tograms of either normal or Lowe’s syndrome serum, their We thank Isa Bernardim for her excellent supervision of patient

origin in urine probably is renal tissue rather than the data and specimens.
blood. They well may represent uniquely tubular proteins. References
Some patients with cystinosis also have y-globulin bands 1. Lowe CU, Terrey M, MacLachan EA. Organicaciduria, de-
(Figure iF). This may reflect the degree of renal tubular creased renal ammonia production, hydrophthalmos, and mental
involvement that is typical of nephropathic cystinosis (3- retardation: a clinical entity. Am J Dis Child 1952;83:778-86.
5). In fact, both Lowe’s syndrome and cystinosis patients 2. Gellis SS, Feingold M. Oculocerebrorenal syndrome. Am J Dis
Child 1972;124:891-2.
display clinical evidence of damage to both glomerular and
3. GahI WA. Cystmosis coming of age [Review]. Adv Pediatr
tubular function; the UPEPs can help to distinguish which 1986;33:95-126.
region of the kidney is primarily responsible for the prob- 4. Gahi WA, Thoene JG, Schneider JA, O’Regan S, Kaiser-Kupfer
lem. MI, Kuwabara T. Cystinosis: progress in a prototypic disease. Ann
Intern Med 1988;109:557-69.
Renal tubular Fanconi syndrome causes urinary losses of
5. Gahi WA, Renlund M, Thoene JG. Disorders of lysosomal
many small molecules besides protein, e.g., glucose, elec- membrane transport. Cystinosis and sialic acId storage diseases.
trolytes, and amino acids. Even though the tubular protein- Chap 107 in: Scriver CR, Beaudet AL, Sly WS, Valle DL, eds. The
uria of Lowe’s syndrome is greater than that of cystinosis, metabolic basis of inherited disease, 6th ed. New York: McGraw-

Downloaded from https://academic.oup.com/clinchem/article/35/11/2233/5662664 by guest on 26 October 2020

children with cystinosis tend to excrete more small mole- Hill, 1989.
6. Waldmann TA, Mogielnicki RP, Strober W. The proteinuria of
cules than do Lowe’s syndrome patients. It is clear from the
cystinosis: its pattern and pathogenesis. In: Schulman JD, ed.
UPEP of the Wilson’s disease and tyrosinemia patients Cyatinosis. Washington, DC: U.S. Govt. Printing OffIce. 1973:55-
that proteinuria is not a major part of their tubular defects. 66 (DHEW Publication #NIH 72-249).
Evidently urine protein electrophoresis can help distin- 7. Papadopoulos NM, Elm RJ, Wilson DM. Incidence of y.globulin
banding in a healthy population by high-resolution electrophore-
guish among the various metabolic diseases causing renal
sis. Clin Chem 1982;28:707-8.
tubular Fanconi syndrome. It can also provide a measure of 8. Costello R, Papadopoulos NM. Oligoclonal banding in hyper-
the severity of glomerular disease in disorders such as gaminaglobulinemia [Abstract]. Cliii Chem 1987;33:905.
Lowe’s syndrome and cystinosis, which have both glomer- 9. Magrath I, Benjamin D, Papadopoulos NM. Serum monoclonal
ular and tubular dysfunction. Further investigation into immunoglobulin bands in undifferentiated lymphomas of Burkitt
and non-Burkitt types. Blood 1983;61:726-31.
the identity and origin of these y-globulin proteins in
10. Buffone GJ, Ellis D. Urinary proteins. In: Ritzmann SE,
Lowe’s syndrome urine may be beneficial in understanding Killingsworth LM, eds. Protein abnormalities. Vol 3. New York:
the basic defect in the disorder. Alan R Lisa, Inc. 1983:117-45.

CLIN. CHEM. 35/11, 2233-2236 (1989)

An Improved Pyrogallol Red-Molybdate Method for Determining Total Urinary Protein

Jean-Luc Orsonneau, PhIlIppe Douet, CatherIne Massoubre, Patrick Lustenberger, and Serge Bernard

We adapted the pyrogallol red-molybdate method for total precision, sensitivity, and practicability, but are also sub-
urinary protein to the Cobas Bio centrifugal analyzer. The ject to variations in binding to different proteins. Among
method is simple, rapid, sensitive, and inexpensive. Addition these, the Coomassie Brilliant Blue method (7) has been
of 25 mg of sodium dodecyl sulfate per liter to the reagent widely studied and markedly improved (9-12). It is very
modifies protein reactivities so that the chromogenicity of sensitive and precise, but the reagent sticks to the wall of
human gamma globulins is the same as that of albumin. cuvettes, so its application is limited. More recently, Wa-
Results by this method and a comparison method that tanabe et al. (8) described a pyrogallol red-molybdate
included gel filtration and a modified biuret reaction corre- (PRM) method in which the reagent does not adsorb onto
lated well (r = 0.951). the wall of cuvettes, but the reactivity with various kinds of
proteins is unequal. We have modified this method so that
reactivities of albumin and gamma globulins are equal,
AdditIon Keyphrasee: centrifugal analyzer . reference interval
response of various proteins and have adapted it to a centrifugal analyzer.

MaterIals and Methods

Many methods for measuring total urinary protein have
been described and compared. The turbidimetric methods Human serum albumin and gamma globulins were pur-
(1-5) have poor precision and sensitivity, limited linearity, chased from Centre National de Transfusion Sanguine,
and variable response to different proteins. The dye- 75731 Paris, France, and the other standard proteins from
binding methods (2, 3, 5-8) are characterized by better Sigma Chemical Co., St. Louis, MO 63178. Pyrogallol red
was from Aldrich Chimie, 67000 Strasbourg, France; so-
dium dodecyl sulfate (SDS) and methanol from Prolabo,
Laboratoire de Chimie A-C.H.R. H#{244}tel-Dieu,44035 Nantes 75526 Paris, France; and the other chemical compounds
Cedex 01, France. from Merck, Darmstadt, F.R.G. Disposable columns con-
Received December 30, 1988; accepted July 19, 1989. taining Sephadex G25M (cat. no. PD-b) were from Phar-

CLINICAL CHEMISTRY, Vol.35, No. 11, 1989 2233

macia, Uppsala, Sweden. The uromucoid was purified from
-J
urine according to Tamm and Horsfall (13). a,
Reagents. The pyrogallol red-molybdate reagent was
prepared as previously described (8), and the comparison ‘ 1.1
method reagent was according to Dcetsch and Gadsden
(14). Solutions of bovine serum albumin in isotonic saline C
a,
(NaCl) were used for standardization. C.)
C
Apparatus. All determinations were performed with a 0
0
Cobas Bio centrifugal analyzer (Hoffmann-La Roche & Co., C
4002 Bale, Switzerland). The settings for the PRM and the a)
modified biuret method (14) are shown in Table 1. The 2
PRM method consists of an end-point measurement (after 3 a-
0
mm of incubation) including a sample and a reagent blank.
The comparison (reference) method consists of an end-point U)
measurement (after 10 mm of incubation) including a Cs
a)

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reagent blank.
0 10 20 30 40 50 60
Results
Reagent SDS Concentration (mg/L)
Effect of SDS. The method described by Watanabe et al. Figure 1. Effect of reagent SDS concentration with various human
(8) is about 30% less sensitive for gamma globulins than for albumin-gamma globulin ratios
albumin. Addition of increasing quantities of SDS to the D albumin 100%, yglobulins 0%;U albumin 80%, yglobulins 20%; albumin
PRM reagent was tested with various albumin/gamma 60%, y globulins 40%; A albumin 40%, y globulins 60%; x albumin 20%, y

globulin ratios prepared with 1 g/L solutions of human globulins 80%; + albumin 0%, globulins 100%
albumin and gamma globulin. The results (Figure 1)
prompted us to choose 25 mg/L as the optimal concentra- V
0
tion of SDS, although this entailed about a 7% loss of -C
sensitivity for albumin. In contrast, the sensitivity for a,
gamma globulins was increased by 25%. Light-chain pro-
teins were assayed under the same conditions (Figure 2). 3 140’
C
The results expressed as a percentage of those for the a,
I..
reference method were 48% and 51%, respectively, for the . 120’
a,
lambda and kappa types with the unmodified PRM re-
agent, but reached 68% and 78% for an SDS concentration 0
of 25 mg/L in the case of the PRM-SDS reagent.
Analytical range. The detection limit was calculated
a’
according to Vassault et al. (15). We assayed a 150 mmol/L U)
C
NaCl solution 30 times; the mean was 3.5 mg/L and the 0
C-
standard deviation 6.7 mg/L. For a and 13 risks fixed at 5%, U)
w
K = 4.65, so the calculated detection limit is 35 mgIL. The
upper limit of linearity, determined with bovine serum
0 10 20 30 40 50 60
Reagent SDS Concentration (mg/L)
Table 1. Cobas BIo Centrifugal Analyzer SettIngs Fig. 2. Reactivity of A (#{149})
and K (0) chains as percent of the
PAM Reference reference method value with various reagent SDS concentrations
method method
Unit 5 5
albumin solutions in 150 mmol/L NaCl, was found to be
Calculating factor 0 0
2.00 g/L. The calibration curve was established with bovine
Standard 1 * *
serum albumin solutions with concentrations ranging from
Standard 2 * *
0.25 to 2.00 gIL, in 0.25 g/L increments, assayed three
Standard 3 * *
times. The linear-regression equation was y = 1.021x -

Limit 2 2 0.022 g/L (r = 0.999).

Temperature, C 37 25 Determination of precision. Within-run precision was
Type of analysis evaluated by using three urine samples. The CVs were
Wavelength, nm 600 440 9.26% (n = 24, i = 0.086 g/L), 1.71% (n = 24, = 1.173
Sample vol, /4. 5 80 g/L), and 1.48% (m = 24, i = 1.924 g/L). Between-run
Diluent vol, L 20 15 precision was evaluated by using two urine controls (Ciba
Reagent, L 350 80 Corning cat. nos. 9036 and 9041). The CVs were 12.5%
Incubation time, s 0 10.0 (n = 20, x = 0.136 g/L) and 7.18% (n = 20, x = 0.348 g/L).
Start reagent vol, pL 0 0 For bovine serum albumin in 150 mmol/L NaCl, the CV
Time of 1st reading, s 1.0 1.0 was 3.10% (n = 45, i = 0.960 g/L).
Time interval, s 30.0 60.0 Determination of accuracy. The reactivity of pyrogallol
Number of readings 7 11
red towards different proteins was further investigated
with pure proteins of various molecular masses, dissolved
Blanking mode
in 150 mmol/L NaCl. Table 2 shows results obtained with
Printout mode 1
the reference method (14) and the PRM-SDS method,

2234 CLINICAL CHEMISTRY, Vol. 35, No. 11, 1989

together with the analytical recovery calculated with re- 2.0
spect to the reference method. Mean recovery was 97.7%.
We assayed 206 urine samples from patients with the -J
reference method (x) and the PRM-SDS method (y). The 0)

results (Figure 3) show a good correlation, r = 0.951. The V

0
equation for the regression line is y = 0.751x 0.026 g/L.
-

a,
Normal range. We assayed 24-h urine specimens from 65
healthy adults, to assess the normal values for total uri- U).
nary protein with the PRM-SDS method. The nonparamet- 0
Cl)
ric normal range determination was 40-50 mg for the lower
limit and 140-180 mg for the upper limit of total urinary
protein excreted for 24 h (95% confidence limits). a-

DIscussIon
0.0
As many publications dealing with total urinary protein

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assays point out, no routine method is really satisfactory. 0.0 1.0 2.0
According to the results of recent investigations (2, 3), the Reference Method (g/L)
turbidimetric methods should be abandoned because of Fig. 3. Comparison between reference and PAM-SOS methods
their high imprecision, variable response to different pro-
teins, limited linearity, susceptibility to interference by
drugs with high renal clearance, and their tendency for the is a competition between pyrogallol red and SDS to bind the
final measurement signal to be unstable. The biuret basic amino acid radicals with their sulfonic acid group at
method or modifications of it, used after protein precipita- the pH (2.5) of the reagent. Besides this, SDS unfolds the
tion or gel filtration, show the best accuracy, not least polypeptidic chains and some additional basic amino acid
because the measurement signal is independent of the groups are exposed. These two mechanisms explain why
protein species. However, they do not have a good practi- increasing the quantities of SDS added to the PRM reagent
cability and are not adapted to routine analysis. Among the leads to an underestimation of albumin, while the reactiv-
dye-binding methods, the Coomassie Brilliant Blue method ity of other proteins, and especially of human gamma
(7) is certainly the most sensitive. It has been studied and
globulins, increases. At the SDS concentration of 25 mg/L,
modified by many authors, and the problem of the different
the sensitivity to albumin and human gamma globulins is
reactivities of various proteins to the reagent has been
equal. We chose this concentration because these two kinds
nearly solved by Macart and Gerbaut (9, 10, 16). Thus this
of proteins predominate in urine. These results agree well
method is frequently used, manually or with automated
with those of Friedenauer and Berlet (20), who demon-
analyzers because of its simplicity of operation (5, 17-19).
strated that addition of SDS to Coomassie Brilliant Blue
The PRM method reported by Watanabe et al. (8) has
good sensitivity for clinical use and good practicability. The reagent lowers the response, concomitant with a somewhat
reagent is not corrosive and does not adsorb onto the wall of closer range for the deviation of measurements of individ-
cuvettes, but the sensitivity depends on the kind of protein. ual proteins.
Pyrogallol red binds basic amino acid groups: for a 0.60 Table 2 shows a satisfactory recovery for alpha and beta
g/L solution of poly-L-lysine (as measured with the refer- globulins but a poor one for the Tamm-Horsfall mucopro-
ence method), the result obtained with the PRM-SDS tein. The 97.7% overall recovery appears satisfactory for
method was 3.40 g/L, whereas, under the same conditions, clinical use.
a 0.20 g/L solution of poly-L-glutamic acid was found to be Results for kappa and lambda chains are clearly better
below the detection limit for the PRM-SDS method. There than those obtained with turbidimetric methods, even
though they still are not perfect. Light chains are easily
detected in urine. Comparison studies point out that the
Table 2. AnalytIcal Recovery for VarIous ProteIns PRM-SDS method gives results that are about 25% lower
Reference PRM-SDS than those of the reference method. It would be the same
method method with any other dye-binding method because some proteins
gIL Recovery, are poorly or not at all detected. The normal range is
lowered in the same proportions. Nevertheless, the princi-
Globulins (Cohn
Fraction IV) 0.47 0.67 142.6 pal proteins found in urine are suitably accounted for, and
Globulins (Cohn the method is adequate for routine clinical use. Results
Fraction IV.I) 0.30 0.31 103.5 clearly are better than with turbidimetric methods, which
Globulins (Cohn are still widely used, and are at least as good as those
Fractions II, III) 0.52 0.61 117.3 obtained with the modified Coomassie Brilliant Blue
Orosomucoid 0.68 0.42 61.8 method (10, 21). More than 100 urine samples have been
Ferritin 0.38 0.31 81.6 assayed every day in our clinical laboratory for two years
Transferrin 0.60 0.63 105.0 without a preliminary semiquantitative screening test.
Apolipoprotein A-I 0.12 0.12 100.0 Results below the analytical range are reported as <0.04
Thyroglobulin 0.27 0.28 103.7 gIL and are not further investigated because they are not of
Thyroxin 0.42 0.51 121.4 clinical interest. The absence of adsorption onto the wall of
f3-Amylase 0.14 0.13 929 cuvettes has made it possible to adapt this method to the
Uromucoid 1.12 0.50 446
________ Hitachi
measures 717 analyzerfunction.so that
of renal it can accompany other

CLINICAL CHEMISTRY, Vol.35, No. 11, 1989 2235

References 12. Wimsatt DK, Lott JA. Improved measurement of urinary total
1. Lustenberger P, Launay-Godard A, Cornu G, Bernard S. Au- protein (including light-chain proteins) with a Coomassie Brilliant
Blue G 250-sodium dodecyl sulfate reagent. Clin Chem
tomatisation en analyse centrifuge et en flux continu du dosage des
1987;33:2100-6.
prot#{233}inestotales urinaires. Clin Chim Acts 1978;84:293-303. 13. Tamrn I, Horsfall F. A mucoprotein derived from human urine
2. McElderry LA, Tarbit IF, Cassells-Smith AJ. Six methods for which reacts with influenza, mumps and Newcastle disease vi-
urinary protein compared. Clin Chem 1982;28:356-60. ruses. J Exp Med 1952;95:71-97.
3. Dilena BA, Penberthy LA, Fraser CG. Six methods for deter- 14. Doetsch K, Gadsden R. Determination of urinary total protein
mining urinary protein compared. Clin Chem 1983;29:553-7. by use of gel filtration and a modified biuret method [Selected
4. Nishi FIN, Elm RJ. Three turbidimetric methods for determin- Method]. Clin Chem 1975;21:778-81.
ing total protein compared. Clin Chem 1985;31:1377-80. 15. Vassault A, Grafrneyer D, Naudin C, et al. Protocole de
validation de techniques (document B, stade 3). Ann Biol Clin
5. Massenet D, Molas J, Pressac D. Dosage des proteines de l’urine
(Paris) 1986;44:686-.745.
et du liquide cephalorachidien par analyse
centrifuge. Comparai- 16. Macart M, Gerbaut L. Optimization of protein measurement
son de trois m#{233}thodes. Ann Biol 1983;41:151-4.
Clin (Paris) in biological fluids by the dye-binding-SDS method [Letter]. Clin
6. Peace MA, Strande CS. A new micromethod for determination Chem 1988;34:998-9.
of protein in cerebrospinal fluid and urine. Clin Chem 17. Fontaine M, Martin-Ponthieu A, Richard MJ, Delescaut MP,
1973;19:1265-7. Baluch L, Porchet N. Dosage de la proteinurie et de la prot#{233}ino-
7. Bradford MM. A rapid and sensitive method for the quantita- rachie. Choix d’une methode en analyse centrifuge. Ann Biol Clin

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tion of microgram quantities of protein using the principle of (Paris) 1987;45:519-26.
protein dye binding. Anal Biochem 1976;72:248-54. 18. Heick HMC, Begin-Heick N, Acharaya C, Mohammed A.
8. Watanabe N, Kaniei 5, Ohkubo A, et al. Urinary protein as Automated determination of urine and cerebrospinal fluid proteins
measured with a pyrogallol red-molybdate complex, manually and with Coomassie Brilliant Blue and the Abbott ABA-100. Clin
in a Hitachi 726 automated analyzer. Clin Chern 1986;32:1551-4. Biochem 1980;13:81-3.
9. Macart M, Gerbaut L. An improvement of the Coornassie Blue 19. FryeR, Lijewski J. Coomassie Brilliant Blue G 250 method for
dye binding method allowing an equal sensitivity to various urinary protein adapted to the Cobas Bio centrifugal analyzer
proteins: application to cerebrospinal fluid. Clin Chiin Acts [Abstract). Clin Chem 1987;33:979-80.
1982;122:93-101. 20. Friedenauer 5, Berlet HH. Sensitivity and variability of the
10. Macart M, Gerbaut L. Evaluation of an improved Coomassie Bradford protein assay in the presence of detergents. Anal Bio-
dye binding method for urinary protein assay. Clin Chim Acta chern 1989;178:263-8.
1984;141:77-84. 21. Urn W, Chirnall W, Stokes Y, Pratt R, Crooke M. Effects of
11. Fermi JM, Mizon C, Dehon B, et a!. Urinary protein determi- sodium dodecyl sulfate, dye concentration and paraprotein on
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dodecyl sulfate. Clin Chirn Acts 1984;143:321-8. Biochem 1988;21:277-81.

CLIN. CHEM. 35/11, 2236-2241 (1989)

Rate Immunonephelometry and Radial Immunodiffusion Compared for Apolipoproteins Al

and B Assay
Paul S. Bachorlk and Teresa A. Cloey

We measured apolipoproteins (apo) Al and B in fresh plasma less well correlated (n = 112, r = 0.74). Frozen control pools
samples and serum control pools by using two rate immuno- were most suitable for apoB analysis.
nephelometric assay (INA) methods (Beckman “Array” and
Behnng) and radial immunodiffusion (RID). Both INA meth- AddItIonal Keyphrases: fresh vs frozen control materials
ods on average gave apoAl values similar to those by RID in intermethod comparison
fresh plasma samples. The coefficients of correlation for the
two INA-RID method pairs were as follows: Beckman INA vs Apolipoproteins Al (apoAl) and B (apoB) arethe major
RID, n = 94, r= 0.73; and Behring INAvs RID, n = 112, r= protein components of high-density lipoproteins (HDL) and
0.66. The bias between the INA and RID methods was low-density lipoproteins (LDL), respectively.’ In the past
reflected reasonably well by either lyophilized or frozen several years, the importance of routine measurement of
apoAl and apoB has become more widely appreciated.
control pools. For apoB, the Beckman INA measurements in
Measurement of these apolipoproteins can aid in assess-
fresh plasma averaged 40% lower than RID values, due
ment of cardiovascular risk
as well as in diagnosis of some
almost entirely to the values assigned to calibration sera, but
forms of dyslipoproteinemia (1, 2). ApoAl and apoB are
results by the two methods were highly correlated (n = 94, r
most commonly measured immunochemically (1, 2), e.g.,
= 0.93). The Behring INA values for fresh plasma averaged
by radioimmunoassay (RIA), enzyme-linked immunosor-
only 13% lower than RID values, but the two methods were
bent assay, radial immunodiffusion (RID), electroimmu-

‘Nonstandard abbreviations: apoAl and apoB, apolipoproteins

Lipid Research-Atherosclerosis Unit, Departments of Pediatrics Al and B, respectively; radial immunodiffusion;
RID, INA, immu-
and Laboratory Medicine, The Johns Hopkins University School of nonephelometric assay; VLDL, IDL, LDL, and HDL, very-low-,
Medicine, Baltimore, MD 21205. intermediate-, low-, and high-density lipoproteins, respectively;
Received June 23, 1989; accepted August 3, 1989. and HDL2 and HDL3, the two major subclasses of HDL.

2236 CLINICAL CHEMISTRY, Vol. 35, No. 11, 1989