Isolation of α2 Macroglobulin Subunits from Human Serum

Subunits of α₂ macroglobulin (α₂M) with sedimentation rates lower than 10 S were found in ultrafiltrates (through macroglobulin-stopping membranes) of normal human sera. Five to six percent of the total α₂M in human sera exists in subunit form. After isolation by means of ammonium sulfate precipitation, curtain electrophoresis, gel filtration and ultrafiltration these subunits were shown to have sedimentation rates of 5–10 S.     

Biochemical properties of canine serum ferritin: iron content and nonbinding to concanavalin A

A sandwich enzyme-linked immunosorbent assay using H-subunit-rich canine heart ferritin as a standard has been developed for measuring canine serum ferritin which is H-subunit-rich. Serum ferritin concentrations in 51 normal dogs ranged from 143 to 1766 ng ml^–1, with a mean value of 479±286 (SD) ng ml^–1. Serum ferritin iron concentrations as determined by an immunoprecipitation technique ranged from 30.4 to 115.9 ng ml^–1 in 15 normal dogs with serum ferritin protein levels of 298 to 959 ng ml^–1. There was a significant linear correlation between the serum ferritin iron and protein levels (r=0.9441, P<0.001), and the mean iron/protein ratio of serum ferritin was 0.112±0.017. When canine sera were incubated with concanavalin A-Sepharose 4B, we observed the apparent binding of serum ferritin to concanavalin A. However, ferritin obtained by heat-treating the sera at pH 4.8 to remove the ferritin-binding proteins did not bind to the lectin. These results suggest that canine serum ferritin contains a considerable amount of iron but no concanavalin A-binding G subunit present in human serum ferritin.     

Dialyzability and binding of digoxin-like immunoreactive factors (DLIF) with serum macromolecules in uremic patients on hemodialysis

Digoxin-Like Immunoreactive Factors (DLIF) which cross-react with antidigoxin antibodies are present in elevated concentrations in patients on hemodialysis, uremia, hypertensives, liver failure, pre-eclampsia and premature birth. DLIF may have a potential role as a natriuretic hormone with a speculated low molecular weight (less than 1000). We studied the dialyzability and bindings of DLIF with serum components in hemodialysis patients. We analyzed DLIF concentrations in sera and protein free ultrafiltrates of 31 patients and 22 normal volunteers using a fluorescence polarization assay for digoxin. The DLIF concentrations were expressed as nmol/L Digoxin Equivalent. The gel filtration analysis was done using three different Bio-Gel columns with molecular weight cut-offs of 10,000, 20,000 and 40,000. Molecules with lower molecular weight than cut-off were absorbed in the column. Only 3 out of 22 normal volunteers (13.6%) showed measurable DLIF. However 23 out of 31 patients (74.2%) showed measurable DLIF. The concentrations of DLIF were significantly higher in patients with renal failure on hemodialysis (P less than 0.05) by both chi-squared and Fisher's exact test. We observed no statistically significant difference in the concentrations of DLIF in pre and post-dialysis sera, indicating that DLIF were not filtered during hemodialysis. We observed no DLIF activity in the protein free ultrafiltrates of any DLIF positive sera (patients and normal volunteers), indicating that unlike digoxin (where we observed 70-80% of total digoxin concentrations in ultrafiltrates), DLIF were strongly bound to serum components. With Bio-Gel filtration experiments (five different serum pools), we recovered all DLIF activities in the fraction equivalent to the void volume of the column with Bio-Gel P6 and P10 columns, indicating that DLIF were almost completely bound to serum components with molecular weight greater than 20,000. On the other hand, we recovered no DLIF activities in the void volume when the same serum pools were passed through the Bio-Gel P30 column, indicating that DLIF were strongly bound to serum macromolecules with molecular weight less than 40,000. In sharp contrast, when serum containing digoxin was subjected to the same series of experiments, we recovered only 20-30% of digoxin concentrations in void volume with all three columns as expected since digoxin is only 25% bound to albumin (MW 67,000).     

High-performance liquid chromatographic analysis of glycoamines in serum

This report describes the development of an HPLC-UV method for studies of glycoamines and glycoamine-like compounds in normal human serum and osteosarcoma patient serum as potential biological markers of cancer. The glycoamines, a newly recognized class of endogenous, low-molecular-mass biopolymers, are conjugates of amino acids and sugar units, containing 5 to 29 amino acid and 1 to 17 sugar units. After ultrafiltration of serum samples, reversed-phase HPLC separation with diode-array detection was used to obtain standard profiles of serum ultrafiltrates below M_r 10 000 in healthy subjects. These highly reproducible profiles utilized two-dimensional peak identification and were used to develop a statistical profile of the major glycoamine peaks in normal serum. This newly developed analytical method was subsequently used to address a key question: whether or not there is a single tumor-specific glycoamine or a family of tumor-specific glycoamines in cancer patient serum. Preliminary results suggest that this method can separate and detect glycoamines and glycoamine-like compounds in various types of cancer patient serum with a high degree of reproducibility on the basis of comparative two-dimensional identification of natural compounds and a panel of synthetic glycoamine analogs. Moreover, the method is useful for following the relative changes in the amount of a given glycoamine over an extended clinical time course. Initial results suggest that a glycoamine or glycoamine-like compound, GA-4.63, may have clinical utility in human osteosarcoma studies.     

Structure and properties of albumin Tokushima and its proteolytic processing by cathepsin B in vitro

Albumin Tokushima is a Japanese genetic variant of human serum albumin. Two homozygous and 6 heterozygous subjects with this variant were found in a family. Albumin Tokushima was purified from sera of the homozygous subjects. Its amino acid composition and amino-terminal sequence were determined and compared with those of a normal serum albumin. Albumin Tokushima with the amino-terminal sequence of Arg-Gly-Val-Phe-His-Arg-Asp-Ala-His-Lys-Ser-Glu-Val-Ala-His-Arg-Phe-Lys- Asp- Leu-Gly-Glu-Glu-Asn-Phe was found to be the same abnormal proalbumin as proalbumin Lille (Abdo, Y. et al. (1981) FEBS Lett. 131, 286-288). The isoelectric points of albumin Tokushima were pH 4.70 and 4.90 as compared with pH 5.05 and 5.25 of a normal serum albumin. Albumin Tokushima was converted to normal serum albumin by purified cathepsin B in vitro. Albumin Tokushima can bind Ni2+ at 4 degrees C but binds little at 37 degrees C.     

Effect of serum on prostaglandin production during the co-culture of human thyroid cells and peripheral blood mononuclear cells

Prostaglandin generation by human peripheral blood mononuclear cells is enhanced during co-culture with human thyroid cells. The objective of the present study was to determine the influence of various sera on this process. Human thyroid adenoma cell monolayers were cultured with normal human peripheral blood mononuclear cells for three days in the presence of a variety of sera, or serum fractions. Prostaglandin E (PGE) in the medium was measured by bioassay or by radioimmunoassay. Significantly more PGE was generated in cultures containing fetal calf serum than in those containing human serum. This difference was not abolished by dialysis of the human serum. When the 50% (NH₄) ₂SO₄ precipitate of the serum was used, PGE generation was similar to that in fetal calf serum, indicating the presence of an inhibitory factor in human serum. The degree of this inhibitory activity was similar in autologous and heterologous human serum, as well as in normal subjects and patients with Graves' disease. Gel filtration and ion-exchange chomatography of human serum showed the inhibitor to co-migrate with albumin. Evidence presented suggests that the inhibitor is not albumin itself but is, instead, a factor tightly bound to albumin. Inhibitory activity was also found in rabbit, goat, rat and cow serum. Prostaglandins are potent modulators of immune-cell function. These data indicate that this process may be modulated by a factor in mammalian serum. The relative absence of this factor in fetal serum may have important implications in regard to the profound changes which occur in the immune system after birth.     

The availability of iron and the growth of Vibrio vulnificus in sera from patients with haemochromatosis

Abstract The ability of the marine bacterium Vibrio vulnificus to grow in sera from patients with haemochromatosis and from normal human volunteers was investigated. Studies showed that although V. vulnificus possesses potential iron uptake systems, the organism is unable to multiply in normal human sera. However, it grows rapidly in serum from patients with haemochromatosis. This growth was found to be due solely to the presence of freely available iron.     

Hyaluronidase in sera of tumour-bearing nude mice

Cancer cell lines often secrete hyaluronidase, suggesting that this enzyme could be used as a marker of growing tumours. We have measured hyaluronidase in the sera of non-grafted mice and mice grafted with human tumour-derived hyaluronidase-secreting H460M and SA87 cells or non-secreting CB 193 cells. Mouse serum hyaluronidase was measured at pH 3.8 using the enzyme-linked sorbent assay (ELSA) technique by reference to human serum whose activity at pH 3.8 was determined by the Reissig technique. The serum hyaluronidase in non-grafted mice ranged from 310-520 mU l^-1 (mean±SD 432±70 mU l^-1, median 440 mU l^-1). Hyaluronidase increased in the sera of tumour-bearing mice grafted with H460M cells or with SA87 cells, but not in the sera of mice grafted with CB 193 cells. Serum hyaluronidase activity in H460M or SA87 tumour-bearing mice correlated with the tumour mass, increased with time, and decreased after tumour removal. Zymography detected two different hyaluronidase forms in the sera of non-grafted mice: type 1 had only one hyaluronidase band and type 2 had five different bands. In both types, enzyme augmentation in tumour-bearing mice correlated with the presence of an additional enzyme band that was not seen in normal sera and that migrated as the cancer cell enzyme did; there was no augmentation of the normal isoform(s). These results show that serum hyaluronidase can be used to follow the development of tumours in mice grafted with hyaluronidase-secreting cells.     

Studies of the binding of different iron donors to human serum transferrin and isolation of iron-binding fragments from the N- and C-terminal regions of the protein.

1. Trypsin digestion of human serum transferrin partially saturated with iron(III)-nitrilotriacetate at pH 5.5 or pH 8.5 produces a carbohydrate-containing iron-binding fragment of mol.wt. 43000. 2. When iron(III) citrate, FeCl3, iron (III) ascorabate and (NH4)2SO4,FeSO4 are used as iron donors to saturate the protein partially, at pH8.5, proteolytic digestion yields a fragment of mol.wt. 36000 that lacks carbohydrate. 3. The two fragments differ in their antigenic structures, amino acid compositions and peptide 'maps'. 4. The fragment with mol.wt. 36000 was assigned to the N-terminal region of the protein and the other to the C-terminal region. 5. The distribution of iron in human serum transferrin partially saturated with various iron donors was examined by electrophoresis in urea/polyacrylamide gels and the two possible monoferric forms were unequivocally identified. 6. The site designated A on human serum transferrin [Harris (1977) Biochemistry 16, 560--564] was assigned to the C-terminal region of the protein and the B site to the N-terminal region. 7. The distribution of iron on transferrin in human plasma was determined.     

An assay for osteoclast-activating factor (OAF) in biological fluids: Detection of OAF in the serum of myeloma patients

A procedure is described for the partial purification and biological assay of a lymphokine, osteoclast-activating factor, in samples of serum. This procedure involves two steps of ultra-filtration of samples, followed by polyacrylamide gel electrophoresis and bioassay of fractions eluted from the gels. The procedure effectively discriminates between osteoclast-activating factor and other agents with similar biological activities (parathyroid hormone, prostaglandin E₂, and 1, 25-dihydroxyvitamin D₃), when each agent is added to normal human serum. Using this procedure as a specific assay for osteoclast-activating factor, serum from normal individuals and myeloma patients was examined. The activity was detected in 11 of 23 myeloma sera but in none of 12 normal sera.     

Effect of pH and oxalic acid on the reduction of Fe3+ by a biomimetic chelator and on Fe3+ desorption/adsorption onto wood: Implications for brown-rot decay

Fenton reaction is thought to play an important role in wood degradation by brown-rot fungi. In this context, the effect of oxalic acid and pH on iron reduction by a biomimetic fungal chelator and on the adsorption/desorption of iron to/from wood was investigated. The results presented in this work indicate that at pH 2.0 and 4.5 and in the presence of oxalic acid, the phenolate chelator 2,3-dihydroxybenzoic acid (2,3-DHBA) is capable of reducing ferric iron only when the iron is complexed with oxalate to form Fe³⁺-mono-oxalate (Fe(C₂O₄)⁺). Within the pH range tested in this work, this complex formation occurs when the oxalate:Fe³⁺ molar ratio is less than 20 (pH 2.0) or less than 10 (pH 4.5). When aqueous ferric iron was passed through a column packed with milled red spruce (Picea rubens) wood equilibrated at pH 2.0 and 4.5, it was observed that ferric iron binds to wood at pH 4.5 but not at pH 2.0, and the bound iron could then be released by application of oxalic acid at pH 4.5. The release of bound iron was dependent on the amount of oxalic acid applied in the column. When the amount of oxalate was at least 20-fold greater than the amount of iron bound to the wood, all bound iron was released. When Fe–oxalate complexes were applied to the milled wood column equilibrated in the pH range of 2–4.5, iron from Fe–oxalate complexes was bound to the wood only when the pH was 3.6 or higher and the oxalate:Fe³⁺ molar ratio was less than 10. When 2,3-DHBA was evaluated for its ability to release iron bound to the milled wood, it was found that 2,3-DHBA possessed a greater affinity for ferric iron than the wood as 2,3-DHBA was capable of releasing the ferric iron bound to the wood in the pH range 3.6–5.5. These results further the understanding of the mechanisms employed by brown-rot fungi in wood biodegradation processes.     

Determination of non-protein-bound iron in rat tissue by ion chromatography with electrochemical detection

A new, rapid, sensitive, and specific method combining ion chromatography with electrochemical detection was developed for measuring non-protein-bound Fe(II) and Fe(III) in biological samples. The procedure was based on the separation of the iron-diethylenetriaminepentaacetic acid complex formed directly on the chromatographic column with anion-exchange resin followed by electrochemical detection. The method enabled more than 0.5 microM Fe(II) and Fe(III) to be determined for injection volumes of 10 microliters. This method was applicable for the determination of Fe(II) and Fe(III) in ultrafiltrates of the rat liver cytosolic fraction. It was found that release of iron from iron-bound proteins was pH dependent and that non-protein-bound iron in the tissues was determined in a ferrous state at low pH values.     

Determination of sialic acids in biological fluids using reversed-phase ion-pair high-performance liquid chromatography

A simple, rapid and sensitive reversed-phase ion-pair high-performance liquid chromatographic method for the determination of N-acetylneuraminic acid and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in biological fluids is described. Determination of N-acetylneuraminic acid released by acidic hydrolysis, in serum, urine and saliva, and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in urine, without hydrolysis, was accomplished by injecting the sample without derivatization, into the chromatograph. Measurements were carried out isocratically within 6 min using a C₁₈ column and a mobile phase of aqueous solution of triisopropanolamine, as ion-pair reagent, 60 mM, pH 3.5 at room temperature with UV absorbance detection. The present method is reported for the first time for the determination of sialic acids in biological fluids. Recoveries in serum, urine and saliva ranged from 90 to 102% and the limits of detection were 60 nM and 20 nM for the two sialic acids, respectively. The method has been applied to normal and pathological sera from patients with breast, stomach, colon, ovarian and cervix cancers, to normal urine and urine from patient with sialuria and to normal saliva.     

Separation of isoenzymatic forms of human serum galactosyltransferase by chromatofocusing and isoelectric focusing. Application to cancerology

Chromatofocusing is used to separate the multiple isoenzyme forms of human serum galactosyltransferase. At least 11 major peaks of activity are observed in normal sera, which are eluted between pH 4.3 and 6.9; a fraction of activity is eluted above pH 7.0. The normal patterns are compared with those obtained with sera from cancer patients and with an ascitic fluid. Chromatofocusing appears as resolutive as agarose isoelectric focusing.     

An assay of dipeptidyl peptidase IV activity in human serum and serum of pregnant women with glycyl-L-proline-1-naphthylamide and other glycyl-L-proline-arylamides as substrates

The authors described a micromethod for measuring dipeptidyl peptidase IV activity in human serum with glycyl-L-proline-1-naphthylamide as substrate. The method requires less than 20 microliters of serum. The pH optimum for cleaving glycyl-L-proline-1-naphthylamine by the enzyme in human serum in Tris-HCl buffer was 8.0 and Km value was established as 7.2 X 10(-4) mol/l. The advantage of this substrate is the absence of spontaneous hydrolysis during the assay of enzyme activity in contrast to glycyl-L-proline-4-nitroanilide. The Km values of the latter substrates and glycyl-L-proline-2-naphthylamide in the same buffer were 1.0 X 10(-4) mol/l and 2.4 X 10(-4) mol/l, respectively. Glycyl-D-proline-4-nitroanilide was not hydrolyzed by the dipeptidyl peptidase IV present in human serum. The activities of dipeptidyl peptidase IV in the sera from 30 healthy human subjects with glycyl-L-proline-1-naphthylamide as substrate were 176.1 +/- 32.8 nkat/l (mean +/- standard deviation; range 100.2-264.1 nkat/l of serum). In this group men had significantly (P less than 0.01) higher activity of the enzyme than women. The cleaving of glycyl-L-proline-1-naphthylamide and glycyl-L-proline-4-nitro anilide by dipeptidyl peptidase IV in human sera was closely correlated (r = 0.86). During normal pregnancy the activity of dipeptidyl peptidase IV in human serum decreases markedly in the first half of pregnancy. After delivery, the serum enzyme activity returns progressively to initial levels.     

ELECTROKINETIC PHENOMENA. III : THE "ISOELECTRIC POINT" OF NORMAL AND SENSITIZED MAMMALIAN ERYTHROCYTES

A survey of the published electrophoretic mobilities of certain mammalian red cells reveals that the isoelectric points accorded to these cells are the result of equilibria incidental to red cell destruction. The electrophoretic mobilities of normal washed sheep and human cells have now been studied in 0.85 per cent NaCl solutions from about pH 3.6 to 7.4. All measurements were made within 2 minutes of the preparation of the suspension of red cells. In no case was reversal of sign of charge observed under these conditions. Reversal of sign of charge occurred only after sufficient time had elapsed to permit sufficient adsorption of the products of red cell destruction. There is little change in mobility as the pH of the medium is decreased. Reversal of sign of charge does occur in the presence of normal and immune (anti-sheep) rabbit sera. The isoelectric point determined under these conditions does not appear to be connected specifically with the immune body but is perhaps associated with phenomena incidental to red cell destruction and the presence of serum. The characteristic lowering of mobility by amboceptor occurs, however, from pH 4.0 to pH 7.4. The curves of mobility plotted against pH for normal and for immune sera support the viewpoint that the identity of the isoelectric points for normal and sensitized sheep cells is not primarily concerned with the immune reaction. It is most unlikely that an "albumin" or a "globulin" surface covers red cells with a complete protein film. Although serum protein reacts with red cells in acid solutions, this is not demonstrable for gelatin. The lowering of mobility usually ascribed to anti-sheep rabbit serum may also occur, but to a lesser degree, in normal rabbit serum. This diminution of mobility is not, in the first place, associated with sensitization to hemolysis induced by complement. This supports the view that only a very small part of the red cell surface need be changed in order to obtain complete hemolysis in the presence of complement.     

Chemiluminescence sandwich enzyme immunoassay for determination of human granulocyte colony stimulating factor (G-CSF)

A chemiluminescence sandwich enzyme immunoassay, using a glucose oxidase (GO) label, was developed for detecting attomole amounts of human granulocyte colony stimulating factor (G-CSF). Purified goat F(ab′)₂ immobilized on a bead and purified goat Fab′ labelled with GO were selected in combination with a chemiluminescent detection system comprising luminol and ferricyanide. The detection limits for G-CSF were 4amol/assay (1 pg/mL) in buffer solution and 10 amol/assay (2.5 pg/mL) in human serum. Coefficients of variation within assay and between assay ranged from 5.5% to 7.8% and from 3.4% to 16.0%, respectively. The G-CSF content of serum from normal healthy individuals was measurable using this method. G-CSF in 24 normal human sera showed a mean value of 19.3 pg/mL and ranged from 3.6 to 83.0 pg/mL.     

Hyaluronidase in sera of tumour-bearing nude mice

Cancer cell lines often secrete hyaluronidase, suggesting that this enzyme could be used as a marker of growing tumours. We have measured hyaluronidase in the sera of non-grafted mice and mice grafted with human tumour-derived hyaluronidase-secreting H460M and SA87 cells or non-secreting CB 193 cells. Mouse serum hyaluronidase was measured at pH 3.8 using the enzyme-linked sorbent assay (ELSA) technique by reference to human serum whose activity at pH 3.8 was determined by the Reissig technique. The serum hyaluronidase in non-grafted mice ranged from 310-520 mU l^?1 (mean±SD 432±70 mU l^?1, median 440 mU l^?1). Hyaluronidase increased in the sera of tumour-bearing mice grafted with H460M cells or with SA87 cells, but not in the sera of mice grafted with CB 193 cells. Serum hyaluronidase activity in H460M or SA87 tumour-bearing mice correlated with the tumour mass, increased with time, and decreased after tumour removal. Zymography detected two different hyaluronidase forms in the sera of non-grafted mice: type 1 had only one hyaluronidase band and type 2 had five different bands. In both types, enzyme augmentation in tumour-bearing mice correlated with the presence of an additional enzyme band that was not seen in normal sera and that migrated as the cancer cell enzyme did; there was no augmentation of the normal isoform(s). These results show that serum hyaluronidase can be used to follow the development of tumours in mice grafted with hyaluronidase-secreting cells.     

Ultrafiltration-light absorption spectrophotometric determination of silicon in blood

An ultrafiltration-light absorption spectrometric method for soluble molybdate-reactive silicon was assessed and applied to bovine and ovine blood plasma and sera, giving precise analytical results. Interfering protein above molecular weight 10,000–25,000 was removed by ultrafiltration, and silicon in ultrafiltrates was quantitated by measuring light absorption at 810 nm of the 1,2,4-aminonaphthol sulfonic acid/ascorbic acid-reduced silicomolybdate. Chemical interferences on the color-forming reaction of remaining blood components were tested by measuring recoveries of silicon added to real blood plasma samples and to synthetic blood plasma solutions, the latter containing typical levels of the major ions Na⁺, K⁺, Ca²⁺, HCO₃^?, and Cl^?, together with varying quantities of the potential interferants (amount per analytical reaction): phosphate (0–0.5 mg P), ferric ion (0–3 mg), fluoride (0–1.25 mg), vanadate (0–0.5 mg V), arsenate (0–10 μg As), and germanate (0–0.5 μg Ge). The mean recovery of added 0.8–9 μg silicon/g of bovine and ovine plasma was 97.7% (SE = 1.0, n = 17); the mean recovery of 1 and 5 μg silicon from synthetic blood plasma solutions with interferant levels up to 50-fold that in normal plasma was 99.2% (SE = 0.3, n = 47). Silicon concentrations found in bovine and ovine blood plasma and sera were typically around 7 μg/ml with procedural reagent blanks consistently low at a mean of 0.12 μg/test (SD = 0.011, n = 20). The silicon level in Center for Disease Control bovine serum (reference specimen Lot R-2274) was found to be (mean ± SE, n = 10) 1.147 ± 0.013 μg/g or 1.172 ± 0.013 μg/ml (25°C). The method detectivity (detection limit) was estimated at 0.03 μg.     

McAbGB2识别的乳腺癌血清抗原性质研究

对抗人乳腺癌血清抗原单克隆抗体GB₂（McAbGB₂）识别的抗原（McAbGB₂抗原）性质及含量进行了研究．结果表明,McAbGB₂抗原是由糖及蛋白质组成的复合蛋白质,不耐热;McAbGB₂抗原决定簇不存在于铁蛋白及癌胚抗原;蛋白质印边检测表明McAbGB₂抗原有两条区带,分子量分别为116及45ku,分布于血液及癌组织;用McAbGB₂进行血清学分析（ELISA试验）,结果表明：乳腺癌阳性符合率达98％（50／51）,正常人及乳腺良性肿瘤病人的假阳性率分别为6％（3／50）及6．7％（1／15）．McAbGB₂抗原可能是新的乳腺癌相关抗原．