Studies on the biosynthesis of cholesterol. IV. Higher counting substances accompanying C14-cholesterol in the intact rat

Studies on the Biosynthesis of Cholesterol. IV. Higher Counting Substances Accompanying W-Cholesterol in the Intact Rat’ Erwin Schwenk and Nicholas T. Werthessen2 From the Worcester Foundation for Experimental Massachusetts

ReceivedAugust

Biology,

Shrewsbury,

12, 1952

U4-cholesterol, synthesized during the perfusion with Cl*-labeled acetate of liver and other surviving organs of swine, always contains radioactive substances part of which are precipitated by digitonin together with the cholesterol (1). The specific count of these companions is considerably higher than that of the CY4-cholesterolafter purificat,ion through 5,6-dibromo3@-cholestanol. It is shown in the following investigation that the formation of higher counting companions (HCC) of cholesterol can also be demonstrated in the intact rat, provided the experiments are carried out appropriately. EXPERIMENTAL Male or female rats weighing from 165 to 185 g. were injected intraperitoneally with 0.2 millicuries (mc.) of (Y-sodium acetate labeled in the carboxyl group and dissolved in 2 ml. of a saline solution. After the time for the experiment had elapsed, the animals were killed, usually by the use of chloroform, and then put into a 2-1. Erlenmeyer flask. Water was added to make a total of 400 g., and 120 g. of KOH in pellets was added to make the alkali concentration 30$& The flask was heated for 15-20 hr. on a steam bath and, after the addition of 300 ml. of 95% ethanol, the contents were filtered from the bones over a glass filter cloth. The residue on the filter was washed with water, and the combined solution and washes were extracted thoroughly with pentane. This extract was washed twice wit,h 10% KOH solution, twice with distilled water and then evaporated to dry____* This work has been supported by grants from the American Cancer Society, the Damon Runyon Fund, the U. S. Public Health Service (Grant No. C. 321) and the Schering Corporation. * Present address: Foundation of Applied Research, San Antonio 6, Texas. 91

92

SCHWENK

AND

WERTHESSEN

ne88in vacuum. Purification of the crude pentane extract through the digitonide and the dibromide of cholesterol and the radioactivity counts were carried out as described for the earlier perfusion work (1, 2). RESULTS

Table I presents part of the results. The figures in this table illustrate that HCC can be observed only when the experiment is concluded after 30 or 60 min. Higher counting TABLE Rate Injected

---

=

Zntraperitoneally 2

Solutione

___--

IX

1

Expt.so.

I with CH~POONa

3

1

4

1

5

Count/min./mg. CH’C”OONa injected

Duration of experiment

after _Digiton. 1 Dibrom. .- -’

blotbcr liqtpr from 1

dlbrom.

1

6 Per cent loss of activity in dibrominaticn

---.

mir.

30 30 30 30 60 246 240 240

10

246 0.1 mc. each day

1

for 10 days

i

314 68 135 98 243 110 373 142 118 312

219 53 79 70 143 114 374 138 121 343

e 77 152 88 203 91 372 130 * o

30 22 41 28 41 0 0 0 0 0

- __--._

0 Not counted.

substances are not found when the animals are killed after 4 hr. or more have elapsed. The figures in col. 5 require explanation. They were obtained in the following way. After brominat.ion, the 5,6dibromo3&cholestanol was filtered; to the filtrate was added 2 g. of zinc dust, and the mixture, with occasional shaking, was allowed to stand for at least an hour. The solution was decanted from the zinc and extracted with ether. The ether was washed with water, dilute alkali, and water and evaporated to dryness. A sample of this residue was counted in the usual way. It is obvious that the count of this residue should be the same as that

BIOSYNTHESIS OF CHOLESTEROL.

93

IV

of the cholesterol from the 5,6-dibromo-3@-cholestanol in those experiments where there are no HCC present. In experiments where HCC are found, they must be in the mother liquor and the residue therefore should have a higher count than the reclaimed cholesterol. The figures in col. 5 are in agreement with this reasoning, and confirm t’he presence of HCC in the short-term runs. CONCLUSIONS

The experiments here reported demonstrate that the production of higher counting digitonin-precipitable substances accompanying cholesterol in its biosynthesis from C4-labeled acetate is not restricted to the perfusion of an isolated organ. During the formation of cholesterol in the intact rat HCC are produced, but they are used up so fast that they can be found only when the experiment is one of short duration. The disappearance of HCC when the experiment is extended for more than 1 hr. is not contradictory to the assumption mentioned before (1) that these companions may be precursors of cholesterol in the biosynthesis from acetate. The experiments demonstrate also that 30 min. suffice to produce an appreciable amount of CY4-cholesterol from C4-acetate. A similar observation was made with intact mice by Hevesy et al. (3), but most of the other work on the biosynthesis of cholesterol with labeled acetate has been made with experiments of longer duration. Hevesy et al., however, did not observe the higher counting substances (HCC) present in the cholesterol as isolated from the digitonide, possibly because the cholesterol was not purified by bromination before counting. STJMMARY

When rats are injected with acetate labeled with Cl4 in the carboxyl group and killed not later than 1 hr. after injection, the CY-cholesterol isolated from the animals in the usual way contains companions precipitable by digitonin with a higher specific count than the C14-cholesterol itself. REFERENCES 1. SCHWENK,

E.,

AND WERTHESSEN,

N. T., Arch.

Biochem.

and Biophys.

40, 334

(1952). 2. WERTHESSEN, N. T., AND SCHWENR, E., Am. J. Physiol., in press. 3. HEVESY, G., RUYSSEX, R., AND BECKMANS, M. L., Esperientia 7, 317 (1951).