a-oxidation of Stearic Acid in the Normal, Starved and Diabetic Rat Liver

Transcription

1 Eur. J. Biochem. 40, (1973) a-oxidation of Stearic Acid in the Normal, Starved and Diabetic Rat Liver Ingemar BJORKHEM Department of Chemistry, Karolinska Institutet, Stockholm, Sweden (Received May 21/August 16, 1973) w-oxidation of stearate, catalyzed by x g supernatant fluid of rat-liver homogenate in the presence of an NADPH-generating system, was stimulated three- to seven-fold by starvation or diabetes. The soluble fraction of liver homogenate was responsible for the stimulation. Thus, addition of x g supernatant fluid of liver homogenate from starved rats stimulated microsomal wl-oxidation of stearate two- to three-fold whereas addition of x g supernatant fluid of liver homogenate from untreated rats had no effect or an inhibitory effect. Evidence was obtained to indicate that the stimulatory effect of the x g supernatant fluid from liver of starved rats was due to the presence in this fraction of liver alcohol dehydrogenase and NAD. The role of alcohol dehydrogenase might be to protect the tui-hydroxylase from product inhibition by oxidation of the 18-hydroxystearic acid formed. The effect of x g supernatant fluid from liver of untreated rats was shown to be due to an inhibition of the ol-hydroxylase reaction and not of the alcohol dehydrogenase reaction. The inhibitory effect of the ioo000 x g supernatant fluid was retained after boiling for 5 min but was partially destroyed after preincubation at 37 "C or after repeated freezing and thawing. The inhibition was shown to be at least not mainly due to interference with formation of stearoyl-coa from stearic acid. Stearoyl-CoA was wi-hydroxylated by the microsomal fraction or by the x g supernatant fluid at a rate about half that of free stearic acid. The possibility is discussed that the low extent of ol-oxidation of long-chain fatty acids observed under normal conditions might be partially due to the presence of the inhibitor(s) in the soluble part of the cell and that disappearance of this inhibition under conditions of starvation and diabetes might be of physiological importance. The microsomal fraction of mammalian liver fortified with NADPH catalyzes ol- as well as w2- hydroxylation of various fatty acids [l-41. ol-hydroxylation is more important, quantitatively, than w2-hydroxylation. The wl-hydroxylated fatty acids can be further oxidized into the corresponding dicarboxylic acids by enzymes in the soluble as well as the microsomal fractions of a rat liver homogenate [4,5-71. It was shown recently that alcohol dehydrogenase is involved in the oxidation of long-chain wl -hydroxy fatty acids into the corresponding dicarboxylic acid in the soluble fraction of rat liver homogenate. The physiological importance of w-oxidation of fatty acids is not known. According to some experiments with liver slices, wl-oxidation accounts for less than 50/, of the total oxidation of stearic acid [8]. However, it is possible that w-oxidation of fatty acids increases under conditions when the over-all utilization of fatty acids is increased. Nomenclature. The carbon atom of the terminal methyl group is designated w1 and the neighboring carbon atom 02. Enzyme. Alcohol dehydrogenase or alcohol : NAD oxidoreductase (EC ). It has been reported in preliminary form that co-oxidation of stearic acid by the io000 x g supernatant fluid of rat liver is increased after starvation and diabetes [9]. No detailed information concerning the magnitude of this increase in available nor is it known whether the microsomal or the soluble fraction of liver homogenate is responsible for the increase in w-oxidation of stearate. In the present report the effect of starvation and diabetes on whydroxylation of stearate has been studied in different subcellular fractions of rat liver from untreated, starved and diabetic rats. EXPERIMENTAL PROCEDURE Haterials [1-14C]Stearic acid was obtained from the Radiochemical Centre (Amersham, England) and was diluted with unlabelled stearic acid to give a specific radioactivity of 20 pci/mg. The acid was purified by silicic acid chromatography [4]. [l-14c]stearoyl-coa (5 pci/mg) was prepared from [i-14c]stearate using

2 ~~~ ~ 416 w-oxidation of Stearic Acid the mixed anhydride procedure [lo]. NAD, NADP, ATP, CoA, isocitric acid dehydrogenase and horseliver alcohol dehydrogenase was obtained from Sigma Chemical Company (St. Louis, Mo.). The alcohol dehydrogenase had a specific activity of 1.6 U/mg protein. Rat-liver aldehyde dehydrogenase was prepared as described previously [7] and had a specific activity of about 30 U/mg protein with acetaldehyde as substrate. Methods White male rats of the Sprague-Dawley strain weighing about 200 g were used. Rats were starved for 72 h or treated with an intravenous injection of alloxan, 60 mg/kg body weight, 48 h before sacrifice. The procedure for preparation of a 20 /, (wlv) homogenate of liver in a modified Bucher medium [I21 and isolation of x g supernatant fluid, I00000 x g supernatant fluid and microsomal fraction were as described previously [4]. Microsomal protein was determined according to Lowry et al. [ll]. [1-14C]- Stearate, 100 pg dissolved in 50 pl acetone, was added to 1.5 ml microsomal suspension or x g supernatant fluid (corresponding to 6-8 mg microsomal protein) together with an NADPH-generating system (I31 in a total volume of 3 ml Bucher medium. In some cases, 0.35 mg [l-14c]stearoyl-coa dissolved in 0.3 ml water was added as substrate. In some experiments 1.5 ml xg supernatant fluid (corresponding to mg protein) 1 pmol NAD, 100 pg horse-liver alcohol dehydrogenase, 1 mg aldehyde dehydrogenase, 10 mg ATP or 3 mg CoA were added to the incubation mixture. Incubations were run at 37 "C for 15 min and were terminated by addition of 3 ml400/, potassium hydroxide in methanol (wlv). The mixture was refluxed for 30 min, acidified, and extracted once with diethyl ether and once with ethyl acetate. The combined ether and ethyl acetate extracts were washed with water until neutral and the solvent was evaporated. The recovery of radioactivity was about 85O/, and no evidence was obtained for selective losses in the extractions. The saponification was found to be necessary in the case of incubations with x g supernatant fluid and incubations with microsomal fraction fortified with I00000 x g supernatant fluid. If the saponification step was omitted in these cases only about half of the incubated radioactivity could be extracted with ether from an acidified water phase (cf. Results). After methylation with diazomethane aliquots of the material were analyzed by radio-gas chromatography as previously [4]. The extent of conversion was calculated assuming no dilution with endogenous substrate (cf. [4]). In most cases, the extent of wloxidation of stearate was expressed as the sum of the wl-oxidized products formed, i.e. 18-hydroxystearic acid and 1,18-octadecadioic acid. Radioactivity was Table 1. Effect of starvation and alloxan diabetes on wl-oxidation of stearate by microsomal fraction and x g supernatant fluid of rat-liver homogenate The figures given are the mean of 4 experiments with standard deviation of the mean. ol-oxidation was expressed as the sum of 18-hydroxystearic acid and 1,18-octadecadioic acid formed. Incubations were 15 min ol-oxidation of stearate by wl-oxidation of stearate in Control Starved Diabetic nmol/mg microsomal protein Microsomal fraction 2.3 f & x g supernatant fluid 1.5 f & & 0.3 assayed with a methane gas-flow counter and 1 pci 14C corresponded to 1.0 x lo6 countslmin. RESULTS Incubations of Stearic Acid with Microsomal Fraction and x g Supernatant Fluid of Homogenate of Liver from Untreated, Starved and Diabetic Rats Table I summarizes results of experiments in which [1-14C]stearate was incubated with the microsomal fraction and the x g supernatant fluid of homogenate of liver of untreated, starved and diabetic rats. The dicarboxylic acid accounted for about 50 /, of the total ol-oxidized products in experiments with the microsomal fraction and /, in experiments with the x g supernatant fluid. Starvation and diabetes had no effect on wl-oxidation of stearate when the microsomal fraction was used as enzyme source. However, when the x g supernatant fluid was used as enzyme source, ol-oxidation of stearate was stimulated about four-fold. The extent of ol-oxidation of stearate was significantly lower with the x g supernatant fluid from untreated rats than with the microsomal fraction from the same rats. Incubations of Stearic Acid with Mixtures of Microsomal Fraction and x g Supernatant Fluid from Liver Homogenate of Untreated and Starved Rats The results of the experiments shown in Table I suggest that the soluble fraction of homogenate of liver from starved rats is able to stimulate microsomal ol-hydroxylation of stearate. This was confirmed by the experiments summarized in Table2. In all these experiments the dicarboxylic acid accounted for 80 /, or more of the total amount of ol-oxidized products formed. Addition of I00000 x g supernatant fluid from homogenate of liver of starved rats stimu-

3 I. Bjorkhem 417 Table 2. Effect of addition of xg supernatant fluid from livers of starved or untreated rats on wl-oxidation of stearate by the microsomal fraction of rat liver The figures given are obtained from one single series of experiments. Three additional series of similar experiments were done which all gave essentially the same results. wl-oxidation was expressed as the sum of 18-hydroxystearic acid and 1,18-octadecadioic acid formed. The amount of microsomal fraction and xg supernatant fluid used was 1.5 ml (cf. Experimental Procedure). No wl-oxidation of stearate occurs in the xg supernatant fluid of a liver homogenate [l-41. Incubations were 15 min Microsomes from starved rats 2.9 Microsomes from untreated rats 3.2 Microsomes from starved rats x g supernatant fluid from starved rats 6.5 Nicrosomes from untreated rats x g supernatant fluid from untreated rats 2.0 Microsomes from starved rats x g supernatant fluid from untreated rats 4.6 Microsomes from untreated rats xq supernatant fluid from starved rats 7.2 wl-oxidation of stearate nmol/mg protein Table 3. Effect of addition of x g supernatant fluid from livers of starved or untreated rats on wl-oxidation of stearate by x g supernatant of rat liver homogenate The figures given are obtained from one single series of experiments. Two additional series of experiments gave essentially the same results. wl-oxidation was expressed as the sum of 18-hydroxystearic acid and 1,18-octadecadioic acid formed. Incubations were 15 min x g supernatant fluid from starved rat x g supernatant fluid from untreated rat x g supernatant fluid from starved rat x g supernatant fluid from starved rat x g supernatant fluid from starved rat x g supernatant fluid from untreated rat x g supernatant fluid from untreated rat x g supernatant fluid from untreated rat x g supernatant fluid from untreated rat x g supernatant fluid from starved rat wl-oxidation of stearate nmol/mg protein lated microsomal ml-oxidation of stearate more than two-fold, regardless of whether the microsomal fraction was obtained from homogenate of liver of untreated or starved rats. Addition of 1OOOOOxg supernatant fluid from homogenate of liver of untreated rats has either a small inhibitory effect (with microsomes from untreated rats) or a small stimulatory effect (with microsomes from starved rats). The degree of inhibition of oj1-oxidation of stearate varied extensively ( /,) with different preparations of I x g supernatant fluid from untreated rats whereas the degree of inhibition with different preparations of microsomal fractions from untreated rats was about the same. The responses to the inhibition was about the same with microsomes prepared as described in Experimental Procedure and with microsomes prepared under conditions in which lipid peroxidation was reduced to a minimum [14]. Incubation of Stearic Acid with Mixtures of x g Supernatant Fluid and x g Supernatant Fluid from Homogenate of Liver of Untreated and Starved Rats The results in Table 3 show that I00000 xg supernatant fluid of homogenate of liver from untreated rats markedly inhibits ol-oxidation of stear- ate by 20000~9 supernatant fluid. The inhibition was most marked with x g supernatant fluid from livers of starved rats. In agreement with the results shown in Table 2, addition of x g supernatant fluid from homogenate of liver of starved rats had a slight stimulatory effect on the col-oxidation of stearate catalyzed by the x g supernatant fluid from homogenate of liver of starved rats. No stimulation was observed when x g supernatant fluid from homogenate of liver of untreated rats was used as enzyme source. Nature of Stimulating Factor in x g Supernatant Fluid of Homogenate of Liver from Starced Rats In the experiments summarized in Table 1-3, it was observed that stimulation of wl-oxidation of stearate by starvation ocurred only when the ratio between 1,18-octadecadioic acid and 18-hydroxystearic acid was high. This finding suggests that the stimulatory effect of the I00000 x g supernatant fluid of liver homogenate from starved rats might at least in part be due to an effect on oxidation of 18-hydroxystearic acid into 1,18-octadecadioic acid. The results given in Table 4 showed that this is the case. Heat treatment of the ioo000 x g supernatant

4 418 w-oxidation of Stearic Acid Table 4. Effect of addition of NAD, alcohol dehydrogenase, aldehyde dehydrogenase and x g supernatant fluid from homogenate of liver of starved rat on microsomal wl-oxidation of stearate The amount of ethanol dehydrogenase activity in the 100OOOxg supernatant fluid added was about 80mU when determined spectrophotometrically according to Dalziel [21]. Incubations were 15 min Microsomal fraction Microsomal fraction x g supernatant fluid from liver homognate 1.1 of starved rats Microsoma1 fraction + boiled xg supernatant fraction from liver 0.9 homogenate of starved rats 0.7 Microsomal fraction NAD 0.8 Microsomal fraction NAD horse-liver alcohol dehydrogenase 0.0 Microsomal fraction + NAD + horse-liver alcohol dehydrogenase + rat-liver aldehyde dehydrogenase 0.0 Microsomal fraction f NAD + horse-liver alcohol dehydrogenase xg supernatant fluid from liver homogenate of starved rats Microsomal fraction + NAD + horse-liver alcohol dehydrogenase + boiled x g supernatant fluid from liver homogenate of starved rats 18-Hydroxystearic 1,18-0ctadecadioic acid formed acid formed nmol/mg rnicrosomal protein fluid resulted in loss of about 75O/, of the stimulatory effect. This experiment indicates that a heat-labile factor is responsible for about 75O/, of the stimulation and a heat-stable factor for about 25O/,. The heat-stable factor might be identical with NAD, as addition of NAD increased wl-oxidation of stearate to about the same extent as boiled x g supernatant fluid. Furthermore the ratio between 1,18- octadecadioic acid and 18-hydroxystearic acid was about the same in the two sets of experiments (Table4). The heat-labile factor is probably identical with aicohoi dehydrogenase as addition of horse-liver alcohol dehydrogenase stimulated wl-oxidation of stearic acid to about the same extent as 10000O~g supernatant fluid from liver of starved rats (Table 4). In both these sets of experiments 1,18-0ctadecadioic acid accounted for more than 85O/, of the total wl-oxidized products formed. Conversion of 18-hydroxystearic acid into 1,18-octadecadioic acid should require aldehyde dehydrogenase in addition to alcohol dehydrogenase. Since no further stimulation of wl-oxidation of stearate was obtained when an aldehyde dehydrogenase preparation from rat liver was added it is apparent that the microsomal fraction contains sufficient amounts of aldehyde dehydrogenase. Addition of boiled x g supernatant fluid of liver homogenate from starved rats did not further increase wl-oxidation of stearate by the microsomal fraction in presence of horse-liver alcohol dehydrogenase and NAD (Table 4). Since wl-hydroxylation of stearate was stimulated under conditions of concomitant and efficient oxidation of 18-hydroxystearic acid into 1,18-0ctadecadioic acid, it is possible that the wl-hydroxylase reaction is subjected to product inhibition. In fact, addition of 18-hydroxystearic acid (15 pg) to a standard incubation mixture with microsomal fraction resulted in an inhibition of cvl-oxidation of stearate although the extent of inhibition (35O/,) was lower than might be expected. Further support for the contention that alcohol dehydrogenase is involved in 01 -oxidation of stearic acid by x g supernatant fluid of homogenate of liver of starved rat was obtained from experiments in which ethanol and pyrazole were used as inhibitors of the alcohol dehydrogenase activity (Table 5). Addition of ethanol as well as pyrazole inhibited the total conversion of stearic acid into wl-oxidized products, and this inhibition ocurred concomitant with an decrease of the ratio between 1,18-octedecadioic acid and 18-hydroxystearic acid. Fig.1 shows that the rate of microsomal 01-oxidation of stearate was linear with enzyme concentration up to about 7.5 mg microsomal protein and with time up to about 30min, regardless of whether NADPH-generating system alone was used or NADPH-generating system combined with NAD and liver alcohol dehydrogenase. Under both conditions 1OOpg stearate was sufficient to saturate the enzyme. Nature of Inhibitory Effect of x g Supernatant Fluid of Homogenate of Liver from Untreated Rats The microsomal fraction contains thiokinase catalyzing conversion of stearate into stearoyl-coa. The inhibition of wl-oxidation of stearate by the x g supernatant fluid from liver of untreated rats could conceivably be due to the possibility that this fraction contained more of the necessary cofactors for t'he thiokinase reaction than the corresponding fraction from starved rats, provided that stearoyl-coa is not a substrate for the wl-hydroxylase. To examine the possibility that there are differ-

5 I. Bjorkhem 419 Table 5. Effect of ethanol and pyrazol on ol-oxidation of (l-14c]stearate catalyzed by xg supernatant fluid of homogenate of liver of starved rats Incubations were 15 rnin 18-Hydroxystearic 1,18-octadecadioic acid formed acid formed nmol/mg microsomal protein x g supernatant fluid of liver homogenate of starved rats (Expt 1) ethanol 5 mm ethanol 20 mm et,hanol 100 mm + ethanol 400 mm 20000xg supernatant fluid of liver homogenate of starved rats (Expt 2) pyrazole 2.5 mm + pyrazole 5.0 mm A 20 B C s - 15 c 10 2 E > Y n n " I 120 Microsomal protein (mg) Time (rnin) Stearlc acid (kg) Fig. 1. Effect of enzyme concentration (A), time (B) and substrate concentration (C) on microsoml ol-oxidation of stearate. Except in (A) 7.5 me: microsomal protein was used. Assav conditions were as described in Experimental Procedure with either NADPH-generuating system alone (04) or NADPH-generating system together with i pmol of NAD and 100 pg liver alcohol dehydrogenase (0-0) ence in thiokinase activities, [ 1-14CIstearic acid was incubated with x g supernatant fluid of homogenates of starved and untreated under the standard incubation procedure. The incubation mixtures were then acidified and extracted with ether without previous saponification [15,16]. Both in the case of incubations with x g supernatant fluid from starved and untreated rats, O/, of the radioactivity could not be extracted from the acidified water phase, indicating a maximal conversion of 50-65O/, of the [1-l4C]stearate or its metabolites into CoA derivatives. When very high concentrations of ATP and CoA were added to the x g supernatant fluid of liver homogenate of starved rats in order to get maximal conversion of stearate into stearoyl-coa, wi-oxidation of stearate was inhibited (Table 6). The inhibition was at least partly due to an effect on the dehydrogenase step as the ratio between 18-hydroxystearic acid and 1,18-octa- decadioic acid was about 1 in these experiments. To examine the possibility that stearoyl-coil is not a substrate for the wl-hydroxylase, [i-14c]stearoyl- CoA was incubated with the microsomal fraction and the x g supernatant fraction of a liver homogenate. The rate of w i-oxidation of stearyl-coa was however about /, of that obtained with stearic acid (Table 7). It should be pointed out that it was not excluded in these experiments that part of the stearoyl-coa had been hydrolyzed prior to the wl-oxidation. Table 8 shows the effect of heat, freezing and thawing on the capacity of x g supernatant fluid of homogenate of liver from untreated rats to inhibit w1-oxidation of stearate. Boiling for 5 min had no effect. Preincubation for 15 min at 37 "C, freezing and thawing or storage at 4 "C for three days resulted in partial destruction of the inhibitory factor(s) in the xg supernatant fluid.

6 420 w-oxidation of Stearic Acid Table 6. Effect of addition of ATP and CoA on wl-oxidation of stearate catalyzed by 200OOxg supernatant fluid of homogenate of liver of starved rats wl-oxidation was expressed as the sum of 18-hydroxystearic acid and 1,18-octadecadioic acid formed. Incubations were 15 min x g supernatant fluid of liver homogenate of starved rats 20000x9 supernatant fluid of liver homogenate of starved rats + 6 mm ATP x g supernatant fluid of liver homogewate of starved rats 1 mm CoA 20000~9 supernatant fluid of liver homogenate of starved rats + 6 mm ATP + 1 mm CoA wl-oxidation of stearate nmol/mg microsomal protein Table I. Incubations of [l-14c]stearoyl-coa with microsomal fraction and X g supernatant fluid of rat-liver homogenate wl-oxidation was expressed as the sum of 18-hydroxystearic acid and 1,18-octadecadioic acid formed. Incubations were 15 min Microsomal fraction + NAD + horse-liver alcohol dehydrogenase pmol [l-14c]stearoyl- CoA 3.2 Microsomal fraction + NAD + horse-liver alcohol dehydrogenase pmol [l-14c]stearate x g supernatant fluid pmol [l-14c]stearoyl-coa xg supernatant fluid 0.35 pmol [l-14c]stearate 3.0 ol-oxidized products formed nmol/mg microsomal protein Table 8. Effect of heating and freezing and tkawing on capacity of X g supernatant fluid of homogenate of liver from untreated rats to inkibit ol-oxidation of stearate wl-oxidation was expressed as the sum of 18-hydroxy stearic acid and 1,18-octadecadioic acid formed. Incubations were 15 rnin x g supernatant fluid of liver homogenate of starved rat x g supernatant fluid of liver homogenate of starved rat x g supernatant fluid of homogenate of untreated rat 20000x9 supernatant fluid of liver homogenate of starved rat O~g supernatant fluid subjected to boiling for 5 mjn x g supernatant fluid of liver homogenate of starved rat x g supernatant fluid subjected to preincubation at 37 "C for 15 min x g supernatant fluid of liver homogenate of starved rat x g supernatant fluid subjected to freezing and thawing five times during 3 days x g supernatant fluid of liver homogenate of starved rat x g supernatant fluid subjected to standing at 4 "C for 3 days wl-oxidation of stearate nmol/mg microsomal protein DISCUSSION The present work confirms the previous finding that wl-oxidation of stearate by a postmitochondrial fraction of rat liver homogenate fortified with an NADPH-generating system is increased after starvation and diabetes [Q, 171. The present results further show that the soluble fraction of liver homogenate is responsible for the stimulatory effect found after starvation. Addition of NAD together with horseliver alcohol dehydrogenase stimulated microsomal ol-oxidation of stearate to about the same extent as did addition of 100OOOxg supernatant fluid from homogenate of liver of starved rats. Addition of NAD and horse-liver alcohol dehydrogenase to a system consisting of microsomal fraction together with x g supernatant fluid from homogenate of liver of starved rats did not further stimulate to1-oxidation of stearate. It can be concluded that the stimulatory effect of the x g supernatant fluid from starved rats is mainly due to the presence of NAD and alcohol dehydrogenase in this cell fraction. The effect of alcohol dehydrogenase and

7 I. Bjorkhem 421 NAD was shown to be due to oxidation of the primary product of the hydroxylation, 18-hydroxystearic acid, into 1,18-0ctadecadioic acid. The mechanism of the stimulation could thus be a release of a product inhibition in the hydroxylase step. Addition of 18-hydroxystearic acid to the incubation mixture was found to inhibit the reaction, but to a lesser extent than expected. It is possible that endogenously formed 18-hydroxystearic acid inhibits 18-hydroxylation of stearate to a greater extent than exogenous 18-hydroxystearie acid. It is noteworthy that NAD stimulated conversion of 18-hydroxystearic acid into 1,18-octadecadioic acid as well as the total conversion of stearic acid into ol-oxidized products (cf. [3,4]). This stimulation must be due to the microsomal col-hydroxy fatty acid dehydrogenase(s) [4,6,18]. It is possible that the activity of the microsomal col-dehydrogenating system is too low to permit a complete release of the microsomal olhydroxylase from product inhibition ; complete disappearance of the product inhibition may be obtained only after addition of ethanol dehydrogenase. Conversion of 18-hydroxystearic acid into 1,18-octadecadioic acid should require aldehyde dehydrogenase in addition to ethanol dehydrogenase. Addition of a preparation of aldehyde dehydrogenase known to be active towards long-chain semialdehydes [7] did, however, not further stimulate microsomal ol-oxidation of stearate. It is probable that oxidation of the hydroxy fatty acid into the semialdehyde is the limiting step and that the microsomal fraction contains sufficient amounts of aldehyde dehydrogenase. The present work show that there must be factor(s) in the soluble part of liver homogenate from untreated rats which inhibits cul-oxidation of stearate. As the ratio between the 1,18-octadecadioic acid and 18-hydroxystearic acid formed under conditions of inhibition was high, it can be concluded that the dehydrogenase step is not inhibited. The inhibitory effect of the 100OOOxg supernatant from liver of untreated rats was at least not mainly due to increased conversion of stearic acid into stearoyl CoA. High concentrations of ATP inhibited wl-oxidation of stearate. This inhibition was probably due to an inhibition of the conversion of 18-hydroxystearic acid into 1,18-octadecadioic acid as the ratio between 1,18-0ctadecadioic acid and 18-hydroxystearic acid formed from stearate was low in the presence of ATP. It has been previously reported that ADP and AMP inhibit the ethanol dehydrogenase [19]. It should be mentioned that the concentration of ATP needed to inhibit the ol-oxidation of stearic acid was much higher than the concentration of ATP normally found in lives homogenates [20]. It is interesting that the inhibitory effect of the x g supernatant fluid of liver homogenate of untreated rats was in general much higher with a x g supernatant fluid than with a microsomal fraction as enzyme source. It is known that when a microsomal fraction is prepared from the x g supernatant fluid as done in the present work lipid peroxides are formed. It is possible that these lipid peroxides might destroy the inhibiting factor(s) in added x g supernatant fluid. This could explain the higher degree of inhibition obtained with the x g supernatant fluid than with the microsomal fraction as enzyme source. However, the degree of inhibition was about the same when the microsoma1 fraction was prepared under conditions in which lipid peroxidation was reduced to a minimum. The ability of the x g supernatant fluid of homogenate of liver from untreated rats to inhibit ol-oxidation of stearate was retained after boiling for 5 min but was partially destroyed after preincubation at 37 "C, after freezing and thawing and after storage for some days at 4 "C. It is evident that the inhibiting factor(s) in the x g supernatant fluid is metabolized or inactivated by enzymes normally present in the x g supernatant fluid. The sensitivity of the inhibitory factor(s) towards freezing and thawing is in accordance with a previous work by Preiss and Bloch [2]. These authors showed that repeated freezing and thawing increased o-oxidation of stearate by the x g supernatant fluid of a rat liver homogenate. In the present work, only ol-hydroxylation of stearie acid was studied. The o2-hydroxylation of stearate was not determined in the different experiments as the very low extent of this hydroxylation is difficult to determine adequately (cf. [4]). Preliminary studies indicate that the effect of starvation and diabetes on ol-oxidation of palmitate is similar to the corresponding effects on cul-oxidation of stearate. The stimulatory effect of starvation on ol-oxidation of palmitate was, however, not completely bound to the soluble fraction of the liver homogenate ; a small increase of the capacity of the microsomal fraction alone to col-hydroxylate palmitic acid was also observed after starvation. oj1-oxidation of lauric acid was also increased after starvation, but in this case the microsomal fraction alone was responsible for most of the stimulation. The effect of starvation and diabetes observed under the conditions used in vitro in the present work may, at least to some extent, reflect conditions in vivo. If this is the case, co-oxidation of long-chain fatty acids might be inhibited under normal conditions and the effect of starvation and diabetes is to release this inhibition. An increased o-oxidation of fatty acids in the starved and diabetic liver could be of physiological importance. It has been suggested that the role of o-oxidation of fatty acids is to convert part of the fatty acids into succinyl-coa [17]. An increased synthesis of succinyl-coil followed by cleavage into succinate might be important under

8 422 I. Bjorkhem: w-oxidation of Stearic Acid conditions of increased utilization of fatty acids. The increased amount of citric acid cycle intermediate thus made available might enhance the oxidation of acetyl-coa, provided that there is a rapid cleavage into succinate [22]. In consonance with the hypothesis that increased amounts of dicarboxylic acid accelerates oxidation of acetyl-coa in the citric acid cycle, Wada et al. [17] have found that administration of long-chain dicarboxylic acids to diabetic rats decrease the concentration of ketone bodies in the blood. The skilful technical assistance of Miss Irene Ferdman and Miss Eva Strindberg is gratefully acknowledged. This work has been supported by the Swedish Medical Research Council (Project 13X-3141). REFERENCES 1. Robbins, K. C. (1961) Fed. Proc. 20, Preiss, B. & Bloch, K. (1964) J. Bid. Chem. 239, Wada, F., Shibata, H., Goto, M. & Sakamoto, Y. (1968) Biochim. Biophys. Acta, 162, Bjorkhem, I. & Danielsson, H. (1970) Eur. J. Biochem. 17, Mitz, M. A. & Heinrikson, R. L. (1961) Biochim. Biophys. Actu, 46, 45. Bjorkhem, I. (1972) Biochim. Biophys. Acta, 260, 178. Bjorkhem, I. (1972) Eur. J. Biochem. 30, 441. Antony, G. J. & Landau, B. R. (1968) J. Lipid Res. 9, Mitsuhashi, 0. & Imai, Y. (1967) Abstr. 7th Int. Congr. Biochem Young, D. L. & Lgnen, F. (1969) J. Biol. Chem. 244, Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. BioZ. Chem. 193, Bergstrom, S. & Gloor, U. (1955) Actu Chem. Scund. 9, Einarsson, K. & Johansson, G. (1969) FEBS Lett. 4, Johansson, G. (1971) Eur. J. Bicchem. 21, Samuel, D. & Ailhaud, G. (1969) PEBB Lett. 2, Pande, S. V. (1972) Biochem. Biophys. Acta, 270, Wada, F., Usami, M., Goto, M. & Hayashi, T. (1971) J. Biochem. (Tokyo) YO, Bjorkhem, I. & Hamberg, 51. (1971) J. Biol. Chem. 246, Theorell, H. & Yonetani, T. (1964) Arch. Biochem. Biophys. 106, Bucher, N. L. R. & Smaffield, M. S. (1966) Biochim. Biophys. Actu, 129, Dalziel, K. (1957) Acta Chem. Scund. 11, Smith, C. M. & Williamson, J. R. (1971) FEBS Lett. 18, 35. I. Bjorkhem, Kemiska Institutionen, Karolinska Institutet, S Stockholm 60, Sweden