91
Clinica Chimica Acta, 60 (1975) 91-96 @ Elsevier Scientific Publishing Company,
Amsterdam
-
Printed
in The Netherlands
CCA 6976
RESISTANCE TO LEVAMISOLE PHOSPHATASES
ANNA
W.-L. CHAN
October
IN HEAT-STABLE
ALKALINE
and J.A. KELLEN
Depariment of Clinical Biochemistry, Center, Toron to (Canada) (Received
(R12456)
UniL’ersity of l’oronto,
Sunnybrook
Medical
25, 1974)
Summary
The inhibitory effect of Levamisole (R12456) on human alkaline phosphatases has been studied. At a concentration of 0.1 mM it is possible to differentiate the source of the enzyme. Enzyme activity relatively resistant to Levamisole (less then 20% loss) is present in serum with the Regan isoenzyme, in placental extracts, in the intestinal “B” preparation and in heat-stable alkaline phosphatases “unmasked” after treatment at high ionic strength.
Introduction
For the last three decades there have been numerous attempts to trace the organ source of serum alkaline phosphatases (EC 3.1.3.1) by selective inhibition studies of the enzyme. Excellent reviews [1,2] cover this subject. Although some amino acids can differentiate purified enzyme preparations from different organs, human serum is such a mixture of isoenzymes in varying proportions that clear-cut inhibition patterns are rare. Even in the case of the most widely accepted, L-phenylalanine, sensitivity to this inhibitor does not necessarily denote the intestinal (or placental) origin of the alkaline phosphatase. Screening with many amino acids and their derivates did not improve the discriminating ability of this approach [ 31 and results in vitro do not necessarily reflect inhibitory effects in vivo [4]. The only widely used drug reported to show inhibitory activity on alkaline phosphatases is aminophylline [ 51 . A chance investigation of the biochemical effects of a group of broad-spectrum antihelmintics indicated that in dogs, these drugs inhibited alkaline phosphatases from all tissues except intestine [6,7]. In our present study, we describe L-(-)-2,3,5,6-tetrahydro-6-phenylimidazo[2,1-6]the effect of Levamisole”, * Levamisole Ontario.
(R12456), Canada.
mol.
weight
240.75.
courtesy
of
McNeil
Laboratories
Canada
Ltd.
Toronto.
92
thiazole . HCl, the levorotatory isomer of Tetramisole, on this enzyme in human tissue extracts and in serum. In view of the increasing use of the heatstable isoenzyme in the diagnosis of malignant growth, we included serum samples from cancer patients in this investigation. Materials and Methods All tissues examined were obtained fresh from autopsy and considered as grossly normal. The alkaline phosphatases were extracted and purified following the procedure of Morton [8] with minor modifications. Minced tissues were homogenized in 0.25 M sucrose, extracted with butanol (2 : 1) and centrifuged. The aqueous layer was precipitated with ice-cold acetone and the 3545% precipitate redissolved in 0.05 M Tris/HCl (pH 7.7). Further purification was achieved by column chromatography on Sepharose 6 B with 0.05 M Tris/ HCl (pH 7.7) as eluant. The active fraction was rechromatographed on DEAE Sephadex A-25 (ascending flow) and eluted with a 0.0-0.3 M NaCl gradient in Tris/HCl. The final purification was approximately lOOO-fold. Under these conditions, placenta and liver gave one fraction; intestinal mucosa yielded two fractions named “A” and “B”. For the enzyme assay, the purified enzyme preparations were diluted with 0.4% human albumin in Tris/HCl; the presence of albumin stabilized the enzyme activity. Serum samples, normal and from patients with cancer, were obtained from the staff and in-patients in the Sunnybrook Medical Centre and Princess Margaret Hospital. Enzyme
assay
Alkaline phosphatase activity was determined according to a modification of the Autoanalyzer method [ 91. The reaction was carried out at 37” C in a carbonate/bicarbonate buffer (pH 10.7) with 72 mM disodium phenylphosphate as substrate. The effect of L-Levamisole was studied by addition of this substance ranging from 0.01 mM to 5 mM (final concentration) to the substrate prior to incubation. Heat-stable alkaline phosphatases were measured after exposing the serum samples to 5 minutes at 65°C in a parallel test. The results are expressed in Placental Isoenzyme units [lo] . In this paper, the heat-stable isoenzyme will be named “Regan” although we are fully aware that this is not a homogeneous protein. Lineweaver-Burk plots were calculated from enzyme activity determinations with substrate concentrations ranging from 72 to 3.6 mM disodium phenylphosphate. In an effort to “unmask” heat-stable alkaline phosphatases which escape determination be being bound to a protein or proteins (possibly antibodies), we applied the approach of Dyce and Haverback’[ll] using high ionic strength. With NaCl, we succeeded in dissociating naturally occurring macromolecules and unmask a heat-stable alkaline phosphatase in a substantial number of sera. Our serum samples were mixed in a roller-shaker overnight at 4”C, with NaCl added to a final concentration of 15% and centrifuged for 30 minutes at 3000 X g. Heat-stability was determined as above.
93
Results Kinetics and the The apparent h, values for the various enzyme preparations serum samples were determined using disodium phenylphosphate concentrations between 3.6 and 72 mM and a fixed Levamisole concentration of 0.1 mM. Plotting the results revealed that the inhibition is uncompetitive for all preparations studied; examples of our observations are presented in Figs 1 and 2, showing the inhibitory effect on the liver isoenzyme and the resistance of the placental type. Inhibition The inhibitory effect of varying concentrations of Levamisole is depicted in Fig. 3, where the critical concentration of 0.1 mM is obvious. It appears that the intestinal “B” enzyme preparations is the most resistant, followed by the “Regan” type and the “unmasked” heat-stable alkaline phosphatase in serum and a placental enzyme preparation (which is probably not homogeneous). The liver and intestinal “A” isoenzyme is inhibited to approximately 50% of its original activity (without Levamisole) at the critical inhibitor concentration.
lineweaver-Burk Liver -‘I-
I
alk. with
Plot
phosphatase 0.M
0-0
Levamisole
2t
T
a2 phenylphosphate
Fig.
1.
mM
Levamisole.
l/u
versus
l/S
plot
for
a liver
alkaline
phosphatase
(A,
preparation
in the presence
and
absence
of 0.1
94
Plot
Lineweaver-Burk Placental
alk.
-“-with
h Fig. 2. 1,”
versus
l/S
O.Zdl
mM
plot
phosphatase
Na2
e---e
Levomisole
o-o
(A,
phenylphosphate
for a placental
alkaline
phosphatase
in the presence
and absence
of 0.1
Levamisole.
Inhibition
-*-•-• --..-____ 8C I-
l-s_
..‘. .--..“..,. l
c .o 2 r C se
‘\
60
‘..
‘.., x.,0
‘l
‘\
‘\
‘\
INTESTINE
(A)
.---INTESTINE .-.
,B)
SERUMtNaCl
.....l ,,\ “‘-*.\_*
;
\
‘. \
.
,
‘\
,
* \*
, a
‘\
‘\
\
‘..,
‘+f * _ .‘.h
I
%
5
‘\
\
\
L
LIVER
,,l,_, , ‘\.
20
-
phosphate
‘\
.
%
REGAN
---
‘\ ‘:::
“:...,
\>
of otk. -
~~~~~~~PLACENTAL
‘\, ‘\,
“...i \
40
source
*\
\
-*q,
Levomisole
*A -.
‘\
. ‘-.
with
‘-‘-:‘-*.
---..I-----_--_,.-___-_____..___________,
2
1
.-
. . . . . . . . . . . . . . ..__._....... __
.Ol
.l mM
Fig. 3. Organ specificity
-,.,m.
4
.OOl
Levomisole
of alkaline phosphatase
inhibition
by Levamisole
at different
concentrations.
mM
95
Discussion In the study of alkaline phosphatases with various inhibitor substances it was shown that this group of enzymes exhibits marked organ spcificities. The inhibitory effect of L-phenylalanine is a typical example; in general, the results are clear-cut when pure enzyme preparations are tested, but there is a considerable over-lap in serum determinations unless overwhelming levels of a single isoenzyme are anticipated. All conclusions drawn from results obtained in serum are also impeded by the presence of known (f.ex. bilirubin, albumin) and unknown inhibitors as well as activators. The identification of isoenzymes in bands separated by electrophoresis is sometimes difficult, as large amounts of alkaline phosphatases from one source (such as liver) may mask the increase or even presence of adjacent weak bands. Heat stability is certainly an additional tool in the differentiation of placental and presumably ectopic alkaline phosphatases. The presence of the “Regan” isoenzyme seems to be of diagnostic value, especially in female genital malignancies [12] . However, heat-stability is a trait which also may show various degrees of heterogeneity in some tissues as confirmed by our observations with purified intestinal enzyme preparations (unpublished results). Although the multiplicity of intestinal alkaline phosphatases is accepted by geneticists [13,14], numerous clinical papers treat this enzyme source as homogeneous. Little is known about the physiologic proportions of different isoenzymes in the intestine and even less about their respective metabolism and appearance in the circulation. In our hands, there was approximately a ratio of 1 : 2 between the intestinal preparations “A” and “B”. Levamisole proved to be a relatively potent, (at 0.1 mM) uncompetitive inhibitor of liver and intestinal “A” alkaline phosphatases, while all heat-stable and intestinal “B”) were resistant isoenzymes (placental, Regan, “unmasked” at the critical concentration. It appears that this approach allows for another marker in proving similarities and identities of isoenzymes. Ruling out pregnancy as a source of heat-stability should not pose difficulties; serum samples obtained from fasting patients have negligible levels of intestinal alkaline phosphatases. The presence of Levamisole-resistant isoenzymes appears to be a strong indicator of ectopic and perhaps tumour-associated alkaline phosphatases. Acknowledgement This work has been supported by a research grant from the Ontario Cancer Treatment and Research Foundation and from the Sunnybrook Hospital University of Toronto Clinic. The skillful technical assistance of Miss Diane Konigsberg is acknowledged with thanks. References 1
W.H.
Fishman,
Am.
2
W.H.
Fishman,
Ann.
J. Med., N.Y.
56 (1974)
Acad.
Sci.,
617650 Art.
2, 166
(1969)
365-819
96 3
W.H.
4
V.
5
L.A.
Fishman
Lustig
Nagode.
Abstr. 6
M.
and
Kellen,
M.R.
Enzymologia,
Enzymologia,
Haussler,
D.W.
41
41
(1971)
Boyce.
M.
(1971)
141-167
336-344
Pechet
and
H.
Rasmussan,
Fed.
Proc..
(1970-1971)
694
Borgers
Duijn,
in
eds),
7
H. van
8
R.K.
9
S. Green,
Electromicroscopy
North-Holland,
Belle.
Morton,
Biochim.
C.L.
and
Cytochemistry,
Amsterdam,
Biophys.
Biochem.
J.,
Acta,
1973, 289
57 (1954)
pp.
(E.
Wissem.
W.T.
(1972)
W.H.
Fishman,
Clin.
Chim.
Acta,
33
(1971)
and
W.H.
Fishman,
Clin.
Chim.
Acta.
33
(1971)
Antigens,
Vol.
11
B.J.
Dyce
12
B.J.
Cadeau.
M.E.
13
B.A.
Walker,
L.C.
14
M.J.S.
and
B.J.
Langman,
Haverback Blackstein Eze, E.
M.C.K.
Leuthold,
in Embryonic and
A.
Malkin.
Tweedie E.B.
and
P. van
158-168
and
Anstiss.
.J. Molenaar
595
Anstiss
C.L.
Dames.
51-54
S. Green
10
41
Sie Hsien-Gieh.
and J.A.
and
Robson,
and
Fetal
Cancer D.A.
Res..
Price
J. Harris,
34
Evans,
(1974) Clin.
J. Luffman
279-286 279--286 2, 1972,
pp.
247-250
729-732 Chin and
Acta.
35
H. Harris,
(1971)
433
Nature,
212
(1966)
Clinica Chimica Acta, 60 (1975) 91-96 @ Elsevier Scientific Publishing Company,
Amsterdam
-
Printed
in The Netherlands
CCA 6976
RESISTANCE TO LEVAMISOLE PHOSPHATASES
ANNA
W.-L. CHAN
October
IN HEAT-STABLE
ALKALINE
and J.A. KELLEN
Depariment of Clinical Biochemistry, Center, Toron to (Canada) (Received
(R12456)
UniL’ersity of l’oronto,
Sunnybrook
Medical
25, 1974)
Summary
The inhibitory effect of Levamisole (R12456) on human alkaline phosphatases has been studied. At a concentration of 0.1 mM it is possible to differentiate the source of the enzyme. Enzyme activity relatively resistant to Levamisole (less then 20% loss) is present in serum with the Regan isoenzyme, in placental extracts, in the intestinal “B” preparation and in heat-stable alkaline phosphatases “unmasked” after treatment at high ionic strength.
Introduction
For the last three decades there have been numerous attempts to trace the organ source of serum alkaline phosphatases (EC 3.1.3.1) by selective inhibition studies of the enzyme. Excellent reviews [1,2] cover this subject. Although some amino acids can differentiate purified enzyme preparations from different organs, human serum is such a mixture of isoenzymes in varying proportions that clear-cut inhibition patterns are rare. Even in the case of the most widely accepted, L-phenylalanine, sensitivity to this inhibitor does not necessarily denote the intestinal (or placental) origin of the alkaline phosphatase. Screening with many amino acids and their derivates did not improve the discriminating ability of this approach [ 31 and results in vitro do not necessarily reflect inhibitory effects in vivo [4]. The only widely used drug reported to show inhibitory activity on alkaline phosphatases is aminophylline [ 51 . A chance investigation of the biochemical effects of a group of broad-spectrum antihelmintics indicated that in dogs, these drugs inhibited alkaline phosphatases from all tissues except intestine [6,7]. In our present study, we describe L-(-)-2,3,5,6-tetrahydro-6-phenylimidazo[2,1-6]the effect of Levamisole”, * Levamisole Ontario.
(R12456), Canada.
mol.
weight
240.75.
courtesy
of
McNeil
Laboratories
Canada
Ltd.
Toronto.
92
thiazole . HCl, the levorotatory isomer of Tetramisole, on this enzyme in human tissue extracts and in serum. In view of the increasing use of the heatstable isoenzyme in the diagnosis of malignant growth, we included serum samples from cancer patients in this investigation. Materials and Methods All tissues examined were obtained fresh from autopsy and considered as grossly normal. The alkaline phosphatases were extracted and purified following the procedure of Morton [8] with minor modifications. Minced tissues were homogenized in 0.25 M sucrose, extracted with butanol (2 : 1) and centrifuged. The aqueous layer was precipitated with ice-cold acetone and the 3545% precipitate redissolved in 0.05 M Tris/HCl (pH 7.7). Further purification was achieved by column chromatography on Sepharose 6 B with 0.05 M Tris/ HCl (pH 7.7) as eluant. The active fraction was rechromatographed on DEAE Sephadex A-25 (ascending flow) and eluted with a 0.0-0.3 M NaCl gradient in Tris/HCl. The final purification was approximately lOOO-fold. Under these conditions, placenta and liver gave one fraction; intestinal mucosa yielded two fractions named “A” and “B”. For the enzyme assay, the purified enzyme preparations were diluted with 0.4% human albumin in Tris/HCl; the presence of albumin stabilized the enzyme activity. Serum samples, normal and from patients with cancer, were obtained from the staff and in-patients in the Sunnybrook Medical Centre and Princess Margaret Hospital. Enzyme
assay
Alkaline phosphatase activity was determined according to a modification of the Autoanalyzer method [ 91. The reaction was carried out at 37” C in a carbonate/bicarbonate buffer (pH 10.7) with 72 mM disodium phenylphosphate as substrate. The effect of L-Levamisole was studied by addition of this substance ranging from 0.01 mM to 5 mM (final concentration) to the substrate prior to incubation. Heat-stable alkaline phosphatases were measured after exposing the serum samples to 5 minutes at 65°C in a parallel test. The results are expressed in Placental Isoenzyme units [lo] . In this paper, the heat-stable isoenzyme will be named “Regan” although we are fully aware that this is not a homogeneous protein. Lineweaver-Burk plots were calculated from enzyme activity determinations with substrate concentrations ranging from 72 to 3.6 mM disodium phenylphosphate. In an effort to “unmask” heat-stable alkaline phosphatases which escape determination be being bound to a protein or proteins (possibly antibodies), we applied the approach of Dyce and Haverback’[ll] using high ionic strength. With NaCl, we succeeded in dissociating naturally occurring macromolecules and unmask a heat-stable alkaline phosphatase in a substantial number of sera. Our serum samples were mixed in a roller-shaker overnight at 4”C, with NaCl added to a final concentration of 15% and centrifuged for 30 minutes at 3000 X g. Heat-stability was determined as above.
93
Results Kinetics and the The apparent h, values for the various enzyme preparations serum samples were determined using disodium phenylphosphate concentrations between 3.6 and 72 mM and a fixed Levamisole concentration of 0.1 mM. Plotting the results revealed that the inhibition is uncompetitive for all preparations studied; examples of our observations are presented in Figs 1 and 2, showing the inhibitory effect on the liver isoenzyme and the resistance of the placental type. Inhibition The inhibitory effect of varying concentrations of Levamisole is depicted in Fig. 3, where the critical concentration of 0.1 mM is obvious. It appears that the intestinal “B” enzyme preparations is the most resistant, followed by the “Regan” type and the “unmasked” heat-stable alkaline phosphatase in serum and a placental enzyme preparation (which is probably not homogeneous). The liver and intestinal “A” isoenzyme is inhibited to approximately 50% of its original activity (without Levamisole) at the critical inhibitor concentration.
lineweaver-Burk Liver -‘I-
I
alk. with
Plot
phosphatase 0.M
0-0
Levamisole
2t
T
a2 phenylphosphate
Fig.
1.
mM
Levamisole.
l/u
versus
l/S
plot
for
a liver
alkaline
phosphatase
(A,
preparation
in the presence
and
absence
of 0.1
94
Plot
Lineweaver-Burk Placental
alk.
-“-with
h Fig. 2. 1,”
versus
l/S
O.Zdl
mM
plot
phosphatase
Na2
e---e
Levomisole
o-o
(A,
phenylphosphate
for a placental
alkaline
phosphatase
in the presence
and absence
of 0.1
Levamisole.
Inhibition
-*-•-• --..-____ 8C I-
l-s_
..‘. .--..“..,. l
c .o 2 r C se
‘\
60
‘..
‘.., x.,0
‘l
‘\
‘\
‘\
INTESTINE
(A)
.---INTESTINE .-.
,B)
SERUMtNaCl
.....l ,,\ “‘-*.\_*
;
\
‘. \
.
,
‘\
,
* \*
, a
‘\
‘\
\
‘..,
‘+f * _ .‘.h
I
%
5
‘\
\
\
L
LIVER
,,l,_, , ‘\.
20
-
phosphate
‘\
.
%
REGAN
---
‘\ ‘:::
“:...,
\>
of otk. -
~~~~~~~PLACENTAL
‘\, ‘\,
“...i \
40
source
*\
\
-*q,
Levomisole
*A -.
‘\
. ‘-.
with
‘-‘-:‘-*.
---..I-----_--_,.-___-_____..___________,
2
1
.-
. . . . . . . . . . . . . . ..__._....... __
.Ol
.l mM
Fig. 3. Organ specificity
-,.,m.
4
.OOl
Levomisole
of alkaline phosphatase
inhibition
by Levamisole
at different
concentrations.
mM
95
Discussion In the study of alkaline phosphatases with various inhibitor substances it was shown that this group of enzymes exhibits marked organ spcificities. The inhibitory effect of L-phenylalanine is a typical example; in general, the results are clear-cut when pure enzyme preparations are tested, but there is a considerable over-lap in serum determinations unless overwhelming levels of a single isoenzyme are anticipated. All conclusions drawn from results obtained in serum are also impeded by the presence of known (f.ex. bilirubin, albumin) and unknown inhibitors as well as activators. The identification of isoenzymes in bands separated by electrophoresis is sometimes difficult, as large amounts of alkaline phosphatases from one source (such as liver) may mask the increase or even presence of adjacent weak bands. Heat stability is certainly an additional tool in the differentiation of placental and presumably ectopic alkaline phosphatases. The presence of the “Regan” isoenzyme seems to be of diagnostic value, especially in female genital malignancies [12] . However, heat-stability is a trait which also may show various degrees of heterogeneity in some tissues as confirmed by our observations with purified intestinal enzyme preparations (unpublished results). Although the multiplicity of intestinal alkaline phosphatases is accepted by geneticists [13,14], numerous clinical papers treat this enzyme source as homogeneous. Little is known about the physiologic proportions of different isoenzymes in the intestine and even less about their respective metabolism and appearance in the circulation. In our hands, there was approximately a ratio of 1 : 2 between the intestinal preparations “A” and “B”. Levamisole proved to be a relatively potent, (at 0.1 mM) uncompetitive inhibitor of liver and intestinal “A” alkaline phosphatases, while all heat-stable and intestinal “B”) were resistant isoenzymes (placental, Regan, “unmasked” at the critical concentration. It appears that this approach allows for another marker in proving similarities and identities of isoenzymes. Ruling out pregnancy as a source of heat-stability should not pose difficulties; serum samples obtained from fasting patients have negligible levels of intestinal alkaline phosphatases. The presence of Levamisole-resistant isoenzymes appears to be a strong indicator of ectopic and perhaps tumour-associated alkaline phosphatases. Acknowledgement This work has been supported by a research grant from the Ontario Cancer Treatment and Research Foundation and from the Sunnybrook Hospital University of Toronto Clinic. The skillful technical assistance of Miss Diane Konigsberg is acknowledged with thanks. References 1
W.H.
Fishman,
Am.
2
W.H.
Fishman,
Ann.
J. Med., N.Y.
56 (1974)
Acad.
Sci.,
617650 Art.
2, 166
(1969)
365-819
96 3
W.H.
4
V.
5
L.A.
Fishman
Lustig
Nagode.
Abstr. 6
M.
and
Kellen,
M.R.
Enzymologia,
Enzymologia,
Haussler,
D.W.
41
41
(1971)
Boyce.
M.
(1971)
141-167
336-344
Pechet
and
H.
Rasmussan,
Fed.
Proc..
(1970-1971)
694
Borgers
Duijn,
in
eds),
7
H. van
8
R.K.
9
S. Green,
Electromicroscopy
North-Holland,
Belle.
Morton,
Biochim.
C.L.
and
Cytochemistry,
Amsterdam,
Biophys.
Biochem.
J.,
Acta,
1973, 289
57 (1954)
pp.
(E.
Wissem.
W.T.
(1972)
W.H.
Fishman,
Clin.
Chim.
Acta,
33
(1971)
and
W.H.
Fishman,
Clin.
Chim.
Acta.
33
(1971)
Antigens,
Vol.
11
B.J.
Dyce
12
B.J.
Cadeau.
M.E.
13
B.A.
Walker,
L.C.
14
M.J.S.
and
B.J.
Langman,
Haverback Blackstein Eze, E.
M.C.K.
Leuthold,
in Embryonic and
A.
Malkin.
Tweedie E.B.
and
P. van
158-168
and
Anstiss.
.J. Molenaar
595
Anstiss
C.L.
Dames.
51-54
S. Green
10
41
Sie Hsien-Gieh.
and J.A.
and
Robson,
and
Fetal
Cancer D.A.
Res..
Price
J. Harris,
34
Evans,
(1974) Clin.
J. Luffman
279-286 279--286 2, 1972,
pp.
247-250
729-732 Chin and
Acta.
35
H. Harris,
(1971)
433
Nature,
212
(1966)
