Changes induced by sarcolysine in vivo in the composition of DNA-bound lipids in sarcoma 37

The two DNA fractions were isolated from sarcoma 37 by the use of the phenol method: supramolecular complex of DNA (SC DNA, 60%) and "phenol" nuclear matrix DNA (PNM DNA, 40%). The lipids in SC DNA represented of light and tightly bound components, the latter was similar to the lipid composition of PNM DNA. SC DNA contains 20 micrograms of neutral lipids (NL) and 6.5 micrograms of phospholipids (PL), while PNM DNA contains 9.8 micrograms of NL and 3.5 micrograms of PL per mg DNA. SC DNA-bound lipids of sarcoma 37 are deficient in free cholesterol (FC, 13%), but rich in cholesterol esters (CE, 39%) and free fatty acids (FFA, 23%); very rich in cardiolipin (CL, 43%) and phosphatidylethanolamine (PE, 28%), but deficient in phosphatidylcholine (PC, 12%). The tumor contains triglycerides (TG) that is absent in DNA of the normal cells. The injection of sarcolysine (10 micrograms/kg) markedly increased (1.5-3 times) the content of all LN and PL fractions in SC DNA, which was accompanied by both the accumulation of FC, TG, PC and the reduction of the remaining lipid fractions in PNM DNA. It is supposed, that DNA-bound lipids may be the target for the action of sarcolysine.     

DNA-bound lipids from eukaryotic (loach spermatozoa, pigeon erythrocytes) and prokaryotic (E. coli B, phage T2) cells

Using thin-layer chromatography, some specific DNA-bound neutral lipids and phospholipids of loach spermatozoa, pigeon erythrocytes, E. coli B and phage T2 cells were studied. These lipids are represented by loosely and firmly bound components. The content of neutral lipids in the above DNAs (per mg of DNA) is 10.6, 4.8, 7.81 and 1.43 micrograms, respectively; that of phospholipids is 4.31, 1.28, 1.14 and 0.54 micrograms, respectively. The eucaryotic DNA-bound lipids are highly deficient of free cholesterol, phosphatidylcholine, phosphatidylinositol and phosphatidylserine but are rich in cardiolipin, phosphatidylethanolamine, cholesterol esters, diglycerides and free fatty acids. The quantitative and qualitative composition of DNA-bound lipids of loach spermatozoa changes during the transition from the superhelical to the relaxed conformation of DNA. Procaryotic DNA-bound neutral lipids are also represented by the free cholesterol, diglyceride and free fatty acid fractions, whereas the DNA-bound phospholipids of procaryotes consist of only two fractions, i.e., cardiolipin and phosphatidylethanolamine. The role of DNA-bound lipids in the structural and functional organization of eucaryotic and procaryotic genomes is discussed.     

Regulation of hepatic secretion of very low density lipoprotein by dietary cholesterol.

Male rats were fed a cholesterol-free diet or the same diet supplemented with either 0.05, 0.1, 0.25, 0.5, 1, or 2% C for 21 days to investigate the effects of cholesterol on secretion of very low density lipoprotein (VLDL). Cholesterol feeding increased plasma and hepatic concentrations of triglyceride (TG) and cholesteryl esters (CE) in a dose-dependent manner. Plasma VLDL and low density lipoprotein (LDL) lipids were elevated by cholesterol feeding, while the high density lipoprotein (HDL) lipids were reduced. The secretion of the VLDL by perfused livers from these cholesterol-fed rats was examined to establish the relationship between the accumulation of lipids in the liver and the concurrent hyperlipemia. Liver perfusions were carried out for 4 h with a medium containing bovine serum albumin (3% w/v), glucose (0.1% w/v), bovine erythrocytes (30% v/v), and a 10-mCi 3H2O initial pulse. Oleic acid was infused to maintain a concentration of 0.6 mM. Hepatic secretion of VLDL-TG, PL (phospholipid), free cholesterol (FC), and CE increased in proportion to dietary cholesterol and was maximal at 0.5% cholesterol in these experiments in which TG synthesis was stimulated by oleic acid. Secretion of VLDL protein and apoB by the perfused liver was also increased. The molar ratios of surface (sum of PL and cholesterol) to core (sum of TG and CE) lipid components of the secreted VLDL, regardless of cholesterol feeding, were the same, as were the mean diameters of the secreted particles. The molar ratios of surface to core lipid of VLDL isolated from the plasma also were not affected by cholesterol feeding. During perfusion with oleic acid of livers from the rats fed the higher levels of cholesterol, the hepatic concentration of CE decreased, while the level of TG was not changed. We conclude that the hypercholesterolemia and hypertriglyceridemia that occur in vivo from cholesterol feeding, concurrent with accumulation of CE and TG in the liver, must result, in part, from increased hepatic secretion of all VLDL lipids and apoB. The VLDL particles produced by the liver of the cholesterol-fed rat are assembled without modification of the surface lipid ratios (PL/FC), but contain a greater proportion of cholesteryl esters compared to triglyceride in the core, because of the stimulated transport of CE from the expanded pool in the liver.(ABSTRACT TRUNCATED AT 400 WORDS)     

Accumulation of cholestatic lipoproteins in ANIT-treated human apolipoprotein A-I transgenic rats is diminished through dose-dependent apolipoprotein A-I activation of LCAT

Administration of alpha-naphthylisothiocyanate (ANIT) to rats induces changes to plasma lipids consistent with cholestasis. We have previously shown (J. Lipid Res. 37 (1996) 1086) that animals treated with ANIT accumulate large amounts of free cholesterol (FC) and phospholipid (PL)-rich cholestatic lipoproteins in the LDL density range by 48 h. This lipid was cleared by 120 h through apparent movement into HDL with concomitant cholesteryl ester (CE) production. It was hypothesised that the clearance was mediated through the movement of the PL and FC into apolipoprotein A-I (apo A-I) containing lipoproteins followed by LCAT esterification to form CE. To test this hypothesis, rats overexpressing various amounts of human apo A-I (TgR[HuAI] rats) were treated with ANIT (100 mg/kg) and the effect of plasma apo A-I concentration on plasma lipids and lipoprotein distribution was examined. In untreated TgR[HuAI] rats, human apo A-I levels were strongly correlated to plasma PL (r(2)=0. 94), FC (r(2)=0.93) and CE (r(2)=0.90), whereas in ANIT-treated TgR[HuAI] rats, human apo A-I levels were most strongly correlated to CE levels (r(2)=0.80) and an increased CE/FC ratio (r(2)=0.62) and the movement of cholestatic lipid in the LDL to HDL. Since LCAT activity was not affected by ANIT treatment, these results demonstrate that the ability of LCAT to esterify the plasma FC present in cholestatic liver disease is limited by in vivo apo A-I activation of the cholestatic lipid and not by the catalytic capacity of LCAT.     

A method of direct measurement for the enzymatic determination of cholesteryl esters

A direct measurement method for the enzymatic determination of cholesteryl esters (CEs) without measuring total cholesterol (TC) and free cholesterol (FC) is described. In the first step, hydrogen peroxide generated by cholesterol oxidase from FC was decomposed by catalase. In the second step, CE was measured by enzymatic determination using a colorimetric method or a fluorometric method. The measurement sensitivity of the fluorometric method was more than 20 times that of the colorimetric method. Optimal conditions of the assay were determined, and examples of measured CE in human plasma, rat liver, and cultured cells are indicated. The method of directly measuring CE was simple and has exceptional reproducibility compared with the technique of subtracting FC from TC using each measured TC and FC.     

Metabolomics (liver and blood profiling) in a mouse model in response to fasting: a study of hepatic steatosis

A metabolomic approach was applied to a mouse model of starvation-induced hepatic steatosis. After 24 h of fasting it appears that starvation reduced the phospholipids (PL), free cholesterol (FC), and cholesterol esters (CE) content of low-density lipoproteins (LDL). In liver lipid profiles major changes were observed using different techniques. High performance thin layer chromatography (HPTLC)-measurements of liver-homogenates indicated a significant rise of FC with 192%, triacylglycerols (TG) with 456% and cholesterol esters (CE) with 268% after 24 h of starvation in comparison with the control group. Reversed phase liquid chromatography coupled to mass spectrometry measurements (LC-MS) of liver homogenate indicated that the intensity of Phosphatidylcholine (PC) in the 24-h starvation group dropped to 90% of the value in the control group while the intensity of CE and TG increased to 157% and 331%, respectively, of the control group. Interestingly, a 49:4-TG with an odd number of C atoms appeared during starvation. This unique triacylglycerol has all characteristics of a biomarker for detection of hepatic steatosis. These observations indicate that in mammals liver lipid profiles are a dynamic system which are readily modulated by environmental factors like starvation.     

Changes in the composition of phospholipids bound to supramolecular DNA of the thymus and liver in gamma-irradiated rats

The composition of supramolecular DNA (SM DNA)-bound phospholipids (PL) of thymus and liver of intact rats and those 2 min, 2, 6 and 24 h after gamma-irradiation (9,7 Gy) was studied. In norm, supramolecular DNA of the thymus was shown to contain 6.7 micrograms PL/mg DNA, and that of the liver, 6.1 micrograms PL/mg DNA, the main components of PL being cardiolipin (CL) and phosphatidylethanolamine (PEA). Substantial changes were detected in the PL composition of SM DNA of gamma-irradiated rat organs. During the postirradiation period the concentration of PEA and CL in thymus SM DNA changed symbatically and irreversibly decreased to traces; whereas in SM DNA of the liver, their concentrations changed antibatically and decreased only to a definite level thus maintaining the necessary "lipid volume". It was shown that PL were not restored in SM DNA of the radioresistant liver.     

Lipid and fatty acid composition of female gilthead seabream during their reproductive cycle: effects of a diet lacking n-3 HUFA

In female sea bream Sparus aurata fed a control diet (C), ovarian levels of neutral lipids (NL) and polar lipids (PL) remained constant between November and March, while a decrease in NL content was observed in liver and muscle. In the same period, liver PL content increased, while no changes were observed in muscle. Between March and June ovarian NL and PL showed a strong decrease, while NL remained constant in liver and muscle. When fish were fed a diet lacking in n-3 highly unsaturated fatty acids, n-3 HUFA (D), the pattern observed was similar to that found in the fish fed diet C, with the exception of liver NL, which increased between March and June. In general, the changes in fatty acid content, in both groups of fish, were highly influenced by the diet given to the broodstock, although these effects were greater on ovarian NL and PL than on liver and muscle lipids. Despite the fact that gilthead seabream females continue feeding during the spawning season, they probably make use of their liver and muscle reserves during the gonadal maturation process. Furthermore, the fatty acid composition of the broodstock diet was reflected in the body composition, especially in the ovaries.     

Intestinal apoB synthesis, lipids, and lipoproteins in chylomicron retention disease

Chylomicron retention disease is characterized by fat malabsorption, hypocholesterolemia, normal fasting triglycerides, and marked intestinal steatosis despite the presence of both plasma and intestinal apoprotein B. The defect remains unknown but presumably involves the synthesis or secretion of chylomicrons. The present investigation examines this hypothesis by studying the biosynthesis of chylomicrons in cultured jejunal explants and by defining the quantitative and qualitative abnormalities of plasma lipids and of circulating lipoproteins. Following 2-3 years of a low fat diet supplemented with medium chain triglycerides, six patients with chylomicron retention disease had significantly higher triglyceride (TG) levels coupled with a decrease in both free (FC) and esterified cholesterol (EC) as well as in essential fatty acids and phospholipids (PL) when compared to healthy controls. The low total plasma cholesterol was largely accounted for by low levels of both low density (LDL) and high density lipoprotein (HDL) cholesterol. VLDL and LDL were characterized by a diminished percentage of CE with an increase of TG while HDL contained relatively more FC as well as PL and less CE. The diameter of VLDL was larger whereas those of LDL and HDL were smaller than in normal controls. Jejunal explants, when incubated with [14C]palmitate, were capable of normal biosynthesis of TG, diglycerides, PL, and CE. These lipids, however, except for PL, were retained in the tissue and could not be secreted into the culture medium. Incubation of intestinal biopsies with [3H]leucine and [14C]mannose resulted in normal protein synthesis and reduced glycosylation. The presence of intestinal apoB-48 was confirmed by immunoblot using 2D8 antibodies. These data suggest that the intestinal defect in this disease results from a disorder of the final assembly of chylomicrons or in the mechanism of their exocytosis.     

Transfer of n-3 and n-6 polyunsaturated fatty acids from yolk to embryo during development of the king penguin

This study examines the transfer of lipids from the yolk to the embryo of the king penguin, a seabird with a high dietary intake of n-3 fatty acids. The concentrations of total lipid, triacylglycerol (TAG), and phospholipid (PL) in the yolk decreased by ~80% between days 33 and 55 of development, indicating intensive lipid transfer, whereas the concentration of cholesteryl ester (CE) increased threefold, possibly due to recycling. Total lipid concentration in plasma and liver of the embryo increased by twofold from day 40 to hatching due to the accumulation of CE. Yolk lipids contained high amounts of C(20-22) n-3 fatty acids with 22:6(n-3) forming 4 and 10% of the fatty acid mass in TAG and PL, respectively. Both TAG and PL of plasma and liver contained high proportions of 22:6(n-3) ( approximately 15% in plasma and >20% in liver at day 33); liver PL also contained a high proportion of 20:4(n-6) (14%). Thus both 22:6(n-3) and 20:4(n-6), which are, respectively, abundant and deficient in the yolk, undergo biomagnification during transfer to the embryo.     

Characterization of monoclonal anti-rabbit apolipoprotein E antibodies and chemical composition of lipoproteins separated by anti-apolipoprotein E immuno-affinity chromatography

Six mouse monoclonal antibodies against rabbit apolipoprotein E (apo E) have been developed. Of these monoclonal antibodies, clone 5 revealed a high affinity for purified apo E, very low density lipoprotein (VLDL) and beta-VLDL. This monoclonal antibody was used to prepare an immunoaffinity column. Coupled to Sepharose 4B, this antibody allowed complete removal of lipoproteins containing apo E from plasma of New Zealand white (NZW) rabbits; 62, 46, 14, and 3% of VLDL-, IDL-, LDL-, and HDL-protein, respectively, were bound to the anti-apo E affinity column. The bound VLDL was significantly rich in free cholesterol (FC) and cholesteryl esters (CE) relative to the unbound VLDL, whereas bound IDL, LDL and HDL were significantly rich in FC only. All of the bound fractions were characterized by significantly increased ratios of FC/phospholipids (PL). These results indicate that the two lipoprotein populations with and without apo E have different lipid compositions. The relatively high content of cholesterol in lipoproteins containing apo E suggests a contribution of apo E to plasma cholesterol transport.     

Aggregated LDL and lipid dispersions induce lysosomal cholesteryl ester accumulation in macrophage foam cells

Macrophage foam cells in atherosclerotic lesions accumulate substantial cholesterol stores within large, swollen lysosomes. Previous studies with mildly oxidized low density lipoprotein (OxLDL)-treated THP-1 macrophages suggest an initial buildup of free cholesterol (FC), followed by an inhibition of lysosomal cholesteryl ester (CE) hydrolysis and a subsequent lysosomal accumulation of unhydrolyzed lipoprotein CE. We examined whether other potential sources of cholesterol found within atherosclerotic lesions could also induce similar lysosomal accumulation. Biochemical analysis combined with microscopic analysis showed that treatment of THP-1 macrophages with aggregated low density lipoprotein (AggLDL) or CE-rich lipid dispersions (DISP) produced a similar lysosomal accumulation of both FC and CE. Co-treatment with an ACAT inhibitor, CP113,818, confirmed that the CE accumulation was primarily the result of the inhibition of lysosomal CE hydrolysis. The rate of unhydrolyzed CE buildup was more rapid with DISP than with AggLDL. However, with both treatments, FC appeared to accumulate in lysosomes before the inhibition in hydrolysis and CE accumulation, a sequence shared with mildly OxLDL. Thus, lysosomal accumulation of FC and CE can be attributable to more general mechanisms than just the inhibition of hydrolysis by oxidized lipids.     

Study of the components of reverse cholesterol transport in lecithin:cholesterol acyltransferase deficiency

Enzymatic and lipid transfer reactions involved in reverse cholesterol transport were studied in healthy and lecithin:cholesterol acyltransferase (LCAT), deficient subjects. Fasting plasma samples obtained from each individual were labeled with [3H]cholesterol and subsequently fractionated by gel chromatography. The radioactivity patterns obtained corresponded to the elution volumes of the three major ultracentrifugally isolated lipoprotein classes (very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL)). In healthy subjects, the LCAT activity was consistently found in association with the higher molecular weight portion of HDL. Similar observations were made when exogenous purified LCAT was added to the LCAT-deficient plasma prior to chromatography. Incubation of the plasma samples at 37 degrees C resulted in significant reduction of unesterified cholesterol (FC) and an increase in esterified cholesterol (CE). Comparison of the data of FC and CE mass measurements of the lipoprotein fractions from normal and LCAT-deficient plasma indicates that: (i) In normal plasma, most of the FC for the LCAT reaction originates from LDL even when large amounts of FC are available from VLDL. (ii) The LCAT reaction takes place on the surface of HDL. (iii) The product of the LCAT reaction (CE) may be transferred to either VLDL or LDL although VLDL appears to be the preferred acceptor when present in sufficient amounts. (iv) CE transfer from HDL to lower density lipoproteins is at least partially impaired in LCAT-deficient patients. Additional studies using triglyceride-rich lipoproteins indicated that neither the capacity to accept CE from HDL nor the lower CE transfer activity were responsible for the decreased amount of CE transferred to VLDL and chylomicrons in LCAT-deficient plasma.     

Free cholesterol is a potent regulator of lipid transfer protein function

This study investigates the effect of altered lipoprotein free cholesterol (FC) content on the transfer of cholesteryl ester (CE) and triglyceride (TG) from very low- (VLDL), low- (LDL), and high-(HDL) density lipoproteins by the plasma-derived lipid transfer protein (LTP). The FC content of VLDL and HDL was selectively altered by incubating these lipoproteins with FC/phospholipid dispersions of varying composition. FC-modified lipoproteins were then equilibrated with [3H] TG, [14C]CE-labeled lipoproteins of another class to facilitate the subsequent modification of the radiolabeled donor lipoproteins. LTP was added and the extent of radiolabeled TG and CE transfer determined after 1 h. With either LDL or VLDL as lipid donor, an increase in the FC content of these lipoproteins caused a concentration-dependent inhibition (up to 50%) of CE transfer from these particles, without any significant effect on TG transfer. In contrast, with HDL as donor, increasing the HDL FC content had little effect on CE transfer from HDL, but markedly stimulated (up to 2.5-fold) the transfer of TG. This differential effect of FC on the unidirectional transfer of radiolabeled lipids from VLDL and HDL led to marked effects on LTP-facilitated net mass transfer of lipids. During long-term incubation of a constant amount of LTP with FC-modified VLDL and HDL, the extent of net mass transfer was linearly related to lipoprotein FC content; a 4-fold increase in FC content resulted in a 3-fold stimulation of the CE mass transferred to VLDL, which was coupled to an equimolar, reciprocal transfer of TG mass to HDL. Since lipid transfer between lipoproteins is integral to the process of reverse cholesterol transport, we conclude that lipoprotein FC levels are a potent, positive regulator of the pathways involved in sterol clearance. FC may modulate lipid transfer by altering the availability of CE and TG to LTP at the lipoprotein surface.     

Effects of high-density lipoprotein(2) on cholesterol transport and acyl-coenzyme A:cholesterol acyltransferase activity in P388D1 macrophages

High-density lipoproteins are the putative vehicles for cholesterol removal from monocyte-derived macrophages, which are an important cell type in all stages of atherosclerosis. The role of HDL(2), an HDL subclass that accounts for most variation in plasma HDL-cholesterol concentration, in cholesterol metabolism in monocyte-derived macrophages is not known. In this study, the dose-dependent effects of HDL(2) on cellular cholesterol mass, efflux, and esterification, and on cellular cholesteryl ester (CE) hydrolysis using the mouse macrophage P388D1 cell line was investigated. HDL(2) at low concentrations (40 microg protein/ml) decreased CE content without affecting cellular free cholesterol content (FC), CE hydrolysis, or cholesterol biosynthesis. In addition, HDL(2) at low concentrations reduced cellular acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity and increased FC efflux from macrophages. Thus, HDL(2) has two potential roles in reverse cholesterol transport. In one, HDL(2) is an acceptor of macrophage FC. In the other, more novel role, HDL(2) increases the availability of macrophage FC through the inhibition of ACAT. Elucidation of the mechanism by which HDL(2) inhibits ACAT could identify new therapeutic targets that enhance the transfer of cholesterol from macrophages to the liver.     

Effect of repeated apoA-IMilano/POPC infusion on lipids, (apo)lipoproteins,and serum cholesterol efflux capacity in cynomolgus monkeys

MDCO-216, a complex of dimeric recombinant apoA-IMilano (apoA-IM) and palmitoyl-oleoyl-phosphatidylcholine (POPC), was administered to cynomolgus monkeys at 30, 100, and 300 mg/kg every other day for a total of 21 infusions, and effects on lipids, (apo)lipoproteins, and ex-vivo cholesterol efflux capacity were monitored. After 7 or 20 infusions, free cholesterol (FC) and phospholipids (PL) were strongly increased, and HDL-cholesterol (HDL-C), apoA-I, and apoA-II were strongly decreased. We then measured short-term effects on apoA-IM, lipids, and (apo)lipoproteins after the first or the last infusion. After the first infusion, PL and FC went up in the HDL region and also in the LDL and VLDL regions. ApoE shifted from HDL to LDL and VLDL regions, while ApoA-IM remained located in the HDL region. On day 41, ApoE levels were 8-fold higher than on day 1, and FC, PL, and apoE resided mostly in LDL and VLDL regions. Drug infusion quickly decreased the endogenous cholesterol esterification rate. ABCA1-mediated cholesterol efflux on day 41 was markedly increased, whereas scavenger receptor type B1 (SRB1) and ABCG1-mediated effluxes were only weakly increased. Strong increase of FC is due to sustained stimulation of ABCA1-mediated efflux, and drop in HDL and formation of large apoE-rich particles are due to lack of LCAT activation.     

A two-step mechanism for free cholesterol and phospholipid efflux from human vascular cells to apolipoprotein A-1

Smooth muscle and endothelial cells in vivo are quiescent yet exposed to high levels of lipoprotein lipids. Phospholipid (PL) and free cholesterol (FC) efflux maintain homeostasis. Smooth muscle cells (SMC) expressed high levels of ABC-1 transporter mRNA, and glyburide-dependent PL and FC efflux to apolipoprotein A-1 (apo A-1), the major protein of high-density lipoprotein. FC efflux was inhibited by vanadate and okadaic acid, while PL efflux was not. Phosphatidylcholine was the major PL transferred by both cell types. Stimulation of phosphatidylserine efflux, redistributed within the membrane by this transporter, was only minimally increased. Umbilical vein and aortic endothelial cells expressed little ABC-1 mRNA, nor did these cells promote either PL or FC efflux in response to the presence of apo A-1. To investigate the mechanism of ABC-1-dependent lipid efflux from these cells, apo A-1 was preincubated in the presence of unlabeled SMC or fibroblasts, and the conditioned medium was then transferred to endothelial cells. This medium catalyzed the efflux of FC but not of PL from endothelial cells. Such FC efflux was resistant to glyburide but inhibited by okadaic acid and vanadate. The data suggest that ABC-1-dependent PL efflux precedes FC efflux to apo A-1 and that the complex of apo A-1 and PL is a much better acceptor of FC than apo A-1 itself. Inhibition of FC but not PL efflux by vanadate and okadaic acid suggests these transfers involve different mechanisms.     

Analysis of cholesteryl esters and diacylglycerols using lithiated adducts and electrospray ionization-tandem mass spectrometry

Cholesteryl ester (CE) and diacylglycerol (DAG) molecular species are important lipid storage and signaling molecules. Mass spectrometric analyses of these lipids are complicated by the presence of isobaric molecular ions shared by these lipid classes and by relatively poor electrospray ionization, which is a consequence of an inherently weak dipole moment in these lipid classes. The current study demonstrates that lithiated adducts of CE and DAG molecular ions have enhanced ionization and lipid class-specific fragmentation in tandem mass spectrometry (MS/MS) scan modes, thereby allowing the implementation of strategies capable of lipid class-specific detection. Using neutral loss (NL) mode for the loss of cholestane from cholesterol esters (NL 368.5) and specific selected reaction monitoring for DAG molecular species, the response of specific molecular species to that of internal standards was determined. CE and DAG molecular species were quantified in human coronary artery endothelial cells (HCAECs) incubated with both palmitic acid and oleic acid. Furthermore, NL 368.5 spectra revealed the oxidation of the aliphatic fatty acid residues of CE molecular species. Taken together, these studies demonstrate a new analytical approach to assessing CE and DAG molecular species that exploits the utility of lithiated adducts in conjunction with MS/MS approaches.