familiar with the determination under study (21),and is thus able to make effective judgements on what factors are likely to be important, and on the reliability of the results. ACKNOWLEDGMENT
The authors thank A. D. Kelmers for providing them with the purified tRNA solutions used in this work. We also wish to thank Miss Rae Lyn Athanacio, Oak Ridge ~~
(21) W. J. Murphy, ANAL.CHEM., 23, 937 (1951).
Associated Universities Student Trainee, University of Nevada, Reno, for her enthusiastic participation and assistance in this project during the summer of 1969; L. B. Rogers, Purdue University, for suggesting this study; and M. A. Kastenbaum, Tobacco Institute, Washington, D. C., for initiating it. RECEIVED for review December 2, 1970. Accepted February 17, 1971. Research sponsored by the U. S. Atomic Energy Commission and the National Institute of General Medical Science under contract with Union Carbide Corporation.
Fluorometric Methods for Analysis of Acid and Alkaline Phosphatase A. Vaughan, G . G . Guilbault,l and D. Hackney Department of Chemistry, Louisiana State University in New Orleans, New Orleans, La. 70122
The fluorometric properties of a series of 3-hydroxy-2naphthanilides (naphthol AS derivatives) were compared. The phosphate esters of these compounds were investigated as fluorogenic substrates for acid and alkaline phosphatase. All the substrates were compared with respect to lowest detectable concentration of enzyme, Michaelis constant, and stability. Naphthol AS-BI phosphate was used as a substrate for the determination of serum alkaline phosphatase by an initial rate method. The results obtained are compared to those obtained from a method employing phenophthalein phosphate as substrate. ALKALINE AND ACID PHOSPHATASE have been assayed by a variety of colorimetric and fluorometric methods. Colorimetric methods include those developed with the use of monophosphate esters of thymol phthalein ( I ) , phenolphthalein (2), and p-nitrophenol(3). More sensitive fluorometric methods have been developed with the use of monophosphate esters of 1- and 2-naphthol (4), flavone (5), and 7-hydroxycoumarin (6). In recent years, a series of naphthol AS phosphates: Naphthol AS (3-hydroxy-2-naphthanilide), naphthol AS-BI (6bromo-3-hydroxy-2-naphthyl-o-anisidine), naphthol AS-D (3-hydroxy-2-naphthyl-o-toluidide),naphthol AS-GR (3hydroxy-2-anthryl-o-toluidide), naphthol AS-LC (4’-chloro3-hydroxy-2‘,5’-dimethoxy-2-naphthanilide),naphthol ASMX (2 ’,4’-dimethyl-3-hydroxy-2-naphthanilide),and naphthol AS-TR (4’-chloro-3-hydroxy-2-naphthyl-o-toluidide) have been developed (7-10) for the cytochemical detection of acid To whom correspondence should be addressed. (1) C.M.Coleman, Clin.Chim. Acta, 13,401 (1966). (2) J. H.Wilkinson and A. V. Vodden, Clin. Chem., 12,701 (1966). ( 3 ) H. Neuman and M. Van Vreedendaal, Clin. Chim. Acta, 17, 183 (1967). (4) D.W.Moss, ibid., 5,283 (1960). ( 5 ) D.B. Land and E. Jackim, Anal. Biochem., 16,481 (1966). (6) G. G. Guilbault, S. H. Sadar, R. Glazer, and J. Haynes, Anal. Lett., 1, 333 (1968). (7) M. S. Burstone, J. Nut. Cancer Znst., 21, 523 (1958). (8) R. S. Barnett, R. Bressler, and A. M. Rutenberg, Anal. Rec., 124, 255 (1956). (9) A. M.Rutenburg, Ann. Histochim., 11, 139 (1966). (10)C.L.Rosales, J. M. Bennett, and A. M. Rutenburg, J. Histochem. Cytochem., 13, 11 (1965).
and alkaline phosphatase. In these cytochemical methods, the naphthol AS derivative liberated from its phosphate ester is coupled with an azo dye to give an intensely colored insoluble dye which serves as a marker for acid or alkaline phosphatase. Ackermann ( 1 1 ) showed that naphthol AS-BI, AS-MX, and AS-TR phosphates were the best substrates for the cytochemical detection of alkaline phosphatase. The intensity of the color produced by these azo dyes has been applied (12, 13) to the determination of alkaline phosphatase immobilized in polyacrylamide gel. Recently, Johnson (14) developed a fluorometric method for the estimation and detection of serum alkaline phosphatase using naphthol AS-MX phosphate as substrate. In the present study, the work of Johnson was extended to cover a wide range of naphthol AS phosphates as substrates for both acid and alkaline phosphatase. Alkaline phosphatase was determined by measuring the rate of formation of the fluorescent naphthol AS. The amount of enzyme is calculated by converting the rate into pmoles of naphthol AS-BI liberated per minute by means of a conversion factor. In acid solution, naphthol AS derivatives are nonfluorescent but calibration curves for acid phosphatase can be obtained by quenching the enzyme reaction after a fixed time with alkali which develops the fluorescence of the liberated naphthol AS. This procedure allows the determination of acid phosphatase. The fluorescent properties of naphthol AS, AS-BI, AS-D, AS-GR, AS-LC, AS-MX, and AS-TR were studied and the phosphate esters of these naphthol AS derivatives were studied as substrates for acid and alkaline phosphatase. Naphthol AS-BI phosphate was found to be the best substrate for both enzymes. It gave the highest rates of hydrolysis and the best K, values of all the substrates investigated. Naphthol AS-BI phosphate was applied to the determination of human serum alkaline phosphatase. The serum alka(11) G. A. Ackermann, Lab. Invest., 11,563 (1962). (12) P. van Duijn, E. Pascoe and M. van der Ploeg, J. Hisrochem. Cytochem., 15, 631 (1967). (13) 2. Lokda, M.van der Ploeg and P. van Duijn, Histochemie, 11, 13 (1967). (14) R. B. Johnson, Clin.Chem., 15, 108 (1969). ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971
721
mpoJH' 9 +
Br
CO
H,O
Phosphataset
. NH
OCH3
Naphthol AS-BI Phosphate (Nonfluorescent)
OCH3
Naphthol AS-BI (Fluorescent) line phosphatase values were obtained first from a standard procedure (15)in which phenolphthalein phosphate was used as a substrate. The initial rate of reaction was determined when naphthol AS-BI phosphate was used as substrate. A plot of initial rate 6s. units of alkaline phosphatase (from phenolphthalein method) is linear and the serum alkaline phosphatase value can be determined directly from this plot. Alternatively, the rate can be converted by means of a factor into naphthol AS-BI International Units (pmoles naphthol AS-BI min-l 1.-' serum). The procedure was found to be accurate, reproducible, and directly applicable to the analysis of alkaline phosphatase in normal, jaundiced, or haemolyzed human serum. EXPERIMENTAL
Apparatus. Fluorescent wavelength measurements and rate determining measurements of serum alkaline phosphatase were made with an Aminco-Bowman spectrofluorometer which utilizes grating monochromators and a xenon arc lamp. A constant temperature was maintained with a thermoelectric cooler. All other rate measurements were made with an Aminco fluoromicrophotometer filter instrument using a Wratten 405 primary filter and Wratten 65A/58 combination secondary filters. A constant temperature of 25 "C was maintained with a circulating water bath. Enzymes. Stock solutions, 1 mg ml-I were prepared from: (a) wheat germ acid phosphatase-nominal activity 0.4 unit (Sigma Chemical Co., St. Louis-one unit will hydrolyze 1 pmole p-nitrophenyl phosphate min-' mg-I at pH 4.8 and 25 "C). (b) Calf intestinal alkaline phosphatasenominal activity 10 units mg-'. (Sigma. One unit will hydrolyze 1 pmole p-nitrophenyl phosphate min-' at pH 10.4 and at 25 "C). These solutions were diluted as required. Buffers. Citrate buffer, O.lM, pH 5.5, was prepared by dissolving sodium citrate in water and adjusting the pH with hydrochloric acid. Tris(hydroxymethy1)amino methane buffer, 0. lM, pH 9.0, was prepared by dissolving the pure compound (Sigma) in water and adjusting the pH with hydrochloric acid. 2-Amino-2-methylpropanol buffer, O.lM, pH 9.8 at 25 "C, was prepared. by dissolving the pure compound (Sigma) in water and adjusting the pH with hydrochloric acid. Naphthol AS Phosphates. The dihydrogen phosphate esters of naphthol AS-BI (Isolab, Akron, Ohio), and AS-GR, AS-MX, and AS-TR (Sigma), were dissolved as 10-2M solutions in methyl cellosolve. The dihydrogen phosphate esters of naphthol AS, AS-D, and AS-LC which were prepared by the method shown below were also dissolved as 10+M solutions in methyl cellosolve. (15) A. L.Babson, Clin. Chem., 11,789 (1965).
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ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971
Preparation of Naphthol AS, AS-D, and AS-LC Phw. phates. Naphthol AS ( 5 g) was dissolved in dry pyridine (100 ml) which had been freshly distilled from barium oxide. This solution was added slowly with stirring to freshly distilled phosphorus oxychloride (1.7 ml) at 0 "C. After 1 hour the reaction mixture was poured over ice (100 g) and stirred for 5 minutes. The resulting mixture was adjusted to pH 8.0 with concentrated sodium hydroxide and then evaporated at 35 "C to dryness under vacuum. The brown solid was extracted with acetone and filtered. The solid remaining was dissolved in water (40 ml), and hydrochloric acid was added to precipitate the phosphate ester which was filtered off and dried. This solid was dissolved in water (40 ml), the pH adjusted to 7.0, and the solid was extracted with 4 : l hexane-butanol mixture (3 X 30 ml) and then with diethylether (2 X 30 ml) to remove colored impurities. The phosphate ester was finally precipitated with hydrochloric acid, filtered, washed with dilute hydrochloric acid, and dried over phosphorus. pentoxide under vacuum. Yield = 40%. The melting points were naphthol AS phosphate, 180-183 "C (decomposition); naphthol AS-D phosphate, 145-148 "C; and naphthol AS-LC phosphate, 182-184 "C. C, H, N, and P found for the naphthol Analysis. The AS-, AS-D, and AS-LC phosphates by elemental analysis agreed with those calculated within experimental error. Naphthol AS-BI-Working Standard. Naphthol AS-BI (0.0372 g) was dissolved in methyl cellosolve (100 ml) and used to standardize the enzyme procedures. This solution which contains 0.1 pmole naphthol AS-BI in 0.1 ml was stored in the dark to prevent photodecomposition. Determination of Acid Phosphatase. Into a 5-ml volumetric flask, place accurately measured volumes of 10-2M naphthol AS-BI phosphate (0.1 ml), and pH 5.5 citrate buffer (3.0 ml). Place in a water bath at 25 "C for 10 minutes and add acid phosphatase (0.20 ml or other convenient volume). Quench the reaction after exactly 3 minutes with 0.2M potassium hydroxide (0.5 ml). Measure the fluorescence (F)in a 1-cm cell (Aex = 405 nm, A,, = 515 nm). Also, measure the fluorescence (F,) of a blank that has been taken through the procedure but to which no enzyme has been added. A solution of working standard, 0.1 ml, is taken through the procedure in place of the phosphate ester and the fluorometer set to read 10 Fluorescence Units. The acid phosphatase activity expressed as pmole naphthol AS-BI mindl mg-I is calculated from the expression ( F - FJ100 X reaction time X mg enzyme taken. Determination of Calf Intestinal Alkaline Phosphatase. In a fluorescence cell, place accurately measured volumes of pH 9.0 tris buffer (3 ml) and 10-ZM naphthol AS-BI phosphate (0.1 ml). Add alkaline phosphatase (0.1 ml or other convenient volume) and measure the rate of change in fluorescence with time (AFimin-l). A blank rate determined using no enzyme should be zero. The activity of the enzyme can be obtained using the method described for serum alkaline phosphatase. Determination of Human Serum Alkaline Phosphatase. In a fluorescence cell, place accurately measured volumes of pH 9.8, 2-amino-2-methyl propanol buffer (3 ml) and 10-2M naphthol AS-BI phosphate (0.1 ml). Serum (0.1 ml) is added and the rate of change in fluorescence is measured (Aex = 405 nm; A,, = 515 nm). The amount of alkaline phosphatase can be determined by two methods. METHOD1. A plot of rate us. enzyme activity determined by an alternative method (in this case the method described by Babson (15) in which phenolphthalein phosphate is used as substrate) gives a straight line. The rate can then be converted directly into phenolphthalein international units where the activity is expressed as pmole phenolphthalein min-' 1.-' serum. The serum alkaline phosphatase values expressed as pmole phenolphthalein min-' 1.-1 serum, can be
Table I. Comparison of Fluorescent Properties of Naphthol AS Derivatives at pH 9.0 Residual fluorescenceb of Wavelength, nm Fluorescencea phosphate Naphthol Excitation Emission coefficient ester AS 388 516 2.2 x 105 0.133 AS-BI 405 515 2.5 x 105 0.11 AS-D 388 515 6 . 6 X 106 0.210 AS-GR 3880 488c AS-GR 26. Id 388 488 1 . 3 x 106 Phosphate 2.8 X lo6 0.09 AS-LC 388 522 2.6 X 106 0.076 AS-MX 388 512 3.1 x 105 0.014 AS-TR 388 512 Fluorescence of hydrolyzed substrate divided by the concentration of original substrate in M . Value for quinine sulfate in 0.1N sulfuric acid at A, = 350 nm and A,, = 450 nm was 2.5 X 106. b Fluorescence of 3.2 X 10-4M naphthol AS phosphates at pH 9.0. e In methyl cellosolve. d 3.5 x 1 0 - 5 ~ . . . I
Table 11. Comparison of Naphthol AS Phosphates as Substrates for Acid Phosphatase Lowest pMole detectable Naphthol Naphthol amount phosphate AS min-Ia (units)* K, AS 4 x 10-4 2 x 10-4 ... AS-BI 6.8 x 2 x 10-5 1.5 x 10-4 AS-D Very slow reaction AS-LC 8 X 8x 1.4 x 10-4 AS-MX 2.3 x 10-3 6x 1.8 x 10-4 AS-TR 7.4 x 10-4 8X 2.5 x 10-4 Measured in a solution containing 5 X 10-3 mg acid phosphatase. * 1 Unit hydrolyzes 1 pnole naphthol AS phosphate min-1 mg-1 at 25 "C.
obtained directly by multiplying the rate by 480. The instrument was calibrated each day as shown below. METHOD2. The rate can be converted into naphthol AS-BI units by calibrating the instrument with a standard solution of naphthol AS-BI. In all experiments, the instrument was calibrated with the naphthol AS-BI working standard (0.1 ml) in buffer solution (3.0 ml) to give a fluorescence reading of 10 F.U. This is the fluorescence value of 0.1 pmole naphthol AS-BI. The units of alkaline phosphatase can be expressed as pmoles naphthol AS-BI min-1 1.-' serum by multiplying the rate by 100. RESULTS AND DISCUSSION Fluorescence of Naphthol AS Derivatives. The effect of pH upon the fluorescence of the naphthol AS derivatives (3.2 X 10-6M) is shown in Figure 1. At pH < 6, these compounds are nonfluorescent and precipitate from solution. At pH = 10, a limiting fluorescence is reached. Naphthol AS-D and AS-TR were found to have the strongest fluorescence. Naphthol AS-GR had no fluorescence in aqueous solution but its phosphate ester had an intense green fluorescence. The fluorescent properties of the naphthol AS derivatives are shown in Table I. All the phosphate esters were found to be stable when stored at 3 "C in the dark, and only slight decomposition of the phosphates occurred over a period of several months. In pH 9.8 buffer solution, the phosphate ester was
I
A
/x aJ
V E
aJ V
g
1.0-
3
i i Q,
5 .c
-
c)
0.5-
Q,
CK
0'
I
I
I
7
8
9
I
10
PH Figure 1. Effect of pH on fluorescence of naphthol AS derivatives (3.2 X 10-6 M ) A . Naphthol AS-D D , Naphthol AS-MX B. Naphthol AS-TR E. Naphthol AS-BI C. Naphthol AS-LC F. Naphthol AS unstable, thus preventing the use of a combined substratebuffer solution except over short periods of time (1 day). Comparison of Naphthol AS Phosphates as Substrates for Acid and Alkaline Phosphatase. The phosphate esters of the naphthol AS derivatives described above were compared as substrates for acid and alkaline phosphatase with respect to stability, spontaneous hydrolysis, rate of enzymatic reaction at optimal pH, Michaelis constant for the enzyme-substrate reaction, and the lowest detectable concentration of enzyme. Table I1 makes a comparison beACID PHOSPHATASE. tween naphthol AS phosphates as substrates for wheat germ acid phosphatase. Naphthol AS and AS-D phosphates were only slowly hydrolyzed. The enzyme reacted optimally at pH 5.5 for all substrates. No blank hydrolysis of the substrates was observed under the condition of the reaction. The calibration curves shown in Figure 2 were obtained by plotting the number of pmoles of naphthol AS liberated in 3 minutes us. the amount of acid phosphatase taken. The enzymatic reaction was quenched by adding excess potassium hydroxide which also developed the fluorescence of the liberated naphthol AS. The fluorescent values were constant even in the presence of a large excess of potassium hydroxide. Lower amounts of enzyme can be determined by increasing the reaction time. Naphthol AS-BI phosphate was found to be the best substrate for wheat germ acid phosphatase. Table I11 shows a comparison ALKALINE PHOSPHATASE. between naphthol AS, AS-BI, AS-D, AS-GR, AS-LC, ASMX, and AS-TR phosphates as substrates for calf intestinal alkaline phosphatase. When calf intestinal alkaline phosphatase was used, the substrates were found to react optimally at pH 9.0. Naphthol AS-GR phosphate which is fluorescent produced a nonfluorescent product when hydrolyzed. The rate of hydrolysis was followed by measuring the decrease in fluorescence with time. Because of the high initial fluorescence, this decrease in fluorescence could not be measured accurately and linear rates were obtained only when a low substrate concentration (3 X 10-6M) was used. It was previously shown (16) that calf-intestinal alkaline phosphatase could be determined over a wide range of concentrations using naphthol AS-BI phosphate as substrate. (16) G. G. Guilbault and A. Vaughan, Anal. L e f t . ,3, 1 (1970). ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971
723
's E
0.055
1 -
0.033
0.017 -
5
15
10
20
Units Acid Phosphatase x IO4 Figure 2. Comparison of naphthol AS phosphates as substrates for acid phosphatase A. B. C. D. E.
Naphthol AS-BI phosphate Naphthol AS-MX phosphate Naphthol AS-TR phosphate Naphthol AS-LC phosphate Naphthol AS phosphate ~
~~
~
~~~~~
Table 111. Comparison of Naphthol AS Phosphates as Substrates for Alkaline Phosphatase Lowest pMole detectable Naphthol Naphthola concentration, phosphate AS min-1 units5 K, AS 8X 5 x 10-4 1.05 X AS-BI 5 x 10-5 2.2 x 10-5 1.55 x 10-3 AS-D 6.9 x 10-4 5 x 10-5 2.3 x 10-4 AS-LC 6 9 x lo-' 7 x 10-5 1.8 x 10-5 AS-MX 6.9 x 10-4 5 x 10-6 2.8 x 10-4 AS-TR 3.9 x 10-4 5 x 10-5 6 x lo-' a pMole naphthol AS liberated min-1 by 5 X mg alkaline phosphatase. 1 Unit hydrolyzes 1 pmole naphthol AS phosphate min-l mg-l at 25 "C. Naphthol AS-BI and AS-D phosphates were found to be the best substrates for alkaline phosphatase. Naphthol ASBI phosphate was deemed to be the better of these two substrates because it had a lower background fluorescence and because it was available commercially (Isolab, Akron, Ohio). The limit of detection of alkaline phosphatase was found to be similar for all the substrates. The naphthol AS-BI phosphate is stable indefinitely, as is the naphthol AS-BI standard. Naphthol AS-BI phosphate was applied to the determination of serum alkaline phosphatase. The optimum pH for serum alkaline phosphatase was found to be 9.8. Figure 3 s h o w the correlation between the serum alkaline phosphatase obtained from the naphthol AS-BI method and the alkaline phosphatase values obtained from a method employing phenolphthalein phosphate. The graph shows good agreement between the two methods over a wide range of serum values. If the serum values are expressed as pmole phenolphthalein liberated min-11.-1 serum, then normal serum has 9-35 units alkaline phosphatase. If the serum values are expressed as pmoles naphthol ASBI liberated min-1 1.-' serum, then normal serum has 1.8-7.0 724
ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971
Naphthol A S - 8 1 units
Figure 3. Correlation between results obtained with naphthol AS-BI phosphate method and phenolphthalein method. Blood serum (100-p1sample) used Table IV. Analysis of Serum Alkaline Phosphatase Units found Units taken phenolphthalein method naphthol AS-BI methodn-O 18 18.2, 17.2 20 19.7, 19.2 24.1, 23.1 24 28 28.0, 28.5 32 33.0, 32.5 37 39.9, 37.1 41 41.8, 41.3 44 42.0, 38.0 48 48.0, 49.2 53 52.6 64.0 65 74 78.1 86 89.2 95 96.5 105 108.2 110 111 134 129 Results expressed as pmoles phenolphthalein min-l serum. b Each value is the average of two measurements. Relative error, &5%.
5
units. Table IV shows some of the results obtained with serum samples. The results are expressed in both cases as phenolphthalein International Units. Over 200 serum samples were analyzed and complete correlation between the two methods was found. The method is applicable to normal, jaundiced, or haemolyzed serum. Methyl cellosolve was found to be noninhibitory to the enzymic hydrolysis at concentrations less than 5 %. CONCLUSIONS
Seven different naphthol AS phosphates were investigated as fluorogenic substrates for acid and alkaline phosphatase. Naphthol AS-BI phosphate was found to be the best substrate for these enzymes, This substrate was applied to the determination of serum alkaline phosphatase and the results compared to those obtained when phenolphthalein phosphate was used as substrate.
RECEIVED for review April 20, 1970. Accepted February 22, 1971. Financial assistance of the National Institutes of Health (Grant ES-00426-03) is gratefully acknowledged.
