Method for the detection of protein in urine

Disclosed is an test for the detection of protein in urine by the use of a reagent system containing a buffer and a protein error indicator. The method involves the use of tartaric acid as cation sensing buffer in combination with a non-cation sensing buffer as the buffer system in order to reduce the incidence of false positive results while using less total buffering material.

BACKGROUND OF THE INVENTION 
The present invention is related to the detection of protein in urine by 
the use of a test strip containing a protein error indicator and a buffer. 
More particularly, it relates to the use of a particular buffer, referred 
to herein as a "cation sensing buffer" which, when used in combination 
with typical buffer systems, helps to alleviate the result distorting 
effects which are observed when high specific gravity (SG) urine is tested 
for protein content in this manner. 
The determination of the presence of protein in a urine sample is important 
in the diagnosis of several pathological conditions affecting the kidney 
and circulatory systems as well as the central nervous system. It is often 
necessary to qualitatively and quantitatively measure protein in urine. 
This is especially true in the diagnosis of diabetes and kidney disease. 
The predominant urine protein associated with diabetes is albumin which is 
the protein most commonly sought out in analyses. 
Various methods for determining the presence of protein in urine are known, 
the most convenient of which involves wetting an absorbant test strip 
impregnated with a protein error indicator and buffer with a small 
quantity of urine. Protein error indicators are pH indicators which 
contain an ionizable group which is displaced in the presence of protein 
to provide a detectable color change. This is the same color change that 
the indicator would undergo under the influence of a pH change, so it is 
important to include buffer in the test strip to thereby avoid a pH 
increase since such an increase could cause the change of color in the 
indicator in the absence of protein thereby resulting in a false positive 
result. 
The tendency toward false positive results is particularly problematical 
with the testing of high SG urine due to the presence in such urine of 
buffers which overwhelm the buffer in the test strip thereby permitting 
the change in pH resulting in the color change of the indicator which is 
indicative of the presence of protein when there is no protein present in 
the urine sample being tested. The primary buffering component in urine is 
phosphate, followed by citrate, uric acid, acetate, glycine and ammonium. 
This sort of false positive is, of course, to be avoided if at all 
possible. An example of such a false positive situation is a test for 
urine albumin in which the protein error indicator is Tetrabromophenol 
Blue, TBPB, which turns from yellow to blue above a pH of 3.7. Another 
common indicator, DIDNTB, turns from colorless to blue at a pH greater 
than 2.1. With the use of prior art buffers the addition of high SG urine, 
which typically has a pH of 5 to 8 and contains buffers which favor the 
maintenance of high pH thereby increasing the pH of the reagent system to 
a level of greater than 3.7, causes the color change which results in a 
false positive reading for protein in the urine sample. 
In Japanese Patent Application No. 3-355044 there is described the use of 
potassium salts to reduce the tendency of false positive responses for 
protein in high SG urine. While it is stated on page 5 of this application 
that the mechanism is unclear, this system may operate on the principle 
that the potassium ion combines with a cation sensing component in urine 
even though the main components in urine are unresponsive cation sensing 
buffers. One of the potassium salts mentioned is potassium citrate. This 
reference also mentions the use of sodium citrate and citric acid as 
buffers. In example 15 of U.S. Pat. No. 5,279,790 there is described the 
preparation of a urine protein strip which involves impregnating a paper 
strip with a mixture of protein error indicators together with a potassium 
citrate buffer. Citric acid, like most anionic buffers possesses the 
ability to release some protons in the presence of metallic cations 
thereby lowering the pH in the presence of such cations. However in order 
for a cation sensing buffer to benefit the urine protein methodology, the 
buffer must release enough protons to lower the reagent pH when exposed to 
cations in the normal physiological ranges found in urine which, even in 
high SG urine, amount to a maximum of 140 mM for potassium and 250 mM for 
sodium. Other cations normally found in urine which contribute to this 
phenomena are calcium, magnesium and ammonium. Citric acid does not 
possess sufficient proton releasing ability to significantly lower pH at 
these cation concentrations. The highest urine buffering capacity is 35 
meq/pH unit. A protein error indicator test should be resistant to this 
level of buffering capacity. A high pH (8-9) and a high buffering capacity 
urine can shift the pH of a citrate buffered reagent by as much as 0.5 
units. The combined Na and K cation sensing ability should be greater than 
0.5 pH units. The increase of 0.5 in reagent pH due to the buffering 
capacity of a high SG urine is offset by a decrease of 0.5 in reagent pH 
due to cation sensing ability of the buffer. The net result is little or 
no increase in reagent pH due to high SG urine, The combined Na and K 
cation sensing was 0.71 for tartaric acid and only 0.25 for citrate. 
SUMMARY OF THE INVENTION 
The present invention involves an improvement to a test strip for the 
detection of protein in urine which test strip comprises an absorbant 
carrier impregnated with a reagent system of a buffer and a protein error 
indicator which undergoes a color change in the presence of protein. The 
improvement involves the use of a buffer system comprising a typical 
buffering agent in combination with. tartaric acid as a cation sensing 
buffer in an amount which renders it capable of releasing sufficient 
protons in the presence of buffers present in the urine to prevent the pH 
of the reagent from being elevated to a level at which the protein error 
indicator will change color in the absence of a significant amount of 
protein in the urine.

The present invention is further illustrated by the following examples: 
Example I (The Relationship of Buffer pKa to Proton Release) 
The release of proton upon the addition of cation was demonstrated with 
oxalic acid. In this experiment the cation was tetramethyl guanidine. This 
cation is not normally found in urine but was selected since it enhances 
the response of oxalate and, therefore, more readily allows the 
relationship of release of proton to cation concentration to be shown. The 
data are presented in Table 1: 
TABLE 1 
______________________________________ 
The pH of Oxalic Acid Solution as a Function 
of Cation Concentration 
Solution pH Molar Ratio of 
Cation 
Oxalic Acid 
Initial Initial Cation to 
mM mM pH of 0.8 pH of 6 Oxalic Acid 
______________________________________ 
0 50 0.80 6.22 0.0 
10 50 0.79 6.26 0.2 
20 50 0.79 6.21 0.4 
30 50 0.74 6.21 0.6 
40 50 0.69 6.20 1.2 
90 50 0.63 6.21 1.8 
120 50 0.61 6.26 2.4 
150 50 0.61 6.26 3.0 
______________________________________ 
The pKa of oxalate is 1.27 for the first carboxylic acid group and 4.29 for 
the second. The release of proton stops once an equivalent number of 
cations are present for each carboxylic acid group. Alkali and alkaline 
earth metals and quaternary ammonium salts also produced a release of 
protons until an equivalent number of cations were present for each 
carboxylic acid group. 
Example II (The Relationship of Buffer to Protein Indicator Dyes) 
The protein reagent paper relies on the detection of protein by using the 
error of a pH indicator in the presence of protein. The principle of this 
assay is that the pH at which the indicator dye changes color is lower in 
the presence of protein, causing the color to appear at a fixed pH. The 
dye is buffered to a constant pH which is low enough to prevent color in 
the absence of protein, but high enough to create color in the presence of 
protein. For example, a protein test based on the TBPB dye is buffered to 
a pH of 3.7 and a test based on the DIDNTB dye is buffered to a pH of 2.1. 
In order for a typical buffer to benefit the protein methodology, the 
buffer must have a pKa in the range of the pH of the protein test. A 
series of buffers with pKa in the 1.80 to 3.0 range were tested with a 
protein reagent based on the DIDNTB dye (cf. Table 3). All of the buffers 
demonstrated buffering capability and maintained the reagent within the 
0.7 pH unit after being dipped in a high SG urine. The buffering 
capacities were dependent upon the individual buffer with the best 
buffering capacity being demonstrated with tartaric acid while the worst 
was observed for maleic acid. 
TABLE 2 
______________________________________ 
The pH of Reagents After Dipping in Water and 
High SG Urine as a Function of Buffer 
Reagent Change 
pH of Reagent 
pH- in 
Buffer 
Dipped in Buffer Reagent 
Buffer pK.sub.1 
Water Urine pK.sub.1 
pH 
______________________________________ 
Citric acid 
3.13 2.23 2.47 -0.90 0.24 
Phosphoric acid 
2.15 2.45 2.77 0.30 0.32 
Citraconic acid 
2.29 2.68 2.97 0.39 0.29 
Diglycolic acid 
2.60 2.42 2.64 -0.18 0.22 
Glycylglycine 
3.22 2.47 2.78 -0.75 0.31 
Glycine 2.34 2.44 2.72 0.10 0.28 
Maleic acid 
1.91 2.39 3.00 0.48 0.61 
Malic acid 
3.40 2.38 2.86 -1.02 0.48 
Lysine 2.18 2.40 2.87 0.22 0.47 
Tartaric acid 
3.04 2.33 2.47 -0.71 0.14 
Sarcosine 2.12 2.68 2.97 0.56 0.29 
Betaine 1.80 2.13 2.51 0.33 0.38 
______________________________________ 
Tartaric acid is the buffer of choice in this example because it exhibits 
the smallest change in pH. This is important because this change is low 
enough to prevent color formation in the absence of protein. The release 
of proton upon the addition of cation was only observed in solutions with 
an initial pH value below or near the pKa of the buffer. The buffer must 
be partially or fully protonated for there to be a cation response. The 
concentration of tartrate needed is, of course, dependent upon its degree 
of protination. A completely deprotonanted buffer would not have any 
response. Therefore, the relationship between test pH and buffer pKa 
requires a buffer with a pKa near to or greater than the reagent pH. All 
of the buffers in Table 3 are at the right conditions, i.e. pKa values of 
1.8 to 3.0 which are greater than or near the reagent pH for cation 
sensing with a pH of 2.1 protein test using DIDNTB dye. 
Buffers typically work best when their pKa is near the pH being buffered, 
i.e. where the buffering capacity is the greatest. However, as can be 
determined from Table 3, a group of buffers having a similar pKa, within 
one pH unit of the test pH, have greatly differing effectiveness. 
Effectiveness is defined as the smallest pH change after contacting the 
formulation with a high SG urine lacking protein. The pH change with 
tartrate was only 0.14 whereas the other buffers exhibited pH changes of 
from 0.22 to 0.61. All of the buffers used have acceptable pKa values for 
buffering capacity, so the difference is explainable by examining the 
cation sensing abilities of the buffer. The change in reagent pH 
represents the combined effects of the buffering capacity and cation 
sensing action. 
The improved buffering capacity of the protein reagent containing tartaric 
acid is explained by the discovery that solutions of KCl lowered the 
protein reagent pH (cf. Table 3). The protein reagent containing betaine 
buffer was not responsive and the citrate buffer was only slightly 
responsive. Accordingly, it can be seen that betaine and citrate are not 
cation sensing. There was a 0.63 unit pH decrease with exposure to 
tartrate while betaine and citrate exhibited virtually no pH decrease due 
to their failure to act as cation sensing buffers. As shown in Table 2, 
the differing cation sensing abilities explain variations in buffering 
capacities between buffers that would be expected to behave in a similar 
manner. 
TABLE 3 
______________________________________ 
The pH of the Protein Reagent After Dipping in a Cation 
Solution 
Cation Reagent Reagent 
Cation mM pH Buffer 
______________________________________ 
None 0 2.14 Tartaric acid 
KCl 50 1.97 Tartaric acid 
KCl 140 1.51 Tartaric acid 
MgCl.sub.2 10 2.15 Tartaric acid 
CaCl.sub.2 10 2.08 Tartaric acid 
None 0 3.56 Citrate 
KCl 50 3.53 Citrate 
KCl 140 3.44 Citrate 
MgCl.sub.2 10 3.50 Citrate 
CaCl.sub.2 10 3.50 Citrate 
None 0 2.04 Betaine 
KCl 140 2.02 Betaine 
______________________________________ 
Most anionic buffers possess some proton release to cations. However, in 
order for a cation sensing buffer to benefit the urinary protein 
methodology, the buffer must release enough protons to lower the reagent 
pH when exposed to cations in the normal physiological ranges. Tartaric 
acid responded with a 0.6 pH unit decrease when exposed to a concentration 
of KCl (140 mM) typical for high SG urines. The cation sensing ability of 
the tartaric acid buffer was further demonstrated by a reagent pH drop of 
1.80 after dipping into a high SG clinical urine containing 140 mM 
potassium and a reagent pH of 2.28 after dipping in a clinical urine 
containing 41 mM potassium. 
The cation sensing agents which are useful in this invention are limited to 
those with a pKa near to or greater than the reagent pH, which are 
responsive to cations in urine at the normal physiological range and which 
provide a decrease in pH capable of offsetting the buffering capacity of 
urine. Only tartaric acid has been shown to exhibit these properties. 
Example III 
An experiment was conducted in which tartaric acid (as cation sensing 
agent) and a typical buffer (citric acid) were tested separately and in 
combination. The tests were carried out using protein reagent as described 
in Example 1, with the reagent pH after dipping reagent strips in water 
and high SG urine determined using a surface PM electrode and pH meter. 
The difference in pH between water and high SG urine is indicated below as 
change in reagent pH. 
The results of this experiment are set out in Table 4. 
TABLE 4 
______________________________________ 
Citric Acid Tartaric Acid 
(Buffer) (Cation Sensing Agent) 
Change in 
Concentration 
Concentration Reagent pH 
______________________________________ 
0 mM 150 mM 0.49 
475 mM 0 mM 0.37 
475 mM 150 mM 0.13 
625 mM 0 mM 0.24 
______________________________________ 
From Table 4 it can be determined that, the combination of 150 mM tartaric 
acid as a cation sensing agent with a non-cation sensing buffer such as 
475 mM citric acid was more effective than either by itself. It is 
unexpected that the addition of 150 mM tartaric acid to 475 mM citric acid 
would produce better SG resistance than 625 mM citric acid. The 150 mM 
tartaric acid concentration is too low to buffer effectively against a 
high SG urine such as that used in this experiment. Accordingly, it can be 
seen that the buffering ability of tartaric acid is not relied upon in 
controlling pH but instead, the cation sensing activity is the key to 
tartaric acid's superior performance in controlling pH when combined with 
a typical buffer. 
The ability of tartaric acid in combination with a typical, non-cation 
sensing buffer in smaller quantities than either agent by itself is 
important because higher concentrations of any buffer can reduce protein 
response. A reduced protein response would mean that a pathological 
protein level would not be detected. This improvement is not limited to 
citric acid as the non-cation sensing buffer since other buffers, such as, 
for example, betaine, phosphoric acid, maleic acid, diglycolic acid and 
citraconic acid, would be expected to provide similar results. 
When using tartaric acid in combination with a noncation sensing buffer, 
the molar ratio of tartaric acid to buffer will typically be from 25 
mM:700 mM to 200 mM:100 mM with a ratio of about 150 mM:475 mM being 
preferred. The cation sensing activity of tartaric acid operates in the 
absence of a non-cation sensing buffer, however, the use of such a buffer 
in combination with tartaric acid is preferred because a typical buffer 
releases protons independent of cations being present in solution. This 
allows buffering in urines low in salt and brings the reagent's pH closer 
to the range where the cation sensing tartaric acid can be effective. 
Example IV (Procedure for the Preparation of Protein Reagent Example) 
The improved protein reagent of the present invention can be made from two 
sequential saturations of filter paper. The first saturation is with an 
aqueous mix containing tartaric acid and a buffer such as citric acid, and 
a background dye such as quinaldine red. The mix is adjusted to pH 2.1 
using sodium hydroxide and/or hydrochloric acid. The second saturation 
involves a toluene mix containing a protein indicator dye such as DIDNTB 
and a polymer like Lutanol M40 which is a poly (vinyl methyl ether). The 
function, preferred concentration and range of operable concentrations are 
set out in Table 5. The mix solutions are used to saturate the strip 
material, e.g. filter paper such as Ahlstrom 204 or 237 whereupon the 
filter paper is dried at 95.degree. C. for 5 minutes after the first 
saturation and at 85.degree. C. for 5 minutes after the second saturation. 
The resultant dry reagent is processed into reagent strips which were 
visually tested. 
TABLE 5 
__________________________________________________________________________ 
Protein Reagent Composition 
Pref. Conc. 
Allowable 
Ingredient 
Function Used Range 
__________________________________________________________________________ 
1st application 
Water Solvent 1000 mL -- 
Tartaric acid 
Cation Sensing Buffer 
93.8 g (625 mM) 
50-750 mM 
Quinaldine red 
Background dye 
8.6 mg (12 .mu.M) 
5-30 .mu.M 
2nd application 
Toluene Solvent 1000 mL -- 
DIDNTB Buffer 0.61 g (0.6 mM) 
0.1-3.0 mM 
Lutonal M40 
Polymer enhancer 
1.0 g 0.5-4 g/L 
__________________________________________________________________________ 
DIDNTB = 
5',5"-Dinitro3',3"-Diiodo-3,4,5,6-Tetrabromophenolsulfonephthalein 
Example V (Relationship of Buffer to the Background Dye) 
Reagent formulations often employ background dyes to achieve better visual 
distinction between test levels. For example, a protein test based on 
DIDNTB as the dye indicator, which undergoes a colorless to blue 
transition, requires a red background dye to change the color transition 
from pink to blue in order to mask detection of normal levels of protein 
to reduce the sensitivity of the test since a faint blue is easily 
detected against white but not under a red background. The background dye 
should be inert to urine components and provide a constant response. The 
differences between an inert and background dye can be demonstrated by 
comparing inert methyl red to reactive quinaldine red as set out in Table 
6. 
Quinaldine red is both pH and light sensitive; its color transition is from 
colorless at pH 1.4 and, with increasing pH up to 3.4, forms various 
shades of red with maximum absorbance at pH 3.4. The quinaldine red pKa of 
2.1 is at the pH of a protein test based on the DIDNTB dye. 
TABLE 6 
__________________________________________________________________________ 
Background Dyes in the Protein Reagent Composition 
.lambda. CLINITEK-200 
Reactivity 
Background Dye 
pH max light Negative Positive 
__________________________________________________________________________ 
Quinaldine Red 
1.7 
528 nm 
Unexposed 
1006 694 
Quinaldine Red 
2.1 
528 nm 
Unexposed 
1005 525 
Quinaldine Red 
2.1 
528 nm 
Exposed 
953 497 
__________________________________________________________________________ 
The positive level as 30 mg/dL albumin. Light exposure to 105 ftcandles 
for 15 minutes. 
Methyl red is neither pH nor light sensitive. No change in reactivity was 
observed after exposure to lighting conditions affecting the quinaldine 
red dye. The methyl red color transition is from red at pH 4.2 to yellow 
at pH 6.4. The methyl red, pKa 5.2, prevented changes in background 
coloration due to pH and, therefore, is a preferred dye. Other inert dyes 
such as FD&C red #3 and Acid Red #40 can also be used. 
Example VI 
The cation sensing abilities of the buffers listed in Table 2 were measured 
and are set out in Table 7 below: 
TABLE 7 
______________________________________ 
pH Changes 
After Adding 
Buffer KCl NaCl 
______________________________________ 
Tartaric Acid -0.50 -0.21 
Citric Acid -0.07 -0.16 
Glycine -0.03 0.03 
Phosphoric Acid -0.08 -0.12 
Maleic Acid -0.05 -0.13 
Malic Acid -0.06 -0.14 
Lysine 0.00 -0.02 
Betaine 0.00 -0.05 
______________________________________ 
This experiment measured the change in pH of a 500 mM buffer solution after 
adding 140 mM KCl or 250 mM NaCl. These salt concentrations were taken as 
the highest urinary values, i.e. the highest physiological concentration 
expected in human urine. The data of Table 7 demonstrate that tartaric 
acid is the most responsive to cations of the buffers tested. The data in 
Table 3 were generated using dry reagents made and tested in a similar 
fashion. The other buffers tested are not useful for cation sensing. 
Oxalate demonstrates a cation response (Table 1), but this response is not 
great enough for use in the present invention.