SHORT COMMUNICATIONS 661

2. Tanaka, R., and K. P. Strickland, Arch. Biochem. Biophys. 111:583 (1965).

3. Fenster, L. J., and J. H. Copenhaver, Jr., Biochim. Biophys. Acta 137:406 (1967).

4. Skou, J. C., Physiol. Rex,. 45:596 (1965). 5. Kleinzeller, A., and J. H. Cort, Nature 180:1124

(1957). 6. Ispolatovskya, M. W., G. A. Levdikova and I. A.

Larina, Biokhimiya 27 : 82 (1962). 7. Manitius, A., K. Bensch and F. H. Epstein, Bio-

chim. Biophys. Acta 150:563 (1968). 8. Bonting, S. L., K. A. Simon and N. M. Hawkins,

Arch. Biochem. 95:416 (1961). 9. Skou, J. C., Biochim. Biophys. Acta 23:394

(1957). 10. Fiske, C. H., and Y. Subbarow, J. Biol. Chem.

66:375 (1925). 11. Jorgensen, P. L., Biochim. Biophys. Acta 151:212

(1968). 12. Bowen, W. J., L. C. Stewart and H. L. Martin, J.

Biol. Chem. 238:2926 (1963). 13. Lowry, O. H., N. J. Rosebrough, A. L. Farr and

R. J. Randall, Ibid. 193:265 (1951). 14. Martonosi, A., J. Donley and R. A. Halpin, Ibid.

243:61 (1968).

[Received March 2, 1970]

Liberation of Aldehydes From AIk-l-enyl Glyceryl Ethers by Acid Hydrolysis

ABSTRACT

Labeled alk-l-enyl glyceryl ethers were used in conjunction with thin layer chromatography to study the liberation of aldehydes from alk-l-enyl glyceryl ethers by two acid hydrolysis procedures. Both methods gave similar results, but n e i t h e r l i b e r a t e d t he a ldehydes quantitatively. Only 75-85% of the alk-l-enyl glyceryl ether radioactivity was liberated as free aldehydes. Several nonaldehyde products were detected and one appeared to be a cyclic acetal.

Synthetic alk-l-enyl ethers (vinyl ethers) are hydrolyzed by acid to aldehydes and alcohols (1). Karnovsky et al. (2) reported the use of methanolic HC1 to release aldehydes from the unsaponifiable lipid fractions of biological origin. Since then, the acid lability of the alk-1- enyl glyceryl ethers has been used to an advantage for the analyses of neutral glycerides and phosphoglycerides containing these ethers. Schmid and Mangold (3) have reported a tech- nique for the selective acid hydrolysis of neutral plasmalogens (alk-l-enyl glyceryl ether diesters) and separation of the products (alde- hydes and diglycerides) on a thin layer chro- matoplate. The technique has since been applied to the phosphatides containing alk-1- enyl glyceryl ethers (plasmalogens) (4-6), alk-1 -enyl acyl glycerides (7) and free alk-l-enyl glyceryl ethers (8,9).

1 Present address: Neuropsychiatric Research Laboratory, V.A. Hospital, Hines, I11. 60141.

2Under contract with the U.S. Atomic Energy Commission.

The liberation of aldehydes from the intact plasmalogens is reported to be quantitative (4,6). However, when free alk-l-enyl glyceryl ethers were exposed to HC1 fumes, the developed chromatoplate showed o~her com- pounds in addition to free aldehydes (10). Unlike the intact neutral plasmalogens and plasmalogens, the free alk-l-enyl glyceryl ethers contain free hydroxyl groups that can lead to the formation of cyclic acetals (11,12). An investigation into the quantitative aspects of the acid hydrolysis of alk-l-enyl glyceryl ethers on adsorbent layers was attempted with a labeled aldehyde, a product of the hydrolysis reaction (I0). However, until now, the lack of radioactive labeled alk-l-enyl glyceryl ethers has not permitted an accurate evaluation of the reaction.

Labeled alk-l-enyl glyceryl ethers were obtained from the experiments with Ehrlich ascites cells in which we reported the route of plasmalogen biosynthesis (13,14). The phospha- tidyl ethanolamine class was reduced with lithium aluminum hydride (12) and the labeled alk-l-enyl glyceryl ethers were separated from the hydrogenolysis products by thin layer chro- matography (TLC) (13,14). The only detect- able contaminant of the alk-l-enyl glyceryl ethers was 6.2% alkyl glyceryl ether, for which the data have been corrected. The dis- appearance of all the alk-l-enyl glyceryl ether radioactivity (Fig. 1 C) after acid treatment indi- cates the absence of other labeled impurities. The tritium and carbon-14 label was located in the hydrocarbon chain as established earlier (13,14). The distribution of radioactivity along the developed TLC plates was determined by counting the radioactivity in successive 2 mm sections of adsorbent layer (13).

LIPIDS, VOL. 5, NO. 7

662 SHORT COMMUNICATIONS

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FIG. 1. Liberation of free aldehydes from radio- active alk-l-enyl glyceryl ethers by acid hydrolysis according to two different procedures: A, hydrolysis extraction procedure of Anderson et al. (10); B, hydrolysis on the chromatoplate as described by Schmid and Mangold (3); and C, hydrolysis by the Schmid and Mangold procedure (3) and development of the chromatoplate in a more polar solvent system (diethyl ether-water, 100:0.5 v/v). Chromatoplates A and B were developed in a hexane-diethyl ether (90:10 v/v) solvent system and all separations were carried out on Silica Gel G adsorbent layers. Peak 1 is dis- cussed in the text. Peak 2 represents the free long chain aldehydes.

When labeled alk-l-enyl glyceryl ethers were spotted on a chromatoplate and exposed to HC1 fumes, as described by Schmid and Mangold (3), and developed, 83.6 + 2.9% of the radio- activity was found in the free aldehyde region of the chromatoplate. Eighty +- 5.3% of the radioactivity occurred in the aldehyde peak when the hydrolysis extract ion procedure by Anderson et al. (10) was used. A comparison of the distribution of radioactivity (Fig. 1A and 1B) indicates that most of the nonaldehyde radioactivity remained near the origin in the 90:10 solvent system. The distribution of radio- activity on a chromatoplate exposed to HC1 fumes and developed in a more polar solvent system (Fig. 1C) showed (a) only a small amount of radioactivity at the origin at tr ibut- able to activity in the glycerol; (b) the contami-

nating alkyl glyceryl ether appearing between samples number 20 and 25; (c) the absence of alk-l-enyl glyceryl ethers opposite the standard appearing between samples number 25 and 31; (d) 10% of the activity appearing in the region of cyclic acetals and long chain alcohols between samples number 33 and 45; and (e) a peak of radioactivity adjacent to the aldehyde peak. The distribution of radioactivity from the hydrolysis extraction procedure (10) was similar.

The results indicate that alk-l-enyl glyceryl ethers are completely converted to other com- pounds when treated either with acid on the TLC plate (3) or by hydrolysis extraction (10). Aldehydes are the primary products of each hydrolysis procedure; however, competing reactions give rise to other compounds. One of the major nonaldehyde compounds appears to be a cyclic acetal. Cyclic acetals that can exist in isomeric forms (15) have been prepared from alk-l-enyl glyceryl ethers (12) and have Rf values corresponding to the radioactivity appearing between samples number 31 and 45 of Figure 1C. Contrary to the conclusions reached by Anderson et al. (10), aldehydes are not quantitatively liberated from alk-l-enyl glyceryl ethers by either method. The dis- crepancy between our results and theirs probably lies in the fact that their conclusions were drawn from studies with a labeled alde- hyde, a product of the acid hydrolysis, which precluded their encountering reactions of the alk-l-enyl glyceryl ethers that would not yield aldehydes. More recently, Bandi (16) has reported the quantitative hydrolysis of alk-1- enyl glyceryl ethers using a procedure almost identical with the procedure described by Anderson et al. (10), except for the longer hydrolysis time. Although we have not tried this slightly modified procedure, it too may not be quantitative. The alk-l-enyl glyceryl ether sample on the thin layer chromatoplate, apparently used to assess the quantitative aspects of the hydrolysis (16), showed a small spot at the origin where we observed radio- activity (Fig. 1A and 1B).

Our results indicate that the quantitative estimation of the percentage of alk-l-enyl glyceryl ethers from the aldehydes liberated from alk-l-enyl glyceryl ethers by either of these methods will be low.

RANDALL WOOD 1 KATHLEEN HEALY The Medical Division 2 Oak Ridge Associated Universities Oak Ridge, Tennessee 37830

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REFERENCES

1. Reppe, W., and collaborators, Justus Liebigs Ann. Chem. 601:81-138 (1956).

2. Karnovsky, M. L., S. S. Jeffrey, M. S. Thompson and H. W. Deane, J. Biophys. Biochem. Cytol. 1:173-182 (1955).

3. Schmid, H. H. O., and H. K. Marigold, Biochim. Biophys. Aeta 125:182-184 (1966).

4. Horrocks, L. A., J. Lipid Res. 9:469-472 (1968). 5. Viswanathan, C. V., M. Basilio, S. P. Hoevet and

W. O. Lundberg, J. Chromatog. 34:241-245 (1968).

6. Viswanathan, C. V., F. Phillips and W. O. Lund- berg, Ibid. 35:66-~71 (1968).

7. Wood, R., and F. Snyder, Arch. Biochem. Biophys. 131:478-494 (1969).

8. Wood, R., R. D. Harlow and F. Snyder, Biochim. Biophys. Acta 176:641-643 (1969).

9. Anderson, R. E., R. B. Cumming, M. Walton and F. Snyder, Ibid. 176:491-501 (1969).

10. Anderson, R. E., R. D. Garrett, M. L. Blank and F. Snyder, Lipids 4:327-330 (1969).

11. Pietruszko, R., and G. M. Gray, Biochim. Bio- phys. A c t a 56:232-239 (1962).

12. Wood, R., and F. Snyder, Lipids 3:129-135 (1968).

13. Wood, R., and K. Healy, Biochem. Biopbys. Res. Commun. 38:205-211 (1970).

14. Wood, R., and K. Healy, J. Biol. Chem. 245:2640-2648 (1970).

15. Aksnes, G., P. Albriktsen and P. Juwik, Acta Chem. Scand. 19:920-930 (1965).

16. Bandi, Z. L., Chem. Phys. Lipids 3:409-412 (1969).

[Received April 3, 1970]

The Effect of Hydrogen Peroxide on Serum Lecithin-Cholesterol Acytransferase Activity 1

ABSTRACT

Glomset" has r epo r t ed an acyltrans- ferase in plasma which ef fec ts the t ransfer of a fa t ty acid f rom the beta pos i t ion of leci thin to choles terol , forming choles- teryl esters. Peroxide is shown to have an inh ib i tory effect on the enzyme activity in a concen t ra t ion of 0.1 M. There is a variable effect wi th a concen t ra t ion o f 0.01 M, wi th a s t imula t ion of activity in some sera, while in o thers , an inhibi t ion. The addi t ion of a syn the t i c sa tura ted leci- th in does no t cause a reversal of the inhib i t ion at 0.1 M H 2 0 2 .

amoun t s of hydrogen perox ide and syn the t i c d ipa lmi toy l L-~-lecithin at 37 C for a per iod of 24 hr.

4-14C-Cholesterol (New England Nuclear Corpora t ion , NEC-018, specif ic act ivi ty: 58 mc /mM) was added to the incuba t ion vial in benzene in the a m o u n t of 0.1 /.tc (0.17 x 10 -3 #M or 0.668 /~g choles tero l ) per 2 ml serum, and the benzene was evapora ted to dryness under a s t ream of n i t rogen, prior to adding the serum. Syn the t i c d ipa lmi toy l L-~-lecithin (No. 10070) was ob ta ined f rom General Biochemi- cals. Hydrogen peroxide , 30% solut ion, analyti- cal reagent grade, was ob ta ined f rom the Mallin- ckrodt Chemical Company .

TABLE I

Exposure of h u m a n serum to 0.1 M hydro - gen perox ide has been s h o w n by Clark et al. to result in a d is rupt ion o f the ~q ipop ro t e in pa t te rn on u l t racent r i fuga t ion (1). Glomset has shown tha t the plasma lec i th in-choles terol acyl- t ransferase react ion is associated wi th the same high dens i ty l ipopro te ins (2), a f inding which is co r robora ted by the a l terat ions in the l ipopro- rein pa t t e rn in the congeni ta l def ic iency of the enzyme (3-5).

The ef fec t of hydrogen peroxide on the acyl- t ransferase react ion was s tudied in vitro. Two milliliter samples of pooled h u m a n serum were incuba ted wi th labelled choles terol , and varying

1presentecl at the Aerospace Medical Association Annual Meeting, San Francisco, May 1969.

Variation in Response to Peroxide a

0.01 M 0.1 M No H20 2, H202, H202,

Experiment % % %

A 30.7 4.9 5.2 B 22.9 27.2 8.8 C 18.6 20.0 4.4 D 33.0 29.3 6.6 E 21.5 24.6 8.6 F 29.0 14.7 -- G 29.9 19.3 --- H 26.5 21.8 --- I 15.6 --- 1.8 J 12.8 --- 3.0

aEach experiment represents an individual or pooled sera. The results are expressed as per cent con- version of 4-14C-cholesterol to ester after 24 hr incu- bation at 37 C.

LIPIDS, VOL. 5, NO. 7