Effects of posture on concentrations of serum proteins in healthy adults. Dependence on the molecular size of proteins

The effect of posture on changes in protein composition of serum and its dependence on molecular size has been investigated in forty individuals classified by age and sex. Blood specimens were drawn (A) at 0700 hours with persons still resting in bed after an overnight sleep; (B) at 0815 hours, in sitting position after 1 h of moderate exercise and again (C) at 0915 hours, lying in bed after 1 h of recumbency. Concentrations of alpha 1-antitrypsin, albumin and alpha 2-macroglobulin in serum were determined with high analytical precision. The mean fractional increases of concentrations from lying (A) to sitting (B) were 0.069 for alpha 1-antitrypsin and 0.063 for albumin, but lower, 0.040, for alpha 2-macroglobulin. Thus these increases, being related to molecular size, were not simply caused by posture induced changes of plasma volume. Considering the results of each individual we found a constant difference between the increase of alpha 1-antitrypsin and alpha 2-macroglobulin of about 0.030 for the whole range of increases (i.e. for alpha 1-antitrypsin 0-0.15). In contrast, the effects of recumbency were independent of molecular size. The mean, fractional decreases in concentration induced by changing of position from sitting (B) to lying (C) were approximately 0.065 for all three proteins.     

alpha 2-Macroglobulin in patients with obstructive lung disease, with and without alpha 1-antitrypsin deficiency

Serum alpha 2-macroglobulin concentrations were measured in 178 patients with emphysema and 115 control subjects of similar age and sex distribution. The study group included 59 PI type Z patients with alpha 1-antitrypsin deficiency, five with the rare alpha 1-antitrypsin null genotype (PI Q0 or --), and seven with alpha 1-antitrypsin deficiency of the rare PI types MmaltonZ or MduarteZ. Individuals with all types of alpha 1-antitrypsin deficiency were found to have significantly increased serum concentrations of alpha 2M (p less than 0.001). These increased concentrations were associated with all types of alpha 1-antitrypsin deficiency, not only with the PI type Z. The highest alpha 2-macroglobulin concentrations were found in the PI Q0 patients (5 with emphysema, 2 with no lung disease), and these patients had almost no circulating alpha 1-antitrypsin. Raised concentrations of serum alpha 2-macroglobulin were not due to emphysema: 86 patients with emphysema, of PI type M, and the normal control subjects had similar average concentrations of alpha 2-macroglobulin. One control subject with an average alpha 2-macroglobulin concentration of only 41% of normal was found.     

Biologic and analytic components of variation of concentration values of selected serum proteins.

The magnitude and statistical significance of the various biologic and analytic sources of variation of the serum concentration values of seven proteins: albumin, alpha1-antitrypsin, transferrin, orosomucoid, IgG, IgM, and IgA, were evaluated in 12 healthy subjects. Blood specimens were obtained at 0800 h, 1100 h, and 1400 h on one particular day. The analytic error was partitioned into pre-instrumental and instrumental components; the biologic variation was separated into inter-subject variation of mean levels and intra-individual within-day variation. The mean levels of the subjects differed significantly for all seven proteins studied. For the group as a whole there were no consistent diurnal variations of significance. However, for all the proteins, except for IgM, significant individual within-day biologic fluctuations were noted. The within-day biologic variation was smaller than the total analytic variation for transferrin, IgG, orosomucoid, and IgM. When the total analytic variation was separated into pre-instrumental and instrumental components, it was found that the pre-instrumental sources of variation were of a relatively greater magnitude for albumin, IgG, IgA, alpha1-antitrypsin, and IgM.     

Synthesis and secretion of α2-macroglobulin by human hepatocytes in culture

Hepatocytes were isolated by application of the two-step collagenase perfusion technique to pieces of human liver. The cells were incubated in serum-free medium or 10% FCS-medium supplemented with insulin, glucagon and dexamethasone, and kept in culture for more than 2 weeks. Seventy-five per cent of the medium was changed regularly and assayed for alpha 2-macroglobulin (alpha 2-M), pregnancy zone protein, alpha 1-antitrypsin and albumin by means of ELISA. Significant amounts of alpha 2-macroglobulin were present in all cultures. During incubation, alpha 2-M accumulated in the medium and the quantity of alpha 2-M released from the cells by far exceeded protein associated with hepatocytes prior to incubation. In 24 h 10(6) hepatocytes secreted 160.5 +/- 82.2 ng of alpha 2-M (mean +/- SD, n = 5). Cell-associated, as well as secreted alpha 2-M appeared to be on native form, as determined by immunoisolates from lysed cells and culture supernatants. Pregnancy zone protein was only detected in about 50% of the cultures and its rate of secretion was less than 2 ng 24 h-1 per 10(6) cells. In contrast, culture medium contained considerable quantities of alpha 1-antitrypsin and albumin. In 24 h, 10(6) hepatocytes released greater than 2 micrograms alpha 1-antitrypsin and greater than 5 micrograms albumin. The present study suggests the hepatocyte to be of major importance for the synthesis of intravascular alpha 2-M.     

Cationic proteins of human granulocytes. V. Interaction with plasma protease inhibitors.

Several cationic proteins of human granulocytes possess chymotrypsin-like and bactericidal activities. The heat-labile chymotrypsin-like activity is inhibited by serum, owing to complex formation with alpha2-macroglobulin and alpha1-antitrypsin. The molar affinity of the cationic proteins for alpha2-macroglobulin is much higher than that for alpha1-antitrypsin. The results indicate that the molar combining ratios are 1:1 for cationic protein to alpha1-antitrypsin and 2:1 for cationic protein to alpha2-macroglobulin. The proteolytic activity against fibrinogen and casein is inhibited by both alpha2-macroglobulin and alpha1-antitrypsin, whereas the activity against small molecular synthetic substrates is inhibited by alpha1-antitrypsin but not alpha2-macroglobulin. The heat-stable bactericidal action of the cationic proteins against Staphylococcus was also inhibited by serum, probably owing to complex formation with alpha2-macroglobulin and alpha1-antitrypsin.     

Concentrations of protease and anti-protease in serum of patients with pancreatic cancer

We measured the concentrations of trypsin, elastase, and three anti-proteases-alpha 1-macroglobulin, alpha 1-antitrypsin, and alpha 1-antichymotrypsin-in serum from 10 patients with pancreatic carcinoma. All 10 showed increased elastase and decreased alpha 2-macroglobulin concentrations, nine had increased alpha 1-antichymotrypsin, and eight had increases in alpha 1-antitrypsin and trypsin. Serial studies during chemotherapy of one patient showed that the protease concentrations decreased during treatment but the concentrations of the anti-proteases remained abnormal.     

Glycated proteins in serum: effect of their relative proportions on their alkaline reducing activity in the fructosamine test

The fructosamine test is considered clinically useful for assessing short-term integrated control of blood glucose, but there are few published data to support this hypothesis. We fractionated glycated and nonglycated proteins by affinity chromatography on phenylboronate columns and, with specific immunochemical methods, determined in the eluted fractions the following proteins, selected according to their biological half-lives and relative concentrations in serum: albumin, IgA, IgG, IgM, apolipoprotein B, haptoglobin, transferrin, alpha 1-antitrypsin, and alpha 2-macroglobulin. We found the following correlations between fructosamine (mmol/L) and, respectively, glycated albumin, IgG, and (albumin + IgG) (each in grams per liter): r = 0.901, 0.702, 0.878. IgM had the highest percentage of glycated molecules (range 11.1-37.5%, mean 22.4%), haptoglobin and alpha 1-antitrypsin the least. This result was almost independent of the proteins' molecular masses and fractional catabolic rate. Albumin evidently contributes most to results of the fructosamine test, confirming conclusions obtained in different ways by others.     

Immunoreactive plasma cholinesterase (EC 3.1.1.8) substance concentration, compared with cholinesterase activity concentration and albumin: inter- and intra-individual variations in a healthy population group

Substance concentrations of plasma cholinesterase (EC 3.1.1.8) were measured in 94 healthy individuals without occupational exposure to known inhibitors (six samples from each individual). Immunoreactive cholinesterase substance concentrations showed an inter-individual variation corresponding to CVtotal = 22% (mean: 5.01 mg/l, SD: 1.11 mg/l). Intra-individual variations of immunoreactive cholinesterase substance concentration were correlated (r = 0.36) to intra-individual variation of albumin. Estimated by a repeated-measures analysis of variance, the observed intra-individual variation of cholinesterase substance concentration corresponded to CV = 8.8% (SD: 0.44 mg/l), which together with a CVerror = 6% (within and between runs), implies a biological intra-individual variation of cholinesterase substance concentration corresponding to CVintra = 6.4%. Specific catalytic activity (kU/mg immunoreactive cholinesterase) was influenced by the ChE-1 phenotype (phenotype U: 1.58 kU/mg, phenotype UA: 1.22 kU/mg), but not by body weight, height, age, and sex. Observed intra-individual variation of specific catalytic activity corresponded to 6.4% (SD: 0.10 kU/mg), which together with an estimated CVerror = 6.2% implies the biological intra-individual variations of specific catalytic cholinesterase activity to be insignificant. The insignificant CVintra makes specific catalytic cholinesterase activity a rational quantity for evaluation of unexpected fluctuations of cholinesterase activity concentrations.     

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.     

Neutral proteases of human granulocytes. IV. Interaction between human granulocyte collagenase and plasma protease inhibitors.

Purified human granulocyte collagenase was inactivated by serum through the formation of complexes with alpha 1-antitrypsin and alpha 2-macroglobulin. A molar combining ratio of 1:1 was observed for each inhibitor. The affinity of alpha 2-macroglobulin was about 10 times that of alpha 1-antitrypsin for granulocyte collagenase. The molar concentration of alpha 1-antitrypsin in the blood exceeds that of alpha 2-macroglobulin by about 12 times, so that the inhibitors may be equally important for defence against granulocyte collagenase.     

Amidolytic and immuno-nephelometric determination of alpha 1-proteinase inhibitor and alpha 2-macroglobulin in serum with calculation of specific inhibitor activities in health and disease

In sera of healthy persons (n = 50) and patients with a variety of diseases (n = 197) the two major proteinase inhibitors, alpha 1-proteinase inhibitor (alpha 1-antitrypsin) and alpha 2-macroglobulin, were measured by two methods: a chromogenic (amidolytic) substrate assay to assess the functional activities, and a laser nephelometric method to determine the immunoreactive concentrations of the respective proteins. The specific proteinase inhibitor activities defined as the number of inhibitor units per g inhibitor protein were calculated. The precision and accuracy of both assays were found to be similar, showing a satisfactory correlation of results for the sera of healthy persons (r = 0.916 for alpha 2-macroglobulin and 0.988 for alpha 1-proteinase inhibitor). In diseased individuals the correlation was lower than in normal persons (0.862 for alpha 2-macroglobulin and 0.907 for alpha 1-proteinase inhibitor). A poor correlation was obtained in patients with liver diseases (r = 0.586 for alpha 1-proteinase inhibitor and 0.852 for alpha 2-macroglobulin). Reference ranges were established for functional and immunological concentrations and for specific inhibitor activities, respectively. Normal values followed a Gaussian distribution. In patients with various diseases including those with acute phase response, the specific inhibitor activities of alpha 1-proteinase inhibitor are reduced significantly; this is because inhibitor activity shows a smaller relative increase than immunoreactivity. Among the various diseases, no significant differences were noted. The specific inhibitor activity of alpha 2-macroglobulin changed significantly only in patients with carcinoma, liver diseases and trauma. Follow up of some patients shows also intraindividual variation of specific proteinase inhibitor activities.     

Urinary plasma protein patterns in acute prostatitis.

We evaluated the diagnostic utility of urinary alpha1-microglobulin, alpha2-macroglobulin and albumin in the diagnosis of acute prostatitis. We studied 133 men (43 +/- 17 years) with, and a reference population (n=36, 41 +/- 16 years) without, urinary tract infection. Prostatectomy samples were used to study the potential interference between prostatic proteins and protein analysis. Urinary alpha2-macroglobulin/albumin ratio was significantly lower in prostatitis compared to the reference population, cystitis or acute pyelonephritis (p < 0.0001). Low alpha2-macroglobulin concentrations in prostatitis are due to inhibition (p = 0.0001) of the immune reaction between alpha2-macroglobulin in presence of polyclonal rabbit antibodies (used for immunonephelometry) by soluble prostatic proteins (+/- 60 kDa) which appear in urine in acute prostatitis. The urinary alpha1-microglobulin/creatinine ratio diagnoses acute pyelonephritis (sensitivity 100% and specificity 87%) and the urinary alpha2-macroglobulin/albumin ratio diagnoses acute prostatitis (sensitivity 100% and specificity of 90%). Stepwise multinomial logistic regression analysis reveals that urinary alpha1-microglobulin, alpha2-macroglobulin, albumin and creatinine provide optimal differentiation between acute pyelonephritis and acute prostatitis (pseudo R2=0.83; Loglikelihood -30.55, p < 0.000001). In conclusion, the combination of hematuria and absence of urinary alpha-2-macroglobulin is diagnostic for acute prostatitis. Even without hematuria, alpha2-macroglobulin remains lower compared to patients without prostatitis.     

Course and blood coagulation findings following systemic short-term fibrinolysis in acute myocardial infarct

A series of 16 consecutive patients with acute myocardial infarction was investigated with respect to changes in coagulatory parameters after intravenous short-term treatment with 1,500 000 IU streptokinase (SK) over a period of 90 minutes. Samples for coagulation assays (fibrinogen, thrombin, time activated partial thromboplastin time (aPTT), normotest, thrombin-coagulase time, platelets, antithrombin III, plasminogen and antiplasmin activity, alpha 2-macroglobulin, alpha 1-antitrypsin, factor X a were collected before and immediately after iv SK, and after 4, 8, 12, 24, 36, 48 and 72 hours. Platelets, antithrombin III, factor X a, alpha 1-antitrypsin and alpha 2-macroglobulin showed no changes over the observed period. The concentrations of fibrinogen and the activities of plasminogen and antiplasmin decreased clearly during the first 24 hours, reaching a minimum immediately after SK administration. Thrombin time and aPTT were prolonged for 36 hours, with a maximum in the first hours after lysis. Conclusions: Invasive diagnostic and/or therapeutic procedures during the first 24 hours after SK lysis should be carried out only for a definite, strict indication and under repeated control of the coagulatory status. After 24-36 hours there is a trend to normalisation of haemostasis. After 36 hours, surgery may be performed without fear of complications due to abnormal coagulability.     

Sequential changes in the concentration of specific serum proteins during typhoid fever infection in man.

An automated immunoprecipitin system has been utilized to quantitate the concentration of 10 specific proteins in the plasma of man. Values obtained by this technique are in agreement with the published concentrations for these specific plasma proteins. This technique was utilized to determine the sequential change s in 10 individual plasma proteins of volunteers exposed to Salmonella typhi. In those volunteers who developed typical typhoid fever, plasma concentrations of the acute phase proteins, alpha1-acid glycoprotein, alpha1-antitrypsin, and haptoglobin, as well as C3 complement were significantly increased with the onset of febrile illness. In contrast, the concentration of plasma albumin and tranferrin were depressed while plasma IgM became elevated during early convalescence from this infection. No significant changes were observed in the plasma concentrations of alpha2-macroglobulin, IgG, or IgA. In the exposed volunteers who did not become ill, the only significant change was a brief depression of alpha1-antitrypsin. During typhoid fever the patterns of change for individual plasma acute-phase globulins were different from those reported for patients with hepatitis, myocaridal infarction, or surgery.     

Factors contributing to intra-individual variation of serum constituents: Physiological day-to-day variation in concentrations of 10 specific proteins in sera of healthy subjects.

Using an automated immunoprecipitin method, we assayed human sera for 10 proteins: haptoglobin, orosomucoid, transferrin, alpha1 antitrypsin, alpha2-macroglobulin, IgG, IGa, IgM, complement C3, and complement C4. Blood from 14 healthy subjects (25-40y) was sampled on six separate days. From each venipuncture serum was divided into four eliquots; two were assayed on the day of venipuncture and two were frozen and kept until the end of the study, when all of the frozen samples were analyzed in one batch. With this experimental design, batch-to-batch analytical variation could be estimated, and we avoided confounding it with the biological variation. Data analysis was based on the analysis of variance technique. The average physiological intra-individual coefficient of variation ranged from 2.5% for transferrin to 11.1% for orosomucoid. THe interindividual variation ranged from 9.5% for transferrin to 70.5% for haptoglobin and the ratio between intra-individual variation and interindividual variation ranged from 0.66 for IgM to 0.26 for orosomucoid and transferrin.     

The acute phase response and its relation to amyloid A degrading activity in serum of patients with rheumatoid arthritis undergoing arthroplasty

The sequential changes in the concentration of specific serum proteins and their relation to amyloid A degrading activity were studied in ten patients with rheumatoid arthritis undergoing arthroplasty of the knee or hip. Serum amyloid A protein increased from a preoperative level of 78 +/- 20 gm/l (mean +/- SEM) to a peak level of 623 +/- 93 mg/l on the third postoperative day (P less than 0.001). The serum amyloid A protein response was greater than that of any other protein including C-reactive protein, to which it was closely related (r = 0.84, P less than 0.001). The concentrations of alpha 1-antitrypsin and alpha 1-antichymotrypsin were highest on the fourth postoperative day (mean changes + 35%, P less than 0.01, and +44%, P less than 0.05, respectively). Serum albumin, pre-albumin and alpha 2-macroglobulin behaved like negative acute phase reactants; the concentrations of albumin and alpha 2-macroglobulin were significantly decreased from the second to sixth and seventh postoperative days, respectively, and the concentration of pre-albumin was significantly decreased on the third and fourth postoperative days. A significant fall in the amyloid A degrading activity of serum occurred during the acute phase reaction. The degradative activity was lowest on the third and fourth postoperative days (P less than 0.001). The results show that the acute phase state in patients with rheumatoid arthritis induces a rise in the concentration of serum amyloid A protein, the putative serum precursor of tissue amyloid A fibrils, and a concomitant reduction in the ability of serum to degrade these fibrils. These factors together may be important in the development of inflammation-associated amyloidosis.     

Biochemical modifications in a case of analbuminemia (author's transl)]

A new case of analbuminemia is described for a six month old child of Algerian origin. The serum albumin concentration was 64 mg/l and its immunochemical action was identical to that of normal albumin. The system reacted by an increase of the synthesis of globulins. For the subject, the alpha1-antitrypsin, ceruloplasmin, haptoglobin, alpha2-macroglobulin, transferrin and immunoglobulins M contents were three times higher than the standard figures. However, it was possible to show that the presence of free bilirubin independent from proteins could be detected at a concentration of 17 mumol/l.     

Intra-individual variation of some analytes in serum of patients with insulin-dependent diabetes mellitus

Biological intra-individual variation in concentrations of 16 clinical biochemical analytes in serum was estimated for 27 patients with insulin-dependent diabetes mellitus (IDDM), and results were compared with those for apparently healthy individuals. In general, the variation was significantly higher in the patients. The ratio of the average intra-individual variation in IDDM patients to that in normal subjects exceeded 2.0 for Na+, K+, creatinine, and alpha-amylase; 1.50 to 2.0 for Cl-, total protein, albumin, cholesterol, and hemoglobin; and 1.2 to 1.5 for urea, uric acid, high-density-lipoprotein cholesterol, and aspartate aminotransferase. This increased variability in IDDM patients may be caused by variations in osmotic diuresis. Average intra-individual variations were greater for women than for men for Na+, total protein, albumin, and hemoglobin. Individual values showed a gaussian distribution for all analytes, including enzymes and triglycerides. No intra-individual variation was time dependent. For practical purposes, decision-making criteria in monitoring IDDM can be derived from the estimated biological component of intra-individual variation and the analytical variation established for each laboratory.     

Plasma Kallikrein Activation and Inhibition during Typhoid Fever

As an ancillary part of a typhoid fever vaccine study, 10 healthy adult male volunteers (nonimmunized controls) were serially bled 6 days before to 30 days after ingesting 10(5)Salmonella typhi organisms. Five persons developed typhoid fever 6-10 days after challenge, while five remained well. During the febrile illness, significant changes (P < 0.05) in the following hematological parameters were measured: a rise in alpha(1)-antitrypsin antigen concentration and high molecular weight kininogen clotting activity; a progressive decrease of platelet count (to 60% of the predisease state), functional prekallikrein (55%) and kallikrein inhibitor (47%) with a nadir reached on day 5 of the fever and a subsequent overshoot during convalescence. Despite the drop in functional prekallikrein and kallikrein inhibitor, there was no change in factor XII clotting activity or antigenic concentrations of prekallikrein and the kallikrein inhibitors, C1 esterase inhibitor (C1-INH) and alpha(2)-macroglobulin. Plasma from febrile patients subjected to immunoelectrophoresis and crossed immunoelectrophoresis contained a new complex displaying antigenic characteristics of both prekallikrein and C1-INH; the alpha(2)-macroglobulin, antithrombin III, and alpha(1)-antitrypsin immunoprecipitates were unchanged. Plasma drawn from infected-well subjects showed no significant change in these components of the kinin generating system. The finding of a reduction in functional prekallikrein and kallikrein inhibitor (C1-INH) and the formation of a kallikrein C1-INH complex is consistent with prekallikrein activation in typhoid fever. The correlation of these changes with the drop in platelet count suggests that a common mechanism may be responsible.     

Howie's antithrombin activity and thrombin inhibitors in the nephrotic syndrome

A prospective study was carried out on a group of 28 patients affected by nephrotic syndrome in order to compare the antithrombin activity, measured by the technique of Howie, the antithrombin III, measured with chromogenic substrate and by radial immunodiffusion, the alpha 2-macroglobulin and the alpha 1-antitrypsin. An increased level of alpha 2-macroglobulin and of the antithrombin activity, measured by the technique of Howie and a reduction of the level of antithrombin III and of alpha 1-antitrypsin was observed. It is suggested that the increased antithrombin activity is related to the increase of alpha 1-antitrypsin was observed. It is suggested that the increased antithrombin activity is related to the increase of alpha 2-macroglobulin concentration in spite of the simultaneous decrease of antithrombin III.