New approach to evaluation of proteinuric states.

We evaluated the use of immunonephelometric methods for measuring specific urinary proteins. Using a nephelometer to detect light scattering (angle, 31 degrees), we measured some proteins immunonephelometrically in serum and aliquots of 24-h urines from 50 apparently healthy children, ages 2-17 years. The mean urinary excretion rate (mg/24h) and the range of values was: for albumin 5.5 (range, 0-13.3), for transferrin 0.5 (0-1.9, for IgG 3.3 0-12), and for alpha 2-macroglobulin 0.6 (0-2.3). Direct comparison of the values for pathological urines with those for a reference population may offer more meaningful information concerning the integrity of the glomerular basement membrane than is provided by protein selectivity indices, and measuring a plasma protein such as albumin in urine may better define pathological proteinuria.     

Renal effects of cardiopulmonary bypass in the elderly

Cardiopulmonary bypass is widely believed to be injurious to renal function. The unknown consequences of renal dysfunction with modern techniques of bypass in the elderly caused us to examine creatinine clearance and the excretion of sensitive marker proteins in older adult patients undergoing CABG. Thirty male patients were divided into three groups: group I with an age up to 60 years, group II with an age between 61 and 70 years, inclusive and group III 71 years and over. Serum creatinine and urea, creatinine clearance, and alpha1-microglobulin (alpha1-MG), N-acetyl-beta-D-glucosaminidase (NAG), Tamm-Horsfall protein (TH) and immunoglobulin G (IgG) were all measured daily, pre- and postoperatively. Creatinine clearance remained lower in the older patients without significant differences. Raised excretion rates of alpha1-MG, and IgG were seen after CPB. The increase in alpha1-MG and NAG during the postoperative period revealed tubular damage in all elderly patients. Measurements of alpha1-MG, NAG and IgG represent useful supplements to standard clinical tests for recognizing early and differentiated changes in renal function.     

Human alpha2-macroglobulin and its antitrypsic and antithrombin activities in serum and plasma.

alpha2-Macroglobulin level, trypsin protein esterase and progressive antithrombin activities were measured in normal and nephrotic sera and plasma. Trypsin protein esterase activity was proportional to the alpha2-macroglobulin concentration in serum and plasma from both normal and nephrotic patients. The results were different, however, with progressive antithrombin activity: in normal plasma, antithrombin III is the main thrombin inhibitor, then alpha2-macroglobulin and alpha1-antitrypsin, whereas in nephrotic syndrome patients, alpha2-macroglobulin is the main thrombin inhibitor.     

Modulation of types I and II acute phase reactants with insulin-like growth factor-1/binding protein-3 complex in severely burned children

OBJECTIVE: To determine whether 0.5 mg/kg insulin-like growth factor (IGF)-1/binding protein (IGFBP)-3, given intravenously, effectively alters the acute phase response in severely burned children. DESIGN: Longitudinal trial with each patient serving as their own control. SETTING: University-affiliated pediatric bum center. PATIENTS: Nine children, 15 yrs of age or less, with burns covering >40% of the total body surface area. INTERVENTIONS: Standard burn care with early burn wound excision and grafting. Blood sampled at defined time points before and after operative procedures. MEASUREMENTS AND RESULTS: Determination of types I and II acute phase reactant proteins, constitutive serum proteins, serum cytokines, serum IGF-1, IGFBP-3, and growth hormone levels. Treatment with IGF-1/BP-3 attenuated increases in type I (complement 3, alpha1-acidglycoprotein) and type II (haptoglobin, alpha1-antitrypsin) acute phase proteins. Further, IGF-1/BP-3 increased constitutive serum protein levels (prealbumin, retinol binding protein, transferrin) and decreased serum IL-6 levels. CONCLUSIONS: Low-dose IGF-1/BP-3 effectively attenuated the type I and type II hepatic acute phase response, increased serum levels of constitutive proteins, and modulated the hypermetabolic response.     

Effects of short-term glycemic control, low protein diet and administration of enalapril on renal hemodynamics and protein permselectivity in type 2 diabetic patients with microalbuminuria

To determine whether each of glycemic control (GC), low protein diet (LPD) or administration of angiotensin converting enzyme inhibitor (ACEI) has beneficial effects on diabetic nephropathy through the different mechanisms, changes in charge and size selectivity of glomerulus and renal hemodynamics were analyzed in microalbuminuric type 2 diabetic patients after additive combination therapy (first period: GC only, second period: GC-LPD, third period: GC+LPD+ACEI). To detect improvement of the impairments of glomerular charge selectivity and size selectivity, changes in the ratio of the renal clearance of two plasma proteins with similar molecular radii and different isoelectric points (pIs) (ceruloplasmin and IgG: CRL/IgG) and changes in the ratio of the renal clearance of two plasma proteins with similar pIs and different molecular radii (alpha2-macroglobulin and albumin: alpha2/Alb) were examined before and after each therapy. Creatinine clearance decreased significantly in the first and third periods although slight but not significant decrease was detected in the second period. Filtration fraction was significantly decreased only in the third period. Although renal clearances of Alb, IgG and CRL were decreased in periods of all three therapies, that of alpha2-macroglobulin with a large molecular radius was decreased significantly only after the third therapy. Neither CRL/IgG nor alpha2/Alb changed during these three therapies. These findings suggest that each of three short-term therapies consisting of GC, GC+LPD and GC+LPD+ACEI, reduced proteinuria in microalbuminuric type 2 diabetic patients not through the improvement of renal size and charge selectivities, but through improvement of renal hemodynamics.     

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.     

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.     

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.     

Measurement of eleven serum proteins by microparticle-enhanced nephelometric immunoassay.

Eleven proteins (immunoglobulins IgG, IgA, IgM, orosomucoid, alpha 1-antiproteinase, haptoglobin, ceruloplasmin, C-reactive protein, transferrin, prealbumin and alpha 2-macroglobulin) in human serum were quantitated by a new microparticle-enhanced nephelometric immunoassay. This is a one step competitive assay, based on the nephelometric measurement of light scattered by clusters of protein-coated microparticles specially synthesized for that use. Statistical evaluation (precision, recovery and method comparison) shows that the determination of serum proteins is reliable and accurate for wide ranges of concentration and that the method is quite adequate for strongly increased concentrations. This microparticle-enhanced nephelometric immunoassay appears to offer an alternative method for routine measurement of a great variety of serum proteins at high and intermediate concentrations, which are usually quantified by radial immunodiffusion or conventional immunonephelometry. On account of its sensitivity, it can also be used for the determination of relatively low concentrations of analytes.     

Blood serum and synovial fluid proteins in Reiter's syndrome

Individual proteins (transferrin, ceruloplasmin, alpha 2-macroglobulin, IgG, IgA and IgM) were examined in blood and synovial fluid of 11 patients with Reiter's syndrome. ARA-1981 was used as a diagnostic criteria. The control group included 40 patients with rheumatoid arthritis and 31 with osteoarthrosis of the knee joint. Statistically significant differences between the concentrations of individual proteins in patients with Reiter's syndrome, rheumatoid arthritis and osteoarthritis were only established in synovial fluid for IgM and for IgM index Csf/CS.     

Identification and quantification of serum proteins secreted into the normal human jejunum

The in vivo transfer of serum proteins to the human intestinal lumen was characterized by crossed immunoelectrophoretic analyses of intestinal perfusates from four healthy volunteers. Serum proteins with molecular masses below 100 kDa and the immunoglobulins were found in human jejunal perfusates. Larger serum proteins were either absent (alpha and beta lipoproteins) or present in small amounts (alpha 2-macroglobulin, haptoglobulin and ceruloplasmin). These results demonstrate the existence of a selective transfer of serum proteins to the intestinal lumen under physiological conditions. The intestinal clearance rate was 0.1 ml serum per hour per 10 cm jejunum for albumin, prealbumin, alpha 1-antitrypsin, orosomucoid, transferrin and haemopexin. The rate of secretion of total protein to the jejunal lumen was 100 mg protein per hour per 10 cm jejunum. About 45% was due to immunoglobulins and further 10-15% due to the remaining serum proteins. It is suggested that the serum proteins pass through the epithelium by a transcellular mechanism.     

Alpha-2-macroglobulin in children with glomerular diseases (author's transl)]

The serum and urine levels of alpha-2-macroglobulin (alpha2-MG) was determined in 33 children with glomerular diseases and in 26 healthy control children. Healthy children showed a minimum level of 275 mg% and maximum level of 337 mg%, with a mean concentration of 301 mg% and a standard deviation of 13 mg%. No alpha2-MG was detected in the urine. Steroid-treated patients with idiopathic nephrotic syndrome displayed elevated inhibitor levels of up to 490 mg%. This might be a direct result of steroid therapy or a consequence of reactively-increased protein synthesis in response to the renal protein loss. In all these patients the urine was found to be alpha2-MG-negative, irrespective of the presence or absence of proteinuria. In the miscellaneous group of glomerulopathies without the nephrotic syndrome, serum levels of alpha2-MG were shown to be normal. The urinary concentrations of alpha2-MG were related to the activity of the disease. alpha2-MG determination in serum and urine seems to be a tool for differential diagnosis and prognosis in some cases of glomerular disease.     

Serum and urinary proteins, lysozyme (muramidase), and renal dysfunction in mono- and myelomonocytic leukemia

Serum levels, urinary excretion, and clearances of several proteins of different molecular weights were studied in 18 patients with mono- and myelomonocytic leukemia. Nine patients had normal renal function (group A) and nine had impaired renal function with azotemia (group B). The majority of patients in both groups had increased concentration of immunoglobulins, particularly IgG, IgA, and IgM; IgD level was normal. Serum transferrin and alpha(2)-macroglobulin were frequently reduced while the level of ceruloplasmin was often increased, especially in patients with azotemia. The activity of lysozyme in the serum was high in all patients, but was considerably higher in group B.Proteinuria was found in most patients but was more prominent in group B. Almost invariably albumin constituted less than 25% of the total protein excreted. Qualitative analysis of various urinary proteins by immunochemical techniques and clearance studies suggested the presence of glomerular as well as tubular dysfunction. Determination of urinary lysozyme frequently showed no direct correlation between the serum level of the enzyme and its concentration in the urine or its clearance by the kidney. In addition to glomerular filtration, impaired tubular reabsorption may account for the high level of lysozyme in the urine. It is postulated that the very high level of lysozyme in the glomerular filtrate and possibly hypergammaglobulinemia may play a role in the induction of tubular damage. Renal impairment has been correlated with histological changes in the kidneys. From a comparative study of various leukemias, it seems that the combined glomerular-tubular dysfunction is a manifestation unique to mono- and myelomonocytic leukemia.     

Parallel increase in urinary excretion rates of immunoglobulin G, ceruloplasmin, transferrin, and orosomucoid in normoalbuminuric type 2 diabetic patients

OBJECTIVE: Increased urinary excretions of several plasma proteins with different molecular radii <55 A and different isoelectric points (pI), such as IgG, ceruloplasmin, transferrin, and orosomucoid, have been independently reported to precede the development of microalbuminuria in diabetic patients. We examined whether increases in urinary excretions of these proteins would be in parallel in the same patient. RESEARCH DESIGN AND METHODS: Urinary excretion rates of proteins mentioned above in timed overnight urine samples were evaluated in 61 normoalbuminuric type 2 diabetic patients (group D) aged 40-60 years and in 17 age-matched control subjects (group C). RESULTS: The excretion rates of these proteins were significantly higher in group D than in group C. These exhibited a strong linear correlation with each other and had a weak correlation with the excretion rate of N-acethylglucosaminidase. The excretion rate of alpha2-macroglobulin with large molecular radii of 88 A was not different between groups C and D, nor did they have any correlations with the excretion rates of the other proteins. Creatinine clearance and blood pressure levels in group D were significantly higher than those in group C. CONCLUSIONS: In normoalbuminuric diabetic patients, excretion rates of plasma proteins with molecular radii <55 A increased in parallel with each other. In view of our previous finding that urinary excretions of these plasma proteins selectively increased in parallel with enhanced glomerular filtration rate after acute protein loading, the present finding may be explained by renal hemodynamic changes, such as increased intraglomerular hydraulic pressure.     

The efficacy of plasma exchange in the removal of plasma components

The efficacy of plasma exchange in the removal of immunoglobulins (IgG, IgM, IgA), complement components (C1q, C4, C3), alpha 1-antitrypsin, alpha 2-macroglobulin, and transferrin was studied by analysis of pre- and postexchange serum samples and the plasma removed both in a healthy volunteer and in five patients undergoing therapeutic plasma exchange. In the healthy volunteer, the measured reduction in serum concentration and the measured amount removed for each component was compared with values predicted by a physical model. For all components except IgM, the sum of the measured amount removed during the procedure and the calculated amount present in the circulation progressively exceeded the calculated intravascular amount present before plasma exchange. This was particularly the case for C4, C3, alpha 1-antitrypsin, and alpha 2-macroglobulin and may be explained by influx from the extra- to the intravascular compartment during the procedure. Influx also occurred in the patients. We conclude that, except for IgM, the actual amount of a component that has been removed should be assessed for proper evaluation of the efficacy of PE.     

Mechanisms of proteinuria in human glomerulonephritis.

We evaluated glomerular barrier function in 28 patients with glomerulonephritis. Neutral dextrans of graded size were used to characterize the size-selective properties of the barrier. Charge selectivity was characterized by electrofocusing excreted urinary proteins. A fractional IgG clearance (relative to freely permeable inulin), smaller or greater than 100 x 10(-5) was used to distinguish patients with minor (group I, n = 13) and major (group II, n = 15) urinary IgG leakage, respectively. Fractional clearances of smaller dextrans (radii 20-50 A) were similar, but those of larger dextrans (radii 52-60 A) were elevated in group II relative to group I patients. A model of solute transport through a bimodal pore size distribution revealed the values for pore radius in the lower mode to approximate 51-55 A in both group I and group II patients. Pore radius in the upper mode, by contrast, was much larger in group II than in group I patients, approximating 87-97 vs. 72-77 A, respectively. Electrofocusing of urinary protein from group I patients revealed mostly albumin (isoelectric point 5.2). In group II patients, however, immunoglobulin excretion was copious. Moreover, the distribution of anionic, neutral, and cationic species (isoelectric points 5.5-8.5) in urinary and plasma eluates of IgG2 and IgG4 was similar. We conclude that when glomerulonephritis is associated with selective albuminuria, as in group I,, there is an isolated reduction of electrostatic retardation of relatively small anionic proteins. Major urinary IgG leakage (group II), however, appears to result from the development in the glomerular membrane of a subpopulation of enlarged pores that are highly permeable towards proteins of large size and varying charge.     

Detection of lysozyme and alpha 2-macroglobulin--lysozyme complexes by immunoblotting

An immunoblotting technique was developed to detect human lysozyme and lysozyme complexes in body fluids. The unoccupied binding capacity of proteins was demonstrated by addition of surplus lysozyme. The sensitivity of immunoblotting to the free enzyme in human albumin solution was less than 5 ng. In serum and pleural fluid, part of exogenous lysozyme was bound to alpha 2-macroglobulin (alpha 2-M). At high concentrations of lysozyme in leukemic sera, part of the enzyme formed an endogenous alpha 2-M complex. On the other hand, the formation of alpha 2-M complexes with exogenous lysozyme was especially striking in sera from nephrotic patients with elevated alpha 2-M. The findings corroborate with previous reports on lysozyme binding to purified alpha 2-M in vitro and suggest that the binding is concentration-dependent with respect to both reaction partners. In vivo the mechanism may provide a pathway for extrarenal lysozyme catabolism medicated by reticuloendothelial cells. No other binding proteins were seen in the present study: lysozyme did not bind to serum immunoglobulins in 35 samples with an immunoglobulin paraprotein, three samples with polyclonally elevated gamma-globulins, 20 other patient sera and 10 normal sera. Neither did lysozyme bind to urinary proteins in five samples from patients with myeloic leukemias nor in 10 samples from myeloma patients with urinary excretion of a monoclonal immunoglobulin light chain.     

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.     

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.     

Abnormality in urinary protein excretion in Japanese men with impaired glucose tolerance.

OBJECTIVE: To examine whether subjects with impaired glucose tolerance (IGT) for more than 2 years have any abnormality in the kidney. RESEARCH DESIGN AND METHODS: We measured urinary excretion rate and clearance of various plasma proteins with different molecular radii and different isoelectric points in 22 Japanese men with IGT (IGT group) and 37 age-matched healthy control subjects (control group). RESULTS: Clearance of ceruloplasmin (molecular radius approximately 45 A; isoelectric point 4.4), IgG4 (molecular radius 55 A; isoelectric point 5.4), and IgG (molecular radius 55 A; isoelectric point 7.4) was significantly higher in the IGT group than in the control group, whereas there were no significant differences in urinary excretion rate of albumin (molecular radius 36 A; isoelectric points 4.8-5.2) and clearance of alpha 2-macroglobulin (molecular radius 88 A; isoelectric point 5.4) between the two groups. CONCLUSIONS: In the present study, we found that clearance of neutral-charged IgG, negatively charged IgG4, and ceruloplasmin with molecular radii of approximately 45-55 A was selectively increased in IGT subjects. This finding does not seem to be explained by impairment of charge and pore-size selectivity in the glomerulus. Therefore, considering the present result together with our recent finding that enhanced glomerular filtration rate (GFR) after acute protein loading in healthy subjects induced a selective increase in clearance of IgG, IgG4, and ceruloplasmin, we suggest that increased intraglomerular hydraulic pressure, although enhanced GFR was not demonstrated, may be at work in these mildly hyperglycemic subjects.