Abstract:
Leucine aminopeptidase (LAP) activity or γ-glutamyltranspeptidase (γ-GTP) activity in a sample is determined by converting a substrate represented by the formula (I): ##STR1## wherein Z represents (CH 3 ) 2  CHCH 2  CH(NH 2 )--or (NH 2 )(COOH) CHCH 2  CH 2  --, R 1  represents hydrogen, alkyl or substituted alkyl, R 2  represents alkylene or hydroxyalkylene, R 3  and R 4  are the same or different and represents hydrogen, halogen, nitro, hydroxyl, sulfo, carboxyl, alkyl, and alkoxy, and salts thereof into a compound represented by the formula (II): ##STR2## wherein R 1 , R 2 , R 3  and R 4  have the same meanings as defined above. The compound (II) is reacted with a chromogen to form a pigment or converted into a diazonium salt which is reacted with a coupling agent to form an azo dye. Absorption of a resultant colored reaction solution is measured.

Description:
This application is a continuation of application Ser. No. 698,623, filed Feb. 6, 1985, now abandoned. 
    
    
     BACKGROUND OF THE INVENTION 
     The present invention relates to a method for the determination of enzyme activity and a composition suitable therefor. In the present specification, the enzymes to be determined are leucine aminopeptidase (hereinafter referred to as LAP) and γ-glutamyltranspeptidase (hereinafter referred to as γ-GTP). 
     Heretofore, as a method for measuring the enzyme activities, it has been known that a substrate for enzymes obtained by binding leucine or glutamic acid with a chromogen is decomposed by the action of the enzyme in a sample to form a colored compound which is determined. 
     As the chromogen, p-nitroanilide, β-naphthylamide, 4-hydroxy-3-carboxyanilide, p-hydroxyanilide, 4-N,N-dialkylamino-3-carboxyanilide, 4-N,N-dialkylaminoanilide and the like are used. 
     The solubility of these compounds is, at most, 6.5 mg/ml and the rate of enzymatic reaction is controlled by the rate of solubility. Further, in the determination of the pigment formed by using these substrates, the results are likely to be influenced by the other components in the sample. Because the determination is made by measuring the absorbancy in the ultraviolet region, a enzymatic reaction must be carried out under the strong alkaline condition which is a dangerous condition. The sensitivity is low because the molecular extinction coefficient of the formed pigments is low a long period of time is required to complete the enzymatic reaction, and thus the accuracy of the results by the method is low. 
     As a result of studies of substrates having few faults, it has been found that p-aminoanilide compounds wherein an alkyl or hydroxyalkyl group containing a sulfo group is bonded to the amino group in the para-position, have excellent solubility and stability of the pigment formed by the enzymatic reaction. It is possible to measure in the visible region because the chromogen has a high molecular extinction coefficient. 
     SUMMARY OF THE INVENTION 
     According to the present invention, LAP or γ-GTP can be determined by converting the substrate for the enzyme to be determined represented by the general formula (I) ##STR3## wherein Z represents (CH 3 ) 2  CHCH 2  CH(NH 2 )-- (hereinafter referred to as Z 1 ) or (NH 2 )(COOH)CHCH 2  CH 2  -- (hereinafter referred to as Z 2 ), R 1  represents hydrogen, alkyl or substituted alkyl, R 2  represents alkylene or hydroxyalkylene, R 3  and R 4  are the same or different and represents hydrogen, halogen, nitro, hydroxyl, sulfo, carboxyl, alkyl, and alkoxy, and salts thereof into the compound represented by the general formula (II) [hereinafter referred to as compound (II)] ##STR4## wherein R 1 , R 2 , R 3  and R 4  have the same meaning as described above by the action of LAP or γ-GTP, (a) reacting the compound (II) with a chromogen to form a pigment or (b) converting the compound (II) into a diazonium salt and reacting the diazonium salt with a coupling agent to form an azo dye, and measuring the absorbancy of the colored reaction solution. 
     DETAILED DESCRIPTION OF THE INVENTION 
     In the definition of R 1  -R 4 , alkyl includes alkyl having 1-5 carbon atoms such as methyl, ethyl, propyl, butyl and amyl, halogen includes chloro, iodo and bromo groups, the alkyl moiety in alkoxy has the same meaning as those of alkyl defined above, alkylene includes alkylene having 1-5 carbon atoms such as methylene, ethylene, tetramethylene and pentamethylene, the alkylene moiety in hydroxyalkylene has the same meaning as those of alkylene defined above, and the substituent of substituted alkyl includes sulfo, hydroxyaryl, nitro, carboxyl, halogen such as chloro and bromo. 
     Compound (I) can be synthesized by the following method. ##STR5## 
     Z&#39; represents the group where the amino group in Z is protected with an amino protecting group and/or the carboxyl group in Z is protected with a carboxyl protecting group. Examples of the protecting group include amino protecting groups such as benzyloxycarbonyl group, tert-butoxycarbonyl (BOC) groups and carboxyl protecting group such as benzyl group and acyl group (acetyl). 
     Examples of compound (III) include N-BOC-L-leucine, N-BOC-L-glutamic acid-α-benzylester. As compound (IV), phenylenediamine or phenylenediamines having substituents corresponding to R 3  or R 4  of the substituents of desired compound may be used. As condensing agent, for example, DCC may be used. 
     The reaction is carried out at room temperature to 60° C. for 1 to 10 hours in an organic solvent. The reaction mixture per se or compound V isolated therefrom is dissolved in an organic solvent such as chloroform and compound VI is added thereto. The reaction is carried out at room temperature to 60° C. for 15-30 hours. 
     As a compound VI, compound corresponding to substituent R 2  of the desired compound is used and examples are shown in reference example 2 described later. The desired compound is obtained by isolating compound VII after completion of the reaction and removing the amino and/or carboxyl protecting group by methods known per se. 
     Examples of the chromogen include amines such as N,N-diethyl-m-toluidine, N-ethyl-N-hydroxyethyl-m-toluidine, N-ethyl-N-3-methylphenyl-N&#39;-acetylethylenediamine (EMAE), N-ethyl-N-(3-methylphenyl)-N&#39;-succinylethylenediamine (EMSE), N,N-di-3-sulfopropyl-m-toluidine, N-ethyl-N-2-hydroxy-3-sulfopropyl-m-toluidine (TOOS), N,N-dimethyl-m-toluidine, N-ethyl-N-2-cyanoethyl-m-toluidine, N-(2-hydroxy-3-sulphopropyl)-m-toluidine, aniline derivatives such as N-ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(2-hydroxy)-3-sulfopropyl-3,5-dimethoxyaniline, N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline, N-ethyl-N-2-cyanoethyl-aniline, N,N-dimethylaniline and N,N-diethylaniline, phenols such as α-naphthol, β-naphthol, o- or p-bromophenol, o- or m-anisole, 2,6-xylenol, 3,5-xylenol, 2,5-xylenol, 2,3-xylenol and o- or m-cresol. 
     These chromogens are used in a concentration of 0.01-5 mg/ml with surfactant such as Triton X-100, Brij-35 and Brij-80 in a concentration of 0.01-2% for chromogens having low solubility. 
     As a chromogen, cinnamaldehydes such as p-N,N-dimethylamino cinnamaldehyde, p-N,N-diethylamino cinnamaldehyde may also be used in a concentration of 0.01-100 mg/ml. In this case, an azo dye is formed as the pigment. 
     As diazonization agent used in the conversion of compound (II) into a diazonium salt, sodium, potassium or ammonium nitrites may be used in a concentration of 0.1-100 mg/ml. As coupling agent, phenols described above such as phenol and 3,5-xylenol may be used in a concentration of 0.01-100 mg/ml. 
     When the formed pigment is not an azo dye, the reaction of compound (II) with a chromogen is usually carried out in the presence of an oxidizing agent including oxidase. 
     Examples of oxidizing agents include inorganic compounds such as hydrogen peroxide, sodium or potassium metaperiodate, sodium or potassium permanganate, sodium or potassium dichromate, sodium or potassium metavanadate, pentacyanoiron complex, peroxides of acids such as peracetic acid, hydrogen peroxide-peroxidase (HRP), and oxidases such as ceruloplasmin and laccase (E C. 1. 10. 3. 2). 
     They may be used in a concentration of 0.01-10 mg/ml for inorganic compounds, 0.1-100 U/ml for oxidases. 
     Tables 1 and 2 show the solubility, maximum wavelength, sensitivity and stability of the formed pigment. 
     
                                           TABLE__________________________________________________________________________ ##STR6##CompoundNo.    Z R.sub.3           R.sub.4                R.sub.1                       R.sub.2__________________________________________________________________________1      Z.sub.2    H      H    (CH.sub.2).sub.3 SO.sub.3 H                       (CH.sub.2).sub.32      Z.sub.1    &#34;      &#34;    &#34;      &#34;3      Z.sub.2    &#34;      &#34;    CH.sub.3                       &#34;4      Z.sub.1    &#34;      &#34;    &#34;      &#34;5      Z.sub.2    &#34;      &#34;    C.sub.2 H.sub.5                       CH.sub.2 CHCH.sub.2OH6      Z.sub.1    &#34;      &#34;    &#34;      &#34;7      Z.sub.2    SO.sub.3 H(2)           &#34;    (CH.sub.2).sub.3 SO.sub.3 H                       (CH.sub.2).sub.38      Z.sub.1    &#34;      &#34;    &#34;      &#34;9      Z.sub.2    Cl(2)  &#34;    &#34;      &#34;10     Z.sub.1    &#34;      &#34;    &#34;      &#34;11     Z.sub.1    CN(2)  &#34;    (CH.sub.2).sub.2 SO.sub.3 H                       (CH.sub.2).sub.212     Z.sub.1    CH.sub.3 (2)           &#34;    &#34;      &#34;13     Z.sub.1    COOCH.sub.3 (2)           &#34;    (CH.sub.2).sub.4 SO.sub.3 H                       (CH.sub.2).sub.414     Z.sub.1    OCH.sub.3 (4)           CH.sub.3 (2)                &#34;      &#34;Control   ##STR7##__________________________________________________________________________ Numbers in parenthesis of R.sub.3 or R.sub.4 show the position of the R.sub.3 or R.sub.4 group on the benzenenucleus. 
    
     
                       TABLE 2______________________________________λ.sub.max (nm)           SE      ST(%)     S (mg/ml)______________________________________1      745          &gt;500    98 &lt;    &gt;2002      745          &gt;500    98&lt;     1303      750          &gt;500    96&lt;     804      750          &gt;500    96&lt;     655      740          &gt;500    98&lt;     1706      740          &gt;500    98&lt;     907      680          237     93      &gt;208      680          237     93      &gt;2009      685          315     98&lt;     &gt;20010     685          315     98&lt;     12511     640          177     95      &gt;20012     715          &gt;500    &gt;98     &gt;20013     705          312     &gt;98     &gt;20014     720          406     &gt;98     &gt;200Control  615          100     90      &lt;20______________________________________ λ.sub.max : Maximum wavelength SE: Sensitivity of pigment ST: Stability of pigment S: Solubility 
    
     Sensitivity 
     50 μl of 1 mM each free compound was added to 3 ml of 0.1M phosphate buffer containing 0.5 mg/ml EMSE and 1 U/ml laccase and the mixture was incubated at 37° C. for 5 minutes. The absorption of the reaction solution at λmax was measured. As a control, 5-aminosalicylic acid was used and the reaction of the compound with 2,6-xylenol was carried out in the presence of metaperiodate under strong alkaline conditions. 
     The results are shown, defining the sensitivity of 5-aminosalicylic acid as 100. 
     The enzymatic reaction is usually carried out in a buffer solution having a pH of 6 to 10 preferably around 8. As the buffer, good buffer, phosphate buffer, acetate buffer, etc. may be used. 
     The present method is very simple and excellent. For example, the solubility of the present substrate is very high and therefor it is not necessary to add a surfactant or organic solvent to increase the solubility. The measurement of absorption is carried out at a wavelength of 560 nm or more and therefore the results are not influenced by the component in the serum. Since the sensitivity and stability of the present substrate are excellent, it is expected that more accurate results will be obtained. 
     Another aspect of the present invention is to provide a test composition for the determination of LAP activity or γ-GTP activity which comprises the substrate represented by the general formula (I) and a chromogen. The composition may also contain an oxidizing agent, an oxidase, a diazonizing agent, a coupling agent or a buffer. 
     Certain specific embodiments of the invention are illustrated by the following representative examples. 
    
    
     EXAMPLE 1 
     
         ______________________________________Reagent solution (pH 7.3)______________________________________Compound No. 2        0.03   g/mlEMSE                  0.5    mg/mlMagnesium nitrate     1      mg/mlGood buffer           8      mg/mlLaccase               1      U/ml______________________________________ 
    
     3 ml of the reagent solution was incubated at 37° C. for ten minutes. To the solution was added 0.05 ml of a serum sample and the mixture was immediately stirred and the change in absorption of the solution at 745 nm was measured for one minute. 
     LAP activity in the sample can be calculated using the following equation: ##EQU1## ΔE means the amount of absorption change per minute. 
     The same procedures as described above were repeated except that the following materials were used. 
     
         ______________________________________(a)  Substrate    L-leucil-3-carboxy-4-hydroxyanilideOxidizing agent             sodium metaperiodateCoupling agent             p-xylenolWavelength for             635 nmmeasurement(b)  Substrate    L-leucil-p-nitroanilideCoupling agent             p-dimethylaminocinnamaldehydeWavelength for             565 nmmeasurement(c)  Substrate    L-leucil-p-N,N-- diethylaminoanilideOxidizing agent             m-periodateCoupling agent             1-naphtol-2-sulfonic acidWavelength for             675 nmmeasurement______________________________________ 
    
     The amounts of absorption change per minute were measured for ten samples and the results are shown in Table 3. 
     
                       TABLE 3______________________________________                          The method accord-                          ing to the present(a)          (b)     (c)       invention______________________________________Serum(1)  0.0188    0.0071  0.0158  0.0454(2)    0.0127    0.0054  0.0095  0.0395(3)    0.0198    0.0090  0.0150  0.0543(4)    0.0203    0.0089  0.0145  0.0541(5)    0.0148    0.0071  0.0116  0.0382(6)    0.0158    0.0071  0.0126  0.0437(7)    0.0153    0.0074  0.0113  0.0400(8)    0.0191    0.0086  0.0150  0.0532(9)    0.0190    0.0082  0.0138  0.0537(10)    0.01840  0.0079  0.0133  0.0476______________________________________ 
    
     EXAMPLE 2 
     The reagent solution wherein laccase was excluded from the reagent solution of Example 1 was used. 
     3 ml of the reagent solution was incubated at 37° C. for 10 minutes and to the mixture was added 0.05 ml of a serum sample. The mixture was immediately stirred and incubated at 37° C. for 10 minutes. After ten minutes passed, 
     (a) 0.03 ml of 10 mg/ml α-naphthol methanol solution and 0.1 ml of 1N-NaOH solution containing 50 mg/ml sodium salt of meta-periodate were added to the solution. The mixture was incubated at 37° C. for 10 minutes and the absorption of the reaction solution at 600 nm was measured. 
     (b) 1 ml of 10 mg/ml p-N,N-dimethylamino cinnamaldehyde ethanol solution and 1 ml of 0.4N-HCl solution were added to the solution and the mixture was incubated at 37° C. for 10 minutes. The absorption of the reaction solution at 570 nm was measured. 
     (c) 0.5 ml of 1N-HCl and 0.5 ml of 1 mg/ml sodium nitrite were added to the solution and the mixture was incubated at 37° C. for 10 minutes. Then 0.1 ml of 1 mg/ml 3,5-xylenol ethanol solution was added thereto and the absorption of the reaction solution at 650 nm was measured. 
     The results are shown in Table 4 
     
                       Table 4______________________________________     (a)  (b)      (c)    Example 1______________________________________Serum 1         0.331  0.386  0.277                              0.04542               0.291  0.336  0.241                              0.03953               0.389  0.466  0.330                              0.05434               0.395  0.460  0.325                              0.05415               0.277  0.320  0.230                              0.03826               0.322  0.372  0.270                              0.04377               0.291  0.333  0.244                              0.04008               0.386  0.453  0.325                              0.05329               0.392  0.460  0.331                              0.053710              0.346  0.405  0.292                              0.0476Ratio of        73     85     61   100sensitivity______________________________________ 
    
     EXAMPLE 3 
     In this example, 500 mg of compound Nos. 1, 3, 5, 7 and 9 each and 500 mg of glycylglycine were dissolved in 100 ml (pH=7.7) and 0.1M phosphate buffer. The resultant solutions were designated A-1, A-3, A-5, A-7 and A-9, respectively. 50 mg of 3-{N-(m-tolyl)-N-ethyl}amino-2-hydroxypropane sulfonic acid was dissolved in 100 ml of phosphate buffer solution to obtain solution B. 50 mg/ml sodium meta-periodate containing 1N-HCl solution was prepared as solution C. 
     6 ml of solution A-1, A-3, A-5, A-7 or A-9 was equally poured to 3 test tubes. The samples were incubated at 37° C. and to the solution was added 20 μg of (a) serum sample (b) standard serum or (c) water. The mixtures were stirred and heated for 5 minutes. After 5 minutes had passed, 1 ml of solution B and 1 ml of solution C were added to each of the mixtures and the mixtures were incubated for 10 minutes after stirring. The absorption of the reaction solutions were measured at the wavelength indicated in Table 2. As the standard serum, a serum having 235 mU/ml of γ-GTP was used. 
     The γ-GTP activity of the serum sample can be calculated by the following equation. ##EQU2## 
     The results are 231, 228, 236, 240, and 242 mU/ml respectively. 
     REFERENCE EXAMPLE 1 
     Production of the hydrobromide of L-leucyl-p-N,N-disulfopropyl aminoanilide. 
     2.49 g of N-BOC-L-leucine (hereinafter referred to as BLL) was dissolved in 100 ml of dioxane. To the solution were added 4.12 g of DCC and 2.16 g of p-phenylenediamine (hereinafter referred to as PDD), and the mixture was incubated at room temperature for 4 hours. The mixture was filtered and the filtrate was concentrated to dryness under reduced pressure. The product was redissolved in methanol/water=80/20 (vol/vol) and the solution was subjected to chromatography using Diaion HP-20 (Produced by Mitsubishi Chemical Industries Ltd.) and methanol/water=80/20 was used as eluting agent. 
     Methanol was removed from the eluate under reduced pressure to obtain N-BOC-L-leucine-p-aminoanilide in the yield of 1.25 g. 1 g of the product was dissolved in 50 ml of chloroform. To the mixture were added 2 ml of triethylamine and 5 g of 1,3-propanesultone and the mixture was incubated at 50° for one day. 50 ml of ethyl ether was added to the mixture which was separated into two layers. To the lower layer were added 5 ml of acetic acid and 20 ml of 25% hydrogen bromide-acetic acid and the mixture was allowed to stand at room temperature. To the mixture was added 50 ml of ethyl ether to remove the BOC group and to form L-leucine-p-N,N-disulfopropyl aminoanilide.HBr as a precipitate. After filtration with a glass filter, the precipitate was dissolved in 50 ml of methanol and the solution was neutralized with 6N-NaOH. After removal of formed NaCl by filtration, ethyl ether was added to form a precipitate. The precipitate was separated by filtration to obtain 0.63 g of desired compound having a melting point of 221°-223° C. 
     
         ______________________________________Elemental analysis          C          H      N______________________________________Found (%)      46.29      6.58   9.07Calculated(%)  46.45      6.67   9.03______________________________________ 
    
     REFERENCE EXAMPLE 2 
     The similar procedures as described in Reference example 1 were repeated except that N-BOC-L-glutamic acid-α-benzylester (BLGB) instead of BLL, or chloro-PPD (CLPPD), sulfo-PPD (SPPD), N-methyl-PPD (NMPPD), N-ethyl-PPD (NEPPD), N-cyano-PPD (CYPPD), methyl-PPD (MPPD), methyloxycarbonyl-PPD (MCPPC) or 2-methyl-5-methoxy-PPD (MOMPPD) instead of PPD and propanesultone (PS), 2-chloroethanesulfonic acid (CESA), 1,4-butanesultone (BS) or 3-chloro-2-hydroxy-propanesulfonic acid (CHPSA) as sulfonizing agent were used to obtain compounds indicated in Table 5. 
     
                       TABLE 5______________________________________Com-                         Sulfo-pound      Compound  Compound                        nizing         R.sub.f ofNo.        III       IV      agent  m.p.    TLC______________________________________1          BLGB      PPD     PS     201-205° C.                                       0.192          BLL       PPD     PS     221-223° C.                                       0.383          BLGB      NMPPD   PS     173-178° C.                                       0.624          BLL       NMPPD   PS     169-172° C.                                       0.805          BLGB      NEPPD   CHPSA  194-197° C.                                       0.446          BLL       NEPPD   CHPSA  180-185° C.                                       0.517          BLGB      SPPD    PS     228-230° C.                                       0.088          BLL       SPPD    PS     217-222° C.                                       0.129          BLGB      CLPPD   PS     225-228° C.                                       0.4510         BLL       CLPPD   PS     201-207° C.                                       0.5211         BLL       CYPPD   CESA   210-215° C.                                       0.4712         BLL       MOMPPD  CESA   153-157° C.                                       0.7013         BLL       MCPPD   BS     169-175° C.                                       0.7714         BLL       MOMPPD  BS     150-153° C.                                       0.63______________________________________