Determination of Urine Protein

H. W. Marlow*

NE of the earliest evidences of serious vascular disease or of

nephritis is proteinuria. It is one of the better single indicators of
renal abnormality. Qualitatively, such tests as the sulfo-salicylic acid,
nitric acid ring, heat and acetic acid tests, and others depend entirely
upon a turbidometric measurement. This utilizes the judgment and
memory of the technician as to progressive increases in turbidity for
a + 1 to + 4 value. Free (4) and his co-workers have explored new
colorimetric tests for proteinuria, taking advantage of the so-called
“protein error of indicators” by utilizing the salicylate buffer (or
other material) and bromphenol blue in tablet form. Another com-
mercial preparation has a thick bibulous paper impregnated with the
dye, tetrabromphenol blue, and citrate buffer. These tests, described
(4) in satisfactory detail by Free and his co-workers, are good screen-
ing procedures but are not quantitative in the sense of obtaining a
result in milligrams per 100 ml. of urine. The method of obtaining
semiquantitative results by these two color-producing tests still
yields a range of values rather than a specific answer, although the
tests are more sensitive and reports are more semiquantitative than
the older procedures such as the salicylate method.
Methods now reported for the quantitative determination of pro-
teins (1) in urine are long and tedious and the results are somewhat
questionable (probably tending to be low), since proteins denature on
standing or with complex treatment. It is therefore desirable that the
Prom the Department of Biochemistry, Veterans Administration Hospital, Downey, Ill.
* Present address: Basic Medical Science Institute, do American Embassy, Karachi,
Pakistan, APO 271, New York, N. Y.
Much credit is due Mrs. Ruby Lewis and Mrs. Harriet Laurie for their technical assistance
and interest in this study.
Received for publication Aug. 14, 1959.
34’
342 MARLOW Clinical Ch.mhfry

method used be ixnniediately applicable as well as sensitive and ac-

curate.
Such a method is desirable when running screening tests on
samples of urine on a large number of patients. It is imperative for
our purposes to follow any changes in urine protein concentration
during the administration of tranquilizing drugs. Serious vascular
damage may be done, or may have begun, before the changes in pro-
tein concentration in the urine will be high enough to be detected by
the usual qualitative procedures.

METHODS
The pigments of the urine may conceivably be thought to interfere
with the color production of protein. It was decided to see if these
might be removed and the test for protein in urine continued. After
trial by usual extraction methods with butyl alcohol, etc., the quantity
of protein was found to be much less, showing that much protein had
been lost in the process. An extraction column was constructed of
Amberlite C.G.-400, Type I (Fisher Scientific), a strong base anion
exchange resin. The urine was slowly drawn through the columns by
slightly positive suction. Protein was determined on the urine before
and after it passed through the column. No color was evident in the
resin-treated urine. Because a test showed the protein content to be
unchanged, it was considered unnecessary to subject the urine to a
resin column, and it was felt that the pigment found in urine was not
interfering with the determination of the protein content according
to the method employed and described below. Since the urine is
diluted 10 times before sampling, the interference was deemed even
less likely because of the even lower concentration of the pigments.
The technic of determination of small quantities of protein in
cerebrospinal fluid has been modified and studied by many investiga-
tors for reproducibility and accuracy in urine protein determination.
To avoid the time-consuming task of seeking out references the en-
tire composite procedure will be outlined in detail, with our thanks
to those who may recognize parts of their own methods (2, 3).
In order to reduce the protein content of the urine to a low value
which may be determined, the following procedure was adopted.
Three solutions are used: (1’) Solution A (an alkaline carbonate solu-
tion prepared by dissolving 10 gm. NaOH, 40 gm. Na2CO3, and I gm.
NaKC4O6 in water to make 1 L.); (2) Solution B (prepared by dis-
solving 5 gm. 0u504 .5 H20 in distilled water and diluting to 1 L.);
Vol. 6, No. 4, 1960 URINEPROTEIN 343

Table 1. REoovxaY or SanuM Pio’ram ADDan TO Uamn

Oaictaated
.jp* % 7 O.D. ra,./lOO mi ,n#{231}JlOO,nl.

Diluted serum (1:100) 90.5 0.043 64

2 S + 1 U (mixture) 84.0 0.076 113 117
1 S + 1 U (mixture) 81.0 0.092 137 143
1 5 + 2 U (mixture) 77.0 0.114 170 169
Diluted urine (1:10) 71.0 0.149 222 ..

* S, serum; U, urine.

and (3) Solution C (the Folin-Ciocalteu phenol reagent diluted 1:2).

After an aliquot of a 24-hour sample of urine (or an overnight sample
diluted to a specific gravity of 1.018) is diluted 1:10 with distilled
water, 0.2 ml. of this diluted urine is measured into a 19-mm. cuvette
with 3.8 ml. of distilled water and 1 ml. of the biuret reagent.#{176} The
mixture is allowed to stand 15 minutes. It is important that a blank
be made at the same time to account for substances that might affect
the optical density of the solntion This is done by mixing 4.8 ml. of
water and 0.2 ml. of the diluted urine in a cuvette. After the unknown
and the blank have stood for 15 minutes, 1 ml. of Folin reagent (Solu-
tion C) is added to each at 30-second intervals, beginning with the
blank, and each is allowed to stand exactly 15 minutes. The spectro-
photometer (Bausch & Lomb) is zeroed with the blank at 700 m
(using a red filter) and each sample is read in turn at 30-second in-
tervals. As a result of multiplying the optical density by the factor
1490, the protein concentration in milligrams per 100 ml. can be
obtained.
The sensitivity of this method was studied a number of ways, but
the results listed below show its ability to detect small changes in pro-
tein concentration.
*The biuret reagent is made by mixing 45 ml. of Solution A with 5 ml. of Solution B.
This is made fresh, just before use.

Table 2. Snwsmvrn or Tjansz PRO!rEIN Tssv

Urine. eeL Reruns, ,,sL losijais 4

(1:10) (1 :100) % 7 O.D. ,npJlOO ml. ,njJlOO eel.

10 0 82.5 0.084 125 ..

8 2 83.5 0.078 116 117

5 5 85.5 0.068 101 104
3 7 87.0 0.061 91 96
0 10 88.0 0.056 83 ..
344 MARLOW Clinical Chemistry

A sample of serum was obtained and diluted 1:100 ml. in 0.9%

saline, and analyzed as above. It gave a reading of 90.5 per cent
transmission. A sample of urine was analyzed by the above method
and the percentage transmission was found to be 71. The solutions
were then mixed as indicated in Table 1. The closeness of agreement
is evident. Other mixtures of different samples were run with the
results shown in Table 2.
It is believed that these random samples of serums and urine mix-
tures, together with their actual determination and calculated values
represent a rather critical analysis, and results are especially good
since these are representative of hundreds of such determinations,
with good duplicate results always obtained.
It is believed that (1) pigments in urine diluted 1:10 (and 1:300 in
the final analysis) do not interfere with the determination of protein
in urine, and (2) that this method is very easily adapted for a routine
quantitative determination of protein in urine in which the protein
is not denatured in the process. The quantity of protein in the urine
is much higher than 71 mg. per day as has been reported. This will
be the subject of a later report on the problem.
REFERENCES
1. McGarry, E., Sehon, A. H., and Rose, B. J., J. Clin. Invest., 34, 832 (1955).
2. Lowa, 0. H., RosxBaouou, N. J., Fsaa, A. L., and RANDALL, R. J., J. Biol. Chem.,
193, 265 (1951).
3. Douaninov, W. H., Lowny, 0. H., Rosxnaouaa, N. J., and FIELDS, WM. S., J. Lab. 4
Clin. Med., 39, 663 (1952).
4. Free, A. H., Bupe, C. 0., and Metzler, I., Clin. Chem. 3, 716 (1957).