Continuous monitoring of prostatic acid phosphatase using self-indicating substrates

Continuous monitoring of prostatic acid phosphatase using

self-indicating substrates

Klaus Lorentz*

Institut fur Klinische Chemie, Medizinische Universitat zu Lubeck, Ratzeburger Allee 160, 23538 Lubeck, Germany

Received 18 March 2002; received in revised form 28 June 2002; accepted 17 July 2002

Abstract

Background: The continuous measurement of acid phosphatase (EC 3.1.3.2) activity in serum represents an analytical task

not yet sufficiently accomplished. Methods: Introducing two novel substrates—2-chloro-4-nitrophenyl phosphate (CNP-P),

which was preferred, and 4-nitronaphthyl-1-phosphate (NN-P)—an alternative assay to measure enzymatic activity was

developed and compared with a modification of Hillmann’s method (azo coupling of released naphth-1-ol with a diazonium

compound). Apart from different substrate concentrations of 2-chloro-4-nitrophenyl phosphate, 4 mmol/l, and naphthyl-1-

phosphate (N-P), 8 mmol/l (with Fast Red TR, 5 mmol/l), respectively, following identical conditions were selected: Citrate, 50

mmol/l, pH 5.75; pentane-1,5-diol, 150 mmol/l; tartrate, 60 mmol/l; 37 jC. Results: Whereas intensity and stability of the azo

dye unpredictably depend on the albumin concentration of the sample, the direct test with 2-chloro-4-nitrophenyl phosphate

resisted sample interferences, showed no intrinsic hydrolysis by albumin, relied on stable reagents and proved superior in

sensitivity, precision and ease of handling. In measuring prostatic phosphatase, the proposed procedure closely correlated with

Hillmann’s method. The preliminary 0.95-reference intervals for adults were 1.2–3.9 kU/l and 5.8–14.8 U/l for total activity,

respectively. Conclusions: The direct assay of the enzyme is suited as an economic, rapid and robust method for mechanized or

manual use.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Prostatic acid phosphatase; Direct assay; 2-Chloro-4-nitrophenyl phosphate; 4-Nitronaphthyl-1-phosphate; Method comparison

1. Introduction

Activity determinations of acid phosphatase (ortho-

phosphoric ester hydrolase, acid optimum, EC 3.1.3.2)

in serum have been mainly continued to assess primary

and metastatic bone disease, especially by the measure-

ment of the tartrate-resistant isoenzyme (band 5) in

serum [1]. The estimation of tartrate-sensitive prostatic

acid phosphatase (PAP) has been undoubtedly super-

seded by that of prostate-specific antigen in the detec-

tion of localized prostatic cancer [2,3], but the enzyme

0009-8981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0009 -8981 (02 )00295 -4

Abbreviations: CNP-P, 2-chloro-4-nitrophenyl phosphate; HSA,

human serum albumin; Km, Michaelis constant; NAC, 2- and 4-(2-

methyl-4-chlorophenylazo)-naphth-1-ol; NbAC, bis-2,4-(2-methyl-

4-chlorophenylazo)-naphth-1-ol; N-P, naphthyl-1-phosphate; PAC,

2-methyl-4-chloro-6-(2-methyl-4-chlorophenyl-azo)-phenol; PAP,

prostatic acid phosphatase; RSD, relative standard deviation; S.D.,

standard deviation; Vmax, maximal velocity.

* Present address: Hugo-Kauffmann-Str.7, D-83209 Prien,

Germany. Tel.: +49-8051-2072; fax: +49-8051-969032.

www.elsevier.com/locate/clinchim

Clinica Chimica Acta 326 (2002) 69–80

has still retained its diagnostic value in monitoring

tumour progression and assessing metastatic spread

[4]. Since immunological procedures and those meas-

uring catalytic concentrations are of equal clinical

value [2,5], the latter should be preferred for economic

reasons including self-indicating reactions which allow

a mechanized measurement.

Although continuous monitoring has been early

achieved by azo coupling of naphth-1-ol [6] liberated

from naphthyl-1-phosphate (N-P), the inherent limi-

tations of the indicator reaction have caused various

modifications to improve the accuracy of measure-

ment [7–12]. Therefore, the following report based

both on enzyme properties and chemical reactions

presents a critical evaluation of this method, and on

the other side, it introduces two novel substrates, viz.

2-chloro-4-nitrophenyl phosphate (CNP-P) and 4-

nitronaphthyl-1-phosphate (NN-P), for the continuous

monitoring of acid phosphatase activity. Although

CNP-P seems to be preferred by the tartrate-resistant

fraction, it is also well suited for the determination of

prostatic phosphatase, for which a method was devel-

oped.

2. Materials and methods

2.1. Instruments

Calibrated glassware corresponding to NBS class A

and SMI-micropettorsR from ScientificManufacturing

Industries (Richmond, CA 94710) were used through-

out. The double-beam spectrometers Lambda 12 Per-

kin-Elmer (Norwalk, CT 06859) and Uvikon 943

Kontron (Zurich, Switzerland) served for method

development, but we applied an EPOS 5060 analyzer

and the spectral line photometer 1101 M both from

Eppendorf (Hamburg, Germany) for assays at 405 and

492 or 546 nm. The latter was equipped with a

computerized multi-cell positioner Megalyzer (MFT,

Hamburg, Germany), enabling the intermittent meas-

urement of absorbance changes of 39 assays in one run.

Photometry was always done using thermostatted 10-

mm light path cuvettes, and pH values were determined

with the glass electrode 405-S7 Ingold (Steinbach,

Germany) attached to a pH meter pH 531 WTW

(Weilheim, Germany) and calibrated with reference

buffers of Merck (Darmstadt, Germany).

2.2. Chemicals and specimens

Fast Red TR (diazotized 2-methyl-4-chloro-amino-

benzene), the purity of which we confirmed by thin

layer chromatography, was from Sigma (St. Louis,

MO 63178). Pentane-1,5-diol came from Fluka

(Buchs, Switzerland), human serum albumin (HSA)

and transferrin from Behringwerke (Marburg, Ger-

many), g-globulin and most surfactants from Serva

(Heidelberg, Germany). Roche Diagnostics (Man-

nheim, Germany) supplied Thesit, the control materi-

als (Precinom UR, Precipath UR) and their test kit

‘Acid Phosphatase’ for method comparison. We pur-

chased basic aluminum oxide from ICN Pharmaceut-

icals (Eschwege, Germany). All other chemicals

including naphthyl-1-phosphate, which met the crite-

ria of acceptance [13] came from Merck. We purified

naphth-1-ol by sublimation and 2-chloro-4-nitrophe-

nol by crystallization [14]. 2-Chloro-4-nitrophenyl

phosphate [15] was used as the biscyclohexylammo-

nium monohydrate (Mr 469.9) like 4-nitronaphthyl-1-

phosphate prepared via 4-nitronaphth-1-ol adapting

currently employed methods [16–18] as follows.

4-Nitronaphth-1-ol [16]: Naphthyl-1-amine was

treated with acetic anhydride in glacial acetic acid

followed by nitration at 15–20 jC in the presence of

sodium nitrite, 10 mmol/mol of pure nitric acid [17].

The brownish solid, after being washed with acetic

acid, 5 mol/l was refluxed in an 30% ethanolic

solution of potassium hydroxide, 2 mol/l. The alcohol

slowly distilling off was replaced by water, and

heating was continued, until the release of ammonia

ceased. The 2-nitronaphth-1-ol potassium salt sepa-

rated on cooling. The 4-isomer was purified with

charcoal, acidified with concentrated hydrochloric

acid, freed from residual 2-isomer by steam distilla-

tion in the presence of ascorbic acid, 10 mmol/l, and

cooled in ice to yield a suspension of crude 4-nitro-

naphth-1-ol. Its filtrate was treated again with charcoal

in 1% ascorbic acid and evaporated. The combined

solids crystallized from ethyl acetate in bright yellow

needles (m.p. 165 jC, yield 60%) which are suited for

syntheses. For spectrometry, the crystals were further

purified under nitrogen by passing a methanolic

solution through ascorbic acid-impregnated basic alu-

minum oxide (10 mg/g) and drying in vacuo (m.p.

166 jC). 2-Nitronaphth-1-ol is less sensitive to oxi-

dation, crystallizes well from acetone or ethyl acetate

K. Lorentz / Clinica Chimica Acta 326 (2002) 69–8070

(m.p. 128 jC, yield 24%) and needs no chromato-

graphic purification.

4-Nitronaphthyl-1-phosphoryl dichloride [18]: To

a vigorously stirred cooled (� 15 jC) mixture of

tetrahydrofuran (42 ml), freshly distilled phosphorus

oxychloride (35 ml = 380 mmol), phosphorus penta-

chloride (50 mg) and well-ground sodium chloride

(200 mg), a solution of 4-nitronaphth-1-ol (24.2

g = 127 mmol) in tetrahydrofuran (50 ml) and anhy-

drous pyridine (11.3 ml = 140 mmol) was added

within 30 min under nitrogen, while the temperature

was allowed to rise to � 5 jC. Stirring was con-

tinued for another 30 min until no further precipita-

tion of pyridinium hydrochloride occurred. The filter

cake was washed thrice with 15 ml of cold tetrahy-

drofuran, and the filtrate was evaporated to a pale

red syrup at � 5 jC with 0.005 Torr to remove the

solvent, excess pyridine and phosphorus oxychloride.

4-Nitronaphthyl-1-phosphate, biscyclohexylam-

monium salt: The phosphoryl dichloride proved ex-

tremely labile, so all subsequent steps had to be

performed below � 15 jC with correspondingly

cooled anhydrous solvents. The oily residue was

stirred with 50 ml of diethyl ether. Another 500 ml

containing 2 ml of sulphuric acid, 50%, were cau-

tiously added in portions of about 50 ml per day. The

solution, clear after two additional days, was dec-

anted from the insoluble pyridinium sulphate, suc-

cessively dried with sodium sulphate and deacidified

with some sodium bicarbonate. Finally, 300 ml of

cyclohexylamine diluted with 2000 ml of diethyl

ether were slowly added, and the stirred white

suspension was allowed to reach 0 jC. The amor-

phous filter cake was washed twice with 100 ml of

diethyl ether and crystallized from absolute ethanol.

This treatment gave 25.2 g (yield 40%) of colourless

monohydrate (found: C, 55.2; H, 7.6; N, 8.5; P, 6.4.

C22H34N3O7P�H2O (Mr 485.5) requires C, 54.42; H,

7.47; N, 8.65; P, 6.38). The hygroscopic substrate

easily undergoes hydrolysis and must be stored over

P4O10 in a desiccator at 5 jC.Alternatively, the compound was converted into

the disodium salt by saturated sodium perchlorate in

ethanolic solution. However, the product is still more

hygroscopic and decomposes rapidly at 5 jC (found:

C, 34.3; H, 2.8; N, 3.9; P, 9.1. C10H6Na2NO6P�2H2O

(Mr 349.2) requires C, 34.40; H, 2.89; N, 4.01; P,

8.87).

Method development was accomplished with hu-

man PAP (CRM 410 from the National Institute of

Biological Standards and Control, Potters Bar, UK) and

sera from the routine laboratory 5–6 h after blood

clotting and partly spiked with highly active super-

natants of prostate and liver homogenates [19]. Icteric,

lipaemic and haemolytic samples were used for inter-

ference studies. For the establishment of preliminary

reference intervals, we selected 80 Sera from appa-

rently healthy males between 25 and 65 (median 42)

years of age without medication, fasted for 10 h and

fulfilling the following conditions: creatinine < 110

Amol/l, g-glutamyltransferase < 28 U/l (25 jC), protein58–73 g/l and glucose < 6.1 mmol/l.

2.3. Reagents

All reagents were made up with bidistilled deion-

ized water, and the calculated weights of pentane-1,5-

diol and Fast Red TR were corrected for the specifi-

cation of their contents. For studies on azo dye

formation, Fast Red TR reagent and naphth-1-ol, 50

Amol/l, were prepared 10–20 min before use, the

latter from an ethanolic stock solution, 0.5 mol/l,

stored under nitrogen for at most 1 day. The following

solutions (1–3 for chromogenic tests; 1, 2 and 4–6

for the comparison method) were selected on the basis

of experiments described later.

(1) Buffer/effectors: L-(+)-tartrate, 66 mmol/l; pen-

tane-1,5-diol, 165 mmol/l; citrate, 55 mmol/l;

adjusted to pH 5.95 at 37 jC.(2) Buffer/effectors: L-(+)-tartrate, 66 mmol/l; pen-

tane-1,5-diol, 165 mmol/l; citrate, 55 mmol/l;

adjusted to pH 4.35 at 37 jC.(3) Substrate: 2-chloro-4-nitrophenyl phosphate (al-

ternatively 4-nitronaphthyl-1-phosphate), 48

mmol/l, in solution 2.

(4) Buffer/effectors/surfactant: Pluronic F 68, 0.61 g/

l, in solution 1.

(5) Diazonium reagent: Fast Red TR, 6.1 mmol/l, in

solution 4.

(6) Substrate: naphthyl-1-phosphate, 96 mmol/l, in

solution 2.

All solutions were stored at 0–5 jC. Solutions 1, 2and 4 are stable unless there is microbial growth.

Solutions 3 and 6 may be stored for 1 week, and

K. Lorentz / Clinica Chimica Acta 326 (2002) 69–80 71

solution 5 must be prepared 10–20 min before use.

For determining total activity L-(+)-tartrate is omitted

from solutions 1 and 2, but in this case, pH must be

adjusted anew to yield pH 5.75 in the assay.

2.4. Procedures

Table 1 presents the analytical system for meas-

urement with CNP-P or NN-P. The comparison

method follows the same protocol, but solution 1 is

replaced by solution 5, and solution 3 by solution 6.

The corresponding factor calculating catalytical con-

centrations in serum is 1150.6 at 492 nm (derived

from e, Table 4). All measurements were carried out

in triplicate at 37F 0.1 jC except procedures to

calculate molar absorption coefficients which were

done in quintuple based on readings between A0.500

and A0.900. Using fixed-time procedures to describe

enzyme characteristics, all assays were normalized to

the same pH of 11.0 by addition of four volumes of

glycine buffer, 0.25–0.5 mol/l, pH 12.6, avoiding

alkaline hydrolysis of the substrates. Concentrations

always refer to the assay volume, if not otherwise

stated, and we used the final assay conditions—

except the variable—for univariate optimization.

Kinetic constants were determined by Woolf–Hanes

linear transformation plots from data of seven sub-

strate concentrations (0.125–8.0 mmol/l), and stabil-

ity studies of reagents were continued for 35 days at

5 jC.The formation of azo dyes from naphth-1-ol, 50

Amol/l, and Fast Red TR, 0.8–5 mmol/l, was inves-

tigated in the presence of HSA and nine nonionic

detergents (in two concentrations each between 0.2

and 5 g/l) at pH 5.25 and 5.75. The colour develop-

ment was initiated by addition of water (for the blank)

or naphth-1-ol, 50 Amol/l, and followed from 2 to 30

min after start by recording spectra in the range 330–

590 nm (with readings at 380-390-400-405-420-520-

540-546 nm). The blank reaction, producing a phenol

azo compound (PAC, I), was measured against the

reagent without Fast Red TR, the developing naphthol

azo compounds (NAC, II; NbAC, III) were read

against the blank, thus subtracting PAC absorbance.

We derived preliminary reference intervals from

nonparametric 0.95-interfractile intervals [20], and

compared the methods by linear regression obeying

conditions reported by Stockl et al. [21]. To avoid

outliers, only means from five determinations show-

ing less than 5% relative standard deviation (RSD)

were accepted.

3. Results and discussion

3.1. Enzyme characteristics relevant in analysis

The choice of an adequate pH value pertinent for

both the enzymatic and the indicator reaction is the

basic step in developing a continuous method. PAP

demonstrated the expected fairly broad optimum

around pH 5.5 without distinctive differences of the

central values of maximal activity for the three sub-

strates: N-P 5.6F 0.10, CNP-P 5.5F 0.25, NN-P

5.4F 0.15 (Fig. 1). The activity did not vary with

citrate concentrations between 50 and 200 mmol/l

Table 1

Assay conditions and protocol for determining prostatic phosphatase activity with 2-chloro-4-nitrophenyl phosphate at 37 jC

Pipette Volume (Al) Measurement conditions and final concentrations

Solution 1 (37 jC) 250 pentane-1,5-diol 150 mmol/l

citrate 50 mmol/l

L-(+)-tartrate 60 mmol/l

Sample 25 volume fraction (v/V) 0.0909 (1:11)

Mix well without removing any of the mixture and incubate to attain 37 jCSolution 3 25 2-chloro-4-nitrophenyl phosphate 4 mmol/l pH 5.75

Mix well, wait for 15 s and record the increasing absorbance at 405 nm for 300 s. Calculate DA/Dt, the average change of absorbance per

minute. No correction for blank reactions is needed in routine measurement procedures.

Using micromolar absorption coefficients (e, l� 10� 6 mol� 1 mm� 1) 1182.2� 10� 6 for sera or 1356� 10� 6 for diluted seminal plasma, the

light path length (l ) 10 mm and the inverse volume fraction (V/v) 11, the catalytic concentration is calculated by U/l =DA/min�V/(v� e� l)

Serum: U/l =DA/min� 930.5 Diluted seminal plasma: U/l =DA/min� 811.2� dilution ratio

Note: With 4-nitronaphthyl-1-phosphate at 436 nm, the factor for serum is 890.6.


with equal values for citrate and acetate. However, in

phthalate, 50 mmol/l, reaction rates were less by 10%

for N-P and by about 5% for NN-P and CNP-P. All

further experiments were, therefore, carried out with

citrate, 50 mmol/l.

Although alcohols preferentially stimulate the

erythrocytic isoenzyme [22], their transphosphoryla-

tion also enhanced, albeit less, the activity of PAP,

pentane-1,5-diol proving most powerful. With CNP-P,

the enzyme is completely activated at 150 mmol/l,

while NN-P needs 200 mmol/l and N-P 250 mmol/l

for maximum activity (Fig. 2A). Equimolar concen-

trations of butane-1,4-diol were by 20% less effective,

but, observed with all substrates, a mixture of pen-

tane-1,5-diol and butane-1,4-diol, 150 mmol/l each,

was equivalent to pentane-1,5-diol, 200 mmol/l.

The response to L-(+)-tartrate, a competitive inhib-

itor of prostatic and lysosomal isoenzymes, was rather

uniform for the tested aryl phosphates (Fig. 2B).

Choosing 60 mmol/l, the residual activity ranged

between 0.06 for both CNP-P and NN-P and 0.09

for N-P because of its higher substrate concentration.

Thus, 120 mmol/l was necessary to attain the same

inhibition of 94% with N-P.

Apart from excess inhibition of CNP-P and NN-P

above 4 mmol/l, substrate dependencies exactly fol-

lowed Michaelis–Menten kinetics, allowing the cal-

culation (n = 5, meanF S.D.) of following apparent

Michaelis constant (Km) values (mmol/l) under the

conditions of the respective assay: 0.377F 0.02

(fixed time) and 0.387F 0.05 (continuously) for N-

P, 0.372F0.05 for CNP-P and 0.370F 0.07 for NN-

Fig. 2. Effect of pentane-1,5-diol (A), L-(+)-tartrate (B) and

substrate concentration (C) on human prostatic phosphatase activity

under the conditions of the assay except the variable: CNP-P (5 - -

5), N-P (o – o, - - , with azo method) and NN-P (. – .).

Fig. 1. Effect of pH on human prostatic phosphatase activity (above)

and reagent blank rate (below) under the conditions of the assay:

CNP-P (5 - - 5), N-P (o – o) and NN-P (. – .).


P. These values were quite dissimilar from those of

0.07 [19], 0.09 [8] and 0.12 mmol/l [7] reported with

N-P or 0.24 mmol/l [19] with CNP-P. Km app increases

with pH and decreases with temperature [23], but the

addition of alcohols proves more important, and thus,

our results conformed well with N-P, 0.30–0.65 mmol/

l, in the presence of some diols, 250 mmol/l [23].

Since Km values increasing with the acceptor con-

centration cogently require higher substrate concen-

trations to maintain maximal reaction velocity,

consequently, incremental tartrate concentrations must

increase to keep the prostatic isoenzyme sufficiently

inhibited. Table 2 summarizes all relevant data for our

choice of assay conditions as a result of mutual

dependencies. Activation energies were calculated

from linear Arrhenius relationship between activity

and temperature observed from 20 to 37 jC. As CNP-P showed lower blanks rates, better stability and

higher sensitivity, we relinquished the use of NN-P

in further studies.

Anticipating a need for surfactants in Hillmann’s

method, only a slight inhibition of N-P cleavage by

human PAP was observed under the conditions of

Table 2 (tartrate omitted) with the following residual

activities: Pluronic F 68 (0.35–1 g/l) 99%, Triton X-

405 (0.5 g/l) 94%, Triton X-100 (0.8 g/l) 90%, Thesit

(0.6 g/l) 91%.

3.2. Azo dye formation and measurement

Depending on the velocity of azo coupling, gov-

erned by factors discussed below and determined by the

solubility of its products, different absorbance spectra

were observed. As exemplarily outlined in Fig. 3, Fast

Red TR couples with its hydrolysis product 2-methyl-

Table 2

Characteristics of three prostatic phosphatase assays applying aryl

phosphate esters in citrate, 50 mmol/l, pH 5.75, at 37 jC

Phosphate ester 1-Naphthyl 2-Chloro-

4-nitrophenyl

4-Nitronaphthyl

Concentration 8 mmol/l 4 mmol/l 4 mmol/l

Relative reaction rate

0.94 Vmax 1.0 Vmax 0.96 Vmax

Reagent blank rates (DA� 10� 3/min) replacing sample by

NaCl, 154 mmol/l 0.66 0.60 1.4

HSA, 40 g/l 0.66 0.65 1.6

Limit of detection (mean of the reagent blank plus 3 S.D.)

U/l (DA� 10� 3/min) 1.31 (1.14) 0.98 (1.05) 2.26 (2.54)

Catalytic concentration of CRM 410 (U/l, meanF 2 S.D.)a

Proposed methods 59.0F 2.8 63.8F 1.5 70.2F 1.8

Without acceptor 32.6F 2.1 32.4F 2.1 36.7F 2.9

Activation by pentane-1,5-diol, 150 mmol/l, above residual

maximum (1.0)a

1.88 2.00 1.95

Inhibition by L-(+)-tartrate, 60 mmol/la

Residual activity 9% 5% 7%

Activation energya

kcal (kJ) 7863 (3.2) 8602 (36.0) not determined

Q10 1.55 1.61 not determined

a CRM 410: 28.0 U/l with 4-nitrophenyl phosphate but without

accelerator [24].

Fig. 3. Absorption spectra of azo compounds generated in the

reaction of Fast Red TR, 2.5 mmol/l, with naphth-1-ol at pH 5.75

in citrate, 50 mmol/l, and pentane-1,5-diol, 150 mmol/l, after 5 min

at 37 jC (different additives). (I) Reaction blank without naphth-1-

ol (PAC) at pH 5.75 in the presence of HSA, 5 g/l: 2-methyl-4-

chloro-6-(2-methyl-4-chlorophenyl-azo)-phenol (—, below). (II)

Intermediate reaction products (NAC) at pH 5.25 with Thesit, 0.6

g/l, in the reaction mixture: (IIa) 2-(2-methyl-4-chlorophenylazo)-

naphth-1-ol; (IIb) 4-(2-methyl-4-chlorophenylazo)-naphth-1-ol

(- - - -). (III) Final reaction product (NbAC) at pH 5.75 in the

presence of HSA, 5 g/l (—, above), or Triton X-405, 0.5 g/l

(- - -, below), as additives: bis-2,4-(2-methyl-4-chlorophenylazo)-

naphth-1-ol.


4-chlorophenol to yield 2-methyl-4-chloro-6-(2-

methyl-4-chlorophenyl-azo)-phenol (PAC, I) which

represents the faintly yellowish reagent blank with a

spectral maximum at 340 nm [11]. The reaction of Fast

Red TR with naphth-1-ol released from N-P leads to 2-

(2-methyl-4-chlorophenylazo)-naphth-1-ol (NAC, IIa)

and its 4-analogue (NAC, IIb), both indicating the

coupling by a yellow colour with a maximum at 375

nm and a shoulder at 420 nm. All these azo compounds

are hydrophobic by lack of more than one hydroxyl

group, and they precipitate from polar solvents in the

absence of surfactants or albumin. However, in solu-

tion, the reaction proceeds by second coupling to

generate the still less-soluble bis-2,4-(2-methyl-4-

chlorophenylazo)-naphth-1-ol (NbAC, III) as already

supposed by Sanders et al. [25]. After extraction with

ethyl acetate, the spectrum of the resulting red complex

shows two distinct peaks at 399 (major) and 531 nm

(minor). However, in assay mixtures, these maxima

varied by F 15 nm depending on the species of protein

or surfactant and the concentration of the additive to

keep NbAC soluble.

The described reaction sequence confirmed earlier

reports on spectral changes paralleling azo coupling of

naphth-1-ol [8,10,11]. Serum catalyzes the reaction

mainly by its albumin contents [10], because modify-

ing effects by additional transferrin, 400 mg/l, and g-

globulin, 2.3 g/l, were missed. Azo coupling is also

accelerated by increasing the pH value up to 5.7 [7]

due to a higher reactivity of the naphthoxide ion

opposite to naphthol [11,26], by addition of surfac-

tants [7,8] and diols [10], to sustain NACs reactive in

micellar solution, and by high concentrations of Fast

Red [11]. These conditions ensure that the formation

of NbAC proceeds so fast that the enzymatic cleavage

becomes the rate-limiting step, and this is met by

methods using Fast Red TR, z 5 mmol/l, together

with a pH value z 5.7. Regarding these conditions,

the final azo complex can either be determined more

sensitively at 405 or near 520 nm with higher specif-

icity, because readings around this wavelength are

almost not affected by both the reaction blank repre-

senting PAC and a delayed coupling with aromatic

amino acids of serum proteins.

Time-dependent changes of the chromophore spec-

trum do not occur after the complete development of

NbAC, but the purported stability of the coloured

complex [11] seemed to be imitated by simultaneous

formation and decay of the azo dye as listed in Table

3, which summarizes a selection of typical results.

Obviously, maximal yield of NbAC, combined with a

bathochromic shift of the minor maximum, is rapidly

attained by HSA, 3 g/l, Brij 35 and 58, 1–5 g/l each,

Rewoquat, 0.5–2.5 g/l, and Thesit, 3 ml/l, using Fast

Red TR>1.5 g/l at pH 5.75. However, the initial

absorbance is followed by a marked decrease over

the whole spectral range. On the other hand, reaction

mixtures containing Triton X-405, 0.5 g/l, Triton X-

100, 0.16–0.8 g/l, and Pluronic F 68, 0.35–2.5 g/l,

display a lower yet constant absorbance with the

minor peak being near 515 nm. Tween 80, 1 g/l,

and Pluronic L 64, 1 g/l, exert similar effects. The

increase of absorptivity changes with concentration by

passing a maximum as previously described [10].

Stability and spectral characteristics are not surfactant

specific as demonstrated by Thesit: 3 g/l react like Brij

35, but 0.6 g/l resemble Pluronic F 68 (Table 3).

Although surfactants in mixtures with serum do not

intensify the absorbance, they reduce its decline with

time. As a consequence, a wavelength should be

selected where the additive does not effect a spectral

shift, viz. between 480 and 510 nm using Pluronic F

68 or Triton X-405 (Fig. 3). Methods working at pH

4.8–5.2 [6,9,23,25,27] and preferring measurements

at 405 nm rely on this decrease combined with a

Table 3

Stability of the naphthol-bis-azo complex (NbAC) generated at 37

jC in the presence of different solubilizers from naphth-1-ol, 50

Amol/l, and Fast Red TR, 2.5 mmol/l

Surfactant—concentration Wavelengths of the maxima

(nm)—e (m2/mol)

Human serum albumin—3 g/l 373—1822 541—1438

1788 (0.98) 1350 (0.94)

Brij 35 —5 g/l 373—2144 539—1640

1924 (0.90) 1478 (0.90)

Thesit —3 g/l 373—1932 536—1490

1414 (0.73) 521–970 (0.65)

—0.6 g/l 372—1694 511—1130

1648 (0.97) 1092 (0.97)

Pluronic F 68— 1 g/l 372—1622 512—1096

1650 (1.02) 1160 (1.02)

Triton X—405– 0.5 g/l 374—1834 516—1284

1852 (1.01) 1276 (0.99)

Molar absorption coefficients after 5 min (above, relative absorp-

tivity = 1.0) and 30 min (below, relative absorptivity in parentheses).


delayed colour development thus imitating steady-

state conditions.

In summary, the inevitable presence of albumin

and its variable concentration in serum samples appa-

rently render reaction conditions not predictable. A

stabilizing addition of Pluronic F 68, Triton X-405 or

X-100 together with strict timing and choice of an

appropriate wavelength, as proposed in our modifica-

tion, improved the reproducibility of Hillmann’s

method, but an absolutely stable chromophore, which

is essential for the accurate performance of a reference

method, could not be achieved.

3.3. Absorbance of reaction products

pH values above 5.5 accelerate as well azo cou-

pling of phenols as the ionization of 2-chloro-4-nitro-

phenol (pK 5.35) and 4-nitronaphthol (pK 5.60), thus

intensifying their absorptivity. However, this concurs

with an incremental spontaneous hydrolysis of their

phosphate esters. Hence, pH 5.75, where 96–99% of

maximum activity were observed (Fig. 1), was uni-

formly chosen as a suitable compromise for all assays.

The absorbance of both indicators slightly increased

with temperature, but NbAC displayed a noticeable

negative thermochromic shift. Its molar absorption

coefficient at the isobestic wavelength of 492 nm

was, admittedly, less than those at 550 nm (1110

m2/mol, pH 5.5, 30 jC) [28] or 585 nm (1120 m2/

mol, pH 6.0, 37 jC) [11]. Table 4 summarizes the

spectrometric data used for calculations and suggests

a high sensitivity by release of 4-nitronaphth-1-ol, if

monitoring at its spectral maximum is available. It

also shows the enhancing effect of albumin on NbAC

absorbance and, conversely, the known negative influ-

ence on that of nitrophenols, which must be consid-

ered in measuring catalytic concentrations in serum.

3.4. Assay characteristics, interferences and analyti-

cal variables

Although azo coupling is speeded up by the

selected reaction parameters, 180 s of preincubation

was necessary to accomplish coupling of protein and

contaminating 2-methyl-4-chlorophenol. This interval

must be extended to 300 s to allow correct measure-

ments of icteric sera containing V 100 Amol/l of

conjugated bilirubin, thus obviating negative interfer-

ence [29,30] with the indicator reaction. Higher con-

centrations require a dilution with HSA, 30 g/l.

Haemoglobin always interfered intensely. The stabil-

ity of Fast Red TR and substrate did not differ from

that of other reports [7,8,11,27,31].

After initiating the reaction with substrate, an

incubation time of 60 s sufficed to convert naphth-

1-ol present in N-P and to cover the lag phase. The

following increase of absorbance was linear with time

for at least 480 s up to 150 U/l. This interval equalled

the more extended zero-order phase at pH 4.8–5.2 as

a result of stabilizing the NbAC absorbance at 492

nm. Accordingly, the sensitivity at this wavelength is

by 25–44% less compared with methods measuring at

405 or 410 nm [6,8,27], which are yet burdened with a

blank of 1–2 U/l depending both on Fast Red con-

centration and albumin contents of the sample.

The directly indicating assay with CNP-P is

devoid of such limitations. A preincubation was only

needed to reach thermal equilibration. There was

virtually no lag phase, but an incubation time of 30

s is recommended to react traces of free 2-chloro-4-

nitrophenol and to attain pH 5.75 and 37 jC with the

precooled substrate solution. Linear conversion rates

were observed for at least 600 s or up to A1.650,

only limited by the capability of the spectrometer.

Thus, the dynamic range of the routine measurement

interval of 300 s ends with 300 U/l. In reference

measurement procedures, the reagent blank has to be

subtracted. Due to its low rate even in the presence of

albumin the detection limit was calculated to 1.51 U/

l. In view of this sensitivity, CNP-P proved superior

Table 4

Molar absorption coefficients of reaction products in various

solutions at 37 jC

Chromophore Wavelength (nm) and e (m2/mol)

NaOH, 20 mmol/l Assay conditions

Naphth-1-ol 332a 743.1F 3.9 334b 735.5F 3.5

NbAC from naphth-1-ol 492 853.6F 5.1

plus HSA, 3.64 g/l 492 956.0F 3.5

2-Chloro-4-nitrophenol 402a 1730.2F 10 403a 1356.8F 6.6

405 1726.5F 12 405 1356.0F 7.2

plus HSA, 3.64 g/l 405 1182.2F 5.5

4-Nitronaphth-1-ol 459a 3040.2F 5.6 459a 2057.4F 8.3

436 2060.1F 5.1 436 1416.2F 4.8

plus HSA, 3.64 g/l 436 1235.1F 5.7

a Spectral maximum.b Fixed-time procedure.


to NN-P which showed a twofold spontaneous

hydrolysis (Table 2). The measurement of a sample

blank is not necessary.

No interference was noted by conjugated bilirubin,

150 Amol/l, and haemoglobin, 1.2 g/l, using 100F 5%

recovery as criterion with acid phosphatase, 10 and 50

U/l. However, haemolyzed specimens may not be

analyzed because of the release of the erythrocytic

isoenzyme. Lipaemic sera with triacylglycerol con-

centrations above 3 mmol/l have to be diluted.

Since the CNP-P solution is stored one pH unit

below the pK value of its chromophore, it showed an

excellent stability at 5 jC with a hydrolysis rate of

0.04% per day. Our preparation of NN-P was less

stable with 0.3% decay per day at pH 5.0.

Table 5 presents the results of mechanized per-

formance in precision testing with two pooled sera of

normal and borderline high catalytic concentration.

Their relative standard deviations correspond to val-

ues obtained with the test kit ‘Acid Phosphatase’ [27]

which applies higher concentrations of substrate,

accelerator, buffer and tartrate.

3.5. Method comparison and preliminary reference

intervals

An activity ratio near 1.0 comparing both methods

without tartrate was observed with supernatants of

prostate homogenates which could be expected from

the same turnover of CNP-P and N-P by CRM 410

(Table 2). Extracts of liver tissue also yielded ratios

between 0.99 and 1.02 and proved equally tartrate

sensitive thus reflecting the high homology of lysoso-

mal and prostatic isoenzymes at their active centres

[32]. However, unselected sera of diseased adults

showed quotients of CNP-P/N-P from 1.5 to 4.5 and

poor correlations (r< 0.5) between both methods and

with regard to the commercial test kit. Highest ratios

were observed in cases of prostatic carcinoma with

metastatic spread.

Hence, the statistical evaluation was confined to

the reference sample group presumed to be more

homogeneous. Table 6 displays the resulting 0.95-

reference intervals and the statistic data derived

thereof demonstrating a closer correlations of methods

than with unselected sera. In the light of these re-

sults—a CNP-P/N-P ratio of 2.15F 0.17 and regres-

Table 5

Imprecision data from 15 mechanized determinations (EPOS 5060 analyzer) of total and tartrate-inhibited acid phosphatase

Total acid phosphatase Tartrate-inhibited fraction

Within series Between series Within series Between series

Mean

(U/l)

RSD

(%)

Mean

(U/l)

RSD

(%)

Mean

(U/l)

RSD

(%)

Mean

(U/l)

RSD

(%)

Substrate naphthyl-1-phosphate

Pool 1 4.65 2.3 4.66 5.5 1.25 5.6 1.26 7.6

Pool 2 8.27 1.6 8.13 3.2 3.21 4.9 3.20 6.3

Substrate 2-chloro-4-nitrophenyl phosphate

Pool 1 10.1 2.1 9.90 4.8 3.82 4.4 3.88 7.1

Pool 2 20.0 1.4 19.6 2.9 5.72 2.8 5.73 5.9

Table 6

Preliminary 0.95-reference intervals and statistical comparison of

total and tartrate inhibited acid phosphatase assays

Method (substrate) Interval

(U/l)

Median

(U/l)

r Slope

(a)

Intercept

(b) (U/l)

Total acid phosphatase (n = 80)

Test kit Roche (N-P) 3.3–6.5 5.2

Proposed method

(CNP-P)

5.8–14.8 11.3 0.891 2.106 0.25a

Comparison method

(N-P)

3.3–6.5 5.0 0.840 0.841 0.69a

Tartrate inhibited fraction (n = 43)

Test kit Roche (N-P) 0.8–3.1 2.2

Proposed method

(CNP-P)

1.2–3.9 2.8 0.976 1.130 0.42a

Comparison method

(N-P)

0.7–2.4 1.6 0.949 0.732 0.05a

Median, r (Pearson’s correlation coefficient) and regression

equation y= axF b (x= test kit) from all values of the reference

interval.a Plots presented in Fig. 4.


Fig. 4. Comparison of total (A) and prostatic (B) acid phosphatase catalytic concentrations as determined by the test kit ‘Acid Phosphatase’

versus the proposed method (CNP-P) and the comparison method (N-P). The lines represent the regression equation y= axF b (see Table 6).


sion equations based on Fig. 4 —it must be inferred

that CNP-P differs in isoenzyme specificity, evidently

by favouring the osteoclastic (macrophagic) compo-

nent. Consequently, a decreasing ratio of 1.35F 0.10

and higher correlation coefficients were observed for

the more selective measurement in the presence of

tartrate.

In Hillmann’s method assay modifications quite

differently influenced total and tartrate-inhibited activ-

ity. The comparison method yielded 0.98 of the total

activity measured with the test kit owing to the

difference of pentane-1,5-diol, 150 mmol/l versus

220 mmol/l, but the ratio decreased to 0.76 for the

tartrate-labile fraction, because using the test kit with

tartrate, 90 mmol/l, the fraction to be subtracted for

calculating the inhibited isoenzymes is smaller.

In contrast to Schiele et al. [33] our frequency

histograms displayed no Gaussian distribution, as can

be roughly demonstrated by projecting the values of

Fig. 4 on the corresponding ordinates. Both ranges for

total and tartrate-inhibited activity using N-P at 37 jCagreed rather well with those of other assays accel-

erated by pentane-1,5-diol [27,34], but reference

intervals established with CNP-P were higher than

ranges with N-P and corresponded to those using 4-

nitrophenyl phosphate [31].

4. Conclusions

Methods based on azo coupling of naphth-1-ol are

multifariously impaired by some unwanted properties

of the diazonium compound, viz. side reactions with

its hydrolytic product, instability and reactions with

sample constituents. Likewise, the generated coloured

products exhibit spectral changes during their forma-

tion, insufficient solubility, or fading in the presence

of albumin. Attempts to eliminate these deficiencies

always lead to unsatisfactory compromises concern-

ing sensitivity versus accuracy of measurement.

Therefore, directly indicating assays have to be

considered with priority, because their reaction con-

ditions are almost exclusively defined by the charac-

teristics of the enzyme and not influenced by the

requirements of an indicator reaction. As a rule,

phosphate esters which release a chromophoric phenol

are ideal substrates, but the introduction of electro-

philic groups to intensify dissociation and colour of

their aromatic constituents renders these more acidic,

and consequently, their phosphate esters represent

mixed anhydrides which undergo incremental sponta-

neous hydrolysis.

Compared with rival methods applying such sub-

strates as 2,6-dichloro-4-acetylphenyl phosphate [35]

or 2,6-dichloro-4-nitrophenyl phosphate [36] CNP-P

is not subjected to a reported hydrolysis by albumin

[37] which causes noticeable blanks according to the

high-volume fraction of the sample necessary in all

assays of acid phosphatases. NN-P presents an alter-

native, but its synthesis proved cumbersome, and the

tested substrate lacked sufficient stability. The advan-

tageous high molar absorption coefficient of its prod-

uct 4-nitronaphth-1-ol at around 459 nm, where no

spectral line is available, allows a highly sensitive

measurement with analyzers operating near this wave-

length. At present, however, CNP-P must be regarded

as the substrate of choice, and the described method

should be considered as a candidate for the certifi-

cation of CRM 410.

Acknowledgements

I thank Barbara Flatter, Barbara Gutschow and

Sandra Rohlf for their skillful technical assistance in

developing the assays, and I am indebted to Prof.

Jurgen Voss (Institut fur Organische Chemie und

Biochemie der Universitat Hamburg) for performing

elemental analyses.

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Continuous monitoring of prostatic acid phosphatase using self-indicating substrates