Laboratory Trial of Protein Determination in Urine Using Different
pH Values of Acetic Acid and Acetate Buffer Method
Dinar Rahayu1, Tuti Rustiana1
1

Department of Medical Laboratory
Technology, Sekolah Tinggi Analis
Bakti Asih, Bandung, Indonesia
Correspondence:
Dinar Rahayu, Jl. Padasuka Atas
No.233, Padasuka, Cimenyan, City
of Bandung, West Java, Indonesia
Zip Code : 40192
Email:
dinarrahayu91071@gmail.com
Received: February 6, 2020
Revised: March 17, 2020
Accepted: April 10, 2020

Abstract
The determination of protein in urine is important in clinical
examination along with other parameters in urine. The
presence of protein in urine can be interpreted that there is a
disorder in kidney. Acid and heat coagulations method is
still widely used in many areas to determine protein in urine.
In this method, the characteristic of protein that will
precipitate in the presence of acid or if exposed to heat is
deployed to gain information about the amount of protein.
The geater amount of protein, the more prominence is the
coagulation. Urine pH also varies according to the
condition, classic acidosis will give an acidic urine and the
presence of ammonium producing bacteria can cause basic
urine. In this research acetic acid method with 6% of
CH3COOH and pH value of 2.9 and buffer acetic with pH
4.5 are used to determine the certain amount of protein (+3
value, corresponds with 2-4 mg/dL protein in urine) in
varied pH values of urine samples. In order to compare the
results, first in control urine with pH 6.8 the results of both
methods is compared with Mann-Whitney test and shows no
significant different, then the Kruskall-Wallis test is used to
compare the results in other pH values to control and the test
is shown also there are no significant difference. This shows
that either acetic acid at pH 2.9 or acetic buffer at pH 4.5 can
be used to determine protein amount in urine.
Keywords
Proteinuria, acetic acid, acetate buffer, urine pH, buffer

molecular

INTRODUCTION
Under normal physiological state, urine

weight

microglobulin

proteins

(β2-M),

(e.g.,

β2-

α1-microglobulin

is expected to be protein free. High molecular

(α1-M), and lysozyme), however, can freely

weight proteins in plasma (e.g., albumin and

pass through the

globulin) could not pass through the filtration

although the filtration amount is low and 95%

membrane due to the effects of the size

of these proteins are reabsorbed when

barrier and charge barrier of the glomerular

entering the proximal convoluted tubule

capillary

(1,2). The final urine protein content is

filtration

membrane.

Low

filtration membrane,

34

Dinar Rahayu, et al.

therefore low (only 30-130 mg/24h) and

practice (4). The proteinuria is commonly

consists primarily of plasma albumin (40%),

assessed by the heat and acetic acid test. Now

immunoglobulin fragments (15%), other

dipstick test is replacing the old methods (5).

plasma proteins (5%), and urinary system-

Heat coagulation test may be used in

originating tissue proteins (40%). The protein

under-resourced settings as an alternative to

concentration in a random urine sample is 0-

dipstick testing or other methods that are

80 mg/L, and the results of qualitative tests

unavailable or too costly. A test tube is filled

for urokinase protein are typically negative.

to two-thirds with urine. A few drops of

When the urine protein exceeds 150 mg/24h

dilute acetic acid are added to make the urine

or the concentration is above 100 mg/L, the

sample acidic. The upper part of the test tube

result for the qualitative protein test becomes

containing urine is heated (but not boiled)

positive. This is known as proteinuria (3).

over a burner. Acidic environment is to

Proteinuria is common finding in chronic

ensure that the coagulation formed is protein

renal failure patients and current evidence

because

indicates that the presence of proteinuria is an

coagulate but dissolves in acidic environment

early marker of an increased risk of

(6).

progessive

kidney

disease,

poor

cardiovascular and death (2).

on

heating,

phosphates

also

The result is considered to be negative
(no protein presents in sample) if there is no

As the measurement and sampling

cloud in solution after the test, a +1 (0.1

procedures for proteinuria assessment have

g/dL) result if there is a definite cloudiness, if

not been standardized yet, it is of clinical

held against a typed papers, letters typed can

importance to take into account different

be seen through the cloud in upper part of the

types

albumins,

test tube, +2 (<0.3 g/dL) if there is definite

laboratory techniques, and urine sampling

cloudiness, if held against the typed paper,

methods in order to have the best approach

letters typed are seen as faint lines in the

for an individual patient (4).

backgound at top of the test tube, +3 (0.3-1.0

of

urinary

proteins,

Total urinary protein can be assessed
using

dipstick,

precipitation,

g/dL) if there is a definite cloudiness, if held

and

against the typed paper, letters typed are seen

electrophoresis methods. Urine specimen for

as faint lines in the backgound at top of the

proteinuria assessment can be obtained either

test tube, +4 (>1.0 g/dL) if there is a thick

from a timed collection or a spot urine

solid precipitation, clot present at top of the

sample. Nevertheless, currently spot urine

test tube (6,7).
The newer method after heat and acid

preferred to a 24-hour urine sample in routine

coagulation is dipstick method to detect

Ina J Med Lab Sci Tech 2020; 2(1): 34-41

35

protein- or albumin-to-creatinine ratios are

Dinar Rahayu, et al.

proteinuria. The reaction is based on the

endogenous acid production from sulfur-

phenomenon known as the ‘protein error’ of

containing amino acids or metabolic acidosis

pH indicators (a dye) where an indicator that

(e.g., chronic diarrhea) while high urine pH

is highly buffered will change color in the

(usually >7) is caused by metabolic alkalosis

presence of proteins (anions) as the indicator

(e.g., vomiting), distal renal tubular acidosis,

releases hydrogen ions to the protein. The dye

urea-splitting organisms (e.g., Proteus), urine

is

tetra

that is infected will become alkaline over

bromophenol blue. When the dye is buffered

time due to formation of ammonia (NH3)

at pH 3, it is yellow; the addition of

from bacterial urease, urine that is exposed to

increasing concentration of protein changes

air for a long time can also have elevated pH

the color to geen then to blue. The developed

due to loss of CO2 from urine (8).

acid-base

indicators

such

as

color is compared with a color chart which

So the aims of this study was to

allows protein concentration to be gaded

examine the results from acetic acid test with

from

to

using 6% of acetic acid (pH 2.9) and acetate

concentrations from 1 to 10 mg/dL to geater

buffer (pH 4.5) of urine in varied pH values

than 500 mg/dL (8).

or urine samples with addition of known

trace

to

+4,

corresponding

At a constant pH, the development of any
geen color is due to the presence of protein.

amount of protein to mimic proteinuria
conditions.

Colors range from yellow to yellow-geen for
negative results and geen to geen-blue for

MATERIALS AND METHODS

36

positive results. But the clinical judgement is

Apparatus used in this research are 100

required to evaluate the significance of trace

mL

results. A color to be defined as proteinuria

photometer, stirrer, micropipette, spiritus

differ from each manufacturer, but usually

burner, test tubes and test tube-rack, Manti

geater than 30 mg/dL indicates significant

Lab MT-103 pH meter, FanMed 80-i

proteinuria (8).

centrifuge, and its centrifuge tubes.

beaker

glasses,

Dirui

DR-7000E

Urine protein mainly consists of albumin.

Materials used are Biuret Reagent. Biuret

This protein can reversibly and drastically

reagent is prepared by dissolved 1.5 g of

change its conformation when exposed to

copper sulfate pentahydrate (CuSO4.5H2O)

changes in solution pH (transitions occurring

and 6 g of sodium potassium tartrate

at pH 2.7, 4.3, 8, and 10) (9).

(KNaC4H4O6.4H2O) in 500 mL of water, and

As for urine, the normal pH range is 5 to

add 300 mL of 10% (w/v) of NaOH, and add

7, with low urine pH can be caused by high

1 g of potassium iodide and make a 1L

protein

solution (10).

diet

because

the

increased

Ina J Med Lab Sci Tech 2020; 2(1): 34-41

Dinar Rahayu, et al.

Acetic acid (CH3COOH) 6%, sodium
acetate (CH3COONa), acetate buffer (pH 4.5)

protein at +3 (4 mg/dL). For each of pH
group, a 40 mL portion of urine is needed.

is made with 1.544 g of sodium acetate

Simulated urine with protein that can be

anhydrous and 0.076 g of acetic acid to make

detected by heat and acid coagulation is

200mL of solution. pH is checked with Manti

achieved by adding serum with the known

Lab MT-103 pH-meter and adjusted with

concentration of protein to urine, the result is

HCl or NaOH to achieve pH of 4.5.

urine sample with protein concentration is 4

Serum and urine were taken from four

mg/dL (+3).

healthy volunteers from students of Sekolah

Urine batch is divided to make urine

Tinggi Analis Bakti Asih wtih normal plasma

samples with different pH by adding

protein level and negative test of protein in

ammonium hydroxide or citric acid (pH

urine.

range is 6, 6.5, 6.8, 7, and 7.5). We made four

To make serum, 3 mL of blood is drawn
from vein and put in centrifuge tube for 10

replications for each method in each pH
value.

minutes before being centrifuged at 1500 rpm

Simulated urine with pH 6.8 is chosen as

for 15 minutes to separate serum from blod

normal pH. To compare the median value

clot. Carefully serum is pipetted and tested

between acetic acid results goup and buffer

for protein with Biuret method. Twenty

acetate results goup at the normal condition

microliters of serum is added to 1mL of

(control at pH 6.8), the Mann-Whitney test is

Biuret reagent. The mixture is incubated for

used, then Kruskal-Wallis test is used for

5 minutes at room temperature (25oC) then

statistical treatment of these data (among

put in photometer. The reading of absorbance

three groups, i.e.; acetic acid results, acetate

at 546 nm is compared to absorbance of

buffer result at each pH , and control) because

protein

protein

it analyzes whether there is a difference in the

concentration in serum is calculated. As

median values of three or more independent

much as 5 g/dL of protein in serum is

samples (11).

standard

solution

and

obtained.

All

experiment

is

conducted

in

Urine sample that is used is a random

Chemistry Laboratory of Sekolah Tinggi

time and collected in a clean and dry plastic

Analis Bakti Asih Bandung, West Java-

container with a lid. The urine specimen is

Indonesia during March until May 2019 and

tested with two methods to check its protein

this study was approved by the Ethics

contents then proceed to the addition of

Committee of Sekolah Tinggi Analis Bakti

serum to make simulated urine that contains

Asih Bandung.

37

Ina J Med Lab Sci Tech 2020; 2(1): 34-41

Dinar Rahayu, et al.

RESULTS
The results of this study can be seen in the following Table 1:
Table 1. Results of Experiments
Treatment

Test

Blank

Acetic Acid
Acetic Buffer
Acetic Acid
Acetic Buffer
Acetic Acid
Acetic Buffer
Acetic Acid
Acetic Buffer
Acetic Acid
Acetic Buffer
Acetic Acid
Acetic Buffer

pH 6.0
pH 6.5
pH 6.8
pH 7.0
pH 7.5

Replicate 1
(-)
(-)
( +++ )
( +++ )
( +++ )
( +++ )
( ++ )
( ++ )
( ++ )
( ++ )
( ++ )
( ++ )

Results
Replicate 2
Replicate 3
(-)
(-)
(-)
(-)
( +++ )
( +++ )
( +++)
( +++ )
( ++)
( +++ )
( +++ )
( +++ )
( ++ )
( ++ )
( +++ )
( +++ )
( ++ )
( ++ )
( ++ )
( ++ )
( ++ )
( ++ )
( ++ )
( ++ )

Replicate 4
(-)
(-)
( +++ )
( +++ )
( +++ )
( +++ )
( +++ )
( +++ )
( +++ )
( +++ )
( +++ )
( ++ )

38

a = (+++)
a = (+++)
a = (+++)
a = (+++)
b = (+++)
b = (+++)
b = (+++)
b = (+++)
a; left test tube is simulated urine sample with acetic acid.
b; right test tube is simulated urine sample with acetic buffer
Fig 1. Results for Simulated Urine Samples at pH 6.0. White Arrows Indicated The
Cloud/Precipitation of Protein.

a = (+++)
a = (++)
a = (+++)
a = (+++)
b = (+++)
b = (+++)
b = (+++)
b = (+++)
a; left test tube is simulated urine sample with acetic acid.
b; right test tube is simulated urine sample with acetic buffer
Fig 2. Results for Simulated Urine Samples at pH 6.5. White Arrows Indicated The
Cloud/Precipitation of Protein.

Ina J Med Lab Sci Tech 2020; 2(1): 34-41

Dinar Rahayu, et al.

a = (+++)
a = (+++)
a = (++)
a = (++)
b = (++)
b = (++)
b = (++)
b = (+++)
a; left test tube is simulated urine sample with acetic acid.
b; right test tube is simulated urine sample with acetic buffer
Fig 3. Results for Simulated Urine Samples at pH 6.8. White Arrows Indicated The
Cloud/Precipitation of Protein.

a = (++)
a = (++)
a = (++)
a = (+++)
b = (++)
b = (++)
b = (++)
b = (+++)
a; left test tube is simulated urine sample with acetic acid.
b; right test tube is simulated urine sample with acetic buffer
Fig 4. Results for Simulated Urine Samples at pH 7.0. White arrows indicated the
cloud/precipitation of protein.

a = (+++)
a = (++)
a = (++)
a = (+++)
b = (++)
b = (++)
b = (++)
b = (++)
a; left test tube is simulated urine sample with acetic acid.
b; right test tube is simulated urine sample with acetic buffer
Fig 5. Results for Simulated Urine Samples at pH 7.5. White Arrows Indicated The
Cloud/Precipitation of Protein

39

Ina J Med Lab Sci Tech 2020; 2(1): 34-41

Dinar Rahayu, et al.

The Mann-Whitney test between acetic

each protein has a definite isoelectric point

acid result and acetate buffer result at control

and albumin which is the dominant protein in

pH (6.8) shows no significant difference,that

urine has isoelectric point of 4.5 then the

means we can say both group gave the same

change of pH nearing to that point will cause

result at normal pH. Then we continued to the

albumin

Kruskall-Wallis test. It was used to compare

correspond to the amount of protein presents

the median values among groups in each pH

in urine (14).

compared to control. The test also shows
there is no significant differences.

to

coagulate

which

we

can

With +3 protein (0.2-0.4 g/dL) in urine
samples, it has been shown in this experiment
that acetic acid test is in accordance with
acetic buffer test which used the prominent

DISCUSSION
The determination of urinary protein is

properties of protein that will coagulate in the

important for its significance use in clinical

presence of heat and/or acid. This process if

diagnosis (12). The rapid test can be

known as denaturation of protein. That means

performed by dipstick but the heat and acid

the change of protein environment will cause

coagulation test are still widely used.

several damages in protein structures. The
itself,

clear sign of denaturation is precipitation in

classically this is done by a 24-hour

which soluble protein has lost several bonds

collection, but as creatinine is excreted at a

in its structure and protein precipitates.

In

determining

proteinuria

constant rate, a ratio of urine albumin to

As for these two methods, it has been

creatinine or protein to creatinine is sufficient

shown that they can be used in many pH

in most patients (13). Nevertheless in

values of the urine samples. A method to be

urinalysis if the protein persists and the

a useful method must endure and still gives a

amount is significant one can suspect the

reliable result in several clinical conditions.

patients has proteinuria symptoms.

40

In this experiments it has been shown

CONCLUSIONS

that heat and acid coagulation test can be used

According to the results of experiments

in wide range of pH (Figure 1-5). Since there

and Kruskall-Wallis test, the determination

can be variation in urine pH from time to time

of protein in urine at pH 6.5 to 7.5 can be

because of many reasons among other the

done with acetic acid test with 6% (pH 2.9)

bacteria that produces ammonium hydroxide

or acetate buffer (pH 4.5). Both of method

that can cause basic urine, or acidosis case

were

which can make urine acidic. This implies to

correspondent of 0.2-0.4 g/dL of protein.

give

+3

positive

value

which

the environment of protein in urine. Since
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Dinar Rahayu, et al.

CONFLICT OF INTEREST
There are no conflicts of interest.

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