Human Plasma Components Research Articles

A simplified method for quantitation of iodine-123 iodobenzamide in human plasma: a technical note

To establish a quantitative single-photon emission tomography (SPET) procedure for imaging CNS D2 dopamine receptors, measurement of unchanged iodine-123 iodobenzamide (123I-IBZM) (a selective D2 ligand) in human plasma was investigated. There are three possible radioactive components in human plasma: hydrophilic compounds (iodide ion, etc.), lipophilic metabolites, and unchanged IBZM. Based on the difference in lipophilicity of IBZM and its lipophilic metabolites (LM), a new quantitative method of analysis of 123I-IBZM using a simple solvent extraction was developed. The selective extraction was achieved with n-octane/phosphate buffer (0.1 M, pH 7.4). Extraction efficiency was 93.2% +/- 0.3% (n = 15) for IBZM from plasma, while 99.3% +/- 0.2% (n = 12) of LM remained in the aqueous plasma fraction. Twenty-one confirmation tests with plasma containing known ratios of IBZM/LM, ranging between 0.39 and 7.60, were performed. The experimental results were very close to the values of the true ratios over the wide range (accuracy approximately 99%, relative standard error 6.6%). The data from this simplified method are comparable to those obtained by the high-performance liquid chromatography method. This improved method provides an easy and accurate way to quantify unchanged IBZM in human plasma. With appropriate kinetic modeling and in conjunction with a dedicated SPET imaging device for measuring quantitative information, it may be possible to develop a practical method for measuring D2 receptor density in vivo.

Partial characterization of galactosyltransferase in human seminal plasma and its distribution in the human epididymis

Galactosyltransferase activity has been partially characterized in human seminal plasma. Km values of 130 mumol l-1 for UDP-galactose and 2.25 mmol l-1 for N-acetylglucosamine were calculated and the enzyme was found to be dependent on temperature and manganese and present as a highly active component of human seminal plasma. Galactosyltransferase was inhibited by nucleotides, glycosylated nucleotides, bovine and human alpha-lactalbumin but not by monosaccharides. Radiation inactivation studies revealed that the biologically active unit of seminal plasma galactosyltransferase has a molecular mass of 45 kDa. Although the majority of galactosyltransferase activity found in seminal plasma is probably of prostatic origin, we report for the first time that it is also present in human epididymal intraluminal fluid. Low activity was detected in the proximal caput region but activity increased to maximum values in the adjacent downstream segment, the intermediate caput region. Specific activity was relatively constant albeit at a lower value in the following epididymal segments and vas deferens. The significance of the epididymal and seminal plasma galactosyltransferase activities is unknown, but the enzyme could be implicated in glycosylation events that are known to be important in gamete interaction.

Role of functional groups of human plasma and luminol in scavenging of NaOCl and neutrophil-derived hypochlorous acid

Hypochlorous acid HOCl/OCl − and other oxidants derived from stimulated polymorphonuclear leukocytes are involved in tissue damage during a number of pathological processes. In order to obtain more detailed information on possible reactions of HOCl/OCl − the effects of both NaOCl and PMN-derived hypochlorous acid on functional groups of amino acid solutions and human plasma are studied. In valine and lysine solutions NaOCl diminishes the number of amino groups in a molar ratio of 1:1 between NaOCl and amino groups. In cysteine and methionine samples the decrease of amino groups starts only after all sulfhydryl or thioether groups are oxidized by NaOCl. If freshly prepared human plasma is treated with increasing amounts of NaOCl all plasma SH groups are oxidized first, then probably the thioether groups and only after this the amino groups are affected. Furthermore, it was found, that the reactivity of luminol against NaOCl is similar to that of amino groups. Increasing amounts of SH groups of components of human plasma are oxidized by incubation with PMA-stimulated polymorphonuclear leukocytes dependent on the incubation time. Plasma amino groups are not affected under the same experimental conditions. The addition of plasma to FMLP-stimulated PMN in the presence of luminol decreases that part of chemiluminescence caused by extracellularly generated hypochlorous acid. Plasma samples pretreated with NaOCl cause a lower inhibition of light generation in FMLP-stimulated PMN only when more than 4 · 10 −8 mol NaOCl per mg protein are used to pretreat plasma. It is assumed that in the development of tissue injuries caused by infiltrated PMN the following sequence of damage occurs in accessible tissue regions. First, the sulfhydryl groups are oxidized, then the thioether groups, and only after this amino and other target groups are affected.

Quantitation of Fansimef Components (Mefloquine + Sulfadoxine + Pyrimethamine) in Human Plasma by Two High-Performance Liquid Chromatographic Methods

Two simple, precise, and selective high-performance liquid chromatographic methods are described for the simultaneous quantitation of mefloquine (MQ) plus pyrimethamine (PYR) or sulfadoxine (SDX) plus its principal metabolite N4-acetylsulfadoxine (N4SDX) in human plasma. After a single-step extraction, MQ plus PYR and SDX plus N4SDX including internal standards were separated using ion-paired and ion-suppression chromatography. Total run times for the assays were less than 12 min. Intraassay and interassay precision of the methods expressed as the coefficients of variation were less than 9% in plasma for the four compounds. The extraction recovery averaged 98% for MQ, 97% for PYR, 96% for SDX, and 81% for N4SDX. Plasma concentrations of the four compounds in a pediatric patient after a single oral dose of Fansimef (MQ + SDX + PYR) were determined to demonstrate the clinical application of the methods.

Isolation of a new actin-binding protein from human seminal plasma

Interaction of human seminal plasma with actin was detected by agar gel immunoelectrophoresis. A major actin-binding protein was isolated from human seminal plasma using an actin-Sepharose 4B column followed by fast-performance liquid chromatography with an anion-exchange Mono-Q column. The protein showed a single band under reduced conditions on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a position corresponding to a molecular mass of 20 kDa. This 20 kDa polypeptide was detected in saliva and extracts of the submandibular gland and seminal vesicles as well as seminal plasma by the method of immunoblotting using monospecific antibody against the 20 kDa antigenic component of human seminal plasma. The protein might be called secretory actin-binding protein (SABP).

Trace metal lung disease: In vitro interaction of hard metals with human lung and plasma components

Hard metal pneumoconiosis is an occupational pulmonary disease caused by long-term exposure to dust produced in the hard metal industry. In vitro experiments have been carried out to study the solubility and metabolic behaviour in human lung tissue and plasma of hard metal alloy constituents such as cobalt, tungsten, tantalum, titanium and niobium. The experiments were carried out using 60Co, 187W, 182Ta, 44Ti and 95Nb radiotracers in combination with neutron activation, radio-release tests and gel filtration techniques. Leaching experiments from neutron-irradiated hard metal dust showed that cobalt was highly soluble, especially in the lung cytosol and plasma, in comparison with tantalum and tungsten. The gel filtration experiments showed three biochemical pools of cobalt in both lung and plasma components, in accordance with the hypothesis that cobalt represents the allergic factor in the development of hard metal disease. High affinity for proteins was observed for Nb, Ta and Ti, but not for W, in agreement with the dissimilar biological half-lives of these elements in the body. The different ability of the metals to interact with biochemical components and to be solubilized in biological media may explain the various degrees of retention in the lung, which would influence the metabolic pathways. This would explain the presence of Co, Ta and W in body fluids, as well as in the pubic hair and toenails of hard metal workers.

Seminal plasma biochemistry III: characterization of coagulum components

Human seminal plasma components involved in coagulum formation have been isolated by liquefaction and reformation of the coagulum in acidic and neutral buffers, respectively. The SDS-PAGE profile of the isolated coagulum (recoagulum) is similar to that reported for the native coagulum immediately following liquefaction. Thus the recoagulum may be considered to represent the native coagulum. Electrophoresis of the recoagulum under non-denaturing conditions reveals the presence of both positively and negatively charged components. These components are sialoglycoproteins that bind copper. Based on these results, a possible mechanism for coagulum formation and liquefaction is discussed.

Gas chromatographic-mass spectrometric identification of four triene monoepoxides of arachidonic acid in human plasma

Four triene monoepoxides of arachidonic acid have been identified as endogenous components of human plasma, the epoxy groups being in the 5,6-, 8,9-, 11,12- and 14,15-positions. Prior to trimethylsilylation and gas chromatographic-mass spectrometric analysis, both the expoxy and ester functions were reduced to hydroxy groups and the double bonds were hydrogenated catalytically. Saturation of the double bonds gave diagnostic spectra that were suitable for elucidating the position of the epoxy group. The shift in the fragmentation of a deuteriated sample verified the presence of the intact epoxides prior to chemical reduction. The presence of the double bonds in the epoxy molecules was demonstrated by reduction using homogeneous catalysis with tris(triphenylphosphine)rhodium(I) chloride and deuterium.

Protein-binding properties of 22-oxa-1.ALPHA.,25-dihydroxyvitamin D3, a synthetic analogue of 1.ALPHA.,25-dyhydroxyvitamin D3.

Protein binding properties of 22-oxa-1 alpha,25-dihydroxyvitamin D3 (22-oxa-1,25-D3), a synthetic analogue of 1 alpha,25-dihydroxyvitamin D3 (1,25-D3), were compared with those of vitamin D3 derivatives. The order of binding affinity to the chick embryonic intestinal receptor was 1,25-D3 greater than 22-oxa-1,25-D3 greater than 25-hydroxyvitamin D3 (25-D3) greater than 24R, 25-dihydroxyvitamin D3 (24, 25-D3) greater than vitamin D3 (D3), while that to the rat plasma vitamin D-binding protein (DBP) was 25-D3 greater than 24,25-D3 greater than D3 greater than 1,25-D3 greater than 22-oxa-1,25-D3. The binding potencies of 22-oxa-1,25-D3 to the receptor and DBP were about 1/8 and 1/600 of the respective values of 1,25-D3. When the distribution of the tritiated compounds in human plasma components was examined by an in vitro polyacrylamide gel electrophoretic method, [3H]-22-oxa-1,25-D3 was found to bind only to the lipoproteins including chyromicron. These results suggest that the replacement of a carbon atom into an oxygen atom in the side chain structure of 1,25-D3 results significant decrease in the binding affinity to DBP and that 22-oxa-1,25-D3 is transported as a complex-form not with DBP but with lipoprotein to the target tissues.

Purification and characterisation of an immunosuppressive factor from normal human seminal plasma

We have previously described the presence of a human seminal plasma component which may prevent the immunologic sensitization of females against sperm and seminal plasma antigens. Purification of this immunosuppressive factor (ISF) by saturated ammonium sulphate precipitation, followed by Sepharose 4-B column chromatography and Con-A Sepharose 4-B affinity chromatography is described here. An apparently single-band protein on SDS gel electrophoresis, having a molecular weight of 35,000, has been isolated. Amino acid analysis of this glycoprotein shows that it is rich in isoleucine, glycine, glutamine and proline, while methionine, tyrosine and asparagine are present in traces.

Demonstration in human plasma of a lectin activity analogous to that of bovine conglutinin.

Evidence of the existence in human plasma of an activity analogous to that of bovine conglutinin is presented. The human plasma component was characterized antigenically and functionally. Human plasma was shown to agglutinate complement-coated erythrocytes in the presence of Ca2+, and this conglutination was inhibited by EDTA. The molecule also binds to complement-reacted solid phase IgG and to zymosan in the presence of Ca2+. The binding to complement is not inhibited by N-acetyl-D-mannosamine, but is inhibited by N-acetyl-D-glucosamine, as previously shown for bovine conglutinin. Antibodies raised against bovine conglutinin cross-react with the human protein. The plasma concentration of the conglutinin-like protein showed a high inter-individual variation between apparently healthy donors. Unlike bovine conglutinin, the human conglutinin activity could not be demonstrated in serum but only in plasma. The activity was more stable in plasma containing metal-ion chelators like EDTA and citrate than in heparin or hirudin plasma.

Isolation and characterization of an inhibitor of factor XIIa from bovine plasma.

An inhibitor of factor XIIa has been purified to homogeneity from bovine plasma. The purification steps included precipitation of contaminating proteins with polyethylene glycol and chromatography on DEAE-cellulose, Affi-Gel blue, and immobilized wheat germ lectin. The apparent molecular weight of the XIIa inhibitor (called INH1) was 85,000, reduced, and 92,000, nonreduced, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The extinction coefficient (E0.1%(280)) of INH1 is 1.3, and the protein contains 17.7% carbohydrate. Purified antibody to INH1 raised in either rabbits or chickens formed a precipitin line of identity with purified INH1 and a component of bovine plasma, but there was no reaction with purified human inhibitors or with any component of human plasma. INH1 inhibits bovine and human XIIa, bovine and human C1-esterase, and human kallikrein, but does not inhibit bovine kallikrein, bovine trypsin, human plasmin, or human thrombin. This activity is similar to that of C1-inhibitor but different from antithrombin III, alpha 2-antiplasmin, or alpha 1-protease inhibitor. INH1 at a physiological concentration (0.47 microM) causes rapid inactivation of XIIa. The two molecules react in a 1:1 stoichiometry with a second-order rate constant of 1.23 X 10(6) M-1 min-1.

A Sensitive Assay, Specific for Endothelial Cell Type Plasminogen Activator Inhibitor in Blood Plasma

A polyclonal antibody raised against plasminogen activator (PA-)inhibitor from endothelial cells fully precipitates the PA-inhibitor in endothelial cell conditioned medium but only a part of the PA-inhibitory activity in blood plasma. This indicates that the PA-inhibitory activity in blood plasma is not due to a single inhibitory component. Performing the assay for PA-inhibitory activity in plasma both in the presence and absence of saturating concentrations of anti-endothelial cell PA-inhibitor antibodies, allows the determination of endothelial cell type PA-inhibitor in plasma. The assay gives a linear dose-response curve of amount of plasma added versus t-PA neutralised. Values for endothelial cell type PA-inhibitor in plasma of a group of 20 healthy individuals are in the range of 0.0-16.8 IU/ml and are not normally distributed (median value 3.0 IU/ml). This method also reveals a second, so far unidentified, PA-inhibitory component in human plasma.

Analysis of the direct effects of prostaglandins on human sperm function

Time-exposure photomicrography and interspecies in-vitro fertilization procedures have been used to examine the influence of prostaglandins on human sperm function. An analysis of variance indicated that the presence of PGs in the incubation media was associated with a significant increase in sperm velocity and the frequency of sperm head rotation, although there were no differences between individual PGs in the degree of stimulation observed. Changes in the penetrating ability of human spermatozoa were detected after exposure to PGs, particularly PGE-1 and PGE-2. PGE-2 induced a sustained increase in penetration rates at all doses of greater than 8.4 micrograms/ml, while exposure to PGE-1 gave a bell-shaped dose-response curve which exhibited a peak between 8.4 and 33.3 micrograms/ml and progressively fell to reach control levels at the maximum concentration tested of 270 micrograms/ml. A combination of PGE-2 and PGE-1 produced a dose-response curve similar to that for PGE-1 alone, while exposure to PGF-2 alpha was without effect. Seminal extracts containing predominantly 19-hydroxy PGE-1, or equal amounts of 19-hydroxy PGE-1 + 2 induced a slight, but significant, rise in penetration rates while a combination of PGs representing the major components of human seminal plasma was without significant effect. We conclude that certain prostaglandins may have a direct action on the functional competence of human spermatozoa.

Expression and regulation of human low-density lipoprotein receptors in Chinese hamster ovary cells.

Low-density lipoprotein (LDL), the major cholesterol transport component of human plasma, delivers cholesterol to mammalian cells via the LDL pathway of receptor-mediated endocytosis. LDL receptor activity and cholesterol biosynthesis are coordinately regulated by cholesterol-mediated feedback suppression. We have developed methods for the isolation of mutant and revertant (M.K., unpublished data) Chinese hamster ovary (CHO) cells having alterations in cholesterol biosynthesis and in the receptor-mediated endocytosis of LDL. The defective locus of one LDL receptor-negative CHO mutant (clone 7a-1) is apparently the structural gene for the LDL receptor (D. Kingsley, M. Segal and M.K., unpublished data). Here we have transfected 7a-1 cells with human DNA and selected colonies which synthesize functional human LDL receptors whose expression is regulated normally. This selection may prove useful for cloning genes required for the receptor-mediated endocytosis of LDL and other ligands and thus for elucidating the molecular defects responsible for familial hypercholesterolaemia, one of the most common genetic diseases in humans.

Comparison of human plasma low- and high-density lipoproteins as substrates for lecithin: Cholesterol acyltransferase

A recent observation that lecithin:cholesterol acyltransferase (EC 2.3.1.43) interacts with both low-density lipoproteins (LDL) and high-density lipoproteins (HDL) in human plasma is in apparent conflict with an earlier finding that the purified enzyme, while highly reactive with isolated HDL, was only minimally reactive with LDL. There is evidence, however, that lecithin: cholesterol acyltransferase may exist physiologically as a component of a complex with other proteins and that studies with the isolated enzyme may therefore provide misleading results. Consequently, interactions of the enzyme with isolated human lipoproteins have been re-examined in incubations containing lecithin: cholesterol acyltransferase as a component of human lipoprotein-free plasma in which a physiologically active complex of the enzyme with other proteins may have been preserved. In this system there was a ready esterification of the free cholesterol associated with both LDL and HDL-subfraction 3 (HDL 3) in reactions that obeyed typical enzyme-saturation kinetics. For a given preparation of lipoprotein-free plasma the V max values with LDL and with HDL 3 were virtually identical. The apparent K m for free cholesterol associated with HDL 3 was 5.6 · 10 −5M, while for that associated with LDL it was 4.1 · 10 −4M. This implied that, in terms of free cholesterol concentration, the affinity of HDL 3 for lecithin:cholesterol acyltransferase was about 7-times greater than that of LDL. When expressed in terms of lipoprotein particle concentration, however, it was apparent that the affinity of LDL for the enzyme was considerably greater than that of HDL 3. When the lipoprotein fractions were equated in terms of lipoprotein surface area, the apparent affinities of the two fractions for the enzyme were found to be comparable.

A species difference in platelet aggregation induced by tumour cells

Ehrlich ascites tumour cells (EATC) induced the aggregation of human platelets but not of sheep or rabbit platelets in native platelet-rich plasma. Aggregation was initiated by the interaction of EATC with a component(s) of human plasma, possibly related to the complement system, which led to the release of cellular ADP, a potent platelet aggregating agent. EATC previously incubated with human platelet-poor plasma induced immediate aggregation in platelet-rich plasma from all three species. The species difference in platelet aggregation by EATC is therefore related to the activity or availability of plasma component(s) responsible for release of cellular ADP rather than to intrinsic differences in platelet responsiveness to the tumour cells.

The endotoxin-induced procoagulant of mouse exudate macrophages: a factor-X activator

The properties of mouse macrophage procoagulant induced by endotoxin in vitro were studied by the acceleration of clotting and by chromogenic assays using as substrates human plasma and bovine components, which are not activated by mouse tissue factor. Maximal macrophage procoagulant activity occurred when activated cells were lysed in culture supernatant fluids, suggesting the interaction of cellular and supernatant factors. This procoagulant was clearly able to activate bovine factor X. The procoagulant also appeared to have prothrombinase activity. However, additional experiments suggested that the bulk of this activity was due to the activation of factor X contaminating the prothrombin. The production of the procoagulant was inhibited by warfarin (5 microM). Its activity was inhibited by 1 mM diisopropylfluorophosphate and unaffected by iodoacetamide, indicating that the procoagulant is a serine protease. Macrophage culture supernatants contained factor-VII-like activity. Neither mouse tissue factor nor macrophage culture supernatants alone activated bovine factor X. The two combined served as an efficient factor-X activator. Active supernatant factor (factor-VII-like) was not produced by macrophages cultured in the presence of warfarin, while the production of the macrophage cellular factor was unaffected by the presence of warfarin. I conclude that exudate macrophages cultured in vitro make and secrete factor VII or a factor-VII-like substance into the culture supernatant. When activated macrophages are lysed in this supernatant, the interaction of a cellular factor (? tissue factor) and factor VII gives rise to a factor-X activator.

The Endotoxin-Induced Procoagulant of Mouse Exudate Macrophages: A Factor-X Activator

AbstractThe properties of mouse macrophage procoagulant induced by endotoxin in vitro were studied by the acceleration of clotting and by chromogenic assays using as substrates human plasma and bovine components, which are not activated by mouse tissue factor. Maximal macrophage procoagulant activity occurred when activated cells were lysed in culture supernatant fluids, suggesting the interaction of cellular and supernatant factors. This procoagulant was clearly able to activate bovine factor X. The procoagulant also appeared to have prothrombinase activity. However, additional experiments suggested that the bulk of this activity was due to the activation of factor X contaminating the prothrombin. The production of the procoagulant was inhibited by warfarin (5 μM). Its activity was inhibited by 1 mM diisopropylfluorophosphate and unaffected by iodoacetamide, indicating that the procoagulant is a serine protease. Macrophage culture supernatants contained factor-VII-like activity. Neither mouse tissue factor nor macrophage culture supernatants alone activated bovine factor X. The two combined served as an efficient factor-X activator. Active supernatant factor (factor-VII-like) was not produced by macrophages cultured in the presence of warfarin, while the production of the macrophage cellular factor was unaffected by the presence of warfarin. I conclude that exudate macrophages cultured in vitro make and secrete factor VII or a factor-VII-like substance into the culture supernatant. When activated macrophages are lysed in this supernatant, the interaction of a cellular factor (? tissue factor) and factor VII gives rise to a factor-X activator.

Rapid isolation of apolipoprotein E from human plasma very low density lipoproteins by molecular sieve high performance liquid chromatography

A rapid (less than 1 hour) and sensitive technique was developed for the isolation of apolipoprotein E from the other main components of human plasma very low density lipoproteins using molecular sieve high performance liquid chromatography with an approximate 80% recovery. The properties of the pure apoprotein were those reported in the literature for products isolated by conventional chromatographic and/or electrophoretic procedures.