A Biuret Method for Determination
Protein in Normal Urine
John Savory,
of
Pin H. Pu, and F. William Sunderman, Jr.
A biuret method has been developed which provides quantitative measurements of
protein in normal urine without interference from drugs or pigments. This method is
intended for use in monitoring clinical trials of new drugs-to detect nephrotoxicity.
Protein is precipitated from duplicate samples of urine by addition of cold ethanolic
phosphotungstic acid. The protein precipitates are separated by centrifugation and
washed with ethanol. Protein from one of the duplicate samples is dissolvel in biuret
reagent. Protein from the second sample is dissolved in an alkaline tartrate reagent
which is identical to the biuret reagent, excepting that copper sulfate has been
omitted. After 20 mm., the differential absorbance of the two samples is measured
at 540 m. The limit of sensitivity for detection of protein in urine is 0.5 mg./100 ml.
The coefficient of variation of replicate analyses of protein in normal urine is 4.2%.
The recovery of protein added to urine averages 103 ± 3%. Analyses of urinary
protein by the biuret procedure provide close correlation with measurements by an
amido black staining method. Systematic search has failed to reveal interference
from urinary pigments, compounds, or drugs which are normally or occasionally encountered in hospitalized patients. In 24-hr. collections of urine from 28 healthy
adults, the protein concentrations averaged 6.2 mg./100 ml. (range 3.0-12.2),
and
the protein excretions averaged 77 mg./day (range 40-150).
R.
INTEREST
IN QUANTITATIVE
METHODS
foi’ deterniination
of prourine from lmealthy subjects
has been stimulated
by tile revised
regulations
of the Food amid Drug Administration
regarding
clinical
trials
of new drugs
(1).
The protocols
for many clinical
trials
currently
specify
analyses
of urinary
protein,
inasmuch
as increased
excretion
of protein
in urine is one of time most dependable
indications
of
nephrotoxicity
(2).
Fnfortunately,
the methods
employed
for measECENT
teill
From
ill
the
Pathology
Department,
University
of
Florida
College
of
Medicine,
Gainesville,
Fla.32601.
Supported
by U. S. Atomic
Energy
Commission
Grant AT-(40-1).3461;
by American
Cancer
Society
Grant E.374B;
amid by U. 5. Public
Health
Service Research
Grant
CA-08783-02
from
the National
Cancer Institute.
Presented
at the scientific
sessions
of the American
Association
of Clinical
Chemists
at the
American
Association
for the Advancement
of Science meeting,
New York, N. Y., Dec. 27, 1967.
Received
for publication
Feb. 2, 1968; accepted
for publication
Mar. 31, 1968.
1160
Vol. 14. No. 12, 1968
URINARY
PROTEIN
1161
urements
of urinary
protein
are often subject
to interference
from
drugs and drug metabolites.
For exanmple, colorimetric
determinations
of protein by the Folin-Lowry phenol reaction may be affected by
streptomycin,
sulfonamides,
salicylates,
p-aminosalicyclic
acid, phenacetin,and chiorpromazine (3).
Similarly,
turbidimetric
estimatioims
of
protein by the sulfosalicyclicacid procedures are subject to interference from
organic
iodine
compounds,
and from
i-butyl-3-carboxyphenylsulfonylurea,
an excretory
product
of tolbimtamide
(3).
The most sensitive
methods
for determinatioll
of urinary
protein
involve drying specimens
of urine on strips of filter paper and staining
the dried proteins
with amido black (4, 5), bromphenol blue (6), or
light green (7) dyes. The dye which becomes
bound to the proteins
is
either estimated directly by densitometry
or is eluted and measured
by colorimetry.
Although
interference
from drugs is apparently
not a
serious problem
with the dye-binding
methods,
such methods
are prone
to marked
day-to-day
variability
and are inconvenient for routine use
in clinical laboratories.
Moreover,
the dye-bindilmg
methods
are subject
to systematic errors owing to differencesin time relative
affinities
of the
protein
fractions
for amido black or bromphenol
blue (8).
Several
investigators
have employed
the biuret reaction
for qllantitative measurements
of urinary
proteins
in patients with a pathologic
process
(9-14),
since the biuret reaction is relativelyspecificfor proteins and yields equivalent
chromogenicity
with albumin
and globulin
fractions
(15). The biuret
reaction
has been reported
by Piscator
(16)
to be applicable
to measurements of protein in normal
urine, and has
been used successfully
by him for the detectiolm of renal toxicity
in
industrial
workers
who are exposed to cadmium. The I’iscator
procedure (16) has been employed
in our laboratory
during the past 2 years;
it has undergone
a lmumber of modifications
which have improved
its
sensitivity
and precision
and which Imave Inilmilnized immterference from
drugs and pigments.
Method
Principle
After
centrifugation
of urine to remove sediment, the protein is
precipitated from duplicate
20-ml. samples
of urine by addition
of an
equal volume of cold ethanolic
phosphotungstic
acid. After ii mm. in
an ice bath to ensure
complete
precipitation,
the protein
precipitates
are separated
by centrifugation
and washed with cold ethanol.
Protein
from one of the duplicate
samples
is dissolved
in 4 ml. of biuret
reagent. Protein
from the second sample
is dissolved
in 4 ml. of an alkaline tartrate
reagent
which is identical
to the biuret
reagent,
except
1162
SAVORY
that copper sulfate
ture, tile differential
540 m.
fT
AL.
Clinical
Chemistry
has been omitted.
After 20 mm. at room temperaabsorbance
of the two samples
is measured
at
Reagents
EthanoliC
phosphotungstie
aCid
Transfer
50 ml. of cone. hydrochloric
acid (s.g. 1.19), 60 ml. of distilled
water,
and 770 ml. of 95c/
(v/v) ethanol
into a 1-L. beaker.
Add 15 gm. of phosphotmmgstic
acid
and dissolve.
The solution
is filtered
and stored
in the refrigerator.
Ethanol,
absolute
Sodium
GlIb ride
Store
solution,
in the refrigerator.
0.85%
(u/v)
Biuret
reagent
Add
9.6 gm. of potassinm
sodium
tartrate
(KNaC4H1O.4H00)
to a 2-L. graduate
cylinder
with ground-glass
stopper and dissolve in 400 ml. of distilled
water.
Add 2.4 gin, of
cupric
sulfate
pentahvdrate
(CuSO4.5H00)
and dissolve.
Slowly add
360 ml. of 2.5 N sodium
hydroxide
followed
by 1.0 gin. of potassium
iodide.
Dilute the solution
to 1500 ml. with distilled
water.
The rcagent is stored
in a plastic
bottle and is stable for several
months
at
room temperature.
Alkaline
tartrate
reagent
This solution
is prepared
in exactly the
same manner
as the biuret
reagent,
except that the cul)ric sulfate
is
omitted.
Protein
standards
The concentration
of protein
nitrogen
in a
sample
of pooled human
serum is determined
by the Kjeldahl
procedure
(17).
The total protein
concentration
is calculated
usilmg time
factor
of 6.54 gm. of protein
per gram of nitrogen
(18).
The serum
protein
standard
is stored in small ampoules
at -10#{176}.An appropriate
dilution
of the protein
standard
is made with 0.85%
(w/v)
sodium
chloride
solution
to achieve
a flumal concentration
of 10-15 mg. of protein per 100 ml. The dilute protein
standard
is stored
at 4#{176}
and is
stable
for 1 week.
SpecialApparatus
Centrifuge
tubes, 50 ml., with ground-glass
stoppers
(Cat. No.
45168, Kimble Glass Co., Toledo, Ohio)
These tubes fit centrifuge
cups
(No. 367), with rubber cushions
(No. 575), metal shields (No. 572), aimd
trunnion
rings
(No. 366)-International
Equipment
Co., Boston,
Mass.
Procedure
The
aliquot
volume of the 24-hr.
of
Urille
is centrifuged
collection
at
of urine is measured.
2000 rpm for 15 mill.
A 50-mi.
The
super-
Vol. 14, No. 12. 1968
URINARY
1163
PROTEIN
liatant urine is decanted
into a beaker alldis used for the analysis.
The
protein concentration in tilecentrifuged urine sample is estimated by
means
of Albustix
test strips
(Ames
Co.).
If the Albustix
reaction
result is read as “negative”
or “trace,”
20 ml. of urine is taken for
analysis.
If the Albustix
test gives qualitative
reaction
results
of 1+,
2+, 3+, or 4+, the following
volumes
of urine should be taken: 10, 5, 2,
and 0.5 ml., respectively.
The requisite
volumes
of urine are transferred
into duplicate
50-mi. centrifuge
tubes and are diluted
(if necessary)
to 20 ml. with 0.85% (w/v)
sodium
chloride
solution.
Into two
additional
pairs of 50-ml. centrifuge
tubes are transferred
(in duplicate) 20 ml. of sodium chloride
solution
(blank samples)
and 20 ml. of
dilute protein
standard
solution
(standard
samples).
The centrifuge
tubes are placed in an ice bath for 5-10 mm.
A 20-ml. portion
of cold ethaumolic phosphotungstic
acid reagent
is
added to each of the tubes.
The contents
of tile tubes
are mixed,
and
the tubes are allowed to stand
in the ice bath for 15 mm., at the end of
which time they are centrifuged
at 2000 rpm for 15 mm. Tile supernatant
liquids are discarded
by decantation,
and the tubes are inverted
to drain onto filter paper.
Cold ethanol
(10 ml.) is added to the tubes,
and the protein
pellets
are dispersed
by means
of a Vortex
rotary
mixer.
The tubes are centrifuged
at 2000 rpm for 10 mm., and the
ethanol
is discarded
by decantation.
The tubes are again inverted
to
drain
Ollto
filter paper.
Biuret
reagent
(4 ml.) is added
to one of each pair of duplicate
tubes; 4 ml. of alkaline
tartrate
reagent
is added
to the remaining
tubes. The protein precipitatesare dissolved
by agitation
with a Vortex rotary mixer.
Time tubes are allowed to stand at room temperature
for 20 mm., which is the time required
for maximum
color development.
The color is stable for at least 2 hr. Time contents
of each tube
are transferred
to a Coleman
spectrophotometer
cuvet (19 mm. diameter).
An adapter
is placed in the bottom
of a Coleman
cuvet holder
so that the absorbance
of a 4-mi. volume can be measured.
A Coleman
Junior
spectrophotometer
is adjusted
to zero absorbance
at 540 m,
using the cuvet which contains
the blank sample aimd alkaline
tartrate
reagent.
The absorbarices
of the remaining
samples
are then measured
and recorded.
Calculations
Protein
cone.
(nmg./100
ml.)
=
X
-
std.
cone.
where:
=
absbi’banc#{234}of urille
sample
treated
with
biuret
reagent
1164
SAVORY
=
A8
A88
=
A88
=
=
absorbance
reagent
absorbance
absorbance
reagent
absorbance
of urine
of protein
of protein
of blank
fT AL.
saml)le
treated
standard
standard
sample
Clinical
treated
treated
treated
with
alkaline
Chemistry
tartrate
with biuret reagent
with alkaline
tartrate
with
biuret
reagent
Results
Modifications of Piscator Method
The following
are the principal
modifications
of the Piscator
method (16) which were introduced
in the present
method:
(1) The volume
of urine was increased
from 2 to 20 ml., in order to increase
the sensitivity of the method.
(2) Protein
precipitation
was performed
in an
ice bath instead
of at room temperature,
since it was found that the
yield of protein
from seven normal
samples
of urine averaged
6.1%
(range
2.6-10.2%)
higher
when precipitation
was performed
at 00
rather
than at 25#{176}.
Prolongation
of the precipitation
period
from 15
mm. to 0.5, 1, 2, 4, or 18 hr. did not ilmcrease the yield of protein.
(3) The protein
precipitate
was washed
once instead
of twice, since a
second ethanol
washing
did not significantly
affect the analytic
results.
(4) Spectrophotometry
was performed
instead
of at 330 m, in order to avoid
violet spectrophotometer
almd to avoid
absorb
light at 330 m1z. (5) Pigment
preparation
of urine “blanks”
which
samples,
except that copper sulfate
is
at the 540 m absorption
peak
the imecessity of using an ultrainterference
from drugs which
interference
was prevented
by
are identical
to the urine biuret
omitted.
Sensitivity, Precision, and Recovery Studies
The limit of detection
for urine protein
(i.e., the protein
concentration which gives a corrected
absorbance
value of 0.01) was 0.5 mg./100
1111.
As illustrated
in Fig. 1, the calibration
curve was linear with protein concentrations
ranging
up to at least 27 mg./100 ml. The coefficient
of variation
of duplicate
analyses
of 24-hr. urine collections
from 28
healthy adults was 4.2% (mean concentration
6.2 mg./100 ml.; standard
deviation
of duplicates
± 0.26).
Measurements
of recovery
of serum
protein
added to seven normal
urine sanlples
in a concentration
of 15
mg./100
ml. averaged
103% (standard
deviation
± 3) with a range
of
98-109%.
Preservation of Urine Specimens
Urine specimens
were stored
in a refrigerator
at 4-10#{176}
during
the
24-hr. collection
period
and until the time of analysis.
No preserva-
Vol. 14, No. 12, 1968
URINARY
1165
PROTEIN
tives were added.
The stability
of urinary
protein
concentration
was
studied
by repeated
analyses
of a specimen
of urine which was kept
for 1 month at 4-10#{176}.
Protein
determinations
were performed
three
times each week for 4 weeks.
The 12 measurements
yielded
a mean
0.6-
0.5-
Fig.
curve
1.
for
of urinary
biuret
Calibration
0.4-
measurementa
protein
by the
procedure.
0.3-
0.2
0.
G
I
0
5
0
15
20
Protein
1mg 1100 ml)
25
30
protein
concentration
of 39.3 mg./100 ml. (S.D. ± 0.5) with a range of
38.5-40.5
mg./100
ml. No consistent
increase
or decrease
in protein
concentration
was observed
during
the period
of observation.
The stability
of protein
concentration
in urine
which
was kept
frozen at -15#{176}
was investigated
by performing
five replicate
analyses
of a urine specimen
before and after freezing
for 1 month.
The mean
protein
concentration
was 40.5 mg./100
ml. before
freezing
and 40.7
mg./100
ml. after freezing.
Comparisons with Other Methods
Concentrations
of protein
lii 24-hr. urine collections
from 14 healthy
adults were measured
by the biuret procedure
and by the amido black
method
of Kaltwasser
et al. (5). As shown in Fig. 2, urinary
protein
levels measured
by the amido black method
averaged
8% higher
than
those measured
by the biuret
procedure.
The correlation
coefficient
was 0.96, and the standard
error of estimate
was 0.77. Protein
concentrations
in 24-hr. urine collections
from 28 healthy
adults were measured by the biuret procedure
and by the sulfosalicyclic
acid method
of
Poortmans
and van Kerchove
(4).
As shown in Fig. 3, there
was
apparently
a random
scattering
of results
of protein
measurements
by these two technics.
1166
SAVORY
fT AL.
Clinkal
Chemistry
Normal Values for Urinary Protein
Collections
of urine
(24 hr.)
female
adults who were judged
history
and physical
examination.
were obtained
from 14 male and 14
to be healthy
on the basis of medical
The mean concentration
of urinary
14
20
Fig.
Slope
of Regression
-1.08
2.
Comparison
of
protein
concentration
measurements
in 24-hr.
urine coflections
from
healthy
adults
by
biuret method and by
amido black procedure
Kaitwasser
et at.
Broken
tine indicates
o
culated
solid
regression
line
theoretical
14
the
the
of
(5).
cal-
line;
represents
the
relationship
of
x=y.
0.
0
2
Protein
4
roy
6
00 roll Biuret
8
10
2
14
Procedure
protein
in the males was 6.4 mg./100
ml. (range
3-12), and the mean
concentration
in the females
was 6.0 mg./100
ml. (range
4-10). The
mean excretion
of urinary
protein
in the males was 79 mg./day
(range
48-150),
and the mean excretion
in the females
was 76 mg./day
(range
40-131).
There was no significant
difference
between
the urinary
protein concentrations
and excretions
in the two sexes, as determined
by
Student’s
t test.
Frequency
distribution
graphs
for urine protein
in
the combined
group of 28 healthy
adults are illustrated
in Fig. 4. The
urinary
protein
concentrations
(milligrams
per 100 ml.) are plotted
in
the graphs
on the left side of the figure, and the urinary
protein
excretions (milligrams
per day) are plotted
on the right side of the figure.
As shown in Fig. 4, the logarithmic
distribution
graphs
adhered
more
closely to time gaussian
conformation
than did time arithmetic
graphs.
Therefore,
the ±2 S.D. limits computed
after logarithmic
transformations are recommended
for use as the normal
ranges
of values.
Vol. 14, No. 12, 1968
URINARY
1167
PROTEIN
Normal Values for Urinary Albumin
Fractionations
of protein
in urine collections
from tile 28 healthy
adults were performed
after concentration
by ultrafiltration
and cellulose acetate
electrophoresis,
as described
by Suimderman
et al. (19).
The mean percentage
of albumin
was 37.7 (S.l). ± 5.8) with a range of
27-50%.
Albumin
excretions
in urine were computed
by multiplying
the total protein
values
(determined
by the biuret
Procedure)
by tile
percentages
of albumin
(measured
by electrophoresis).
Time mean excretion
of albumin
was 29.0 mg./day
(S.D. ± 12.0) with a range
of
18-52 mg./day.
Interfering
Drugs and Pigments
In an endeavor
to detect sources
of interference,
time biuret
procedure was employed
for measurements
of protein
in 24-ilr. collections
of urine from patients
on the medical,
surgical,
pediatric,
and psychi14
2
Fig.
3. Comparison
protein
of
concentration
measurements
urine collections
healthy
adults
in
#{176}-
24-hr.
from 28
by
the
biuret
procedure
and by
the sulfosalicylic
acid
method of Poortmans
and
van
Kerchove
0
<
E 8
6
(4).
.
Protein
#{149}#{149}:
#{149}, .
#{149}#{149}
1mg / tOO ml)
Biuret
Procedure
atric services
of the hospital.
Systematic
search failed to reveal
any
interference
from pigments,
including
porphyrins
and biliruhin,
or
from a wide variety
of drugs, including
aimtibiotics,
sedatives,
analgesics, vitamins,
tranquilizers,
and roentgenographic
contrast
media.
a
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c’J
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Vol. 14, No. 12, 1968
URINARY
1169
PROTEIN
Discussion
The normal
values for urinary
protein,
which were obtained
in the
present
study, are contrasted
in Table 1 with the values reported
by
previous
investigators.
The range of protein
colmcentrations
observed
in this study
(3-12 mg./100
ml.) agrees
with the findings
of most
previous
authors.
It may be noted that the ranges
of protein
excretions which have been obtained
by the Folin-Lowry
phenol
reaction,
in general,
have been higher
than the ranges
obtained
by the other
methods
of analysis.
The present
finding that there is no significant
difference
between
normal
excretion
of urinary
protein
in male and
female adults is consistent
with the observations
of Tidstrom
(6) and
Jorgensen
(28).
Time normal
values for urinary
albumin
obtained
in
the present
study are compared
in Table 2 with the values
reported
by investigators
who have employed
a variety
of electrophoretic
technics and various
methods
for quantitation
of total protein.
In the present study, measurements of protein concentration in normal urine by the biuret procedure
provided
reasonable
correlation
with
analyses
by an amido black staining
method, but did not correlate
with
protein
estimations
by a sulfosalicylic
acid method.
The absorbances
obtained
with normal
urine by use of the sulfosalicylic
acid method
TABLE
1.
NoRMAL
\ALIJES
FOR
EXCRETION
OF PROTEIN
IN
Urinary
Au/Sore
M#{246}rner
(20)
Gunton
&
Burton
(21)
Tarnoky
(22)
Rigas & Heller
Year
(23)
Boyce et al. (24)
Melo et al. (25)
Coye &
Rosandich
(26)
Piscator (16)
Tidstrom
(6)
Saifer &
Gerstenfeld (27)
Jorgensen (28)
Hemmingsen
&
Skov (29)
Present
authors
Medhodof
quantitation
No.of
iubjecte
Gravimetry
1947
1951
1951
100 d’
42
15
1934
1959
Surface activity
Surface
activity
Refraction
gradient
scanning
Biuret reaction
Folin-Lowry
reaction
1960
1961
1963
Folin-Lowry
reaction
Biuret reaction
Bromphenot
blue
10 d’
22
29
29 9
Folin-Lowry reaction
Folin-Lowry
reaction
1968
Folin-Lowry
reaction
Nitrogen
analyses
Biuret reaction
1968
ml.
-
2-
& range)
8)
7)
34(l-
-
50)
45(4253)
126 (90-168)
-
55(22-130)
5.5(3-12)
-
-
36(
-
29(10-
9-
73)
80)
-
194 (S.D. ±53)
122(91-138)
115(85-145)
-
2 16(38-394)
-
-
d’
9
-
39(30-
-
-
d’
9
mg/day
37(0-l2)
5.6(2-12)
13
15
8
17
18
49
49
14
14
(mean
-
mg./100
1895
1964
1967
URCNE
protein
6.4(3-12)
60(4-lO)
96( 2-190)
79 (48-150)
76(40-131)
1170
SAVORY
Table
2.
NORMAL
BASED
\ALUES
UPON
FOR
ET AL.
Clinical
EXCRETION
ELECTROPHORETIC
OF ALBUMIN
IN URINE
FRACTIoNATIoNS
Urinary
Au/liar,
Year
Rigas & lleller(2.i)
Boyce ci a!. (24)
MeGarry
ci a!. (30)
Well) ci a!. (31)
Hemmingsen
Skov
Present
(211)
authors
1951
1954
1953
1958
Me/hod
of
electrophoreeis
No. of
oubjects
Tiselills
Tiselius
15
13
7
Tiselius
Tiselius
Filter paper
Chemistry
%
ablumi
n (mean
of total
36(S.D±5)
38
& range)
mg/day
13(S.D±2)
17
39 (32-54)
11
23(13-27)
31(15-45)
11
27
36
&
1968
1968
Aciylamide
Cellulose
gel
acetate
49
28
53(22-77)
38
(27-50)
29(18-52)
ranged
from 0.005 to 0.045. Therefore,
it is apparent
that the sulfosalicylie
acid method
was not sufficiently
sensitive
to permit
quantitative measurements
of protein
in normal
urine.
The biuret method
described
in this paper is particularly
intended
for use in monitoring
clinical
trials
of new drugs-as
a means
of
detecting
drug-induced
nephrotoxicity.
The method
should also have
clinical
application
as a quantitative
means
of assessing
orthostatic
(32, 33) and exercise-induced
(26, 34) proteinuria.
Moreover,
the investigations
of Harlan
et at. (35) indicate
that quantitative
measurements
of urinary
protein
may be valuable
as an index of immunologic rejection
in patients
who have received
renal transplants.
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