192 A. L. LEHNINGER, G. D. GREVILLE VOL. 12 (1953) 



report. The important facts concerning citrate formation from the isomers of BOH in 

 the liver systems are that ^-BOH readily undergoes oxidation via the tricarboxylic acid 

 cycle, as evidenced by extra oxygen uptake and extra citrate formation, whereas Z-BOH 

 forms considerably less or no citrate under the same circumstances and is largely oxidized 

 to acetoacetate. 



Tests with washed kidney preparations (expt. 4, Table I) showed that both isomers 

 cause extra oxygen uptake in the presence of fumarate and both form extra citrate, 

 indicating that both isomers are oxidized via the tricarboxvlic acid cycle, and at approxi- 

 mately equal rates. Such kidney preparations are known to oxidize acetoacetate via the 

 cycle quite readily^, in contradistinction to liver mitochondrial preparations. Differences 

 in mechanism of oxidation of the two isomers of /3-hydroxybutyrate are thus apparent 

 only in the liver preparations, because of the very limited ability of these to utilize free 

 acetoacetate. 



Other experiments showed that the citrate formation from (f-/3-hydroxybutyrate 

 in both liver and kidney mitochondrial systems is almost completely inhibited by low 

 concentrations {0.0005 M) of sodium fluoride, although such amounts of fluoride do not 

 interfere with citrate formation from pyruvate (see Table III). Fluoride is known to 

 inhibit fatty acid oxidation^^' ^'^, suggesting that there is a locus of fluoride inhibition in 



TABLE III 



EFFECT OF FLUORIDE ON CITRATE FORMATION FROM ^-BOH. 



Each vessel contained 0.005 ^I MgClj, o.ooi M ATP, 0.014 ^^ phosphate buffer pH 7.5, 0.06 M 

 KCl, o.oi M fumarate and washed particles from 0.45 g (wet wt.) rat liver. Total volume 3.0 ml. 

 Pyruvate (o.oi M final), d-BOH (o.oi M), and fluoride (0.002 M) were added as indicated. Tempera- 

 ture, 22°. Time, 65 minutes. 



c- I . I Fluoride Oxygen uptake Citrate formed 



(0.002 M) microatoms microtnoles 



None — 



None + 



^-BOH — 



d-BOU + 



pyruvate — 



pyruvate + 



citrate formation which may be common to the oxidation pathways taken by d-BOH 

 and the saturated fatty acids. Cheldelin and Beinert have observed that 0.033 ^ 

 fluoride prevents utilization of dl-BOH beyond the acetoacetate stage in rabbit liver 

 mitochondrial systems^^. 



Although the rf-isomer formed little acetoacetate under any circumstances in the 

 experiments with liver particle systems described above, acetoacetate formation from 

 i-BOH was found to occur if relatively small concentrations of a tricarboxylic acid cycle 

 intermediate were present in the system. With low concentrations of fumarate 

 {1-5 -10"* M), d-BOH gives rise to significant amounts of acetoacetate, although these 

 never approached the yield expected from the extra oxygen uptake if equation (i) also 

 represented the oxidation of d-BOH (See Table IV). It is also seen that with increasingly 

 higher concentrations of fumarate, acetoacetate no longer appears as product. It may 

 be concluded that the formation of acetoacetate from d-BOH depends on "priming" 

 or "sparking" by low concentrations of cycle intermediates. The /-isomer, on the other 

 hand, requires no "sparking" to be oxidized to acetoacetate. 



References p. 202. 



