FI88310C - FOERFARANDE OCH MEDEL FOER BESTAEMNING AV UREA - Google Patents

Description

1 883: 0

Method and test apparatus for the determination of urea

The invention relates to a method and a test device for determining urea in a sample by means of urease, a base and an indicator. The method is particularly suitable for the rapid determination of the urea content of milk.

The metabolism of a ruminant, such as a cow, is substantially affected by the functions of the rumen and foreskin. Feed utilization is affected by the intake of microbial energy and protein in the rumen. If energy and protein intakes are out of balance, feed protein utilization will deteriorate. This can be seen e.g. increase in blood urea levels after feeding.

In several studies, the amount of urea in milk 15 has been found to follow variations in the amount of urea in the blood (e.g., Oltner & Wiktorsson 1983, Livestock Prod.Sei. 10: 457, Refsdal 1983, Acta Vet.Scand. 24: 518). Fluctuations in the amount of urea in the blood and also in milk are largely due to changes in feeding (e.g. Refsdal and Oltner & 20 Wiktorsson, supra). For this reason, the urea content of milk has been used in feeding monitoring as an indicator of feeding success. For normal feeding, the urea range has been found to be 20-30 mg of urea in 100 ml of milk (Setälä et al. 1987, Karjatalous 10:52 and 11:54).

Urea can be determined by a method in which urea is decomposed by urease to ammonium carbonate and ammonium ions are reacted with phenol and hypochlorite to give a blue compound to be determined spectrophotometrically (Fawcett & Scott 1960, J. Clin. Path. 13: 156). 30 The method has also been applied to milk (eg Rajamäki & Rauramaa 1984, Finnish Chemical Letters 2: 47-48).

In order to make the most of the urea test, the urea test should be performed at the cows' barn. as a "cow side" quick test. The method mentioned above 35 is not suitable because it has to be

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laboratory.

Urea can also be determined by reacting a sample containing urea with urease and detecting a change in pH caused by the hydrolysis product with a suitable indicator. The method is used, for example, to determine urea levels in blood, plasma and urine (GB Patent Publication No. 922,665). A strip test for milk based on this is described in Tierärtzl. Prax. 1985, 13: 559.

A disadvantage of strip tests in general is that they are able to analyze only an extremely small number of samples, which often leaves the result unreliable. The result varies due to the small number of samples and the way the tester pipettes the sample onto the strip or dips the strip into the test solution. This problem is exacerbated by the fact that when the test strip is dipped in milk, the milk itself interferes with the accurate interpretation of the color change of the strip. In order to eliminate the disturbing effect of the milk, strips have been developed in which the reaction zone which comes into contact with the milk is placed next to the indicator zone 20 (FI application publication 891920). The reaction zone contains urease, base, glucose and surfactant. The strip is held only in one closed tube until the ammonia released from the reaction zone has diffused into the indicator zone, which is slow. The strips are preferably read after only two hours.

A method for the determination of urea has now been invented which is more reliable than milk immersion strips and faster than a strip in which an indicator zone is placed next to a milk immersion reaction zone and which is kept in a closed tube at one end during the reaction time.

The method and test device according to the invention are characterized by what is stated in the claims. In the method, the sample is reacted with urease and a base, and at least the reaction with the base is performed in a closed state in which the indicator system is placed 3 88310 so that it is not in contact with other reagents. The sample can be reacted first with urease and then only with a base, whereby at least the latter reaction is carried out in said closed state. If, instead, the sample is reacted with urease and base simultaneously, the whole reaction is carried out in a closed state. The test device comprises a closed space in which at least the reaction with the base is carried out and in which the indicator system is located so that it is not in contact with other reagents.

Reaction reagents include urease and a base. Urease cleaves the test sample into urea ammonium salts and the base releases ammonia from the salts. Ammonia is absorbed into the indicator system and causes the pH indicator-15 to change color over a period of time.

The indicator system comprises one or more pH indicators. The indicator is usually dissolved in a suitable buffer. It is also possible to use several such buffer / indicator mixtures which change color at different pH values. The determination of the urea content of the milk is preferably performed in a so-called the double test. Ko. The buffer / indicator mixtures of the duplicate test are preferably such that a urea density <20 mg / 100 ml does not yet cause a color change in either buffer / indicator mixture, but at a concentration of 20 to 20 mg / 100 ml the other buffer / indicator reacts. mixture and when the urea is more than 30 mg / 100 ml, both buffer / indicator mixtures react.

Preferably, the buffer / indicator mixtures each contain phosphate buffer, but at different concentrations. Preferably, the phosphate buffers contain the sodium salt of phenol red and the sodium salt of bromothymol blue, for example in a weight ratio of 1: 1. The second phosphate buffer is preferably 0.00025M and the second 0.00230M sodium dihydrogen phosphate buffer.

the indicator system can be in either liquid 35 or solid form. The 4,88310 indicator system in liquid form can be separated from other reagents by a gas permeable membrane. The membrane can be, for example, a Teflon tube tape or a Teflon filter membrane with a pore size of 0.5. The indicator system can preferably be solidified by means of alginate and / or by impregnation on filter paper and drying.

The indicator system is preferably placed on the lid of the container to be closed. The lid may be two-part, in which case two different buffer / -10 indicator mixtures are placed in it. Add urease, sample and base to the bottom of the vessel and close the lid tightly. After a certain time, the colors of the indicator system are read. The method according to the invention is particularly suitable for determining the urea content of milk, but other samples such as urine may also be considered.

The invention is illustrated by the following examples.

Example 1.

Buffer / indicator mixture I containing 25 mg of 20 phenol red sodium salt, 25 mg of bromothymol blue sodium and 3 g of alginate in 100 ml of 0.00025 M sodium dihydrogen phosphate is absorbed on a filter paper. Buffer / indicator mixture II similar to I except that the sodium dihydrogen phosphate content is 0.00230 M is absorbed into the second filter paper. The papers are dried at 90 ° C for 60 min. and then placed in a two-part lid of a 10 ml container with a sealed lid so that the papers are separated by a partition in the lid. Urea (about 60 to 250 U) is applied to the bottom of the vessel as a solid or solution. Add 1 ml of milk to the vessel li-30 and, after 2.5 minutes, 0.5 to 1 ml of 0.1 M NaOH and close the lid. After 2.5 minutes, read the colors of the buffer / indicator mixtures I and II formed on the lid of the test vessel. The results are compared to the color chart and interpreted as follows: i 5 88319

Test vessel lid Sample urea color combination mg / 100 ml yellow / yellow <20 5 orange-red / yellow 20 - 30 orange-red / orange-red> 30

Example 2 Urea concentrations were determined from 118 milk samples by the method of Example 1 and the spectrophotometric reference method (Fawcett & Scott 1960, J. Clin. Path. 13: 156). The results are shown in Table 1.

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Table 1. Distribution of milk samples (pieces) into different urea content categories

Urea mg / 100ml <20 20-30> 30 20 _ <20 17 3 20-30 2 34 3> 30 - 8 51 • 25 Ref. Method total / pcs 19 45 54 118

The method according to the invention evaluated 86.4 30% of the samples in the correct category compared to the reference method. Table 2 shows the corresponding sample distribution, taking into account the internal scatter of the reference method (+ 5%). In this case, the proportion of samples evaluated in the correct category is 94.1%.

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Table 2. Distribution of milk samples (pieces) into different urea content categories

Urea mg / 100ml <20 20-30> 30 5 _ <20 18 1 20-30 1 40 1> 30 - 4 53 10 Re f.method total / pcs 19 45 54 118

Example 3.

15 With test strips for space use (S)

Azostix (Tierärtzl. Prax. 1985, 13: 559), the urea concentrations of 64 milk samples were determined by the above-mentioned reference method and the method according to the invention. The results are shown in Tables 3 and 4.

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Table 3. Distribution of milk samples (pcs) into different urea content classes with test strip 25 Urea mg / 100ml <20 20-30> 30 <20 7 20-30 6 21 5> 30 - 1 24 30 _

Ref. Method total / pcs 13 22 29 64 35 82.8% of the samples (53/64) were evaluated with the test strip in the correct category 7 88310.

Table 4. Distribution of milk samples into different urea content classes by the method according to the invention

Urea mg / 100 ml <20 20-30> 30 <20 13 2 10 20-30 - 17 2> 30 - 3 27

Ref. Method tot. / pcs 13 22 29 64 15

The method according to the invention evaluated 89.1% of the samples (57/64) in the correct category.

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Example 4.

To a 4 ml vessel was added 0.5 ml of the buffer / indicator mixture I mentioned in Example 1, which was in liquid form, i.e. it did not contain alginate. The mouth of vessel 25 was covered with a Teflon film having a pore size of 0.5 μm. To another similar vessel was added 0.2 ml of milk and about 15 U of urease. After 2.5 minutes, 200 μΐ of 0.1 M NaOH was added. The vessel containing the buffer / indicator mixture and the vessel 30 containing the sample and other reagents were joined to the mouths with tape so as to provide a completely sealed state in which the gas permeable membrane separated the buffer / indicator mixture from the other reagents. The vessels thus connected were stirred for 1 minute, after which the color of the indicator mixture was read. A parallel assay was performed using the liquid β 88310 buffer / indicator mixture II mentioned in Example 1. The results were interpreted as in Example 1. The urea concentrations of 24 milk samples were determined. 87.5% of the samples were evaluated in the correct category. The results are shown in Table 5.

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Table 5. Distribution of milk samples (pieces) into different urea content categories 10 Urea mg / 100ml <20 20-30> 30 <20 6 20-30 18 1> 30 - 1 7 15

Ref method total / pcs 79 8 24

Claims (10)

Method for determining the urea content of milk by means of urease, base and indicator, characterized in that at least the reaction with the base is carried out in a closed space, where the indicator system is placed so that it is not in contact with the other reagents. 2. A method according to claim 1, characterized in that at least the reaction with the base 10 is carried out in a closed vessel in which the indicator system is placed. 3. A method according to claim 1, characterized in that the determination is carried out as a double test using an indicator system comprising two buffer / indicator mixtures which change color at different pH values. 4. A method according to claim 3, characterized in that one buffer / indicator mixture changes color at a urea content of at least 20 mg / 100 ml and the other buffer / indicator mixture changes color where the urea content exceeds 30 mg / ml. 100 ml. Process according to claim 4, characterized in that the buffer / indicator mixtures contain two phosphate buffers of different concentration. 25 6. A process according to claim 5, characterized in that the phosphate buffers contain the sodium salt of phenol red and the sodium salt of bromothymol bleach in the weight ratio of 1: 1. 7. Test means for determining the urea content of milk by means of urease, base and indicator, characterized in that it comprises a closed space, where the at least one reaction with the base is carried out and where the indicator system is located so that it does not is in contact with the other reagents. 12 8831 0 8. Test means according to claim 7, characterized in that the indicator system is in solid form. 9. Test means according to claim 7, characterized in that it comprises a vessel which can be closed, where the indicator system is located in the lid and the other reagents on the bottom. 10. Testing agent according to claim 7, characterized in that it comprises the reagents mentioned in claim 10, 3, 4, 5 or 6, which are needed for dual testing.