Lipid phosphorus determination

The inclusion of phosphoric acid in the mobile phase increases the potential for error in subsequent quantitation by lipid phosphorus determinations and has been replaced with a mobile phase containing acetonitrile-methanol-sulphuric acid (100 3 0.05) to provide resolution of phosphatidylinositol, phosphatidylserine, phosphatidyl-ethanolamine, phosphatidylcholine, lysophosphatidylcholine and sphingomyelin (Kaduce et al., 1983). The authors reported that a reduction in the sulphuric acid content of the mobile phase caused a broadening of the eluted peaks and an increase in the retention of phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine, while if omitted, these components did not elute. It was also noted that if the methanol content of the mobile phase was increased then the retention times of all the phospholipids were decreased. Samples were therefore injected in chloroform-diethyl ether (1 1) to avoid altering the concentration of methanol in the mobile phase. 

The phospholipid content of milk and milk products is given in Table 4.5 (Kurtz 1974). Total phospholipid is usually determined by measuring the lipid phosphorus content of the product and multiplying by 26 (AOCS 1975). As the total milk lipid increases in a milk product, so does the phospholipid concentration. However, the ratio of phospholipid to total lipid varies greatly. Referring to Table 4.5 skim milk contains the smallest concentration of phospholipid but the highest ratio of phospholipid to total lipid. The opposite relationship is seen in cream and butter. 

Friend leukemia cells (FLCs) and cell variants resistant to different levels of doxorubicin were grown to the saturation level in drug-free medium. The phospholipid composition was determined in duplicate or triplicate from two separate experiments. The values are expressed as percent of total lipid phosphorus. 

We prepared specimens of the membrane fraction from each cell type and determined two parameters the lipide content gravimetri-cally, and the lipide phosphorus colorimetrically (Table I). The lipide phosphorus is in all three cases of the order of 3% of the lipide. Then we determined the same two parameters, lipide gravimetrically and lipide phosphorus colorimetrically, on the whole cell substance. Again the lipide phosphorus is of the order of 3% of the lipide. If both parameters belong exclusively to the membrane substance, membrane percentage can be calculated independently by means of the two parame-... 

As described by Albro and Dittmer (1968), periodate oxidation of the alkenylglycerols proceeded smoothly to completion in a buffered mixture within 3 hr at room temperature. Basing calculations on the amount of lipid phosphorus used in the hydrogenolysis experiment just discussed, 1 mol of the 1-O-alkenylglycerol will yield 1 mol of formaldehyde. As little as 0.1 iimol of alkenylglycerol can be determined. 

Subsequent to recovery of the total lipids of a cellular preparation as a chloroform-soluble fraction, the total phosphorus content can be determined (see Chapter 3) and then, depending on the amount of lipid phosphorus (or whether the preparation is radiolabeled or not, see below), analytical and/or preparative thin-layer chromatography can be undertaken. In either case, if the experimental protocol is centered on a signal-transduction process, then there may be insufficient material for a phosphorus analysis. In the latter instance, the cellular preparation is prelabeled with 32P or [3H]inositol and the labeled products are located by autoradiography. A preferred type of adsorbent (for thin-layer chromatography) is Merck silica gel 60 (oxalate impregnated). An effective solvent for separation of the phosphatidylinosi-tols and other lipids is chloroform-acetone-methanol-acetic acid-water (80 30 26 24 14, v/v). The approximate / values of cellular phospholipids under these conditions are presented as follows ... 

Rouser G, Fleisher J, Yamamoto A (1970) Two dimensional thin layer chromatographic separation of plant lipids and determination of phospholipids by phosphorus analysis of spots. Lipids 5 494-496... 

The concentration of phospholipids in a lipid mixture can be estimated as phosphate after ashing the sample. The ash can be treated with nitric acid, dissolved in hydrochloric acid, and the phosphorus determined by inductively coupled plasma-atomic emission spectrometry at 214.9 nm. Alternatively, the phosphorus can be determined by atomic absorption spectrometry at 213.547nm with a graphite furnace. Traditional methods for phosphorus determination include the precipitation of phosphate as quinoli-nium molybdophosphate and gravimetric determination, or colorimetric methods such as the reaction with ammonium molybdate and acidic ammonium vanadate solution followed by determination of the absorbance at 460 nm. 

Detection with iodine vapour before quantitative determination has been used frequently, e.g. analysis of lipid esters in conjunction with gas chromatographic determination [187] or with colorimetric determination after conversion to ferric hydroxamate complexes [731] to detect analgesic drugs [215] (cf.Fig. 79) and steroids [217, 437] with subsequent spectrophotometric determination in the visible or UV regions to detect phospholipids, where phosphorus determination followed [6, 79, 637]. 

This overlapping of fractions shows the need for controlling the elution by qualitative or quantitative methods, such as phosphorus determination, total lipid determination, spot tests on silica thin-layers with subsequent sulfuric acid spray and charring,... 

Total phospholipids are quantitated by determination of the phosphorus content of lipid extracts from which non-lipid phosphorus has been removed by purification procedures. For this purpose chloroform-methanol extracts subjected to diffusion purification (Folch et al. 1951, Sperry 1955) are suitable. Lipid phosphorus is determined in aliquots of these extracts after digestion. Most procedures for determination of phosphorus are based on the method of Fiske and Subbarow (1925) which utilizes conversion of phosphate to phosphomolybdate and its subsequent reduction to molybdic blue. Modifications of this method were reviewed by Lindberg andERNSTER (1956). Avery convenient phosphorus assay was described by Bartlett (1959). Total phospholipids are calculated by multiplication of the lipid phosphorus values with 25. These values are only approximations since phosphorus does not represent exactly 4% of each phospholipid molecule. 

The proportion of each phospholipid was determined 24 hours after addition of elicitors to the cell suspension and in control untreated cells (Fig. 1). Phosphatidylcholine (PC) and phosphatidylethanoiamine (PE) were the two major phospholipids, representing respectively 43% and 28% of the total lipidic phosphorus content of control cells. In elicitor-treated cells, these yields decreased to 34% and 21%, respectively. In parallel, there was an increase in the amount of lysoPC and lysoPE. The other phospholipids, which showed only minor changes, were not further investigated. 

A time course study showed that, following an initial increase probably due to subculturing, there was always less lipidic phosphorus in elicitor-treated cells than in controls, 24 hours after the beginning of the treatment. Over three independent experiments, the observed decrease averaged -15%. The amounts of the two major phospholipids, PC and PE, were determined upon elicitor treatment. In control cells their levels increased for the first 10 hours after beginning of the experiment, /.e. after transfer of the cells to the fresh medium. In response to the elicitor, their levels varied moderately, as shown for PC in figure 2. These results are consistent with the net decrease observed at 24 hours. It thus appears that elicitor treatment... 

Phospholipids assay Polar lipids were determined using colorimetric method according to [12]. After acidic hyckolysis, phosphorus was complexed with ammonium molybdate, leading to the formation of a chromogen which absorbed at 820 nm. Standard... 

A culture of B. subtilis glycerol auxotroph strain B42 was filtered, washed and resuspended in medium, one-third was harvested by centrifugation, half the remainder was incubated with 20 jxg glycerol/ml, and the other half was incubated without glycerol for 60 min. Membranes were prepared, and protein and lipid phosphorus were determined. The values for 168.2 are shown to enable comparison with a wild-type culture. 

Lipid phosphorus was determined after extraction with chloroform-methanol (1 2, vol/vol). A mol. wt. of 750 was used for phospholipid. 

Strain NCIB 8253 grown as in Broglie et al. (1980) to mid-exponential phase was brought to 15 C and pulse labeled 1 min with 2-[ H]glycerol (1.3 yCi/ml). Lipid phosphorus was determined as in Table 1. 

Briefly, liposomes (10mM) were incubated for 30minutes at 37°C for egg phosphatidylcholine (EPC) and at 60°C for HSPC-based liposomes with 50 X 10 dpm of methylamine (1 x 10 dpm/mole). At the end of incubation an aliquot of this mixture was passed down a Sephadex G-50 minispin column equilibrated in 10 mM histidine-sucrose buffer 10%, pH 6.7 buffer. Liposomes were eluted at the column void volume and separated from the unencapsulated methylamine. The concentration of liposomes in the original liposomal dispersion and in the void volume fraction was determined from the organic phosphorus (phospholipid) concentration (see section Lipid Quantification and Chemical Stability above) (10,49,53). 

The obtained STPP liposomes were characterized by size distribution analysis, P NMR spectroscopy (Fig. 3), and by zeta potential measurements (Fig. 4). The size of liposomes with 20 mol% incorporated STPP was determined to be 132 zb 59 nm, which did not change significantly upon storage at 4°C over several days. The P NMR spectrum of STPP liposomes shows two chemical shifts correlating to the phosphorus in the lipid s phosphate groups and to the positively charged phosphorus of STPP. No differences in both chemical shifts between the free compounds (i.e., free STPP and free... 

This reagent is composed of 0.3% ninhydrin in 2-propanol-acetic acid-pyridine (90 10 4 10, v/v). The plate is sprayed at room temperature with this reagent, allowed to dry, and then placed in an oven at 100°C. Within 3-5 min at this temperature a purple color will indicate a positive reaction. Though this test is approximately two to three times less sensitive than the phosphorus spray, it is, nevertheless, an important adjunct in determining the presence of primary or secondary amines. However, a disadvantage is that the lipid sample cannot be recovered subsequent to the spray reaction. 

NMR is a valuable technique in the analysis of lipid phases. More specifically, proton, deuterium, carbon-13, fluorine-19, and phosphorus-31 NMR have been utilized for analysis of the dynamic and motional properties of lipids, lipid diffusion, ordering properties, head-group hydration, lipid asymmetry, quantitation of lipid composition, and head-group conformation and dynamics. Cullis et al. and Gruner et al. have shown the importance of P-31 NMR as a tool in the determination of phase properties and lipid asymmetry and the identification of bilayer, hexagonal, and isotropic phases. 

Phospholipid fatty acid analysis (PLFA) is based on the determination of signature lipid biomarkers from the cell membranes and walls of microorganisms. Phospholipids are an essential part of intact cell membranes, and information from the lipid analysis provides quantitative insight into three important attributes of microbial communities viable biomass, community structure, and nutritional status. Phospholipid fatty acid prohles have been used to show the response of the microbial community to phosphorus availability (Keinanen et ah, 2002). Signature lipid biomarker analysis may not detect every species of microorganism in an environmental sample accurately, because many species have similar PLFA patterns. 

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