Chemistry: analytical and immunological testing – Peptide – protein or amino acid
Reexamination Certificate
1999-04-30
2001-10-02
Snay, Jeffrey (Department: 1743)
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
C436S095000, C436S164000
Reexamination Certificate
active
06297057
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a method of measuring a concentration of a specific constituent in a liquid sample, more particularly a method of measuring a protein level (albumin concentration) and a sugar level (glucose concentration) of urine collected from humans or animals, and an apparatus for measuring them. The urine sugar level and urine protein level reflect the health conditions. So, an easy-to-use and accurate measuring method has been sought.
Hitherto, a urine test has been conducted by dipping a test paper impregnated with a reagent by a testing constituent such as sugar and protein into urine, and examining a color reaction of the test paper by a spectrophotometer. This test method requires different kinds of test papers for different testing constituents, and a new test paper is required for each test, thereby leading to a high running cost. There also has a limit to automatizing the process for labor saving.
When the urine test is done by this method at home, a layman should mount and replace test papers. It is not so pleasant a job and has been a block to the spread of the urine testing unit among the households.
Then, a urine test requiring no such expendable supplies as a test paper is proposed in an international patent application with a given international publication No. W097/18470. The idea of this application is that the urine sugar and urine protein levels are quantified by measuring the angle of rotation of the urine since glucose and albumin are optically active, while other urine constituents show little optical rotatory power.
When a light is propagated through a liquid containing an optically active substance, the direction of polarization rotates in proportion to the concentration of the optically active substance. It is expressed in the following equation.
A=L×&agr; (1)
where:
L: measuring optical path length
A: angle of rotation [degree]
&agr;: specific angle of rotation of optically rotatory substance
If, for example, a light with a wavelength of 589 nm is propagated 100 mm through a glucose aqueous solution of 100 mg/dl in concentration, the direction of polarization of the light rotates 50×10
−3
degrees.
Utilizing such property, the sugar and protein levels in urine are calculated by using the above equation. Shown in Table 1 are the specific angles of rotations of aqueous solutions of glucose and albumin at 20° C.
TABLE 1
Wavelength
589 nm
670 nm
Glucose
50 degrees
40 degrees
Albumin
−60 degrees
−43 degrees
In a case N kinds of optically active substances are contained in a solution, the angle of rotation of the solution is given as follows:
A=L×(&agr;
1
×C
1
+&agr;
2
×C
2
+ . . . +&agr;
N
×C
N
) (2)
where:
L: measuring optical path length
A: angle of rotation [degrees]
&agr;
r1
(n=1, 2, . . . , N): specific angle of rotation of substance n (N: natural number)
C
r1
: concentration of substance n [kg/l]
a
r1
: specific angle of rotation of substance n
As is evident from equation (2), the measured angle of rotation of the solution has an information about the concentrations of the plural optically active substances dissolved in the solution. That is, the angles of rotation measured of urine is a sum of the angle of rotation caused by glucose and that caused by albumin. Since the specific angle of rotation differs with the wavelength of propagated light, the angles of rotation are measured with different wavelengths of light in this method. And the sugar and protein levels in urine are calculated by simultaneous equations of equation (2).
In this method, with one wavelength of a light source, either the sugar or protein level in urine can be calculated if the concentration of the other is known. But if neither the sugar or protein level in urine is known, two or more light sources are required. Another shortcoming is that because there is not much difference between the change in specific angle of rotation of glucose which occurs with the change in light wavelength and that of albumin as shown in Table 1, no accurate determination of the sugar and protein levels in urine can be hoped for even if a plurality of light sources are used. Especially because the protein level in urine is smaller in one order of the magnitude than the sugar level, the accuracy in determination is low.
BRIEF SUMMARY OF THE INVENTION
It is accordingly an object of the present invention to provide a method of determining a concentration of a specific constituent in a liquid sample, especially a protein concentration with high accuracy, which is suitable for urinalysis, eliminating the problems mentioned above.
According to the present invention, a liquid sample containing protein is opacified by heating, and a light is then projected on the sample and the intensity of the light which passed through or scattered in the sample is measured. In this way, the concentration of protein in the sample can be determined accurately. This method is most suitable for urine test.
The healthy adult usually discharges 1,000 to 1,500 ml of urine a day. Of that amount, the total solid contents come to 50 to 70 g. Of the solid contents, about 25 g are inorganic substances which consist mainly of sodium chloride, potassium chloride and phosphoric acid. Most of them are dissolved and ionized in urine. The rest are organic substances, mainly urea and uric acid, but sugar or glucose and protein are also present through small in quantity. The protein in urine is essentially albumin. Glucose is discharged into urine usually in 0.13 to 0.5 g a day. From this and the amount of urine, the glucose level in urine or the urine sugar level is estimated at not larger than 50 mg/dl on the average. However, with diabetics, the figure is several hundred mg/dl and can be as high as several thousand mg/dl. In other words, the values for diabetics can be 10 to 100 times as high as the normal level. On the other hand, albumin is usually still less than glucose and discharged into the urine in 3 to 60 mg a day. Calculated from that and the amount of urine, the albumin concentration in urine or the urine protein level is estimated normally at not larger than 6 mg/dl. But the urine protein level in patients with kidney disorder can reach 100 mg or more/dl −10 times as high as the normal level.
Such abnormal protein levels in urine can be detected on the basis of the intensity of the transmitted or scattered light. Furthermore, measurement of the angle of rotation of urine provides information on optically active substances, that is, glucose and albumin. If, therefore, the angle of rotation of urine is determined in advance and the urine is then heated thereby being opacified, the urine sugar level can be determined with high accuracy by measuring the degree of the white turbidity. Urine samples which are difficult to be opacified by heating are mixed with a bivalent metal ion or acid before heating so as to facilitate whitening.
Thus realized is a urine test method ease to maintain and control, requiring no expendable supplies.
In the present invention, the intensity of the transmitted or scattered light is measured on a heat treated protein containing liquid sample. Determination can be made in another way in which a light is projected on the liquid sample being heated, and the intensity of the transmitted or scattered light is measured thereby determining the protein concentration from the change with the temperature of the liquid sample in those light intensities.
In still another method according to the present invention, while the sample is heated, the intensity of the transmitted or scattered light is measured at two different temperatures. The protein concentration can be calculated on the basis of intensity ratio in the two measurements of the transmitted or scattered light.
While the novel features of the invention are set forth particularly in the appended claims, the invention, both as to organization and cont
Kawamura Tatsurou
Onishi Hiroshi
Sonoda Nobuo
Matsushita Electric - Industrial Co., Ltd.
McDermott & Will & Emery
Snay Jeffrey
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