Automated multicapillary electrophoresis for analysis of human serum proteins

BACKGROUND: We evaluated a new, automated multicapillary zone electrophoresis (CE) instrument (Capillarys), 4.51 software version; Sebia) for human serum protein analysis. METHODS: With the Capillarys beta1-beta2+ reagent set, proteins were separated at 7 kV for 4 min in 15.5 cm x 25 micro m fused-silica capillaries (n = 8) at 35.5 degrees C in a pH 10 buffer with online detection at 200 nm. Serum samples with different electrophoretic patterns (n = 265) or potential interference (n = 69) were analyzed and compared with agarose gel electrophoresis (AGE; Hydrasys)-Hyrys, Hydragel protein(e) 15/30 reagent set; Sebia). RESULTS: CVs were <3.5% for albumin, <11% for alpha(1)-globulin, <4.1% for alpha(2)-globulin, <7.4% for beta-globulin, and <5.8% for gamma-globulin (3 control levels); measured throughput was 60 samples/h. In patients without paraprotein (n = 116), the median differences between CE and AGE were -5.4 g/L for albumin, 4.0 g/L for alpha(1)-globulin, 0.7 g/L for alpha(2)-globulin, 0.6 g/L for beta-globulin (P <0.001 for all fractions), and -0.1 g/L for gamma-globulin (not significant). More samples had at least one gamma-migrating peak detected by CE (n = 135 vs 130; paraprotein detection limit, approximately 0.5-0.7 g/L), but fewer were quantified (n = 84 vs 91) because of gamma- to beta-migration shifts. There was a 1.2 g/L median difference between CE and AGE for gamma-migrating paraprotein quantification (n = 69; P <0.001). Several ultraviolet-absorbing substances (lipid emulsion, hemoglobin) or molecules (contrast agent, gelatin-based plasma substitute) induced CE artifacts. CONCLUSIONS:The Capillarys instrument is a reliable CE system for serum protein analysis, combining advantages of full automation (ease of use, bar-code identification, computer-assisted correction of alpha(1)-globulins) with high analytical performances and throughput.     

慢性肺心病患者自血紫外线照射治疗后血浆蛋白的变化及其临床意义

５０例慢性肺心病患者接受紫外线照射充氧自血回输治疗（ＵＢＩＯ），另以５０例作对照。治疗前后测定血浆白蛋白、前白蛋白、铜蓝蛋白、转铁蛋白、连株蛋白、α１－抗胰蛋白酶、α１－酸性糖蛋白、Ｃ－反应蛋白、载脂蛋白Ｂ、血浆结合蛋白、α２－巨球蛋白、纤维蛋白溶解酶原。结果表明，ＵＢＩＯ组治疗后前白蛋白、转铁蛋白、纤维结合蛋白明显升高，α１－抗胰蛋白酶、α１－酸性糖蛋白、连株蛋白、铜蓝蛋白、Ｃ－反应蛋白显著降低     

Quantitative immunological determination of 12 plasma proteins excreted in human urine collected before and after exercise

Urine was collected from 6 healthy male adults at rest and from 20 male adults after a marathon race (25 miles). The concentrated urines were quantitatively analyzed, by single radial immunodiffusion, for their content in 12 different plasma proteins: tryptophan-rich prealbumin, albumin, alpha(1)-acid glycoprotein, alpha(1)-antitrypsin, ceruloplasmin, haptoglobin, Gc-globulin, transferrin, hemopexin, beta(2)-glycoprotein I, gammaA-globulin, and gammaG-globulin.Albumin, gammaA-globulin, and gammaG-globulin represent the major part of the plasma proteins detected in normal urine excreted by humans at rest (12, 0.5, and 2.5 mg respectively, out of a total excretion of 17.5 mg of plasma proteins per 24 hr). The other plasma proteins were excreted at a lower rate (< 0.4 mg/24 hr). The relative content of tryptophan-rich prealbumin, alpha(1)-antitrypsin, Gc-globulin, transferrin, and gammaG-globulin was lower in normal urine than in normal serum, whereas that of alpha(1)-acid glycoprotein, beta(2)-glycoprotein I, and gammaA-globulin was higher. The ratio of gammaG-globulin to gammaA-globulin was 4.9:1. When plotted on a logarithmic scale, no direct relationship between the molecular weight of a protein and the value of its renal clearance could be observed.Strenuous exercise increased (up to 50-fold) the excretion of plasma proteins which represent 82% of the total proteins found in urine, instead of 57% in urine collected from humans at rest. There was particularly a significant rise of tryptophan-rich albumin, albumin, alpha(1)-acid glycoprotein, transferrin, gammaA-globulin, and gammaG-globulin (0.26, 127, 11.8, 3.3, 1.2, and 2.0 mug respectively, out of a total excretion of 167 mug of plasma proteins per min). The ratio of gammaG-globulin to gammaA-globulin was 16:1. After exercise, the renal clearance of proteins increased from 2 to 40 times, but, as for the urine of normal subjects at rest, no direct relationship between molecular weight and renal clearance could be observed.     

Plasma protein contents determined by Fourier-transform infrared spectrometry

BACKGROUND: Fourier-transform infrared (FT-IR) spectrometry has been used to measure small molecules in plasma. We wished to extend this use to measurement of plasma proteins. METHODS: We analyzed plasma proteins, glucose, lactate, and urea in 49 blood samples from 35 healthy subjects and 14 patients. For determining the concentration of each biomolecule, the method used the following steps: (a) The biomolecule was sought for which the correlation between spectral range areas of plasma FT-IR spectra and concentrations determined by comparison method was greatest. (b) The IR absorption of the biomolecule at the most characteristic spectral range was calculated by analyzing pure samples of known concentrations. (c) The plasma concentration of the biomolecule was determined using the FT-IR absorption of the pure compound and the integration value obtained for the plasma FT-IR spectra. (d) The spectral contribution of the biomolecule was subtracted from the plasma FT-IR spectra, and the resulting spectra were saved for further analyses. (e) The same method was then applied to determining the concentrations of other biomolecules by sequentially comparing the resulting FT-IR spectra. RESULTS: Results agreed with those obtained by clinical methods for the following biomolecules when analyzed in the following order: albumin, glucose, fibrinogen, IgG(2), lactate, IgG(1), alpha(1)-antitrypsin, alpha(2)-macroglobulin, transferrin, apolipoprotein (Apo)-A(1), urea, Apo-B, IgM, Apo-C(3), IgA, IgG(4), IgG(3), IgD, haptoglobin, and alpha(1)-acid glycoprotein. CONCLUSION: FT-IR spectrometry is a useful tool for determining concentrations of several plasma biomolecules.     

Identification of specific plasma proteins determining the agarose gel electrophoresis by the immunosubtraction technique

The immunosubtraction technique was used to identify the proteins in electrophoretically separated plasma. The technique consists of forcing the plasma through a layer of monospecific antibody purified by electrophoresis; the specific protein is thereby precipitated and is consequently absent from the subsequent electrophoretogram. This technique was successfully applied to the following proteins (in order of distance from the anode): prealbumin, albumin, alpha-lipoproteins, alpha 1-antitrypsin, Gc-globulins, alpha 2-macroglobulin, haptoglobin, fibronectin, transferrin, beta-lipoproteins, C3, fibrinogen. The method may be applied to the study of protein polymorphism.     

Computer-supported interpretation of protein profiles after capillary electrophoresis

BACKGROUND: Electrophoretic patterns of proteins in serum/plasma are useful in the diagnosis and evaluation of many diseases. Capillary zone electrophoresis (CZE) allows rapid and automated protein separation and produces digital absorbance data, appropriate for mathematical analysis. We previously demonstrated success in detection of monoclonal immunoglobulins in such a system. This study tests new algorithms to produce rapid standardized computer-supported interpretation of the entire electropherogram. METHODS: Data from Beckman Paragon CZE 2000 electropherograms were compared with quantitative protein data from >800 routine clinical samples. Algorithms were designed to produce semiquantitative analyses of major proteins and to define different patterns of inflammation based on the electropherogram. RESULTS: The algorithms produced reliable semiquantitative evaluations of prealbumin, albumin, alpha1-antitrypsin, haptoglobin, and transferrin, but were less accurate for alpha1-acid glycoprotein. Some genetic variants of albumin and deficiency variants of alpha1-antitrypsin were easily recognized. Complex clinical traits such as degree and type of inflammation could be evaluated. When used together with previously developed algorithms addressing immunoglobulins, the new algorithms provide relevant clinical interpretation. Selected outputs indicate the need for reflex testing or evaluation by specialists. CONCLUSIONS: Automation of both electrophoresis and interpretation can provide a rapid, inexpensive, standardized analysis that can hopefully improve the diagnostic information and clinical outcome for large groups of patients. It also provides objective criteria for clinical interpretations, to be validated or adjusted in future clinical studies.     

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.     

Automated serum protein electrophoresis by Capillarys.

Capillary zone electrophoresis (CZE) of serum proteins is increasingly gaining impact in clinical laboratories. In this report, we evaluate automated capillary zone electrophoresis by Capillarys (Sebia, France). Within-run and between-run imprecision for the five electrophoretic fractions was <2% and <6%, respectively. Data obtained with Capillarys correlated with results obtained with agarose gel electrophoresis and Paragon CZE 2000 (Beckman Coulter, USA). Analysis of serum obtained from patients with inflammation, nephrotic syndrome, bisalbuminemia, and alpha1-antitrypsin deficiency revealed that Capillarys was able to detect these abnormalities. Two hundred thirty eight samples were analyzed by agarose gel electrophoresis, Capillarys, capillary electrophoresis using Paragon CZE 2000 system, and immunofixation. Sample selection was based on the presence of a disturbed morphology (e.g., spike) of the protein profile or hypogammaglobulinemia on agarose gel electrophoresis and/or Capillarys. Immunofixation revealed the presence of a monoclonal protein, oligoclonal bands, polyclonal pattern, and a normal profile in, respectively, 89, 66, 19, and 64 samples. With Capillarys, Paragon, and agarose gel electrophoresis, a spike and/or disturbed morphology of the profile was found in 222, 182, and 180 samples, respectively. In these samples, immunofixation was negative in 73 (33%), 46 (25%), and 39 (22%) samples, respectively. These data indicate that Capillarys has a lower specificity than agarose gel electrophoresis and Paragon 2000. Of the 89 samples with a monoclonal protein, Capillarys, Paragon, and agarose gel electrophoresis failed to detect, respectively, three, three, and one monoclonal protein(s). Interferences by radio-opaque agents, complement degradation products, fibrinogen, and triglycerides are described. In conclusion, automated capillary zone electrophoresis with Capillarys provides for reproducible, rapid, and reliable serum electrophoresis.     

Acute-phase proteins from the liver and enzymes from myocardial infarction; a quantitative relationship.

In 14 patients with acute myocardial infarction (M.I.) not having any other disease, the possible quantitative relationship between enzymes from M.I. and changes in concentration of acute phase reactants coming from the liver were studied. The patients were followed up until 1 1/2 years after M.I. and comparison of baseline-protein values took place using a control group of 18 healthy individuals. Quantitation of protein changes was done by planimetric determination of the area under the concentration curve and by taking peak values. The myocardial infarction was quantitatively estimated by mathematical analysis of the time course of alpha-hydroxybutyrate dehydrogenase (alpha-HBDH) plasma concentrations and by taking peak values. A quantitative relationship with enzymatic infarct size was found for haptoglobin, alpha1-acid glycoprotein, alpha1-antitrypsin, C-reactive protein, fibrinogen and E.S.R. Albumin and transferrin did not show a negative quantitative relationship with enzymatic infarct size. Humoral factors originating from the site of tissue injury and evoking in proportion a positive acute phase reaction by the liver are probably the basis for this observed quantitative relationship.     

Reduction of hepatic acute phase response after partial hepatectomy in elderly patients

The hepatic capacity for acute phase protein synthesis after partial hepatectomy in the elderly patients was prospectively studied. Forty-one patients who consecutively underwent a partial hepatectomy were grouped according to age of greater or less than 70 years; 12 were in the older group and 29 in the younger. The changes in the levels of serum interleukin-6,α ₁-antitrypsin,α ₁-acid glycoprotein, haptoglobin, and plasma fibrinogen were measured after surgery. The postoperative changes in standard liver function tests were also measured. The incidence of postoperative infected complications was 25% in the older group and 7% in the younger (P=0.28). Although postoperative levels of serum interleukin-6 were similar between the two groups, those of serumα ₁-antitrypsin,α ₁-antitrypsin glycoprotein, and haptoglobin were significantly lower in the elderly (P<0.05). Postoperative levels of serumα ₁-antitrypsin and plasma fibrinogen showed an increase of about 30% compared with the preoperative values (P<0.05) in the younger group, but no significant increasein the older. Postoperative deterioration of serum albumin levels and hepaplastin test values was also significantly more severe in the older group (P<0.05). We conclude that in the older patients, a reduction of acute phase protein synthesis occurs after partial hepatectomy as a result of a global deterioration of liver function, and may render patients liable to infection.     

Characterization of plasma proteome in biliary atresia

BACKGROUND: Biliary atresia (BA) is a disorder during infancy with unknown etiology in which progression frequently leads to liver cirrhosis. Plasma proteome is characterized in this study. METHODS: Twelve paired plasma samples from 6 children with BA who received surgical correction at early stage and then liver transplantation at late stage of liver cirrhosis were studied. Plasma samples from 2 subjects without liver disorder were used as normal reference for 2-dimensional gel electrophoresis and for identification of protein spots by mass spectrometric analysis. Plasma samples from another 3 normal subjects (with a total of 5) were used for nephelometric quantification of immunoglobulin kappa light chain in comparison with patients' samples. RESULTS: Among the protein spots detected, ranging from 6 to 200 kDa mass with pIs of 3-10, significant up-regulation of immunoglobulin kappa light chain was found at the late stage of BA, which was subsequently confirmed by nephelometric analysis. Conversely, significant decrease of apolipoprotein (Apo) A-I and C-II, haptoglobin alpha2 and beta chain, and transthyretin were detected during the progression of BA. CONCLUSIONS: Increased immunoglobulin kappa light chain detected in late-stage BA characterizes adverse immune modulation in this disorder. Decreased apolipoproteins, haptoglobin and transthyretin levels might be potential markers of progressive liver injury, fibrosis and defective lipid metabolism in BA.     

Luminometric assays of seven acute-phase proteins in minimal volumes of serum, plasma, sputum, and bronchioalveolar lavage

We describe immunoluminometric assays for seven acute-phase proteins, which can be determined in minimal volumes of plasma, serum, sputum, and bronchioalveolar lavage. The theoretical volume of serum or plasma required to measure all seven analytes in duplicate is 130 nL, although in practice the smallest volume of sample was enough to fill a hematocrit tube (about 25 microL of blood), collected from neonates by the heel-prick method. The assays could be performed with 10 microL of sputum or with 100 microL of bronchioalveolar lavage. We measured alpha 1-antitrypsin, alpha 2-macroglobulin, alpha 1-acid glycoprotein, thyroxin-binding prealbumin, C-reactive protein, and total and secretory immunoglobulin A. The assays are rapid enough for all results to be returned to the ward on the same day and are suitable for monitoring neonatal sepsis. All coefficients of variation, derived from compound precision profiles, were less than 7% for clinically relevant analyte concentrations. Correlation with commercially available nephelometric assays was good.     

Magnetic resonance spectroscopy of serum and acute-phase proteins revisited: a multiparametric statistical analysis of metabolite variations in inflammatory, infectious and miscellaneous diseases

Proton MR spectra and biochemical assays have been recorded on the sera of 40 patients and ten controls in order to document the correlation between spectroscopic and biochemical variations in selected pathologies (cancer, inflammatory and infectious diseases, diabetes). N-acetyl proton resonances are essentially generated by the N-acetyl residues of the glucidic moieties borne by the most abundant acute-phase proteins (alpha1-acid glycoprotein, alpha1-antitrypsin and haptoglobin). These resonances are not correlated to immunoglobulins A, G and M levels. Principal component analysis shows that variations in spectroscopic and biochemical data are independent markers of the inflammatory status of patients but no additional sensitivity or specificity is obtained when the two sets of data are combined.     

Protein binding of disopyramide in liver cirrhosis and in nephrotic syndrome

The plasma protein binding of disopyramide (D) was determined in seven patients with cirrhosis, six with nephrotic syndrome, and seven healthy subjects. Plasma samples containing concentrations of 0.2 to 12.0 micrograms/ml were ultrafiltered and the free fractions were measured with fluorescence polarization immunoassay. The mean free fractions at D concentrations ranging from 1 to 6 micrograms/ml were significantly (P less than 0.01) greater in patients with cirrhosis than in healthy subjects. No difference was observed between patients with nephrotic syndrome and healthy subjects. The free fraction at D 3 micrograms/ml correlated better with alpha 1-acid glycoprotein (r = -0.77) than with albumin (r = -0.46). Patients with cirrhosis had significantly (P less than 0.01) lower capacity constants as compared with the other two study groups. There was a significant (P less than 0.01) correlation between capacity constant and alpha 1-acid glycoprotein (r = 0.71). Our results suggest that the D therapeutic range measured as the total plasma concentration in cirrhosis, but not in nephrosis, should be approximately 50% lower than previously believed.     

The relationship between the erythrocyte sedimentation rate (ESR) and plasma proteins in clinical materials and models

The relationship between the erythrocyte sedimentation rate (ESR) and plasma proteins was studied within homogenous clinical material and in vitro models. In acute phase reactions, fibrinogen was the likely cause of the ESR-elevation, but there were significant associations between the ESR and the concentrations of alpha 1-antitrypsin, C3, haptoglobin and albumin. In chronic diseases, the ESR-elevation was probably caused by fibrinogen, mono- or polyclonal increase of IgG, IgA, IgM alone or in combinations. In multiple myeloma of the IgG and IgA subtypes, significant correlations were found between the ESR and the monoclonal proteins or between the ESR and the percentage of plasma cells in bone marrow. Model studies showed that the ESR increased linearly with the concentrations of fibrinogen or gammaglobulin (IgG) when these exceeded normal thresholds. The ESR was slightly decreased by increasing concentrations of albumin. Albumin had a synergistic effect on the ESR together with gamma-globulin, but not together with fibrinogen.     

Protein markers in evaluation of nephroprotective effects of antihypertensive drugs in patients with arterial hypertension

Fifty patients with stable slight and moderate uncomplicated essential hypertension, treated by ramipril, atenolol, or isradipine, were examined. Total protein and urinary excretion of individual proteins were studied before and after treatment. Urinary concentrations of apolipoproteins A1 and B1, alpha 1-acid glycoprotein, alpha 1-antitrypsin, prealbumin, albumin, beta 2-microglobulin, transferrin, haptoglobin, IgG and IgA, and C3 and C4 complement components were measured. Index of proteinuria selectiveness was calculated for each portion of urine. All three drugs exerted a nephroprotective effect, atenolol being the most active of them. Apolipoproteins, IgG, and complement components were the most valuable for diagnosis. Their excretion correlated with the severity of arterial hypertension and efficiency of treatment. Use of protein markers helps reliably assess the renal function and monitor the treatment efficiency.     

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.     

Analysis of HbA1c on an automated multicapillary zone electrophoresis system

Hemoglobin A1c (HbA1c) is a frequently requested laboratory test and there is thus a need for high throughput instruments for this assay. We evaluated a new automated multicapillary zone electrophoresis instrument (Capillarys 3 Tera, Sebia, Lisses, France) for analysis of HbA1c in venous samples. Routine requested HbA1c samples were analyzed immunologically on a Roche c6000 instrument (n?=?142) and then with the Capillarys 3 Tera instrument. The Capillarys 3 Tera instrument performed approximately 70 HbA1c tests/hour. There was a strong linear correlation between Capillarys 3 Tera and Roche Tina-Quant HbA1c Gen 3 assay (y?=?1.003x – 0.3246 R^2?=?.996). The total CV for the 12 capillaries varied between 0.8 and 2.2% and there was a good agreement between duplicate samples (R^2?=?.997). In conclusion, the Capillarys 3 Tera instrument has a high assay capacity for HbA1c. It has a good precision and agreement with the Roche Tina-Quant HbA1c method and is well suited for high volume testing of HbA1c.     

Leucocyte-associated plasma proteins. Association of prealbumin, albumin, orosomucoid, alpha 1-antitrypsin, transferrin and haptoglobin with human lymphocytes, monocytes, granulocytes and a promyelocytic leukaemic cell line (HL-60)

The distribution of prealbumin, albumin, orosomucoid, alpha 1-antitrypsin, haptoglobin and transferrin, including their electrophoretic heterogeneous variants, was studied in isolated lymphocytes, monocytes and granulocytes and in a human promyelocytic cell line (HL-60) by crossed immunoelectrophoresis. Prealbumin, albumin and transferrin were present in lymphocytes, monocytes and granulocytes, whereas the cellular variants of orosomucoid and haptoglobin were present only in granulocytes. alpha 1-Antitrypsin was present in four electrophoretic variants which were differently distributed among the various cell types. Synthesis of alpha 1-antitrypsin by monocytes, granulocytes and HL-60 cells was demonstrated by 14C-leucine incorporation. The six plasma proteins could not be removed from intact cells by incubation with the respective antibodies at 0 degrees C, or iodinated by lactoperoxidase catalysed iodination at 23 degrees C. They were, however, readily solubilized by freeze-lysis of the cells, suggesting an intracellular localization. Compared to their plasma counterparts none of the proteins differed in their hydrophobic properties but the carbohydrate residues of orosomucoid, alpha 1-antitrypsin and haptoglobin were different. The pattern of disappearance of the proteins from the cells during incubation suggested that the localization of albumin and transferrin in relation to the cells differed from that of the other proteins.     

Response of plasma lipoproteins and acute phase proteins to myocardial infarction.

Plasma concentrations of lipoprotein-lipids, apolipoprotein B (apoB) and of seven other proteins have been estimated serially in 27 patients up to three months following myocardial infarction. Results were compared with those from age- and sex-matched control subjects. At three months the mean total, low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol concentrations were higher than those of the control subjects, whereas very low density lipoprotein (VLDL) cholesterol, total and VLDL triglyceride, and total and LDL apolipoprotein B concentrations were not significantly different. Relative to concentrations at three months total and LDL cholesterol and apolipoprotein B concentrations fell markedly, and a slight fall occurred in HDL cholesterol following infarction. VLDL cholesterol and total and VLDL triglyceride were decreased only on day one. Albumin and transferrin concentrations were higher and alpha 1-acid glycoprotein was lower at three months than in the control subjects; alpha 2-macroglobulin, caeruloplasmin, haptoglobin and immunoglobulin IgM were not significantly different. Following infarction albumin and transferrin fell, alpha 2-macroglobulin did not change, and alpha 1-acid glycoprotein, caeruloplasmin, haptoglobin and IgM rose. The changes in both lipids and protein are probably part of the general metabolic response to trauma.