Albumin-like material in urine
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Kidney International, Vol. 66, Supplement 92 (2004), pp. S65–S66 Albumin-like material in urine WAYNE D. COMPER and T ANYA M. OSICKA Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia, and AusAm Biotechnologies, Inc., New York, New York Albumin-like material in urine. Recent studies have demon- strated the presence, in both rat and human urine, of a modified form of albumin not detected by conventional antibodies. This modified albumin behaves physicochemically as intact albumin under nondenaturing conditions. We have demonstrated this form of albumin to be disproportionately excreted in microal- buminuric states in diabetes. Quantitation of this modified form of albumin leads to the prediction of the onset of microalbumin- uria in diabetic patients on average 3 to 4 years earlier than when measured by conventional immunoassays. Recent studies have demonstrated that diabetic patients produce urine that contains relatively large quantities of an intact modified form of albumin that is undetected by conventional albumin antibodies. Im- munounreactive albumin behaves physicochemically in terms of size, shape, and charge like native albumin. This study examines in more detail the nature of this intact modified form of albumin. METHODS Intact modified form of albumin, found at relatively high levels in the urine of diabetic patients, has been examined by HPLC, native polyacrylamide gel elec- trophoresis (PAGE), immunoassays, and capillary elec- trophoresis. The modified form of albumin, isolated from urine using affinity chromatography, has also been ana- lyzed. RESULTS AND DISCUSSION Figure 1 shows native PAGE profiles of urine col- lected from diabetic patients over 24 hours. This study demonstrates that the major band in the urine comigrates with the albumin standard. The bands have been con- firmed as albumin by liquid chromatography coupled to an MS/MS instrument (Q-TOF) (performed by the Aus- tralian Proteome Analysis Facility, Sydney, Australia). Key words: diagnostic, early kidney disease, diabetes. C 2004 by the International Society of Nephrology The concentrations of albumin in each of these samples was determined by high performance liquid chromatog- raphy (HPLC, which measures total intact albumin = immunoreactive + immunounreactive). Immunoreactive albumin was determined by radioimmunoassay (RIA). The study clearly indicates that for samples where the immunoassay would measure low or negligible levels of albumin, considerable albumin is present as seen by gel staining and HPLC. A good linear correlation was found between quantitation of the amount of material in the band by densitometric analysis and HPLC, but not RIA. Figure 2 demonstrates that other plasma proteins run quite differently to albumin on native PAGE. Tamm- Horsfall glycoprotein was not detected in diabetic urine, as determined by capillary electrophoresis. Purified mod- ified albumin preparations have also been shown to have nondetectable quantities of these plasma proteins, as well as Na + /K + -ATPase beta subunit, as determined by enzyme-liked immunosorbent assay (ELISA). Various experiments to test the stability of immunoun- reactive albumin have demonstrated that it is stable at both −20 ◦ C and −70 ◦ C for up to a month, and is also stable for at least six freeze-thaw cycles. The potential usefulness of detecting immunounreac- tive albumin has been demonstrated recently in a ret- rospective study [1]. The retrospective study examined the differential lead-time for the development of mi- croalbuminuria as measured by both conventional RIA (measures immunoreactive) and HPLC (measures total albumin = immunoreactive plus immunounreactive). For type 1 progressors, the mean lead-time for the HPLC as- say versus the RIA was 3.9 years, with a 95% confidence interval of 2.1 to 5.6 years. For type 2 progressors, the mean lead-time was 2.4 years with a 95% confidence in- terval of 1.2 to 3.5 years. There was no significant differ- ence between the lead-time analysis between type 1 and type 2 diabetic patients. For the type 1 nonprogressors there was no significant difference (P = 0.056) between the false positives for the HPLC assay (6/280 urines, 2.1%, CI of 0.8–4.6) and RIA (1/280 urines, 0.4%, CI of 0–2.0). For the type 2 nonprogressors, the false positives for the HPLC assay (28/265 urines, 10.6%, CI of 7.1–14.9) were significantly higher (P < 0.0005) than those for the RIA S-65
Transcript of Albumin-like material in urine
Kidney International, Vol. 66, Supplement 92 (2004), pp. S65–S66
Albumin-like material in urine
WAYNE D. COMPER and TANYA M. OSICKA
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia, and AusAm Biotechnologies,Inc., New York, New York
Albumin-like material in urine. Recent studies have demon-strated the presence, in both rat and human urine, of a modifiedform of albumin not detected by conventional antibodies. Thismodified albumin behaves physicochemically as intact albuminunder nondenaturing conditions. We have demonstrated thisform of albumin to be disproportionately excreted in microal-buminuric states in diabetes. Quantitation of this modified formof albumin leads to the prediction of the onset of microalbumin-uria in diabetic patients on average 3 to 4 years earlier than whenmeasured by conventional immunoassays.
Recent studies have demonstrated that diabeticpatients produce urine that contains relatively largequantities of an intact modified form of albumin thatis undetected by conventional albumin antibodies. Im-munounreactive albumin behaves physicochemically interms of size, shape, and charge like native albumin. Thisstudy examines in more detail the nature of this intactmodified form of albumin.
METHODS
Intact modified form of albumin, found at relativelyhigh levels in the urine of diabetic patients, has beenexamined by HPLC, native polyacrylamide gel elec-trophoresis (PAGE), immunoassays, and capillary elec-trophoresis. The modified form of albumin, isolated fromurine using affinity chromatography, has also been ana-lyzed.
RESULTS AND DISCUSSION
Figure 1 shows native PAGE profiles of urine col-lected from diabetic patients over 24 hours. This studydemonstrates that the major band in the urine comigrateswith the albumin standard. The bands have been con-firmed as albumin by liquid chromatography coupled toan MS/MS instrument (Q-TOF) (performed by the Aus-tralian Proteome Analysis Facility, Sydney, Australia).
Key words: diagnostic, early kidney disease, diabetes.
C© 2004 by the International Society of Nephrology
The concentrations of albumin in each of these sampleswas determined by high performance liquid chromatog-raphy (HPLC, which measures total intact albumin =immunoreactive + immunounreactive). Immunoreactivealbumin was determined by radioimmunoassay (RIA).The study clearly indicates that for samples where theimmunoassay would measure low or negligible levels ofalbumin, considerable albumin is present as seen by gelstaining and HPLC. A good linear correlation was foundbetween quantitation of the amount of material in theband by densitometric analysis and HPLC, but not RIA.
Figure 2 demonstrates that other plasma proteins runquite differently to albumin on native PAGE. Tamm-Horsfall glycoprotein was not detected in diabetic urine,as determined by capillary electrophoresis. Purified mod-ified albumin preparations have also been shown to havenondetectable quantities of these plasma proteins, aswell as Na+/K+-ATPase beta subunit, as determined byenzyme-liked immunosorbent assay (ELISA).
Various experiments to test the stability of immunoun-reactive albumin have demonstrated that it is stable atboth −20◦C and −70◦C for up to a month, and is alsostable for at least six freeze-thaw cycles.
The potential usefulness of detecting immunounreac-tive albumin has been demonstrated recently in a ret-rospective study [1]. The retrospective study examinedthe differential lead-time for the development of mi-croalbuminuria as measured by both conventional RIA(measures immunoreactive) and HPLC (measures totalalbumin = immunoreactive plus immunounreactive). Fortype 1 progressors, the mean lead-time for the HPLC as-say versus the RIA was 3.9 years, with a 95% confidenceinterval of 2.1 to 5.6 years. For type 2 progressors, themean lead-time was 2.4 years with a 95% confidence in-terval of 1.2 to 3.5 years. There was no significant differ-ence between the lead-time analysis between type 1 andtype 2 diabetic patients. For the type 1 nonprogressorsthere was no significant difference (P = 0.056) betweenthe false positives for the HPLC assay (6/280 urines, 2.1%,CI of 0.8–4.6) and RIA (1/280 urines, 0.4%, CI of 0–2.0).For the type 2 nonprogressors, the false positives for theHPLC assay (28/265 urines, 10.6%, CI of 7.1–14.9) weresignificantly higher (P < 0.0005) than those for the RIA
S-65
S-66 Comper and Osicka: Albumin-like material in urine
Albumin concentration measured by RIA, mg/L
Albumin concentration measured by HPLC, mg/L
25.4
0.95
5.7
181.
44.
25 24.5
3.76
1.03 20
.421
0.8
Albumin
50.4
6.7
17.9
196.
821
.550
.441
.38.
768
.625
2.2
Fig. 1. Native PAGE analysis of diabetic urines from different indi-vidual patients containing various amounts of immunoreactive intactalbumin and immunounreactive intact albumin. The amount of albu-min determined by HPLC and RIA are shown, as well as the positionof the albumin monomer.
(0/265 urines, 0%, CI of 0–1.4). For the type 1 progressorsthe false negatives for the RIA (67/121 urines, 55.4%, CIof 46.1–64.4) were significantly higher (P < 0.0005) thanthose for the HPLC (0/121 urines, 0%, CI of 0–3.0). Sim-ilarly, for the type 2 progressors the false negatives forthe RIA (78/190 urines, 41.1%, CI of 33.9–48.4) were sig-nificantly higher (P < 0.0005) than those for the HPLC(5/190 urines, 2.6%, CI of 33.9–48.4).
CONCLUSION
These results demonstrate that measurement of totalalbumin including immunounreactive albumin may allow
Transferrin
α1-antitrypsin
α2-hs-glycoprotein
α1-acid glycoprotein
Albumin
Fig. 2. Native PAGE of albumin compared to position of other plasmaprotein standards. Albumin runs differently to a1-antitrypsin, transfer-rin, a1-acid glycoprotein, a2-HS-glycoprotein. IgG did not enter thegel.
earlier detection of microalbuminuria associated with di-abetic nephropathy.
Reprint requests to Wayne D. Comper, AusAm Biotechnologies, 645Madison Avenue, New York, NY 10022.E-mail: [email protected]
REFERENCE
1. COMPER WD, OSICKA TM, CLARK M, et al: Earlier detection of mi-croalbuminuria in diabetic patients using a new urinary albumin as-say. Kidney Int 65:1850–1855, 2004
