IE913920A1 - A process and a test means for the determination of urea - Google Patents

Abstract

Determination of the concentration of urea in a sample, especially milk, is by means of urease, alkali and an indicator. The sample is reacted with urease and alkali in a closed space in which the indicator system is placed such that it is not in contact with the other reagents.

Description

lfr A process and a test means for the determination of urea The invention relates to a process and a test 5 means for the determination of urea in a sample by means of urease, alkali and an indicator. The process is especially suitable for rapid determination of the concentration of urea in milk.

The metabolism of a ruminant, such as a cow, is 10 essentially affected by the functions of the rumen and the forestomachs. The energy and protein supply of rumen microorganisms affects the feed utilization. If the supplies of energy and proteins are not balanced, the utilization of feed proteins is deteriorated. This is manifested e.g. in increased urea levels in blood after feeding.

Several researches have shown that the amount of urea in milk varies with the level of urea in blood (e.g. Oltner & Wiktorsson 1983, Livestock Prod.

Sci. 10:457, Refsdal 1983, Acta Vet. Scand. 24:518). Variation in the level of urea in blood as well as in milk is largely due to changes in feeding (e.g. Refsdal and Oltner & Wiktorsson, as above). For this reason, the concentration of urea in milk has been used as an indicator in the assessment of successful feeding. In feeding according to standard requirements, a urea concentration range of 20 to 30 mg of urea in 100 ml of milk has been observed (Setaia et al. 1987, Karjatalous 10:52 and 11:54).

Urea can be determined by a process in which it is degraded by urease into ammonium carbonate, and the ammonium ions are reacted with phenol and hypochlorite to achieve a blue compound which can be determined spectrophotometrically (Fawcett & Scott 1960, J. Clin. Path. 13:156). The process has also been applied to milk (e.g. RajamSki & Rauramaa 1984, Finnish Chemical Letters 2:47-48).

To get the maximum benefit from a urea determination, the determination should be performed as a rapid cow-side determination in the cowhouse. The above-mentioned process is unsuitable for this purpose, because it has to be performed in a laboratory.

Urea can also be determined by reacting a sample containing urea with urease and observing the change in pH caused by the hydrolysis product by means of a suitable indicator. This process is utilized in the determination of the concentration of urea e.g. in blood, plasma and urine (GB Patent Specification 922 665). A strip test of this type for milk is disclosed in Tier&rtzl. Prax. 1985, 13:559.

A common drawback of strip tests is that they are able to analyze only an extremely small amount of sample, and so the obtained result often remains un20 reliable. The result varies on account of the small amount of sample, but it is also affected by the way the performer of the test pipettes the sample on the strip or dips the strip into the solution to be analyzed. The problem is further aggravated by the fact that when the test strip is dipped into milk, milk as such disturbs accurate reading of the color change in the strip. To eliminate the disturbing influence of milk, strips have been developed in which a reaction zone to be brought into contact with milk is positioned beside an indicator zone (FI Patent Application 891920). The reaction zone contains urease, alkali, glucose and tenside. The strip is kept in a tube closed at one end only until ammonia liberated from the reaction zone has diffused into the indicator zone, which takes place slowly.

The strips are preferably read only after two hours.

A determination process has now been discovered which is more reliable than strips to be immersed in milk and more rapid than a strip in which the indicator zone is positioned beside the reaction zone to be immersed in milk and which is kept in a tube closed at one end during the reaction time.

The process and the test means according to the invention are characterized by what is disclosed in the claims. In the process, the sample is reacted with urease and alkali and at least the reaction with the alkali is carried out in a closed space in which the indicator system is placed in such a way that it is not in contact with the other reagents. The sample can first be reacted with urease and then with alkali, and at least the last-mentioned reaction is carried out in the above-mentioned closed space. If the sample, instead, is reacted with urease and alkali simultaneously, the reaction is entirely carried out in a closed space. The test means comprises a closed space in which at least the reaction with alkali is carried out and in which the indicator system is placed in such a way that it is not in contact with the other reagents.

The reaction reagents comprise urease and alkali. Urease degrades urea contained in the sample to be analyzed into ammonium salts, and the alkali liberates ammonia from the salts. Ammonia is absorbed into the indicator system and it causes a color change in the pH indicator within a predetermined period of time.

The indicator system comprises one or more pH indicators. The indicator is generally dissolved in a suitable buffer. It is also possible to use several buffer/indicator mixtures which change color at difIE 913920 ferent pH values. The determination of the concentration of urea in milk is preferably carried out as a so-called double test. The buffer/indicator mixtures of the double test are preferably such that a urea concentration below 20 mg/100 ml does not yet cause a color change in either one of the buffer/indicator mixtures, whereas one of the mixtures reacts at a concentration between 20 and 30 mg/100 ml and both mixtures react when the urea concentration is greater than 30 mg/100 ml. Preferably, the buffer/indicator mixtures both contain a phosphate buffer, though in different concentrations. The phosphate buffers preferably contain sodium salt of phenol red and sodium salt of bromtymol blue at a weight ratio of 1:1, for instance. One phosphate buffer is preferably 0.00025M and the other 0.00230M sodium dihydrogenphosphate buffer.

The indicator system may be in liquid or solid form. An indicator system in liquid form can be separated from the other reagents by means of a gaspermeable membrane. The membrane may be e.g. of Teflon tube tape or Teflon filter membrane with a porosity of 0.5 pm. The indicator system is preferably solidified by using alginate and/or by absorbing in filter paper and drying.

The indicator system is preferably placed in the cover of a closable container. The cover may comprise two parts, in which case two different buffer/indicator mixtures are placed in it. Urease, sample and alkali are introduced to the bottom of the container, and the cover is closed tightly. After a predetermined period of time, the colours of the indicator system are read. The process according to the invention is especially suitable for the deter35 mination of the concentration of urea in milk but it can also be applied to other samples, such as urine.

The invention will be illustrated by means of the following non-restrictive examples.

Example 1 Buffer/indicator mixture I containing 25 mg of sodium salt of phenol red, 25 mg of sodium salt of bromtymol blue and 3 g of alginate in 100 ml of 0.00025M sodium dihydrogenphosphate is absorbed in filter paper. Buffer/indicator mixture II otherwise similar to the mixture I except that the concentration of sodium dihydrogenphosphate is 0.00230M is absorbed in another filter paper. The papers are dried at 90°C for 60 min and then placed in a twopart cover of a tightly closable 10 ml container in such a way that a partition wall of the cover separates the papers from each other. Urease (about 60 to 250 U) is introduced to the bottom of the container in liquid or solid form. 1 ml of milk is added to the container, and 0.5 to 1 ml of 0.1M NaOH is added after 2.5 minutes, and the cover is closed. After 2.5 minutes, the colours of the buffer/ indicator mixtures I and II formed in the cover of the test container are read. The results are compared with a color chart and interpreted as follows: Color combination of Urea concentration of the cover of the test sample, mg/100 ml container yellow/yellow orange-red/yellow orange-red/orange-red to 30 > 30 Example 2 Urea concentrations were determined from 118 milk samples as described in Example 1 and by a spectrophotometric reference method (Fawcett & Scott 1960, J. Clin. Path. 13:156). The results are shown in Table 1.

Table 1. Distribution of milk samples (number of samples) into different urea concentration classes Urea mg/100 ml < 20 20 -30 > 30 < 20 17 3 - 20 to 30 2 34 3 >30 8 51 Reference process Total/samples 19 45 54 118 As compared with the reference process. 86.4% of the samples fell into the right class when determined by the process according to the invention. Table 2 shows the corresponding sample distribution when the internal deviation of the reference process (± 5%) is taken into account. The proportion of samples fallen into the right class is then 94.1%.

Distribution of milk samples (number of samples) into different urea concentration classes Table 2.

Urea mg/100 ml < 20 20-30 > 30 < 20 18 1 - 20 to 30 1 40 1 > 30 - 4 53 Reference process Total/samples 19 45 54 118 Example 3 15 The concentration of urea in 64 milk samples was determined by using AzostixR test strips intended for farm use (Tierartzl. Prax. 1985, 13:559), by the above-mentioned reference process and by the process according to the invention. The results are shown in Tables 3 and 4.

Table 3. Distribution of milk samples (number of samples) into different urea con25 centration classes by the test strip Urea mg/100 ml < 20 20-30 > 30 <20 7 to 30 6 21 5 >30 - 1 24 Reference process Total/samples 13 When using the test strip, 82.8% of the samples (53/64) fell into the right class.

Table 4. Distribution of milk samples (number of samples) into different urea concentration classes by the process of the invention Urea mg/100 ml < 20 20-30 > 30 < 20 13 2 - 20 to 30 - 17 2 > 30 - 3 27 Reference process Total/samples 13 22 29 64 When using the process of the invention, 89.1% of the samples (57/64) fell into the right class.

Example 4 0.5 ml of the buffer/indicator mixture I mentioned in Example 1 was introduced into a 4 ml container in liquid form, that is, the mixture did not contain alginate. The mouth opening of the container was covered with a Teflon membrane having a porosity of 0.5 pm. To another similar container, 0.2 ml of milk and about 15 U of urease were added. After 2.5 minutes, 200 pi of 0.1M NaOH was added to it. The container with the buffer/indicator mixture and the container with the sample and the other reagents were combined with their mouth openings against each other with tape into a fully closed space in which the gasIE 913920 permeable membrane separated the buffer/indicator mixture from the other reagents. The containers so combined were shaken for one minute, whereafter the color of the indicator mixture was read. A duplicate determination was made by using the liquid-form buffer/indicator mixture II mentioned in Example 1. The results were interpreted as in Example 1. The concentration of urea in 24 milk samples was determined. 87.5% of the samples fell into the right class. The results are shown in Table 5.

Table 5. Distribution of milk samples (number of samples) into different urea con15 centration classes Urea mg/100 ml < 20 20-30 > 30 < 20 6 - - 20 to 30 1 8 1 > 30 - 1 7 Reference process Total/samples 79 8 24

Claims (2)

Claims: 1. A process for the determination of the concentration of urea in a sample, especially milk, by 5 means of urease, alkali and an indicator, characterized in that at least the reaction with the alkali is carried out in a closed space in which the indicator system is placed in such a way that it is not in contact with the other reagents. 10 2. A process according to claim 1, char- acterized in that at least the reaction with 15 the alkali is carried out in a closed container, the indicator system being placed in the cover of the container. 3. A process according to claim 1, c h a r a c- terized in that the determination is carried out as a double test by using an indicator system comprising two buffer/indicator mixtures changing color at different pH values. 20 4. A process according to claim 3, characterized in that one buffer/indicator mixture changes color at a urea concentration of at least 20 mg/100 ml and the other buffer/indicator mixture changes color at a urea concentration greater than 30 25 mg/100 ml. 5. A process according to claim 4, characterized in that the buffer/indicator mixtures contain a phosphate buffer of two different concentrations . 30 6. A process according to claim 5, characterized in that the phosphate buffers contain sodium salt of phenol red and sodium salt of bromtymol blue at a weight ratio of 1:1. 7. A test means for the determination of urea by means of urease, alkali and an indicator, c h a r35 acterized in that it comprises a closed space in which at least the reaction with the alkali is carried out and in which the indicator system is placed in such a way that it is not in contact with 5 the other reagents. 8. A test means according to claim 7, characterized in that the indicator system is in solid form. 9. A test means according to claim 7, char10 acterized in that it comprises a closable container, the indicator system being placed in the cover of the container and the other reagents on its bottom. 10. A test means according to claim 7, 15 characterized in that it comprises the reagents mentioned in claim 3, 4, 5 or 6 required for a double test. 11. A process for the determination of the concentration of urea in a sample according to any preceding claim substantially as hereinbefore described and exemplified.

1

2. A test means for the determination of urea according to any preceding claim substantially as hereinbefore described and exemplified.