CH625833A5 - Method for the determination of acid phosphatase - Google Patents

Description

The present invention relates to an improved method for determining the acid phosphatase of human origin, in which a reagent for activating the acid phosphatase is used.

The acidic phosphatase is an enzyme which has the ability to optimally cleave phosphomonoesters in the acidic pH range. The acid phosphatase of human origin is coming

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

3rd

625 833

in various organs, such as B. in the prostate, in the liver, in the spleen or in blood cells and passes into the serum in case of cell damage. The determination of acid phosphatase in body fluids, especially in serum or plasma, therefore plays an important role in the diagnosis of 5 diseases of the organs in question.

The activity of the acid phosphatase in serum or plasma is significantly increased by everything in the presence of prostate cancer or prostate hypertrophy and the determination of this prostate phosphatase activity enables the diagnosis of these specific diseases.

In Chemical Abstracts Vol. 42, No. 22,1948,8838g it is mentioned that alcohols with up to 3 carbon atoms are able to increase the activity of the acid phosphatase. Appropriate experiments have shown that this is not the case and that 15

especially methanol, ethanol and n-propanol have no influence on the activity of the acid phosphatase. In contrast, it has now been found within the scope of the present invention that the activity of the acid phosphatase of human origin can be significantly increased by adding a straight-chain alcohol having 4 to 6 20 carbon atoms. Since the activity of acidic phosphatase of human origin in body fluids is relatively low, an increase in this activity means a clear technical advance with regard to the determination of this important enzyme. 25th

The present invention thus relates to an improved method for determining the acid phosphatase using an activator containing a straight-chain alcohol having 4 to 6 carbon atoms.

More specifically, the present invention relates to a method for determining an acid phosphatase of human origin in a body fluid, characterized in that a solution containing 50 μmol / 1 to 50 mmol / 1 of a phosphomono ester, 50 to 300 mmol / 1 of a buffer substance for adjusting a pH 35 between 4.5 and 6.5 and 10 to 300 mmol / 1 of an activator containing at least one straight-chain alcohol with 4 to 6 C atoms at an incubation temperature between 20 ° C and 45 ° C the phosphatase effect in the body fluid to be examined and measures the substrate conversion. 40

The phosphomonoester used as the substrate for the enzymatic reaction has the general formula

R-O-PO3H2

45

where R is any organic residue.

However, R is preferably a chromophore, such as. B. 4-nitrophenyl, thymolphthalein, phenolphthalein, 2-chloro-4-nitrophenyl; or a fluorophore such as e.g. B. 1-naphthyl. Adenosine, glycerol or phenyl can also be used as R 50.

For the amount of substrate used, an optimal concentration is preferably selected, which is different for the individual substrates and is also influenced by the pH. The optimal substrate concentration, which causes the maximum possible enzyme activity at a specific pH value, can be read off using a substrate activity curve.

The substrate activity curve for a specific substrate is obtained by measuring the enzyme activity with different substrate concentrations under otherwise identical conditions.

According to the invention, the substrate concentration for the acid phosphatase is between 50 jimol / 1 and 50 mmol / 1; preferably 60 (imol / 1 and 25 mmol / 1. Thus, at a pH of 5.5, the optimal substrate concentration is approximately 25 mmol / 165 for β-glycerophosphate, 2.5 mmol / 1 for 4-nitrophenyl phosphate and 100 jimol / I for phenolphthalein diphosphate.

In the method according to the invention, the pH is adjusted between 4.5 and 6.5, preferably between 5 and 6, by means of a buffer substance. Suitable buffer substances are e.g. B. citrate buffer and acetate buffer. The buffer concentration is usually between 50 mmol / 1 and 300 mmol / I, preferably 100 mmol / I.

According to the present invention, the activator used contains a straight-chain alcohol having 4 to 6 carbon atoms, preferably n-butanol, n-pentanol and 1,5-pentanediol. 1-Pentanol and 2-pentanol are particularly preferred.

The concentration of the activator is 10 to 300 mmol / 1, preferably 100 to 200 mmol / 1.

The activator can contain one or more straight-chain alcohols with 4 to 6 carbon atoms. However, the activator preferably consists of a uniform, straight-chain alcohol having 4 to 6 carbon atoms.

After mixing a sample of the body fluid to be analyzed with the test solution, the enzymatic reaction is carried out by incubating the solution at a defined reaction temperature which is between 20 and 45 ° C., preferably between 30 and 37 ° C. The incubation period is usually between 5 and 60 minutes. However, the reaction is preferably carried out after 30 minutes by means of a suitable additive such as. B. stopped by means of sodium hydroxide solution or a trisodium phosphate solution.

When choosing a suitable substrate such. B. 2-chloro-4-nitrophenyl phosphate, it is also possible to measure the enzymatic reaction kinetically.

The substrate turnover is the measure of the enzyme activity. It is measured by determining the decrease in the phosphomonoester used or the increase in the organic residue released. Since the activation of the acid phosphatase by transphosphorylation, i.e. H. a phosphate transfer from the phosphomonoester used to the alcohol used as an activator, the amount of free phosphate after the enzymatic reaction does not correspond to the substrate conversion and accordingly cannot be taken as its measure.

The type of measurement depends on the substrate used. For example, when using 4-nitrophenylphosphate, thymolphthaleinphosphate, phenolphthaleinphosphate or when using naphthylphosphate, the increase in the organic residue released can be determined directly photometrically or fluorimetrically after the enzymatic reaction has been appropriately stopped.

When using other substrates such. B. β-glycerophosphate, phenyl phosphate or adenosine monophosphate, the substrate conversion is measured in such a way that the released organic residue is converted chemically or enzymatically into a substance that can be measured photometrically.

So z. B. when using phenyl phosphate, the released phenol is converted into a blue dye using the phenol reagent [Folin-Ciocalteus, Merck 9001].

The method according to the invention is suitable for both manual and automatic determinations of acid phosphatase. As a destination, z. B. serum, plasma, blood, cerebrospinal fluid or urine can be used.

The reagents necessary for carrying out the method according to the invention, namely phosphomonoesters, buffers and activators, can be used for diagnostic purposes, such as suitable additives such. B. Detergents such as Brij-35 (Polyoxyäthylenlau-ryläther) or stabilizers such as magnesium ions can be added.

The reagents are preferably packaged in a reagent set. A reagent set suitable for the purposes of the present invention advantageously contains a phosphomonoester in one or more containers a),

625833

4th

b) a buffer substance for establishing a pH value between 4.5 and 6.5,

c) a straight-chain alcohol with 4 to 6 carbon atoms as an activator.

The reagent set can also additionally contain a container with a control or calibration solution and / or a container with a reagent to stop the reaction and / or a container with one for further conversion of the released organic residue and / or a container with the mentioned, for suitable additives for diagnostic purposes such as detergents or stabilizers.

However, it is not necessary to store reagents and additives in separate containers. The present invention also comprises a reagent set, in which some of the components necessary for carrying out the method according to the invention are kept together in the same container.

So z. B. the buffer substance can be stored together with the activator in dissolved form in a single container.

With the exception of the activator, which is always in liquid form, the various reagents and additives can be in both liquid and solid form, e.g. B. packed as powder, granules, tablets or lyophilisate.

The invention is illustrated by the examples below.

example 1

To 2.0 ml of substrate buffer solution, consisting of 0.1 mol / 1 citrate buffer (pH 5.25), 5 g / 1 Brij-35 and 5 mmol / 14-nitrophenyl phosphate, with different contents of 1-pentanol, 1 ml of acidic prostate phosphatase solution (isolated according to the method of Lam et al., Clin. Chem. 19, 483, 1973) was mixed in and incubated at 37 ° C. for 30 minutes. The reaction is stopped by adding 1.0 ml of 1N sodium hydroxide solution and the released 4-nitro-phenolate is measured photometrically at a wavelength of 405 nm.

The test results are shown in Table I below in% of the prostate phosphatase activity without the addition of 1-pentanol.

1-pentanol

Activity of

concentration

Prostate phosphatase in the test solution

0 mmol / 1

100%

50 mmol / 1

137%

100 mmol / 1

173%

150 mmol / 1

189%

200 mmol / 1

139%

Example 2

The procedure of Example 1 is repeated with 2-penta nol instead of 1-pentanol. The test results are shown in Table I below

I reproduced in the same way.

2-pentanol

Activity of

concentration

Prostate phosphatase in the test solution

0 mmol / 1

100%

50 mmol / 1

138%

100 mmol / 1

172%

150 mmol / 1

190%

200 mmol / 1 139%

Example 3

The procedure of Example 1 is repeated with 1,5-pentanediol instead of 1-pentanol. The test results are shown in Table III below.

1,5-pentanediol concentration in the test solution

Prostate phosphatase activity

0 mmol / 1

100%

50 mmol / 1

120%

100 mmol / 1

131%

150 mmol / 1

140%

200 mmol / 1

148%

Example 4

The procedure of Example 1 is repeated with 1-butanol instead of 1-pentanol. The test results are shown in Table IV below.

1-butanol concentration in the test solution

Alactivity of prostate phosphatase

0 mmol / 1

100%

50 mmol / 1

119%

100 mmol / 1

131%

150 mmol / 1

131%

200 mmol / 1

116%

Example 5

The procedure of Example 1 is repeated with 1-hexanol instead of 1-pentanol. The test results are shown in Table V below.

1-hexanol

Activity of

concentration

Prostate phosphatase in the test solution

0 mmol / 1

100%

25 mmol / 1

107%

50 mmol / 1

114%

100 mmol / 1

120%

Example 6

0.05 ml of prostate phosphatase solution are added to 2.0 ml of 0.1 mol / 1 acetate buffer, pH 5.5 with 62 μmol / l of phenolphthalein di-phosphate with different contents of 1-pentanol, and the solution over 15 minutes incubated at 37 ° C. To stop the reaction and to develop the color, 2.0 ml of 0.4 mol / 1 sodium phosphate buffer, pH 10 are mixed in and the color intensity is measured at the wavelength of 546 nm.

The test results are shown in Table VI below.

1-pentanol concentration in the test solution

Prostate phosphatase activity

0 mmol / 1

100%

50 mmol / 1

154%

100 mmol / 1

174%

150 mmol / l

177%

200 mmol / 1

131%

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

5

625 833

Example 7

0.1 ml of erythrocyte phosphatase is mixed into 2.0 ml of 0.1 mol / 1 acetate buffer (pH 5.5), 5 mmol / 14-nitrophenyl phosphate and 5 g / l Brij-35 with different contents of 1-pentanol. After 30 minutes of incubation at 37 ° C., the enzyme reaction is stopped by adding 1.0 ml of 1 N sodium hydroxide solution and the released 4-nitrophenolate is determined photometrically at the wavelength 405 nm.

The test results are shown in Table VII below.

1-pentanol concentration in the test solution

Activity of

Erythrocyte phosphatase

0 mmol / 1

100%

50 mmol / 1

132%

100 mmol / 1

153%

150 mmol / 1

162%

200 mmol / 1

136%

Example 8

0.1 ml of serum is added to 0.5 ml of 0.1 mol / 1 citrate buffer, pH 5.5 and 5 mmol / 14-nitrophenylphosphate. After 30 minutes of incubation at 37 ° C., the enzyme reaction is stopped by adding 2.5 ml of 0.1N sodium hydroxide solution and the released 4-nitrophenolate is measured photometrically at a wavelength of 405 nm. This activity determination of the acid phosphatase is carried out once without 1-pentanol and once with 150 mmol / 11-pentanol.

The test results are shown in Table VIII below.

sample

Activity without 1-pentanol (U / 1)

Activity with 150 mmol / 1 of 1 -pentanol (U / 1)

Serum 1

1.12

2.10

Serum 2

16.63

24.55

Serum 3

18.41

31.53

Serum 4

20.65

33.59

Example 9

0.1 ml of prostate phosphatase solution is added to 2.0 ml of 0.1 mol / l acetate buffer, pH 5.5 and various concentrations of phenyl phosphate. After 5 minutes of incubation at 37 ° C., 0.5 ml of phenol reagent [Folin-Ciocalteus] and 1.0 ml of 20% NazCOa solution are pipetted in and the mixture is incubated at 37 ° C. for a further 10 minutes. The intensity of the blue dye, which corresponds to the concentration of released phenol, is determined at the wavelength of 578 nm. The experiments are carried out in parallel once without 1-pentanol and once with 150 mmol / 1 pentanol-1 in the substrate buffer solution.

The test results are shown in Table IX below.

Phenylphosphate - without 1-pentanol AE 578 with 150 mmol / 1 concentration in nm / 5mjn pentanol-1 AE 578 nm / 5min of the test solution

0.078 mmol / 1

0.105

0.169

0.155 mmol / 1

0.150

0.280

0.312 mmol / 1

0.198

0.390

0.625 mmol / 1

0.245

0.490

1.25 mmol / 1

0.275

0.550

2.50 mmol / 1

0.280

0.590

5.00 mmol / 1

0.280

0.590

Example 10

0.05 ml of purified prostate phosphatase solution are mixed into 1.0 ml of 0.1 mol / 1 acetate buffer (pH 5.5) and 0.5 mmol / 1 phenolphthalein monophosphate with different contents of 1-pentanol. After 10 minutes of incubation at 37 ° C., the enzyme reaction is stopped by adding 2.0 ml of 0.4 mol / 1 sodium phosphate buffer and the released phenolphthalein is determined photometrically at a wavelength of 546 nm.

The test results are shown in Table X below.

1-Pentanol concentration Activity of the prostate in the test solution phosphatase

0 mmol / 1 100%

50 mmol / 1 125%

100mmol / l 135%

150 mmol / 1 140%

Example 11

To 1.0 ml of 0.1 mol / 1 citrate buffer (pH 5.75), 1.2 mmol / 1 of thymolphthalein phosphate, 5 g / l of Barij-35 with different contents of 1-pentanol, 0.05 ml of purified prostate phosphatase solution added. After 10 minutes of incubation at 37 ° C., the enzyme reaction is stopped by adding 2.0 ml of 0.2 mol / 1 bicarbonate buffer of pH 10 and the released thymolphthalein is measured photometrically at the wavelength of 578 nm.

The results are summarized in Table XI below.

1-Pentanol concentration Activity of the prostate in the test solution phosphatase

0 mmol / 1 100%

50 mmol / 1 117%

100 mmol / 1 125%

150 mmol / 1 140%

5

10th

15

20th

25th

30th

35

40

45

50

55

Claims (40)

625 833 1. A method for determining an acidic phosphatase of human origin in a body fluid, characterized in that a solution containing 50 µmol / l to 50 mmol / 1 of a phosphomono ester, 50 to 300 mmol / I of a buffer substance as a substrate for adjusting a pH value between 4.5 and 6.5 and 10 to 300 mmol / l of an activator containing at least one straight-chain alcohol having 4 to 6 carbon atoms at an incubation temperature between 20 ° C. and 45 ° C. and subjecting the body fluid to be examined to the Measures substrate sales. 2. The method according to claim 1, characterized in that the acid phosphatase of human origin is prostate phosphatase. 2nd PATENT CLAIMS 3. The method according to claim 1, characterized in that the acid phosphatase of human origin is erythrocyte phosphatase. 4. The method according to any one of claims 1 to 3, characterized in that 4-nitrophenyl phosphate is used as the phosphomonoester. 5. The method according to any one of claims 1 to 3, characterized in that is used as the phosphomonic ester thymolphthalein phosphate. 6. The method according to any one of claims 1 to 3, characterized in that phenol phthalein phosphate is used as the phosphomonoester. 7. The method according to any one of claims 1 to 3, characterized in that naphthyl phosphate is used as the phosphomonoester. 8. The method according to any one of claims 1 to 3, characterized in that β-glycerophosphate is used as the phosphomonoester. 9. The method according to any one of claims 1 to 3, characterized in that adenosine monophosphate is used as the phosphomonoester. 10. The method according to any one of claims 1 to 3, characterized in that phenylphosphate is used as the phosphomonoester. 11. The method according to any one of claims 1 to 3, characterized in that 2-chloro-4-nitrophenyl phosphate is used as the phosphomonoester. 12. The method according to any one of claims 1 to 11, characterized in that a buffer is used to adjust a pH of about 5.5. 13. The method according to claim 12, characterized in that a citrate buffer is used as the buffer sutotance. 14. The method according to claim 12, characterized in that an acetate buffer is used as the buffer substance. 15. The method according to any one of claims 1 to 13, characterized in that the activator used consists of n-butanol. 16. The method according to any one of claims 1 to 13, characterized in that the activator used consists of 1-pentanol. 17. The method according to any one of claims 1 to 13, characterized in that the activator used consists of 2-pentanol. 18. The method according to any one of claims 1 to 13, characterized in that the activator used consists of 1,5-pentane diol. 19. The method according to any one of claims 1 to 6 and 11 to 18, characterized in that the conversion of phosphomonoester is determined by direct photometric measurement of the released organic residue. 20. The method according to any one of claims 1 to 3.7 and 12 to 18, characterized in that the conversion of phosphomonoester is determined by direct fluorometric measurement of the released organic residue. 21. The method according to any one of claims 1 to 3.8 to 10 and 12 to 18, characterized in that the conversion of phosphomonoester is determined by chemically or enzymatically converting the released organic residue into a substance that can be measured photometrically . 22. Reagent set for carrying out the method according to claim 1, comprising in one or more containers a) a phosphomonoester, b) a buffer substance for adjusting a pH value between 4.5 and 6.5, c) a straight-chain alcohol with 4 to 6 carbon atoms as an activator. 23. Reagent set according to claim 22, containing 4-nitrophenyl phosphate as phosphomonoester. 24. Reagent set according to claim 22, containing Thy-Molphthaleinphosphat as Phosphomonoester. 25. Reagent set according to claim 22 containing phenolphthalein phosphate as a phosphomonoester. 26. Reagent set according to claim 22 containing naphthyl phosphate as a phosphomonoester. 27. Reagent set according to claim 22 containing β-glycerophosphate as the phosphomonoester. 28. Reagent set according to claim 22 containing adenosine monophosphate as phosphomonoester. 29. Reagent set according to claim 22 containing phenyl phosphate as phosphomonoester. 30. Reagent set according to claim 22 containing 2-chloro-4-nitrophenyl phosphate as a phosphomonoester. 31. Reagent set according to one of claims 22-30 containing a citrate buffer as a buffer substance. 32. Reagent set according to claim 31, containing an acetate buffer as a buffer substance. 33. Reagent set according to one of claims 22-32 containing n-butanol. 34. Reagent set according to one of claims 22-32 containing 1-pentanol. 35. Reagent set according to one of claims 22-32 containing 2-pentanol. 36. Reagent set according to one of claims 22-32 containing 1,5-pentanediol. 37. Reagent set according to one of claims 22-36 additionally containing a container with a control or calibration solution. 38, additionally containing a container with a reagent suitable for further conversion of the released organic residue. 40. Reagent set according to one of claims 22 to 38. Reagent set according to one of claims 22 to 37, additionally containing a container with a reagent for stopping the enzymatic reaction. 39, additionally containing a container with a detergent. 41. Reagent set according to one of claims 22 to 39. Reagent set according to one of claims 22 to 40, additionally containing a container with a stabilizer.