Interaction of prostaglandins with low-density lipoproteins in human blood

Interaction of prostaglandins (PG) with human plasma low density lipoproteins (LDL) was studied, using fluorescent spectroscopy and photoreactive labeling. It was demonstrated that PGE1 at low concentrations (less than 10(-9) M) induces specific lipid rearrangements on the surface of LDL globules. It was assumed that these rearrangements are brought about by the interaction of PG with apolipoprotein B to form short-living complexes. A possible mechanism and biological significance of the observed phenomenon are discussed.     

Radioimmunoassay of prostaglandins A and B in human blood

A highly sensitive radioimmunoassay for the measurement of plasma prostaglandins A and B, expressed in equivalents of PGA₁, is described. This method was used for the measurement of prostaglandins A and B (PGA/B) in 23 healthy volunteers and 25 hypertensive patients.The PGA/B concentration in peripheral venous plasma of 23 healthy normotensive subjects is 115 ± 15 pg/ml. The repeated measurement of the same plasma samples kept frozen for 60 days at ?20°C shows mean 194% increase of PGA/B concentration.The major site of synthesis of PGA/B seems to be the kidney. However in two patients PGA/B concentration in arterial blood was greater than in venous blood suggesting the possibility of cardio-pulmonary synthesis. The major site of inactivation is the hepatic circulation, as PGA/B concentration in hepatal venous blood is by 30% lower than in vena caval blood.The arterial concentration is 3% lower than venous PGA/B demonstrating very low pulmonary inactivation. Therefore the prostaglandins of the A and B series may represent a “circulating hormone”.The plasmatic PGA/B is significantly increased in renovascular and essential hypertension.     

Synthesis of prostaglandins by subpopulations of human peripheral blood monocytes

The ability of monocytes/macrophages to regulate various aspects of immunologic responses may in part depend on their release of soluble substances such as prostaglandins. Using quantitative gas-liquid chromatography/mass spectrometry, prostaglandin E₂ was found to be the major prostaglandin synthesized in culture by human peripheral blood monocytes. Subjecting these cells to discontinuous density gradient fractionation demonstrated significant differences in the synthesis of prostaglandins E₂ and E₁ among the resulting monocyte subpopulations.     

Regulation of human peripheral blood monocyte collagenase by prostaglandins and anti-inflammatory drugs

Macrophages, which produce the collagenolytic enzyme collagenase, are commonly found at sites of connective tissue destruction in chronic inflammatory lesions. Since tissue macrophages are derived from circulating peripheral blood monocytes, we used these less-differentiated, more readily available cells to examine the production and regulation of collagenase. Human monocytes, isolated in large quantities by counterflow centrifugal elutriation, were shown to produce substantial amounts of collagenase when stimulated by concanavalin A (Con A) and to a lesser extent with lipopolysaccharide, while unstimulated monocyte cultures produced negligible collagenase. Collagenase was detected in the culture media within the first 24 hr of culture after activation with peak production at 48 hr. Analysis of the intracellular regulation of collagenase revealed that synthesis of this enzyme required a prostaglandin (PGE₂)-dependent step since indomethacin-inhibited enzyme synthesis was reversed by PGE₂. Additionally, dibutyryladenosine cyclic monophosphate (dBcAMP) restored collagenase synthesis in indomethacinblocked cultures, indicating a PGE₂-dependent generation of cAMP requirement for collagenase production similar to that demonstrated in experimental animals systems. In additional studies, anti-inflammatory drugs which are known to modulate connective tissue destruction were analyzed for their influence on monocyte-derived collagenase. Dexamethasone, colchicine or retinoic acid all inhibited collagenase synthesis by monocytes in a dose-dependent manner although the effect of these drugs on monocyte PGE₂ synthesis differed. Dexamethasone inhibited PGE₂ synthesis, which resulted in the suppression of collagenase. However, PGE₂ production was unaffected by colchicine whereas retinoic acid caused a significant increase in PGE₂ levels. Inhibition of collagenase synthesis by dexamethasone, but not colchicine or retinoic acid, could be reversed by PGE₂ or phospholipase A₂. These findings provide insight into the intracellular events regulating monocyte collagenase synthesis and also implicate monocytes as a target of anti-inflammatory agents which ameliorate connective tissue degradation associated with chronic inflammatory lesions.     

Extraction and purification of prostaglandins and thromboxane from biological samples for gas chromatographic analysis

An efficient extraction procedure for the isolation of prostaglandins (PGs) from biological samples for their subsequent quantification by gas chromatography-electron capture detection (GC-ECD) is described. PGs were extracted from lung, kidney, spleen and stomach fundus into ethyl acetate at different pHs. The highest recovery and least extraction of contaminating pigments was obtained at pH 4.5. Pigments and other contaminants are removed by thin-layer chromatography using a solvent system chloroform-isopropyl alcohol-ethanol-formic acid (45:5:0.5:0.3). The isolated PGs were determined by GC-ECD after appropriate derivatization. The overall recovery of PGs using this procedure is 60%.     

Extraction and identification of different prostaglandins in Allium cepa

Green onions (Allium cepa) were homogenized in a blender and extracted by normal extraction methods except that diethyl ether was used as the first extracting solvent. Different analytical procedures were used for the identification of the prostaglandins separated. TLC was applied using silica gel 60 F254 plates and a mixture of benzene, dioxane and acetic acid (20:10:1) as eluent, and the Rf values were compared with those of authentic samples. GC analysis on an SE 30 packed column and FID was applied; relative retention times of the onion extract components were measured and matched with authentic prostaglandin samples using cholesterol as an internal standard. GC-MS analyses using the same conditions adopted for GC analysis were conducted on a Finnigan MAT 112S instrument. Four peaks were identified. The prostaglandins identified were F1 alpha, E1, B1 and A2.     

Role of prostaglandins in human fertility

One function of prostaglandins in semen may be suppression of an anti-sperm immune response.     

Red blood cell deformability and the prostaglandins

The effects of Prostaglandin E₁ and Prostaglandin E₂ on the deformability of the human red blood cell have been studied using the glass micropipette method. In the range of concentrations 10^?13 to 10^?5 M, neither PGE₁ nor PGE₂ alters the red cell deformability.     

In vitro synthesis of prostaglandins and related lipids by populations of human peripheral blood mononuclear cells

This report focuses on the identification of the human peripheral blood mononuclear cells that do or do not produce prostaglandins (PGs) and related arachidonic acid metabolites. Our results, using two different assay systems, indicate that the monocyte/macrophage (MØ) is the major and possibly sole source of thromboxane (TXB₂) and prostaglandin E₂ (PGE₂) among peripheral blood mononuclear cells. Adherent peripheral blood monocytes (> 95% esterase positive) produced substantial amounts of these compounds. Quantitation of products which had incorporated exogenous ¹⁴C-arachidonic acid and radioimmunoassay of adherent cell culture fluids demonstrated that the amount of TXB₂ produced by these cells was appreciably greater than the amount of PGE₂ produced. Additional confirmation of TXB₂ synthesis was shown by abolishing the TXB₂ peak on TLC and TXB₂ activity detected by RIA by treating cells with a specific inhibitor of thromboxane synthetase. In contrast, non-adherent T cells failed to synthesize either PGE₂ or TXB₂. Non-adherent B cells (95% Ig positive) incubated with ¹⁴C-arachidonic acid produced a small peak of radioactivity co-chromatographing with TXB₂, and no PGE₂. All three cell populations incorporated similar amounts of ¹⁴C-arachidonic acid into hydroxy-fatty acids. We were unable to detect 6-keto-F_1α, the hydrolysis product of prostacyclin (PGI₂) in any of the cell types tested. The absence of PG synthesis among normal peripheral blood T and B cells was also noted among established human lymphoid cell lines. Neither a human T (CCRF), nor a human B-cell line (GM-130), produced PGE₂ or TXB₂. Three murine macrophage cell lines, P388D₁, J774.2, and WHI-3 produced PGE₂ and the latter TXB₂ as well.     

Extraction of cholesterol from the blood using multiple emulsions

The process of cholesterol extraction from the blood of rabbits with experimental atherosclerosis was investigated, with the following reinjection of the blood with reduced amount of cholesterol. Liquid membranes were applied to extract free cholesterol. Emulsion consisting of blood-compatible membrane (oil and sorbitan oleate) and the inner phase (water solution of digitonin) was used as an extractant. The average degree of cholesterol extraction was 23.4%. Pretreatment of the emulsion, its composition and the way of preparation make it possible to perform the extraction from the blood without a considerable increase in the degree of hemolysis.     

Biosynthesis of prostaglandins in human ovarian tissues

Experiments in vitro demonstrate, that there are different patterns of PG-biosynthesis in the corpus luteum and in the follicles containing cortical substance of the human ovary. In the follicles 6-keto-F₁α. the transformation product of prostacyclin, is the main fraction; prostaglandins F₂α and E 2 being of inferior importance with regard to their amounts. The formation of the corpus luteum is in close correlation with a strongly increased prostaglandin E₂ and a diminished prostacyclin biosynthesis; prostaglandin F₂α hardly seems to be involved in this process.By means of indomethacin the formation of all three examined prostaglandins can be prevented almost completely, in the cortical substance as well as in the corpus luteum. LH (or HCG) at concentrations ranging from 2 ng to 20 μg per ml homogenate produce no stimulating effect of statistical significance on the rate of biosynthesis in both tissues.     

Quantification of prostaglandins in human seminal fluid

A method is described for measurement of the prostaglandins present in human seminal plasma. The PGEs and the 19-hydroxy-PGEs are converted to the respective PGB-compounds. They are determined with UV-absorbance after purification with two chromatography steps. The PGFs and the 19-hydroxy-PGFs are determines by gas chromatography, which also allows measurement of the 8β-isomers. Recovery of added PG was in the average 99.2% (range 89.9–107.7). Mean variation between duplicate analyses was 5.6% (range 0.9–9.1). The method has been simplified to allow at least limited clinical use.     

Use of ion-exchange resins for removing prostaglandins from human urine prior to radioimmunoassay

Investigation of prostaglandin and prostaglandin metabolite levels in human urine by radioimmunoassay requires the removal of prostaglandins from urine in a predictable, reproducible manner. Screening of eighteen ion-exchange resins for their ability to remove tritiated prostaglandins from human urine was undertaken. Two resins, Amberlite IRP-64M and Amberlite CG-50 were found acceptable.