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A gas-chromatography combustion isotope ratio mass spectrometry (GCC-IRMS) method using carbon 13 (13C)-stable isotope to trace n-3 polyunsaturated fatty acids (PUFA) turnover in vivo is presented. Natural 13C abundance of commercial n-3 PUFA was measured from 100 to 300 ng of fatty acids and was −27.58, −27.83, and −28.16 for 22:6n-3, 22:5n-3, and 20:5n-3, expressed as δ13C‰ versus Pee Dee Belemnite (PDB), respectively. Precision of δ13C‰ values was comparable for the three PUFA and gave relative standard deviations of 0.95-0.97%. Isotope enrichment of 0.0010 at.% could be detected. Triglycerides enriched in [13C]22:6n-3 ([13C]22:6-TG) were synthesized by growing a microalgae on [l-13C]glucose. [13C]22:6n-3 represented 36 wt.% of total triglyceride fatty acids and had an isotope enrichment of 2.0420 at.%, which was the double of natural abundance. The isotope enrichment of 22:6n-3 in lipids from rat lipoproteins and red cells could be followed as a function of time after ingestion of 3 mg [13C]22:6-TG and showed specific patterns according to the lipid compartments. The retroconversion of [13C]22:6n-3 was also detected in HDL phosphatidylcholine by the appearance of [13C]22:5n-3 and [13C]20:5n-3. On the other hand, 22:6n-3 natural 13C abundance in human lipid classes of lipoproteins and blood cells has been measured using 10 ml plasma, even for the more limiting lipid compartments in terms of 22:6n-3 dose size. It is concluded that 13C-enriched n-3 PUFA along with GCC-IRMS can be used to study fatty acid fluxes in vivo, and that this method represents a convenient alternative to the utilization of radiotracer fatty acids in humans.

Summary A crude extract (EAP) is prepared on an industrial scale from human term placental tissue by acid extraction and alcohol fractionation. The major proteins [(gammaglobulins (11%), transferrin (3%), albumin (6%), peptides The concentrations of the different known proteins, particularly plasma proteins, lipoproteins, transport, and adhesion proteins, were determined by immunodiffusion. The lipids were identified by thin-layer chromatography. The extracted lipids were essentially composed of phospholipids (56%), free fatty acids (12%), and free cholesterol (29%). The steroid hormones were determined by RIA titrations and only low levels (under ng) could be detected. The EAP was mainly manufactured to get a growth factor extract. Its content in growth factors was estimated by specific techniques designed for each factor. The lowest levels were detected for IGF-2 (15–140 ng/ml), while EGF/TGF-alpha and bFGF concentrations were in the same range, i.e., 100–250 ng/ml. The highest levels were detected for IGF-1 (350–1300 ng/ml) and especially TGFβ (0.9–4.8 μg/ml). The growth promoting capacities of EAP were determined by cell culture assay. EAP stimulates proliferation, of various cells (MRC5 and various fibroblasts, corneal endothelial cells). The ED5O (half maximal stimulation) has been found to be in the range of 100–200 μg/ml. These results were obtained on three different pilot batches of EAP which ensure the reproducibility of the results. The growth factor content of this extract is of some interest in different fields as purification of isolated growth factors, serum replacement, and as a potentially useful pharmacological agent in wound healing.

The appearance of 13C in rat lipoprotein, blood cells, and brain lipids was followed as a function of time after the ingestion of triglycerides (TG) containing [13C]22:6n-3. The time course of 13C abundance in 22:6n-3 of various lipid pools, measured by gas chromatography combustion-isotope mass spectrometry, established precursor-product relationships within lipids. The [13C]22:6n-3 was rapidly incorporated into very low density lipoprotein-chylomicron-TG and unesterified fatty acids bound to albumin, with a concomitant maximal appearance at 3 h and further decline. Lysophosphatidylcholines (lysoPC) bound to albumin were also enriched in [13C]22:6n-3, and their labeling appeared to be mainly due to hepatic secretion at the earliest time points. From 12 h postingestion, the synthesis of [13C]22:6n-3-lysoPC was twice as high as that of unesterified [13C]22:6n-3, making lysoPC a potential source of 22:6n-3 supply for tissues. The labeling of platelets, red blood cells, and brain phospholipids presented different kinetics, presumably involving the two lipid forms of [13C]22:6n-3 bound to albumin, to different extents. We conclude that [13C]22:6n-3 esterified in TG is rapidly redistributed within blood lipoproteins and the albumin fraction and that its incorporation in lipid species bound to albumin influences its uptake by target tissues.

The exchange of docosahexaenoic acid (22:6n-3) within lipid pools in rat and human has been followed as a function of time after the ingestion of triglycerides (TG) containing 22:6n-3 labeled with 13C(13C 22:6n-3). The 13C abundance in the fatty acid was measured by gas-chromatography-combustion isotope ratio mass spectrometry which allowed the detection of 0.001 atom 13C percent 12C. The 13C 22:6n-3 appearance was rapid in the TG of very low density lipoprotein plus chylomicron fraction, in which the maximal labeling was observed at 3 and 2 h after ingestion in rat and human, respectively. Concomitant with the TG utilization of this fraction by lipoprotein lipase from tissues, unesterified 13C 22:6n-3 appeared in the plasma albumin. 13C 22:6n-3 bound to albumin was mostly present in unesterified form before 12 h post-ingestion while after that period, lysophosphatidylcholine (lysoPC) bound to albumin carried higher 13C 22:6n-3 concentrations. These lyso-PC were mostly from hepatic origin and might represent a potential source of 22:6n-3 redistribution to tissues. The 13C 22:6n-3 uptake into rat brain PC and phosphatidylethanolamine was still increasing when the concentration of plasma unesterified 13C 22:6n-3 had already dropped to a minimal plateau value and during the period of maximal plasma circulation of 13C 22:6n-3-lysoPC bound to albumin. In contrast, the uptake of 13C 22:6n-3 into blood platelet PC occurred during the phase of important circulation of 13C-22:6n-3 bound to albumin, suggesting the in vivo efficiency of the Lands pathway for this fatty acid. It is concluded that 13C 22:6n-3 esterified in TG is rapidly absorbed and redistributed within plasma lipoproteins and that its redistribution within the two lipid species bound to albumin might influence its uptake by platelets and rat brain.

In order to specifically modify the fatty acid composition of cell membrane phospholipids, we have developed an original method based on the transfer of pure phospholipid molecular species to membranes. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) subclasses containing 18:2n-6 and 22:6n-3 at the sn-2 position were incorporated into human platelet membranes using the endogenous phosphatidylinositol/PC transfer protein (PI/PC-TP) and the phospholipid transfer protein from maize (L-TP), respectively. PI/PC-TP was shown to catalyze a strict exchange of phospholipids between platelet membranes and unilamellar vesicles containing 1,2-diacylglycerophosphocholine (diacyl-GPC; 16:0/18:2-GPC, or 16:0/22:6-GPC). The proportions of 18:2n-6 and 22:6n-3 in diacyl-GPC of platelet membranes were gradually increased from 10.7 to 16.9% and from 0.8 to 10.1%, respectively, whereas the PE and PI fatty acid compositions were not changed. The diacyl-GPC enrichment in 22:6n-3 and 18:2n-6 did not induce changes in membrane fluidity parameters measured by electron-spin resonance of 5- and 16-nitroxy stearic acids. Similarly, 18:2n-6 and 22:6n-3 esterified in 1,2-diacylglycerophosphoethanolamine (diacyl-GPE) have been incorporated in platelet membranes by an apparent exchange process under conditions where donor vesicles had a phospholipid composition equivalent to that of platelet membranes. The proportions of 18:2n-6 and 22:6n-3 were selectively and progressively increased from 6.0 to 21.2% and from 2.2 to 17.2%, respectively, in diacyl-GPE of platelet membranes. Thus, the L-TP- and PI/PC-TP-catalyzed enrichment can be used for studying the modulation of membrane biological activities by defined changes of fatty acid composition of specific phospholipid classes or subclasses.

The phospholipid molecular species from a large-scale preparation of human placenta lipids were analyzed. The major placental phospholipids were choline glycerophospholipids (CPL) (53.2 wt%), sphingomyelin (21.7 wt%) and ethanolamine glycerophospholipids (EPL) (14.6 wt%). 1,2-Diacyl-glycerophosphocholine was the most abundant subclass of CPL (91.7 mol%), while EPL contained 1,2-diacyl (54.6 mol%) and 1-alk-1'-enyl-2-acyl (43.8 mol%) subclasses. The level of polyunsaturated fatty acids (PUFA) in total phospholipids was remarkably constant (38.4-39.9 mol%) within all placental batches tested. The long-chain PUFA, mainly 20:4n-6 and 22:6n-3 of the n-6 and n-3 series, respectively, were found in high proportion in all phospholipid classes, especially in EPL (46.7 mol%) and in inositol glycerophospholipids (IPL) (39.9 mol%). CPL and serine glycerophospholipids were much richer in 18:1n-9 and 18:2n-6. High levels of molecular species with arachidonic acid in the sn-2 position were found particularly in 1-alk-1'-enyl-2-acyl-glycerophosphoethanolamine (with 24.0 mol% 16:0 and 22.0 mol% 18:0 in sn-1 position) and in 1,2-diacyl glycerophosphoinositol with 42.6 mol% 18:0 in sn-1 position. EPL subclasses were rich in 22:6n-3, which occurs mainly as 16:0/22:6n-3 (11.7 mol%) in the plasmalogen form and as 18:0/22:6n-3, 16:0/22:6n-3 and 18:1/22:6n-3 in the diacyl forms. Based on their availability and composition, placental phospholipids could be of interest, for example, for supplementing artificial milk preparations with n-3 and n-6 long-chain PUFA for newborn infants with insufficiently developed 18:2n-6 and 18:3n-3 desaturation/elongation.