Human plasma albumin transports [13C]docosahexaenoic acid in two lipid forms to blood cells.

Docosahexaenoic acid (22:6) decreases blood platelet function and is highly concentrated in the brain where its depletion leads to functional impairments. Because the platelets and blood brain barrier capillary endothelium cannot hydrolyze the complex lipids for fatty acid (FA) uptake, nonesterified FA (NEFA) bound to albumin are assumed to be the delivery route of FA to these cells. The supply of 13C-labeled 22:6 to blood cells by plasma albumin was studied in humans after a single ingestion of this FA esterified in a triglyceride (TG). The 22:6 13C/12C ratio, measured by gas chromatography combustion-isotope ratio mass spectrometry was measured in lipid classes from albumin, platelets, leukocytes, and erythrocytes (taken as a tentative index of the brain uptake). Nonesterified [13C]22:6 bound to albumin was rapidly produced after ingestion, as a result of the hydrolysis of very low density lipoprotein (VLDL) plus chylomicron TG. We found that albumin carried another source of 22:6, lyso-phosphatidylcholines (lyso-PC), in which [13C]22:6 accumulated while the nonesterified [13C]22:6 reached its minimal plasma concentrations. Computation of the relative contribution of NEFA and lyso-PC for the [13C]22:6 delivery to platelets and erythrocytes showed that the [13C]22:6 supply to platelets occurred uniquely through NEFA, whereas this pool was weakly involved in the delivery to erythrocytes. In contrast, lyso-PC was uniquely concerned with the 22:6 delivery to erythrocytes and represented the major part of this supply. We conclude that plasma albumin carries 22:6 in two lipid forms that are involved differently in the delivery of this FA to target cells.

Retroconversion and metabolism of [13C]22:6n-3 in humans and rats after intake of a single dose of [13C]22:6n-3-triacylglycerols.

The apparent retroconversion of docosahexaenoic acid (22:6n-3) to eicosapentaenoic acid (20:5n-3) and docosapentaenoic acid (22:5n-3) was studied in vivo, in rats and humans, after they ingested a single dose of triacylglycerols containing [13C]22:6n-3 ([13C]22:6-triacylglycerol), without 22:6n-3 dietary supplementation. The amount of apparent retroconversion and the distribution of the three n-3 polyunsaturated fatty acids (PUFAs) in plasma lipid classes were followed as a function of time by measuring the appearance of 13C in these PUFAs with gas-chromatography combustion-isotope ratio mass spectrometry. This [13C]22:6n-3 retroconversion, calculated by summing the amounts of [13C]22:5n-3 and [13C]20:5n-3 in plasma lipids, was lower in humans than in rats, reaching a maximum of approximately 9% of the total plasma [13C]22:6n-3 in rats, but only 1.4% in humans. The incorporation of [13C]22:6n-3 and [13C]22:5n-3 in lipid classes followed their endogenous distribution with a maximal accumulation in phospholipids, but a low incorporation into cholesterol esters (CEs), whereas [13C]20:5n-3 was equally present in phospholipids and CEs. The ratio of the amount of HDL-CE to HDL-phosphatidylcholine for [13C]20:5n-3 was higher than for [13C]22:6n-3, indicating a selectivity of the lecithin-cholesterol acyltransferase enzyme with regard to these PUFAs, which may be related to the differences in their biological properties after fish oil feeding. The occurrence of a weak basal 22:6n-3 retroconversion in humans supports feeding this pure PUFA in cases in which 20:5n-3 presents undesirable side effects and when specific alterations of blood lipids are expected.

Metabolic fate of an oral tracer dose of [13C]docosahexaenoic acid triglycerides in the rat.

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.

In vivo compartmental metabolism of 13C-docosahexaenoic acid, studied by gas chromatography-combustion isotope ratio mass spectrometry.

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.

Stable isotope tracer and gas-chromatography combustion isotope ratio mass spectrometry to study the in vivo compartmental metabolism of docosahexaenoic acid.

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 delta 13C /1000 versus Pee Dee Belemnite (PDB), respectively. Precision of delta 13C /1000 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 [1-13C]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.(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipid molecular species from human placenta lipids.

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.

Isolation and characterization of two different molecular forms of basic fibroblast growth factor extracted from human placental tissue.

Basic fibroblast growth factor (bFGF) was purified to homogeneity from human placental tissue on a semi-large scale. Placental bFGF consists of two proteins of apparent molecular masses 16,000 and 18,000 dalton, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis under non-reducing conditions. Microsequence analysis showed that both proteins have the same N-terminal sequence Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly-Ser-Gly-Ala-Phe..., which is identical with that of (1-146) bFGF extracted from human brain. After reduction by dithiothreitol or mercaptoethanol, placental bFGF appears as a single protein of 16,000 dalton. The reduced protein displays the same ability to stimulate the proliferation of CCL39 fibroblasts as the non-reduced doublet. These data indicate that bFGF extracted from placental tissue consists of two proteins with different apparent molecular masses which do not differ in their N-terminal sequence but in their oxidation state.

Growth suppression of transformed cells by a human placental extract not related to transforming growth factor beta.

We have examined whether human placental extracts contain tumour-growth-inhibitory factors. One fraction (EAP) from such extracts inhibited growth, in soft agar, of Ha-ras-transformed BALB/c 3T3 cells and human squamous lung carcinoma A-2182 cells. However, this fraction had no effect on the anchorage-dependent growth of these cells, although there was a slight mitogenic activity on nontransformed cells. These data together with those on plating efficiency indicated no significant cytotoxicity of EAP on transformed cell lines. Although this fraction contained transforming growth factor beta (TGF beta), this cannot account for its inhibitory activity, since (a) pure TGF beta does not inhibit anchorage-dependent growth of Ha-ras-transformed BALB/c 3T3 cells, (b) EAP retains its inhibitory activity in the presence of antibodies against TGF beta and (c) the inhibitory activity did not copurify with TGF beta. Partial characterization of our inhibitory factor suggests that the inhibitory factor is a new tumour-growth-inhibitory factor.

Fibroblast behavior on gels of type I, III, and IV human placental collagens.

Various collagens were extracted and purified from human placenta after partial pepsin digestion. We prepared type III + I (57:43), enriched type I, type III, and type IV collagens on an industrial level, and studied their biological properties with MRC5 fibroblast cells. Using the process of contraction of a hydrated collagen lattice described by Bell, we found tha the contraction rate was dependent on collagen type composition. The contraction was faster and more pronounced with pepsinized type I collagen than with pepsinized type III + I (57:43) collagen; the lowest rate was obtained with the pepsinized type III collagen. Using a new technique of collagen cross-linking, a gel was made with type IV collagen. This cross-linking procedure, based on partial oxidation of sugar residues and hydroxylysine by periodic acid, followed by neutralization, resulted in an increased number of natural cross-link bridges between oxidized and nonoxidized collagen molecules, without internal toxic residues. The fibroblasts were unable to contract type IV/IVox collagen gels. The type IV/IVox collagen gel was transparent and its amorphous ultrastructure lacked any visible striated fibrils. Fibroblast cells exhibited atypical behavior in these type IV/IVox collagen gels as evidenced by optical and electron microscopy. The penetration of fibroblasts could be measured. Fibroblasts penetrated faster in type IV/IVox collagen gels than in untreated type III + I collagen gels. The lowest rate of penetration was obtained with cross-linked type III + I gels. Fibroblast proliferation was similar on untreated or cross-linked type III + I collagen gels and slightly increased on type IV/IVox collagen gels, suggesting that this cross-linking procedure was not toxic.

Towards large-scale purification of natural CSF-1 from human placenta tissue extracts.

The monocyte-macrophage colony-stimulating factor (colony-stimulating factor 1) is characterized and partially purified from industrially processed human tissues for the first time. A five-step purification procedure using placenta tissue extracts furnished a 13,620-fold enrichment of biological activity. This procedure includes a "pilot" scale anion-exchange chromatography at pH 4.5, gel permeation, and lectin affinity separation followed by HPLC steps (hydrophobic interaction and C18 reverse-phase chromatographies). The purified bioactive material, which stimulates only monocyte-macrophage progenitors and mature cells, showed an Mr of 58,000-62,000 (gel filtration) and an isoelectric point of 3.8-4.0. The hydrophobicity of the molecule was low, and the biological activity was eluted at 50% acetonitrile on a C18 reverse-phase HPLC column. It was totally inactivated by 2-beta-mercaptoethanol reduction and heat treatment. Immunoprecipitation and neutralization of biological activity with specific anti-CSF-1 antibodies (not shown) demonstrated that this material was CSF-1. Step 5 of this protocol yielded two silver-stained bands on 12.5% SDS-PAGE: a major 55-kDa band (96%) and a minor 33-kDa band (4%). CSF-1 was detected exclusively in a band of 52-62 kDa by both Western immunoblotting and bioassays. Immunoaffinity techniques using antibodies directed against selective epitopes on the placental CSF-1 are now considered to purify this material to homogeneity. This approach to the mass production of natural CSF-1 from human tissue has advantages with respect to both the difficulty of post-translational processing of bioactive material in procaryotes and the cost of eucaryotic cell cultures.

[Epikeratoplasty without sutures using collagen IV lens in monkeys: description of the surgical technic]. / Epikératoplastie sans suture avec lentille de Collagène IV chez le singe: description de la technique chirurgicale.

The authors describe their original epikeratoplasty technic without sutures which is compatible with the use of collagen IV lens. They describe the first four observations of primates operated on using this technic. At first the epithelium is removed at the cornea center and a trepanation is made of 4 mm diameter and 0.1 mm depth. The bottom of the trepanation is then cut horizontally, and the periphery of the lens is put in the cornea stroma. Later the epithelium will recover the collagen lens. The lens is perfectly set in the cornea. We don't use any suture and so we avoid astigmatism and neovascularisation. The follow-up consisted of biomicroscopic examination photography, specular microscopy, pachymetry, photokeratoscopy (Nidek System) tonometry and histology. Clinical observance showed a perfect lens tolerance. The cornea is immediately transparent and within a week epithelial cells recovered the lens of three animals out of four. The photokeratoscopy study proved the important cornea refraction modification. This technic is reversible and the lens can be exchanged. A study of histology has begun and already shows a pluristratified epithelium. Further studies will test the biomaterial stability and ultra structural relations between the collagen IV lens and epithelial cells.

[Use of human placental type IV collagen in tympanoplasty. Results of initial animal experimentation]. / Utilisation du collagène de type IV réticulé humain placentaire dans les tympanoplasties. Résultats de l'expérimentation préliminaire animale.

This study presents the use of human type IV collagen film in myringoplasty in dogs. Evaluation criteria were clinical (repeated otoscopies) and histological (histological analysis of cicatrised membranes). This new biomaterial should seem to be a technological advance on temporal bone aponevrosis or perichondria auto-grafts. This study constitutes the necessary preliminary research to any application in man.

[Human collagen IV lenses and epikeratoplasy in monkeys. Study of epithelial cicatrization]. / Lentilles de collagène IV humain et épikératoplastie chez le singe. Etude de la cicatrisation de l'épithélium.

A previous study showed the good tolerance of the human type IV collagen lenses implanted by epikeratoplasty in 8 monkeys. It also showed type IV collagen is a good material for the reepithelialization. But we have been interested in the stability of the epithelial healing. The ultrastructural study found anchoring structures. However these were inconstant. So the authors try to understand better the mechanisms of this synthesis of the anchoring structures and describe the future ways of this experimental study.

Biocompatibility of human collagen type IV intracorneal implants.

A new biomaterial made from human placental collagen type IV has been developed in corneal inlays for refractive keratoplasty. Eight dogs received intracorneal lenses to investigate biocompatibility. Postoperative follow-up observation of 2 years revealed that the eyes remained clear and without inflammatory reaction. Endothelial cells were unaltered. Histological and ultrastructural studies showed no signs of inflammation, ulceration, or encapsulation. No fibroblastic activity was evident at the lens stroma interface.

[Viscous human collagen IV. Physical properties and experimental tolerance]. / Le collagène IV visqueux d'origine humaine. Propriétés physiques et tolérance expérimentale.

Viscous solutions of collagen IV have been used experimentally on rabbits and monkeys. The physical properties of the solution allow a first class visco-surgery. Tolerance is studied: degradation and absorption in the anterior chamber, intraocular pressure, endothelial effects, biological sensitivity. Good tolerance and mechanical properties are said excellent and use during human surgery has started.

Hemoglobin solutions coupled with polyethylene glycol 1900: preparation, purification, quality control and pharmacological trials by hemorrhagic shock.

The usefulness of hemoglobin solutions as oxygen transporters is limited by their high affinity for oxygen and rapid elimination from the circulation. Various chemical modifications of hemoglobin aimed at overcoming these two handicaps have been suggested. We have developed a conjugate of pyridoxylated human hemoglobin with monomethoxypolyoxyethtylene 1900, whose preparation and properties are described. We present comparative results on short-term or definitive survival of Wistar rats which, during hemorrhagic shock due to the loss of 60 or 80% of their blood mass, were given a solution of native or modified hemoglobin, in some cases purified by ion-exchange chromatography to remove non-heme proteins, lipids, and some endotoxins. The more complex the treatment used to improve the properties and the purity of the hemoglobin solutions, the longer the animals survived. The loss of hemoglobin in the urine was greatly reduced after conjugation: after 20 h, less than 6% of the total infused.

Modulation of cellular phorbol ester binding and/or protein kinase C activity by human placental fractions.

We describe two factors in human placenta that modulate the interaction of phorbol ester tumor promoters with cell membranes or with protein kinase C. One, phorbol ester binding inhibitory factor, can inhibit binding of [3H]phorbol-12,13-dibutyrate to cultured cells or to a membrane fraction but does not inhibit its binding to a homogeneous C kinase preparation (phorbol ester binding sites). The other, C kinase activating factor, stimulates C kinase activity in a calcium-dependent manner. We separated these two biochemical activities from a crude human placental fraction by gel filtration.

Large scale use of Spherosil ion exchangers in plasma fractionation.

Spherosil microbeads are spherical and made of porous silica. Their surface is coated with hydrophilic and/or hydrophobic polymers. They are specially designed for the separation of proteins on an industrial scale either by ion exchange or by bioaffinity chromatography. Since 1980, large columns have been used in Institut Mérieux for the purification of placental albumin and several vaccines. Here we describe a chromatographic process for the purification of human albumin and immunoglobulins (IgG) from 25 1 of plasma per cycle. First the plasma was freed of the coagulation factors and then clarified at pH 5.25. The corresponding supernatant was filtered and processed in sterile conditions. Albumin was then purified by ion exchange on 3 successive columns, respectively containing: 6.25 kg of DEAE SPHEROSIL W-1000; 3.5 kg of QMA SPHEROSIL PH-1000; 8 kg of COOH-SPHEROSIL W-1000. The concentration and pH of the buffers were selected to reduce, as much as possible, the total quantity of ion exchangers required per cycle. IgG were then purified from the filtrate of the first of the previous columns. 1 column of 6.25 kg of DEAE SPHEROSIL W-1000 was used at pH 6.8. The selected chromatographic parameters allowed us to demonstrate a total elimination of HBs Ag and HB Virus when voluntarily added to an initial sample (RIA determination and HBV-DNA analysis by molecular hybridization). A second column of a large pore anion exchanger: DEAE SPHEROSIL LP-3000 was added at the end of the IgG purification, as a final security to avoid any risk of transmitting the Hepatitis B virus. The yield and quality of the final products will be presented.