Automated

Alkaline

Phosphatase

Determination
Monophosphate

III. Evaluation
Bernard

of Phenolphthalein
Klein and James

H. Kaufman

An are: (2) ent

automated

procedure

for

the

determination as the substrate.

of serum Important

alkaline assets

phosphatase

uses

phenolphthalein

monophosphate

of this substrate directly; analysis. compared.

(1) A chromogenic Bilirubin does not agreement method and by the

product is enzymically produced and can be measured interfere, thus eliminating the need for a control when the enzyme activities determined procedure automated p-nitrophenylphosphate are

Excellent

is obtained

by the pres-

FHE

SYNTHESIS

of

phenolphthalein of selective prompted in

monophosphate inhibition of an evaluation an automated automated

(1,

2)

and

a confor report using

templated

investigation isomers phosphatase development

alkaline

phosphatase substrate This

by amino acid serum alkaline presents this the substrate.

of this adaptation. assay

of a simple

procedure,

Materials
Reagents

and Methods
The Morris Plains, substrate N..J.) is T)iagiiostics containing available as Division, 65 mM warmed lot and be fails use, should a

Phenol concentrated Warner-Chilcott

phi hal em solution

mono Laboratories,

phosphate (Phosphastrate,

General

phenolpllthalein pH 10.15. The to room of concentrated only those lots accepted this
Prom 10468.
Thanks Mrs. Jean

nionophosphate stock solution before

in 7.8 M 2-amino-2-methyl-l-propanol, is stored under refrigeration and use. The baseline should of about Concentrated analysis
Veterans
for contributions; his

temperature

noise be A (2).
Administration

level prior 0.001-0.003

of each to

substrate solution giving a noise level automated usable

Research
Mr.
for

tested
=

for is still
the
are
Me.ver

analysis. for manual

Laboratory,
Morgenstern clerical

substrate

which

test

Automation
due

Hospital,
to the preliminary

Bronx,

N.
studies;

Y.

Stanley her capable

contributions Dr.

Research Institute, for permitting us to review his manuscript Jacob Levine, Technicon Instruments Corp., for suggesting used in examiniag the manifold and flow system. fleceived for publication July 22, 1966; accepted for publication

Arthur Babson, Warner-Lambert prior to publication; and Mr. and providing the A-fl fittings Oct. 4, 1966,

Vol.

13, No.

4,

1967

ALKALINE

PHOSPHATASE:

Ill

291

Buffer diluent 1. 2-Amino-2-rnethyl-1-propanol, solution (3) is diluted to about 800 to 11.0 (20-25#{176}) with 6 N HC1 and 2. Phosphate buyer, 0.1 M, pH 1120 and 20.3 water. When the 5006-4 Enzyme solution in the gm. Na,HPO4 reagents are is diluted Phosphastrate The the calculation were described standards to
. 7 1120 completely

0.2 ml. diluted 11.2: with

Ill: with

38 ml. water. to 1 L. 1)issolve The

stock pH

50%

(w/v)

is adjusted Na3PO4. 12

9.3 gm.

magnetic dissolved is also of

bar stirring in 800 ml. the pH is checked and available enzyme as Item standards of from W-

1 L. This kit.

buffer

preparation of in

serum

and controls, and serum specimens, this laboratory

(3).

alkaline phosphatase activity a previous communication

Instrumental
The Analyzer specimen/hr. a single preferred
Operating

Considerations
and flow diagram for use with II the Technicon Autoare shown in Fig. (1:1) sampling glass simplifies coil. 1. The Sampler cam. The incubation chart paper is fitted module ruled in with the 40 is fitted with absorbance is

manifold

20-ft. and

Recorder calculations.

Procedure

With through

the the

sample manifold

line and

aspirating flow

water, system,

the until

reagents the combined

are

pumped reaction

SAMPLER

TUBE

SIZE

(INCHES) SMC SUBSTRATE

0.010 0.045 0.056 0.0 5

H20
AIR SAMPLE

BUFFER F/C,

0.081 0.081

Fig.

1. metal

Manifold probe into

and

flow

diagram concentrated

for

use

with

AutoAnalyzer.

Substrate

tube

is

inserted

with

vial

of

substrate.

292

KLEIN

& KAUFMAN

Clinical

Chemistry

stream

reaches

the

flow

cell

and are with

the

recorder

baseline and by m

is set then passage and

at

0.01

A.

Standard enzyme Each sample is the ance single of the 20-ft. pink

solutions incubated coil color produced

aspirated diluted diluted is measured

first, substrate with alkaline at 550

specimens. through The absorbrecorded.

at 37#{176}, and

buffer.

Results
A typical cial curve line lyophilized upon is obtained phosphatase cordnigs calibration enzyme which when activity curve standard it is based saline are using dilutions (Versatol is shown dilutions assayed I.U. E) in Fig. of a serum (3).
(2).

obtained and the pool

with strip with

a commerchart calibration high alkato phosre-

2. A similar

Linearity assayed showed

is maintained for alkaline

about 25 Shinowara When 26 unselected phatase

(3)

units, about 120 serum specimens by the units automated method, (S.D., 0.19).

were

activity and by the Shinowara

p-nitrophenylphosphate comparison a mean

procedure difference

present

of 0.21

> 0

0 00%

H__
C) It)
-

750/s

w U z
CJ

U, >

cJ

0 hO ALKALINE Fig. 2. At left, PHOSPHATASE calibration units). Also curve shown (sHIN0wARA based is a on dilutions 15 UNITS) of Versatol E (100% at Versatol right. E equals

16.6

Shinowara

sample

interaction

experiment,

V01.

13, No. 4, I61

ALKALINE

PHOSPHATASE:

III

293

Sampling

and Interaction

Sampling em that

at the recorded

40/hr. at 95%

(1:1 of the

wash

ratio)

rate

produced

phenolphthalSpeci-

continuous

sampling

absorbance.

men interaction, when a serum with an activity of 3 Shinowara units, followed a specimen assayed at 21 Shinowara units, was 5%. The same specimens analyzed at the 40/hr. (2:1) rate yielded a slightly increased absorbance action was produced teraction studies, with good (97% of the steady-state 7%. At the 60/hr. (1:1) was the 86% 40/hr. precision of the (1:1) the the continuous serums rate rate (1:1) sampling 60/hr. between 2 test rate, sampling) but the the phenolphthalein sampling increased is advocated; can be used. absorbance, to 11%. for specimen interabsorbance and For rapid the precise assay in-

Discussion
Several phthalein termination correlation years ago, data were as presented which indicated that phenoldiphosphate served of serum alkaline (4). An important a sensitive substrate for the rapid dephosphatase activity with good clinical advantage was the ability of the substrate own chromogen removed from which the absorbed absorbance maximally maximum automated and flow excellent by important and pheacl1ieved. diboth

to generate enzymically its at a wavelength considerably of the serum pigments. operation was an attractive system were assembled agreement procedures, between using the the

Adapting this simple procedure to extension. Accordingly, a manifold and preliminary trials demonstrated serum same enzyme substrate. activities However, as an determined equally

criterion, demonstration nolphthalein formation Re-examination of the phosphate hydrolysis, the automated system, (5, 6) who demonstrated tory, further studies the automated were reluctantly mated With
*Data

of linearity (expressed kinetics of

between enzyme activity as absorbance) was not the enzymatic phenolphthalein

using experimentally determined optimums for confirmed recent findings by other investigators a sequential two-step reaction. In this laboraon phenolphthalein of serum although could still diphosphate as a substrate for alkaline phosphatase activity the substrate in a rapid autobe used.* monophosphate
on an request. automated phenolphthalein A recent flow

determination abandoned, procedure synthesis

used
in this

screening the
on the

successful
flow system

of phenolphthalein
laboratory are 263, 1966) Nonlinear available describes enzymatic

and
paper system product

(Comfort and Campbell, using phenolphthalein is evident, from that

Cliii. CAirn. diphosphate report.

A eta, 14, substrate.

294
its availability The as elements
and Flow

KLEIN

& KAUFMAN
solution, system are tile

Clinical

Ckems+ry

a stable of the
System

conceiitrated automated

question

was below.

re-

opened.
The

examined

Manifold

In Babson
were

preliminary et al. assembled 0.1 coil ml. at

(2)

experiments,

the

optimum

conditions

determined and flow substrate 14 miii. buffer, in a and 11

by system (1 :26) single the

were applied. Thus, whereby a mixture were incubated diluted the with

a simple manifold of 1.2 ml. diluted approximately 5.0 ml. of pH

and 40-ft.

serum

37#{176}, and

absorbance The results tion was phthalein S.D., 0.03),

at 550 m of were expressed obtained between monophosphate and the

phenolphthalein produced in terms of Shinowara units. determination (average with deviation,

was recorded. Good correlathe

the manual substrate (2)

phenol0.2 units; (average

p-nitrophenylphosphate

procedure

(3)

deviation, 0.4 units; but continued studies related to the analytical of the procedure. First, strate drops pipette cessive it was solution of the inconvenient required concentrate

S.D., 0.05). These results were very encouraging exposed incidental problems which, though unprocess, nevertheless detracted from the utility to prepare for numerous into water. into of the large volumes it of diluted sub-

analyses Second,

by squeezing was very to Third,

counted difficult to insure by suceither

the viscous substrate

liquid solutions

the diluent identical

reproducibly, composition.

method of dilution, the total volume much less than anticipated, since centrate could not be expressed by of the pipette and was lost. Fourth, posed; this was indicated initially after 1 hr., by the These problems centrate effected probe ing in situ by the (internal vial tube appearance were solved (Fig. placement diameter, dispensing in the substrate 1), prior

of diluted substrate some indeterminate squeezing, or adhered the diluted substrate by a gradually the with drifting buffered the stainless insert

obtained was volume of conto the outside slowly decombaseline, substrate sample. This and conwas

of a pink tint. by diluting to mixing

of a stock Technicon 0.034 in.) with a 0.016-in, cap, and connection customary manner. Thus, was readily aspirated, fitting, and effect could internal sometimes concentrated rinsing.

steel sample into the opento the 0.8 sub0.05 ml.

of the

of the

probe

strate pump ml. concentrated

approximately diluted with thoroughly be obtained of substrate curled back

air-segmented water a glass double-mixing tion into in the tnalytical of the polyethylene dispensing and even runs, container,

via an 11-3 glass coil. The same tubing cap, after but tended with this thorough to clog an tubing with

mixed in by inser0.01-0.02 on between in. itself

diameter

Vol. 13, No. 4, 1967

ALKALINE

PHOSPHATASE:

Ill

295
fitting, fitting, tubing, of entrance mixed not with small with listed the as a short liquid a

The diluted Technicon platinum volumes

serum substrate,

sample and item, stock

was

introduced The

via A-6 length allows flowing

an of smooth stream.

A-6 glass

incubated. is a 25-mm. This

2 mm.

capillary into the

insert. larger-volume

The Enzyme Reaction

In sitivity 40-ft. tion early were experiments, obtained by good enzyme kinetic the was data given However, and at remarkable senconducting incubations 30#{176}, in a single of

coil (Fig. 3). of the procedure

Serious consideration at that temperature. flow their analytical heating

to the standardizaas most users have was adjustable reevalu-

automated continuous thermoregulators on ated at 37#{176}. It (about 6 mill.) mately was gave

equipment baths, the

do not incubation

determined that incubation good sensitivity-e.g., units, and maintained

in a single 20-ft. coil 0.75-0.85 A for approxikinetics (See Fig. 3).

20 Shinowara

linear

Fig. m of nolphthalein cubation lengths. fer; buffer.

3.

Absorbances formed varying buffers, symbols symbols,

with

at

550 phein-

enzymically times, Solid open

and indicate

coil 2. buf.
IL) 0

amino-2-methyl-1-propanol phosphate

z cr
0
U)

VERSATOL

E(I0O%r21

SHINOWARA

UNITS

The

Colorimetric

Reaction

After baited

incubation,

the

reaction

stream

was

brought

to

pH

11, which

em

further enzyme activity and generated quinoid anion. This was accomplished by

the colored phenolphthaladding either 0.1 M phos-

296 phate buffer phenolphthaiein phate only Both propanol, is permitted fords action Third, in this preparation
Interferences

KLEIN

& KAUFMAN

Clinical

Chemistry

or

0.2 M 2-amino-2-methyl-1-propanol absorbaiices were produced the was considered reasons. the quieter specimens flow system baseline having other buffer an greater incubated suitable. First, as absorbance.
in

by

each coil at

buffer. buffer;

Dissimilar the phosoccurred Fig. activity buffer less in activity. reagent thus tile afinter3).

buffer when the for consistently between buffers

afforded reaction are several with

Coincidence 37#{176} (see prefer range and

a 40-ft. We

2-amino-2-methyl-1of enzyme the 2% used assays; Second, exhibits difference

a greater described. 8- to 16-fold patterns

2-amino-2-methyl-1-propanol laboratory for several of an additional

buffer is a frequently automated enzyme solution is avoided.

One addition

of

the principal to providing

advantages a chromogenic

of

the present substrate is that in reaction product whose absorboccurs contributed alkaremoval of the at

ance is linear 550 m, well by the natural line phosphatase of interfering nitrophenolate

with enzyme removed from serum assay serum anion:

x

activity, the absorbance maximum the absorbances at 410-460 m By contrast, in the selective

chromogens.

automated

with p-nitrophenylphosphate chroinogens required

substrate, dialysis

pH>

10

400

(3).

To rubin, lowing ously

determine what contribution made to the absorbance experiment assayed serum amounts was performed pool equivalent were

the recorded diluted to 5-20

serum pigments, after enzyme 4): with mg./100 1-ml. 1 nil. ml.,

particularly reaction, portions of bilirubin and reassayed. of

the

bilifol-

(Fig.

a previsolution It is

containing

apparent that no increased 96% of the expected activity ings produced by enzymic quantities by repeating ances did not did of bilirubin. the assay not exceed Below in the 0.01.

absorbance due to hilirubin was retained. Also shown assay of solutions containing them are the control absence of substrate. was error concluded, in the under pigment osmotic processing, assay the

was recorded; are the recordthe indicated obtained absorbbilirubin nor developed which a did

recordings The control that measurement, conditions is hemoglobin,

It

therefore,

contribute

a significant

it inhibit serum alkaline phosphatase, for this automated procedure. The other common interfering blood may cytes appear during because specimen of mechanical collection or and

disruption or be

of erythropresent as

Vol. 13, No. 4, 1961

ALKALINE

PHOSPHATAS:

Iii

297

symptom

of hemolytic

disease.

To

evaluate

the

effect

of this

pignielit

several specimens ysis. Some cleat clot produce specimens serums activity. was partly different were originally The results

of blood were carefully serum was separated disintegrated degrees recentrifugeci. withdrawn are shown of mechanically visual The were
ni

collected so as to avoid hemolrapidly from the clot. Then the for discolored for I. varying after serums alkaline which and intervals tile the phosphatase blood clear to hemolysis;

now Table

assayed

Fig.
on

4.

Effect at formed At pool,

of

of 550

bilirubin m phenolof

absorbances left,

enzymically phthalein. of of serum hiliruhin recorded recortll,igs all

absorbances a(ldition At Iii m. control s(ihHtrato) 550 right, sol Low.

U.

before hO Irul at show

solutions.

absorbanees timis, er for sorbnnecs

(without specimens.

Iable

1.

EtPECT

1)11 liIMOt,Yl4(N
(lv, r (on?

ON
av ni to

ltiovoMlTItIe

4t()NI4(

(I

itioi
liv ,no1g,:.vi

A ID4(IKIIAN)IP4)
50 ,,Il

no

it, N..?

,\u.

.lonisgi

Ill)

N tI

A sony

((In?

I 2 :1 4 5 (1

0.176 0.200 0.184 9.255 0.125 0.250 0.160 ((.190 0.287 0.347

0,111)7 ((.010 0.016 0.011 0.012 0.010 0.00$ 0,009 (1.006 0.007

0,11)))

((.240 0,220
((.25(1

(1.01(1) 0,0:10

1)15(1

0,100 1.16$ ((.244 ((.118 ((.24)) ((.152 0.181

0,281

0,184 (1.15$ 0,23(1

0,100 0,272 ((.1511 0,188

0.01(2 ((.1(14 (1,15(1 (((((10

0.050 ((.052

((.340 ((.25(1

0.3:12
0.206 ((.240 0.144
0,31)4

7
8 9 10

0,066
0,040

1)276
((354

0.840

298
Significant differences were

KLEIN

& KALJFMAN
which indicated A control (as shown that

Clinical

Ckems+ry

found

hemolysis

did was absorb60 of

un-

cause increased and spurious required to bring the enzyme ance values) into agreement. serums assay did the absorbance with the serum was hemolyzed, present nonicteric automated

absorbances. assay results Control analyzed not exceed baseline. phenolphthalein assayed.

determination by the net among over

absorbances

during the development 0.02 A; most values Hence, Icteric a control monophosphate serum could

were

the usu-

ally 0.01 A above usually unnecessary when without

Units

analysis was substrate be processed

unhemolyzed interference.

Babson tivity, ate 1 mole

et al. defined of

(2)

recommended instance, phosphatase U. (mean, clinical for present

the as the per 22;

International amount minute activity S.D., usage

Unit of enzyme per liter.

of enzyme that They will found

acliberthe

in this

phenolphthalein

range of serum alkaline ( 2 S.D.) to be 9-35 recommendation. traditional for human

among 6.6 U.). the in

50 normal We concur rooted enzyme Shinowara units to discussed

subjects in this more activities units. product,

(3, 4).

However, unit expressions; serums in the

is deeply

in the

this reason alone, report are given of enzyme activity has been previously

Determination of the relationship as based on different substrates,

References
1.
.

Babson, Cliii. Babson,

3.

4. 5.

A. L., Phenolphthalein monophosphate, a new substrate for alkaline phosphatase. Chem. 11, 789 (1965). A. L., Greeley, S. J., Coleman, C. M., and Phillips, 0. E., Phenolphthalein monophosphate as a substrate for serum alkaline phosphatase. Cliii. Chem. 12, 482 (1966). Morgenstern, S., Kessler, G., Auerbach, J., Flor, R. V., and Klein, B., An automated pnitrophenyiphosphate serum alkaline phosphatase procedure for the AutoAnalyzer. Clin. Chem. 11, 876 (1965). Klein, B., Reed, P. A., and Babson, A. L., Rapid method for the quantitative determination of serum alkaline phosphatase. Cliii. Chem. 6, 269 (1960). Borst Pauwels, G. W. F. W. Occurrence of phenolphthalein monophosphate as an intermediate in the enzymatic hydrolysis of phenolphthalein diphosphate to phenolphthalein and orthophosphate. Rzhekhina, N. I., action of alkaline The Naf are kinetics phosphatase.

202,
of

190
hydrolysis Biokhirniya

(1964).
of sodium 27, 359 phenolphthalein (1962). phosphate by the

6.