IL34773A - A reagent for the determination of blood sugar - Google Patents

Description

A REA&EHT FOR SHIS D322E∑l' ΙΪΙΑ2Ι0Η OF BLOOD SUGAR The present invention is concerned, with a reagent for the determination of "blood sugar by the o-toluidine method, which reagent does not contain glacial acetic acid.

The determination of blood sugar with o-toluidine in glacial acetic acid is a standard method in clinical laboratories. According to this method, the blood sample, after deproteinisation, is mixed with about the tenfold amount of a reagent solution which contains o-toluidine and thiourea dissolved in glacial acetic acid. The mixture is heated, a green coloured material being formed which is measured in a photometer at 630 nm.

This* known method suffers from two important disadvantages which present considerable problems, especially in large routine laboratories. On the one hand, the sensitivity is comparatively low, i.e. the possibility of error in the lower region of sensitivity is increased. On the other hand, the use of glacial acetic acid as the reaction medium gives rise to a strong and very unpleasant .. smell, not only when pipetting the reagents but also when heating the samples in a boiling waterbath, as well as when cleaning the glass reaction vessels used. Furthermore, the corrosion of the photometer by the glacial acetic acid vapours is increased considerably.

Attempts have, therefore, already been made to overcome these disadvantages by the use of another solvent and reaction medium. Thus, it has already been proposed to use semi-concentrated acetic acid. However, this does not result in the disadvantages being overcome.

It has been proposed by A.Hartel et al. (Z. klin.

.V* Chem. u. klin. Biocbem., »' /1 969) to replace the glacial acetic acid by a mixture of glycollic acid and malic acid, water, methanol and ethylene glycol monomethyl ether. A reagent of this type is also commercially available.

In the case of another commercially available reagent, the glacial acetic acid is replaced by organic esters, and salicylic acid.

The two above-mentioned glacial acetic acid-free reagents admittedly overcome the problem of malodour and also improve the sensitivity of the test. This sensitivity is, in the case of -the usual normal values for the blood sugar content in whole blood of 60 - 1 00 mg.%, below the value of E -^, 0.1 00 at the measurement wavelength of 578 nm usually employed. In the case of very low blood sugar values, such as occur in cases of hypoglycaernia, the extinction differences thus are of the order of the blank. Therefore, there is a need for the provision of a reagent which still further increases the sensitivity of the method.

A further disadvantage of the two above-mentioned known reagents is the instability of the solvent mixture v/hich is due to the content of readily volatile components. The increased evaporation rate is of significance because the reagent must be heated for 8 minutes in a boiling water bath for the development of the coloration. In the case of the known salicylic acid-containing reagent, even after standing for about 1 hou„r in the air, the dissolved substances start tc crystallise out.

Finally, another disadvantage is that the substances used instead of glacial acetic acid are considerably more expensive and thus the economic competitiveness of such reagents is impaired.

It is, therefore, an object of the present invention to provide a new reagent for the determination of blood sugar which avoids the above-mentioned disadvantages of the known reagents and is dependable, very sensitive and economical .

The reagent according to the present invention for the determination of blood sugar contains -toluidine , thiourea, an ethylene glycol mono-lower alkyl ether and an acid and is characterised in that it contains phthalic acid as the acid and dimethyl formamide as a solubilising agent.

From A. Haertel .& H. Lang (Arztl. Lab., 1J5, 60/1966), it was known that non-hydroxylated dicarboxylic acids could not replace the glacial acetic acid because they prevented the formation of the desired coloured material. It is, therefore, most surprising that phthalic acid is an outstanding catalyst for the aldose-specific colour reaction of o-toluidine, whereas, for example, isophthalic acid and terephthalic acid are useless.

The reagent according to the present invention preferably comprises 0.1 - 5 parts by weight thiourea, IO - 65 parts by weight phthalic acid, 50 - 130. parts by volume dimethyl formamide, 20 - 100 parts by volume of an ethylene glycol mono-lower alkyl ether and 5 - 30 parts by volume o-toluidine. The sum total of the dimethyl formamide and ethylene glycol mono-lower alkyl ether advantageously An especially preferred composition consists of 0.1 - 0.3 parts by weight thiourea, 0 - 50 parts by weight phthalic acid, 70 - 110 parts by volume dimethyl formamide, -+0 - 80 parts by volume of an ethylene glycol mono-lower alkyl ether and 15 - 20 parts by volume o~ toluidine .

Within the scope of the present invention, the term "lower aUvyl" is to be understood to mean an alkyl radical containing up to 6 carbon atoms.

The reagent according to the present invention can contain up to about 5% water, without its usefulness being thereby impaired.

The reagent according to the present invention does not result in any unpleasant smell nor is there any danger of a loss of solvent when heating in order to bring about formation of the coloured material. The reagent is colour-less to pale yellow in colour and the viscosity is low so that there is no impairment of the pipetting due to air bubbles or after-runnings. The after-runnings in the case of a normal pipetting amount to <^ i.e. in the case of 2.0 ml. of reagent, are 0.02 ml. The reagent is also storage stable although, in the case of compa atively long periods of storage at an elevated temperature (33°C), an intensification of the yellow colour can occur. However, this influences neither the measurement sensitivity to any considerable extent nor the stability of the colour formed.

An especial advantage of the reagent according to the present invention is its outstanding sensitivity, even in the case of low glucose contents. Thus, even in the case of the range of 20 - 400 mg.% (and even up to 1 g.;¾') glucos content, there is obtained a very good linear proportionality "between the extinction values at 578 nm and the glucose concentration. In this regard, reference is made to Pig.1 of the accompanying drawings. When the glucose content is over 2+00 mg.%, then the sample is expediently diluted, for example, in the ratio of 1 : 1 0 , since even in the case of this content, an extinction of 0 .65 is measured, The sensitivity of the reagent according to the present inventioai is considerably greater than the sensitivity of the known reagents, as can be seen from the following Table 1 : T A 6 L E 1 determination methods = The superior sensitivity of the reagent according to the present invention, which can be seen from the last column of the above Table 1 , is of great practical importance since it is hereby possible, even in cases of hypoglycaemia, as well as in cases in which only very little blood is available, i.e. in case of blood sugar measurement values of 30 - 50 mg.%, to obtain extinctions of the order of 0.050 to 0.082 at 578 nm, measured against a blank, so that errors of measurement in the reading off of the photometer are very considerably reduced.

A further advantage of the reagent according to the-present invention lies in the fact that in the region of the especially preferred composition, it represents a so-called "optimum composition". This means that all individu components of the mixture are present in the concentrat o in which the sensitivity, the period of incubation of the test and the stability of the resultant colour have their maxima and a slight alteration of the conditions does not cause any significant differences in the course of the test or in the measurement operation. The working errors are, therefore, in the case of this .composition, extremely low.

The colour which arises in the case of the o-toluidin reaction is, in principle, unstable. It is assumed that an unknown secondary reaction follows by means of which the extinction at 62+0 nm, the maximum, and at the measurement wavelength of 578 nm, decreases in the course of time. This instability of the colour is- reduced in the case of the reagent according to the present invention. Fig.2 of the accompanying drawings shows the remaining colour intensity as a percentage of the original value, in depend- ence. upon time, which the sample had at ambient temperature at the end of the incubation. The glycollic acid reagent was used as comparison reagent. There can clearly be appreciated the superior stability of the reagent according to the present invention. This improvement represents an important advantage v/hen a large series of tests are to be measured one after the other.

A further advantage of the reagent according to the present invention lies in the fact that the stability is independent of the glucose concentration in the sample.

The possibilities of error of the method are hereby minimised.

A further advantage of the reagent according to the present invention is its cheapness. Thus, the cost of phthalic acid is only about one ninth of the cost of glycollic acid.

The following Examples are given for the purpose of illustrating the present invention: -Example 1. 0.2 g. thiourea and 5.0 g. phthalic acid are dissolved in 100 ml. dimethyl formamide (b.p. 152 - 155°C.) 50 ml. ethylene glycol monoethyl ether are then added thereto and finally 18.0 ml. o-toluidine are stirred in.

For the purpose of deproteinisation, 0.1 ml. of blood is pipetted into 1 c0 ml. % trichloroace ic acid, mixed and after standing for a few minutes, cent ifuged.

From the supernatant, there is taken 0.2 ml. and this is mixed with 2.0 ml. of the above-described reagent accord ing to the present invention. The mixture of supernatant and reagent is heated for 8 minutes on a boiling waterbath.

After cooling in cold water, the resultant green coloured material is measured in a photometer at 578 run against a blank (trichloroacetic acid and reagent) . A glucose standard ( 1 00 mg. glucose/1 00 ml . 0.2% benzoic acid solution) is treated in the same manner. The blood sugar content is calculated from the following equation: sample glucose content of the sample (mg. ) = < ■ — x 1 00 standard The determination was repeated with a series of blood samples of differing glucose content. The results obtained are set out in Fig.1 of the accompanying drawings. They show the outstanding linear proportionality of the results which are obtained v/ith the reagent according to the presen invention.

In the, above-described manner, the standard deviation of the determined blood sugar values from one another was ascertained by means of 1 0 determinations on the same serum The standard deviation, which serves for the assessment of the distribution of the individual values around the arithmetic mean, is calculated from the following equation: In the present case, s = 1 .25 mg.%. As arithmetic mean value (x) in the case of the ten determinations, there was found 1 63.3 mg.J¾ glucose. The exactitude which can be achieved with the reagent according to the present inventio can also be expressed by the following equation: The variation coefficient (VC) , which is an expressio of the standard deviation in percent of the mean value, amounts to i 0.lG , calculated from the following equation: s x 100 VC = —= X Example 2.

The o-toluidine method, wit the use of the reagent according to the present invention, was compared with the known enzymatic, highly specific blood sugar determination method, using the system hexokinase/intermediate enzyme. In this case, 25 different whole blood samples were tested in the manner described in Example 1. The s me number of blood samples were determined enzymatically with hexokinase intermediate enzyme. The blood sugar values obtained by both methods were compared. The results obtained are shown in Pig.3 of the accompanying drawings. The good agreement of the values which were obtained by the two methods is obvious. The distribution of the comparable individual values is, on average, t 2,0%.

Claims (6)

1. What .we claim is :-1. A reagent for the determination of "blood sugar, which comprises £-toluidine , thiourea, an ethylene glycol mono-lower alkyl ether, phthalic acid and dimethyl formamid as a solubilising agent.

2. A reagent according to claim 1 , which comprises 0.1 - 5 parts by weight thiourea, 10 - 65 parts by weight phthalic acid, 50 - 30 parts by volume dimethyl formamide, 20 - 100 parts by volume of an ethylene glycol mono-lower alkyl ether and - 30 parts by volume _o-toluidine .

3. A reagent according to claim 2, which comprises 0.15 - 0.3 parts by weight thiourea, kO - 50 parts by weigh phthalic acid, 70 - 110 parts by volume dimethyl formamide, 1+0 - 80 parts by volume of an ethylene glycol mono-lower alkyl ether and 15 - 20 parts by volume -toluidin©.

4. A reagent according to claim 2 or 3, wherein the sum total of dimethyl formamide and of the alkylene glycol mono-lower alkyl ether does not exceed 150 parts by volume.

5. A reagent according to any of the preceding claims, whenever containing water in an amount of up to 5%*

6. A reagent according to claim 1 for the determination of blood sugar, substantially as hereinbefore described an exemplified.