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CLIN. CHEM.

29/3, 553-557 (1983)


CLINICALCHEMISTRY, Vol. 29, No. 3, 1983 553
Six Methods for Determining Urinary Protein Compared
Beverly A. Dllena, Lloyd A. Penberthy, and Callum G. Fraser
Inter-laboratory surveys have shown that routine methods
of urinary protein determination are often unsatisfactory.
Therefore, we compared six frequently used methods for
determination of protein in urine with respect to linearity,
within-batch and between-batch precision, comparative bias,
and practicability. We assayed dilutions of human and bovine
albumin and serum, and fresh and lyophilized human urine.
We find that the AACC Selected Method has poor practicabil-
ity and poor precision under routine conditions, but good
linearity. The sulfosalicylic acid/biuret technique is also im-
practicable, requires a large sample, and is not linear at low
concentrations of urine protein. The Coomassie Brilliant Blue
technique has a narrow range of linearity and poor precision.
The sulfosalicylic acid/sodium sulfate turbidimetric method is
not precise and cannot be standardized with bovine materi-
als. The Ponceau-S technique has good performance char-
acteristics and practicability, and we recommend it for routine
laboratory use.
Inter-laboratory quality-assurance surveys of the stan-
dard of performance of urinary protein analyses in the
United States (1-4) and in Australia (5-7) have shown that
the biases within and between methods are high; the range
between low and high values for the same specimen varied
from normal to distinctly abnormal. In addition, many
techniques have poor intra-laboratory precision. It has been
suggested (1) that disease states may well go undetected
because of this poor performance; in addition, healthy
individuals may also be labeled as diseased.
Even though a Selected Method for the quantitation of
urinary protein (8) and an Australian recommended method
(5) have been published, no single method has been widely
adopted by laboratories. Recently, McElderry et al. (9)
compared the linearity, accuracy, precision, sensitivity, and
specificity of six methods: the Coomassie Brilliant Blue
method in two commercial forms, Ponceau-S dye binding,
ferric chloride/tannic acid turbidimetry, turbidimetry with
benzethonium chloride in alkali, and trichloroacetic acid!
biuret colorimetry. However, many other methods are wide-
ly used by laboratories. We therefore report here our studies
in which we have compared the Selected Method of the
American Association for Clinical Chemistry (8), the recom-
mended method of the Royal College of Pathologists of
Australia (5), the manual Coomassie Brilliant Blue tech-
nique of Heick et al. (10), sulfosalicylic acidlsodium sulfate
turbidimetry (11), trichloroacetic acid turbidimetry (12),
and Ponceau-S dye binding (13). For each method we
assessed linearity, within-batch and between-batch preci-
sion, comparative bias, and practicability.
Materials of animal origin, materials primarily designed
for use for serum analyses, and lyophilized materials are
widely used both as calibration materials and in intra- and
inter-laboratory quality-control and assurance programs.
Because such materials may not be truly representative of
Department of Clinical Biochemistry, Flinders Medical Centre,
Bedford Park, South Australia, 5042.
Received Aug. 9, 1982; accepted Dec. 23, 1982.
specimens from patients (14), we therefore examined the
precision and bias of the six methods with a variety of such
materials.
Our aims were to investigate the possible reasons for the
documented poor performance of six frequently used meth-
ods and to recommend a single method that would be
suitable for wide use in the clinical laboratory.
Materials and Methods
Materials
Bovine serum albumin (Cohn Fraction V powder) and
human serum albumin (crystallized and lyophilized) were
obtained from Sigma Chemical Co., St. Louis, MO 63178.
Stock 35 g/L solutions were prepared and, after determina-
tion of total protein by the biuret method with a Technicon
6-Plus AutoAnalyzer (Technicon Instruments Corp., Tarry-
town, NY 10591), with use of Ortho Automated Reference
Serum (Ortho Diagnostics Inc., Raritan, NJ 08869) as
standard, solutions in saline (9 g of NaC1 per liter) of
approximately 0.25 and 0.50 g/L were prepared. Aliquots
were stored at -20 #{176}C.
Fresh bovine serum was obtained by centriftigation of
freshly clotted blood obtained at slaughter; fresh human
serum was obtained by centrifugation of freshly clotted
blood obtained from apparently healthy laboratory person-
nel. After determination of total protein by the biuret
method, both sera were diluted with saline to prepare
solutions of approximately 0.25 and 0.50 g/L. Aliquots were
stored at -20 #{176}C.
Monitrol Ii was supplied by Dade Division, American
Hospital Supply Corp., Miami, FL 33152; and Weilcome
Unassayed bovine serum material from Wellcome Reagents
Ltd., Beckenham, Kent, U.K. Vials of each were reconstitut-
ed each day and dilutions to approximately 0.25 and 0.50 g
of protein per liter were prepared in saline.
The primary clinical standard used throughout this study
was a human urine to which a known amount of human
serum albumin had been added. This material was specially
supplied by Commonwealth Serum Laboratories, Parkville,
Victoria, 3052, Australia, and had been prepared from a
pool of random urine samples collected from apparently
healthy males. Except for protein, the base pool and the
standard did not have any tests positive on qualitative
urinalysis, and ascorbate concentrations were undetectable.
The concentration of protein in the base pool was not
significantly different from the detection limits of the meth-
ods used, and the relative density was 1.015. The standard
had an assigned value (by weight) of 0.50 g of protein per
liter. We assigned concentrations of protein in each of the
materials used in this study by using this standard and the
Selected Method (8); analyses were performed in 10 separate
analytical batches.
Ortho Control Urine II was supplied by Ortho Diagnostics
Inc., Raritan, NJ 08869. Several vials were reconstituted,
the material was pooled, and aliquots of it were stored frozen
at -20#{176}C.
Fresh human urine samples with low, medium, and high
protein concentrations were obtained from patients in the
Flinders Medical Centre. These were analyzed by use of the
Selected Method (8) in duplicate on three occasions. Values
of 0.22, 2.48, and 20.40 g/L were obtained. The latter two
urines were diluted fourfold and 20-fold, respectively, with
saline. Aliquots of these dilutions and of the undiluted urine
were stored at -20 #{176}C.
All other reagents used were as stated in the published
methods or were equivalent Analar grade.
The performance characteristics of the methods with
materials stored at -20 #{176}C were not significantly different
from those achieved with similar fresh materials.
Methods
All absorbances were measured in 1-cm cuvettes with a
Model 300-Ti spectrophotometer (Gilford Instrument Labs.
Inc., Oberlin, OH 44074). Samples were transferred with
Eppendorf pipettes. In addition, in separate experiments,
the Selected Method, as recommended (8), was performed
with use of glass volumetric pipettes throughout.
The six procedures examined were done exactly as de-
scribed in the references except for minor modifications to
the Selected Method (8). Disposable syringes (Terumo Corp.,
Tokyo, Japan) of 5-mL capacity were used as chromatogra-
phy columns. A single glass bead was used as the bed
support. In this study, the Selected Method when performed
with glass pipettes is termed the Selected Method, and when
performed with air-displacement pipettes is termed the
Selected Method (routine).
Results and Discussion
Analytical Variables
Linearity. Linearity was assessed by duplicate analysis, in
each of two batches, of at least nine samples of different
concentrations prepared by dilution of the materials recom-
mended as standard with saline. In addition, linearity was
Table 1. Maximal Useful Limit of Linearity with
Samples Used for Assessment of Linearity and
with Urine
Maximal limit of
linearity, g/L
Method
Selected Method
SSA/biuret
Coomassie Brilliant
Blue
SSA/sodium sulfate
TCA turbidimetry
Ponceau-S
Sample
Human albumin
Human serum
Human albumin
Human serum
Human serum
Ortho serum
Tables1-5: SSA, sulfosalicylic acid;TCA, trichloroacetic acid.
554 CLINICALCHEMISTRY, Vol. 29, No. 3, 1983
assessed by duplicate analysis, in each of two batches, of
nine dilutions with normal human urine of a human urine
with a high protein concentration. Table 1 shows the
samples and the maximal useful limits of linearity.
The Selected Method showed the widest range of linear-
ity. The trichloroacetic acid turbidimetry technique had the
narrowest useful range of linearity; use of this method
would entail dilution and reanalysis for a substantial pro-
portion of specimens from patients.
We assayed, in duplicate, six samples of urine to which
albumin was added in various amounts up to a concentra-
tion of 2.50 g!L. The maximum limit of linearity with these
samples was identical to that found by the corresponding
method for urines containing protein.
Within-batch precision. For each method, within-batch
precision was assessed by analysis in duplicate of thawed
aliquots of the three human urine specimens, with low,
medium, and high protein concentrations, in 10 batches.
The standards recommended for each method were used.
These had values assigned by the Selected Method. Values
for precision calculated by use of the paired results, are
shown in Table 2, both as standard deviation (SD) and
coefficients of variation (CV).
When the precision of the Selected Method was used as
the comparison point for application of the F-test, all meth-
ods had similar precision at a low urinary protein concentra-
tion (0.22 gIL). At a medium (0.62 gIL) concentration of
urinary protein, only the sulfosalicylic acid/sodium sulfate
method was significantly more precise than the Selected
Method, whereas the Selected Method (routine) had signifi-
cantly poorer precision than the Selected Method. At a high
(1.02 g/L) concentration of urinary protein, all methods
except the Coomassie Brilliant Blue technique had signifi-
cantly better precision than the Selected Method and, again,
the Selected Method (routine) had the poorest precision. The
sulfosalicylic acid/biuret, sulfosalicylic acid/sodium sulfate
turbidimetry, and trichloroacetic acid turbidimetry tech-
niques were all precise for all concentrations examined.
Between-batch precision. Between-batch precision was
assessed by 10 replicate analyses, in separate batches, of
each of the eight calibration materials; each material was
studied at concentrations of approximately 0.25 and 0.50 g/
L. In addition, two urine samples from patients, with protein
concentrations of 0.22 and 0.62 g/L, were analyzed 10 times.
Sample Urine The precision for each method, as SD and CV, calculated
4.0 4.0 with the primary standard used in this study, is shown in
3.5 3.5 Table 3.
1.0 0.7 The conclusions we drew from this study were:
1 6 1 6 #{149} The poorest overall precision was found with Coomassie
0:6 0.6 Brilliant Blue technique and sulfosalicylic acid/sodium sul-
1.6 1.6 fate turbidimetry.
#{149} There did not appear to be any direct relationship be-
tween the precision achieved and the type of material
Method
Table 2. WIthin-Batch
Rd
Imprecision
ative protein concn of sample
Low Medium High
SD, g/L CV, % SD, g/L CV, % SD, g/L CV, %
Selected Method 0.013 5.9 0.023 3.7 0.035 3.4
Selected Method (routine) 0.015 6.8 0.043 6.9 0.064 6.3
SSA/biuret 0.011 5.0 0.0118 1.8 0.0198 1.9
Coomassie Brilliant Blue 0.015 6.8 0.022 3.5 0.030 2.9
SSA/sodium sulfate 0.008 3.6 0.016 2.6 0.01 48 1.4
TCAturbidimetry 0.014 6.4 0.016 2.6 0.0148 1.4
Ponceau-S 0.011 5.0 0.020 3.2 0.0148 1.4
8Precision significantly better than
that obtainedby the Selected Method(p 0.05).
Method Material
0.089(17.8) 0.080(15.4) 0.064(12.8) 0.038(8.3) 0.065(13.8) 0.065(13.8) 0.071(15.1) 0.065(18.6) 0.063(10.2)
CLINICAL CHEMISTRY, Vol. 29, No. 3, 1983 555
Table 3. Between-Batch Imprecision (SD in g/L, CV % in Parentheses) for Eight Calibration Materials at
Two Different Concentrations and for Two Urine Samples from Patients
Bovine Human Bovine Human Dade Ortho Wellcome Ortho Human
albumin albumin serum serum serum serum serum urine urine
Selected method 0.039(13.9) 0.027(10.8) 0.045(20.5) 0.036(16.4) 0.024(10.9) 0.037(16.1) 0.041(18.6) 0.026(15.3) 0.028(12.7)
(routine)
SSA/biuret 0.021(9.1) 0.021(8.4) 0.035(16.4) 0.026(11.8) 0.044(20.0) 0.028(12.2) 0.030(13.0) 0.036(21.2) 0.051(23.2)
0.041(8.2) 0.043(8.3) 0.058( 11.6) 0.055( 12.0) 0.051(10.9) 0.041(8.7) 0.036(7.7) 0.025(7.1) 0.061(9.8)
Coomassiebrilliant 0.023(10.0) 0.041(16.4) 0.050(22.7) 0.047(21.4) 0.024(10.9) 0.042(18.3) 0.034(15.5) 0.048(28.2) 0.037(16.8)
blue
0.049(9.8) 0.046(8.8) 0.046(9.2) 0.052(11.3) 0.030(6.4) 0.030(6.4) 0.032(6.8) 0.049(14.0) 0.087(14.0)
SSA/sodium sulfate 0.047(20.4) 0.028(11.2) 0.043(19.5) 0.038(17.3) 0.028(12.7) 0.027(11.7) 0.026(11.8) 0.044(25.9) 0.040(18.2)
0.100(20.0) 0.047(9.0) 0.085(17.0) 0.060(13.0) 0.077(16.4) 0.047(10.0) 0.070(14.9) 0.060(17/1) 0.070(11.3)
TCA turbidimetry 0.022(9.6) 0.018(7.2) 0.043(19.5) 0.025(11.4) 0.019(8.6) 0.012(5.2) 0.031(14.1) 0.012(7.1) 0.024(10.9)
0.050(10.0) 0.026(5.0) 0.058(11.6) 0.047(10.2) 0.042(8.2) 0.029(6.9) 0.046(9.8) 0.047(13.4) 0.046(7.4)
Ponceau-S 0.026(11.3) 0.022(8.8) 0.039(17.7) 0.012(5.5) 0.015(6.8) 0.011(4.8) 0.022(10.0) 0.025(14.7) 0.027(12.3)
0.052(10.4) 0.020(3.8) 0.057(11.4) 0.024(5.2) 0.016(3.4) 0.024(5.1) 0.027(5.7) 0.030(8.6) 0.036(5.8)
analyzed; this implies that use of frozen or lyophilized,
bovine or human albumin, serum or urine would be, in
general, satisfactor.y as quality-control materials to monitor
precision.
#{149} The sulfosalicylic acid/biuret, trichloroacetic acid turbi-
dimetry, and Ponceau-S methods had the best overall preci-
sion.
#{149} The Selected Method had significantly better between-
batch precision than the Selected Method (routine).
We calculated the precision found when using the stan-
dards recommended for the methods. The Coomassie Bril-
liant Blue technique had better precision when human
albumin diluted in saline was the standard, but the preci-
sion of the sulfosalicylic acid/sodium sulfate turbidimetry
technique was poorer with human serum diluted with saline
as the standard. We consider it likely that, as shown
previously in our laboratory (17, 18), detailed examination
of mode of standardization and type and concentration of
standard by the individual laboratory could significantly
improve the precision of the method chosen by that labora-
tory.
Comparatiue bias. Peters (19) has suggested that the
method of choice for measurement of protein should be a
version of the biuret reaction and that bovine or human
serum albumin, assayed by Kjeldahl analysis, should be
used for standardization. However, the composition of uri-
nary proteins can vary very widely (20), a primary standard
for such a complex and variable mixture of components
cannot truly be defined, and neither definitive nor reference
methods exist. It is therefore difficult at this time to define
the accuracy of urinary protein methods in strict numerical
terms as deviation from the true value.
In this study, we assigned values to all albumin and
serum-based materials and urine samples by using the
Selected Method because such a method has been thorough-
ly evaluated and verified by independent workers before
being accepted. Human albumin weighed in to a normal
urine obtained from healthy men was used as the standard.
The means of 10 replicate analyses on each of eight calibra-
tion materials at concentrations of approximately 0.25 and
0.50 g/L and on two urine samples are shown in Table 4. The
same technique and standard having been used to assign
values, we believe that the data obtained are valid for
assessing comparative bias among methods and materials.
The results obtained for the different proteins showed, in
general, little difference between methods. This implies
that, for most methods, human or bovine albumin, serum, or
urine could be used for standardization and quality control,
provided that the protein concentration was adequately
assessed.
However, for the sulfosalicylic acid/sodium sulfate meth-
od, results for bovine serum albumin solutions were falsely
increased; therefore, bovine serum albumin should not be
used as a calibration material for this method, and material
supplemented with bovine serum albumin should not be
used in inter-laboratory quality-assurance surveys. Human
albumin has also been reported to give increased results in
this method, but the composition of the precipitating rea-
gent appears to be important, the absorbance varying with
the amount of sodium sulfate added (21). In addition, higher
results were found with the Ortho urine material. This
material is stated to be human urine supplemented by
material of nonhuman origin; bovine albumin has been
shown (22) to be present. In contrast, bovine serum and
Wellcome serum, a bovine serum-based material, appeared
to be satisfactory as calibration materials.
The sulfosalicylic acid/biuret technique gave increased
results for all albumin- and serum-based materials but gave
correct results for urine materials. This is because this
method, recommended for Australian laboratories, requires
that a blank be performed with urine samples submitted for
analysis but does not recommend using a blank for the
saline dilutions of human serum to be used as standards.
Although McElderry et al. (9) showed considerable differ-
ences in response between albumin and globulin in the
Coomassie Brilliant Blue technique, we did not find, in this
study, any particular problems of bias with serum-based
materials.
albumin albumin serum urine urine
Table 5. Practicability Scores for the Methods Evaluated
Turnaround Reagent Technical Sample
Method time Equipment preparation skill size Costs Total
Selected Method 1 3 2 1 2 1 10
SSA/biuret 4 4 2 4 1 3 18
CoomassieBrilliantBlue 5 5 5 5 5 4 29
SSAlsodium sulfate 5 5 5 4 2 4 25
TCA turbidimetry 5 5 5 4 1 5 25
Ponceau-S 5 4 4 3 4 4 24
556 CLINICAL CHEMISTRY, Vol. 29, No. 3, 1983
Method
Table 4. Mean Protein Concentrations (g/L) for Albumin- and Serum-Based Materials at Two
Concentrations and for Two Urine Samples from Patients
Material
Bovine Human Bovine Human Dade Ortho Wellcome Ortho Human
serum serum serum serum
Selected method (routine) 0.23 0.25 0.22 0.22 0.22 0.23 0.22 0.17 0.22
SSA/biuret
0.50 0.52 0.50 0.46 0.47 0.47 0.47 0.35 0.62
0.27 0.31 0.31 0.30 0.30 0.29 0.30 0.19 0.17
0.54 0.58 0.62 0.59 0.63 0.59 0.59 0.34 0.60
Coomassie brilliant blue 0.31 0.28 0.19 0.22 0.21 0.25 0.23 0.21 0.23
0.54 0.52 0.36 0.42 0.39 0.45 0.41 0.35 0.51
SSA/sodium sulfate 0.42 0.25 0.17 0.23 0.24 0.25 0.21 0.22 0.20
1.05 0.52 0.51 0.48 0.51 0.54 0.55 0.61 0.57
TCA turbidimetry 0.22 0.22 0.28 0.26 0.29 0.28 0.31 0.17 0.23
Ponceau-S
0.47 0.43 0.58 0.51 0.56 0.56 0.59 0.43 0.50
0.28 0.29 0.21 0.25 0.25 0.26 0.25 0.18 0.24
054 0.55 0.43 0.49 0.47 0.50 046 0.34 0.55
For turbidimetnc methods, there were no apparent prob-
lems of comparative bias between human albumin and
serum-based materials and urines used in this study. Per-
haps this was because all materials were diluted in 9 g/L
saline: turbidimetric methods generally suffer from failure
of standards and samples to form precipitates identically,
and precipitation may not occur at low protein concentra-
tions in urines of high ionic strength. It is therefore possible
that results on urine samples from patients do have consid-
erable bias.
Practwability. Turnaround time, equipment required, dif-
ficulty of reagent preparation, technical skill required,
sample size, and costs were judged on a scale of 1 to 5, 5
being the best possible score (Table 5). The Selected Method
had the worst score and the method recommended for
Australian laboratories the second worst score; the other
four methods had comparable high scores, with the Coomas-
sie Brilliant Blue technique being the most practicable.
Conclusions
The Selected Method was the least practicable of the
methods studied. The precision of the method was poor when
performed under routine laboratory conditions. The method
had the widest linear range and little apparent bias. The
best use of this method would perhaps be as a reference
method.
The sulfosalicylic acidibiuret technique was also some-
what impracticable and, specifically, requires a large sam-
ple volume. The method had good precision and linear
range, and little apparent bias with urine samples; however,
it overestimated the protein concentration of albumin and
serum-based materials by not requiring blanks to be per-
formed with such samples.
The Coomassie Brilliant Blue technique was most practi-
cable, but the relatively low linear range and poor precision
were disadvantages.
The sulfosalicylic acid/sodium sulfate turbidimetry tech-
nique was simple to perform but a large sample size is
required. The method had poor precision, and bovine mate-
rials could not be used as calibration material.
The trichioroacetic turbidimetry technique had generally
good practicability but required a large sample and had a
narrow linear range. The precision was good, and there was
little apparent bias with the samples used in this study.
The Ponceau-S technique had good practicability, re-
quired little sample, had good precision, and little apparent
CLINICAL CHEMISTRY, Vol. 29, No. 3, 1983 557
bias. We recommend this technique for routine clinical
chemistry laboratory use, particularly because human or
bovine albumin, serum, or urine appeared to be satisfactory
for calibration and quality-control or assurance. McElderry
et al. (9) also recommend this method as one that has
considerable advantages.
We thank Miss M. E. Coles, Division of Clinical Chemistry,
Institute of Medical and Veterinary Science, Adelaide, for her
advice, and the referees of the manuscript for their detailed helpful
critiques.
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