Quantitative 31P NMR Method for Individual and Concomitant Determination of Phospholipid Classes in Polar Lipid Samples.

Herein, to achieve individual and concomitant quantifications of phospholipid classes, an absolute quantification 31P NMR method using an internal standard was examined. Phospholipid standards and dietary foods were dispersed to prepare test solutions in an anionic surfactant (sodium cholate) solution containing EDTA, as a modification based on a reported method. Each phospholipid class showed a reproducible chemical shift at a near-neutral test solution pH of 6.90±0.04 and temperature of 30.0±0.1°C. The quantity of synthetic phosphatidylcholine measured using 31P NMR was consistent with that measured by 1H NMR using an internal standard. As the principal phospholipid class of soybean and egg yolk lecithin is phosphatidylcholine, the measurement conditions of 31P NMR (pulse interval time and number of scans) were optimized such that minor phospholipids, including lysophospholipids, also present in lecithin could be quantified simultaneously. Phospholipid classes in commercial polar lipid samples derived from porcine brain, yeast, and soybean were individually quantified using the above conditions. Using phosphoserine as the internal standard material allowed the absolute molar quantity of the phospholipid class to be precisely determined with traceability to the SI. The determined molar amounts of phospholipid classes were then translated to the weight amount by assuming that each phospholipid class contained two stearic acid molecules as the constituent fatty acid. The calculated total contents of each phospholipid class by 31P NMR were in good agreement with those obtained by molybdenum blue colorimetry. Furthermore, the quantitative values of the principal phospholipid classes in the polar lipid samples obtained by 31P NMR corresponded in a broad view, however, was more informative for the separation of individual phospholipid species rather than the quantitative 2D thin-layer chromatography.

Marine omega-3 phospholipids: metabolism and biological activities.

The biological activities of omega-3 fatty acids (n-3 FAs) have been under extensive study for several decades. However, not much attention has been paid to differences of dietary forms, such as triglycerides (TGs) versus ethyl esters or phospholipids (PLs). New innovative marine raw materials, like krill and fish by-products, present n-3 FAs mainly in the PL form. With their increasing availability, new evidence has emerged on n-3 PL biological activities and differences to n-3 TGs. In this review, we describe the recently discovered nutritional properties of n-3 PLs on different parameters of metabolic syndrome and highlight their different metabolic bioavailability in comparison to other dietary forms of n-3 FAs.

[Effect of tangshen formula on phospholipids metabolism in diabetic nephropathy patients].

This study was to report the effect of Tangshen Formula on phospholipids metabolism in diabetic nephropathy patients. A normal phase-HPLC-TOF/MS method was used in this study for the determination of seven species of phospholipids in human plasma. Then, the concentration changes of potential phospholipids biomarkers were discussed in diabetic nephropathy phase III and phase IV patients among different groups, including before and 3, 6 months after administration of Tangshen Formula. Significant increases of PE750, PI885, PC792, PC826, PC830, PC854 and PC802 levels were observed 6 months after administration of Tangshen Formula and conventional western medicine, as well as a decrease of LPC540 level, when compared with those before medication. Concentrations of all the potential phospholipids biomarkers showed a tendency towards normal levels; however, both the improvement degree and onset time of these compounds were not same. Additionally, Tangshen Formula treatment based on conventional western medicine treatment was more efficient in adjusting the levels of these compounds when compared with western medicine treatment alone, especially for the phase IV patients. These results indicated that Tangshen Formula was capable in regulating and improving phospholipids metabolism in diabetic nephropathy patients, which may be related with the direct or indirect inhibition of protein kinase C pathway and the corresponding reduction of phospholipase A2 activity. Therefore, Tangshen Formula may be used as an effective drug for diabetic nephropathy therapy, at least as an adjunctive therapeutic drug.

Serum fatty acid imbalance in bone loss: example with periodontal disease.

Among the numerous factors of bone remodelling, the local action of arachidonic acid metabolites together with cytokines, is particularly important, especially that of prostaglandin PGE2. It has been suggested that the alveolar bone destruction in periodontal disease and osteoporosis can be treated by reducing the ratio of arachidonic acid in phospholipids, which would diminish prostaglandin production. The aim of this study was to evaluate the main serum polyunsaturated fatty acids and a possible alteration in the level of arachidonic acid in patients suffering from periodontal bone loss. Of the 105 patients who participated the study, 78 were suffering from periodontal bone loss and 27 served as a control group. The fatty acids were measured in serum by gas-chromatography. The results showed that the level of fatty acids of the n-6 pathway was higher in our patients with bone loss than in the control group, whereas the reverse was observed with fatty acids of the n-3 pathway. In conclusion, our patients' bone losses are linked with an imbalance between n-6 and n-3 fatty acids, which seems to justify a diet increase in 20- and 22-carbon fatty acids.

Effects of dietary gamma-linolenic acid-rich borage oil combined with marine fish oils on tissue phospholipid fatty acid composition and production of prostaglandins E and F of the 1-, 2- and 3-series in a marine fish deficient in delta5 fatty acyl desaturase.

The effects of gamma-linolenic acid-rich borage oil (BO), in combination with different marine oils, namely an eicosapentaenoic acid (EPA) rich oil (MO) or a DHA-rich oil (TO), on tissue fatty acid composition and prostaglandin production were investigated in turbot, a species which lacks appreciable delta5 fatty acyl desaturase activity. The juvenile turbot grew well on the experimental diets and there were no significant differences in final weights between dietary treatments. Irrespective of the marine oil component, both the BO-containing diets increased tissue phospholipid levels of 18:2n-6 and 18:3n-6, and their respective elongation products, 20:2n-6 and 20:3n-6, compared to fish fed a control diet containing a standard Northern hemisphere fish oil. Both the BO-containing diets increased the production of 1-series prostaglandins (PG), this being observed across all tissues investigated with PGF and especially PGE. The BO/MO diet also reduced 20:4n-6 in tissue phospholipids without affecting 20:5n-3, whereas the BO/TO combination decreased 20:5n-3 but increased 20:4n-6. The production of 2-series and 3-series PGs was also altered by the dietary treatments but the changes were less dependent upon the tissue levels of their respective precursor fatty acids, 20:4n-6 and 20:5n-3. The BO-containing diets had very significant effects on gross fatty acid compositions of the phospholipids including increased proportions of saturated fatty acids and n-6 polyunsaturated fatty acids (PUFA) and decreased proportions of monounsaturated fatty acids and n-3 PUFA. Overall, this study shows that eicosanoid production in turbot tissues can be influenced by dietary fatty acids, not only by changes in the absolute and relative levels of specific eicosanoid precursor PUFA in tissue phospholipids, but also by general effects on membrane composition, structure and function induced by gross fatty acid compositional changes.

High-performance liquid chromatography separation of phospholipid classes and arachidonic acid on cyanopropyl columns.

An HPLC method for the separation and analysis of arachidonic acid and eight phospholipid classes is described: phosphatidylglycerol, phosphatidylinositol, cardiolipin, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and 2-lysophosphatidylcholine. The separation is carried out at 60 degrees C on 2 cyanopropyl columns using a gradient of acetonitrile and 5 mM sodium acetate (pH 5.0). Cyanopropyl columns require a lower proportion of water in the mobile phase to elute the more polar phospholipids than other types of columns and are thus less prone to equilibration problems. The method is highly reproducible (average coefficient of variation for each retention time less than or equal to 3.5%) and permits analysis of peaks by phosphorus content. Data obtained by analyzing lipid extracts from rat alveolar macrophages prelabeled with [G-3H]-arachidonic acid were analyzed by this HPLC method and compared to standard analysis by TLC. There was a significant correlation between the radioactivity profiles obtained with the two chromatographic methods (HPLC versus TLC) by linear regression analysis [HPLC = 0.83 (TLC) + 3.58, n = 25, r = 0.95, P less than 0.001].

Quantitative analysis of phospholipids by 31P-NMR.

High-field 31P nuclear magnetic resonance spectroscopy was used to quantitate phospholipids in mixtures in organic solvents. The sample is dissolved in chloroform-methanol and analyzed at 161.7 MHz with decoupling of the protons. Signals were identified using authentic compounds, and their relative distribution was measured in mole percent. The method has good accuracy and reproducibility, and was used to analyze phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylinositol, cardiolipin, and phosphatidic acid in egg lecithin. Four commercial egg phospholipids and the phospholipids from a total lipid extract of rat liver were analyzed. The method could be utilized to analyze phospholipids from other sources.

Ureteral occlusion decreases phospholipid and cholesterol of renal tubular membranes.

Unilateral ureteral obstruction of 24 h duration in the dog results in a 20% decrease in the amount of total phospholipids present in basolateral membranes of renal tubular cells obtained from the experimental kidney as compared with the amount of phospholipid in basolateral membranes prepared from the contralateral kidney of the same dogs or from the kidneys of normal sham-operated dogs. There was also a decrease in the content of cholesterol and cholesterol esters of the basolateral membranes from the experimental kidney. By contrast, no significant change in lipid content was observed in brush border membranes obtained from the experimental kidney of dogs with unilateral ureteral obstruction. The decrease in phospholipid content of basolateral membranes was accompanied by a 40% fall in the content of phosphatidylcholine and a 12% fall in sphingomyelin. There was a small (12%) but significant increase in the content of phosphatidylethanolamine in basolateral membranes. The mechanisms responsible for the selective decrease in phospholipid content of basolateral membranes remain to be established. It is postulated that changes in solute transport and altered response to hormones observed in the postobstructed kidney of animals with unilateral ureteral obstruction may be explained, at least in part, by changes in the lipid composition of basolateral membranes.