1-s2.0-S0009898196064467-main

ELSEVIER Clinica Chimica Acta 258 (1997) 3-20

Serum and urine albumin: a progress report on their measurement and clinical significance

Basil T. Doumas a,*, Theodore Peters Jr. b

aMedical College of Wisconsin, Department of Pathology, P.O. Box 26509, Milwaukee, WI 53226-0509, USA

bResearch Institute, Bassett Healthcare, Cooperstown, NY 13326, USA

Received 10 June 1996; accepted 17 June 1996

Abstract

For about 25 years, bromcresol green and bromcresol purple have been the basis for most of the measurements of serum albumin in the US and perhaps in the world. The longevity of the methods is due to their being simple, sensitive, specific, inexpensive and relatively free from interferences. The lack of change in the serum albumin methodology is balanced by two important developments. First, the recognition of the importance of serum albumin in the maintenance of good health, and the association of decreased concentrations with increased risk of morbidity and mortality. Second, the association of albuminuria with diabetic nephropathy, which without medical intervention could lead to end-stage renal disease. The development of accurate and precise methods for urinary albumin has provided a tool to physicians to extend the length and improve the quality of life of many diabetic individuals. Copyright 1997 Elsevier Science B.V.

Keywords: Serum albumin; Urinary albumin; Measurement; Bromcresol green; Bromcresol purple

1. Introduction

The 25 years since the appearance of the widely used bromcresol green method for albumin by Doumas et al. [1] have seen major advances in our knowledge of this abundant and familiar plasma protein. The findings have

* Corresponding author.

0009-8981/97/$17.00 Copyright 1997 Elsevier Science B.V. All rights reserved PII S0009-8981 (96)06446-7

B.T. Doumas, T. Peters / Clinica Chimiea Acta 258 (1997) 3-20

concerned its structure, its genetics, its metabolism, and its clinical significance. They are reviewed in detail in a recent monograph devoted to albumin [2].

In the 1970s the complete amino acid sequences of human and bovine albumins, 585 and 583 residues, respectively, were finally determined by chemical means. At that time this was a formidable task, albumin being the longest peptide chain sequenced to date. By 1996 cDNA sequences of a total of 13 albumin species had appeared, covering all chordate classes from primates to the lamprey [3]. The long single chain was found to be formed into a series of three homologous domains, each with ~ 190 amino acids and each consisting of three disulfide-bonded loops. The serial loop-link configuration allows the albumin molecule to expand and yet readily reassume its native structure, explaining its well-known resistance to denat- uration by acidity or heat.

This flexibility has frustrated efforts to obtain high-precision crystals of albumin, and only recently has the tertiary structure of albumin succumbed to the efforts of the X-ray crystallographer [4]. Surprisingly, its configuration in a crystal was shown to be heart-shaped, not a linear ~irrangement of its three domains as predicted by decades of hydrodynamic studies of ultracentrifugation, diffusion, and viscosity. It is possible that both struc- tures are tenable, and that the pliable albumin molecule assumes a linear shape under conditions of flow but packs more firmly into a triangular form in a solid crystal.

The X-ray studies have delineated two major binding sites for hydropho- bic ligands; Site I in the middle domain which binds bilirubin, salicylate, warfarin, penicillins, and many other therapeutic drugs, and Site II in the carboxyl-terminal domain which binds chiefly tryptophan and benzodi- azepines. The sites for as many as 6 long-chain fatty acids await resolution; the two most avid sites are apparently in the carboxyl-terminal region. Copper(II) and nickel(II) bind at a highly specific site at the amino-termi- nus, and glutathione and nitric oxide bind at the lone thiol group at cysteine-34.

The complete gene sequence of human albumin has been uncovered [5]. Its ~ 18000 bp are divided by 14 introns into 15 exons which reflect the internal homology of its three domains. Three other closely related proteins have been found to form the albumin superfamily - - a-fetoprotein, vitamin D-binding protein (also termed the group-specific or Gc globulin), and the recently discovered a-albumin [6] or afamin [7] which appears to be the adult counterpart of a-fetoprotein. The albumin superfamily genes lie in the order 5' - - albumin - - a-fetoprotein - - a-albumin - - vitamin D-binding protein - - 3', close to the centromere in the long arm of human chromosome 4, at 4ql 1-22.

B.T. Doumas, T. Peters / Clinica Chimiea Acta 258 (1997) 3-20

Polymorphism of serum albumin is rare except among some inbred tribes or isolated family groups, but 53 variants causing bisalbuminemia have been traced to mutations at specific sites in the albumin mRNA, usually a single-base substitution [2]. Only one has an important clinical concern, that of familial dysalbuminemic hyperthyroxinemia, in which a substitution of histidine for arginine-218 creates a strong binding site for thyroxine [8]. This change causes high total thyroxine concentrations in euthyroid subjects; the free thyroxine level remains normal and is the assay of choice. It is the only variant detected by a functional change rather than by appearance of a second albumin band upon electrophoresis.

Other research has shown that albumin is synthesized in the liver as preproalbumin, bearing an amino-terminal 18-residue signal peptide which guides the growing molecule into secretory channels. The peptide chain is assembled in less than 2 min, and its 17 disulfide bonds are completed in about 0.5 min more. The chief intracellular form is proalbu- min, carrying a cationic amino-terminal hexapeptide which is normally cleaved immediately prior to secretion, but which may escape into the circulation in cases of severe liver damage or in the presence of mutation to the hexapeptide sequence.

Intravenous administration of commercially prepared human serum albumin, Cohn Fraction V (HSA V) developed during World War II, has continued and even increased to the point where its production is now a billion-dollar international business and a major part of most hospital pharmacies' budgets. Although transmission of a virus such as hepatitis or HIV is effectively blocked by pasteurization of the final albumin product, major efforts are in progress to prepare albumin industrially by recombinant production in yeast.

The value of albumin levels in assessing the chronic state of a patient's nutritien has been increasingly recognized. Even further, albumin has been found to be an indicator of future good health. Thus, albumin concentration is positively correlated with length of survival of patients undergoing renal dialysis [9], and renal transplant [10] patients, with prognosis for rehabilitation of elderly stroke patients [11], and with decreased morbidity and mortality in myeloma, biliary cirrhosis, and fol- lowing cardiac surgery [2]. The benefit of albumin is proportional to its concenlLration even within the normal range; thus, to have levels of > 43 g/1 compared to 41-43 g/1 was related to 20-40% reductions in mortality among populations > 71 years old [12]. Whether the beneficial effect of albumin is achieved through maintenance of the blood volume, through its action as an antioxidant, or through other metabolic functions remains lco be discovered.

B.T. Doumas, T. Peters / Cliniea Chimiea Acta 258 (1997) 3 20

2. Earlier albumin methods

Not until the early 1950s did dye-binding methods for albumin supplant the salt precipitation technique of Howe [13]. In this procedure globulins were precipitated in 1.5 mol/1 sodium sulfate and the albumin was determined in the supernatant by a Kjeldahl or biuret assay. There was an obvious need for a more direct technique.

Methyl orange [14] was the first of the dyes, followed quickly by 2-(4'-hydroxyphenylazo)benzoic acid (HABA) [15]. These methods are gen- erally based on a change in color of an organic dye upon binding to albumin; the dyes are usually pH indicators as well, so that control of the pH of the reaction mixture by buffering is essential. HABA fell into disrepute when it was discovered to be susceptible to interferences from bilirubin and various drugs [16], and was gradually superseded by bromcresol green (BCG).

Rodkey had introduced BCG binding as an albumin assay in 1965 [17], but at pH 7.1 the absorbance of the reagent blank was very high. The application at pH ~ 4 is attributable to Bartholomew and Delaney in the same year [18]. Refinements of reagent concentrations, addition of surfac- tant to prevent precipitation of the albumin-dye complex, and improved calibration formed the now-classical procedure of Doumas et al. in 1971 [1].

New developments in albumin methodology include immunochemical methods, modifications of the BCG and bromcresol purple (BCP) methods to improve their specificity and linearity, development of sensitive and specific methods for measuring urinary albumin in diabetic patients, and the recognition of serum albumin as a predictor of morbidity and mortality in hospitalized patients and in patients on hemodialysis. An excellent review on the measurement of albumin in serum and plasma was published about 10 years ago by Hill [19].

2.1. Bromcresol green

The lack of specificity of the BCG method for albumin and the two-step reaction (fast and slow) with serum specimens were reported in the original publication of Doumas et al. [1]. Despite its adverse effect on specificity, a 10 min reaction time was chosen so that results by the BCG method would be close to those of electrophoresis [1], which at that time was considered the standard for the measurement of serum albumin. The lack of specificity, especially at low albumin and high globulin concentrations, was confirmed by many investigators [20-23] and was found to be due to the reaction of BCG with 'acute phase reactants' [23] and ~- and /q-globulin serum fractions [24].

B.T. Doumas, T. Peters / Clinica Chimica Acta 258 (1997) 3-20

The specificity of the BCG method was improved substantially by Gustaf,;son [23] who made absorbance measurements immediately after mixing serum and reagent, thus preventing the binding of other serum proteins. Gustafsson's findings were confirmed by several investigators who used his approach in a variety of clinical laboratory instruments [25-29]. Improved specificity was also obtained by a modified BCG reagent containing 0.3 mmol/1 BCG, 50 mmol/1 succinate buffer, pH 4.20, and 8 ml/1 of 30% Brij-35 [30], or by adding NaC1 (0.8 mol/1) to the BCG reagent [31].

Albumin values of calibrators for clinical instruments are assigned by comparison to standards made from pure human albumin. With human serum-based calibrators albumin values are not affected by small variations in the pH or the Brij-35 concentration of the BCG reagent. This is not the case with calibrators based on bovine serum or BSA. There exists an inverse relationship between albumin values and Brij-35 concentration in the BCG reagenl:; a procedure for avoiding inaccuracies in assigning albumin values has been described [32].

2.2. Bromcresol purple

The short report on the measurement of serum albumin with BCP by Louderback et al. [33] was followed by a more detailed publication by Carter [34]. An automated version of the method and evaluation of its accuracy was published by Pinnell and Northam [35]; serum albumin concentrations with the BCP method were in good agreement with those obtained with Laurell's electroimmunoassay [36] while results with a BCG method [37] were higher by 6.8 g/1 (constant bias). The specificity of BCP for albumin was confirmed by several investigators [38-40], although several shortcomings of the method have been reported. BCP underesti- mates serum albumin bound covalently to bilirubin (6-bilirubin) [41,42]. In pediatric patients on chronic hemodialysis albumin values by BCP were on the average lower by 7 g/1 compared with those obtained by electroimmunoassay [40]; lower albumin values were also obtained in patients with renal insufficiency [43]. The BCP method measured accurately the albumin concentration in the serum of patients with chronic renal failure treated with continuous ambulatory peritoneal dialysis (CAPD), but underesti- mated the plasma albumin concentration by 3 g/1 in patients treated with hemodialysis [44].

The underestimation of serum albumin with BCP in patients undergoing hemodialysis is due to 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), a major endogenous ligand substance present in uremic serum [45]; the extent of albumin underestimation was correlated to the serum concentration of CMPF [46]. Patients undergoing CAPD or hemodialysis


had serum CMPF levels twice as high and 3.5 times as high, respectively as healthy subjects [47].

Heparin has been reported to interfere in both the BCG and BCP methods [48,49]. In the BCG method heparin at a concentration of 90 units/ml of blood decreases serum albumin by an average of 1.3 g/1 while at a concentration of 20 units/ml, which obtains when blood is collected in 7-ml heparinized tubes, heparin does not interfere [49]. The interference, which is due to the development of turbidity when heparinized plasma instead of serum is used, can be eliminated in the BCP procedure by increasing the molarity of acetate buffer to 0.25 mol/1 or by the use of 0.1 mmol/1 acetate buffer containing 0.15 mol/1 NaC1 [50].

2.3. Bromcresol green or bromcresol purple?

Depending on the conditions of the assay, the positive bias of BCG methods in the determination of serum albumin varies from 1.5 g/1 [29,51] to over 11 g/1 [19]. The average bias for methods using short reaction times ( _< 25 s) has been reported to be between 3 and 5 g/l and it is fairly constant over the range of serum albumin concentration [25,52]. The overestimation of a serum albumin by BCG prompted calls for replacing this method by BCP [53,54].

Both methods have advantages as well as shortcomings, and the BCP method has no clear advantage over the BCG method. A potential short- coming of the BCP method is that with different albumin preparations the absorptivity of the BCP-HSA complex is more variable than the absorptivity of the BCG-HSA complex. In the laboratory of one of the authors (BTD) seven different HSA preparations from various sources (all showing purity > 98% by electrophoresis) were analyzed for total protein [55] and for albumin by the BCP [49] and BCG (25 s incubation time) procedures. Results shown in Table 1 indicate that the response of the HSA preparations was more variable with BCP than with BCG and one would expect the uncertainty of albumin values assigned to calibrators to be smaller for the BCG than for the BCP method.

The question, however, is not purely academic in view of the importance of serum albumin in patients treated with hemodialysis [56-58]. Lowrie and Lew reported that in a sample of more than 12000 hemodialysis patients treated at 237 dialysis facilities in the US, of the laboratory variables studied, a serum albumin of less than 40 g/1 was the one most highly associated with the likelihood of death [56]. Churchill et al. found that a serum albumin concentration of 30 g/1 or lower in hemodialysis patients is associated with increased probability of pulmonary edema, infections, hos- pitalizations associated with cardiovascular disease, septicemia and pneu-


monia [57], and according to the study of Owen et al. [58] the serum albumin concentration was a more powerful (21 times greater) predictor of death than the urea reduction ratio.

Considering these findings, the End-Stage Renal Disease (ESRD) Net- work Administration branch of the US Health Care Financing Administra- tion (HCFA) and 'a panel of practising ESRD health care professionals' included serum albumin when they developed quality assurance screening criteria for dialysis facilities [59]. In adult hemodialysis patients the criterion screen :is a screen albumin > 35 g/1. For peritoneal dialysis patients and for all ped!iatric patients the criterion is a serum albumin > 30 g/1. Blagg et al. [59] compared serum albumin concentrations in 235 unselected dialysis patients and found mean values of 38 g/l, by the BCG, and 33 g/l, by the BCP and immunonephelometric methods; a mean difference of 5 g/1 in serum albumin between BCG and BCP was obtained in 121 samples from healthy adults. In view of these findings Blagg et al. believe that the HCFA criteria are valid only for the BCG method, and propose that the criteria for serum albumin concentration by the BCP method be 30 g/1 for hemodialysis patients and 25 g/1 for peritoneal dialysis patients.

Table 1 Ratios of albumin (g/l) to total protein (g/l) for various albumin preparations obtained by the BCF' and BCG methods

HSA Albumin/total protein x 100

BCP BCG

Hyland, HSA V # 1 100.8 99.8 Hyland, HSA V # 2 91.4 97.7 Hyland, HSA V # 3 93.2 98.0 Hyland, HSA V # 4 100.1 99.2 Hyland, HSA V # 5 100.1 99.5 Kabi, tq!SA 95.6 98.4 Miles, HSA, monomer 100.0 98.3

Data in this table are mean values from analyses performed at 2 different times. Albumin standards for the BCP and BCG methods were prepared from HSA V, 100 g/1 solution from Miles Laboratory Inc. This HSA solution (Pentex, Human Albumin, 10% solution, catalog No. 81-017-1) may be obtained from ICN Biomedicals, P.O. Box 5023, Costa Mesa, CA 92626. The exact protein concentration of this solution and the other HSA solutions (all containing about 50 g/1 protein), shown in the left column, was determined by a biuret method and use of a reagent that provides identical absorptivities (color yield) for HSA and BSA solutions prepared from lyophilized powders. The procedure was calibrated with Standard Reference Material (SRM) 927 from the National Institute of Standards and Technology. The composition of the biuret reagent is: CuSO4.5H20, 2.0 g/l; NaOH, 0.12 mol/1; KNa tartrate, 9,0 g/l; KI, 5 g/1 [55].

10 B.T. Doumas, T. Peters / Clinica Chimica Acta 258 (1997) 3-20

Table 2 Albumin mean values (g/l) by the BCG and BCP methods reported in the CAP 1993-1995 Chemistry Surveys

Specimen no.

1993 C06 C07 C08 C09 C10 BCG 29.7 47.5 33.5 40.3 27.4 BCP 31.6 50.8 35.9 42.8 29.0 BCP-BCG 1.9 3.3 2.4 2.5 2.4

1994 C11 C12 C13 C14 C15 BCG 26.2 27.7 39.8 44.7 43.3 BCP 28.5 30.0 43.4 48.1 47.2 BCP-BCG 2.3 2.3 3.6 3.4 3.9

1995 C06 C07 C08 C09 C 10 BCG 43.5 39.8 26.1 40.3 43.8 BCP 47.1 43.0 28.9 43.4 47.2 BCP-BCG 3.6 3.2 2.8 3.1 3.4

Before such criteria are established it would be of interest first to take a look at the albumin values obtained by major analyzers in clinical laboratories and assess the difference between the BCG and BCP methods.

Data from the Chemistry Survey of the College of American Pathologists (CAP) reveal that in 1995 64% of the 6100 participating laboratories use the BCG and 34% the BCP method while fewer than 10 laboratories, if any, use another method. Owing to the better specificity of the BCP method one would expect albumin values on the survey specimens to be about 3-5 g/1 lower by the BCP than by the BCG method. That, however is not the case; surprisingly, values by BCG are from 1.9-3.9 g/l lower than those of BCP (Table 2). The most likely explanation for the paradox is that albumin values of calibrators used for the BCP method are assigned by the BCG method. This may explain why reported normal values (reference intervals) are in many cases identical for both methods (Table 3).

Clinical chemists are obliged to provide reliable (not necessarily accurate) results to clinicians, so that they can make the right decisions with regard to treatment of patients. In the case of serum albumin the simplest and perhaps the least costly way to accomplish this is for manufacturers to establish reliable normal values (reference intervals) for albumin for their instrument(s)/reagent system and for laboratories to verify the manufactur- er's normal values. Guidelines on how to establish such values have already been published [60]. Consistency in the calibration of instrument systems would ensure that the normal values will not 'change' with time. Further-

B.T. Doumas, T. Peters / Clinica Chimica Acta 258 (1997) 3-20 11

more, for those who use the BCG method we recommend short incubation times (25 s or less) to improve the specificity of the measurement.

2.4. Immunochemical assays

Despite their specificity, immunochemical assays are scarcely used for the measurement of serum albumin. The requirement for prediluting the serum and the relatively high cost may have discouraged instrument manufacturers from incorporating such assays in routine clinical analyzers. Such assays are more appropriate for other body fluids in which the albumin concentration is low, e.g. urine and cerebrospinal fluid. Of the laboratories participating in the CA]? Chemistry Survey fewer than 10 (methods used by fewer than 10 laboratories are not listed in the CAP reports) are measuring serum albumin by immunochemical methods.

Laurell's electroimmunoassay [36], selected by the Expert Panel on Protein,; of the International Federation of Clinical Chemistry as the reference method for serum albumin [61], was thoroughly investigated by Pascucci et al. [62]; modifications of certain steps of this technique improved the precision of the method to a long-term CV of 3%. Serum albumin as well as other serum proteins have been measured by immunoturbidimetry [63], laser-nephelometry [64,65], and fluoroimmunoassay [66,67].

Although the importance of selecting appropriate reference materials has been emphasized by several investigators [54,62,63], problems with standardization still persist. Results for serum albumin reported by two groups of inve,;tigators, both using exactly the same equipment, were substantially different; from 35.7-64.0 g/1 with a median of 52 g/1 by one group [64], and from 311.9-50.8 g/1 with a median of 45.1 g/1 by the other group [65].

Table 3 Normal 'values (reference intervals) for serum albumin (g/l) provided some laboratory analyzers

by manufacturers of

Method Albumin

ACA (duPont) Dimension (duPont) Ektachem (Johnson and Johnson) Hitachi (Boehringer-Mannheim) Paramax (Dade) Spectrum (Abbott) Spectrum (Abbott) Synchron (Beckman)

BCP 37- 50 BCP 37-50 BCG 39-50 BCG 35-50 BCG 35-48 BCG 38-50 BCP 39-49 BCP 35-50


The main use of immunochemical assays has been in the evaluation of the specificity of dye-binding methods.

2.5. Urinary albumin ('microalbumin')

Diabetic nephropathy develops in about 45% of the patients with insulin- dependent diabetes (Type I) [68,69], and in 30-35% of patients with non-insulin-dependent diabetes (Type II) [70,71]. In the past, nephropathy was detected by a positive test for protein in routine urinalysis. The interval between the onset of clinical proteinuria and end-stage renal failure requir- ing dialysis or transplantation is about 5 years [72]. The rate of deteriora- tion, although continuous, may be diminished or stabilized by restricting the intake of dietary protein [73], use of angiotensin-converting enzyme (ACE) inhibitors [74], and good control of the blood glucose level [75,76].

Before the development of sensitive and specific methods for urinary albumin, erroneously referred to as microalbumin (the excreted albumin molecule is of normal size), diabetic nephropathy was recognized when urine was positive for protein by the dipstick, which has a sensitivity of about 150 mg/l of albumin [77]. Now, we know that urine albumin concentrations well below the detection limit of the dipstick are associated with progression to overt nephropathy [78-81]. The risk of albuminuria increases with the level of hemoglobin A~ [81,82], and with high blood pressure in patients with non-insulin dependent diabetes [79]. It appears that strict glycemic control and maintenance of normal blood pressure are essential in preventing albuminuria and reduce the risk of nephropathy.

2.6. Measurement

Urinary albumin is measured by a variety of immunochemical methods including immunoturbidimetry [83], immunofluorescence (FIA) [84], enzyme-linked immunosorbent assay (ELISA) [85], radioimmunoassay [86,87], and zone-immunoelectrophoresis [88].

2. 7. Urine specimen

There is little agreement in the literature as to what is the most appropriate specimen for measuring albumin. Choices include: the first morning specimen, timed [81,89] and untimed [90,91]; a random (untimed) specimen [90,92]; a 24-h specimen [93,94]. The reasons for the different opinions are: the variability of the albumin excretion rate (AER) during a 24-h period (it is higher during the day than during the night), the volume of urine varies with the water intake, the inconvenience in collecting timed or 24-h specimens, and the inaccuracy of such collections.

B.T. Doumas, T. Peters / Clinica Chimiea Aeta 258 (1997) 3-20 13

There is also no consensus on how to express urinary albumin results. For timed specimens results are expressed as AER in/tg/min [93-95]; for untimed specimens, in mg/l [91,96] and as albumin/creatinine ratio (mg of albumin per g or per mmol of creatinine) [90,92,97,98].

2.8. Normal values (reference intervals)

The upper limit of the AER for healthy individuals is about 7-8/tg/min (10-12 mg/24 h) [72], but higher values have been reported [96]. Values from 8-20/zg/min (30 mg/24 h) are considered borderline, and values over 20/zg/min are indicative of diabetic nephropathy [79].

2.9. Recommendations for screening

Current guidelines for screening for albuminuria are as follows [79]: a timed AER, either a 24-h or overnight (8-12 h) collection, is deafly the most sensitive of assays. If this is not possible, an untimed urine specimen may be used. A urinary albumin/creatinine ratio >_ 30 mg/g and < 300 mg/g has been identified as a range that places men and women at increased risk for diabetic nephropathy and its complications. If a normal ratio < 30 mg/g Jis obtained, then the same assay need only be repeated yearly. If the ratio i~s > 30 mg/g the abnormality needs to be confirmed. At least two albumin/creatinine ratios in the range of 30-300 mg/g indicates the presence of albuminuria and incipient diabetic nephropathy. If the ratio exceeds 300 mg/g on two occasions, overt clinical nephropathy is present. Routine use of dipsticks as a method of screening is not recommended.

2. I0. Analytical performance

The interlaboratory performance of urinary albumin measurements was evaluated in 5 laboratories using 5 different methods [99]. For urine specimens having concentrations of 2, 15 and 29 mg/1 the corresponding total CVs were 33, 18 and 19%, respectively. Individual within-laboratory CVs for the 3 specimens ranged from 5.1-12.4% by zone immunoelectrophoresis, and from 15.8-44.8% by immunoturbidimetry. Deviations from target values ranged from -51.9-19.1% for the 15 mg/1 specimen, and ~rom -56.4-20.5% for the 29 mg/1 specimen. On the basis of the intraindividual biological variation, which is 36% (coefficient of variation), an analytical CV of 18% would be considered as desirable performance [91]. This goal was met by some of the methods used in this study, and even lower analytical CVs have been reported by other investigators [87,91].

14 B.T. Doumas, T. Peters / Clinica Chimiea Acta 258 (1997) 3 20

2.11. Stability of albumin in urine

Despite numerous studies [87,100-104] there is no agreement on how stable albumin is in urine and what is the best way to store urine specimens. Urine albumin was found to be stable when stored for 2 days at room temperature or 2 weeks at -20C [97,98]. Similar results were obtained by Osberg et al. [87] who reported that the albumin concentration was not affected by storage of the urine for 8 weeks at 4C, but storage at -20C for 8 weeks or longer caused a decrease, especially at albumin concentrations below 30 mg/l. The authors concluded that, to prevent loss of albumin, urine specimens should be analyzed fresh or stored at 4C and assayed as soon as possible. In another study [99] urine albumin was found stable in 5 pooled specimens stored for 160 days at -70C; at -20C the average decline in albumin concentration was 0.27% per day. Other investigators [103,104] found that urinary albumin was not affected when stored at - 20C for 2-6 months, but losses of 5% per year occurred when specimens were kept frozen for as long as 5 years. Sorensen [105] suggested that the loss of albumin upon freezing, especially in specimens with low concentrations, could be due to its adsorption on the container walls, but experiments failed to demonstrate such adsorption on glass, polystyrene or polypropy- lene tubes [104]. Until the issue of specimen storage is settled, it appears that the safest way is to analyze urines as soon as possible or, if analysis is delayed for a few days, to keep them in the refrigerator.

3. Summary

For about 25 years, bromcresol green and bromcresol purple have been the basis for most of the measurements of serum albumin in the US and perhaps in the world. The longevity of the methods is due to their being simple, sensitive, specific, inexpensive and relatively free from interferences. The situation is analogous to enzymatic methods for cholesterol and triglycerides or ion selective electrodes for sodium, potassium, and chloride. Reliable methods stay around for a long time. If light absorption or emission continues to be the basis for measuring the concentration of many body fluid constituents, we do not expect much change in albumin methods for the foreseeable future.

The lack of change in the serum albumin methodology is balanced by two important developments; firstly, the recognition of the importance of serum albumin in the maintenance of good health, and the association of decreased concentrations with increased risk of morbidity and mortality and secondly, the association of albuminuria with diabetic nephropathy, which without medical intervention could lead to end-stage renal disease.


The development of accurate and precise methods for urinary albumin has provided a tool to physicians to extend the length and improve the quality of life of many diabetic individuals.

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