Characterization of the urinary albumin degradation pathway in the isolated perfused rat kidney

This study examines the existence of the urinary albumin degradation pathway and the proposed role of receptor-mediated endocytosis in this process using the isolated perfused rat kidney (IPK) model. Albumin-derived peptides in IPK urine are analyzed in terms of their relative size distribution using radioactivity and absorbance at 214 nm, and their susceptibility to trypsin digestion. The effects of perfusing kidneys with concanamycin A and myristoyl trimethyl ammonium bromide (MTMAB), inhibitors of the receptor-mediated endocytosis regulators vacuolar-type H(+) ATPase (v-ATPase) and dynamin GTPase, respectively, are examined. Normal IPK urine contains mildly degraded (defined as approximately 10-40 kDa; 43.0 +/- 8.3%) and heavily degraded (defined as <10 kDa; 22.6 +/- 7.7%) albumin peptides as well as intact albumin (34.5 +/- 4.1%). The relative size distribution of the peptides is similar by radioactivity and absorbance at 214 nm, and both profiles are reduced to very small peptides following trypsin digestion. Administration of concanamycin A or MTMAB causes a significant increase in the proportion of intact albumin (concanamycin A: 55.8 +/- 11.6%; MTMAB: 50.0 +/- 11.9%) excreted compared with normal IPK urine. This coincides with a reduction in the proportion of mildly (concanamycin A: 27.6 +/- 9.8%; MTMAB: 39.9 +/- 11.5%) and heavily degraded (concanamycin A: 16.6 +/- 7.4%; MTMAB: 10.0 +/- 2.5%) albumin present and is not associated with changes in glomerular permeability to albumin because no significant change is observed in the fractional clearance of Ficoll (radius range 20-60 A) in the presence of concanamycin A. This study demonstrates the existence of albumin peptides in IPK urine and suggests that receptor-mediated endocytosis plays a role in urinary albumin degradation.     

尿中免疫化学非反应性白蛋白在糖尿病肾病早期诊断中的意义

糖尿病患者尿中的微量白蛋白（mAb）的检测对早期发现糖尿病肾病的意义已为临床所广泛重视。近期发现糖尿病（可能也包括心血管病、高血压）患者尿中的一部分白蛋白用常规的免疫化学检测手段（放射免疫分析，免疫浊度法、免疫光散射法）不能检出，且在尿中出现得比mAb早数年。这种蛋白暂且被命名为免疫化学非反应性白蛋白（immunochemically nonreactive urinary albumin，INUA）。尿中的白蛋白可存在多种形式。2001年Greive等发现患糖尿病鼠的尿中白蛋白量增高，但用常规的免疫化学法测定不出来。     

Immunoturbidimetry of urinary albumin: prevention of adsorption of albumin; influence of other urinary constituents

I describe a simple, rapid immunoturbidimetric assay for low concentrations of albumin in urine (2 to 260 mg/L). However, in this assay, the human serum albumin (HSA) in the standards binds nonspecifically to the polystyrene or glass tubes. This nonspecific binding cannot be prevented by adding bovine serum albumin (BSA) to standards, because the anti-HSA antibody cross reacts with BSA. Adding Triton X-100 (1 mL/L) to standards effectively prevents this nonspecific binding of HSA from standards to both polystyrene and glass tubes. High concentrations of compounds found in urine from normal and diabetic subjects do not interfere with this assay if pH extremes can be avoided. The between-day CV is 4.8% at means = 18.8 mg/L and 2.0% at means = 183.1 mg/L. Measurements by this immunoturbidimetric method (y) correlate well with those obtained by a radioimmunoassay (x): y = 1.078x - 0.141 mg/L (n = 98; r = 0.984) and with those obtained by a radial immunodiffusion method (x'): y = 1.026x' - 0.117 mg/L (n = 98; r = 0.983). Urinary excretion of albumin by 25 healthy, nondiabetic subjects was less than 8 micrograms/min.     

Radioimmunoassay for urinary albumin.

We describe a rapid, sensitive, and precise radioimmunoassay for urinary albumin (Ualb). Aliquots of diluted urine were incubated at room temperature for 1 h with 125I-labeled albumin and a rabbit antiserum monospecific for human albumin. Phase separation was effected by the double-antibody technique. The dose-response curve as linear in the range of 15.6-10000 ng, equivalent to 4 to 3000 mg/liter of urine. The limit of sensitivity was 16 ng of albumin. The coefficient of assay variation was 4.8%, both at 44 mg/liter and at 1304 mg/liter. A displacement curve obtained with a serially diluted urine sample of high albumin concentration was completely superimposable with the curve for which human albumin was used as a standard. In 26 normal individuals the range for Ualb was 2.2--12.6 mg/24h, and for albumin clearance (Calb, 1.8 x 10(-5)-19.6 x 10(-5) ml/min. After renal homografts in 25 patients, Ualb ranged from 16.9 to 9928 mg/24 h, and Calb from 2.7 x 10(-4) to 1.7 x 10(-1) ml/min. Both increased Ualb and Calb correlated well with the severity of renal homograft rejection.     

Enzyme immunoassay for urinary albumin

We describe an enzyme-linked immunosorbent assay for urinary albumin, performed on microtiter plates with use of commercially available antisera and peroxidase conjugate. The assay range is 3-1000 micrograms/L, the sensitivity 625 pg. The method is suitable for measurement of albumin excretion in either normal or pathological urine. For 20 normal children, the range of urinary albumin excretion was 1.7-22.9 mg/24 h.     

Heterogeneity of urinary albumin from diabetic patients

A fundamental cause of diabetic microalbuminuria, heterogeneity of normal and diabetic urinary albumin was shown by affinity chromatography on Cibacron Blue F3GA. By changing the properties of interaction with the matrix, the protein was separated into six fractions. Samples of urinary albumin from proteinuria patients showed the same elution profiles as those of serum albumin, whereas those from controls or normoalbuminuria diabetic patients exhibited different elution patterns. The relative percentage of the resin unbound fraction of urinary albumin was ten or more times higher than that of serum albumin, and the ratio decreased with increasing albumin excretion into urine. More than 6 mol fatty acid/mol albumin combined with the unbound fraction. It is suggested that microalbumin excretion into urine is the result of excessive unesterified fatty acid binding to the protein.     

尿微量白蛋白的测定及其临床意义

目的使用Beckman ICS—Ⅱ免疫分析仪测定尿微量白蛋白,对该测定方法及临床应用进行评价。方法运用手动速率散射比浊法测出抗原和抗体结合的速率峰值,其高低与抗原的含量成正相关。结果通过对糖尿病、高血压、动脉硬化、健康人群检测尿微量白蛋白,证明该法具有准确,快速,经济,操作简单,效果满意,其相关系数在0．97以上。结论Bekman ICS—Ⅱ免疫分析仪测定尿微量白蛋白,结果可靠,可用于对糖尿病、高血压等相关疾病的病情监测,评估,疗效观察等。     

Microplate measurement of urinary albumin and creatinine

We describe microplate methods for measurement of human urinary albumin (HUA) by competitive enzyme-linked immunosorbant assay (ELISA) and creatinine with a modified commercial enzymatic kit. Incorporation of substrate mixing into the competitive ELISA changes the dynamic absorbance-concentration response, greatly simplifying calculations and improving sensitivity and accuracy. Measurement of creatinine in urine and plasma samples with a commercially available enzymatic kit modified for analysis by use of an inexpensive microplate reader produced values comparable in precision and accuracy to those obtained by an automated kinetic Jaffé method.     

Reactivity of urinary albumin (microalbumin) assays with fragmented or modified albumin

BACKGROUND: Controversy exists regarding occurrence and measurement of structural variants of albumin in urine. In this study, we examined cross-reactivity of in vitro modified albumins in assays for urine albumin (microalbumin). METHODS: We analyzed albumin modified by reagents, trypsin, or physical treatments or differing in primary sequence (animal albumins) with an immunoturbidimetric assay (Beckman LX20) using goat antiserum and a competitive immunoassay (Siemens Immulite) using a monoclonal antibody. We assessed occurrence of albumin fragments in urine by use of Western blotting of 24 specimens. RESULTS: Chemical modification, modest sequence substitution (gorilla albumin), or cleavage of albumin by cyanogen bromide (CNBr) had little effect on reactivity in the LX20 assay. Albumin extensively cleaved with trypsin retained partial reactivity. The Immulite assay generally was affected more severely by albumin modifications and sequence changes. Western blots of fresh urine specimens or specimens stored at -80 degrees C showed little albumin fragmentation, but some specimens stored for 3 years at -20 degrees C had extensively fragmented albumin that was detected by the LX20 but not the Immulite assay. CONCLUSIONS: Nearly equivalent reactivity of intact albumin and CNBr fragments in the immunoturbidimetric assay indicates reactivity of antibodies with multiple epitopes throughout albumin. Therefore, it is difficult to abolish reactivity of albumin in this type of urine albumin assay. Differential sensitivity of 2 assays to albumin modification identifies a potential source of assay nonequivalence in measuring urinary albumin, particularly for specimens stored at -20 degrees C.     

人群调查中尿白蛋白检测方法的探讨

目的 探讨人群调查中尿白蛋白标本留取及检测方法.方法 选取659名北京市居民,收集其24 h尿测定UAER;并留取次日随机尿和晨尿,分别采用尿ACR及半定量尿白蛋白试纸法测定尿白蛋白.以24 h UAER作为标准,建立应用两种方法检测晨尿和随机尿白蛋白的ROC曲线,比较敏感度、特异度及ROC曲线下面积.结果 晨尿和随机尿ACR分别为9.36(5.12～33.29)mg/g及11.29(6.34～41.29)mg/g,组间比较差异无统计学意义(t=-1.382,P＞0.05),相关分析显示两者高度相关(r=0.932,P＜0.01).晨尿和随机尿ACR与24 h UAER具有高度相关性,相关系数分别为0.853及0.874,P均＜0.01.随机尿ACR诊断白蛋白尿的敏感度为77.9%,特异度为91.0%,晨尿ACR诊断白蛋白尿的敏感度为78.4%,特异度为95.7%.随机尿白蛋白试纸法诊断白蛋白尿的敏感度为90.3%,特异度为41.1%,特异度明显低于ACR法.随机尿与晨尿ACR的ROC曲线下面积分别为0.918±0.012及0.929±0.015,组间比较差异无统计学意义(χ2=2.13,P＞0.05).随机尿白蛋白试纸法ROC曲线下面积0.661±0.021,低于随机尿ACR(χ2=248.41,P＜0.01).结论 随机尿的ACR兼具简便及准确的特点,是人群调查中诊断白蛋白尿的良好指标.

Abstract：

Objective To evaluate the spot urine sample collection method and value of urinary albumin measurement in population survey. Methods Six hundred and fifty-nine Beijing residents were requested to collect 24 h urine for detection of UAER, as well as random spot urine samples and morning urine samples in the next day. Rapid semi-quantitative urinary albumin-specific dipstick and ACR were measured in each spot urine specimen. The 24 h UAER was measured as golden standard to generate ROC curves and evaluate the sensitivity, specificity and AUC of each method. Results The value of ACR in the morning spot urine samples and random spot urine samples were 9. 36(5. 12-33.29) mg/g and 11.29(6. 34-41.29) mg/g respectively and there was no significant difference between these two groups (t = - 1. 382,P＞0.05). The correlation was significant in the two groups (r = 0.932, P ＜ 0.01). The correlation coefficient between ACR in the morning spot urine samples and UAER was 0. 853 (P ＜ 0. 01). The correlation coefficient between ACR in the random spot urine samples and UAER was 0. 874 (P ＜0. 01).The sensitivity and specificity of ACR for diagnosis of albuminuria in the random urine samples were 77. 9% and 91.0%. The sensitivity and specificity of ACR for diagnosis of albuminuria in the morning urine samples were 78. 4% and 95.7%. Concerning the semi-quantitative urinary albumin-specific dipstick, sensitivity and specificity were 90. 3% and 41.1% , respectively. The specificity was much lower than that of ACR. The area under the ROC curves of ACR in the random urine specimens and the morning urine specimens was 0. 918 ±0. 012, 0. 929 ± 0. 015, respectively. There was no statistical difference between these two groups (χ2 =2. 13, P＞0. 05). The area under the ROC curves of semi-quantitative urinary albumin-specific dipstick in the random urine specimens was 0.661 ±0.021, lower than that of ACR (χ2 = 248.41, P＜0.01).Conclusion Measurement of ACR in random urine samples is a reasonable method with simplicity and accuracy for the detection of albuminuria in general population screening program.     

Evaluation of a new radioimmunoassay for urinary albumin

We have evaluated a new commercially available radioimmunoassay kit for albumin determination in urine. The assay is precise; within-run precision (CV) in the clinically significant ranges is 1.8-3.5%, between-run, 1.2-8.5%. The minimum detection limit was 0.6 micrograms/ml. Analytic recovery of different concentrations of albumin added to urine ranged from 98% to 103%. Samples, stored in plastic containers, were stable at room temperature for periods up to 7 days. Mean albumin excretion rates, measured in 20 normal volunteers for 3-h and 24-h periods during the same day were similar (7.1 +/- 4.6 [SD] versus 6.5 +/- 5.0 micrograms/min). In 8 normal subjects, 3-h excretion rates measured daily for 5 days showed no significant variability. In eight insulin-dependent diabetic subjects, albumin excretion measured in short periods of urine collection (3 h) were also in close agreement with 24-h collections (24.7 +/- 28.9 versus 17.6 +/- 18 micrograms/min). From these results it appears that this commercially available kit is suitable for conveniently monitoring microalbuminuria in large numbers of patients in research studies as well as for office practice. Such widespread use should make it possible to better determine the clinical usefulness of this test in the management of diabetic patients.     

A simplified enzyme-linked immunosorbent assay for urinary albumin

A sensitive and simplified competitive binding enzyme linked immunosorbent assay (ELISA) has been developed for quantification of urinary albumin. It was found that, probably because of a high antibody dilution, a conventional ELISA with three incubation steps could be simplified to an assay with only one incubation step without losing sensitivity or precision. The technical conditions of the assay are described. Albumin was coated to the walls of polystyrene microtitre plates. Diluted urine was mixed with rabbit antiserum against albumin and then with alkaline phosphatase conjugated anti-rabbit immunoglobulins. The mixture was then incubated in the microtitre plate for 3 h. After washing the plate, substrate was added and the enzyme activity was measured. Detection limit of the assay was 15 micrograms/l or 1.5 ng. The intra-assay and inter-assay coefficients of variation were 6 and 9%, respectively. The range of 24-h excretion of urinary albumin in apparently healthy subjects was 0.6-27.2 mg.     

类风湿关节炎患者尿微量白蛋白与NAG测定及其意义

笔者对60例尿蛋白定性和肾功能正常的类风湿关节炎患者及60例健康人分别进行尿微量白蛋白、NAG的含量测定，结果类风湿关节炎组尿微量白蛋白和NAG均比对照组明显升高。提示：类风湿关节炎患者肾小球及肾小管有损伤，尿微量白蛋白与NAG测定比尿定性检查更为灵敏，可作为类风湿关节炎患者早期肾损害的敏感指标。     

Methodological evaluation and comparison of five urinary albumin measurements

Background: Microalbuminuria is an indicator of kidney damage and a risk factor for the progression kidney disease, cardiovascular disease, and so on. Therefore, accurate and precise measurement of urinary albumin is critical. However, there are no reference measurement procedures and reference materials for urinary albumin. Methods: Nephelometry, turbidimetry, colloidal gold method, radioimmunoassay, and chemiluminescence immunoassay were performed for methodological evaluation, based on imprecision test, recovery rate, linearity, haemoglobin interference rate, and verified reference interval. Then we tested 40 urine samples from diabetic patients by each method, and compared the result between assays. Results: The results indicate that nephelometry is the method with best analytical performance among the five methods, with an average intraassay coefficient of variation (CV) of 2.6%, an average interassay CV of 1.7%, a mean recovery of 99.6%, a linearity of R=1.00 from 2 to 250 mg/l, and an interference rate of <10% at haemoglobin concentrations of <1.82 g/l. The correlation (r) between assays was from 0.701 to 0.982, and the Bland–Altman plots indicated each assay provided significantly different results from each other. Conclusion: Nephelometry is the clinical urinary albumin method with best analytical performance in our study. J. Clin. Lab. Anal. 25:324–329, 2011. © 2011 Wiley‐Liss, Inc.     

Analytical validation of an HPLC assay for urinary albumin

BACKGROUND: Microalbuminuria is the earliest clinical finding for renal disease. Diabetic individuals often produce modified forms of albumin, perhaps due to impaired lysosomal processing, that are undetectable by common immunoassays but accurately measured by HPLC. METHODS: We evaluated the performance of a commercially available, FDA-approved HPLC assay (AusAm Biotechnologies, NY) and compare results to our immunoturbidimetric assay (ITA, Beckman-Coulter, CA) using random urine specimens from 32 nondiabetic and 60 type 1 and 2 diabetic subjects. RESULTS: The HPLC assay was linear to 963 mg/l with a limit of detection of 6.1 mg/l. Within-run and between-run precision was <2% and 7-10%, respectively. Unpreserved urine was stable for at least 3 days at room temperature and 10 days at 4 degrees C. In both diabetic and nondiabetic subjects urinary albumin concentrations were higher by HPLC than by ITA, and many more diabetic and nondiabetic individuals were classified as microalbuminuric by HPLC than by ITA. The HPLC assay showed acceptable performance; however, because urinary albumin concentrations are higher in apparently healthy nondiabetic as well as diabetic subjects, different cutpoints will be necessary to accurately differentiate microalbuminuria. CONCLUSIONS: Prospective studies are necessary to determine whether the HPLC assay can effectively detect microalbuminuria earlier than current assays without a concomitant increase in the false positive rate.     

Relationship between urinary albumin and albumin/creatinine ratio during normal pregnancy and pre-eclampsia

Pre-eclampsia is a serious complication of pregnancy and it is important to detect the condition as early as possible. Albuminuria is an important symptom of pre-eclampsia and repeated urine analyses to screen for the condition are part of the standard antenatal care. The purpose of this study was to investigate whether measurement of the urine albumin/creatinine ratio in spot samples could be a complement to the dipstick method and could reduce the need for 24-h urine collections. Urine samples were collected for 24 h in weeks 12, 24 and 36 of pregnancy from both normotensive women and women who developed hypertension or who had pregnancy-induced hypertension (PIH) when they entered the study. The 24-h albumin excretion was significantly correlated to the albumin/creatinine ratio in all measurements (Pearson correlation coefficient). In week 12, the values were: n = 44, r = 0.964, p < 0.001 (normotensive group) and in the PIH group: n = 8, r = 0.789, p < 0.05. In week 24, the correlation values were r = 1.0 and p < 0.001 in both the normotensive group (n = 41) and in the PIH group (n = 11). In week 36 the correlation values were r = 0.791 and p < 0.001 in the normotensive group (n = 39) and r = 1.0 and p < 0.001 in the PIH group (n = 16). Microalbuminuria was defined as urine albumin excretion higher than 30 mg/24 h and this corresponded to an albumin/creatinine ratio of 2.9. Microalbuminuria was found in three persons in the PIH group and in two persons in the normotensive group. Overt albuminuria (> 300 mg/24 h) was found in one of the 46 normotensive women (2%) and in 3 of the 19 PIH women (16%). In all these women the high albumin values had been detected by using the albumin/creatinine ratio method. In conclusion, it has been found that the albumin excretion in urine correlates significantly to the albumin/creatinine ratio during pregnancy. The urinary albumin/creatinine ratio appears to be a good alternative to the dipstick method and to 24-h urine collections.