Abstract:
Transferase and protease activity in homogenates and biological solutions is fluorometrically determined at wavelengths corresponding generally to those used for fluorometric NADH-linked determinations by utilizing novel substrate compositions consisting essentially of certain fluorogenic 5-aminoisophthalic acid derivatives coupled to amino acid constituents specific to the transferases and proteases under investigation.

Description:
RELATED APPLICATION 
     This application is a continuation-in-part of co-pending application Ser. No. 709,720, filed July 29, 1976, now abandoned. 
    
    
     BACKGROUND 
     Enzyme substrates with naphthylamines as chromogenic groups linked to other amino acids have been described in the literature for the determination of transferases and proteases, such as γ-glutamyl transpeptidase, lecuine aminopeptidase, oxytocinase, and trypsin. Orlowski et al, Clin. Chim, Acta, 7:755-760 (1962), and references cited therein. The determination of transferase and protease activity in human serum, urine, and tissues may have diagnostic significance; for example, the assay of γ-glutamyl transpeptidase activity in human serum may be useful in the differential diagnosis of liver diseases, because the enzyme activity is particularly high in obstructive jaundice and liver cancer while lower activities are observed in viral hepatitis and liver cirrhosis. Orlowski et al, supra. See also Rosalki et al, Ann. Clin. Biochem. 7:143 (1970). The majority of studies with respect to γ-glutamyl transpeptidase determinations have been carried out using naphthylamines in formulating the substrates and, unfortunately, the products (i.e., naphthylamines) are both toxic and carcinogenic, presenting undesirable risks for general laboratory use. 
     Many of the enzyme assays commonly performed in clinical laboratories are NADH linked; that is, they involve a series of reactions which ultimately result in the reduction of nicotinamide adenine dinucleotide (NAD) to its reduced form, NADH. The NADH is then detected spectrophotometrically or fluorometrically. The more recent fluorometric procedures have the characteristic advantages of simplicity, speed, and economy, and often have the further advantage of greater sensitivity. Typically, a fluorometric NADH-linked test involves the use of a filter fluorometer which directs ultraviolet light at a wavelength of about 340 nm against the surface of the sample and which measures the fluorescence, or rate of change of fluorescence at an emission wavelength of about 465 nm. 
     Other references illustrating the state of the prior art are U.S. Pat. Nos. 3,979,447, 3,862,011, 3,773,626, 3,591,458, 3,878,048, 3,892,631, and Wildes et al, J. Am. Chem. Soc., 95:8, 2610 (1973), and Bayley et al, Eur. J. Biochem. 56 (2), 455-65 (1975). 
     SUMMARY 
     This invention involves the discovery of certain compositions of matter useful as enzyme substrates in the fluorometric determination of transferase (or transpeptidase) activity in homogenates and biological fluids. Such substrates are believed novel and are relatively safe for laboratory use. An especially important advantage is that such substrates yield, upon cleavage by the enzymes under investigation, fluorogenic moieties which have peak fluorometric excitation and emission values approximately those of NADH-linked tests and, therefore, assays for fluorometrically determining transferase or protease activity by the use of such substrates may be conducted with standard fluorometers using the same filters intended for conventional NADH-linked assays. Thus, a transferase such as γ-glutamyl transpeptidase may be measured using the same fluorometric equipment and filters used for conducting assays of other enzymes such as SGOT, SGPT, CPK, LDH, and HBD. 
     DESCRIPTION 
     The enzyme substrates involved in this invention are 5-aminoisophthalic acid derivatives of the general formula ##STR1## and wherein each of R 1  and R 2  is --OH, --NH 2 , --NHCH 3 , --NHC 2  H 5 , --N(CH 3 ) 2 , --N(C 2  H 5 ) 2 , --N(CH 3 ) (C 2  H 5 ), --OCH 3 , or --O(CH 2 ) n  CH 3 , n is an integer from 1 through 4, and wherein R 3  is an amino acid moiety capable of being cleaved from the remainder of the substrate when exposed to a transferase or protease having activity specific to that substrate, in some cases in the presence of glycylglycine or some other appropriate acceptor such as glutamate, glycine, or glycylglycylglycine. Such substrates which have amino acid moieties (that may comprise several amino acid groups) and which are specific to various transferases and proteases are as follows: 
     
         __________________________________________________________________________Substrate        Enzyme__________________________________________________________________________(A)-lys-ala      DAP-II(A)-Z-ala-arg-arg            Catheps in B 1(A)-BZ-val-lys-lys-arg            Cathepsin B 1a(A 2-HCl)CBZ-arg-arg            Cathepsin B 1(A-diacetate)-N-CBZ-arg-arg-arg            Trypsin(A 3-HCl)-L-arg-arg            DAP III(A)-Z-gly-gly-arg            Anionic Trypsin, Plasminogen Activator,            Proinsulin Converting Enzyme(A)-pro-arg      DAP-I or Cathepsin C(A)-α-BZ-phe-val-arg            Thrombin(di-A)-L-cystine Oxytocinase(A)-γ-glutamyl            γ-Glutamyl Transpeptidase(A formate)-L-leu-gly-gly(A)-leu          Aminopeptidase(A)-BZ-arg-pro-gly-phe-phe-leu            Cathepsin D(A)-phe-pro-ala-met            Cathepsin B 1b(A)-glutaryl-gly-L-phe(A)-gly-pro      DAP-IV(A)-CBZ-pro-ala-gly-pro            Collagenase(A)-his-ser      DAP I or Cathepsin C(A)-N-CBZ-L-pro-L-phe-L-his-L-leu-L-leu-L-val-L-tyr-L-ser(A)-N-CBZ-gly-L-met            Renin(A)-glutaryl-ala-ala            Elastase(A)-BZ-arg-pro-gly-phe-phe-pro            Cathepsin D(A)-ala          Aminopeptidase B(A)-BZ-arg       Trypsin/Cathepsin B 1(A)-BZ-arg-gly-leu(A)-met(A)-BZ-arg-gly-tyr            DAP-I(A)-ser-tyr      Cathepsin C__________________________________________________________________________ 
    
     In the above, the designations constitute established abbreviations as follows: ala (alanine), arg (arginine), BZ (benzoyl, CBZ and Z (carbobenzoxy), gly (glycine), his (histidine), leu (leucine), lys (lysine), met (methionine), phen (phenylalanine), pro (proline), ser (serine), tyr (tyrosine), val (valine). To increase solubility rates, all of the substrates may, if desired, be converted to salts such as, for example, the hydrochloride, hydrobromide, acetate, or formate salts of the amino acids. 
     Each of the substrates, when exposed to its corresponding enzyme, is cleaved, the amino acid moiety being released or coupling with a suitable acceptor such as glycylglycine, to leave the fluorogenic primary amine (i.e., substrate (A), as identified above, in which the substituent for R 3  is a hydrogen atom). All of such fluorogenic aromatic amines have peak excitation and emission characteristics, when exposed to ultraviolet light, which are sufficiently close to those of an NADH-linked test (λex=340 nm; λem=465 nm) to permit fluorometric activity measurements using the same equipment and filters employed for such standard NADH tests. Specifically, such chromophores have peak excitation characteristics at a wavelength within the range of 320 to 380 nm and peak emission characteristics at a wavelength within the range of 420 to 480 nm. For example, if substrate (A) has methoxy groups as R 1  and R 2 , then the resulting chromophore will have a peak excitation wavelength of about 335 nm and a peak emission wavelength of about 445 nm. 
     In practicing the method of this invention, substrate is first dissolved in a sterile aqueous solution which preferably contains a suitable buffer to insure that the pH will be maintained at or near the optimum pH of the enzyme of interest. For example, where the enzyme to be measured is γ-glutamyl transpeptidase, the reaction may be measured over a broad range of pH values from about 7.5 to 9.0, a pH of 8.2 yielding maximum activity in the fluorometric assay system. The substrate solution is mixed with the sample (suspension or solution) and transferred to a suitable cuvet with any suitable fluorometer being used to measure front-surface fluorescence. The rate of production of the fluorogenic compound is directly proportional to the amount of transferase present in the sample. 
    
    
     The following examples are further illustrative of the invention: 
     EXAMPLE 1 
     Serum γ-glutamyl transpeptidase may be measured fluorometrically by utilizing γ-(L-glutamyl)-5-aminoisophthalic acid, dimethyl ester, hydrochloride salt, as the substrate. Such substrate has the structural formula: ##STR2## 
     The reagent solution contained 5 mM substrate, 55 mM glycylglycine, and 100 mM Tris buffer (pH 8.2 at 25° C.), the solution volume being 1.5 ml. The reagent solution was warmed to 37° C., sample was added (volume at 0.05 ml), the reactants were mixed and pumped into a flow-through cuvet. The rate of increase in fluorescence was then measured for a minimum of 4 minutes using a front-surface instrument (λex=365 nm; λem=465 nm). By such a procedure, the rate of change in fluorescence of the end product (5-aminoisophthalic acid, dimethyl ester) resulting from the hydrolysis of the substrate was measured, and the slope was calculated as the change in fluorescence per minute of reaction. 
     EXAMPLE 2 
     The results of serum samples tested in accordance with Example 1 were compared with the results of colorimetric assays run on the same patient samples, using GGTP reagent as marketed by Dade Division of American Hospital Supply Corporation and following the method set forth in the package instructions. To facilitate interpretation of data, the ΔF/min. was changed to International Units per liter (IU/L) by totaling IU/L and ΔF/min. and deriving a factor IU/ΔF. The sera was tested in two groups of 14, one group representing undiagnosed conditions and the other diagnosed conditions, and the following results were obtained: 
     
         ______________________________________Glutamyl TranspeptidaseActivity (IU/L)Sample           Fluorometric                        Colorimetric______________________________________1                47          452                52          513                12.5        174                85.4        795                113.4       1106                196.9       1957                14.5        178                14.5        189                345         34410               43.4        4811               48.8        7212               236.4       22513               212.3       19814               259.6       26015               80.9        8716 Metastatic Cancer            87.5        11917 Gastritis     89.5        9418 Dehydration   166.4       17219 Obst. Jaundice            23          2620 Colostomy     181         18421 Hepatomegaly  250.4       26422 Cancer of Bladder            146.7       14823 Jaundiced     164.0       16724 Hip Problem   215.4       20025 Hodgkins      14.7        1426 Chest Pain, Hypertension            62.8        4727 Pulm. Embolus 174.2       15028 Sarcodosis    193.5       178______________________________________ 
    
     The data demonstrate excellent correlation between the fluorometric method and the conventional colorimetric method for the determination of serum levels of γ-glutamyl transpeptidase. 
     EXAMPLE 3 
     The γ-(L-glutamyl)-5-aminoisophthalic acid, dimethyl ester, hydrochloride salt, used as the substrate in Example 1 may be prepared by mixing phthaloyl glutamic anhydride (13.2 g, 0.051 mole) and 5-aminoisophthalic acid, dimethyl ester (10.4 g, 0.050 mole) in 60 ml of dioxane, and stirring same at 55°-60° C. (bath temperature) for 1.5 hours. After evaporation of the solvent, the residue is then dissolved in 200 ml of methanol and hydrazine hydrate (7.5 g, 0.15 mole). The solution should then be filtered and allowed to stand at room temperature (2 days). A resulting white precipitate is then collected, washed with 100 ml of water and 25 ml of ethanol, agitated in 100 ml of 0.5 N hydrochloric acid, and filtered. The filtrate is treated with sodium bicarbonate to give a pH of 6.5 to 7.0, and the precipitate (8 g) is collected and dried. The hydrochloride salt may then be prepared by dissolving 1  gram of the glutamyl derivative in a solution of 0.3 ml of concentrated hydrochloric acid and 6 ml of methanol. After evaporation of the methanol, the solid is then dried under reduced pressure. 
     EXAMPLE 4 
     The following process may be used to prepare other 5-aminoisophthalic acid derivatives which may then be coupled to appropriate amino acid constituents as indicated. ##STR3## 
     Where an amide is to be formed, RNH 2  is substituted for ROH in this equation. In either event, the end product is then reacted with the particular amino acid desired in the appropriate form (as illustrated in Example 3 in connection with phthaloyl glutamic anhydride) to produce the final amino acid derivative of aminophthalic acid to be used as a substrate for determining transpeptidase and/or protease activity. 
     While in the foregoing we have disclosed the invention in considerable detail for purposes of illustration, it will be understood by those skilled in the art that many of these details may be varied without departing from the spirit and scope of the invention.