A competitive enzyme-linked immunosorbent assay for alpha 1-acid glycoprotein in serum and urine samples

In this solid-phase competitive enzyme-linked immunosorbent assay for alpha 1-acid glycoprotein in serum or urine, antiserum to human alpha 1-acid glycoprotein is incubated with solid-phase-bound alpha 1-acid glycoprotein in the presence of standard or sample. Incubation with second antibody labeled with alkaline phosphatase then follows, before development with substrate. Results obtained correlate well with a fluorescent assay involving the dye Auramine O (r = 0.953) and with radial immunodiffusion (r = 0.921). The present assay covers the range 0.2 to 5 mg/L and 16 samples take 2.5 h to complete. This assay is useful for measuring concentrations of alpha 1-acid glycoprotein in serum and also in urine, for which other assay methods are not sufficiently sensitive.     

SKF 525A displaces drugs from serum alpha 1-acid glycoprotein binding sites

In vivo treatment of rats with beta-diethylaminoethyl-diphenylpropylacetate hydrochloride (SKF 525A), an inhibitor of hepatic monooxygenases, decreases the serum binding of oxprenolol and propranolol, which bind mainly to alpha 1-acid glycoprotein, but not that of phenytoin, which is bound to albumin. The effect was maximal 40 min after treatment and had disappeared after 6 hr, when enzyme inhibition was still present. A displacing effect was also observed when SKF 525A was added in vitro to serum of rats, dogs and humans and to human alpha 1-acid glycoprotein, whereas binding to human serum albumin was not affected. SKF 525A was found to be equipotent with tris(2-butoxyethyl)phosphate, a known displacer of binding of drugs from alpha 1-acid glycoprotein. When studying the pharmacokinetics or the effects of drugs after SKF 525A pretreatment, the possibility that altered protein binding may influence the findings should be considered.     

Changes in alpha 1-acid glycoprotein serum concentrations and glycoforms in the developing human fetus.

alpha 1-Acid glycoprotein concentrations and reactivity to concanavalin A were measured in maternal and fetal serum and amniotic fluid obtained from 24 women undergoing diagnostic cordocentesis at 20 to 33 wk gestation and in 30 additional fetal sera (19 to 34 weeks gestation). Maternal alpha 1-acid glycoprotein serum levels were five to ten times higher than fetal and amniotic levels. Fetal alpha 1-acid glycoprotein levels were found to increase with advancing gestational age. Using crossed immunoaffino electrophoresis with concanavalin A, alpha 1-acid glycoprotein patterns were identical in maternal serum and amniotic fluid but totally different in fetal serum. The fetal concanavalin A pattern changed progressively during fetal life towards that of the newborn. These data confirm earlier assumptions of fetal synthesis of alpha 1-acid glycoprotein and provide normal reference values for alpha 1-acid glycoprotein in fetal serum. In addition, the specific fetal concanavalin A pattern indicates that the alpha 1-acid glycoprotein glycosylation process during fetal life differs from that in post-natal life.     

SOME BIOCHEMICAL PROPERTIES OF THYMUS LEUKEMIA ANTIGENS SOLUBILIZED FROM CELL MEMBRANES BY PAPAIN DIGESTION

Thymus leukemia (TL) alloantigenic activity was solubilized by papain proteolytic digestion from intact RADA1 tumor cells. If the cells were labeled with amino acids and fucose, the TL alloantigen could be isolated as a doubly labeled glycoprotein fragment by indirect precipitation from the papain digest. This TL glycoprotein fragment was approximately the same mol wt as the papain-digested H-2.4 alloantigen fragment as judged by chromatography on Sephadex G-150 in sodium dodecyl sulfate. The carbohydrate chain of the TL glycoprotein obtained by exhaustive pronase digestion behaved as a glycopeptide of approximately 4,500 mol wt, as compared with the glycopeptide of the H-2.4 alloantigen that had a mol wt of about 3,500. Thus, the TL alloantigen can be solubilized by papain digestion as a glycoprotein fragment similar in mol wt to the H-2 alloantigen glycoprotein fragment. The carbohydrate chain of the TL glycoprotein is larger than the H-2 carbohydrate chain.     

A solid-phase enzyme-linked immunosorbent assay for the quantitation of human plasma alpha 1-acid glycoprotein.

We describe a solid-phase enzyme-linked immunosorbent assay for alpha 1-acid glycoprotein in human plasma. Plasma samples are incubated with alkaline phosphatase-linked, purified alpha 1-acid glycoprotein in alpha 1-acid glycoprotein-specific antibody-coated polystyrene tubes. The alkaline phosphatase that becomes attached to the tube via an immunological reaction between the alpha 1-acid glycoprotein and the specific antibody is measured spectrophotometrically. This assay is accurate reproducible, simple, and economical. As little as 4 microgram of alpha 1-acid glycoprotein per liter can be detected. The normal range for alpha 1-acid glycoprotein in the plasma of healthy adults, as measured by this method, is 0.48-1.27 g/L; the range is significantly different, 0.29-0.73 g/L, for women who are taking oral contraceptive pills.     

Inheritance of human α1-acid glycoprotein (orosomucoid) variants

Although variants of sialic acid-free alpha(1)-acid glycoprotein have been described in human beings, the mode of inheritance of these types has not been reported previously. With the use of a new technique of immunofixation after agarose gel electrophoresis of neuraminidase-treated whole serum, the present study demonstrates that the types of alpha(1)-acid glycoprotein variants in family members are consistent with inheritance as autosomal traits with codominant expression. Gene frequencies have been determined for several ethnic groups. Of a total of 11 maternal-cord serum pairs, seven were discordant types, indicating that the fetus synthesizes alpha(1)-acid glycoprotein and confirming a previous report that there is no significant transplacental passage of this protein.     

Alpha 1-acid glycoprotein decreases recovery of total protein in urine when trichloroacetic acid is used to precipitate the proteins

Total urine protein was measured in 132 samples by an automated benzethonium chloride method and the Ponceau-S/trichloroacetic acid (PS/TCA) method. Of these, 27% gave a result 0.1 g/L or more higher by the benzethonium chloride method. Of this 27%, most contained an abnormally high concentration of the acute-phase reactant, alpha 1-acid glycoprotein. By assaying urine containing added alpha 1-acid glycoprotein and albumin, we found that alpha 1-acid glycoprotein causes the PS/TCA method to underestimate the total urine protein concentration, whereas the benzethonium chloride method is unaffected. Not all urinary albumin was precipitated by TCA when alpha 1-acid glycoprotein was present. Therefore, protein methods in which trichloroacetic acid is used as a concentrating step before the assay will underestimate total urine protein when the concentration of alpha 1-acid glycoprotein is high.     

Sensitive chemiluminescence immunoassay for the determination of rat serum alpha1-acid glycoprotein.

We present the establishment of a sensitive immunoassay for the determination of alpha1-acid glycoprotein (orosomucoid, AGP) in rat serum. The assay is based upon antigen capture by surface-immobilized specific polyclonal rabbit anti-AGP antibodies with biotinylated rat AGP (rAGP) as the tracer, and formatted as competitive enzyme immunoassay. Signaling is performed by streptavidin-conjugated horseradish peroxidase. Enzyme activity is quantified by an enhanced chemiluminometric method, allowing the sensitive detection of rAGP serum levels in small sample volumes.     

Alterations of branching and differential expression of sialic acid on alpha-1-acid glycoprotein in human seminal plasma

BACKGROUND: The degree of branching and types of fucosylation of glycans on alpha(1)-acid glycoprotein (AGP) have been found to be associated with alpha(1)-acid glycoprotein concentrations in human seminal plasma. The glycosylation pattern of alpha(1)-acid glycoprotein in seminal plasma obtained from men living in infertile couples can undergo alterations in relation to sperm analysis and/or alpha(1)-acid glycoprotein concentrations. METHODS: The glycosylation of alpha(1)-acid glycoprotein was studied upon the reactivity with specific lectins by crossed affinity immunoelectrophoresis (concanavalin A), and by glycoprotein lectin immunosorbent assay (Maackia amurensis and Sambucus nigra lectins), as well as high pH anion-exchange chromatography with pulsed amperometric detection. RESULTS: Nonsignificant differences in alpha(1)-acid glycoprotein glycan branching and degree of its sialylation were observed among the AGP derived from seminal plasmas in relation to spermiogram and sperm morphology. However, significant concentration-dependent differences were found in extent of branching and type of sialylation. CONCLUSIONS: The presence in seminal plasma of high concentrations of aberrantly glycosylated AGP molecules might be indicative for a chronic inflammatory condition in the reproductive tract, and can be used as additional tool to subdivide the seminal plasmas of men living in infertile couples.     

Incidence of varying factors on the immunochemical behavior of alpha 1-acid glycoprotein

Electroimmunodiffusion methods of Laurell and radial immunodiffusion method of Mancini are compared for the qualitative and quantitative analysis of native and desialylated alpha 1-acid glycoprotein. Samples are incubated under different conditions at decreasing pH (3.5 to 0.5 pH units), with increasing ionic strength and with neuraminidase during different time intervals. Results show a pronounced decrease in electrophoretic mobility of alpha 1-acid glycoprotein treated either with acidic reagents or with neuraminidase (ionic strength has no effect). Such a procedure might involve chemical or enzymatic hydrolysis by which sialyl residues are removed. This hydrolysis implicates lower results in the estimation of the desialylated glycoprotein by electroimmunodiffusion. On the other hand, the amounts of alpha 1-acid glycoprotein evaluated by radial immunodiffusion are not modified after incubation. This is expected since diffusion and antigenic properties are not related to the sialic acid content. The data suggest that radial immunodiffusion, less accurate and sensitive than electroimmunodiffusion, is nevertheless more adequate for estimating native and desialylated alpha 1-acid glycoprotein.     

Novel surface antigen expressed on dividing cells but absent from nondividing cells

Radioimmunoprecipitation followed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis was used to study the distribution on human lymphoid cells of a previously undescribed surface antigen recognized by several heteroantisera. A glycoprotein with a 90,000 mol wt (under reducing conditions) was detected on all cell lines tested including T, B, null, and myeloid cell lines, although the amount of antigen present varied considerably. The antigen was absent from normal peripheral blood lymphocytes (PBL), B and T cells, monocytes, granulocytes, thymocytes, and erythrocytes. After stimulation with lectins or allogeneic B cells, the antigen was induced on PBL T cells. A limited number of leukemic T cells tested all expressed the antigen, as did melanoma cell line and human embryonic lung fibroblasts. Hence, the antigen was present only on dividing lymphoid cells and absent from nondividing cells, but was also present on the two examples of dividing non-lymphoid cells tested. Under nonreducing conditions, the 90,000-mol wt band normally present disappeared to be replaced by another at approximately 200,000 mol wt. The glycoprotein bound to lectins from lentil and ricin, but not to wheat germ agglutinin. It could be readily labeled metabolically by [35S]methionine or by surface iodination, and appeared to be a major membrane protein on some cell lines.     

Changes in serum acute phase proteins in ovarian cancer patients receiving cis-diamminedichloroplatinum (CDDP) infusion therapy

Nine patients with adenocarcinoma of the ovary were given a number of courses of Cisplatin, by I.V. infusion. In four patients a complete clinical response was observed and these patients are disease-free one year after treatment. There was no clinical response in the remaining five patients who have subsequently died. Serial determinations of three acute-phase reactant proteins (alpha 1-acid glycoprotein, haptoglobin, alpha 1-antitrypsin) were performed before every infusion and after therapy. Constantly high or rising serum levels of haptoglobin and alpha 1-acid glycoprotein were associated with progression of the cancer, whereas in patients who were disease-free after therapy, these glycoproteins remained essentially in the normal range. The results suggest that serial measurements of haptoglobin and alpha 1-acid glycoprotein may have clinical value as aids in deciding the effectiveness of drug therapy in these patients.     

Preclinical investigation of alpha 1-acid glycoprotein (orosomucoid)

The molecular properties of alpha 1-acid glycoprotein are briefly discussed. This molecule has been shown in in vitro experiments to have both a stabilizing effect on vascular permeability and antiinflammatory properties. We were able to demonstrate these two effects in vivo in guinea pigs (skin, Evan's Blue extravasation) and in rats (paw, carrageenan induced inflammation). Further experiments were performed in rats relating to possible therapeutic indications for alpha 1-acid glycoprotein: (1) inhibitory effect on brain edema formation after experimental stroke, (2) therapeutic effect in the puromycin aminonucleoside-induced minimal change nephrosis, (3) improvement of vital parameters in hemorrhagic-hypovolemic shock, (4) increase in survival rate in septic peritonitis, and (5) promising effects in burn-induced remote lung injury. The high content of sialic acid and the high negative charge of alpha 1-acid glycoprotein are believed to be major contributors to its stabilizing effect on vascular permeability. The protein is bound to the glycocalyx of the endothelial cells (and presumably to structures of the glomerular basement membrane), thereby hindering the passage of other polyanionic molecules through the vascular wall. The antiinflammatory/immunomodulatory effect of alpha 1-acid glycoprotein appears mainly due to suppression of polymorphonuclear neutrophils. This action is dependent on the glycan part of the molecule, which is highly variable (microheterogeneity). It is obvious that there are differences between the different glycan forms as far as the antiinflammatory property of the protein is concerned. Together with data in the literature, the results presented here suggest a variety of potential indications for therapeutic use of alpha 1-acid glycoprotein in humans.     

Alteration of sugar chains on alpha(1)-acid glycoprotein secreted following cytokine stimulation of HuH-7 cells in vitro

In inflammation, hepatocytes secrete several proteins into serum, the so-called acute phase proteins. In addition to increased serum levels of alpha(1)-acid glycoprotein (AGP), there are also changes in the composition of its sugar chains. To investigate the cytokine-stimulated alteration of sugar chains on AGP, we cultured the human hepatoma cell line HuH-7 cells in serum-free medium (IS-RPMI) with and without IL-1beta and IL-6, and analyzed AGP secretion into the medium. AGP was increased during stimulation with both IL-1beta and IL-6, although the effect of IL-1beta was more pronounced. Lectin-binding assay for secreted AGP also indicated significant increases in the binding activities to Aleuria aurantia lectin (AAL), Sambucus sieboldiana agglutinin (SSA), and concanavalin-A (ConA). In particular, AAL binding activity increased with higher expression of the sialyl Lewis X (sLe(X)) antigen, NeuAc alpha2-3 Gal beta1-4 (Fuc alpha1-3) GlcNAc-R. Thus, the increase in AGP fucosylation may be correlated with the increase of sLe(X). The present results indicate that the serum-free culture of HuH-7 cells provides a useful model for investigating the secretion of proteins from hepatocytes, and the effects of cytokines on the changes in sugar chains of glycoproteins in vitro.     

Influence of bupivacaine on mepivacaine protein binding

To elucidate the mechanism of any drug displacement interaction, we examined the protein binding of mixtures of mepivacaine and bupivacaine in serum and solutions of albumin or alpha 1-acid glycoprotein. Protein binding data with mepivacaine alone were best described by a model with one class of binding site and a partitioning constant in serum and by a model with one class of binding site in both isolated protein solutions. Binding affinity of mepivacaine in serum was reduced in the presence of bupivacaine. Displacement of mepivacaine by bupivacaine was observed when an alpha 1-acid glycoprotein solution was studied. Classic competitive inhibition was demonstrated. Bupivacaine reduced mepivacaine binding to albumin, but the degree of displacement was not significant. When administered simultaneously, these two amino-amide local anesthetics interact synergistically to produce a higher than expected free concentration of mepivacaine. This interaction increases the risk of toxicity.     

D-galactosamine-induced liver injury: a rat model to study the heterogeneity of the oligosaccharide chains of alpha 1-acid glycoprotein

The effect of D-galactosamine on the structure of the glycan moiety of alpha 1-acid glycoprotein was studied throughout a nine days experiment. It was shown that: D-galactosamine led to an alteration of the Concanavalin A crossed immunoelectrophoresis pattern and to a decreased sialic acid content of alpha 1-acid glycoprotein. The undersialylation of alpha 1-acid glycoprotein was not linked to a change in the relative ratio of various Concanavalin A forms. At the end of the experiment (9 days after galactosamine injection), the Concanavalin A non-reactive forms of alpha 1-acid glycoprotein remained elevated whereas alanine transaminase activity, total protein and alpha-acid glycoprotein had returned to a control level. D-galactosamine-treated rats seem to be a suitable model for the study of the very fast cyclic modulations of the synthesis of the glycan moiety of glycoproteins.     

Purification and characterization of a major human Pneumocystis carinii surface antigen.

Previous studies of Pneumocystis carinii have identified the major surface antigen of rat and human isolates as proteins of 116,000 and 95,000 mol wt, respectively, that are antigenically not identical. In this study both rat and human P. carinii proteins were purified by solubilization with zymolyase followed by molecular sieve and ion exchange chromatography. The native proteins had an apparent mol wt of 290,000 or greater, based on molecular sieve studies as well as cross-linking studies. Both proteins were glycoproteins; treatment with endoglycosidase H resulted in a 9% decrease in mol wt. The carbohydrate composition of the rat P. carinii glycoprotein was distinct from the human isolate; glucose, mannose, galactose, and glucosamine occurred in approximately equimolar ratios in the human P. carinii protein, whereas glucose and mannose were the predominant sugars of the rat P. carinii protein. To evaluate humoral immune responses to the human P. carinii protein, an enzyme-linked immunosorbent assay using purified protein was developed. Some, but not all, patients who subsequently developed P. carinii pneumonia demonstrated a serum antibody response to the surface antigen. Nearly all subjects without a history of P. carinii pneumonia had no detectable antibodies. Purified P. carinii proteins will greatly facilitate the investigation of host-P. carinii interactions.     

Microheterogeneity of the carbohydrate moiety of human alpha 1-acid glycoprotein in two benign liver diseases: alcoholic cirrhosis and acute hepatitis

To determine whether alterations of the carbohydrate moiety of human alpha 1-acid glycoprotein constitute a marker of hepatic damage we studied purified alpha 1-acid glycoprotein from healthy individuals and two groups of patients with benign liver diseases: alcoholic cirrhosis and acute viral hepatitis. The results indicate: (1) increased concanavalin A-non reactive forms in cirrhosis and hepatitis, (2) a markedly increased proportion of fucosyl residues in all cirrhotic and some hepatitis patients. Although hyperfucosylation is generally considered to be a tumor marker, the observation here in the two benign liver diseases indicates that an increased fucosyl content should be considered as a more general expression of pathological glycoconjugate metabolism.     

Bound homocysteine, cysteine, and cysteinylglycine distribution between albumin and globulins

BACKGROUND: Major portions of homocysteine (Hcy), cysteine (Cys), cysteinylglycine (CysGly), and glutathione in serum are covalently bound to proteins via disulfides. Albumin has been considered the dominant binding protein. METHODS: Pooled serum and plasma from healthy adults were fractionated into albumin and globulins by affinity columns. Content of Hcy, Cys, CysGly, and glutathione was determined for serum and plasma fractions and purified proteins by an HPLC method before and after incubation with excess CysGly, Hcy, or glutathione RESULTS: Of protein-bound amino acids in pooled serum, 12% of Hcy, 21% of Cys, and 33% of CysGly were bound to globulins, with the remainder bound to albumin. Slightly higher proportions were bound to globulins in pooled plasma. Globulins had approximately 16% of total exchangeable disulfide and thiol groups in serum based on results of loading with CysGly. These results agree with expected abundance of unpaired Cys residues in globulins relative to albumin. Significant amounts of disulfide-linked amino acids were detected for HDL and alpha1-acid glycoprotein but not for transferrin. Exchange of disulfide-linked amino acids on exposure to excess Hcy or glutathione was much faster for albumin than for alpha1-acid glycoprotein. CONCLUSIONS: Approximately 10%-30%, of protein-bound Hcy, Cys, and CysGly are disulfide-linked to globulins. Amino acids disulfide-linked to albumin are rapidly exchangeable, while exchange of disulfide-linked amino acids from globulins, such as alpha1-acid glycoprotein, is much slower. Consequently, the pools of Hcy, Cys, and CysGly bound to albumin and globulin may represent kinetically and functionally distinct pools. Plasma concentrations of total Hcy and Cys, which are dominated by albumin-bound pools, may not reflect the abundance of functionally significant modifications of globulins.     

An immunological study of nine proteins in CSF and serum of a group of epileptic patients.

The concentrations of nine proteins, alpha-1-acid glycoprotein, antitrypsin, prealbumin, transferrin, albumin, IgG, ceruloplasmin, IgA and alpha-2-macroglobulin, have been determined in the serum and CSF of two groups of patients, one control and one experimental, by an immunological method. The experimental group were patients suffering from grand mal epilepsy. The control group showed no detectable neurological disorder. In the group of grand mal epileptics, only prealbumin showed a significant elevation in CSF when compared with the control group. In contrast, the rest of the proteins are decreased with respect to the controls except for alpha 1-acid glycoprotein and transferrin. The results from this study also suggest that something more than an ultrafiltration process dependent upon molecular weight, is important in determining the concentration of some serum proteins in the CSF.