 63 & I I
150 k 2 5 I5 k 5**
2 77
k 27*
51
83 f 13
-
Acetyl-CoA Butyryl-CoA Octanoyl-CoA Palmitoyl-CoA Crotonyl-CoA Acetoacet yl-CoA
Controls (n = 4)
51 18.6 13.8 9.7 17000 1840
Acetoacetyl-Co A 3-Oxohexyl-CoA 2-MethylacetoacetylCoA
reversible inhibition by high concentrations of mercaptoacetyl-CoA which might occur in the mitochondria1 matrix is possible. The inhibition of j-oxidation of palmitoylcarnitine observed may be explained by the inhibition of the thiolases and of 3-hydroxybutyryl-CoA dehydrogenases. Quandt & Huth (1984) found that incubation of acetoacetylCoA thiolase with CoASH led to partial inactivation; CoASH combines with an -S.S- link to form HS-enzymeSSCoA complexes with less activity, and acetoacetyl-CoA thiolase partly occurs in vivo as such complexes. It is possible that mercaptoacetyl-CoA may react similarly forming less active HS-enzyme-S.S.CH,CO-CoA complex with both acetoacetyl and 3-oxoacyl-CoA thiolases. Mitochondrial fractions were incubated with 1 mM[U-'4C]-3-methyl-2-pentanoate (ketoisoleucine) and the ''C-labelled products analysed by h.p.1.c. (Causey et al., 1986). The I4C-labelledcompounds detected were 2-methylbutyryl-CoA, 2-methylbut-2-enoyl-CoA, propionyl-CoA, acetyl-CoA, succinyl-CoA and methylmalonyl-CoA. The amounts of labelled intermediates in mitochondria fractions from mercaptoacetate-treated and control rats were in the normal range (Causey et al., 1986). No labelled 2-methyl3-hydroxybutyryl-CoA or 2-mercaptoacetyl-CoA were detected in any sample. These results suggest that the remaining activity of 2-methylacetoacetyl-CoA thiolase does not appear to be rate-limiting when 3-methyl-2-
& 5 k 5.6 & 3.5 f 2.5 & 2,900 k 225
307 k 51 242 38 948 +_ 182
42
("/I
43 f 21 20.4 k 4.6 14.7 k 1.9 9.9 k 1.8 17500 f 4,100 1227 k 448*
33
75 f 14** 38 f 12** 283 41**
76 77 70
16
oxypentanoate is the sole substrate in mitochondria t'rom mercaptoacetate-treated rats. Alternatively, metabolism of 3-methyl-2-pentanoate beyond the stage of 2-methyl-2enoyl-CoA could involve an enzyme insensitive to mercaptoacetate or mercaptoacetyl-CoA. Mitochondrial fractions from mercaptoacetate-treated rats were incubated with 25 pM-octanoyl-CoA plus 1 mM-L-carnitine. These showed decreased concentrations of acetyl-CoA compared with controls consistent with decreased activities of the 3-oxoacyl-CoA thiolases. We thank the Wellcome Trust for financial support. Bauche, F., Sabourault, D., Guidicelli, Y., Nordmann. J. & Nordmann, R. (1981) Biochem. J. 1%, 803-809 Bauche, F., Sabourault, D., Guiducelli, Y.. Nordmann, J. & Nordmann, R. (1982) Biochem. J. 206, 53-59 Causey, A. G . . Middleton, B. & Bartlett. K . (1986) Biochem. J . 235, 343-350 Furata, S., Miyazawa, S. & Hashimoto, T. (1981) J. Biochem. 90, 1739- 1750 Holland, P . C., Senior, A. E. & Sherratt, H. S. A. (1973) Biochm J . 136, 173-184 Middleton, B. & Bartlett, K. (1983) Clin. Chem. Actu 128, 291 -305 Osmundsen, H. & Bremer, J. (1977) Biochem. J . 164, 621433 Quandt, L. & Huth, W. (1984) Biochim. Biophys. Actu 784. 169 176 Turnbull, D. M., Bartlett, K., Younan, S. I. M. & Sherratt. H. S. A. (1 984) Biochern. Phurmucol. 33, 475-481.
Intrasynaptosomal free calcium and potassium-depolarization R. H. ASHLEY* School of Biological Sciences, University of Sussex, Falmer, Brighton BNI 9QG, U.K. Abbreviations used: [CaZf],, intracellular concentration of free Ca2+; [Ca2+],, extracellular concentration of free Ca" ; [KCI],, extracellular concentration of KCI. *Present address: The Cardiac Department, Cardiothoracic Institute, 2 Beaumont St, London W I N 2DX, U.K.
The temporal resolution offered by the chemiluminescent detection of synaptosomal acetylcholine release (Israel & Lesbats, 1981) has recently been matched by similar advances in the monitoring of intrasynaptosomal free Ca2+ (Heinonen et al., 1985). While probes like arsenazo 111 (op. cit.) remain tricky to introduce, and their responses difficult to calibrate, these are the very strengths of the fluorescent indicator quin2 (Tsien er a/., 1982). However, at the near millimolar intracellular concentrations required, 1986
