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.     

Three turbidimetric methods for determining total protein compared

We used human serum protein fractions to evaluate the sensitivity and bias of three turbidimetric methods for determining concentrations of proteins. Each fraction (Cohn Fractions II, III, IV, and V) was assigned a protein concentration value that was determined by the biuret method, which we calibrated with purified monomer of human serum albumin. All three turbidimetric methods (those involving sulfosalicylic acid/sodium sulfate, trichloroacetic acid, and alkaline benzethonium chloride) gave acceptable results for Fraction V with crystallized human serum albumin as the reference material, but there was bias by each of the three methods for the three globulin fractions. The method involving alkaline benzethonium chloride with measurement at 450 nm had the best sensitivity within the range of linearity and the most consistent bias among the three globulin fractions. These results define the dilemma for valid calibration of these methods for total serum protein in cerebrospinal fluid and urine.     

尿血红蛋白对苄索氯铵法定量测定尿蛋白的干扰评价

目的评价尿血红蛋白对苄索氯铵法定量测定尿蛋白的影响。方法参照美国临床和实验室标准化协会(CLSI)的EP7-A2方案,配制不同浓度血红蛋白的尿液标本做剂量-效应试验,同时收集50例不同隐血程度的尿液标本做临床标本的偏倚试验;尿蛋白定量在DPP罗氏生化工作站采用苄索氯铵法检测,与磺基水杨酸法进行比较。结果剂量-效应试验显示,当尿血红蛋白浓度达0.2g/L时即对苄索氯铵法测定尿蛋白产生显著性干扰(P0.05);临床标本的偏倚试验显示,不同程度的隐血对苄索氯铵法测定尿蛋白产生明显的正干扰。结论重视尿隐血阳性和/或含有红细胞的尿液标本,在采用苄索氯铵法测定尿蛋白时应评价其准确性,必要时使用磺基水杨酸法进行确认。     

Protein determination in urine--a critical review

Seven methods for protein determination in urine were systematically checked, viz. turbidimetric assay with benzethonium chloride, turbidimetry with trichloroacetic acid of various concentrations, micellar complex formation with Coomassie Brilliant Blue G-250, formation and gel filtration of protein complexes with copper, reaction of protein tannin compounds with iron, and biuret reaction after different precipitation procedures. The evaluation was mainly based on the following criteria: identical reaction with various proteins, stable and linear absorbance with concentration, complete recovery, and greatest possible freedom from interferences. On this basis, the biuret reaction must now be regarded as the method of choice. Albumin and gamma-globulins are reliably estimated after precipitation by perchloric acid or by an ethanol-diethylether mixture, whereas all proteins, including uromucoid, may be measured after precipitation by Tsuchiya's reagent. Interference from urinary pigments can be avoided by reading versus a sample blank.     

Total protein determination in urine: elimination of a differential response between the coomassie blue and pyrogallol red protein dye-binding assays

BACKGROUND: The total protein content of urine is a good index of renal function, but its determination is unreliable. Protein dye-binding assays are simple, but they characteristically lack a uniform response to different proteins. METHODS: We investigated a differential response of the Sigma Microprotein Coomassie Brilliant Blue (CBB) and Pyrogallol Red-molybdate (PRM) protein dye-binding assays to urine, using human albumin, albumin/globulin, or urinary protein as calibrator. RESULTS: The urine protein values (n = 60) obtained with the CBB assay were 110-13 500 mg/L (mean, 2390 mg/L) compared with 160-18 300 mg/L (mean, 3470 mg/L) obtained with the PRM assay (CBB:PRM protein concentration ratio, 0.46-0.88, mean, 0. 69 +/- 0.10). The differential response was highly reproducible as indicated by Sigma urine control Level 1 (within-day CBB:PRM ratio, 0.68 +/- 0.02; between-day CBB:PRM ratio, 0.67 +/- 0.04) and Sigma urine control Level 2 (within-day CBB:PRM ratio, 0.60 +/- 0.01; between-day CBB:PRM ratio, 0.59 +/- 0.02). The use of urinary protein as a calibrator (rather than human albumin) greatly improved the agreement between the assays when applied to urine (y(CBB) = 0. 972x(PRM) - 16 vs y(CBB) = 0.685x(PRM) + 17). In studies using urine controls, this calibrator also improved agreement between the CBB, PRM, trichloroacetic acid (TCA), and benzethonium chloride protein methods and, to a lesser extent, agreement with the TCA-Ponceau S method. CONCLUSION: The use of a urinary protein calibrator improves the agreement between different methods used to determine total protein in urine.     

Assessment of the benzethonium chloride method for routine determination of protein in cerebrospinal fluid and urine

We have tested the characteristics of the method of Iwata and Nishikaze (Clin Chem 25: 1317, 1979). The linearity, sensitivity, and precision are satisfactory and the reactivity of benzethonium chloride with various proteins (albumin, immunoglobulins) is the same. The method has been compared with Meulemans's technique (Clin Chim Acta 5: 757, 1960), routinely used in our laboratories, by analysis of 82 samples of cerebrospinal fluid (CSF) and 119 samples of urine. Our results for cerebrospinal fluid agree well with those of Iwata and Nishikaze (r = 0.976; y = 0.992x - 0.013), but we find their method unsuitable for urinary protein determination, probably because of interfering compounds in urine.     

Variability of standard clinical protein assays in the analysis of a model urine solution of fragmented albumin

OBJECTIVES: This study investigates the sensitivity of various standard clinical techniques in the detection of albumin fragments. The significance of this work is in the detection of urinary proteins, such as albumin, which has recently been discovered to be excreted as mainly peptide fragments as a result of filtered albumin undergoing degradation during renal passage. All filtered proteins undergo a similar degradation process. DESIGN AND METHODS: Albumin digested with trypsin was used as a model urine solution. The solution was assayed for albumin concentration by various methods including the biuret assay that is known to detect urinary albumin fragments. The digest solution was also analyzed by various clinically used chromagen assays, electrophoretic and chromatographic methods to determine whether they are able to detect the fragmented protein. RESULTS: The benzethonium chloride, Coomassie blue, and pyrogallol red assays for urine protein, the immunoassay for human albumin and sodium dodecyl sulfate polyacrylamide gel electrophoresis with Coomassie blue staining were unable to detect the albumin fragments. Capillary electrophoresis was sensitive to the fragments but with low resolution. High-performance liquid chromatography gave the best results. CONCLUSIONS: Many techniques utilized to assay patient urine samples are unable to detect fragmented albumin and, hence, will severely underestimate albumin and protein excretion.     

A micro-method for measuring total protein in cerebrospinal fluid by using benzethonium chloride in microtiter plate wells

By using a benzethonium chloride concentration 12-fold that described originally (Clin Chem 1979;25:1317-9), we developed a reliable method suitable for routine measurement of total protein in cerebrospinal fluid. Only 10 microL of sample is required. Reactivity to immunoglobulin G (IgG) and to albumin (Alb) is similar, as is necessary for specimens that can have very varied IgG/Alb ratios. The assay, performed in a microtiter plate for ease of use, has a between-batch coefficient of variation of 3.4% for a protein concentration of 450 mg/L. This contrasts with a dye-binding technique with Ponceau S, for which the CV was 9% at the same concentration.     

An alternative method for assaying cerebrospinal fluid protein in the presence of methotrexate

Therapeutic concentrations of methotrexate can cause significant positive interference in cerebrospinal fluid (CSF) protein values when assayed in the Du Pont aca. Conversely, our modified turbidimetric method, in which trichloroacetic acid (TCA) plus a sample blank containing dilute hydrochloric acid is used in place of TCA, exhibits little or no interference from methotrexate. This was verified by assaying solutions that contained a constant amount of protein (approximately 430 mg/L) and various amounts of methotrexate (0.0-2.3 x 10(-4) mol/L) by both the Du Pont aca and the manual turbidimetric method. As expected, the aca results showed increasing protein values with increasing methotrexate, whereas the manual method gave results approximating the expected protein value irrespective of the methotrexate concentration.     

Is standardization more important than methodology for assay of total protein in cerebrospinal fluid?

Four manual micromethods for protein determination, two turbidimetric (trichloroacetic and sulfosalicylic acid-sodium sulfate) and two colorimetric (Lowry and Coomassie Brilliant Blue--sodium dodecyl sulfate, CBB-SDS) were used to compare the standard curves for total protein (0.30 to 3 g/L) produced with three reference materials: bovine serum albumin, human serum albumin, and diluted human serum. We measured the apparent protein content of a sample of pooled human cerebrospinal fluid by all four methods and with use of all three standards. The only reference material that gave similar results with all four methods was diluted human serum; the CBB-SDS was the only method that gave identical results with all three reference materials. We then measured the protein concentration of 28 individual cerebrospinal fluid samples by the four methods, with diluted human serum as standard. Results by all methods correlated well, but only the sulfosalicylic acid and the CBB-SDS methods gave equivalent results. We conclude that the choice of standard is more important than the method used. However, the CBB-SDS method may be the preferred method because it produced identical standard curves with all three protein standards.     

Bedside hemoglobin measurements: Sensitivity to changes in serum protein and electrolytes

Objective. Our objective was to compare the effect of protein and electrolyte changes associated with hemodilution on the accuracy of photometric and conductivity hemoglobin determination methods.Methods. Blood samples from 10 patients with normal preoperative serum electrolytes and total protein levels were studied. From an indwelling arterial line, 20 ml of blood were removed; hemoglobin values were measured pre-(Baseline) and postdilution by Coulter counter, conductivity, and photometric methods. Blood samples were diluted by placing 4 ml of blood into three test tubes, and adding 1 ml of either 25% albumin, 0.9% sodium chloride, or 5% dextrose in water.Results. Blood sample dilution resulted in a reported conductivity hemoglobin that was significantly different from the Coulter value (p=0.0004) when 25% albumin, 0.9% sodium chloride, and 5% dextrose in water solution was used. Using the same dilutions, the photometric method accurately reflected Coulter hemoglobin values. The correlation between photometric and Coulter hemoglobin measurements was R²=0.97,p=0.0001. Correcting the conductivity hemoglobin values for changes in total protein, chloride and sodium significantly improved correlation with Coulter hemoglobin (R² of uncorrected versus corrected=0.37 and 0.72;p=0.0001).Conclusions. In the range of electrolyte and protein concentrations found in this study, the photometric method of hemoglobin assessment was more accurate than either corrected or uncorrected conductivity hemoglobin determinations, as compared to Coulter-based measurements.Eight percent of the expenses for this research project were provided by HemoCue Inc, Helsinburg, Sweden. This study was presented, in part, at the 1992 meeting of the American Society of Anesthesiologists, New Orleans, LA. The authors would like to acknowledge Jeffrey Weiss, DO, and Brian Wales, MD, for their assistance with this study.     

New micro-turbidimetric method for determination of protein in cerebrospinal fluid and urine.

We report a new micro-scale (0.1-mL sample) turbidimetric method for determination of protein by use of benzethonium chloride in alkali. The method is highly specific for protein, has a higher sensitivity than the classic method of Lowry et al., and shows satisfactory reproducibility and recovery. The turbidity produced in our method is the same for albumin and gamma-globulin and is more stable than in Meulemans' method (in which sulfosalicylic acid is used) or in the method of Bossak et al. (in which trichloracetic acid is used). In contrast to Pesce and Strande's method, there is no manipulative loss of protein.     

苄索氯铵比浊法测定脑脊液蛋白质的方法学评价

目的对一种新的测定脑脊液蛋白质含量的方法——苄索氯铵比浊法进行方法学评价。方法采用罗氏（Roche）公司的质控品、c．f．a．s定值血清和试剂在Roche P800全自动生化分析仪上,参照美国临床实验室标准化委员会（NCCLS）评价方案对苄索氯铵比浊法的灵敏度、精密度、线性、抗干扰性进行评价,并与强生（Johnson）公司的干化学法进行比对。结果经实验得出苄索氯铵比浊法的测量灵敏度为42．7mg／L,标本浓度在85mg／L与2685mg／L之间线性关系良好,精密度检查低、中、高浓度标本的批内、批间、日间、总变异系数（CV）〈5％,干扰试验除了血红蛋白在浓度达到0．1g／L开始产生明显干扰外,葡萄糖、维生素C、胆红素在试验浓度内均未产生明显干扰,与强生公司的干化学法进行比对,Passing—Bablok回归方程为y=0．931X＋12．5,线性回归的Cusum检验证实偏倚无统计学意义（P〉0．05）。一元直线回归方程为y=0．922X＋13．8,计算得出相关系数（r）=0．986,表明两种方法相关性良好。结论苄索氯铵比浊法稳定性好、灵敏度高、线性范围宽、能对抗多种物质的干扰,是一种适合于临床应用的较理想的方法。     

Identification and quantification of Bence-Jones proteinuria by automated nephelometric screening

A two-step screening of urine samples for Bence-Jones proteins is described. The proposed method is fast and fully mechanized; quantitative results are obtained within minutes. As a first step alpha 1-microglobulin, albumin, transferrin and IgG are measured immunonephelometrically. Then the cumulative concentration of the four markers is compared with that of total protein, which is determined by nephelometry during trichloroacetic acid protein precipitation. Bence-Jones proteinuria is indicated by large concentrational differences (greater than 31%) between the four markers and total protein. As a second step, Bence-Jones proteins are assessed directly if they are present. Immunoglobulin light-chains are measured immunonephelometrically and the kappa:lambda ratio is used to discriminate between monoclonal and polyclonal forms. Using this strategy, urine samples from 84 patients with monoclonal gammopathia or multiple myeloma were screened. Bence-Jones proteinuria was detected in 40 cases. In a reference collective (69 patients with different types of renal proteinuria) Bence-Jones proteinuria was not observed. Comparing the results with those obtained by immunofixation, the nephelometric method has a sensitivity of 100% and a specificity of 97%, differing only in a single false-positive result. Additional information about renal forms of proteinuria is supplied by the first screening step. This permits an assessment of the renal involvement in Bence-Jones proteinuria, and the method can also be used for nephrological diagnosis.     

苄索氯胺法——一种新的尿蛋白定量检测方法

目的 对苄索氯胺法这种新的尿蛋白检测方法进行评价。方法 收集150例门诊就诊患者尿液,用苄索氯胺法检测其尿蛋白含量,以此评价该方法的线性范围、精确度、灵敏度、回收试验、重复试验。结果该方法线性范围上限达2.014g／L,回收率手工法为86．95％,自动法为106．85％,有良好的精密度（CV分别为4．97％和2．63%）,灵敏度为单一钨酸比浊法的19～34倍,该法与单一钨酸比浊法的相关性良好,相关系数r为0．9846。两法结果进行t检验,t=0．436,P〉0．05,结果 差异无统计学意义。结论 该方法有灵敏度高、重复性好的优点,结果稳定,标本用量少,适用于自动分析,是值得在临床上推广的方法。     

An enzymatic method for the measurement of glycated albumin in biological samples

BACKGROUND: In order to determine glycated albumin more easily and rapidly, we developed a new enzymatic method for glycated albumin in blood samples. METHODS: The method involves use of albumin-specific proteinase, ketoamine oxidase and serum albumin assay reagent. In the assay, glycated albumin is hydrolyzed to glycated amino acids by proteinase digestion, and ketoamine oxidase oxidizes the glycated amino acids to produce hydrogen peroxide, which is quantitatively measured. Glycated albumin is calculated as the percentage of glycated albumin in total albumin. RESULTS: The calibration curve for glycated albumin concentration was linear (r(p)=0.999) between 0.0 and 50.0 g/l and that for albumin concentration was linear (r(p)=0.999) between 0.0 and 60.0 g/l. The analytical recoveries of exogenous glycated albumin added to serum were 100-102.5%. The within-run and between-run CVs were 0.45-0.67% and 1.09-1.26%, respectively. This method was free from interference by bilirubin, chyle, glucose, globulins and labile intermediate. Weak interference by hemoglobin and ascorbic acid was observed. Glycated albumin detected by the present method was significantly correlated with glycated albumin detected by high-performance liquid-chromatographic (HPLC) method (serum: r(s)=0.989, plasma: r(p)=0.992). CONCLUSIONS: This new enzymatic method is simple, rapid, allows multiple determinations and enables quantitative analysis of glycated albumin.     

The normal range for total protein and protein fractions in the cerebrospinal fluid of children (author's transl)]

In order to establish normal values, samples of cerebrospinal fluid from 109 healthy children between the ages of 0 and 13 years were analyzed for total protein, different protein fractions after electrophoretic separation, and IgG. Total protein was determined by the biuret method after precipitation with trichloroacetic acid. For the determination of the different protein fractions, the fluid was concentrated by high pressure filtration; the fractions were separated by microzone elektrophoresis on cellulose acetate membranes, then assayed photometrically after staining with amido black B. IgG was determined by radial immunodiffusion.     

Fingerstick glycosylated hemoglobin, plasma protein, and albumin

We have developed techniques that permit the affinity-chromatographic determination of glycosylated hemoglobin, plasma protein, and albumin on fingerstick samples of whole blood. The fingerstick glycohemoglobin technique takes advantage of the high sensitivity of measurement of hemoglobin by absorbance at 414 nm. The glycosylated plasma protein is assayed by a highly sensitive method based on binding of Coomassie blue. An enzyme-linked immunosorbent assay is used to measure albumin in the bound and nonbound fractions of an aminophenylboronic acid chromatographic separation. The fingerstick method for assay of glycosylated plasma albumin gives results that are approximately 40% higher than comparable values obtained on the same patient with a 1-ml plasma sample determined with the bromcresol green technique. There is good correlation of fingerstick glycoalbumins with fingerstick glycohemoglobins and glycosylated plasma protein values. These procedures should be useful for children and for large-scale ambulatory screening for diabetes mellitus.     

Assay of cerebrospinal fluid protein: a rate biuret method evaluated

We evaluated a rate colorimetric method (Beckman) for measuring total protein in cerebrospinal fluid. The automated instrument we used was Beckman's ASTRA TM. A 100-microL sample of spinal fluid is introduced into the biuret reagent in the reaction cell and the increase in absorbance at 545 nm is monitored for 20.5 s. Solid-state circuits determine the rate of alkaline biuret-protein chelate formation, which is directly proportional to the total protein concentration in the sample. The linear range of measurement is 120 to 7500 mg/L. Day-to-day precision (CV) over the range of 150 to 1200 mg/L ranged from 15.2 to 2.3%. The method was unaffected by radical alteration of the albumin/globulin ratio, but there is a positive interference in the presence of hemoglobin, a suppression in the presence of bilirubin, and no effect by xanthochromia. The method is precise, accurate, rapid, and convenient. The method was compared with the trichloroacetic acid method as performed on the Du Pont aca III, giving a correlation coefficient (r2) of 0.9693. The method is precise, accurate, rapid, and convenient.     

Hemoglobin screening in prospective blood donors: comparison of different blood samples and different quantitative methods.

Portable photometers are increasingly used for hemoglobin screening in blood centers. In a total of 500 unselected prospective blood donors, venous hemoglobin concentrations were measured using a hematology analyzer, and fingerstick hemoglobin concentrations were measured on both a HemoCue B and a Biotest hemoglobin photometer. In 100 of the donors, earstick hemoglobin levels were also measured on the HemoCue system. The mean hemoglobin levels in the fingerstick samples were slightly higher than those in the venous samples. The deviation exceeded the limit of +/-10g l(-1) in 9% of samples tested by HemoCue and in 20% of those investigated by Biotest. The use of earstick samples led to considerable overestimation of the actual hemoglobin concentration.