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Clinical Chemistry 43: 996-1002, 1997;
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(Clinical Chemistry. 1997;43:996-1002.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

Microalbuminuria and borderline-increased albumin excretion determined with a centrifugal analyzer and the Albumin Blue 580 fluorescence assay

Manfred A. Kessler1, Andreas Meinitzer2, Walter Petek2 and Otto S. Wolfbeis1,a

1 Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, 93040 Regensburg, Germany.

2 Faculty of Medicine, Departments of Clinical Chemistry and Laboratory Medicine, BL I, Karl-Franzens University, 8010 Graz, Austria.
a Author for correspondence. Fax 49-941-943-4064; e-mail Otto.Wolfbeis{at}chemie.uni-regensburg.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We report a new automated fluorescence assay for determination of albumin in urine. The dye Albumin Blue 580 specifically binds to albumin with exhibition of strong red fluorescence. The albumin concentration is calculated from emission intensity at 616 nm (excitation at 590 nm) and a calibration curve. Two Cobas Fara programs cover working ranges of 2–200 and 1–50 mg/L with detection limits of 1.4 and 0.4 mg/L, respectively. Within-run CVs (n = 10) ranged from 1.7% (189 mg/L) to 8.9% (7.2 mg/L) for 2–200 mg/L and from 2.9% (43.3 mg/L) to 5.7% (2.3 mg/L) for the 1–50 mg/L range. A test of urine samples (n = 100) submitted to routine analysis gave results that agreed well with those by the Behring nephelometric assay: AB 580 = 0.922 (± 0.010) BNA + 4.16 (± 0.78). No interference was detected from other urine components, including several proteins and 46 drugs. The high specificity and sensitivity make the method ideal for determination of microalbuminuria. In addition, the method is fast, inexpensive, and well-suited for clinical laboratory application and thus may be used instead of immunoassays.


Key Words: indexing terms: urine • diabetes • renal function • hypertension • atherosclerosis • nephropathy • cardiovascular disease


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Microalbuminuria (MAU) is defined as a urinary albumin excretion between 30 and 300 mg/24 h, i.e., increased above normal but still not detectable by commonly used urine dipstick methods (1).1 During the past decade, detection and determination of MAU have become integral in various areas of healthcare, given numerous studies studies linking MAU and various diseases (for a comprehensive overview, see references 1 and 2).

MAU is an early sign of incipient renal disease and a marker of its progression. Furthermore, MAU is a key indicator of the need for intensified treatment in diabetes mellitus and hypertension (3) and is one of the most powerful predictors of cardiovascular disease in nondiabetic individuals (1)(4)(5).

Patients with insulin-dependent diabetes mellitus are susceptible to developing nephropathy, the peak incidence occurring as late as 15–20 years after the onset of diabetes (6). As is now well established, even a slightly increased excretion of albumin (15–30 mg/24 h) is both diagnostic for incipient renal disease and prognostic for the development of end-stage renal disease and mortality (7)(8)(9). Recent studies show that also in patients with non-insulin-dependent diabetes mellitus, persistent MAU is clearly associated with increased mortality (10)(11).

Apart from its use in monitoring renal function loss and renoprotection in diabetes mellitus, MAU plays a key role as a potential cardiovascular and atherosclerotic risk factor (12)(13). In association with endothelial dysfunction, it is considered a generalized indicator of cardiovascular vulnerability. Hence, the detection of MAU is an early alert to undertake efforts to reduce other cardiovascular risk factors (14).

Broad screening for MAU is justified by a favorable cost-benefit ratio, because it can help identify patients who would benefit from renoprotective therapy and antihypertensive treatment (15). Moreover, evidence indicates that, if detected at an early stage, MAU can be stabilized or even reduced with antihypertensive intervention (16). The American Diabetes Association (17) and the American Kidney Foundation (18) are in consensus that all persons with diabetes should have their urine tested annually for MAU.

In addition to the qualitative detection of overt MAU by dipstick methods, precise quantitative determination of albumin is essential for diagnosis of incipient MAU, for assessing the renal state, for optimizing diabetes care, and for monitoring the success of therapy. Therefore, precise assays for urinary albumin are now becoming inevitable in laboratory medicine (19).

For determination of MAU, the urinary albumin concentration range of interest is 2–200 mg/L, particularly the 15–40 mg/L region that covers the usual cutoff limits between normal and increased albumin excretion (20). Several formats of immunoassays are available that are sufficiently responsive and reliable for albumin determinations at these concentrations, and some are fully automated to analyze large numbers of samples. However, using these methods to assay only a few samples is impractical and expensive, which limits their application in local laboratories. In recent years, handy immunoassay-based test kits have emerged but are designed for screening rather than precise quantification of MAU (21); routine quantifying methods are not yet common.

Recently, the Albumin Blue (AB) series of fluorescent albumin dyes has been described (22). These dyes are capable of targeting albumin highly specifically by forming a strongly fluorescent complex. Unlike the case with other common albumin probes, the fluorescence of the AB complex is enhanced by more than an order of magnitude upon binding to albumin and is not subject to interference by other urinary proteins. The properties of derivatives AB 633 and AB 670 have been exploited to assay urinary albumin at trace concentrations (23)(24). Although that method is comparable with immunonephelometry in terms of detection limits and performance, the rather short shelf lives of AB 633 and AB 670 stock solutions obviate their routine application.

To overcome poor reagent stability, we developed a new derivative (AB 580) with that has better stability without compromising assay performance. The derivative has already been used to determine serum albumin (25). In this work we have implemented the AB 580 assay on the Cobas FaraTM II (Hoffmann-La Roche, Basel, Switzerland) centrifugal analyzer for urine analysis and report our evaluation of this combination as a fast and simple low-cost assay especially designed for precise quantification of MAU.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
instrumentation
The assay was performed with a Cobas Fara II equipped with a 616-nm interference filter (no. 9001042). The nephelometric assay was performed with a Behring Nephelometric Analyzer (BNA; Behring, Marburg, Germany). Absorbance was measured in 1-cm cuvettes with a Beckman Instruments (Fullerton, CA) DU 640 spectrophotometer. Uncorrected fluorescence spectra were recorded with a Shimadzu (Kyoto, Japan) RF-5001 PC spectrofluorophotometer. All measurements were performed at 25 °C.

reagents
We obtained AB 580, ([2-chloro-3-(2,2-dicyanoethenyl)-2-cyclohexen-1-ylidene]methylpropanedinitrile potassium salt (Mr = 306.8), from Molecular Probes (Eugene, OR).

The dye stock (AB 580 71 µmol/L in isopropanol) was prepared as follows: Dissolve solid AB 580 in isopropanol (cat. no. 405-7; Sigma Chemical Co., St. Louis, MO) to give a concentration of ~30 mg/L in a dark blue solution (crude stock). Check the concentration of AB 580 by diluting an aliquot 10-fold with isopropanol and measuring the absorbance at 580 nm. If A580 = 1.0 ± 0.05 (1-cm cuvette, blanked against isopropanol), use the crude stock as the dye stock. Otherwise, dilute the crude stock with isopropanol or add more solid AB 580 and recheck the concentration. The dye stock remains stable for at least 2 years when stored at 4 °C in the dark.

To prepare the working dye solution, dilute 5.0 mL of dye stock to a final volume of 100 mL with an equivolume solution of isopropanol/water. The A580 of this solution is 0.5 ± 0.05, and the reagent is stable for at least 6 months when stored at -20 °C in the dark.

The working buffer was prepared by mixing 3.0 g of 3-N-morpholinopropanesulfonic acid (MOPS free acid; Sigma M-1254), 9.0 g of MOPS sodium salt (Sigma M-9381), 12.0 g of sodium chloride, and 1.0 g of EDTA disodium salt (Sigma E-4884) with 900 mL of distilled water, and 100 mL of isopropanol. The resulting pH is 7.4 ± 0.2.

calibrators and controls
The calibrator diluent was prepared by mixing 2.7 g of KH2PO4, 0.9 g of K2HPO4, 4.5 g of sodium chloride, 0.5 g of EDTA disodium salt, and 50 mg of human IgG (Sigma G-4386), in 500 mL of distilled water. The resulting pH is 6.0 ± 0.5. IgG is a stabilizer, preventing nonspecific adsorption of albumin to the wall of the container. The albumin stock calibrator (2000 mg/L) was prepared by dissolving 10 mg of human serum albumin (essentially fatty acid- and globulin-free, prepared from Cohn Fraction V; Sigma 3782) in 5 mL of calibrator diluent. The concentration was finely adjusted until the A280 was 1.080 ± 0.010 (26) blanked against calibrator diluent. Calibrators were prepared by diluting calibrator stock with calibrator diluent to final concentrations of 2.0, 10, 30, 100, and 200 mg/L (working range 2–200 mg/L) and 1.0, 10, 30, and 50 mg/L (working range 1–50 mg/L), respectively.

N-Protein Standard (SY/OWXI 12/13, lot no. 083604 D; Behring) was used as a calibrator in the nephelometric assay. N/T-Protein Control Serum (SY/M OWXK 12/13, lot no. 083703; Behring) was diluted 1:1000 with calibrator diluent to contain 42.0 mg/L; this solution was used as control material for all assays.

urine samples
We selected centrifuged urine samples from a bulk of samples submitted to routine analysis. After initial measurement by the Cobas Fara, the samples were divided into five categories according to albumin concentrations: A: 0–30 mg/L; B: 30–60 mg/L; C: 60–100 mg/L; D: 100–200 mg/L; and E: >200 mg/L. For method comparison, we drew 30 samples each of categories A and B and 20 samples each of categories C and D by random selection and analyzed with the BNA.

assay procedure
The AB 580 working dye solution and the working buffer were placed in the start reagent cup and the main reagent cup of the Cobas Fara reagent rack, respectively. Before the first assay run, we adjusted the lowest detection limit of the photomultiplier according to the instructions, with the 100 mg/L calibrator. The assay program is summarized in the Appendix.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
properties of ab 580
Like other derivatives of the AB series (22)(23)(24), AB 580 undergoes extensive changes in its fluorescence properties on binding to albumin. Under the conditions of the Cobas Fara assay (excitation at 590 nm, emission at 616 nm), the relatively weak fluorescence in absence of albumin is increased by 17-fold upon binding to albumin. Fig. 1 shows the fluorescence excitation and emission spectra of AB 580 (0.2 µmol/L) both before and after addition of albumin (11.1 mg/L in the cuvette solution). The solutions have the same composition as the cuvette solutions of the Cobas Fara for assaying samples with albumin content of 0 and 200 mg/L, respectively.



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  		Figure 1. Fluorescence excitation (1, 3) and emission (2, 4) spectra of AB 580 in pH 7.4 buffer before (1, 2) and after addition of human serum albumin, 11.1 mg/L (3, 4).

The solutions have the same composition as the cuvette solutions of the Cobas Fara after running albumin samples of 0 and 200 mg/L, respectively.

Compared with derivatives AB 633 and AB 670, dye AB 580 has much better stability as indicated by the times to decomposition of one-half of the amount of dye in pH 7.4 aqueous buffer: 3 days for AB 633, 3 h for AB 670, and 50 days for AB 580 at 20 °C. Moreover, the degradation product of AB 580 does not interfere in the assay. Nonetheless, the improved stability of aqueous stock solutions is still insufficient for routine application. Fortunately, isopropanol solutions are much more stable. Thus, an isopropanol stock of AB 580 did not show any notable degradation after 2 years of storage at 4 °C.

assay performance
Working range and calibration curve.
Two Cobas Fara programs were developed with working ranges of (a) 2–200 mg/L (injected sample volume 15 µL) for a general test covering normoalbuminuria and the full concentration range of MAU, and (b) 1–50 mg/L (injected sample volume 60 µL), for a test with an enhanced detection limit and greater precision at low concentrations, covering normoalbuminuria and albumin concentrations in early MAU (Fig. 2 ). The greater precision is advantageous for determining increased albumin excretion.



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  		Figure 2. Calibration curve of the AB 580 fluorescence assay for determining urinary albumin with the Cobas Fara II.

The working range of 2–200 mg/L covers urinary albumin concentrations encountered in normoalbuminuria and microalbuminuria.

Precision.
For the working range 2–200 mg/L, within-run CVs (n = 10) were 8.9% (7.2 mg/L), 3.0% (26 mg/L), 3.1% (52.2 mg/L), 2.5% (98.9 mg/L), and 1.7% (189 mg/L). Between-run CVs (n = 10; calibration with each run) were 6.4% (15 mg/L), 2.6% (39 mg/L), 3.2% (62 mg/L), and 3.1% (104 mg/L). The detection limit for albumin (3 SD of the blank signal, n = 10, within-run) was 1.4 mg/L in the sample, equivalent to 0.07 mg/L in the cuvette solution.

For the working range 1–50 mg/L, within-run CVs (n = 10) were 5.7% (2.3 mg/L), 2.5% (6.5 mg/L), 3.2% (24.9 mg/L), and 2.9% (43.3 mg/L). Between-run CVs (n = 10; calibration with each run) were 10.1% (2.3 mg/L), 7.1% (6.5 mg/L), 3.8% (24.9 mg/L), and 3.7% (43.3 mg/L). The detection limit for albumin, defined as above, was 0.4 mg/L in the sample.

Recovery.
Values obtained for controls were within the specified reference range, with recoveries between 95% and 100% of the reference value. For analysis of supplemented samples, we added various volumes of calibrator stock to aliquots of urine pools. Analytical recovery of albumin is summarized in Table 1 .\


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  		Table 1. Recovery of albumin in supplemented sample analysis.

Method comparison.
Comparison of the AB 580 fluorescence assay (y) with the BNA immunonephelometric assay (x) gave y = 0.922(± 0.010) x + 4.16 (± 0.78) (n = 100, r = 0.989) with mean y = 63.8 mg/L and mean x = 63.0 mg/L (Fig. 3 ).



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  		Figure 3. AB 580 fluorescence assay (y) and Behring immunonephelometric assay (BNA; x) compared for determination of urinary albumin.

Regression analysis gives y = 0.922 (± 0.010) x + 4.16 (± 0.78); n = 100; r = 0.989.

Interference.
We tested assay interference by supplementing aliquots of urine pools (15 mg/L albumin) with common drugs, vitamins, proteins, and urine metabolites (Table 2 ). The concentrations added were well above the maximum concentrations encountered in clinical practice. The overall mean albumin concentrations of 71 samples tested was 14.95 mg/L (CV 7.9%). Because the albumin concentrations measured were all within 2 SD of the reference value, i.e., 15 ± 1.9 mg/L, we considered that none of the substances interfered significantly. Metformin gave the greatest interference of all substances tested, the albumin concentration in its presence being 13.1 mg/L.


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  		Table 2. Drugs, vitamins, urine metabolites, and proteins tested for interference.

Human proteins globulin, transferrin, Bence Jones protein, plasmin, plasminogen, antitrypsin, fibrinogen, trypsin, and chymotrypsin, as well as soybean inhibitor, bovine pancreatic trypsin inhibitor, and poly(D-lysine) did not interact substantially with AB 580. The presence of hemoglobin at concentrations >100 mg/L (between first visible and overt hematuria) lowered the measured albumin values by as much as 20%. Similarities in amino acid sequence and tertiary structure mean that human and bovine serum albumin as well as other albumin species (except ovalbumin) responded almost equally. Also, micellar solutions of detergents strongly enhanced the fluorescence of AB 580 and therefore strongly interfered in the assay.

Fatty acids displace AB 580 (and vice versa) by competitive binding. Nevertheless, because of the low solubility of free fatty acids in aqueous solution, free AB 580 is always in excess, and the equilibrium is shifted towards bound AB 580. This is indicated by only minor signal changes upon transition from essentially fatty acid free to fatty acid saturated albumin.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Reagent composition.
AB 580 is the first derivative of the Albumin Blue series that provides sufficient stability of its stock solution to allow application in the clinical routine laboratory. Isopropanol was chosen as a solvent because of its stabilizing effect, low toxicity, and lack of effect on the assay. It also is an effective antifreezing agent for dye solutions at -20 °C. The isopropanol content of the working dye solution provides sufficient stability and, at the same time, is low enough to prevent dissolution of the plastic components of the Cobas Fara.

We specify the concentration of the dye stock by A580 rather than weight per volume. The former is more accurate and practical and is independent of the purity of solid AB 580. The concentration of stock solution and the protocol presented here may be changed to improve the detection limit of the assay or to get a wider working range. The assay is performed at pH 7.4, which is within the pH region of optimum performance. Although the assay is not extremely pH-sensitive, the strong buffer used prevents pH shifts from samples with extreme pH values.

Calibration and assay procedure.
The binding of AB 580 to albumin obeys the Law of Mass Action. Therefore, calibration curves are nonlinear and approach a point of saturation at high albumin concentrations. When performed in the upper flat region of the calibration curve, the assay gave unreliable results with unacceptable high CVs. To prevent this situation, we use excess AB 580, which makes the calibration curve steeper and provides an extended reliable working range up to 200 mg/L. Moreover, excess AB 580 suppresses the competitive binding of potential interferents at the albumin binding sites. Apart from the logit-log4 function used by the Cobas Fara, the calibration curve may be approximated by the function y = [Ax/(1 + Bx)] + C, in which x = concentration of albumin in the sample; y = relative fluorescence intensity; and A, B, and C are parameters obtained from a curve fit.

Because the assay was found to depend strongly on temperature, good temperature control is essential. This is achieved by the thermostating feature of the Cobas Fara. Errors arising from minor temperature deviations are compensated for by running calibrators with each run.

Method comparison.
Given the different principles of the methods compared, the fluorescence assay agrees well with the independent BNA immunonephelometric method. In addition, the correlation coefficient (0.989) is comparable with that obtained from comparing two immunological methods (27). Regression analysis gives a y-intercept of 4.2 ± 0.78, which is significantly different from zero, and a slope (0.922 ± 0.010) significantly different from 1.0. Both deviations may be attributed to the different calibrators used in the two methods.

In some cases, low values obtained with the BNA were determined to be falsely negative after dilution and reexamination. These samples were easily identified by the Cobas Fara from their high fluorescence reading and the out-of-range indicator (>200 mg/L).

Features and advantages of the method.
Given a detection limit and a selectivity about equal to those of immunological methods, the AB 580 assay performed with the Cobas Fara has several advantages. (a) It is fast and easy to perform without incubation or pretreatment of urine samples. (b) The extended working range of 2–200 mg/L covers the entire scale of >=MAU without dilution of samples. (c) With no need for expensive antibodies, reagent costs are low, and no laboratory animals are needed for antibody production. (d) Unlike immunoassays, the AB 580 method does not report false negatives if albumin concentrations highly exceed the upper limit of the working range. (e) There is no lot-to-lot variation of the reagent, and the quality of the reagent can be easily checked by measuring its absorption spectrum.

In conclusion, the method presented here is ideally suited for assaying urinary albumin in determining MAU. Furthermore, this method also appears potentially useful for determining albumin at low concentrations in other biological fluids. It is easy to perform, fully automated, and comparable with existing immunoassays in terms of detection limits and performance. Besides the Cobas Fara, the assay may also be performed with other fluorescence instruments, including microplate readers, an approach we are currently investigating.


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  		Table 3. Appendix. Cobas Fara program for urinary albumin (working range 2–200 mg/L).


   Acknowledgments
 
This project was supported by the Austrian Program for Advanced Research and TEchnology (APART) of the Austrian Academy of Sciences. We also thank R. A. Magnotti, Chemistry Laboratory, Department of Pathology and Laboratory Medicine, University of Cincinnati (Ohio) Hospital, Chemistry Laboratory, for stimulating discussions.


   Footnotes
 
1 Nonstandard abbreviations: MAU, microalbuminuria; AB, Albumin Blue; and BNA, Behring Nephelometric Analyzer.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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