Henry Rakoff

Distribution of deuterium-labeledcis-andtrans-12-octadecenoic acids in human plasma and lipoprotein lipids

Triglycerides containingcis- andtrans-12-octadecenoic acid (12c-18∶1 and 12t-18∶1) andcis-9-octadecenoic acid (9c-18∶1) labeled with deuterium were fed to 2 young adult male subjects. These fatty isomers each contained a different number of deuterium labels, which allowed mass spectrometric analysis to distinguish among them after they were fed as a mixture. This approach results in a direct comparison of the absorption and distribution of these 3 monoenoic acids into blood plasma and lipoprotein lipids. Plasma lipid data indicated that all phospholipid fractions selectively incorporate 12c-18∶1 and 12t-18∶1 in preference to 9c-18∶1. Discrimination against 12c-18∶1 and 12t-18∶1 compared to 9c-18∶1 was found in the plasma neutral lipids, with a strong discrimination against 12t-18∶1 incorporation into the cholesteryl ester fraction. Considerable reduction in the percentage of linoleic and arachidonic acid was observed when 12–18∶1 isomers were incorporated in plasma triglyceride, phosphatidylcholine and sphingomyelin samples. Chylomicron lipid analyses indicated that all isomers were well absorbed. Variation was observed in the relative distribution of 12c-18∶1, 12t-18∶1 and 9c-18∶1 between the very low density, low density and high density lipoprotein lipid classes. No desaturation of 12c-18∶1 to linoleic acid was detected.

Metabolism in humans of cis-12,trans-15-octadecadienoic acid relative to palmitic, stearic, oleic and linoleic acids

Mixtures of triglycerides containing deuterium-labeled hexadecanoic acid (16∶0), octadecanoic acid (18∶0),cis-9-octadecenoic acid (9c–18∶1),cis-9,cis-12-octadecadienoic acid (9c, 12c–18∶2) andcis-12,trans-15-octadecadienoic acid (12c,15t–18∶2) were fed to two young-adult males. Plasma lipid classes were isolated from samples collected periodically over 48 hr. Incorporation and turnover of the deuterium-labeled fats in plasma lipids were followed by gas chromatography-mass spectrometry (GC-MS) analysis of the methyl ester derivatives. Absorption of the deuterated fats was followed by GC-MS analysis of chylomicron triglycerides isolated by ultracentrifugation.Results were the following: (i) endogenous fat contributed about 40% of the total fat incorporated into chylomicron triglycerides; (ii) elongation, desaturation and chain-shortened products from the deuterated fats were not detected; (iii) the polyunsaturated isomer 12c,15t–18∶2 was metabolically more similar to saturated and 9c–18∶1 fatty acids than to 9c,12c–18∶2 (iv) relative incorporation of 9c,12c–18∶2 into phospholipids did not increase proportionally with an increase of 9c,12c–18∶2 in the mixture of deuterated fats fed; (v) absorption of 16∶0, 18∶0, 9c–18∶1, 9c,12c–18∶2 and 12c,15t–18∶2 were similar; and (vi) data for the 1- and 2-acyl positions of phosphatidylcholine and for cholesteryl ester fractions reflected the known high specificity of phosphatidylcholine acyltransferase and lecithin:cholesteryl acyltransferase for 9c,12c–18∶2.These results illustrate that incorporation of dietary fatty acids into human plasma lipid classes is selectively controlled and that incorporation of dietary 9c,12c–18∶2 is limited. These results suggest that nutritional benefits of diets high in 9c,12c–18∶2 may be of little value to normal subjects and that the 12c,15t–18∶2 isomer in hydrogenated fat is not a nutritional liability at the present dietary level.

Preparation of fatty acids and esters containing deuterium.

INTRODUCTION A. Reaction sequences DEUTERIUM EXCHANGE A. Preparation of nonanal-2,2-d2 B. Preparation of tetradecanoic-2,2-d2 acid C. Preparation of perdeuterated octadecanoic acid D. Preparation of perdeuterated hexadecanoic acid MULTIPLE BOND DEUTERATION A. Reduction with Lindlars catalyst 1. Preparation of 7-chloro-cis-3-heptene 2. Preparation of deuterated Lindlars catalyst 3. Preparation of methyl cis-9-octadecenoate-9,10-d2 B. Reduction with Wilkinsons catalyst 1. Deuteration with tris(triphenylphosphine) chlororhodium 2. Preparation of methyl threo-12,13-dihydroxyoctadecanoate-9,10-d2 C. Deuterohydrazine reduction--preparation of methyl octadecanoate-9,10-d2 D. Hydroboration--preparation of methyl cis-9-octadecenoate-9,10-d2 E. Lithium aluminum hydride--preparation of trans-2-octen-7-ynol CHEMICAL REDUCTION A. Lithium aluminum deuteride reduction--preparation of eicosanol-l,l-d 2 B. Clemmensen reduction--Preparation of methyl octadecanoate-5,5,6,6,7,7-d 6 CHEMICAL SYNTHESIS A. Chain extension 1. With deuterated formaldehyde--preparation of 2-octynol-l,l-d2 2. Via the nitrile (a) Preparation of pentadecanenitrile-3,3-d2 (b) Preparation of pentadecanoic-3,3-d2 acid (c) Preparation of methyl cis-lO-nonadecenoate 3. With malonic ester (a) Preparation of docosanoic-3,3-d 2 acid (b) Preparation of cis-I l-eicosenoic acid (c) Preparation of cis-9,cis-12-octadecadienyl methanesulfonate B. Anodic coupling--preparation of tetradecanoic-10,10-d2 acid C. Acetylenic coupling 1. Preparation of 3-nonynol-7,7,8,8-d4 2. Preparation of 2-(9-decynyloxy)-tetrahydropyran 3. Preparation of 4-decynoic acid 4. Preparation of 10-tetradecynoic acid 5. Preparation of 2-(8-nonen-5-ynyloxy)-tetrahydropyran 6. Preparation of 11-dodecen-9-ynyl acetate D. Wittig reaction 1. Preparation of methyl cis-9-octadecenoate-14,14,15,15,17,18-d6 2. Preparation of methyl 12,15-octadecadienoates-9,10-d 2 (a) Without stereoselective control (b) With stereoselective control 3. Preparation of phosphonium salts (a) Phosphonium bromides--preparation of cis-3-hexenyltriphenylphosphonium bromide (b) Phosphonium iodides 4. Preparation of halides (a) Iodide from chloride--preparation of 1-iododecane-4,4,5,5-d4 (b) Iodide from tetrahydropyranyl ether--preparation of 1-iodononane-5,5,6,6,8,9-d6 (c) Preparation of tetrahydropyranyl ethers (d) Bromide from alcohol with PBra--preparation of l-bromononane-2,2-d 2 (e) Bromide from alcohol with triphenylphosphine dibromide (1) Preparation of l-bromo-cis-3-hexene (2) Preparation of l-bromo-cis-3-nonene-7,7,8,8-d4

Partial argentation resin chromatography (PARC): I. Effect of percent silver on elution and separation of methyl octadecadienoate isomers

Partial argentation resin chromatography (PARC) for the separation of octadecadienoate ester isomers was investigated. In comparison to saturated silver resin chromatography, the time necessary to elute methylcis,cis-octadecadienaotes was dramatically shortened when columns containing sulfonic acid ion exchange resin silvered in the range of 60~90% of theoretical (meaning 60~90% of the sulfonic acid protons in the resin were replaced by silver ions) were used. Methods for preparation and silvering of the resin are discussed. The XN1010 resin (Rohm and Haas) was analyzed for total sulfonic acid groups and the amount of silver that can be incorporated by one or 2 treatments with silver nitrate was determined. A series of partially silvered resin columns was prepared and samples of methyl linoleate were eluted to study the effect of the percentage silvering on elution volumes and peak shapes. Twenty-gram samples of mixtures ofcis,trans- andtrans,trans- and oftrans,cis- andcis,cis-methyl 12,15-octadecadienoates were separated on a 91% PARC (91% silvered) column.

Synthesis and properties of methyl 9,12,15-octadecatrienoate geometric isomers

Abstract The eight geometrically isomeric methyl 9,12,15-octadecatrienoates were prepared by using the Wittig reaction to couple cis- or trans-3-hexyenyltriphenylphosphonium bromide and methyl 12-oxo-cis- or trans-9-dodecenoate. Pairs of geometric triene isomers formed were separated by partial silver resin chromatography. Physical constants including melting points, percent trans by infrared, equivalent chain lengths (ECL), and 13C nuclear magnetic resonance (NMR) chemcial shifts are tabulated for the individual isomers.

δ8 desaturationin vivo of deuterated eicosatrienoic acid by mouse liver

In vitro evidence has been reported for an alternate pathway that involves δ8 desaturation of n-6 and n-3 polyunsaturated fatty acids (PUFA). The present study was designed to allow detection of δ8 desaturationin vivo and to provide an estimation of the relative contribution of δ8 desaturation to thein vivo synthesis of n-3 fatty acids. Male adult ICR mice were fed a semisynthetic fat-free diet for eight days, and then the diets were supplemented for three days with deuterated 11,14,17-eicosatrienoic acid (20:3-d8) labeled at the 3,3,4,4,8,8,9,9 carbon positions. Analysis of liver total lipid by gas chromatography/mass spectroscopy indicates that the total deuterated fatty acids contained 22.3% 20:3n-3-d8 and 28.9% of metabolites formed by elongation and δ5 desaturation of 20:3n-3-d8. Deuterated metabolites resulting from retroconversion to 18:3-d6 and subsequent metabolism by classical pathways represented 35.3% of the total deuterated fatty acids. The retroconversion product (18:4n-3-d6) of 20:4n-3-d6 and/or-d8 was 9.0% of the total. A minor percentage (4.4%) of the products identified (20:4n-3-d6, 20:5n-3-d6, 22:5n-3-d6, 22:6n-3-d5 and 24:6n-3-d5) were formed by δ8 desaturation. This study provides the first clear evidence of δ8 desaturationin vivo in the mouse liver. Whether δ8 desaturation would have a greater importancein vivo when the δ6 desaturase pathway is disrupted remains to be determined.

Preparation of methylcis-9,cis-12,cis-15-octadecatrienoate-15,16-d 2 and methylcis-9,cis-12,cis-15-octadecatrienoate-6,6,7,7-d 4

Methylcis-9,cis-12,cis-15-octadecatrienoate-15,16-d2 was obtained from Wittig coupling of methyl 12-oxo-cis-9-dodecenoate,18, and 3,4-dideutero-cis-3-hexenyltriphenylphosphonium bromide,16. Compound18 was obtained by periodic acid oxidation of methyl 12,13-dihydroxy-cis-9-octadecenoate,17, obtained fromVernonia oil. Compound18 also was synthesized from methyl oleate as the starting material. The deuterated fragment,16, was prepared from 3-hexynol and using Lindlars catalyst and deuterium gas to introduce the deuterium atoms.Methylcis-9,cis-12,cis-15-octadecatrienoate-6,6,7,7-d4 was prepared by Wittig coupling of 3,6-nonadienyltriphenylphosphonium iodide,5, with methyl 9-oxononanoate-6,6,7,7-d4,11. Deuterium atoms were introduced during the synthesis of11 from 3-butynol and 5-bromopentanoic acid with deuterium gas in the presence of [Ph3P]3-RhCl. For the preparation of5, the 3,6-nonadiynol intermediate was reduced to 3,6-nonadienol with P-2 Nickel and hydrogen.The final products were separated from isomers formed during the synthetic sequences by silver resin chromatography.

13C nuclear magnetic resonance of mono-and dihydroxy saturated and unsaturated fatty methyl esters

Abstract13C nuclear magnetic resonance spectra were obtained for methyl esters oferythro- andthreo-9,10-dihydroxystearates, for 12-hydroxy-cis- andtrans-9-octadecenoates, and forthreo-12,13-dihydroxy-cis-andtrans-9-octadecenoates.Erythro andthreo compounds may be distinguished easily by the difference in the chemical shifts of the carbons alpha to the hydroxy-bearing carbons. Monohydroxy compounds are easily distinguished fromvicinal dihydroxy compounds by differences in chemical shifts of both the hydroxy-bearing carbons and of the carbons alpha to them. The presence of a hydroxy-bearing carbon beta to a double bond results in the two carbons of the double bond of a hydroxy-bearing carbon beta to a double bond results in the two carbons of the double bond having different chemical shifts, with the numerical values being different for thecis andtrans isomers. The chemical shift of a carbon alpha to both a doubly bonded carbon and a hydroxy-bearing carbon is influenced both by the geometry of the double bond and the number of hydroxy-bearing carbons.

Preparation of deuterated methyl 6,9,12-octadecatrienoates and methyl 6,9,12,15-octadecatetraenoates

Methyl 6,9,12-octadecatrienoate-15,15,16,16-d4 was obtained by Wittig coupling between 6,6,7,7-tetradeutero-3-nonenyltriphenylphosphonium iodide, 8, and the aldehyde ester, methyl 9-oxo-6-nonenoate. Methyl 6-oxohexanoate, obtained by ozonolysis of cyclohexene, was coupled in a Wittig reaction with [2-(1,3-dioxan-2-yl)ethyl]triphenylphosphonium bromide to give methyl 8-dioxanyl-6-octenoate. This compound was transacetalized to methyl 9,9-dimethoxy-6-nonenoate, which was then hydrolyzed to the aldehyde ester. For the preparation of compound 8, the tetrahydropyranyl ether of 2-pentynol was deuterated with deuterium gas and tris-(triphenylphosphine)chlororhodium. The tetradeuterated tetrahydropyranyl ether was converted to the bromide with triphenylphosphine dibromide, and the bromide was coupled with 3-butynol by means of lithium amide in liquid ammonia to give 3-nonynol-6,6,7,7-d4. Hydrogenation over Lindlars catalyst converted the deuterated alkynol to 3-nonenol-6,6,7,7-d4. This deuterated alkenol was converted to the bromide with triphenylphosphine dibromide, then to the iodide with sodium iodide in acetone, and finally to 8 with triphenylphosphine in acetonitrile. Methyl 6,9,12,15-octadecatetraenoate-12,13,15,16-d4 was obtained by Wittig coupling between methyl 9-oxo-6-nonenoate and 3,4,6,7-tetradeutero-3,6-nonadienyltriphenylphosphonium iodide, 15. For the preparation of compound 15, the bromide obtained from the reaction of 2-pentynol with triphenylphosphine dibromide was coupled with 3-butynol with lithium amide in liquid ammonia. The resulting 3,6-nonadiynol was deuterated with deuterium gas in the presence of P-2 nickel, and the resultant deuterated nonadienol was converted to 15 through the bromide and iodide. The final products were separated from isomers formed during the synthetic sequences by silver resin chromatography.

Silver resin chromatographic separation of methyl cis- and trans- mono- and dihydroxy fatty esters

Column chromatography on silver ion-saturated Amberlyst XN 1010 cation exchange resin gave very good separation of a mixture of methyl 12-hydroxy-cis-andtrans-9-octadecenoates and of methylthreo-12, 13-dihydroxy-cis- andtrans-9-octadecenoates. Comparison of the retention volumes of nonhydroxy, monohydroxy, and dihydroxy saturated and monoenoic methyl esters and of dienoic methyl esters shows that the hydroxy group interacts with the column packing to slow passage of the compound through the column, although the effect of a hydroxy group is less than that of atrans double bond. The effects of the hydroxy groups are additive; the ratio of retention volumes of dihydroxy ester to monohydroxy ester is slightly larger that that of monohydroxy ester to nonhydroxy ester. The retention volume of a cis monoenoic ester is equal to that of a hydroxytrans monoenoic ester and that of a hydroxycis monoenoic ester is equal to that of a dihydroxytrans monoenoic ester.