CUN. CHEM. 21/3, 398-401 (1975)
Improved Biuret Procedure for Routine Determination
of Urinary Total Proteins in Clinical Proteinuria
Eugene W. Rice
This communication describes and evaluates an improved routine methodology for quantitating clinical proteinuria. Based on investigations of Piscator and of Savory et al., a modified Tsuchiya’s reagent (ethanolic
HCI-phosphotungstic acid) is used to precipitate proteins at 56 #{176}C,
followed by biuret spectrophotometry at
540 nm. The accuracy of the proposed procedure was
assessed by comparisonswith results obtained by using
an ultrafiltration
membrane thatretainssoluteswithan
average molecularweightinexcess of 10 000 forseparating of urinary proteins before they are measured with
the biuret reaction. Precision of the method (coefficient
of variation) is typically 2-3%.
Additional Keyphrases:
phosphotungstlc acid)
tration
Tsuchiya ‘s reagent (ethanol/c HCI#{149}
u/trail!-
#{149}
spectrophotometry
Contemporary reportsgenerallyindicatethat the
total quantity of proteins in the urine of normal
healthyadultsaverages less than 100 to 150 mg per
liter (1). When the excretion is greater than about
250 mg per day, it is usually concluded that the
subject has “clinical” proteinuria of potential pathological significance. More than 0.3 g per day indicates
renal disease, unless associated with transient conditions such as proteinuria resulting from vigorous exercise. Accurate appraisal of clinical proteinuria
is
important not only for evaluation of certain renal diseases, but also for their early detection.
The status of determination
of proteins
in urine
leavesmuch to be desired. It isestimatedthata third
of the clinical laboratories in the U. S. do not offera
quantitativeprocedure for urinary total proteins,
and, of those thatdo,a fifth are using obsolete or inappropriate methods, and intra- and interlaboratory
variation is high for those, that are using “reasonably
good” methods.
Pathology
Department,
Allentown Hospital Association, Allentown, Pa. 18102.
Presented, in part, at the 26th National Meeting of the AACC,
Las Vegas, Nev., August 1974.
Received June 10, 1974; accepted Dec. 30, 1974.
398
CLINICAL CHEMISTRY, Vol. 21, No.3, 1975
The deficiencies of such analytical procedures, reviewed elsewhere (2), are such that it is enough to relate here that widely divergent results have been recorded repeatedly in comparative studies of various
existing procedures (1, 2-7). Thus, a more reliable
routine method for assessingclinical proteinuria
clearly is needed.
In the present discussions “clinical proteinuria”
should be interpreted medically as the presence of
excess plasma proteins in the urine. It should be
noted,however,thatthe proposed Tsuchiya’s reagent
willprecipitate
the normal “Tamm-Horsfall” glycoprotein (8), which isthe most abundant of the proteins derived from the urinary tract itself, as well as
urinary proteins that originate from the plasma.
After testing several reported procedures from the
literature, I concluded thatTsuchiya’sreagent(ethanolic HC1-phosphotungstic acid) for precipitating
urinaryproteins,followedby biuretspectrophotometry,was the most reliable.
In 1923 Shevky and Stafford used this reagent to semiquantitatively
estimate
urine proteins (9), and it was later slightly modified
(1, 10, 11). In the method described by Piscator (11),
urine and reagent are allowed to react for 15 mm at
room temperature before removal of the precipitated
proteins by centrifugation.
Savory et al. (1) recommend precipitation for 15 mm in an ice bath because
they found that values for seven normal samples of
urineaveraged6.1% (range, 2.6 to 10.2%) higher when
precipitation
was performed at 0 #{176}C
rather than 25
OC.
I decided to investigate precipitation
of proteins
from proteinuricsamples atan elevatedtemperature,
and I used 56 #{176}C,
forconvenience,becausemost clinical laboratories have water baths or heating blocks
adjusted to this temperature for various serological
tests.I found that most urinary protein precipitates
adsorb less tan-colored
pigments
at 56 #{176}C
than 0 #{176}C.
Moreover, any color adsorbed during 15 mm at 56 #{176}C
is more nearly removed by ethanol washing, resulting
in both lower spectrophotometric
blanks and less interference with the color produced by the biuret reac-
Table 1. Effect of Precipitation Temperature
on Determination of Urinary Total Proteins
with Tsuchiya’s Reagent (10 Specimens)
Urinary
Absorbancos
proteins,
Temp., C
0
56
0
56
0
56
0
56
0
56
0
56
0
56
0
56
Bluret
-
bk.
Corr.
g/Iit.r
0.110-0.010
0.086 0.002
0.210-0.027
0.100
0.084
0.183
0.46
0.38
0.83
0.156-0.008
0.036-0.005
0.023-0.000
0.437-0.022
0.353
0.013
0.148
0.031
0.023
0.415
0.340
0.68
0.14
0.11
1.90
1.55
0.192-0.032
0.132-0.023
0.087 0.000
0.076 0.000
0.160
0.109
0.087
0.73
0.50
0.490-0.056
0.076
0.434
0.35
1.98
0.453
0.041
0.333-0.005
0.412
0.328
1.88
1.50
0.290
0,404
0.449
0.110
0.107
0.328
1.32
1,84
2.05
0.50
0.49
-
-
-
-
0.295-0.005
Q.4-Q.Q52
56
0.481 = 0,032
0
0.132=0.022
56
0.128= 0.021
Std,(1.5guItar) 0.328= 0.000
0
0.40
-
0 ‘C mean, 1,03/Oter; 55‘C mesn, 0.53/Oter.
10 000. Pollak et al. (12) found protein recoveries to
be excellent when serum or urine was concentrated
through this membrane. Moreover, Diaflo ultrafiltration had little effect on the electrophoretic,
immunoelectrophoretic,
or quantitative
immunodiffusion
behavior of serum or urine proteins, confirming the
results of Blatt et al. (13). Such urinary protein studies represent the continuance of numerous earlier investigations (14, for example) on the characterization
of total nondialyzable solids of normal human urine,
of which proteins form a part.
Reagents
Tsuchiya’s reagent, as modified by Lehmann
(1).
In a screwtop bottle containing 5.0 ml of concentrated hydrochloric acid, 6.0 ml of distilled water,
and 77 ml of ethanol:water (95:5 by vol), dissolve 1.5
g of phosphotungstic acid.
Benedict’s qualitative
solution, as cited by Piscatom (11). In a beaker containing
about 60 ml of distilled water, dissolve 17.3 g of sodium citrate dihydrate and 10.0 g of anhydrous sodium carbonate. In
another beaker dissolve 1.73 g of’ cupriesulfatepen=
tahydrate in about 10 ml of distilled water, Mix the
two solutions and dilute the final ragent to 100 ml
with distilled water. Stable indefinitely at room tam.
perature.
Alkaline citrate “blank” solution, Prepare this re=
agent in the same manner as the Benedict qualitative
tion. The typical magnitude of the statistically signif
kant bias between precipitation
at 0 and 56
was
assessed with 10 urine samples. The data (Table 1)
showthat samples precipitated at 0 #{176}C
averaged 0.10
e 0,11 (SD)g of protein/liter (range, =0,21 to +0.34;t
2.86) higher than duplicates precipitated at 58 e.
That thissignificant
difference is principally attrlb’
utable to adsorbed urine pigments is further substan.
tiated by the finding that corrected absorbance values
for protein standards dissolved in physiological saline
are unaffected by the temperature of precipitation.
The heating step constitutes the most important im.
provement in the proposed methodology for pro=
teinuric specimens,
Matarials and Mithods
Apprntue
I used a Bausch & Lomb ‘Spectronic 70” spectro=
photometer with matched 1.27 cm (1j=inch) round cu
vets. Comparative data were obtained by an ultrafil=
tration technique involving repetitive concentration
and dilution of urine samples at room temperature
by use of a nitrogen.pressurixed stirred ultrafiltra=
tion cell, Model 12 (capacity, 10 ml), and “Diaflo” ul=
trafiltration
membranes, type PM1O, 25 mm diame=
tar (both from Amicon Corp., 21 Hartwell Ave., Lex.
ington, Mass, 02173). These membranes are com=
posed of inert, nonionic polymers and retain solutes
with an average molecular weight in excessof about
solution, omitting the cupric sulfate, Stable indefl=
nitelyat room temperature.
Protein
standard solutio,te, The biuret reaction
given by a phosphotungsticacid precipitate of uri=
nary proteinsreportedly
yields the same color lnten.
sity with an equivalent weight of albumin or globu=
lins (1), as I confirmed
with use of either bovine
serum albumin,
human serum albumin,
or pooled
human sara diluted appropriately with sodium chlo=
ride solution (8.5 g/liter) to achieve a known eoneen=
tration of about 1,5g/liter. “Abnormal Urine Chemis=
try Control” (Product No. 2920=80; Lederle Lnhora=
tories, Pearl River, N. V. 10965), “2=Pak=Chemistry
Urine Control, Supplemental”
(listed No. 045=426;
Hyland, Costa Mesa, Calif. 92628), or “Urine Con=
trol” (cat. No. U3001=2; Dade Division American
Hospital Supply Corp., Miami, F’la. 88152) have
served equally well as daily quality=control materials,
These freeze=dried products are prepared from
human urine and many constituents are assayed, in=
eluding proteins in concentration of 0.9 to 1.5 g/liter.
When 2.0 ml of standard or urine Is used in the pro=
cedure to be described, Beer’s law is followed up to
2.0 g of protein per liter, Greater protein eoncantra=
tions require more than 3.15 ml of biuret reagent for
complete color production. The diluted protein stan=
dards are stable about one weak at 4 #{176}C
Procedures
Proposed routine biuret
total proteins.
procedure
CL,INICALCHtMISTnY
Vol. 21 No
for
,
urinary
1575
3S
hydroxide
solution (30.0 g/liter)
plus 0.15 ml of alka-
line citrate “blank” solution. Record the absorbances
of the standard and sample pairs containing alkaline
0.1450
citrate “blank” reagent.
9. Adjust to zero absorbance with a cuvet containing 3.0 ml sodium hydroxide solution (30.0 g/liter)
plus0.15ml ofBenedict’ssolution.
Record the absorbances of the remaining pairs, which contain Ben-
0.350
edict’s solution.
Calculations
0.250
0.150
0.050
0.50
URABT
1.50
AL
2.50
pso’rEms(g/u.t.r)
Fig. 1. Typical
standardization
curveforurinary
proteins
in
clInical protelnurla
2 n of standard soiutons of human serum albumin used
1. Into one pair of test tubes or cuvets add 2.0 ml
of centrifuged urine sample that contains not more
than “2+” protein by qualitative testing(see “Calculations”). Into a second pair add 2.0 ml of protein
standard solution.
2. Add dropwise, with mixing, 2.0 ml of Tsuchiya’s
reagent to all tubes, heat at 56 #{176}C
for 15 mm, coolto
room temperature with tap water, centrifuge for 10
mm, carefully decant the clear supernatant liquids,
and invert the tubes on filter paper to drain.
3. Add 1 ml of absolute ethanol to all tubes, disperse the protein pellets by means of a mechanical
mixer, centrifuge the tubes rapidly for 5 mm, decant
the ethanol and again invert the tubes on filter paper
to drain thoroughly.
4. Repeat step 3 once.
5. Add 3.0 ml of sodium hydroxide solution(30.0
g/liter) to all tubes,and mix to dissolvethe washed
protein precipitates.
6. Add 0.15ml of alkaline citrate “blank” reagent
to one of each pair of standard and urine duplicates,
0.15ml ofBenedict’s solution to the other.
7. After mixing, leave the tubes at room temperature for 20 mm for maximum development of the biuret color, which is stable for at least an hour.
8. Adjust the spectrophotometer
to zero absorbance at 540 nm with a cuvetcontaining 3.0 ml sodium
400
CLINICAL
CHEMISTRY,
Vol. 21, No.3,
1975
Calculate “corrected” absorbances by subtracting
the alkalinecitrate“blank” absorbances (step 8)
from the respectivebiuretabsorbances(step9).
Urinary totalproteins(ing/liter) = (corr.A sample/corr. A std.) X concn.of std.soln. The sensitivity
is about 20 to 30 mg/liter.If the protein concentration is greater than 2.0 g/liter, repeat the analysis,
using 2.0 ml of urine previously diluted appropriately
with water. A typical standardization graph, in which
2 ml of standard solutions of human serum albumin
are used, is given in Figure 1.
Comparative
ultra lilt ration-biuret
procedure
for
urinary total proteins.
1. Add 10.0 ml of centrifugedurine to the assembled ultrafiltration
cell and ultrafilter the sample to a
volume ofabout 0.1-0.2 ml.
2. Add 10 ml water to the celland ultrafilter again
as above.
3. Repeat step 2 twice.
4. Carefullyadd sodium chloride solution (8.5 g/
liter)
to the cell’s
10-mi graduationmark, stirrapidly
for about 15 s,promptly transferthe solutionto a
testtube,and centrifugerapidlyforabout 5 mm.
5. Pipet 2.0ml of the centrifugedsolution of urinary proteins into each of two cuvets and add 1.0 ml
of sodium hydroxide solution (90.0 g/liter).
6. Complete the procedure and calculations as described above, starting with step 6.
Results and Discussion
The standard deviationsforproteinin threeurine
specimens, each determined 10 times each, but on
separate days, by the proposed routine procedure,
were ±40, 20,and 40 mg/liter; the coefficients of variation were ±2.0, 2.4, and 2.1%,respectively.
In another four-week study of day-to-day precision, 30 urine samples, each analyzed in duplicate on
a different day, showed a CV of ±3.1% [1.50 ± 0.046
(SD) g/liter].
Duplicate recoveries of albumin solutions added to
three proteinuric urine samples in final concentrations of 0.62, 1.51, and 2.34 g/liter averaged 102, 97,
and 101%, respectively. Recovery of proteins from,
urine specimens is difficult to establish with certainty, because of the problems inherent in quantitating
urine proteins accurately at low concentrations (12).
Accuracy was evaluated by measuring the protein
concentrations of 11 urine samples by the proposed
procedure and by the ultrafiltration
methodology
outlined above. Values by the ultrafiltration
method
averaged only 10 ± 49 (SD) mg/liter higher than the
precipitation
technique. The CV was ±3.4%, the
standard error ±15 mg/liter. These correlation data
establish that the routine procedure yields results
that are not statistically significantly different from
those obtained by the more laborious ultrafiltration.
In this investigation no quantitative
measurements
of proteins in normal urine were undertaken. However, 2-mi portions of 10 urine samples giving “negative” results with sulfosalicylic
acid were carried
through the procedure. The “corrected” absorbances
ranged from 0.000 to 0.010 (mean, 0.003). If desired,
the method may be adapted to studies of normal
urine by analyzing a larger volume of sample (20 ml),
as proposed by Savory et al. (1).
Proposals for standardization
of total protein assays in serum as detailed by Peters (15) state “total
protein shall be defined as the totalnondialyzable
(molecular weight greater than 10 000) polypeptide
substances.” Also, because results for serum by biuret techniques are known to correspond closely with
values obtained by the Kjeldahl technique, specification of an established version of the biuret reaction,
with use of standards prepared from either bovine or
human serum albumin, is advised for intra-laboratory use (15, 16). Savory et al.(1) preferreda standard
solution
of pooled human sera for their studies on
normal
urine.
Although reports on serum to date (15, 16) do not
includespecificdiscussionof urinary total proteins,
it would seem desirable to adopt the same analytical
principles
as far as possible. This communication
describes and evaluates such a routine technique for assessing clinical proteinuria.
I believe that the method
providesa valid basis for comparison and investiga-
tion of other procedures for urinary “total proteins”.
For example, a brief preliminary study indicates that
the acid-acetone method (17) for precipitating
urinary total proteins (including mucoproteins) generally may yield similar values. However, even if further
data should support this tentative observation, the
method described here is more convenient.
References
1. Savory, J., Pu, P. H., and Sunderman, F. W., Jr., A biuret method for determination
of protein in normal urine. Clin. Chem. 14,
1160(1968).
2. Lancaster,
R.G.,Quantitative Urinary Protein, Speciali’opics
No. ST-63 (1973), American Society of Clinical Pathologists,
Chicago, Ill., 19’73.
3. (a) Doetach, K., Determination
of total urinary protein by combined gel filtration and automated biuret reaction. Clin. Chem. 18,
296 (1972); (b) Doetsch, K., and Gadsden, R. H., Further on urinary protein determination.
Clin. Chem. 20, 1384 (1974). Letter to
the Editor.
4. Hemmingsen, L., A simple and rapid method for quantitative
determination
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L. J., Lim, K. L., Beng, C. G., and Lau, K. S.,
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10. Lehmann, J., Proteinbestamning
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of cadmium proteinuria.
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C. A., Protein solutions: Concentration
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Total nondialyzable solids (TNDS) in human urine. I. The amount
and composition of TNDS from normal subjects. J. Clin. Invest.
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15. Peters, T., Proposals for standardization
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16. Doumas, B. T., and Wang, T. W., Standardization
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proteins. Clin. Chem. 20,882 (1974). Abstract.
#{163}7.
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401