Phosphatidyl alcohols: Effect of head group size on domain forming properties and interactions with sterols

In this study, we have examined the membrane properties and sterol interactions of phosphatidyl alcohols varying in the size of the alcohol head group coupled to the sn-3-linked phosphate. Phosphatidyl alcohols of interest were dipalmitoyl derivatives with methanol (DPPMe), ethanol (DPPEt), propanol (DPPPr), or butanol (DPPBu) head groups. The Phosphatidyl alcohols are biologically relevant, because they can be formed in membranes by the phospholipase D reaction in the presence of alcohol. The melting behavior of pure phosphatidyl alcohols and mixtures with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or cholesterol was assessed using high sensitivity differential scanning calorimetry (DSC). DPPMe had the highest melting temperature (∼ 49 °C), whereas the other phosphatidyl alcohols had similar melting temperatures as DPPC (∼ 40-41 °C). All phosphatidyl alcohols, except DPPMe, also showed good miscibility with DPPC. The effects of cholesterol on the melting behavior and membrane order in multilamellar bilayer vesicles were assessed using steady-state anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and DSC. The ordering effect of cholesterol in the fluid phase was lower for all phosphatidyl alcohols as compared to DPPC and decreased with increasing head group size. The formation of ordered domains containing the phosphatidyl alcohols in complex bilayer membranes was determined using fluorescence quenching of DPH or the sterol analogue cholesta-5,7,(11)-trien-3-beta-ol (CTL). The phosphatidyl alcohols did not appear to form sterol-enriched ordered domains, whereas DPPMe, DPPEt appeared to form ordered domains in the temperature window examined (10-50 °C). The partitioning of CTL into bilayer membranes containing phosphatidyl alcohols was to a small extent increased for DPPMe and DPPEt, but in general, sterol interactions were weak or unfavorable for the phosphatidyl alcohols. Our results show that the biophysical and sterol interacting properties of phosphatidyl alcohols, having identical acyl chain structures, are markedly dependent on the size of the head group.     

Chemical and physicochemical studies of lysylphosphatidylglycerol derivatives. Occurrence of a2' yields 3' lysyl migration

Syntheses of 1,2-didodecanoyl-sn-glycero-3-phosphoryl-1′-(3′-O-L-lysyl)-sn-glycerol (IV) and 1,2-didodecanoyl-sn-glycero-3-phosphoryl-1′-(2′-O-L-lysyl)-sn-glycerol (VIII) as well as 1,2-didodecanoyl-sn-glycerol-3-phosphoryl-1′-sn-glycerol (XII) are described. 2′- and 3′-lysylphosphatidylglycerol are obtained as pure isomers and can be distinguished spectroscopically (infrared, 100 and 300 MHZ NMR). By these criteria a migration of the lysyl group from the 2′ to the 3′ position of the glycerol occurs in the presence of a strong acid catalyst such as HCl. On the other hand, a weak acid such as acetic acid appears ineffective in inducing lysyl migration, even at very high concentrations.Spectroscopic analysis furthermore demonstrated that lysylphosphatidylglycerol extracted from the Staphylococcus aureus membrane, is a 3′-isomer.     

Synthesis of phospholipids. III. Synthesis of 1,2-dipalmitoyl-rac-glyceryl-3-phosphoryl-3′β-cholesterol and 1,2-dipalmitoyl-rac-glyceryl-3-phosphoryl-20′-(3β-hydroxy norpregn-5-ene)

The chemical synthesis of two new glycerophosphatide analogues containing steroid groups, i.e., 1,2-dipalmitoyl-rac-glyceryl-3-phosphoryl-3′β-cholesterol and 1,2-dipalmitoyl-rac-glyceryl-3-phosphoryl-20′-(3β-hydroxy norpregn-5-ene) is described.     

Synthesis and characterization of deoxy analogues of diphytanylglycerol phospholipids

Novel analogues of diphytanyl phospholipids, 2,3-diphytanyl sn-1-glycerol-1-phosphoryl-1'-(1',3'-propanediol) (dPG), 2,3-diphytanyl-sn-glycerol-1-phosphoryl-1'-propanol (ddPG) and 2,3-diphytanyl-sn-glycerol-1-phosphoryl-1'-(1',3'-propanediol-3'-p hosphate) (dPGP), were synthesized according to modifications of previously published procedures. The samples were TLC and analytically pure and were characterized by 13C- and 1H-NMR and negative FAB/MS. The pK values of dPGP in aqueous dispersions or in methanol/water (1:1, v/v) were determined by potentiometric titration and compared with those of 2,3-diphytanyl-sn-glycerol-1-phosphoryl-3'-sn-glycerol-1'-phosphat e (PGP). The dissociation constant of the third ionizable POH group of dPGP was more than 2 pK units higher than that of PGP, indicating that the free glycerol hydroxyl group plays an important role in headgroup conformation and stabilization, perhaps through hydrogen bonding with the phosphate group(s).     

The essential role of specific Halobacterium halobium polar lipids in 2D-array formation of bacteriorhodopsin.

The mechanism whereby bacteriorhodopsin (BR), the light driven proton pump from the purple membrane of Halobacterium halobium, arranges in a 2D-hexagonal array, has been studied in bilayers containing the protein, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and various fractions of H. halobium membrane lipids, by freeze fracture electron microscopy and examination of optical diffractograms of the micrographs obtained. Electron micrographs of BR/DMPC complexes containing the entire polar lipid component of H. halobium cell membranes or the total lipid component of the purple membrane, with a protein-to-total lipid molar ratio of less than 1:50 and to which 4 M NaCl had been added, revealed that trimers of BR formed into an hexagonal 2D-array similar to that found in the native purple membrane, suggesting that one or more types of the purple membrane polar lipids are required for array formation. To support this suggestion, bacteriorhodopsin was purified free of endogenous purple membrane lipids and reconstituted into lipid bilayer complexes by detergent dialysis. The lipids used to form these complexes are 1,2-dimyristoyl-sn-glycerol-phosphocholine (DMPC) as the major lipid and, separately, each of the individual lipid types from the H. halobium cell membranes, namely 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol 1'-phosphate (DPhPGP), 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol 1'-sulphate (DPhPGS), 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol (DPhPG) and 2,3-di-O-phytanyl-1-O-[beta-D-Galp-3-sulphate-(1----6)-alpha-D- Manp-(1----2)-alpha-D-Glcp]-sn-glycerol (DPhGLS). When examined by freeze-fracture electron microscopy, only the complexes containing 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol- 1'-phosphate or 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol-1'-sulphate, at high protein density (less than 1:50, bacteriorhodopsin/phospholipid, molar ratio) and to which 4 M NaCl had been added, showed well defined 2D hexagonal arrays of bacteriorhodopsin trimers similar to those observed in the purple membrane of H. halobium.     

Aluminum-induced lipid phase separation and membrane fusion does not require the presence of negatively charged phospholipids

The interaction of Aluminum with phosphatidyl serine lipid vesicles containing variable amounts of phosphatidyl ethanolamine, phosphatidyl choline and cholesterol has been studied by lipid phase separation monitored by fluorescence quenching. The interaction of Al3+ with neutral phospholipid membranes has also been investigated. Maximal lipid phase separation can be demonstrated in mixed phosphatidyl ethanolamine-cholesterol vesicles when using concentrations of aluminum between 87.5 and 125 microM. Millimolar concentrations of Ca2+, Mn2+, Cd2+ and Zn2+ were without any effect. Aluminum also induced fusion of phospholipid membranes monitored by resonance energy transfer between N-(7-nitro-2,1,3, benzoxadiazol-4 yl) phosphatidyl ethanolamine and N-(lissamine Rhodamine B-sulfonyl) phosphatidyl ethanolamine, either when containing low amounts of phosphatidyl serine (12.5%) or without any negatively charged phospholipid. Aluminum-induced fusion of liposomes was also monitored by the fluorescence of the terbium-dipicolinic acid complex (Tb-DPA3-) formed during fusion of vesicles containing either Tb-(citrate)6- complex or sodium salt of dipicolinic acid.     

Shikimic acid complexes of platinum. Preparation, reactivity, and antitumor activity of (R,R-1,2-diaminocyclohexane) bis(shikimato) platinum(II). Evidence for a novel rearrangement involving platinum-carbon bond formation

The complex (R,R-1,2-diaminocyclohexane)bis(shikimato)platinum(II) (shikimato = the anion of 3R,4S,5R-trihydroxy-1-cyclohexene-1-carboxylic acid), I, has been synthesized and purified by high performance liquid chromatography (HPLC). The complex is only moderately stable in aqueous solution. Its major hydrolysis product, also purified by HPLC, is proposed to be a unique complex type in which a single shikimate group is coordinated through both the carboxylate oxygen and the C(2) vinylic carbon of the shikimate moiety [Pt(R,R-dach)(O,C-shikimato)], II. In vitro, complex I is active against L1210 leukemia and against an L1210 cell line with acquired resistance to cisplatin. In vivo, the complex is active against L1210, P388, and B16 melanoma; this activity is highly schedule-dependent. Complex II is also active against L1210 leukemia.     

Synthesis and Herbicidal Activity of Phenoxypropionic Acid Derivatives with Imidazo[1,2-α]pyridine Moiety

Phenoxypropionic acid derivatives (Ia-s) with an imidazo[1,2-a]pyridine moiety were synthesized and their herbicidal activities were examined. The activities were affected dramatically by the substituents on the imidazo[1,2-a]pyridine ring, and good substituents to enhance the herbicidal activity were a cyano group at the 3-position and a chlorine atom at the 6-position. Among the compounds, n-propyl 2-[4-(6-chloro-3-cyano-2-imidazo[1,2-a]pyridinyloxy)phenoxy]propionate(Iq) was most active against gramineous weeds and the activity was comparable to that of the commercial herbicide fluazifop-butyl.     

Evaluation of the cyclopentane-1,2-dione as a potential bio-isostere of the carboxylic acid functional group

Cycloalkylpolyones hold promise in drug design as carboxylic acid bio-isosteres. To investigate cyclopentane-1,2-diones as potential surrogates of the carboxylic acid functional group, the acidity, tautomerism, and geometry of hydrogen bonding of representative compounds were evaluated. Prototypic derivatives of the known thromboxane A₂ prostanoid (TP) receptor antagonist, 3-(3-(2-((4-chlorophenyl)sulfonamido)-ethyl)phenyl)propanoic acid, in which the carboxylic acid moiety is replaced by the cyclopentane-1,2-dione unit, were synthesized and evaluated as TP receptor antagonists. Cyclopentane-1,2-dione derivative 9 was found to be a potent TP receptor antagonist with an IC₅₀ value comparable to that of the parent carboxylic acid. These results indicate that the cyclopentane-1,2-dione may be a potentially useful carboxylic acid bio-isostere.     

Synthesis of phosphatidylinositol mannosides (PIMs)

Two strategies towards the synthesis of phosphatidylinositol mannosides (PIMs) were elaborated which permit selective access to the O-1-, O-2-, and the O-6 position of the myo-inositol residue. Starting materials are 1,2:5,6- and 1,2:4,5-di-O-cyclohexylidene-DL-myo-inositol, respectively. In the latter case, the required assignment to the D- or L-series is based on the transformation of one enantiomer into known (-)-liriodentritol. The efficiency and potential versatility of the two approaches is exemplified in the synthesis of PIMs (D)-1a and its pseudoenantiomer (L)-1b, both having myristoyl residues as part of the phosphatidyl moiety.     

The effect of gamma irradiation at sub- and supralethal doses on the lipid composition of the mucosa and of the brush border membrane of the small intestine in rats]

A study was made of the amount of cholesterol and its ethers (phosphatidyl choline, phosphatidyl ethanolamine, sphingomyelin, phosphatidyl serine, and phosphatidyl inositol) in mucosa and membrane of the small intestine brush border 24 h after 4 Gy and 2 h after 20 Gy irradiation. No changes in the lipid content of mucosa and membrane of the brush border were noted after 4 Gy irradiation. Exposure to 20 Gy radiation doubled the number of cholesterol ethers and made the number of individual phospholipids and cholesterol increase by nearly 1.5 times. The amount of phosphatidyl serine in the brush border membrane increased by almost 3 times; the concentration of other lipids increased by nearly 1.5 times; cholesterol/phospholipid ratio was unchangeable.     

Antioxidant Properties of the Major Polyphenolic Compounds in Broccoli

We have examined the antioxidant activity of the major phenolic compounds in Broccoli: two flavonol glycosides (quercetin 3-O-sophoroside and kaemp-ferol 3-O-sophoroside) and four hydroxycinnamic acid esters (1,2'-disinapoyl-2-feruloyl gentiobiose, 1-sinapoyl-2-feruloyl gentiobiose, 1,2,2'-trisinapoyl gentiobiose and 1,2-disinapoyl gentiobiose). The Trolox C equivalent antioxidant capacity (TEAC) and inhibition of iron/ascorbate-induced lipid per-oxidation of phosphatidyl choline vesicles were measured. In the aqueous phase TEAC assay, the two flavonol glycosides were less active than their respective aglycones. TEAC values for the hydroxycinnamic acid esters were less than the sum of their constituent hydroxycinnamic acids on a molar basis. Quercetin 3-O-sophoroside was a potent inhibitor of lipid peroxidation, in contrast to kaempferol 3-O-sophoroside. The hydroxycinnamic acid esters were highly effective at preventing lipid damage with the exception of 1,2,2'-trisinapoyl gentiobiose. The six compounds analysed herein demonstrate the antioxidant activity of the major phenolics in broccoli and indicate the effect on antioxidant activity of sugar substitutions in the phenolic B ring.     

Glycerol reorientation during the conversion of phosphatidylglycerol to bis(monoacylglycerol)phosphate in macrophage-like RAW 264.7 cells

Bis(monoacylglycero)phosphate (BMP) has the unique stereoconfiguration of 3-acyl-sn-glycero-1-phosphoryl-1'-sn-[3'-acylglycerol] (Brotherus, J., Renkonen, O., Herrmann, J., and Fischer, W. (1974) Chem. Phys. Lipids 13, 178-182) which differs from other known mammalian phospholipids that have the sn-glycero-3-phosphoryl configuration. This stereochemistry may contribute to its physiologic function. Here we describe studies using the macrophage-like cell line RAW 264.7 designed to determined how this unique stereoconfiguration occurs. These studies show that the stereoconfiguration of BMP produced from exogenous phosphatidylglycerol (PG) by RAW 264.7 cells has the expected stereoconfiguration of 3-acyl-sn-glycero-1-phosphoryl-1'-sn-[3'-acylglycerol]. Experiments using diacyl-sn-[2-3H]glycero-3-phosphoryl-sn-1'-[2-3H]glycerol demonstrate that this unique stereoconfiguration is not produced due to an oxidation/reduction mechanism involving the sn-2-glycerol carbon. When dioleoyl-sn-[1-14C]glycero-3-phosphoryl-rac-glycerol was converted to 14C-labeled BMP, the 14C label was found esterified to the phosphate moiety. These results suggest that a stereospecific enzyme is capable of reorienting the radiolabeled glycerol backbone of this PG substrate, effectively changing the stereochemistry of the lipid. We also show that this enzyme is stereoselective with regard to the base glycerol moiety of the substrate PG used. Finally, we propose a new pathway for the synthesis of BMP from PG.     

Disulfide bond involvement in the maintenance of the cryptic nature of the cross-reacting determinant of metacyclic forms of Trypanosoma congolense

The variable surface glycoprotein (VSG) of African trypanosomes possesses a 1,2-dimyristoylglycosylphosphatidylinositol at the carboxy terminus. Cleavage of the 1,2-dimyristoylglycerol (1,2-DMG) moiety from the VSG reportedly results in a higher apparent molecular mass and an increased binding of antibodies against the "cross-reacting determinant" (CRD), a cryptic epitope present on most VSGs. Using metacyclic forms of Trypanosoma congolense, we show that the processes involved are more complex than heretofore presumed and that the removal of the 1,2-DMG moiety may not be necessary for binding of anti-CRD antibodies (RxCRD). Among other findings, we observe the following: (1) in sonicated samples of trypanosomes metabolically labeled with [3H]myristate, the binding of RxCRD on Western blots is coincident with bands containing labeled (membrane form) VSGs; (2) disulfide reduction of trypanosome sonicates suffices to promote RxCRD binding in the presence or absence of inhibitors of a glycosylphosphatidylinositol-specific phospholipase C; (3) trypanosomes directly solubilized in detergents show quantitative and qualitative differences in RxCRD binding which depend upon the detergent used and the order of addition of disulfide reducing agents. We conclude that the binding of RxCRD to T. congolense metacyclic VSGs depends upon the degree of unfolding of the molecule and is clearly a complex, multistep process in which structural changes and disulfide reduction play pivotal roles.     

Biochemical changes accompanying apoptotic cell death in retinoblastoma cancer cells treated with lipogenic enzyme inhibitors

Retinoblastoma (RB) is a malignant intra-ocular neoplasm that affects children (usually below the age of 5 years). In addition to conventional chemotherapy, novel therapeutic strategies that target metabolic pathways such as glycolysis and lipid metabolism are emerging. Fatty acid synthase (FASN), a lipogenic multi-enzyme complex, is over-expressed in retinoblastoma cancer. The present study evaluated the biochemical basis of FASN inhibition induced apoptosis in cultured Y79 RB cells. FASN inhibitors (cerulenin, triclosan and orlistat) significantly inhibited FASN enzyme activity (P < 0.05) in Y79 RB cells. This was accompanied by a decrease in palmitate synthesis (end-product depletion), and increased malonyl CoA levels (substrate accumulation). Differential lipid profile was biochemically estimated in neoplastic (Y79 RB) and non-neoplastic (3T3) cells subjected to FASN inhibition. The relative proportion of phosphatidyl choline to neutral lipids (triglyceride + total cholesterol) in Y79 RB cancer cells was found to be higher than the non-neoplastic cells, indicative of altered lipid distribution and utilization in tumor cells. FASN inhibitor treated Y79 RB and fibroblast cells showed decrease in the cellular lipids (triglyceride, cholesterol and phosphatidyl choline) levels. Apoptotic DNA damage induced by FASN inhibitors was accompanied by enhanced lipid peroxidation.     

Electrical conductivity in lipid bilayer membranes induced by pentachlorophenol.

Electrical conductivity induced in thin lipid bilayer membranes by pentachlorophenol has been studied. The membranes were formed from phosphatidyl choline, phosphatidyl ethanolamine, or phosphatidyl glycerol and various amounts of cholesterol. The position and the magnitude of the maximum of the conductivity vs. pH curve depend on the type of lipids and cholesterol content. At low pentachlorophenol concentrations and low pH the concentration dependence of conductivity is quadratic and becomes linear at higher pH. Above 10(-5) M of pentachlorophenol the concentration dependence of the membrane conductivity tends to saturate. Presence of pentachlorophenol enhances membrane transport of nonactin-K+ complex. Increase of cholesterol content increases pentachlorophenol induced conductivity in all membranes and shifts the conductivity toward lower pH. For phosphatidyl choline the largest rate of change of membrane conductivity with cholesterol occurs at 1:1 phospholipid to cholesterol molar ratio. Pentachlorophenol is found to be a class II uncoupler and the experimental results are consistent with the hypothesis that the membrane permeable species are dimers formed by combination of neutral and dissociated pentachlorophenol molecules. Several schemes of membrane conduction, including dimer formation in the aqueous phase as well as at the membrane-water interface have been considered. Arguments are given in favor of the formation of dimers within the membrane surface.     

Detergent solubilization and hydrophobic chromatography of rat brain phosphatidylinositol kinase

Rat brain microsomal phosphatidylinositol kinase activity was maximally activated in the presence of either 3 mM sodium deoxycholate, 2% Triton-X-100, or 30–40 mM octylglucoside. Among these detergents, 1% Triton-X-100 was most effective in solubilizing the enzyme, and after treatment with, this agent, 100% of the activity was recovered in the high speed supernatant. Octylglucoside solubilized 40% of the enzyme at concentrations below its critical micelle concentration of 25 mM and up to 80% at higher levels. Solubilized phosphatidylinositol kinase failed to adsorb to adenosine nucleotide affinity resins. However, when the Triton-X-100 extract was chromatographed on an uncharged hydrophobic resin, consisting of dodecyl chains attached to Sepharose 4B by ether bonds, nearly all the enzyme activity was retained, and from 44–85% could be eluted with 8 mM sodium deoxycholate. Solubilization followed by hydrophobic chromatography resulted in several-fold purification of phosphatidylinositol kinase and may have disrupted interactions of the enzyme with other hydrophobic proteins sufficiently to allow its substantial purification by conventional or affinity chromatography techniques.The abbreviations used are phosphatidylinositol 1,2-diacyl-sn-glycero-3-phosphoryl-1-l-myo-inositol - phosphatidylinositolphosphate 1,2-diacyl-sn-glycero-3-phosphoryl-1-l-myo-inositol-4-monophosphate - phosphatidylinositolbisphosphate 1,2-diacyl-sn-glycerol-3-phosphoryl-1-l-myo-inositol-4,5-bisphosphate - octylglucoside 1-0-n-octyl-d-glucopyranoside     

Investigating the structural and dynamic properties of n-doxylstearic acid in magnetically-aligned phospholipid bilayers by X-band EPR spectroscopy

X-band electron paramagnetic resonance (EPR) spectroscopy has been employed to investigate the dynamic properties of magnetically-aligned phospholipid bilayers (bicelles) based on the molecular order parameters (S(mol)), the hyperfine splitting values and the line shapes of the EPR spectra. For the first time, a series of EPR spectra of n-doxylstearic acid spin-labels (n = 5, 7, 12, and 16) incorporated into Tm3+-doped parallel-aligned, Dy3+-doped perpendicular-aligned, and randomly dispersed 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dihexanoyl-sn-glycero-3-phosphocholine (DMPC/DHPC) bicelles with respect to the direction of the static magnetic field have been investigated as a function of cholesterol content and temperature variation to characterize the orientational aspects along the hydrocarbon acyl chains. Important general observations are that under conditions for which the bicelle is poised in the liquid crystalline phase, the degree of ordering decreases as the nitroxide moiety is transferred toward the end of the stearic acid acyl chains. The addition of cholesterol increases the phase transition temperature and alignment temperature of the DMPC/DHPC phospholipid bilayers and increases the chain order. However, increasing the temperature of the bicelle system decreases the chain order. This report reveals that the dynamic properties of DMPC/DHPC bicelles agree well with other biological and model membrane systems. The results indicate that magnetically-aligned phospholipid bilayers are an excellent model membrane system.     

Fluorescent probe--a cholesterol analog: localization in plasma lipoproteins and in model lipid particles

The absorption spectrum of fluorescent probe--a cholesterol analog cholesta-5,7, 9(11)-trien-3 beta-ol has a vibrational structure with the maximum 326 nm. Its fluorescence spectra maximum is 370 nm. The localization of the probe in lipoproteins of high, low and very low density and in lipid spheres is studied. There are measured the areas, which occupied one molecule of cholesterol and phosphatidyl choline on the surface of lipid spheres and the radius of the lipid spheres. The localization of the probe in lipoproteins and lipid spheres is determined. The areas which occupied one molecule of phosphatidyl choline on the surface of lipid sphere is equal to the same area in mono- and bilayers. Cholesterol has the same condensing action on phosphatidyl choline in lipid spheres as in mono- and bilayers. All the probe molecules are localized on the surface of lipid spheres and lipoproteins and the B-ring of the molecule is immersed on 1.3 +/- 0.2 nm relative to polar groups. The hydroxyl group of cholesterol is arranged near the carbonyl group of phospholipid and the formation of the H-bond between these groups is possible.     

Entry of [(1,2-13C2)acetyl]-L-carnitine in liver tricarboxylic acid cycle and lipogenesis: a study by 13C NMR spectroscopy in conscious, freely moving rats.

The biochemical pathways involved in acetyl-L-carnitine utilization were investigated in conscious, freely moving rats by 13C NMR spectroscopy. Following 4-h [(1,2-13C2)acetyl]-L-carnitine infusion in fasted animals, the free carnitine levels in serum were increased, and an efflux of unlabelled acetyl-L-carnitine from tissues was observed. [(1,2-13C2)Acetyl]-L-carnitine was found to enter biosynthetic pathways in liver, and the acetyl moiety was incorporated into both cholesterol and 3-hydroxybutyrate carbon skeleton. In accord with the entry of [(1,2-13C2)acetyl]-L-carnitine in the mitochondrial acetylCoA pool associated with tricarboxylic acid cycle, the 13C label was also found in liver glutamate, glutamine, and glutathione. The analysis of the 13C-labelling pattern in 3-hydroxybutyrate and cholesterol carbon skeleton provided evidence that the acetyl-L-carnitine-derived acetylCoA pool used for ketone bodies synthesis in mitochondria was homogeneous, whereas cholesterol was synthesized from two different acetylCoA pools located in the extra- and intramitochondrial compartment, respectively. Furthermore, cholesterol molecules were shown to be preferentially synthesized by the metabolic route involving the direct channelling of CoA-activated mitochondria-derived ketone bodies into 3-hydroxy-3-methylglutarylCoA pathway, prior to equilibration of their acyl groups with extramitochondrial acetylCoA pool via acetoacetylCoA thiolase.