Rapid measurement of plasma free fatty acid concentration and isotopic enrichment using LC/MS

Measurements of plasma free fatty acids (FFA) concentration and isotopic enrichment are commonly used to evaluate FFA metabolism. Until now, gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) was the best method to measure isotopic enrichment in the methyl derivatives of ¹³C-labeled fatty acids. Although IRMS is excellent for analyzing enrichment, it requires time-consuming derivatization steps and is not optimal for measuring FFA concentrations. We developed a new, rapid, and reliable method for simultaneous quantification of ¹³C-labeled fatty acids in plasma using high-performance liquid chromatography-mass spectrometry (HPLC/MS). This method involves a very quick Dole extraction procedure and direct injection of the samples on the HPLC system. After chromatographic separation, the samples are directed to the mass spectrometer for electrospray ionization (ESI) and analysis in the negative mode using single ion monitoring. By employing equipment with two columns connected parallel to a mass spectrometer, we can double the throughput to the mass spectrometer, reducing the analysis time per sample to 5 min. Palmitate flux measured using this approach agreed well with the GC/C/IRMS method. This HPLC/MS method provides accurate and precise measures of FFA concentration and enrichment.     

New method for GC/FID and GC-C-IRMS analysis of plasma free fatty acid concentration and isotopic enrichment

A simple, direct and accurate method for the determination of concentration and enrichment of free fatty acids (FFAs) in human plasma was developed. The validation and comparison to a conventional method are reported. Three amide derivatives, dimethyl, diethyl and pyrrolidide, were investigated in order to achieve optimal resolution of the individual fatty acids. This method involves the use of dimethylamine/Deoxo-Fluor to derivatize plasma free fatty acids to their dimethylamides. This derivatization method is very mild and efficient, and is selective only towards FFAs so that no separation from a total lipid extract is required. The direct method gave lower concentrations for palmitic acid and stearic acid and increased concentrations for oleic acid and linoleic acid in plasma as compared to methyl ester derivative after thin-layer chromatography. The [(13)C]palmitate isotope enrichment measured using direct method was significantly higher than that observed with the BF(3)/MeOH-TLC method. The present method provided accurate and precise measures of concentration as well as enrichment when analyzed with gas chromatography combustion-isotope ratio-mass spectrometry.     

Measurement of stable isotopic enrichment and concentration of long-chain fatty acyl-carnitines in tissue by HPLC-MS

We have developed a new method for the simultaneous measurements of stable isotopic tracer enrichments and concentrations of individual long-chain fatty acyl-carnitines in muscle tissue using ion-pairing high-performance liquid chromatography-electrospray ionization quadrupole mass spectrometry in the selected ion monitoring (SIM) mode. Long-chain fatty acyl-carnitines were extracted from frozen muscle tissue samples by acetonitrile/methanol. Baseline separation was achieved by reverse-phase HPLC in the presence of the volatile ion-pairing reagent heptafluorobutyric acid. The SIM capability of a single quadrupole mass analyzer allows further separation of the ions of interest from the sample matrixes, providing very clean total and selected ion chromatograms that can be used to calculate the stable isotopic tracer enrichment and concentration of long-chain fatty acyl-carnitines in a single analysis. The combination of these two separation techniques greatly simplifies the sample preparation procedure and increases the detection sensitivity. Applying this protocol to biological muscle samples proves it to be a very sensitive, accurate, and precise analytical tool.     

Simultaneous gas chromatographic determination of concentration and isotopic enrichment of fatty acids in human plasma using flame ionization and mass spectrometric detection

Free fatty acids (FFAs) are important not only because they provide substrate for oxidation but also because they have the potential to regulate several metabolic and hormonal processes. Using stable isotope tracers, these processes can be studied. Here we present a gas chromatographic method to measure FFA concentrations and enrichments after extraction from plasma and subsequent derivatization in one analytical run, using both flame ionization and mass-selective detection. For concentration determinations intra-assay variation ranged from 1.5 to 4.9%, inter-assay variation ranged from 3 to 11%. Intra- and inter-assay variations of the enrichment determination of palmitic acid were 1.4 and 0.9%, respectively.     

Validation of a procedure for exogenous isotopic labeling of lipoprotein triglyceride with radioactive triolein.

A procedure has been developed for the exogenous isotopic labeling of triglyceride-rich lipoproteins (chylomicrons and very low density lipoproteins) using high specific activity radioactive triglyceride in the presence of aqueous dimethyl sulfoxide. The labeled product lipoproteins showed unchanged chemical and physical properties. When the particles had also been labeled biologically by incorporation of unesterified fatty acids into the triglycerides of lipoproteins secreted by liver or intestine, both endogenous and exogenous labels were removed at the same rates in the isolated perfused heart and liver or in intact or functionally hepatectomized rats. These experiments additonally indicated that the triglyceride fatty acid composition of chylomicrons and very low density lipoproteins was unchanged during triglyceride depletion in the peripheral tissues. Using such labeled lipoproteins it has been shown that uptake of remnant lipoprotein cholesteryl ester and triglyceride by the liver is simultaneous. The labeling procedure described should prove suitable for kinetic studies of the disposition of the various lipoprotein non-polar ('core') lipids.     

Effect of plasma free fatty acid concentration on the content and composition of the free fatty acid fraction in rat skeletal muscles.

Skeletal muscles contain a fraction of free (unesterified) fatty acids. This fraction is very small, but important since it contributes to the creation of the plasma-myocyte free fatty acid concentration gradient. Maintenance of this gradient is necessary for blood-borne fatty acids to be transported into the cell. There are no data on the regulation of the content and composition of the free fatty acid fraction in the cell. The aim of the present study was to examine the effect of an elevation and a reduction in the plasma-borne free fatty acid concentration on the content and composition of the free fatty acid fraction in different skeletal muscle types. The experiments were carried out on male Wistar rats with 280 - 310 g body weight. They were divided into four groups - 1, control; 2, exercised 3 h on a treadmill moving with a speed of 1,200 m/h and set at + 10 degrees incline; 3, treated with heparin; and 4, treated with nicotinic acid. Samples of the soleus as well as the red and white sections of the gastrocnemius muscles were taken. These muscles are composed mostly of slow-twitch oxidative, fast-twitch oxidative-glycolytic and fast-twitch glycolytic fibres, respectively. Lipids were extracted from the muscle samples and from the blood; the free fatty acid fraction was isolated by means of thin-layer chromatography. The individual free fatty acids were identified and quantified using gas-liquid chromatography. The plasma concentration of free fatty acids was as follows: control group, 236.1 +/- 32.9; after exercise, 407.4 +/- 117.5; after heparin, 400.8 +/- 36.8; and after nicotinic acid, 102.5 +/- 26.1 micromol/l (p < 0.01 vs. control values in each case). The total content of the free fatty acid fraction in the control group was as follows: white gastrocnemius, 27.6 +/- 7.3; red gastrocnemius, 52.2 +/- 13.9; soleus, 72.3 +/- 10.2 nmol/g. Elevation in plasma free acid concentration during exercise increased the total content of free fatty acids in the white gastrocnemius (38.7 +/- 13.9) and in the soleus (103.4 +/- 15.9 nmol/g; rest-exercise: p < 0.05 and p < 0.01, respectively), but had no effect in the red gastrocnemius. Neither elevation in the plasma free fatty acid concentration with heparin nor reduction with nicotinic acid affected the total content of the free fatty acid fraction in the muscles examined. The ratio of plasma concentration of individual acid to muscle concentration for the same acid varied greatly, depending on acid, muscle type and experimental group. The ratio was positive (above unity) for each acid almost in all cases with the exception of certain acids in the nicotinic acid-treated group where it was below unity. We conclude that the skeletal myocytes maintain a stable level of free fatty acid fraction in the wide range of plasma free fatty acid concentrations.     

Long chain fatty acid binding to human plasma albumin.

The binding of six physiologically important long chain fatty acids to defatted human plasma albumin was measured at 37 degrees in a calcium-free Krebs-Ringer phosphate buffer, pH 7.4. The data were analyzed in terms of multiple stepwise equilibria. With the saturated acids, the magnitude of the equilibrium (association) constants, Ki, increased as the chain length increased: laurate smaller than myristate smaller than palmitate smaller than stearate. Oleate was bound more tightly than stearate; by contrast, linoleate was bound less tightly than stearate. The equilibrium constants, K1 through K12, ranged from 2.4 times 10-6 - 3.5 times 10-3 m-1 for laurate to 2.6 times 10-8 - 3.5 times 10-5 m-1 for oleate. Successive values of Ki decrease for each of the acids, indicating that major cooperative binding effects do not occur over the physiological range of fatty acid concentrations. In no case could the Ki be segregated into distinct classes, suggesting that any grouping of albumin binding sites is somewhat arbitrary. The results were inconclusive concerning whether premicellar association of unbound fatty acid occurs. Although corrections for premicellar association produced very little change in the Ki values for myristate, they raised the Ki for palmitate and stearate by 300 to 700 per cent. A sigmoidal relationship was obtained when the logarithm of Ki was plotted against chain length for the saturated fatty acids containing 6 to 18 carbon atoms, indicating that the binding energy is not simply a statistical process dependent only on the fatty acid chain length. This selectivity that albumin contributes to the binding process may be due to varying degrees of configurational adaptability of its binding sites as the fatty acid increases in length.     

Simultaneous assay of isotopic enrichment and concentration of guanidinoacetate and creatine by gas chromatography-mass spectrometry

A gas chromatography-mass spectrometry (GC-MS) method for the simultaneous measurement of isotopic enrichment and concentration of guanidinoacetate (GAA) and creatine in plasma sample for kinetic studies is reported. The method, based on preparation of the bis(trifluoromethyl)pyrimidine methyl ester derivatives of GAA and creatine, is robust and sensitive. The lowest measurable m₁ and m₃ enrichment for GAA and creatine, respectively, was 0.3%. The calibration curves for measurements of concentration were linear over ranges of 0.5 to 250 μM GAA and 2 to 500 μM for creatine. The method was reliable for inter- and intraassay precision, accuracy, and linearity. The technique was applied in a healthy adult to determine the in vivo fractional synthesis rate of creatine using primed-constant rate infusion of [1-¹³C]glycine. It was found that isotopic enrichment of GAA reached a plateau by 30 min of infusion of [1-¹³C]glycine, indicating either a small pool size or a rapid turnover rate (or both) of GAA. In contrast, the tracer appearance in creatine was slow (slope = 0.00097), suggesting a large pool size and a slow rate of synthesis of creatine. This method can be used to estimate the rate of synthesis of creatine in vivo in human and animal studies.     

Serum selenium concentration related to myocardial infarction and fatty acid content of serum lipids.

A longitudinal case-control study of 33 patients with one or more risk factors for coronary heart disease and 64 controls showed that the serum selenium concentration (range 0.63-1.33 mumol/l (50-105 micrograms/l] was not associated with development of clinical manifestations of coronary heart disease during a follow up of five to seven years. The content of polyunsaturated fatty acids, especially eicosapentaenoic acid, in serum cholesterol esters and phospholipids was positively correlated with selenium concentration. As a low content of polyunsaturated fatty acids in serum lipids was an independent risk factor for coronary heart disease in these subjects it may be hypothesised that the high coronary risk in subjects with a very low serum selenium concentration (less than 0.57 mumol/l (less than 45 micrograms/l] might be due not to selenium deficiency but to the coexisting low concentrations of polyunsaturated fatty acids in serum.     

Validation of a new procedure for calculating tissue/blood distribution coefficient

An economic procedure for calculating the tissue/blood distribution coefficient (Kp) for physiological models of pharmacokinetics is substantiated. It stems from evaluation of Kp vs. the drug concentration in tissue and blood specimens. The estimate of the time for collecting biosubstrates for assay of the drug content in them is inverse of the constant of the blood drug elimination rate: Kp = Ct/Cb, where Ct and Cb are the drug concentrations in tissues and blood at the time moment equal to 1/kel. In this procedure the estimates of Kp agree with the values calculated by the AUC procedure which is the most exact but much more labour-consuming.     

Effect of electrical stimulation of the hypothalamus on plasma free fatty acid concentration in cats

The effect of electrical stimulation of various hypothalamic regions on levels of plasma free fatty acids, glucose, triglycerides, and cholesterol was studied in fasted cats. Appreciable changes were observed in plasma free fatty acids and glucose but not in plasma triglycerides or cholesterol. These changes appeared to be dependent upon small differences in the placement of electrodes and could not be related to a distinct hypothalamic locus. The results indicate that there is a dissociation between hypothalamic neurons that may affect plasma glucose concentration and those that may affect the plasma free fatty acids. It is suggested that the hypothalamus of the cat contains neurons that may influence autonomic discharge to adipose tissue and thus affect the plasma free fatty acid level and other neurons that may influence autonomic discharge to the liver and thus affect glucose output into the circulation. The distribution of both types of neurons is not limited to a distinct region of the hypothalamus in cats.     

A simplified procedure to determine tryptophan residues in proteins.

A procedure is described to determine tryptophan residues in proteins using a tryptophan reagent, 2-hydroxy-5-nitrobenzyl bromide. The method involves the treatment of the unfolded protein with the reagent in 9 m urea at acid pH; incubation of the mixture at room temperature for 2 hr and the removal of the excess reagent by centrifugation and gel filtration. The amount of tryptophan in a protein is determined from the optical density of the labeled protein at 280 and 410 nm, and from the known optical density of 1 mg/ml of the protein at 280 nm and of the reagent at 280 and 410 nm. The efficacy of the method was tested with eight proteins whose tryptophan content is known.     

Changes in fatty acid branching and unsaturation ofStreptomyces cinnamonensis as a response to NaCl concentration

Three differentStreptomyces cinnamonensis strains (viz. standard, regulatory, and non-differentiating) were grown in a synthetic medium containing 0–4% NaCl and the composition of total cellular fatty acids was analyzed. The increasing salt concentration resulted in an increasing proportion of even-numbered straight-chain fatty acids, mostly at the expense of branched-chain fatty acids. Unsaturated fatty acids, palmitoleic acid in particular, represent the major proportion of straight-chain fatty acids.     

Noninvasive methods to determine the critical micelle concentration of some bile acid salts

In this work the critical micelle concentrations (cmc) of four bile salts, sodium cholate, sodium glycocholate, sodium deoxycholate, and sodium glycodeoxycholate, are determined and presented. Three independent noninvasive methodologies (potentiometry, derivative spectrophotometry, and light scattering) were used for cmc determination, at 25 degrees C with ionic strength adjusted to 0.10 M with NaCl. Spectrophotometric and potentiometric studies of some bile salts were also executed at various ionic strength values, thus allowing the influence of the ionic strength on the cmc value of the bile salt to be assessed. A critical comparison of the cmc values obtained with data collected from the literature is presented. Furthermore, this work makes an evaluation of the conceptual bases of different methodologies commonly used for cmc determination, since variations in the results obtained can be related mainly to different intrinsic features of the methods used (such as sensitivity or the need to include tracers or probes) or to the operational cmc definition applied. The undoubted definition of the experimental bile salt concentration that corresponds to cmc (operational cmc) is essential since in the case of these amphiphiles the formation of micelles is not as abrupt as in the case of ordinary association colloids. The biphasic nature of their aggregation leads to a "round-shaped" variation of the experimental parameters under analysis, which makes difficult the evaluation of the cmc values and can be responsible for the different results obtained.     

Phospholipid and fatty acid enrichment of Phanerochaete chrysosporium INA-12 in relation to ligninase production

Summary Ligninase activity of Phanerochaete chrysosporium INA-12 was increased when vegetable oils emulsified with sorbitan polyoxyethylene monooleate (Tween 80) were added to growth medium. Maximal enzyme yield was 22.0 nkat·ml^-1 in olive oil cultures after 4 days incubation. P. chrysosporium INA-12 was also able to utilize tall oil fatty acids for ligninase synthesis. An extracellular lipase activity was detected during the primary phase of growth in culture containing vegetable oils. On the other hand, ligninase production was 1.5-fold enhanced when olive oil cultures were supplemented with soybean asolectin as a phospholipid source. In cultures supplied with olive oil plus asolectin, P. chrysosporium INA-12 mycelium exhibited a preferential enrichment of oleic acid (C18:1), phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) as compared to lipid-free medium. PC and LPC enrichment was associated with an increased ratio of saturated versus unsaturated fatty acids of phospholipids.