Phospholipids quantitative measurement

An additional example for the usefulness of IR spectroscopy in studying drug interactions with phospholipid vesicles is the quantitative determination of acyl chain conformation in gramicidin-DPPC mixtures [63]. The technique provides a quantitative measure of the extent to which membrane-spanning peptides induce disorder of phospholipid gel phases and order in liquid crystalline phases. 

Quantitative measurement of phospholipids is rare in routine clinical practice but more common in research (e.g., in studies of dietary influences). The choline-containing phospholipids lecithin, lysolecithin, and sphingomyelin, which account for at least 95% of total phospholipids in serum, are readily measured by an enzymatic reaction sequence using phospholipase D, choline-oxidase, and horseradish peroxidase. Kit methods with this enzymatic sequence are available commercially. Before the availability of enzymatic reagents, the common quantitative method involved extraction and acid digestion with analysis of the total lipid-bound phosphorus. ... 

NMR can be used to provide determination of chemical purity and quantitative measurements of impurities in materials. The accuracy and precision of quantitative NMR measurements are comparable to other instrumental analyses methods. Major components can be accurately determined with precisions better than 1 % RSD while impurities in materials may be quantified at 0.1 % or lower (Maniara et al.). The most common nuclei used for quantitative analyses are H, C, and P. Quantitative P NMR has been used to determine phospholipids, inorganic phosphorus, and organophosphorus compounds, such as phosphorus-based insecticides (Maniara et al.). 

The metabolism of choline as it is related to the phospholipid fraction of tissues has been the subject of numerous papers, and various procedures have been devised for the isolation of this fraction and the estimation of its components. In this connection choline has been used as a quantitative measure of the lecithin and sphingomyelin contents of tissues, and the choline phosphorus ratio (theoretical value of unity) as an indication of the purity of these fractions. Considerable variations in the values obtained on the same tissue by different laboratories may indicate that the methods employed for the extraction, purification, and analysis are not entirely satisfactory. Indeed, the deviations observed in the same labo-... 

Koryta et al. [48] first stressed the relevance of adsorbed phospholipid monolayers at the ITIES for clarification of biological membrane phenomena. Girault and Schiffrin [49] first attempted to characterize quantitatively the monolayers of phosphatidylcholine and phos-phatidylethanolamine at the ideally polarized water-1,2-dichloroethane interface with electrocapillary measurements. The results obtained indicate the importance of the surface pH in the ionization of the amino group of phosphatidylethanolamine. Kakiuchi et al. [50] used the video-image method to study the conditions for obtaining electrocapillary curves of the dilauroylphosphatidylcholine monolayer formed on the ideally polarized water-nitrobenzene interface. This phospholipid was found to lower markedly the surface tension by forming a stable monolayer when the interface was polarized so that the aqueous phase had a negative potential with respect to the nitrobenzene phase [50,51] (cf. Fig. 5). 

A procedure for determination of lipid hydroperoxides in human plasma is based on kinetic measurement of the CL of luminol (124) with hemin (75a) catalysis . CLD of microperoxidase-catalyzed oxidation of luminol (124) or isoluminol (190) was applied to detection and determination of amino acid hydroperoxides after exposure to UV and y-irradiation A method for determination of hydroperoxides in the phospholipids of cultured cells uses isoluminol (190) and microperoxidase as catalyst " . Simultaneous determination of phosphatidylcholine hydroperoxides and cholesteryl ester hydroperoxides in human serum is carried out by quantitative extraction of the lipids, HPLC separation by column switching and CLD using isoluminol (190) with microperoxidase catalysis . ... 

J McHowat, JH Jones, MH Creer. Quantitation of individual phospholipid molecular species by UV absorption measurements. J Lipid Res 37 2450-2460, 1996. 

The amount of disorder in the fatty acid side chains in a phospholipid bilayer, as a function of temperature. The side chains become much more disordered as the temperature increases through the 7m. The solid curve represents a bilayer that does not contain cholesterol the dotted curve, a bilayer with the same phospholipid plus about 25% cholesterol. The amount of random, disorderly motion of the fatty acid chains can be measured quantitatively by deuterium- or l3C-NMR. At any given temperature, the disorder is greater near the tips of the chains (toward the middle of the bilayer) than it is close to the head-groups. 

The NMR measurements were made on sonicated phospholipid vesicles to obtain relatively narrow 31P-NMR signals while the electrophoretic mobility measurements were made on unsonicated vesicles. Since there are differences between the two systems (e.g., area per molecule (13,14)), we do not attempt to quantitatively compare the 31P-NMR data with the electrophoretic data, but rather use the 31P-NMR data as an independent demonstration of the difference between the binding of calcium and magnesium. We note, however, that the linewidth ratio for the outer monolayer of the sonicated vesicles were identical within experimental error (see Table II). This implies that the Ca++/Mg++ selectivity of the two monolayers is identical. We had expected the selectivity to be greater for the inner monolayer because the polar head groups of the lipids in this monolayer occupy a smaller area (13). 

Figure 7 shows the response of the redox potential in a perfused hamster liver to the addition of 45 mN ethanol. Instead of the vitro labeling strategy Just described. the pyridine nucleotide pools in this hamster liver were labeled vivo by intraperitoneal injection of 35 mg [5- C] nicotinamide 5 hours prior to sacrifice. The bottom two spectra (2.6 min and 12.8 min) were obtained prior to addition of ethanol. They show resonances from labeled NAD, natural abundance glycogen and natural abundance choline methyl groups of phospholipids but no resonance from reduced pyridine nucleotides. After addition of 45 mM 10% [1- C] ethanol (at 17.9 min), resonances from C-1 of ethanol and NADH are detectable. These data demonstrate that the pyridine nucleotide pools labeled by intraperitoneal injection are metabolically active and that addition of 45 mN ethanol results in a marked change in the redox potential of the liver as measured by NNR. Furthermore, the observation of separate resonances for the oxidized and reduced pyridine nucleotides indicates that chemical exchange between oxidized and reduced forms is slow on the NNR time scale, and demonstrate that NNR may be used to quantitate the redox potential of free pyridine nucleotides situ. 

In practice, the majority of TLC separations are qualitative or semiquantitative (visual comparison) in nature. However, modern computer-controlled densitometers are now available that scan sample and calibrator chromatograms in tracks on HPTLC plates and provide quantitative capabilities. Clinically relevant analytes that have been measured by TLC include amino acids, bile acids, carbohydrates, drugs, fipids, glycolipids, phospholipids, porphyrins, prostaglandins, steroid hormones, purines, pyrimidines, derivatives of nucleic acid, and urinary organic acids. The advantages of TLC include simphcity, rapidity, versatility, ability to process a large number of samples... 

The determination of PG is a commonly used qualitative method used to assess FLM in the United States. Measurement of PG was classically performed by thin-layer chromatography. It could be performed alone or in combination with other amniotic fluid phospholipids. The later test was loiown as a lung profile since its introduction in the early 1970s. Although thin-layer chromatography is still offered by some reference and hospital laboratories, most hospital laboratories use a rapid slide method for qualitative PG detection. Several enzyme-based tests for quantitative PG have also been published, but none is widely... 

Penetration of Human Serum Albumin into Phosphatidylserine Monolayers. We have tried to analyze quantitatively the results of Kimelberg and Papahadjopoulos (7) of protein penetration in phospholipid monolayers. These authors did not measure the surface density of the penetrating protein. They studied the system phosphatidylserine monolayer—human serum albumin (HSA) at different pH values. When the pH was 7.4, the protein and the lipid repelled each other. When the... 

In this assay (Demel et al., 1977 Demel et al., 1982), a monolayer, containing 14C-labeled phospholipid, is formed at an air—water interface. Vesicles and exchange protein are injected into the subphase. The loss of radioactivity from the surface is monitored continuously with a gas flow detector. Alternatively, the rate of transfer of radiolabeled lipids is measured by recovering the subphase or monolayer and quantitating the radiolabeled lipids. The difficulty in preparing the monolayers and the... 

Recent experimental advances have made quantitation of weak membrane adhesion possible in concentrated solutions of macromolecules. We report direct measurements of the free energy potential for adhesion of phospholipid Dilayers in solutions of two plasma proteins (fibrinogen and albumin) over a wide range of volume fraction (Q-0.1). Tne results are consistent with a thermodynamic model for adhesion based on depletion of macromolecules from the contact zone. 

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