Portal de Periódicos da CAPES

Detection of urinary albumin

2005; Elsevier BV; Volume: 12; Issue: 2 Linguagem: Inglês

10.1053/j.ackd.2005.01.012

1548-5609

Wayne D. Comper, Tanya M. Osicka,

Renal function and acid-base balance

Microalbuminuria is an important clinical marker in patients with diabetes and cardiovascular disease. The concentration of albumin in urine has traditionally been measured by semiquantitative dipsticks or by various quantitative immunochemical methods such as immunonephelometry, immunoturbidimetry, and radioimmunoassay. However, until recently, urinary albumin not reabsorbed by proximal tubular cells was assumed to be excreted intact. Studies have now revealed that the nature of urinary albumin is complex and is excreted as a mixture of intact albumin, albumin-derived peptides that are not detected by routine dipstick and antibody-based tests, and a species of intact albumin (immunounreactive albumin), also not detected by dipstick and antibody-based tests. A new test, Accumin, based on high-performance liquid chromatography analysis, is able to detect all the immunoreactive intact albumin and immunounreactive intact albumin (total intact albumin) in urine. The advantage in the use of Accumin over a conventional dipstick test or antibody-based laboratory method for detecting microalbuminuria is that false negatives are reduced and a relatively earlier diagnosis of incipient kidney disease can be achieved. The introduction of Accumin has, therefore, highlighted the need for a global standard in the detection and measurement of microalbuminuria. By detecting all of the immunoreactive and immunounreactive intact albumin in urine, Accumin has virtually invalidated the use of dye and immunologically-based dipstick tests and immunologically-based laboratory methods in screening for microalbuminuria in diabetic patients and in identifying microalbuminuria as a risk factor for cardiovascular disease. Microalbuminuria is an important clinical marker in patients with diabetes and cardiovascular disease. The concentration of albumin in urine has traditionally been measured by semiquantitative dipsticks or by various quantitative immunochemical methods such as immunonephelometry, immunoturbidimetry, and radioimmunoassay. However, until recently, urinary albumin not reabsorbed by proximal tubular cells was assumed to be excreted intact. Studies have now revealed that the nature of urinary albumin is complex and is excreted as a mixture of intact albumin, albumin-derived peptides that are not detected by routine dipstick and antibody-based tests, and a species of intact albumin (immunounreactive albumin), also not detected by dipstick and antibody-based tests. A new test, Accumin, based on high-performance liquid chromatography analysis, is able to detect all the immunoreactive intact albumin and immunounreactive intact albumin (total intact albumin) in urine. The advantage in the use of Accumin over a conventional dipstick test or antibody-based laboratory method for detecting microalbuminuria is that false negatives are reduced and a relatively earlier diagnosis of incipient kidney disease can be achieved. The introduction of Accumin has, therefore, highlighted the need for a global standard in the detection and measurement of microalbuminuria. By detecting all of the immunoreactive and immunounreactive intact albumin in urine, Accumin has virtually invalidated the use of dye and immunologically-based dipstick tests and immunologically-based laboratory methods in screening for microalbuminuria in diabetic patients and in identifying microalbuminuria as a risk factor for cardiovascular disease. Microalbuminuria, defined as an albumin excretion rate (AER) between 20 and 200 μg/min (30–300 mg/d), is an important clinical marker in patients with diabetes because of its established association with progressive kidney disease.1American Diabetes AssociationDiabetic nephropathy.Diabetes Care. 2003; 26: S94-S98PubMed Google Scholar, 2The ATLANTIS Study GroupLow dose ramipril reduces albuminuria in type 1 diabetic patients without hypertension.Diabetes Care. 2000; 23: 1823-1829Crossref PubMed Scopus (100) Google Scholar, 3Parving H.-H. Lehnert H. Bröchner-Motensen J. et al.The effect of irbesartan on the development of diabetic nephropathy in patients with type 2 diabetes.N Engl J Med. 2001; 345: 870-878Crossref PubMed Scopus (2948) Google Scholar However, microalbuminuria is also becoming increasingly recognized as an independent risk factor for cardiovascular disease in patients with or without diabetes.1American Diabetes AssociationDiabetic nephropathy.Diabetes Care. 2003; 26: S94-S98PubMed Google Scholar, 4Heart Outcomes Prevention Evaluation (HOPE) Study InvestigatorsEffects of ramipril on cardiovascular and microvascular outcomes in people with diabetes mellitus Results of the HOPE study and MICRO-HOPE substudy.Lancet. 2000; 355: 253-259Abstract Full Text Full Text PDF PubMed Scopus (3277) Google Scholar, 5Mogensen C.E. Microalbuminuria and hypertension with focus on type 1 and type 2 diabetes.J Intern Med. 2003; 254: 45-66Crossref PubMed Scopus (200) Google Scholar Importantly, reduction of AER with angiotensin-converting enzyme (ACE) inhibitors and, more recently, with angiotensin II receptor antagonists correlates with a plateau of or a decrease in the rate of decline of kidney function that is independent of the antihypertensive effect of these agents.2The ATLANTIS Study GroupLow dose ramipril reduces albuminuria in type 1 diabetic patients without hypertension.Diabetes Care. 2000; 23: 1823-1829Crossref PubMed Scopus (100) Google Scholar, 3Parving H.-H. Lehnert H. Bröchner-Motensen J. et al.The effect of irbesartan on the development of diabetic nephropathy in patients with type 2 diabetes.N Engl J Med. 2001; 345: 870-878Crossref PubMed Scopus (2948) Google Scholar, 4Heart Outcomes Prevention Evaluation (HOPE) Study InvestigatorsEffects of ramipril on cardiovascular and microvascular outcomes in people with diabetes mellitus Results of the HOPE study and MICRO-HOPE substudy.Lancet. 2000; 355: 253-259Abstract Full Text Full Text PDF PubMed Scopus (3277) Google Scholar, 6Mogensen C.E. Viberti G. Halimi S. et al.Effects of low-dose perindopril/indapamide on albuminuria in diabetes: Preterax in albuminuria regression: PREMIER.Hypertension. 2003; 41: 1063-1071Crossref PubMed Scopus (166) Google Scholar Therefore, routine screening to detect patients at risk for microalbuminuria is critical to patient care. The American Diabetes Association recommends that annual microalbuminuria testing should be performed in diabetic patients without clinical proteinuria 5 years after the diagnosis of type 1 diabetes and at the time of diagnosis of type 2 diabetes.7Sacks D.B. Bruns D.E. Goldstein D.E. et al.Guidelines and recommendations for laboratory analysis in the diagnosis and management of diabetes mellitus.Clin Chem. 2002; 48: 436-472PubMed Google Scholar Microalbuminuria has traditionally been screened in the office setting by use of a variety of semiquantitative dipsticks. These tests involve wetting a chemically impregnated test strip with urine. For example, Clinitek Microalbumin dipsticks (Bayer Corporation, Tarrytown, NY) detect microalbuminuria by albumin binding to a sulfonephthalein dye, which produces a color reaction that is read reflectometrically in a Clinitek 50 portable urine chemistry analyzer (Bayer Corporation, Tarrytown, NY). Comparisons of this dipstick to reference laboratory tests have shown that the sensitivity (true positive rate = true positives/true positives + false positives) ranges from 79% to 95.4% and that the specificity (true negative rate = true negatives/true negatives + false positives) ranges from 73% to 81%.8Le Floch J.P. Marre M. Rodier M. et al.Interest of Clinitek Microalbumin in screening for micro-albuminuria Results of a multicenter study in 302 diabetic patients.Diab Metab. 2001; 27: 36-39PubMed Google Scholar, 9Meinhardt U. Ammann R.A. Fluck C. et al.Microalbuminuria in diabetes mellitus Efficacy of a new screening method in comparison with timed overnight urine collection.J Diabetes Comp. 2003; 17: 254-257Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 10Parsons M. Newman D.J. Pugia M. et al.Performance of a reagent strip device for quantitation of the urine albumin:creatinine ratio in a point of care setting.Clin Nephrol. 1999; 51: 220-227PubMed Google Scholar Another dipstick test is the Micral-Test II test strip (Boehringer Mannheim, Indianapolis, IN). In this test, albumin passes via a wick fleece into a conjugate fleece, where it binds to specific, gold-labeled antibodies, and then flows to a detection pad.10Parsons M. Newman D.J. Pugia M. et al.Performance of a reagent strip device for quantitation of the urine albumin:creatinine ratio in a point of care setting.Clin Nephrol. 1999; 51: 220-227PubMed Google Scholar, 11Mogensen C.E. Viberti G.C. Peheim E. et al.Multicenter evaluation of the Micral-Test II test strip, an immunologic rapid test for the detection of microalbuminuria.Diabetes Care. 1997; 20: 1642-1646Crossref PubMed Scopus (53) Google Scholar A chemical reaction in the detection pad produces a color that is compared visually with color blocks. Comparisons of this test to reference laboratory results have shown that the sensitivity ranges from 93% to 97.1%, and the specificity ranges from 33.3% to 81%.10Parsons M. Newman D.J. Pugia M. et al.Performance of a reagent strip device for quantitation of the urine albumin:creatinine ratio in a point of care setting.Clin Nephrol. 1999; 51: 220-227PubMed Google Scholar, 11Mogensen C.E. Viberti G.C. Peheim E. et al.Multicenter evaluation of the Micral-Test II test strip, an immunologic rapid test for the detection of microalbuminuria.Diabetes Care. 1997; 20: 1642-1646Crossref PubMed Scopus (53) Google Scholar, 12Gilbert R.E. Akdeniz A. Jerums G. Detection of microalbuminuria in diabetic patients by urinary dipstick.Diabetes Res Clin Pract. 1997; 35: 57-60Abstract Full Text PDF PubMed Scopus (19) Google Scholar Traditionally, 3 laboratory methods, immunonephelometry, immunoturbidimetry, and radioimmunoassay, have been used for the confirmation and quantitative measurement of microalbuminuria. The performance characteristics of these methods are listed in Table 1. Briefly, the immunonephelometry technique involves albumin in the urine sample coming into contact with an anti-human albumin antibody to produce an antigen-antibody reaction. An increase in light scatter from this reaction is analyzed optimetrically to provide a urinary albumin concentration. The immunoturbidimetry technique involves albumin in the urine sample and human albumin, bound to latex particles, competing for a monoclonal antibody, which aggregates the latex particles. Consequently, the amount of aggregation that results is in inverse proportion to amount of albumin in the urine sample. The aggregation amount is measured optimetrically and converted mathematically to urinary albumin concentration. The radioimmunoassay technique involves albumin in the urine sample displacing isotopically labeled human albumin that has an antibody bound to it. Consequently, the amount of labeled albumin that remains bound to the antibody is in inverse proportion to the amount of albumin in the sample. The “free” and “bound” labeled albumin can be separated in several ways for radioactivity measurement. Radioactive counts are compared with a calibration or a standard curve to provide urinary albumin concentration.Table 1Performance Characteristics of Immunonephelometry, Immunoturbidimetry, and Radioimmunoassay Methods Used for the Detection and Measurement of Microalbuminuria in Persons With DiabetesAssaysInterassay Coefficients of VariationDetection LimitFalse Negatives v HPLCFalse Positives v HPLCRadioimmunoassay9.2% at 12.2 mg/L16 μg/L23%0%4.8% at 33 mg/LImmunonephelometry by use of Beckman Array Analyser4.2% at 12.1 mg/L2 mg/LND (1)ND5.3% at 45 mg/LImmunoturbidimetry by use of Dade-Behring Turbitimer4.1% at 10.6 mg/L6 mg/L36%0%2.2% at 43.2 mg/L4.2% at 77.9 mg/LImmunoturbidimetry by use of Dade-Behring Dimension RxL8.5% at 8 mg/L6 mg/LNDNDChemistry Analyser3.4% at 35 mg/LHPLC2.4% at 95.8 mg/L2 mg/LAbbreviation: ND, not determined.Data from Osicka and Comper.36Osicka T.M. Comper W.D. Characterization of immunochemically nonreactive urinary albumin.Clin Chem. 2004; 50: 2286-2291Crossref PubMed Scopus (93) Google Scholar Open table in a new tab Abbreviation: ND, not determined. Data from Osicka and Comper.36Osicka T.M. Comper W.D. Characterization of immunochemically nonreactive urinary albumin.Clin Chem. 2004; 50: 2286-2291Crossref PubMed Scopus (93) Google Scholar Comparisons of these laboratory methods for the detection and measurement of albumin in the urine of persons with diabetes have demonstrated that the results from these methods can vary considerably from one another. For example, a greater than 6-fold difference was found for urinary albumin concentrations measured by immunoturbidimetry (Urin-Pak Immuno Microalb, Miles Laboratories, UK) compared with radioimmunoassay (Albumin RIA, Pharmacia, Sweden),13Giampietro O. Penno G. Clerico A. et al.Which method for quantifying “microalbuminuria” in diabetics? Comparison of several immunological methods (immunoturbidimetric assay, immunonephelometric assay, radioimmunoassay and two semiquantitative tests) for measurement of albumin in urine.Acta Diabetol. 1992; 28: 239-245Crossref PubMed Scopus (35) Google Scholar a 2.5-fold difference was found by use of immunoturbidimetry (Albufast, Amplifon Divisione Biomedica, Italy) compared with radioimmunoassay (Albumin RIA, Pharmacia, Sweden),13Giampietro O. Penno G. Clerico A. et al.Which method for quantifying “microalbuminuria” in diabetics? Comparison of several immunological methods (immunoturbidimetric assay, immunonephelometric assay, radioimmunoassay and two semiquantitative tests) for measurement of albumin in urine.Acta Diabetol. 1992; 28: 239-245Crossref PubMed Scopus (35) Google Scholar a 1.4-fold difference was found by use of immunonephelometry (Istituto Behring, Italy) compared with radioimmunoassay,13Giampietro O. Penno G. Clerico A. et al.Which method for quantifying “microalbuminuria” in diabetics? Comparison of several immunological methods (immunoturbidimetric assay, immunonephelometric assay, radioimmunoassay and two semiquantitative tests) for measurement of albumin in urine.Acta Diabetol. 1992; 28: 239-245Crossref PubMed Scopus (35) Google Scholar and a 1.6-fold difference was found by use of immunonephelometry (Array 360 analyzer loaded with reagents from Beckman, Ireland) compared with immunoturbidimetry (aca IV analyzer loaded with reagents from Dade, USA).14Roberts W.L. Calcote C.B. Cook C.B. et al.Comparison of four commercial urinary albumin (microalbumin) methods Implications for detecting diabetic nephropathy using random urine specimens.Clin Chim Acta. 1998; 273: 21-33Crossref PubMed Scopus (29) Google Scholar Numerous other studies have found similar levels of variation between different immunoassays.15Watts G.F. Bennett J.E. Rowe D.J. et al.Assessment of immunochemical methods for determining low concentrations of albumin in urine.Clin Chem. 1986; 32: 1544-1548PubMed Google Scholar, 16Tiu S.C. Lee S.S. Cheng M.W. Comparison of six commercial techniques in the measurement of microalbuminuria in diabetic patients.Diabetes Care. 1993; 16: 616-620Crossref PubMed Scopus (50) Google Scholar The concentration of albumin in urine has traditionally been measured by semiquantitative dipsticks or by various quantitative immunochemical methods (see above). However, until recently, albumin not reabsorbed by proximal tubular cells was assumed to be excreted intact and detected by specific immunoassays and dye-based dipsticks. This assumption, however, has proved to be incorrect. Studies in rats and in humans have demonstrated that albumin that appears in urine is significantly degraded (>95%) to peptide fragments.17Osicka T.M. Comper W.D. Protein degradation during renal passage in normal kidneys is inhibited in experimental albuminuria.Clin Sci. 1997; 93: 65-72Crossref PubMed Scopus (56) Google Scholar, 18Osicka T.M. Houlihan C.A. Chan J.G. et al.Albuminuria in patients with type 1 diabetes is directly linked to changes in the lysosome-mediated degradation of albumin during renal passage.Diabetes. 2000; 49: 1579-1584Crossref PubMed Scopus (138) Google Scholar, 19Osicka T.M. Pratt L.M. Comper W.D. Glomerular capillary wall permeability to albumin and horseradish peroxidase.Nephrology. 1996; 2: 199-212Crossref Scopus (82) Google Scholar, 20Burne M.J. Panagiotopoulos S. Jerums G. et al.Alterations in renal degradation of albumin in early experimental diabetes in the rat A new factor in the mechanism of albuminuria.Clin Sci. 1998; 95: 67-72Crossref PubMed Scopus (58) Google Scholar, 21Gudehithlu K.P. Pegoraro A.A. Dunea G. et al.Degradation of albumin by the renal proximal tubule cells and the subsequent fate of its fragments.Kidney Int. 2004; 65: 2113-2122Crossref PubMed Scopus (83) Google Scholar Size exclusion chromatography and differential filtration through size-selective membranes has revealed that the albumin peptide fragments in urine are in the molecular weight range of 200 to 10,000 Da. A representative size-exclusion chromatographic profile from Sephadex G-100 of tritium labeled albumin ([3H]albumin) in urine collected from a healthy volunteer (Fig 1A) and from a diabetic patient with albuminuria (Fig 1B) analyzed 24 hours after intravenous injection of [3H]albumin into the circulation is shown in Figure 1. Figure 1A reveals that most of the [3H]albumin derived material is degraded to small fragments in the molecular weight range of 200 to 10,000 Da. Figure 1B reveals that the intact albumin to albumin peptide fragment ratio increases in urine collection from diabetic patients with albuminuria. Immunochemical-based assays as well as dye-binding dipsticks cannot measure albumin peptide fragments in urine. The ultimate effect of such underestimation of the net amount of albumin excreted in the urine is enormous. Conventional immunoassays suggest that normal humans would only excrete less than 30 mg/d of albumin, whereas, in reality, the amount of albumin-derived material excreted may range from 1 to 3 g/d.18Osicka T.M. Houlihan C.A. Chan J.G. et al.Albuminuria in patients with type 1 diabetes is directly linked to changes in the lysosome-mediated degradation of albumin during renal passage.Diabetes. 2000; 49: 1579-1584Crossref PubMed Scopus (138) Google Scholar, 22Greive K.A. Balazs N.D. Comper W.D. Protein fragments in urine have been considerably underestimated by various protein assays.Clin Chem. 2001; 47: 1717-1719PubMed Google Scholar, 23Eppel G.A. Nagy S. Jenkins M.A. et al.Variability of standard clinical protein assays in the analysis of a model urine solution of fragmented albumin.Clin Biochem. 2000; 33: 487-494Crossref PubMed Scopus (43) Google Scholar. These studies demonstrate that large quantities of low-molecular-weight proteinaceous material, derived from postfiltration processing, exists in urine that had not been recognized previously. Although the presence of peptides and trichloroacetic acid (TCA) soluble radiolabelled material originated from circulating proteins such as albumin were previously identified in urine,24Katz J. Rosenfeld S. Sellers A.L. Role of the kidney in plasma albumin catabolism.Am J Physiol. 1960; 198: 814-818PubMed Google Scholar, 25Lou M.F. Hamilton P.B. Separation and quantitation of peptides and amino acids in normal human urine.Meth Bioch Anal. 1979; 25: 203-271Crossref PubMed Google Scholar, 26Strobel J.L. Cady S.G. Borg T.K. et al.Identification of fibroblasts as a major site of albumin catabolism in peripheral tissues.J Biol Chem. 1986; 261: 7989-7994Abstract Full Text PDF PubMed Google Scholar, 27Yedgar S. Carew T.E. Pittman R.C. et al.Tissue sites of catabolism of albumin in rabbits.Am J Physiol. 1983; 244: E101-E107PubMed Google Scholar it was generally considered that these had been derived through extra renal degradation. The degradation process occurs in cells distal to the glomerular basement membrane, most probably in tubular epithelial cells, where albumin is endocytosed and trafficked to lysosomes. Once degraded, albumin is exocytosed into the tubular lumen and excreted in urine. The degradation products are exclusively found in urine and not in blood. The degradation process appears to be partially impaired in diabetes. The ratio of intact to albumin peptides increases with increasing albuminuria in diabetes both in rats20Burne M.J. Panagiotopoulos S. Jerums G. et al.Alterations in renal degradation of albumin in early experimental diabetes in the rat A new factor in the mechanism of albuminuria.Clin Sci. 1998; 95: 67-72Crossref PubMed Scopus (58) Google Scholar, 28Osicka T.M. Yu Y. Panagiotopoulos S. et al.Prevention of albuminuria by aminoguanidine or ramipril in STZ-diabetic rats is associated with the normalisation of glomerular protein kinase C.Diabetes. 2000; 49: 87-93Crossref PubMed Scopus (121) Google Scholar, 29Russo L.M. Osicka T.M. Brammar G.C. et al.The renal processing of albumin in diabetes and hypertension in rats Possible role of TGF-β.Am J Nephrol. 2003; 23: 61-70Crossref PubMed Scopus (36) Google Scholar, 30Russo L.M. Brammar G.C. Jerums G. et al.The effect of ramipril on excessive albumin fragment excretion in diabetes and hypertension The role of increased lysosomal activity and decreased TGF-β expression.J Hypertens. 2003; 21: 1-10Crossref PubMed Scopus (23) Google Scholar and in humans (Fig 1B).18Osicka T.M. Houlihan C.A. Chan J.G. et al.Albuminuria in patients with type 1 diabetes is directly linked to changes in the lysosome-mediated degradation of albumin during renal passage.Diabetes. 2000; 49: 1579-1584Crossref PubMed Scopus (138) Google Scholar At present, no diagnostic tools routinely measure albumin peptide fragments in urine. However, contemporary studies that utilize capillary electrophoresis coupled with mass spectrometry have demonstrated a variety of peptides in normal urine and in urine of patients with kidney disease.31Wittke S. Fliser D. Haubitz M. et al.Determination of peptides and proteins in human urine with capillary electrophoresis-mass spectrometry, a suitable tool for the establishment of new diagnostic markers.J Chromatography. 2003; 1013: 173-181Crossref Scopus (177) Google Scholar, 32Weissinger E.M. Wittke S. Kaiser T. et al.Proteomic patterns established with capillary electrophoresis and mass spectrometry for diagnostic purposes.Kidney Int. 2004; 65: 2426-2434Crossref PubMed Scopus (195) Google Scholar This technique is likely to provide new information as a diagnostic tool because changes in the peptide profile, or “fingerprint,” can reflect renal disease and possibly other diseases, such as cancer. In addition to albumin peptide fragments, intact albumin excreted by the kidney may exist as immunoreactive intact albumin and immunounreactive intact albumin. Unlike the albumin peptide fragments, the immunounreactive intact albumin is approximately the same molecular weight as the immunoreactive intact albumin molecule. This observation has been highlighted in studies of streptozotocin-induced diabetic rats in which radiolabeled albumin was infused into each rat via an osmotic pump. Urinary albumin was found to contain albumin that was undetectable by conventional immunoassays (immunounreactive intact albumin) but detectable by the radioactive tag.33Greive K.A. Eppel G.A. Reeve S. et al.Immuno-unreactive albumin excretion increases in streptozotocin diabetic rats.Am J Kidney Dis. 2001; 38: 144-152Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar Immunounreactive intact albumin has also been found in urine from diabetic patients by use of high-performance liquid chromatography (HPLC) analysis, which measures both immunoreactive intact albumin and immunounreactive intact albumin (total albumin).34Comper W.D. Osicka T.M. Jerums G. High prevalence of immuno-unreactive intact albumin in the urine of diabetic patients.Am J Kidney Dis. 2003; 41: 336-342Abstract Full Text PDF PubMed Scopus (94) Google Scholar, 35Comper W.D. Osicka T.M. Clark M. et al.Earlier detection of microalbuminuria in diabetic patients using a new urinary albumin assay.Kidney Int. 2004; 65: 1850-1855Crossref PubMed Scopus (107) Google Scholar Immunounreactive intact albumin has been isolated from diabetic urine through the use of antialbumin immunosubtraction chromatography and HPLC purification.36Osicka T.M. Comper W.D. Characterization of immunochemically nonreactive urinary albumin.Clin Chem. 2004; 50: 2286-2291Crossref PubMed Scopus (93) Google Scholar The purified preparation was found to contain less than 1% contamination by common urinary proteins (including transferrin, immunoglobulin G, Tamm Horsfall glycoprotein, α1-antitrypsin, and α1-acid glycoprotein) and is stable to freezing and frequent freeze/thaw cycles.36Osicka T.M. Comper W.D. Characterization of immunochemically nonreactive urinary albumin.Clin Chem. 2004; 50: 2286-2291Crossref PubMed Scopus (93) Google Scholar The immunochemical nonreactive nature of this immunounreactive intact albumin molecule may result from the fact that the epitope is altered by a conformational change caused by incomplete processing in the lysosomal pathway, which is compromised in diabetes but not in healthy subjects,18Osicka T.M. Houlihan C.A. Chan J.G. et al.Albuminuria in patients with type 1 diabetes is directly linked to changes in the lysosome-mediated degradation of albumin during renal passage.Diabetes. 2000; 49: 1579-1584Crossref PubMed Scopus (138) Google Scholar, 20Burne M.J. Panagiotopoulos S. Jerums G. et al.Alterations in renal degradation of albumin in early experimental diabetes in the rat A new factor in the mechanism of albuminuria.Clin Sci. 1998; 95: 67-72Crossref PubMed Scopus (58) Google Scholar or from the attachment of ligands such as glucose or fatty acids, which are increased in diabetes. In fact, immunounreactive intact albumin has been shown to consist of a limited number of polypeptide-chain scissions, held together by noncovalent intrachain and disulfide bonds, by native polyacrylamide gel electrophoresis (PAGE) and reducing sodium dodecyl sulfate (SDS)-PAGE analysis.36Osicka T.M. Comper W.D. Characterization of immunochemically nonreactive urinary albumin.Clin Chem. 2004; 50: 2286-2291Crossref PubMed Scopus (93) Google Scholar In light of the findings that dye, immunologically-based dipstick, and immunologically-based laboratory methods do not analyze all intact albumin in urine, the potential for false negatives in detecting and measuring microalbuminuria by conventional tests is increased. Accumin (AusAm Biotechnologies, Inc., New York, NY), which is based upon HPLC analysis, substantially lowers this potential for error, particularly in individuals with diabetes mellitus, by detecting and measuring all intact urinary albumin. Accumin works by separating the intact albumin molecules in urine from other proteins by passing the urine through a column that contains size-selective gel material. The size (and shape) of the proteins determine their rate of flow through the column; thus, albumin is separated from other proteins on the basis of its size. The ability of protein to absorb ultraviolet light is exploited to directly quantify the levels of albumin present in the urine as it leaves the end of the column. The Accumin test performs well. It has an interassay coefficient of 2.4% at a urinary albumin level of 95.8 mg/L and an albumin detection limit of 2 mg/L.37Comper W.D. Jerums G. Osicka T.M. Differences in urinary albumin detected by four immunoassays and high performance liquid chromatography.Clin Biochem. 2004; 37: 105-111Crossref PubMed Scopus (81) Google Scholar The advantage of the use of Accumin over conventional dipstick and laboratory methods for the detection of microalbuminuria has been investigated. False negatives for the detection of microalbuminuria by the Clinitek Microalbumin dipsticks versus Accumin was determined to be 42.9% for urine collected from 115 diabetic patients.38Comper W.D. Jerums G. Osicka T.M. Deficiency in the detection of microalbuminuria by urinary dipstick in diabetic patients.Diabetes Care. 2003; 26: 3195-3196Crossref PubMed Scopus (19) Google Scholar The detection of microalbuminuria by immunoturbidimetry versus Accumin resulted in 36% false negatives for urine samples collected from 115 diabetic patients,37Comper W.D. Jerums G. Osicka T.M. Differences in urinary albumin detected by four immunoassays and high performance liquid chromatography.Clin Biochem. 2004; 37: 105-111Crossref PubMed Scopus (81) Google Scholar and microalbuminuria detected by radioimmunoassay versus Accumin resulted in 23% false negatives for urine collected from 96 diabetic patients.37Comper W.D. Jerums G. Osicka T.M. Differences in urinary albumin detected by four immunoassays and high performance liquid chromatography.Clin Biochem. 2004; 37: 105-111Crossref PubMed Scopus (81) Google Scholar The differential lead times for detecting microalbuminuria for Accumin versus radioimmunoassay were determined for type 1 and type 2 diabetic patients.35Comper W.D. Osicka T.M. Clark M. et al.Earlier detection of microalbuminuria in diabetic patients using a new urinary albumin assay.Kidney Int. 2004; 65: 1850-1855Crossref PubMed Scopus (107) Google Scholar Analysis was performed on 511 urine samples collected over a 13-year period from a total of 42 patients with type 1 diabetes, 17 progressors and 25 nonprogressors. The mean leadtime for Accumin versus radioimmunoassay for these patients was 3.9 years (95% confidence interval of 2.1 to 5.6 years). Analysis was also performed on 634 urine samples collected over the same period from a total of 49 patients with type 2 diabetes, 24 progressors and 25 nonprogressors. The mean leadtime for Accumin versus radioimmunoassay for these patients was 2.4 years (95% confidence interval of 1.2 to 3.5 years). These results not only identify that progression from normoalbuminuria to microalbuminuria in diabetic patients is associated with an increase in urinary immunochemically nonreactive albumin but also raise the possibility that measurement of total albumin may allow earlier detection of progression to kidney disease.35Comper W.D. Osicka T.M. Clark M. et al.Earlier detection of microalbuminuria in diabetic patients using a new urinary albumin assay.Kidney Int. 2004; 65: 1850-1855Crossref PubMed Scopus (107) Google Scholar Urinary albumin concentration measured by immunonephelometry and HPLC were compared by analysis of 24-hour urine samples collected from 1,484 subjects in the PREVEND study.39Brinkman J.W. Bakker S.J. Gansevoort R.T. et al.Which method for quantifying urinary albumin excretion gives what outcome? A comparison of immunonephelometry with high-performance liquid chromatography.Kidney Int. 2004; 66: S69-S75Crossref Scopus (81) Google Scholar Whereas 13 of 666 subjects (2.0%) who were classified as normoalbuminuric by HPLC were classified as microalbuminuric by immunonephelometry, 337 of 986 subjects (34.2%) who were classified as normoalbuminuric by immunonephelometry were classified as microalbuminuric by HPLC. Mean urinary albumin concentrations for the 998 subjects who would have been considered normoalbuminuric by immunonephelometry were 6.78 mg/L for immunonephelometry and 17.6 mg/L for HPLC. Furthermore, logistic regression analyses with an abnormal ankle-brachial index as outcome parameter revealed adjusted odds ratios of 1.78 (95% CI 1.01–3.12, P < 0.05) and 4.67 (95% CI 1.68–12.9, P < .05), respectively, for microalbuminuria and macroalbuminuria as determined by HPLC, compared with 1.37 (95% CI 0.77–2.41, P = NS) and 3.85 (95% CI 1.53–9.67, P < .05), respectively for nephelometry.39Brinkman J.W. Bakker S.J. Gansevoort R.T. et al.Which method for quantifying urinary albumin excretion gives what outcome? A comparison of immunonephelometry with high-performance liquid chromatography.Kidney Int. 2004; 66: S69-S75Crossref Scopus (81) Google Scholar The introduction of Accumin has highlighted the need for a global standard in the detection and measurement of microalbuminuria. The discrepancy between Accumin and immunochemical-based assays demonstrates that Accumin may provide a relatively early diagnosis of incipient kidney disease.35Comper W.D. Osicka T.M. Clark M. et al.Earlier detection of microalbuminuria in diabetic patients using a new urinary albumin assay.Kidney Int. 2004; 65: 1850-1855Crossref PubMed Scopus (107) Google Scholar, 39Brinkman J.W. Bakker S.J. Gansevoort R.T. et al.Which method for quantifying urinary albumin excretion gives what outcome? A comparison of immunonephelometry with high-performance liquid chromatography.Kidney Int. 2004; 66: S69-S75Crossref Scopus (81) Google Scholar By detecting all of the immunoreactive and immunounreactive intact albumin in urine, it has virtually invalidated the use of dye and immunologically-based dipstick tests and immunologically-based laboratory methods in screening for microalbuminuria in diabetic patients and in identifying microalbuminuria as a risk factor for cardiovascular disease.40Busby D.E. Bakris G.L. Comparison of commonly used assays for the detection of microalbuminuria.J Clin Hypertens. 2004; 6: 8-12Crossref Scopus (36) Google Scholar Further, its measurement of all intact albumin enables clinicians to assess disease severity and monitor therapeutic effectiveness.