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Context: Peripheral and central endocannabinoids and cognate acylethanolamides (AEs) may play important but distinct roles in regulating energy balance., Objective: We hypothesized that in humans central/peripheral endocannabinoids are differently associated with adiposity and energy expenditure and differ by race., Design: We examined associations of arachindonoylethanolamide, 2-arachidonoylglycerol, palmitoylethanolamide, and oleoylethanolamide (OEA) assayed in plasma and cerebrospinal fluid (CSF) with race, adiposity, and energy expenditure., Setting/participants: In this monitored clinical inpatient study, CSF was obtained by lumbar puncture in 27 individuals (12 Caucasian, 11 American Indian, and four African-American). Twenty-four hour and sleep energy expenditure were measured by indirect calorimetry in a respiratory chamber., Main Outcome Measure: Samples were analyzed from a previous study originally designed to test a blood-brain barrier leptin transport deficit in human obesity., Results: CSF (but not peripheral) 2-arachidonoylglycerol was significantly increased in American Indians compared with Caucasians (18.48 ± 6.17 vs. 10.62 ± 4.58 pmol/ml, P < 0.01). In the whole group, peripheral AEs were positively but in CSF negatively associated with adiposity. However, in multivariate models adjusted for the other peripheral and CSF AEs, peripheral arachindonoylethanolamide was the only AE significantly associated with adiposity. Interestingly, CSF OEA concentrations were positively associated with adjusted 24 hour and sleep energy expenditure (r = 0.47, P < 0.05; r = 0.42, P < 0.05), but peripheral OEA was not., Conclusions: These data indicate a central alteration of the endocannabinoid system in American Indians and furthermore show that AEs in both compartments play an important but distinct role in human energy balance regulation.

Dopaminergic therapies remain the most efficacious symptomatic treatments for Parkinson's disease (PD) but are associated with motor complications, including dyskinesia, and nonmotor complications, such as psychosis, impulse control disorders (ICD), and dopamine dysregulation syndrome (DDS). Nondopaminergic neurotransmitter systems, including the endocannabinoid system, are probably critical to the development of these complications. The role of fatty acid amide hydrolase (FAAH) in mediating l-3,4-dihydroxyphenylalanine (L-DOPA)-induced behaviors was explored in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned marmoset model of PD. Pharmacodynamic and locomotor effects of the selective FAAH inhibitor [3-(3-carbamoylphenyl)phenyl] N-cyclohexylcarbamate (URB597) were assessed via bioanalytical (liquid chromatography-tandem mass spectrometry) and behavioral observation approaches. URB597 (3, 10, 30, or 60 mg/kg p.o.) increased plasma levels of the FAAH substrates N-arachidonoyl ethanolamide (anandamide), N-oleoyl ethanolamide, and N-palmitoyl ethanolamide by 10.3 ± 0.3-, 7.8 ± 0.2-, and 1.8 ± 0.1-fold (mean of URB597 groups ± S.E.M.), respectively, compared with vehicle (all p < 0.001) 4 h after administration. Treatment with L-DOPA (20 mg/kg s.c.) alleviated parkinsonism but elicited dyskinesia, psychosis-like-behaviors and hyperactivity, a potential correlate of ICD and DDS. During the 2 to 4 h after L-DOPA, corresponding to 4 to 6 h after URB597 administration, URB597 reduced total L-DOPA-induced activity and the magnitude of hyperactivity by 32 and 52%, respectively, to levels equivalent to those seen in normal animals. Treatment with URB597 (10 mg/kg p.o.) did not modify the antiparkinsonian actions of L-DOPA or L-DOPA-induced dyskinesia and psychosis. URB597 did not alter plasma L-DOPA levels and was without behavioral effects when administered alone. Inhibition of FAAH may represent a novel approach to reducing L-DOPA-induced side effects, such as ICD and DDS, while maintaining the antiparkinsonian benefits of L-DOPA treatment.

An LC-MS/MS-ESI method has been validated for simultaneous estimation of the three endocannabinoids; N-arachidonoylethanolamine (AEA), N-oleoylethanolamine (OEA) and palmitoylethanolamide (PEA), in surrogate matrix using AEA-d (4) as an internal standard with highest sensitivity over the existing methods. Simple precipitation was used to extract analytes and these were subsequently analyzed on a monolithic column. Linear response function was established over the concentration range 12.3 to 1225 pg/ml for AEA (r > 0.994); 0.70 to 641 ng/ml for OEA (r > 0.999) and 0.54 to 321 ng/ml (r > 0.998) for PEA. The intra- and inter-day precision values met the acceptance to criteria as per US FDA guidelines. Analytes were found to be stable in the battery of stability studies. The method was applied to quantify endogenous levels of analytes in rat plasma.

Endocannabinoids including N-acylethanolamides (NAEs) are a family of lipid-related signaling molecules implicated in many physiological and disease states which elicit their activities via the cannabinoid receptors. Anandamide (N-arachidonoylethanolamine, AEA) is the most characterized endocannabinoid and has been detected in many tissues and bio-fluids including human plasma and the central nervous system. The endocannabinoid-like NAEs, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) are described as entourage compounds because they illicit similar physiological effects to AEA but have little or no affinity for cannabinoid receptors. As entourage compounds, levels of these NAEs can greatly influence the efficacy of AEA yet there are few studies which measure these compounds in bio-fluids. Here we describe a rapid, highly sensitive, specific and highly reproducible ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the analysis of AEA, OEA, and PEA in human bio-fluids including plasma, serum, breast milk, and amniotic fluids. This validated method using deuterated (AEA-d(8), OEA-d(2), and PEA-d(4)) internal standards, represents an improvement over previous analyses in terms of run time (4 min), limit of detection (0.9 fmol on column for AEA and PEA and 4.4 fmol on column for OEA), precision (relative standard deviations of peak areas: 3.1% (AEA), 2.9% (OEA), and 5.4% (PEA) for 133 fmol on column) and accuracy (95.1-104.9%). The sensitivity and precision of the validated method described here suggests that this method is suitable for the analysis of AEA, OEA, and PEA in clinical samples and may be utilized for the investigation of bio-matrices containing limited amounts of NAEs.

Background: Endocannabinoids (eCBs) are ubiquitous lipid mediators that act on specific (CB1, CB2) and non-specific (TRPV1, PPAR) receptors. Despite many experimental animal studies proved eCB involvement in the pathogenesis of stroke, such evidence is still lacking in human patients. Our aim was to determine eCB peripheral levels in acute stroke patients and evaluate their relationship with clinical disability and stroke volume., Methods: A cohort of ten patients with a first acute (within six hours since symptoms onset) ischemic stroke and a group of eight age- and sex-matched normal subjects were included. Groups were also matched for metabolic profile. All subjects underwent a blood sample collection for anandamide (AEA), 2-arachidonoylglycerol (2-AG) and palmitoylethanolamide (PEA) measurement; blood sampling was repeated in patients on admission (T0), at 6 (T1) and 18 hours (T2) thereafter. Patients neurological impairment was assessed using NIHSS and Fugl-Meyer Scale arm subitem (FMSa); stroke volume was determined on 48 h follow-up brain CT scans. Blood samples were analyzed by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry., Results: 1)T0 AEA levels were significantly higher in stroke patients compared to controls. 2)A significant inverse correlation between T0 AEA levels and FMSa score was found. Moreover a positive correlation between T0 AEA levels and stroke volume were found in stroke patients. T0 PEA levels in stroke patients were not significantly different from the control group, but showed a significant correlation with the NIHSS scores. T0 2-AG levels were lower in stroke patients compared to controls, but such difference did not reach the significance threshold., Conclusions: This is the first demonstration of elevated peripheral AEA levels in acute stroke patients. In agreement with previous murine studies, we found a significant relationship between AEA or PEA levels and neurological involvement, such that the greater the neurological impairment, the higher were these levels.

Background: Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS. Therapies that affect the endocannabinoid (EC) system may have immunomodulatory, symptomatic and neuroprotective effects., Aim: The aim of this study was to determine how levels of EC and related compounds are altered in MS., Methods: Plasma and whole blood were collected from 24 MS patients (10 relapsing-remitting (RR); 8 secondary-progressive (SP); 6 primary-progressive (PP); 19 females; 25-66 years) and 17 controls (10 females; 22-62 years). Plasma EC and related compounds were quantified by liquid chromatography-tandem mass spectrometry. Fatty acid amide hydrolase (FAAH), cannabinoid receptors CB(1) and CB(2) mRNA were measured by quantitative reverse transcriptase-polymerase chain reaction., Results: Anandamide (AEA) and palmitoylethanolamide (PEA) were higher in RRMS compared to controls (p=0.001 and p=0.027). AEA, PEA and oleoylethanolamide were also increased in SPMS plasma (p=0.001, p=0.004, and p=0.005). PPMS patients had higher AEA plasma levels compared to controls (p=0.009). FAAH mRNA was decreased in SPMS (p=0.04) but not in RRMS or PPMS blood. CB(1) (p=0.012) and CB(2) mRNA (p=0.003) were increased in the PPMS., Conclusion: The EC system is altered in MS. It may be dynamically modulated depending on the subtype of the disease, but further studies with larger subgroups are needed to confirm this.

A simple and rapid analytical method is described for the simultaneous quantitative analysis of three different N-acylethanolamides in human biological samples: anandamide (AEA), oleoylethanolamide (OEA), and palmitoylethanolamide (PEA). The method is based on a new hybrid solid phase extraction-precipitation technology followed by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) analysis using d(4)-AEA as the internal standard. The method is linear up to 100ng/ml with a limit of quantitation of 50pg/ml for AEA and 100pg/ml for OEA and PEA. Good reproducibility, accuracy, and precision were demonstrated during the method validation. Application of this new methodology to the analysis of clinical study samples is presented.

The global metabolite profiles of endogenous compounds of Wistar rats from 12 to 20 weeks old were investigated to take deep insight into and get better understanding of the pathogenesis of development and aging. Plasma from Wistar rats at 12, 14, 16, 18, and 20 weeks old were analyzed using GC/TOFMS. Multivariate data analysis was then used to process the metabonomic data which indicated excellent separation between different weeks and showed that the metabolic profiles of the samples changed with age, enabling age-related metabolic trajectories to be visualized. Decreased concentrations of citric acid, cis-aconitic acid, 9-(z)-hexadecenoic acid along with increased levels of hexanedioic acid, alpha-tocopherol, 3-indole propionic acid, etc contributed to the separation. Several major metabolic pathways were identified to be involved in metabolic regulation. This suggests that GC/TOFMS-based metabonomics is a powerful alternative approach to identifying potential biomarkers and investigating the physiological developments of aging and it is important to employ suitable age-match control group in metabonomic study of physiological monitoring, drug safety assessment, and disease diagnosis, etc.

Central endocannabinoid signaling is known to be responsive to stressful stimuli; however, there is no research to date characterizing the effects of stress on peripheral endocannabinoid content. The current study examined serum content of the endocannabinoid ligands N-arachidonylethanolamide (anandamide; AEA) and 2-arachidonoylglycerol (2-AG), and the non-cannabinoid N-acyl ethanolamine (NAE) molecules palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) under basal conditions, immediately following the Trier Social Stress Test (TSST), and 30 min thereafter, in 15 medication-free women diagnosed with major depression, and 15 healthy matched controls. Basal serum concentrations of AEA and 2-AG, but not PEA or OEA, were significantly reduced in women with major depression relative to matched controls, indicating a deficit in peripheral endocannabinoid activity. Immediately following the TSST, serum 2-AG concentrations were increased compared to baseline; serum AEA concentration was unchanged at this time point. Serum concentrations of PEA and OEA were significantly lower than baseline 30 min following the cessation of the TSST. The magnitude of these responses did not differ between depressed and control subjects. These are the first data to demonstrate that the peripheral endocannabinoid/NAE system is responsive to exposure to stress.

The endocannabinoid, arachidonoylethanolamide (AEA), and the peroxisome proliferator-activated receptor (PPAR)-alpha ligand, oleylethanolamide (OEA) produce opposite effects on lipogenesis. The regulation of OEA and its anti-inflammatory congener, palmitoylethanolamide (PEA), in adipocytes and pancreatic beta-cells has not been investigated. We report here the results of studies on acylethanolamide regulation in these cells during obesity and hyperglycaemia, and provide an overview of acylethanolamide role in metabolic control. We analysed by liquid chromatography-mass spectrometry OEA and PEA levels in: 1) mouse 3T3F442A adipocytes during insulin-induced differentiation, 2) rat insulinoma RIN m5F beta-cells kept in 'low' or 'high' glucose, 3) adipose tissue and pancreas of mice with high fat diet-induced obesity (DIO), and 4) in visceral fat or blood of obese or type 2 diabetes (T2D) patients. In adipocytes, OEA levels remain unchanged during differentiation, whereas those of PEA decrease significantly, and are under the negative control of both leptin and PPAR-gamma. PEA is significantly downregulated in subcutaneous adipose tissue of DIO mice. In RIN m5F insulinoma beta-cells, OEA and PEA levels are inhibited by 'very high' glucose, this effect being enhanced by insulin, whereas in cells kept for 24 h in 'high' glucose, they are stimulated by both glucose and insulin. Elevated OEA and PEA levels are found in the blood of T2D patients. Reduced PEA levels in hypertrophic adipocytes might play a role in obesity-related pro-inflammatory states. In beta-cells and human blood, OEA and PEA are down- or up-regulated under conditions of transient or chronic hyperglycaemia, respectively.

Context: Underlying mechanisms explaining the effects of osteopathic manipulative treatment (OMT) are poorly defined. The authors evaluate various nociceptive (pain) biomarkers that have been suggested as important mediators in this process., Objective: To determine if OMT influences levels of circulatory pain biomarkers., Methods: In a prospective, blinded assessment, blood was collected from 20 subjects (10 with chronic low back pain [LBP], 10 controls without chronic LBP) for 5 consecutive days. On day 4, OMT was administered to subjects 1 hour before blood collection. Blood was analyzed for levels of beta-endorphin (betaE), serotonin (5-hydroxytryptamine [5-HT]), 5-hydroxyindoleacetic acid (5-HIAA), anandamide (arachidonoylethanolamide [AEA]), and N-palmitoylethanolamide (PEA). A daily questionnaire was used to monitor confounding factors, including pain and stress levels, sleep patterns, and substance use., Results: Increases from baseline in betaE and PEA levels and a decrease in AEA levels occurred immediately posttreatment. At 24 hours posttreatment, similar biomarker changes from baseline were observed. A decrease in stress occurred from baseline to day 5. The change in PEA from baseline to 24 hours posttreatment correlated with the corresponding changes in stress. Subgroup analysis showed that subjects with chronic LBP had significantly reduced 5-HIAA levels at 30 minutes posttreatment (P=.05) and 5-HT levels at 24 hours posttreatment (P=.02) when compared with baseline concentrations. The increase in PEA in subjects with chronic LBP at 30 minutes posttreatment was two times greater than the increase in control subjects., Conclusion: Concentrations of several circulatory pain biomarkers were altered after OMT. The degree and duration of these changes were greater in subjects with chronic LBP than in control subjects without the disorder.

We developed a new selective liquid chromatography-electrospray ionization-tandem mass spectrometry method for the identification and quantification of anandamide (AEA), an endogenous cannabinoid receptor ligand, and other bioactive fatty acid ethanolamides (FAEs) in biological samples. Detection limit (0.025 pmol for AEA and 0.1 pmol for palmitoylethanolamide (PEA) and oleoylethanolamide (OEA)) and quantification limit (0.2 pmol for AEA and 0.4 pmol for OEA and PEA) were in the high fmol to low pmol range for all analytes. Linear correlations (r(2)=0.99) were observed in the calibration curves for standard AEA over the range of 0.025-25 pmol and for standard PEA and OEA over the range of 0.1-500 pmol. This method provides a time-saving and sensitive alternative to existing methods for the analysis of FAEs in biological samples.

Background: Fatty acid ethyl esters (FAEEs) are generated by the nonoxidative metabolism of alcohol and correlate positively with blood alcohol levels (BAL). As FAEEs are produced predominantly in the liver and bind to albumin in plasma, blood FAEE concentrations may be affected by serum albumin levels. The aim of this exploratory study was to define the relationship of FAEE levels with BAL after adjustment for serum albumin concentration., Methods: Fatty acid ethyl ester, BAL, and albumin concentrations were measured from de-identified, ethanol-containing serum samples (N=18). The assay focused on 3 FAEE species ethyl palmitate, ethyl stearate, and ethyl oleate. The relationships of individual and total FAEE concentrations with BAL using albumin as a covariate were analyzed. Values for Pearson's r between the BAL and the natural log-transformed FAEE (ln FAEE) levels were calculated, and then compared with partial correlation coefficients when controlling for albumin. The impact of serum albumin levels on the relationship between ln FAEE and BAL was evaluated by simple linear regression analysis., Results: Concentrations of total FAEE ranged from 632 to 20,166 nM, and BAL ranged from 9 to 375 mg/dL. In the 18 samples, the Pearson correlation coefficient between BAL and total FAEE levels was 0.868, and increased to 0.909 when controlling for albumin (p<0.0001). Similar statistically significant increases in the partial correlation coefficient occurred for each of the individual species of FAEE when controlling for albumin. In simple linear regression, albumin significantly reduced the variability in the model correlating BAL and FAEE for each of the individual species, as well as for total FAEE., Conclusions: The association between FAEE levels and BAL is enhanced by consideration of serum albumin concentrations. Our findings suggest that serum albumin should be included in any model attempting to define the relationship between BAL and FAEE.

Fatty acid ethanolamides (NAEs) are naturally occurring hydrophobic molecules usually present in a very small amount in many mammalian tissues and cells. Moreover, these compounds have been isolated in mammalian biological fluids, such as blood. Palmitoylethanolamide (C16:0) (PEA) is a fully saturated NAE, which presents some possible pharmaceutical activities, such as anti-inflammatory and antinociceptive effects. PEA is physiologically present in the mammalian blood at concentrations ranging from 9.4 to 16.7 pmol/ml. Since increasing evidence indicates that oxidative modification of low-density lipoproteins (LDL) is an important determinant in atherogenesis, the aim of this study was to evaluate the effect of physiologically relevant concentrations of PEA on Cu2+-induced LDL oxidation (measured as conjugated dienes formation). Our experiments indicate both anti-oxidative and slightly pro-oxidative effects of PEA. The anti-oxidative effect is obtained at low PEA concentrations (0.01 and 0.1 microM), while the pro-oxidative effect is obtained at a higher PEA concentration (1 microM). Fluorescence and circular dichroism data indicate that the effect of PEA occurs mainly by affecting the conformational features of ApoB-100.

The purpose of the present study was to investigate the possible regulation of plasma fatty acids by an acute isotonic-isooncotic central volume expansion. We measured the levels of nonesterified fatty acids (NEFA) in plasma from 12 essential hypertensive patients subjected to water immersion (WI). Central hypervolemia by WI over 2 hours caused the levels of most NEFA to increase, concomitantly with a marked natriuretic and kaliuretic response. With respect to baseline values, serum insulin levels did not change during WI, while there was a profound suppression of plasma renin activity (PRA) and plasma aldosterone. In addition, when individual NEFA percent increase was expressed as a function of salt-sensitivity index (calculated as the change in mean arterial pressure [MAP] divided by the change in urinary sodium excretion rate), a greater percent increase in stearic acid (r =.72, P <.009), palmitic acid (r =.83, P <.001), and palmitoleic acid (r =.58, P <.048) was found during WI in those hypertensive subjects showing higher salt-sensitivity index. Thus, by demonstrating that an acute isotonic-isooncotic volume expansion may induce a significant increase of most NEFA plasma levels, we suggest that volume expansion per se could be included among the well-recognized risk factors for cardiovascular morbid events., (Copyright 2003 Elsevier, Inc. All rights reserved.)

Familial hypobetalipoproteinemia (FHBL) subjects may develop fatty liver. Liver fat was assessed in 21 FHBL with six different apolipoprotein B (apoB) truncations (apoB-4 to apoB-89) and 14 controls by magnetic resonance spectroscopy (MRS). Liver fat percentages were 16.7 +/- 11.5 and 3.3 +/- 2.9 (mean +/- SD) (P = 0.001). Liver fat percentage was positively correlated with body mass index, waist circumference, and areas under the insulin curves of 2 h glucose tolerance tests, suggesting that obesity may affect the severity of liver fat accumulation in both groups. Despite 5-fold differences in liver fat percentage, mean values for obesity and insulin indexes were similar. Thus, for similar degrees of obesity, FHBL subjects have more hepatic fat. VLDL-triglyceride (TG)-fatty acids arise from plasma and nonplasma sources (liver and splanchnic tissues). To assess the relative contributions of each, [2H2]palmitate was infused over 12 h in 13 FHBL subjects and 11 controls. Isotopic enrichment of plasma free palmitate and VLDL-TG-palmitate was determined by mass spectrometry. Non-plasma sources contributed 51 +/- 15% in FHBL and 37 +/- 13% in controls (P = 0.02). Correlations of liver fat percentage and percent VLDL-TG-palmitate from liver were r = 0.89 (P = 0.0001) for FHBL subjects and r = 0.69 (P = 0.01) for controls. Thus, apoB truncation-producing mutations result in fatty liver and in altered assembly of VLDL-TG.

Acetyl trimethylaminoethyl ester iodide derivatives have been used to selectively analyze isomeric long-chain hydroxy fatty acids by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The binary derivatives of 2-, 3-, 12- and 16-hydroxypalmitic acids afford remarkably different product ion spectra. Further discrimination between isomers is possible by acylating with pivaloyl chloride. 2- and omega-Hydroxy long-chain fatty acids form pivaloyl esters in quantitative yield whereas other secondary alcohols only partially react. Cotton-based filter paper used for blood collection contains substantial amounts of esterified long-chain hydroxy fatty acids. From the product ion spectra of the acetyl trimethylaminoethyl esters the hydroxydocosanoic and -tetracosanoic acids are >90% omega-hydroxy. All remaining saturated and unsaturated hydroxy acids are >90% 2-hydroxy acids. A method for the quantification of free 3-hydroxypalmitic acid in plasma by ESI-MS/MS for the diagnosis of long-chain 3-hydroxyacyl CoA dehydrogenase deficiency (LCHAD) is described. Median plasma concentrations of 0.43 micromol/L (control, n = 22) and 12.2 micromol/L (LCHAD, n = 3) were obtained from 5 microL plasma samples., (Copyright 2002 John Wiley & Sons, Ltd.)

The objective of this study was to examine the ocular absorption behavior of an amphiphilic prodrug after instillation onto the cornea of rabbits. A micellar solution of O-palmitoyl tilisolol (PalTL), an amphiphilic prodrug, was prepared. After instillation of tilisolol (TL) and PalTL, the drug concentrations in the tear fluid, cornea, aqueous humor, iris-ciliary body, vitreous body, and blood were measured. In addition, in situ ocular absorption behavior was also evaluated. After instillation of TL, the concentration of TL in the tear fluid quickly decreased. After instillation of PalTL, prolonged retention and high concentrations of PalTL in tear fluid and the cornea were observed. In addition, more prolonged retention of the TL concentration after instillation of PalTL than after instillation of TL was observed in the cornea, aqueous humor, and iris-ciliary body. In situ experiments demonstrated that PalTL was mainly absorbed by the corneal route and the improvement effects of PalTL under in vivo conditions was due to an enhanced transit time of PalTL in ocular tissues. PalTL, an amphiphilic prodrug, exhibited increased retention in the precorneal area compared with the parent drug, TL, resulted in improved ocular absorption of the parent drug., (Copyright 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association)

The biological actions of the endogenous cannabinoid anandamide are terminated by carrier-mediated transport into neurons and astrocytes, followed by enzymatic hydrolysis. Anandamide transport is inhibited by the compound N-(4-hydroxyphenyl)arachidonylamide (AM404). AM404 potentiates several responses elicited by administration of exogenous anandamide, suggesting that it may also protect endogenous anandamide from inactivation. To test this hypothesis, we studied the effects of AM404 on the plasma levels of anandamide using high-performance liquid chromatography/mass spectrometry (HPLC/MS). Systemic administration of AM404 (10 mg kg(-1) intraperitoneal, i.p. ) caused a gradual increase of anandamide in rat plasma, which was significantly different from untreated controls at 60 and 120 min after drug injection. In plasma, both AM404 and anandamide were associated with a plasma protein, which we identified as albumin by non-denaturing polyacrylamide gel electrophoresis. AM404 (10 mg kg(-1), i.p.) caused a time-dependent decrease of motor activity, which was reversed by the cannabinoid CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide.hydrochloride (SR141716A, 0.5 mg kg(-1), i.p). These results are consistent with the hypothesis that AM404 inhibits anandamide inactivation in vivo.

Determination of free fatty acids (FFAs) in control serum and human plasma was carried out by high-performance liquid chromatography with electrochemical detection. Peak height for palmitic, palmitoleic, stearic, oleic, linoleic, and arachidonic acids at a detection potential of -415 mV vs a saturated calomel electrode showed a linear relation to acid amount in the range 50-1600 pmol. The present method for plasma free fatty acid determination required only 10 microL of plasma sample. The method is simple and the time for blood pretreatment is short. Change in plasma FFA with blood glucose level was monitored before and after meal ingestion by this method, using one male and one female subject. It was possible to rapidly reduce plasma FFA and increase blood glucose subsequent to the meal. The present method is thus shown to have potential for clinical application., (Copyright 2000 Academic Press.)

Background: Cholestatic liver disease develops in 30% to 70% of neonates receiving total parental nutrition (TPN). We analyzed the fatty acid composition of serum phospholipids from control and TPN-fed rabbits to determine if TPN altered the fatty acid profile., Methods: Eleven male New Zealand White rabbits aged 9 to 11 weeks received TPN, whereas 11 other rabbits were offered standard laboratory rabbit chow ad libitum. After 14 days on the prescribed diet, serum samples were analyzed for their phospholipid fatty acid content by gas chromatography., Results: The proportions of palmitolenic (16:2n7), alpha-linolenic (18:3n3), arachidic (20:0), and eicosaenoic (20:1n9) acids were significantly lower in the serum phospholipids of the TPN-fed animals compared with the control group. The proportion of docosahexaenoic acid (22:6n3), a fatty acid that is critical to the development of the nervous system, was increased two- to threefold., Conclusions: The differences in proportions of fatty acids observed between control and TPN-fed animals indicate that a fatty acid elongation and desaturation pathways are perturbed in rabbits on TPN.

Fatty acid ethyl esters (FAEEs) are nonoxidative products of ethanol metabolism and have been implicated as mediators of ethanol-induced organ damage. Previous studies have demonstrated that FAEEs bind to lipoproteins and albumin in human plasma after ethanol ingestion. Analysis of human serum with varying blood ethanol levels and endogenously formed FAEEs revealed a positive correlation between serum FAEE concentration and the percentage of FAEEs associated with lipoproteins, predominantly very low density and low density lipoprotein. Similar results were obtained when increasing amounts of FAEEs were added to serum with zero blood ethanol. Additional studies indicated that free fatty acids and FAEEs do not compete for binding to albumin or lipoproteins. Data support the conclusion that the distribution of FAEEs among their carriers in the serum is dependent on serum FAEE concentration.

The possibility that fatty acid ethyl esters (FAEEs), esterification products of fatty acids and ethanol, are mediators of ethanol-induced organ damage was suggested by an autopsy study in which individuals who died while acutely intoxicated were found to have FAEEs predominantly in the organs damaged by ethanol abuse. We initially observed in human subjects after ethanol consumption that there is a marked preference for the synthesis of ethyl palmitate and ethyl oleate over other FAEEs. To investigate the basis for this relative fatty acid specificity for FAEE synthesis, we used an in vitro system of Hep G2 cells incubated with ethanol. The cells were capable of synthesizing FAEEs upon exposure to ethanol and they showed a preference for synthesis of ethyl palmitate and ethyl oleate, as was found in human plasma after ethanol ingestion. This finding allowed us to explore the metabolic preference for palmitate and oleate for FAEE synthesis at the biochemical level using intact cells. We demonstrated that the preferential selection of palmitate and oleate for FAEE synthesis was not likely to be the result of specificity for palmitate and oleate by FAEE synthase or preferential uptake of palmitate and oleate by Hep G2 cells. In studies to determine whether the preference for ethyl palmitate and ethyl oleate synthesis was a result of higher concentrations of palmitate and oleate in the extracellular medium, we observed that the synthesis of ethyl oleate, ethyl linoleate, and ethyl arachidonate, but not ethyl palmitate, is influenced by the extracellular concentration of its corresponding fatty acid. The results of our studies indicate that ethyl palmitate and ethyl oleate are the predominant ethyl esters synthesized, that there is no preferential uptake or enzyme affinity for their fatty acid precursors to explain the predominance, and that ethyl palmitate synthesis is uniquely unaffected by the concentration of palmitate in the extracellular medium.

This project was designed to test the hypothesis that long-chain saturated fatty acids (myristate, palmitate, and stearate) are metabolized differently in human subjects, and that these differences may therefore account for the changes in plasma lipoprotein composition when these fatty acids are altered in the diet. Ethyl esters of each of the stable-isotope-labeled fatty acids (2H3- or 2H4-myristate, 13C16-palmitate, and 13C18-stearate) were fed to five nonhyperlipidemic men. The concentration of each labeled fatty acid was monitored for up to 72 hours as the fatty acids were assimilated into the lipid components (phospholipid [PL], triglyceride [TG], and cholesteryl ester [CE]) of the plasma lipoproteins (TG-rich lipoproteins [TRL], intermediate-density [IDL], low-density [LDL], and high-density lipoprotein [HDL]). Over 95% of the myristate was incorporated into TG, whereas 33% and 9% of the stearate and 18% and 7% of the palmitate were incorporated into PL and CE, respectively. The mean residence times (MRTs) for myristate in TG (8.6 to 9.9 hours) and PL (6.7 to 10.9 hours) in the individual lipoprotein subfractions were significantly shorter than for either palmitate (TG, 12.7 to 15.3 hours; PL, 19.6 to 21.3 hours) or stearate (TG, 10.7 to 15.5 hours; PL, 17.8 to 19.9 hours). The MRTs for stearate were shorter than for palmitate in PL. These data indicate that TG fatty acid in general, and myristate TG in particular, is the most rapidly cleared of the saturated fatty acids. There was a rapid transfer of labeled TG and PL between the lipoproteins. We were unable to detect any significant amount of stearate desaturation or elongation. In conclusion, these data demonstrate that myristate, palmitate, and stearate are metabolized in unique ways, and that it may therefore be inappropriate to continue to regard all "saturated fatty acids" as metabolically similar in clinical studies. Rather, it is important that we elucidate more clearly the specific metabolic pathway of each fatty acid to understand the mechanisms by which it alters plasma lipoprotein concentrations and composition and influences atherogenesis.

Background: Portacaval anastomosis has an hypolipemic effect in familial hypercholesterolemia and in healthy animals. In cirrhosis, it raises serum cholesterol, but there is no information on its effect upon plasma fatty acids. However, indirect data suggest that portacaval shunting might contribute to the polyunsaturated fatty acid deficit of these patients. We assessed the effect of portacaval anastomosis on plasma fatty acid profile in cirrhosis., Methods: Forty-four Child-Pugh class A/B bleeding cirrhotics were randomized to be treated with portacaval anastomosis (n = 20) or nonsurgical therapy (n = 24). Fatty acid profile in plasma total lipids, alcohol intake, anthropometry, Child-Pugh score, serum cholesterol, triglycerides, and antioxidant micronutrients were assessed before and 3, 6, 12, 18, and 24 months after surgery or the start of nonsurgical therapy. Time course of plasma fatty acids was assessed using unbalanced repeated measures models with the above mentioned variables acting as covariates., Results: No changes in the time course of percent plasma saturated, monounsaturated, and essential fatty acids were found between groups. Percent long-chain omega-6 and omega-3 polyunsaturated fatty acids decreased during follow-up in shunted patients compared with controls (p = .007 and p < .0005). However, this was not due to a true decrease in polyunsaturated fatty acid levels but to greater increases in saturated and monounsaturated fatty acid concentrations in shunted patients compared with control patients (p = .047 and p = .006)., Conclusions: Portacaval anastomosis does not worsen plasma polyunsaturated fatty acid deficiency in cirrhosis. However, by increasing saturated and monounsaturated fatty acids, it further decreases plasma lipid unsaturation.

Recent studies have shown that, in addition to free steroid hormones, those bound to albumin in plasma may also be available to tissues. In this report, the effects of free fatty acids (FFA) on the binding of steroids to albumin were compared for the cases of bovine serum albumin (BSA) and human serum albumin (HSA). The apparent association constant, Ka, was estimated from the changes in the equilibrium partition coefficient of steroids between the aqueous/hexane phases caused by the addition of albumin to the aqueous phase. In the case of BSA, Ka for progesterone and testosterone increased upon binding of FFA (myristic, palmitic and stearic acid) to BSA and the maximum value of Ka for these steroids could be attained by 3--4 mol of FFA bound per mol BSA. Furthermore, the elution profiles of gel-filtration chromatography clearly showed that progesterone and testosterone are easily liberated from the steroid/BSA complexes and that FFA potentiates the binding of these steroids to BSA. In the case of HSA, the binding affinities of progesterone and testosterone were not greatly affected by bound FFA. On the other hand, the affinities of ethynylestradiol to both BSA and HSA were unaffected below 4 mol of FFA binding per mol.

Objectives: Low concentrations of alpha tocopherol are claimed to be associated with an increased prevalence of coronary heart disease. This study was undertaken to see whether measurements of serum tocopherol concentrations can contribute to discrimination between subjects with and without coronary heart disease., Setting: All patients had been referred to the department of cardiology of the University Hospital in Uppsala, Sweden., Subjects: Male patients (n = 69) below 60 years of age with coronary heart disease (CHD) and healthy age-matched reference subjects (n = 138) were compared., Results: Lipid-corrected alpha tocopherol concentrations did not differ significantly between the groups, but the CHD group had a lower mean concentration of gamma tocopherol and a higher alpha/gamma ratio. In a stepwise logistic regression analysis, the LDL/HDL ratio was the best independent discriminator between the groups, followed by the proportion of palmitic acid in the cholesterol esters and the alpha/gamma tocopherol ratio., Conclusions: The lower gamma tocopherol concentration and the high ratio between alpha and gamma tocopherol in the CHD group indicate a difference in antioxidative status between CHD patients and healthy subjects. The lipid-lowering treatment of these CHD patients is far from optimal.

Potential promoting effects of alpha-linolenic, linoleic and palmitic acids were investigated in a two-stage urinary bladder carcinogenesis model. In experiment 1, male F344 rats were given 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in their drinking water for 4 weeks and then basal diet containing 10% alpha-linolenic, 10% linoleic or 10% palmitic acid along with 0.2% butylated hydroxyanisole (BHA) as an antioxidant for 24 weeks. The development of tumors in the urinary bladder was not increased by treatment with any of the fatty acids. In experiment 2, male F344 rats were given 10% alpha-linolenic, 10% linoleic or 10% palmitic acid along with 0.2% BHA in their diet for 8 weeks without prior BBN treatment. The administration of fatty acids was not associated with any increase in the 5-bromo-2'-deoxyuridine labeling index of the urinary bladder epithelium. Serum and/or urine fatty acid levels increased in the cases of alpha-linolenic and linoleic acid treatments, but not with palmitic acid. Under the present experimental conditions neither the two polyunsaturated nor the one saturated fatty acid exerted any promoting effect on urinary bladder carcinogenesis.

Determinants of basal lipolysis were investigated in a group of 23 volunteers of both sexes. Body composition was measured by underwater weighing and resting metabolic rate with indirect calorimetry. Insulin sensitivity was determined by using the minimal model of Bergman. The rate of appearance of palmitic acid in the blood was measured with 14C-1-palmitate as the tracer administered as a nonprimed constant infusion. Simple and stepwise-regression analyses were performed to determine correlations and to generate a multivariate model to examine the determinants of basal lipolysis. Fat intake (as a percent of total energy intake) was correlated with basal lipolysis (r = 0.57, P < 0.005) as well as protein intake (as a percent of energy intake) (r = -0.46, P = 0.03) in univariate analysis. Body weight (r = 0.56, P = 0.005) explained a greater portion of the variance in lipolysis than fat-free mass (r = 0.44, P < 0.05) or fat mass (NS). Independent predictors of basal lipolysis were determined by stepwise regression. The best model generated included fat intake, fat-free mass, carbohydrate intake, and daily energy intake as significant determinants of lipolysis (r = 0.89, P < 0.001). The insulin sensitivity index and sex were not independent predictors of basal lipolysis. Thus, our data support either body weight or fat-free mass as more appropriate indexes than fat mass to normalize basal rates of lipolysis in humans. Furthermore, the macronutrient composition of the diet is an important determinant of lipolysis and thus should be considered in future experimental designs.

Protein 4.2 is a major protein of the human erythrocyte membrane. It has previously been shown to be N-myristoylated. After labeling of intact human erythrocytes with [3H]palmitic acid, radioactivity was found to be associated with protein 4.2 by immunoprecipitation of peripheral membrane proteins extracted at pH 11 from ghosts with anti-(4.2) sera, followed by SDS/PAGE and fluorography. The fatty acid linked to protein 4.2 was identified as palmitic acid after hydrolysis of protein and thin-layer chromatography of the fatty acid extracted in the organic phase. Protein 4.2 could be depalmitoylated with hydroxylamine, suggesting a thioester linkage. Depalmitoylated protein 4.2 showed significantly decreased binding to protein-4.2-depleted membranes, compared to native protein 4.2.

Spur cell anemia is a hemolytic anemia seen in severe alcoholic cirrhosis that is characterized by unusual morphology and a decreased ratio of phospholipids to cholesterol in the erythrocyte membrane. We hypothesized that defective phospholipid repair may contribute to the red blood cell (RBC) phospholipid abnormalities of spur cell anemia. Therefore, we compared RBCs from normal control subjects with RBCs from spur cell anemia patients. The incorporation of [14C] arachidonic acid into the phospholipids and acylcarnitine (acyl-Cn) of spur cells and normal RBCs was analyzed by a direct-phase high performance liquid chromatography column to separate both the phospholipids and acyl-Cn. There was less uptake of the [14C] arachidonate into phosphatidylethanolamine of spur cell RBCs (12.9% +/- 1.0%) compared with normal RBCs (20.5% +/- 2.8%; P = .0245). However, more arachidonate was incorporated into the acyl-Cn of spur cells (spur cell acyl-Cn [24.5% +/- 2.9%] v normal control acyl-Cn [10.1% +/- 1.9%]; P = .0018). We conclude that phospholipid biosynthesis is inhibited and that acyl-Cn formation is spared in spur cell anemia RBCs. These metabolic changes may help account for the lipid abnormalities seen in spur cell anemia RBCs and contribute to the hemolytic process.

The fatty acid composition of the erythrocyte membrane was determined in 22 hypercholesterolaemic patients managed by dietary restriction, and compared with that of 22 normocholesterolaemic controls, roughly matched for age and sex with the patient group. The patients exhibited higher relative proportions of palmitic (P < 0.01) and stearic (P < 0.005) acids and lower relative proportions of linoleic acid (P < 0.05) in the erythrocyte membrane, compared with controls, which could be due to presumed dietary differences between the groups. In the patients, the degree of hypercholesterolaemia was poorly correlated with erythrocyte linoleic acid. Measurement of erythrocyte linoleic acid might prove useful in the routine management of hypercholesterolaemia.

Turnover of plasma free fatty acids (FFAs) can be determined from the palmitate enrichment of plasma after administration of analogues labeled with stable isotopes. We studied the conditions to measure both the concentration and the 13C enrichment of plasma palmitate by gas chromatography-mass spectrometry (GC-MS) using crude extracts. The method used plasma extraction after addition of heptadecanoic acid as internal standard and methylation with diazomethane. Subsequently the samples were analyzed by GC-MS. Plasma palmitate levels determined with this simplified method did not differ statistically from those obtained by a more "classical" procedure using FFA separation from other plasma lipids. Palmitic acid turnover rates (Ra) were evaluated in the steady-state period, in two normal subjects after 90 min infusion with [1-13C]palmitate bound to human albumin. The rate of appearance (Ra) was found to be 0.92 and 1.08 mmol kg-1 min-1, which is in good agreement with the turnover rate previously reported for normal subjects. Sample preparation and GC-MS analysis by the proposed procedure are simple and rapid and thus the method appears to be particularly useful in clinical studies where numerous samples have to be analyzed.

To determine whether estrogen directly affects effective adipose lipolysis, palmitate rate of appearances ([14C]palmitate) was measured in 15 postmenopausal women. Each volunteer was studied after > or = 2 mo of estrogen treatment and again after > or = 2 mo of estrogen deficiency. Plasma hormone concentrations were controlled and identical on the 2 study days with use of the pancreatic clamp technique, and the lipolytic response to epinephrine and epinephrine + phentolamine was assessed. Results showed that overall palmitate flux was greater (10-20%, P < 0.05) during the estrogen-deficient than during the estrogen-replete study. Adrenergic stimulation of lipolysis was not specifically influenced by estrogen treatment, and control of plasma hormone concentrations did not eliminate the difference in palmitate flux between the estrogen-deficient and estrogen-replete study days. We conclude that estrogen deficiency is associated with increased plasma free fatty acid availability and that estrogen likely has direct, albeit small, effects on adipose tissue lipolysis.

Palmitic acid shows a very low and unknown solubility at neutral pH. Binding equilibria of palmitate to human serum albumin accordingly cannot be investigated by measuring free and bound ligand concentrations as in conventional binding studies. It is feasible, on the other hand, to describe the binding equilibria in relative terms, by measuring the concentration, p, of reserve albumin, previously defined as the concentration of a purified standard albumin preparation which in buffered solution binds a trace amount of palmitate as tight as it is bound in the sample. Palmitate availability is calculated as C/p, when C is the concentration of bound palmitate. The general binding equation is modified to contain the availability beside relative binding constants, Li = Ki/K1,St, where K1,St is the first stoichiometric binding constant for palmitate to the standard albumin preparation. Availabilities and relative binding constants can replace free concentrations and usual binding constants in considerations of biochemical transport and enzymatic mechanisms. A method is described for measuring the concentration of reserve albumin for binding of palmitate, based upon determination of dialytic exchange rates for palmitate among identical equilibrium samples. A technique for reproducibly adding radiolabelled palmitate to the samples is given.

The aim of the study was to ascertain the metabolic and dietary determinants of changes in serum lipids during a 15-month diet therapy of obese patients (n = 71, 41 males, 30 females) with recently diagnosed Type 2 (non-insulin-dependent) diabetes. The subjects lost weight and improvement in glycaemic control was observed, but due to variation in individual responses the mean serum total cholesterol or non-HDL cholesterol did not change significantly. The proportion of palmitic acid decreased and that of linoleic acid increased in serum lipids during the study, and serum triglycerides decreased and HDL-cholesterol increased. In univariate analyses, decreased serum triglyceride level was associated with serum triglycerides at baseline, decreases in body mass index, fasting blood glucose and palmitic acid proportion of serum triglycerides, and the intake of saturated fats and dietary fibre, but in multiple regression analyses the determinants for decreased serum triglycerides were high serum triglycerides at baseline and a decreased proportion of palmitic acid in serum triglycerides. In univariate analysis, increased HDL-cholesterol was associated with the baseline HDL-cholesterol, decrease in the triceps/subscapularis ratio and the intake of saturated and mono-unsaturated fatty acids, but none of these variables had an independent contribution to the increase in serum HDL-cholesterol in multiple regression analysis. In conclusion, a reduction of palmitic acid in the serum lipids, which was probably due to reduction of dietary saturated fatty acids, had beneficial effects on serum lipids in obese patients with Type 2 diabetes, independently of weight loss and improvement in glycaemic control.

To assess whether ursodeoxycholic acid (UDCA) treatment has any beneficial effect on essential fatty acid (EFA) deficiency in patients who have had a Kasai operation for extrahepatic atresia (EBA), responses of serum fatty acids to UDCA administration (15 mg/kg/d) were investigated in eight jaundice-free patients and in eight patients with jaundice (serum total bilirubin > or = 1.0 mg/dL). All patients were also given taurine supplementation (100 mg/kg/d). Serum fatty acid composition was determined before and 6 months after UDCA treatment. Serum total bile acid concentration and serum total bilirubin value, as a part of conventional liver function tests, were measured before and during UDCA therapy. Before UDCA treatment, the concentrations of linoleic acid and arachidonic acid were significantly lower (P > .05 for the former; P > .01 for the latter) in both the jaundice and jaundice-free groups than in the controls. After 6 months of treatment, the linoleic acid concentration significantly increased (P > .05), to the normal range, in the jaundice-free group, but not in the jaundice group. The arachidonic acid concentration did not increase significantly in either group. The serum total bile acid concentration was lower in six of the eight jaundice-free patients and in four of the eight jaundice patients. The serum total bilirubin value decreased in six of the eight jaundice-free patients and in four of the eight jaundice patients; however, the degree of improvement was not statistically significant in either group. No side effects developed, and there were no changes in blood chemistry values unrelated to liver disease.(ABSTRACT TRUNCATED AT 250 WORDS)

The alpha subunits of GTP-binding regulatory proteins (G proteins) are subject to lipid modifications required for anchorage to membrane and/or interactions with other proteins. With the knowledge that alpha subunits are palmitoylated, which we demonstrate here for human platelets, we sought to determine whether these subunits also bind arachidonate and myristate in a covalent, post-translational manner. All alpha subunits examined were found to incorporate radioactivity upon incubation of human platelets with [3H]palmitate, [3H]arachidonate, and [3H]myristate. The identity of [3H]palmitate and [3H]arachidonate as covalently bound fatty acids was confirmed by high pressure liquid chromatography following alkaline methanolysis. With [3H]myristate, however, the bound fatty acid proved to be [3H]palmitate, presumably generated by a 2-carbon chain elongation. Protein-bound [3H]palmitate and [3H]arachidonate were released by hydroxylamine at neutral pH, implying a thioester linkage between protein and fatty acid. Thus, post-translational modifications of G protein alpha subunits include palmitoylation and arachidonoylation, but not myristoylation. Given the different physical properties of saturated and unsaturated fatty acids and the large-scale release of arachidonate during platelet activation, changes in arachidonate incorporation may serve as an important regulator of alpha subunit function.

Patients with Crohn's disease may become zinc-deficient and, in such patients, an altered metabolism of radiolabelled long-chain fatty acids has been reported. We have investigated the possible reversal by zinc supplementation of altered long-chain fatty acid profiles of red cells in Crohn's disease. Twenty patients with long-standing Crohn's disease in clinical remission received 200 mg of zinc sulphate daily for 6 weeks. Phospholipid fatty acid profiles of washed red cells were analysed before and after zinc treatment and compared to those of 20 unsupplemented healthy controls. Plasma zinc levels in Crohn's were 72 +/- 8 micrograms/dL before zinc treatment and increased to 114 +/- 10 micrograms/dl after the therapy. Prior to zinc supplementation, the percentage of palmitic, stearic and oleic acids was significantly higher in Crohn's disease, while linoleic, arachidonic and n-3 fatty acids were reduced in Crohn's disease compared to healthy controls. Zinc supplementation abolished these pre-treatment differences in red-cell long-chain fatty acid profiles but did not affect plasma fatty acid values. Further studies are needed to clarify whether these fatty acid changes can be related to the clinical course of the disease.

Both glucose and insulin are important regulators of glucose uptake and hepatic glucose release. Because insulin concentrations rarely if ever increase under daily living conditions, unless glucose concentrations also increase, we sought to determine whether hepatic and extrahepatic responses to changes in insulin and glucose concentration are impaired in patients with non-insulin-dependent diabetes mellitus (NIDDM). To address this question, glucose metabolism was measured in diabetic and nondiabetic subjects. A computer-driven infusion system was used to produce a nondiabetic postprandial insulin profile in both groups while sufficient exogenous glucose was infused to mimic nondiabetic postprandial glucose concentrations. Although NIDDM was associated with greater (P < 0.05) hepatic glucose release both before and during the prandial insulin infusion, suppression did not differ in the diabetic and nondiabetic subjects (-1.06 +/- 0.20 vs. -0.86 +/- 0.15 mmol/kg every 4 h). In contrast, stimulation of both glucose disappearance (0.77 +/- 0.27 vs. 1.68 +/- 0.27 mmol/kg every 4 h) and forearm glucose uptake (187 +/- 81 vs. 550 +/- 149 mumol/dl every 4 h) was lower (P < 0.05) in diabetic than in nondiabetic subjects. Thus, despite increased basal rates of glucose production, obese individuals with NIDDM had decreased stimulation of glucose disappearance but normal suppression of hepatic glucose release in response to nondiabetic prandial glucose and insulin concentrations. These data indicate that the increase in glucose that occurs with carbohydrate ingestion is likely to compensate for hepatic but not extrahepatic insulin resistance.

To examine whether hyperglycemia is an independent regulator of adipose tissue lipolysis, we measured palmitate flux ([3H]palmitate) on two occasions in eight volunteers with insulin-dependent diabetes. On one. occasion, euglycemia was maintained for 4 h continuously; on a different occasion, hyperglycemia (plasma glucose, 12 mmol/l) was induced after 2 h of euglycemia. Palmitate flux decreased from 1.39 +/- 0.22 to 1.25 +/- 0.18 mumol.kg-1 x min-1 during sustained euglycemia and from 1.43 +/- 0.24 to 1.13 +/- 0.19 mumol.kg-1 x min-1 during the transition from the euglycemic to the hyperglycemic study intervals. There were no significant differences between the changes in palmitate flux from the first to the second study interval on the control (euglycemia-euglycemia) and experimental (euglycemia-hyperglycemia) study days and no difference between palmitate flux on different study days. Thus, in the face of euinsulinemia, euglucagonemia, and the absence of somatostatin, no effect of hyperglycemia on free fatty acid metabolism could be detected in humans.

Stable isotope tracers and indirect calorimetry were used to evaluate the progressive alterations in lipid and glucose metabolism after 12, 18, 24, 30, 42, 54, and 72 h of fasting in six healthy male volunteers. The rates of appearance (Ra) of glycerol and palmitic acid in plasma doubled from 2.08 +/- 0.22 and 1.63 +/- 0.20 mumol.kg-1 x min-1, respectively, after 12 h to 4.36 +/- 0.36 and 3.26 +/- 0.40 mumol.kg-1 x min-1, respectively, after 72 h of fasting (P < 0.01). Of the total increase in lipid kinetics, 60% occurred between 12 and 24 h of fasting; the greatest interval change occurred between 18 and 24 h of fasting. Glucose Ra and plasma concentration decreased by approximately 25% between 12 h (11.0 +/- 0.4 mumol.kg-1 x min-1 and 5.58 +/- 0.08 mmol/l, respectively) and 72 h (8.3 +/- 0.3 mumol.kg-1 x min-1 and 4.14 +/- 0.10 mmol/l, respectively) of fasting (P < 0.01), but no statistically significant changes occurred between 18 and 24 h of fasting. Plasma insulin decreased by approximately 50% between 12 h (64.6 +/- 12.9 pmol/l) and 72 h (30.1 +/- 7.9 pmol/l) of fasting (P < 0.001). Of the total decline in plasma insulin, 70% occurred within the first 24 h of fasting. These results demonstrate that the mobilization of adipose tissue triglycerides increases markedly between 18 and 24 h of fasting in young adult men. The early alterations in lipid metabolism are associated with a decline in circulating insulin but do not seem to be regulated by changes in glucose kinetics or plasma glucose concentrations.

We have previously presented a precursor-product stable isotopic technique for measuring in vivo the fraction of very low-density lipoprotein-fatty acids (VLDL-FA) derived from de novo lipogenesis (fractional DNL). Here, we propose a technique for converting fractional DNL into absolute rates of DNL and describe its explicit underlying assumptions. The technique combines the fractional DNL method with a modification of the method of S. Klein, V. R. Young, G. L. A. Blackburn, B. R. Bistrain, and R. R. Wolfe (J. Clin. Invest. 78: 928-933, 1986), for estimating hepatic reesterification of free fatty acids (FFA). Infusions of [1,2,3,4-13C]palmitate and [1-13C]acetate are performed concurrently with indirect calorimetry in human subjects. Fractional DNL (based on mass isotopomer distribution analysis of VLDL-FA), the rate of appearance of plasma FFA (Ra of FFA), and net fat oxidation in the whole body are measured. Equations from the hepatic reesterification model, modified to include the contribution from DNL, allow calculation of absolute DNL (= fractional DNL x [Ra of FFA - net whole body fat oxidation], when respiratory quotient < 1.0). Sample results from human subjects with different dietary energy intakes are presented, with calculations of absolute DNL, absolute reesterification, and absolute fat oxidation rates. The assumptions of this technique (in particular, that all fat oxidized is derived at steady state from circulating FFA and that DNL and reesterification of FFA both occur exclusively in liver) are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Total plasma fatty acids were analysed in 75 first trimester pregnant women, 32 second trimester pregnant women, 27 women at delivery and 38 non-pregnant women. Eleven fatty acids were studied by capillar gas chromatography SP-column. Compared with non-pregnant women, in the first trimester there was a significant percentual increase in palmitic, palmitoleic, stearic and docosahexaenoic acids, whereas linoleic and eicosapentaenoic percentually decreased. Between the first and second trimester a significant increase in the proportion of palmitic acid and a significant percentual decrease in arachidonic acid was detected. Between the second trimester and delivery the aforementioned changes were more marked. Between the second trimester and delivery there was also a significant percentual increase in oleic and linolenic acids, whereas the proportions of stearic, linoleic, dihomogammalinolenic and docosahexaenoic acids significantly decreased. It is suggested that the intake in polyunsaturated fatty acids should be increased during pregnancy, specially in the third trimester.

Plasma free fatty acid (FFA) levels tend to be lower and the plasma lipolytic hormone response to prolonged exercise of the same intensity is blunted after endurance exercise training. To determine whether training elicits a corresponding decrease in plasma FFA turnover and metabolism during prolonged exercise, we measured plasma [1-13C]palmitate kinetics and oxidation and respiratory gas exchange in 13 subjects during the latter portion of a 90- to 120-min bout of cycle ergometer work performed before and after 12 wk of alternate-day cycling and running. Training increased total fat oxidation during prolonged exercise by 41% (P < 0.005). However, for the final 30-60 min of the cycle ergometer protocol, the rate of 13CO2 production from [1-13C]palmitate oxidation was 27% lower (P < 0.05), the rate of palmitate turnover was 33% less (P < 0.05), and plasma FFA and glycerol concentrations were 32 and 20% lower (P < 0.05), respectively, than in the untrained state. Thus endurance exercise training results in decreased plasma FFA turnover and oxidation during a 90- to 120-min bout of submaximal exercise because of a slower rate of FFA release from adipose tissue.

Binding of fibrinogen to platelets washed from the blood of patients with hypercholesterolemia and hypertriglyceridemia (n = 25) and control donors (n = 12) was compared. In addition, the content of platelet glycoprotein IIb was determined by radioimmunoassay. Fibrinogen was bound in significantly higher amounts (P < 0.02) to hyperlipidaemic platelets activated by ADP than to control ones (107,112 +/- 16,371 and 45,612 +/- 6495 molecules per platelet, respectively). The mean content of GPIIb was the same in hyperlipidaemic and in control platelets (2.06 +/- 0.16 and 1.94 +/- 0.21 micrograms/10(8) platelets, respectively). The amount of fibrinogen bound to the activated hyperlipidaemic platelets showed a positive correlation with total plasma cholesterol and LDL (r = 0.45 and 0.47, respectively) whereas a negative correlation with plasma HDL was found (r = -0.50). The increased expression of fibrinogen binding sites similar to that of hyperlipidaemic platelets could be produced by preincubation of normal platelets with palmitic acid. This was evidenced by a significant increase of fibrinogen binding sites in control platelets. This suggests that either palmitoylation of the receptor or microenvironment changes in the membrane lipid bilayer may be responsible for the enhanced platelet receptor capacity to bind fibrinogen.

Fatty acid composition of phosphatidylinositols was analyzed in peripheral blood lymphocytes from nine alcoholic patients who were well nourished and without severe acute and chronic liver disease, before and after stimulation with anti-CD3 antibody. Six comparable nondrinkers were studied as controls. A reduction in unsaturated fatty acid (mainly arachidonic) and an increase in palmitic and stearic acid molar content were observed in phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2) in unstimulated samples from alcoholic patients in comparison with normal subjects, leading to a significant decrease in the saturated/unsaturated ratio. In controls, anti-CD3 stimulation caused a marked decrease in arachidonic acid relative molar content counterbalanced by an increase in other polyunsaturated fatty acid relative molar content in PI, PIP, and PIP2 fractions. Interestingly, after anti-CD3 stimulation, alcoholic patients show the same trend of modification in the fatty acid composition resulting in a sharp reduction of arachidonic acid relative molar content. These results support the hypothesis of an alteration in nutrients being responsible for immune derangement in alcoholics.

Binding of valproate and palmitate to serum albumin was studied in 29 valproate-treated epileptic patients. The results were compared with similar observations in a reference group of 43 non valproate-treated individuals. The binding affinity for palmitate was decreased (P < 0.0001) resulting in increased availability of long-chain fatty acids (P = 0.008) due to competitive valproate binding in the valproate-treated patients. The findings support a hypothesis on the pathogenesis of obesity as a complication of valproate treatment of epilepsy.

The posttranslational modification of proteins by fatty acids has been shown to involve long chain-saturated fatty acids, predominantly palmitate. In the present study, we demonstrated by metabolic labeling of human platelets with [3H]arachidonate and [3H]eicosapentaenoate that these polyunsaturated fatty acids can also become covalently linked to proteins. The extent of binding of arachidonate to proteins was somewhat less than that of palmitate. Arachidonate binding to platelet proteins was not significantly influenced by the inhibition of cyclooxygenase and lipoxygenase. This finding and the high performance liquid chromatography analysis of radiolabeled products removed from proteins by selective cleavage techniques established that arachidonate, and not its metabolic products, was the protein-linked radiolabeled moiety in [3H]arachidonate-labeled platelets. A 7.5-fold higher concentration of unlabeled palmitate competed to a small extent with [3H] arachidonate for protein labeling. Both arachidonate and eicosapentaenoate were bound to proteins almost exclusively through ester linkages. It was further demonstrated that 61 and 66% of total protein-linked arachidonate and eicosapentaenoate, respectively, were bound via thioester bonds. In contrast, 91% of the binding of palmitate to proteins occurred via thioester linkages. As demonstrated by SDS-polyacrylamide gel electrophoresis and fluorography, the patterns of palmitoylated and arachidonoylated proteins were similar but not identical, with selected proteins only palmitoylated or only arachidonoylated. [3H]Eicosapentaenoate labeled the same set of proteins as [3H]arachidonate. The fluorographic pattern of 3H-arachidonoylated proteins was not changed by cyclooxygenase and lipoxygenase inhibitors. The binding of a polyunsaturated fatty acid to a protein in place of a saturated fatty acid could significantly influence the hydrophobic interactions of the protein and, thereby, have important functional implications.

Glucagon may regulate FFA metabolism in vivo. To test this hypothesis, six healthy male volunteers were infused with somatostatin, to inhibit endogenous hormone secretion, and insulin, glucagon, and GH to replace endogenous secretion of these hormones. In the hypoglucagonemia experiments, the glucagon infusion was omitted, and in the hyperglucagonemic experiments glucagon was infused at 1.3 ng/kg.min, to produce physiological hyperglucagonemia. In two sets of control experiments, glucagon was infused at 0.65 ng/kg.min, in order to maintain peripheral euglucagonemia, and the plasma glucose concentrations were clamped at the levels observed in either the hypo- or hyperglucagonemic experiments. Rates of FFA and glycerol (an index of lipolysis) appearance (Ra) were estimated with the isotope dilution method using [1-14C]palmitate and [2H5] glycerol. Plasma glucagon concentrations decreased during the hypoglucagonemic experiments (85 +/- 12 vs. 123 +/- 22 ng/L, P < 0.05) and increased during the hyperglucagonemic experiments (186 +/- 20 vs. 125 +/- 15 ng/L, P < 0.05), whereas other hormone concentrations remained the same. Hypoglucagonemia resulted in equivalent suppression of FFA Ra (3.7 +/- 0.2 vs. 5.9 vs. 0.3 mumol/kg.min, P < 0.01) and glycerol Ra (1.2 +/- 0.2 vs. 2.2 +/- 0.5 mumol/kg.min, P < 0.05). Similarly, hyperglucagonemia resulted in equivalent stimulation of FFA Ra (5.2 +/- 0.4 vs. 3.7 +/- 0.3 mumol/kg.min, P < 0.05) and glycerol Ra (1.5 +/- 0.3 vs. 1.1 +/- 0.1 mumol/kg.min, P < 0.05). These results indicate that glucagon has a physiological role in the regulation of FFA metabolism in vivo.