82 SHORT COMMUNICATIONS

Isolation of 3~ , 7~, 12~-Trihydroxycoprostanic Acid From Baboon Bile

T RIHYDROXYCOPROSTANIC ACID (3~, 7~, 12~) (THCA) has been implicated as an inter-

mediate in the biological formation of bile acids from cholesterol (1,2). This compound can be converted to cholic acid by suitably for- tified rat liver preparations (3,4), a n d it has been demonstrated that cholesterol can be con- verted to trihydroxycoprostanic acid in the al- ligator (5) and in man (6). The conversion of 26-~4C-cholesterol to labeled THCA in the baboon is the subject of this communication.

A young male baboon (4-5 years) which had been maintained on a nutritionally ade- quate semi-synthetic diet (7) was given a dose of 26-~4C-cholesterol (0.025 ~c) in corn oil by stomach tube. The animal was fasted for 18 hr, bled by venipuncture and killed. The serum, liver, aorta and bile were taken for analysis. Aliquots of liver and aorta were ho- mogenized in chloroform-methanol (2:1) and the extracts assayed for free and ester choles- terol. The bile (13 ml) was deproteinized with ethanol, and the supernatant fluid con- centrated to a small volume and subjected to hydrolysis with NaOH at 125 C under pres- sure (6). The hydrolysate was acidified with HC1 and the bile acids extracted with ethyl acetate.

The total serum cholesterol level and the and fl serum lipoprotein cholesterol levels (Table I) were of the same order of magni- tude as those of other baboons fed the same diet (7). The liver contained appreciable quantities of radioactive free (634 cpm/mg) and ester (1872 cpm/mg) cholesterol, but there was no detectable radioactivity in the aorta.

Thin layer chromatography (TLC) of the biliary bile acids on Silica Gel G, using ethyl acetate-acetic acid (95:5) as the developing solvent, yielded major spots corresponding to cholesterol (Rr = 0.69) THCA (Rf = 0.58) and chotic acid (Rf = 0.21). The spots were visualized with an anisaldehyde-H2SO4-HOAc spray reagent (10). Another aliquot of the bile acid extract was subjected to TLC, and the areas corresponding to cholic acid and THCA were scraped from the plate and the extracts assayed for radioactivity and subjected to fur- ther purification by TLC. The cholic acid thus obtained was not radioactive, which would be expected since the precursor was 26-~4C - cholesterol. The THCA fraction contained 2,600 cpm. This material was shown to be

LIPIDS, VOL. 4, NO. 1

FIG. I. TLC of authentic 3a-, 7a-, 12ct-trihy- droxycoprostanic acid (THCA) and of baboon bile. System: Ethyl acetate-acetic acid (95:5) on Silica Gel G. Detection: Anisaldehyde in H..,SOr HOAc.

identical with authentic THCA by its Rr value in TLC (Fig. 1) and by its retention time when subjected to gas-liquid-chromatography

SHORT COMMUNICATIONS

TABLE I Level and Specific Activity of Serum Cholesterol in a Baboon Fed 26-14C-Cholesterol

83

Free a Estera Total a Sample mg/100 ml cpm/mg rag/100 ml cpm/mg rag/100 ml cpm/mg

Serum 35 679 79 776 114 703 a Lipoproteinb 18 679 41 838 59 740 /~ Llpoproteinb 13 632 38 696 50 608

aMethod Ref. 8. bMethod Ref. 9.

(GLC) (SE-30 supported on 1% QEF) . Pooled bile from 24 baboons, half of whom

had been fed either sodium 1-14C-acetate or 4-14C-cholesterol (7), yielded 0.96 g of crude mixed bile acids (676,000 cpm). Preparative FLC on Silica Gel G, using isopropyl ether. isooctane-acetic acid (50:25:30) as the devel- oping solvent, yielded areas corresponding to cholic acid (40,800 cpm) and T H C A (15,600 cpm). The two bile acids were shown to be similar to authentic samples by TLC and GLC.

The T H C A fraction was further purified by crystallization from ethyl acetate to yield 0.10 mg of pure 3~-, 7~-, 12~(-trihydroxycoprostanic acid with a specific activity of 647 cpm/mg. The material had a melting point of 184 C and did not depress the melting point of an authentic sample of THCA.

P. P. SHAH EZRA STAPLE (deceased) IRWIN L. SHAPIRO a

DAVID KRITCHEVSKY a Department of Biochemistry, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

aWistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania.

ACKNOWLEDGMENTS

Supported in part by grants AM-07202; HE-03299 and HE-05209 from the National Institutes of Health and Re- search Career Award 4-K6-HE-734.

REFERENCES

I. Haslewood, G. A. D., Physiol. Re','. 35, 178-196 (1955).

2. Bergstrom, S., in "Ciba Foundation Symposium on Biosynthesis of Terpenes and Sterols," G. E. Wolsten- holme and M. Connor, eds., Little, Brown and Co., Boston, 1959, pp. 185-205.

3. Bridgwater, R. J., and S. Lindstedt, Acta. Chem. Scand. 11, 409-413 (1957).

4. Briggs, T., M. W. Whitehouse and E. Staple, J. Biol. Chem. 236, 688-4591 (1961).

5. Briggs, T., M. W. Whitehouse and E. Staple, Arch. .Biochem. Biophys. 85, 275-277 (1959).

6. Staple, E. and J. L. Rabinowitz, Biochim. Biophys. Acta 59, 735-736 (1962).

7. Kritchevsky, D., E. S. Kritchevsky, P. P. Nair, J. A. Jastremsky and I. L. Shapiro, Nutr. Dicta 9, 283-299 (1967).

8. Sperry, W. M. and M. Webb, J. Biol. Chem. 187, 97-110 (1950).

9. Castaigne, A. and A. Amselem, Ann. Biol. Clin. 17, 336-347 (1959).

10. Kritchevsky, D., D. S. Martak and G. H~ Rothblat, Analyt. Biochem. 3, 388-392 (1963).

[ R e c e i v e d J u l y 12, 1968]

A Rapid Micro Technique for Differentiating Between Iso, Anteiso and Other Mono Methyl Branched Fatty Chains

A VARIETY OF TECHNIQUES are available which give either complete or partial in-

formation on the location of a methyl branch in a fatty chain. Techniques discussed in (1) in- clude mass spectra, x- ray diffraction, infrared spectra, oxidative degradation, retention data in GLC, melting points of binary mixtures of branched with normal acids, and others. Nuclear magnetic resonance (2) , and x-ray diffraction of single crystals of urea adducts (3) have also been used. Except for GLC, all the above techniques are either complex or re- quire at least milligram amounts of material or expensive apparatus, and the hazards in-

volved in identifying branched fatty chains solely on the basis of GLC retention data have been discussed (4) .

This report describes a method capable of differentiating between as little as 1 t~g of iso or anteiso fatty chains by degrading them with acidic KMnO 4 respectively to acetone or 2- butanone, and analyzing the ketones by GLC on a Poropak QS column. In the iso chain the methyl branch is on the (o minus 1 C-atom (alternately designated as ~02) and in the an- teiso chain the methyl branch is on the o~ minus 2 C-atom (alternately designated as (03) where o~ is the terminal C-atom of the fatty chain.

LIPIDS, VOL. 4, N o . 1