Dual-Labeled Technique for Human Lipid Metabolism ... - naldc - USDA

Reprinted from LIPIDS, Vol. 11, No.2, Pages: 135-142 (1976)

Dual-Labeled Technique for Human Lipid Metabolism Studies Using Deuterated Fatty Acid Isomers' E.A. EMKEN, W.K. ROHWEDDER, and H.J. DUTTON, Northern Regional Research Laboratory, ARS, USDA, Peoria, Illinois 61604, R. DOUGHERTY and J.M. IACONO, Lipid Nutrition Laboratory: Nutrition Institute, ARS, USDA, Beltsville, Maryland 20705, and J. MACKIN, Department of EndOCrinology, Georgetown Medical School, Washington, DC 20005

ABSTRACT

Two deuterated fatty acids, elaidated 2 and oleate-d 4 , were fed simultaneous-

ly to a human subject as a mixture of trielaidin-d 6 and triolein-d 12' Periodically, blood samples were drawn, and red blood cells were separated from the plasma. Red blood cells and plasma lipids were fractionated and analyzed by combined gas chromatography-multiple ion mass spectroscopy. Dual deuterium-labeling allows rate and extent of fatty acid incorporation to be followed in various plasma and red cell neutral and phospholipid fractions. Maximum amount of deuterated fat varied from 4% in cholesterol ester to 64% in phosphatidyl ethanolamine. The highest levels of deuterated fat occurred in either 6-, 8-, or 12-hr samples; generally, .. 30

/ x

Monoene in Sample

20 10

of"T-"'T""T.,--,--,---=r--===;=--F=~ 024681216

24 32 Time Ihr)

40

48

FIG. 2. Incorporation of EI-d 2 (elaidate-d 2 ) and OI-d 4 (oleate-d 4 ) into plasma free fatty acids.

and then returned to 44% in the 24-hr sample. The 6-hr plasma FFA fraction contained the maximum amount (64.2%) of deuterated fat found in any neutral lipid or phospholipid fraction. Total deuterated fat increased rapidly bu t cleared just as rapidly from the plasma FFA (Fig. 2). Initially, OI-d4 and El-d 2 are incorporated into plasma FFA at similar rates, but El-d 2 is incorporated considerably more than LIPIDS, VOL. 11, NO.2

E.A. EMKEN ET AL.

138

70 r----:--------------, Mixture Fed: 48.7% EI·d2 51.3% 01·d4 o

porated into all plasma phospholipids Extent of incorporation varied from a maximum of 61.8% in PE down to 11 % in SM. 60 The GS-MIMS analyses of plasma PE fraclabel in Monoene tions are plotted in Figure 3. The 6-hr plasma 50 PE fraction contained the largest percentage of deuterated fat (61.8%) of any phospholipid fraction. Because deuterated monoene in 6- and -= 40 8-hr PE fractions contained about 60% El-d 2 ... ~ compared to 48.7% in the mixture fed, prefer~ 30 ential incorporation of El-d 2 over Ol-d 4 is indicated. El-d 2 increased to 85% of the deuterated monoene in the 24-hr PE sample due to 20 a more rapid removal of 0l-d 4 from plasma PE. Total monoene in plasma PE increased from 9.1 % (0 hr) to 23.8% (6 hr) and then decreased to 5.6% in the 24-hr sample. Total monoene curve in Figure 3 resembles the curve showing the total deuterated fat content of the samples. 40 24 32 48 Obviously, this increase in total monoene occurs because deuterated monoene has been Time Ihr] incorporated into plasma PE. FIG. 3. Incorporation of EI-d 2 (elaidate-d 2 ) and In Figure 3, the peaks of the curves repreOI-d 4 (oleate-d 4 ) into plasma phosphatidyl ethanolamine. senting labeled fatty acids have been drawn on the basis that these curves must fit a continuous 0l-d 4 in the 6-hr sample. Data on the 12- and mathematical function. 24-hr samples indicate that El-d 2 is cleared Analysis of the plasma PS + PI samples is from FFA faster than Ol-d 4 . Octadecenoate given in Table II. The PS + PI 12-hr sample may also increased from an initial 34% to 54% in the have contained a higher percentage of deuter6-hr sample and then decreased to 17% in the ated fat than the 8-hr fraction (39.2%), but the 48-hr sample. PS + PI 12-hr sample was lost during processing. Relatively low levels of deuterated fat were A selective incorporation of 0l-d 4 was noted in found in all plasma. CE samples compared to plasma PS + PI. The 6- and 8-hr samples conplasma phospholipids. The 24-hr plasma CE tained 60.7% and 57.0% Ol-d 4 compared to fraction contained the most deuterated fat 51.3 % Ol-d 4 in the mixture fed. The percent(4.1%), of which 85.4% was 0l-d 4 . Thus the ages for Ol-d 4 and El-d 2 (Table II) indicate enzymes, such as phosphatidyl choline:choles- both fatty acids were removed at similar rates terol acyltransferase (EC 2.3.1.43) and acyl- from the PS + PI fraction. CoA:cholesterol O-acyltransferase (EC Human blood plasma phospholipids contain 2.3.1. 26), which are responsible for cholesterol ca. 70% PC (26). Selection of El-d over 0l-d 4 2 esterification appear to be less active but much in plasma PC is apparent from the curves in more selective than those enzymes responsible Figure 4. The percentage of El-d in PC is 2 for phospholipid formation. 9-11 % higher than the 48.7% El-d 2 in the Plasma Phospholipids mixture fed. The rate and extent of incorporaDeuterated oleate and elaidate were incor- tion of deuterated oleate and elaidate into

~

TABLE II Analysis of Phosphatidyl serine and Phosphatidyl Inositol Samples a from Human Blood Plasma Phospholipids Sample time (hr)

Total label in monoene (%)

1 4 6 8 24 48

0.0 0.0 26.5 39.2 24.5 3.6

EI-d2 in label (%)

39.2 43.0 46.4 43.1

(%)

Total monoene in sample (%)

60.8 57.0 53.6 56.9

11.3 13.1 14.9 15.0 12.4 10.4

01·d4 in label

aMixture fed: 48.7% EI-d2 (elaidate-d2) and 51.3% 01-d4 (oleate-d4).

LIPIDS, VOL. 11, NO.2

METABOLISM OF ELAIDATE AND OLEATE

plasma PC were slower than into plasma PE. Maximum deuterated monoene content in PC was 24.8% for the l2-hr sample compared to 61.8% for the 6-hr PE sample. Clearing of deuterated fat from plasma PC was also slower than from plasma PE. The 24-hr PC sample still contained 15.5% labeled monoene, whereas the 24-hr PE sample contained 7.8% labeled monoene. This difference indicates that the 8-hr PE fraction, which hau more than three times as much deuterated fat as the 8-hr PC fraction, clears deuterated fat much more rapidly than the PC fraction. GC analysis of plasma TG, FFA, and PE showed that the percentage total monoene increased as the percentage deuterated fat increased. This correlation was not so dramatic in PC samples. The percentage total monoene in PC remained constant between 18.5-21.5%, whereas the percentage deuterated fat increased from 0.0 to 24.8% in the l2-hr sample. These results demonstrate that deuterated monoenes are directly replacing nondeuterated monoenes. Phospholipase-A was used to hydrolyze the fatty acid in the 2 position of plasma PC. Analysis of the remaining fatty acid in the 1 position of plasma PC is given in Figure 5. Preferential incorporation of El-d 2 into the 1 position of PC, as seen in Figure 5, has been observed in other studies (27,28). A maximum of 35% deuterated fat was found in the 1 position of the 12-hr PC fraction (PC-I), which is 11 % more label than was in the total PC sample. Because El-d 2 in the 8-, 12-, and 24-hr PC-l samples averaged ca. 75%, elaidate incorporation is mainly in the I-position. GC analysis of PC-l did not indicate a major replacement of saturates by EI-d 2 as others (29) have reported; instead, unlabeled monoene was apparently replaced by El-d 2 . GC-MIMS data were not consistently satisfactory for the fatty acids obtained by phospholipase-A hydrolysis of PC samples. The fatty acids in the 2 position of the l2-hr PC sample

139

50 r - - - - - - - - - - - - - - , Mixture Fed: 48.7% EI·d2 51.3% OI·d4 40 I-

8 12

024 6

16

I

I

40

48

FIG. 4. Incorporation of EI-d 2 (elaidate-d2 ) and OI-d 4 (oleate-d 4 ) into plasma phosphatidyl choline.

50r-------------....., Mixture Fed: 48.7% EI·d2 51.3% 01·d4

401-

r

0

o

_301-

~

'" Q.

~ Total label in Mcnoene

f, r-""',~I-d2 ~ ~ 0~

... ,,~ / Monoene '-"'-" /':'i in Sample \ "'" ~x"" c:;'x",.-"'x-----x__ -0 101----....:::~ o-.- o _._. 01.f :':';"-o-._._._._._._._

20 I-

t 1/

4

.0

o"'";'-~"

I

I

02468 12 16

'---'11)

I

I

24 32 Time [hr)

I

I

40

48

FIG. 5. Incorporation of EI-d 2 (elaidate-d 2) and OI-d 4 (oleate-d 4 ) into the one position of plasma phosphatidyl choline.

(PC-2) contained 15.9% total deuterated fat, of which 56% was 0l-d 4 . This 56% 0l-d 4 is 4.7% higher than that fed and demonstrates that

TABLE III Analysis of Lysophosphatidyl Choline Samples a from Plasma Phospholipids Sample time (hr) 0 2 4 6 8 12 24 48

Total label in monoene (%) 0.5 5.1 13.0 21.0 19.5 9.6 6.0

EI- 60% deuterated monoene was incorporated into these fractions 6 hr after ingestion of the deuterated fat. In contrast, the PC fraction, which constitutes ca. 70% of plasma phospholipids, contained a maximum of only 25% deuterated monoene 12 hr after ingestion of labeled fat. Such slow incorporation and slow clearing of the labeled monoene from PC reflect either its less active role in metabolic processes or a large PC pool in body tissue. Analysis of phospholipase-A treated PC showed that plasma-PC-l fractions were unique. These fractions were the only set of samples that showed a strong preference for incorporation of El-d 2 but did not show a large increase in total monoene content. The change in El-d 2 :01-d 4 ratio in the lyso-PC fractions

141

shown in Table III may be caused by an experimental artifact or may reflect an actual selective incorporation of Ol-d 4 at low monoene concentrations. Lyso-PC data illustrate that small differences in metabolism of similar compounds can be compared by dual deuterium labeling. Normally these extremely small differences at low levels of isotope incorporation would be lost because of experimental variation if the labeled fats were fed separately. Good GC-MIMS data were not obtained for all PC-2 and SM fractions because of insufficient sample size or extraneous and interfering impurities. Such problems are not expected to recur and should not be a factor in future experiments. RBC lipids incwporated little labeled fat into RBC membrane, although esterification or exchange of human RBC lipids with plasma lipids has been reported (30) and mature erythrocytes from rats are known to incorporate labeled fat into their phospholipids (31). The RBC-TG fraction was the only RBC lipid that contained appreciable amounts (23% maximum) of deuterated fat. Other RBC lipid fractions had < 5% deuterated fat, an amount which limits the effectiveness of dual deuterium labeling for following lipid incorporation into RBC. In summary, we have demonstrated successfully that human metabolism of two fatty acid isomers can be studied directly under identical biological and experimental conditions. The natural fatty acid isomer, oleic acid, was fed as triolein and compared with the unnatural fatty acid isomer, elaidic acid, fed as trielaidin. Total monoene content of plasma lipids paralleled the total deuterated fat incorporation. FFA, TG, and PE fractions contained the largest amounts of deuterated fat. PE and the one acyl position of PC were the most selective fractions for preferential accumultion of El-d 2 . The PS + PI fraction was the most selective for Ol-d 4 incorporation. Comparisons made between the metabolism of elaidate and oleate in human plasma and RBC are intended only to demonstrate how dual deuterium labeling can be applied to solve the problem of human lipid metabolism. Data from additional experiments will be presented in a subsequent paper and oleate-elaidate metabolism in human blood lipids will be compared in detail. ACKNOWLEDGMENTS W. L. Everhart provided technical aid involving GCr-.UMS analyses and D.l. Wolf assisted in computer programming.

LIPIDS, VOL. 11, NO.2

142

E.A. EMKEN ET AL. REFERENCES

1. Mounts, T.L., E.A. Emken, W.K Rohwedder, and H.J. Dutton, Lipids 6:912 (1971). 2. Okita, G.T., and J.L. Spratt, in HTritium in the Physical and Biological Sciences," Vol. II, Edited by the International Atomic Energy Agency, Vienna, Austria, 1962, p. 85. 3. Steinberg, J.S., and M. Gold, Lipids 5:988 (1970). 4. LeKim, D., H. Betzing, and W. Stoffel, HoppeSeyler's Z. Physiol. Chern. 353:949 (1972). 5. Langley, R., "Practical Statistics Simply Explained," Dover Publications, Inc., New York, NY, 1971, p. 117. 6. Butterfield, R.O., and H.J. Dutton, JAOCS 45:635 (1968). 7. Lindlar, H., Helv. Chim. Acta 35:446 (1952). 8. Osbond, J.M., P.G. Philpott, and LC. Wickins, J. Chern. Soc. 2779 (1961). 9. Litchfield, C., R.D. Harlow, A.F. Isbell, and R. Reiser, JAOCS 42:78 (1965). 10. Emken, E.A., C.R. Scholfield, and H.J. Dutton, Ibid. 41:388 (1964). 11. Emken, E.A., Lipids 7:459 (1972). 12. Stein, R.A., and N. Nicolaides, J. Lipid Res. 3:476 (1962). 13. Wheeler, D.H., R.W. Riemenschneider, and C.E. Sando, J. BioI. Chern. 132:687 (1940). 14. Mikolajczak, KL., C.R. Smith, Jr., M.O. BagbY, and LA. Wolff, J. Org. Chern. 29:318 (1964). 15. Robertson, P.W., W.E. Dasent, R.M. Milburn, and W.H. Oliver, J. Chern. Soc. 1628 (1950). 16. Jackson, J.E., and W.O. Lundberg, JAOCS 40:276 (1963). 17. Dutton, H.J., C.R. Scholfield, E. Selke, and W.K Rohwedder, J. Catal. 10:316 (1968). 18. Thomas, A.F., "Deuterium Labeling in Organic Chemistry," Appleton-Century-Crofts, New York,

LIPIDS, VOL. 11, NO.2

NY, 1971, p. 290. 19. Rohwedder, W.K, D.L Wolf, and W.L. Everhart, "G as Chromatography-Mass Spectrometry of Deuterium-Labeled Blood Lipids by Multiple Ion Monitoring," presented at the North Central Regional ACS meeting, West Lafayette, IN, June 3-5,1974. 20. Enselme, J., "Unsaturated Fatty Acids in Atherosclerosis," Vol. 16, International Series of Monographs on Pure and Applied Biology - HDivision: Modern Trends in Physiological Sciences," MacMillan Co., New York, NY, 1962, p. 43. 21. Ways, P., C.F. Reed, and D.L Hanahan, J. Clin. Invest. 42: 1248 (1963). 22. Folch, J., M. Lees, and G.H. Sloane-Stanley, J. BioI. Chern. 226:497 (1957). 23. Mangold, H.K, and D.C. Malins, JAOCS 37:383 (1960). 24. Skipski, V.P., R.F. Peterson, and M. Barclay, Biochern. L 90:374 (1964). 25. Nutter, L.J., and O.S. Privett, Lipids 1:258 (1966). 26. Phillips, G.B., and J.T. Dodge, J. Lipid Res. 8:676 (1967). 27. Lands, W.E.M., M.L. Blank, L.J. Nutter, and O.S. Privett, Lipids 1:224 (1966). 28. Selinger, Z., and R. T. Holman, Biochim. Biophys. Acta 106:56 (1965). 29. Okuyama, H., W.E.M. Lands, F.D. Gunstone, and J.A. Barne, Biochemistry 11 :4392 (1972). 30. Winterbourn, C.c., and R.D. Batt, Biochim. Biophys. Acta 202:9 (1970). 31. Mulder, E., and L.L. Van Deenen, Ibid. 106: 106 (1965).

[Received September 8, 1975]