137
Clinica Chimica Acta, 62 (1975) 137-142 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7141
A COMPARISON OF CANINE NODAL HEPATIC ALKALINE PHOSPHATASE AND VARIANT ALKALINE PHOSPHATASES AND LIVER
WALTER
E. HOFFMANN
and JOSEPH
OF SERUM
L. DORNER
Department of Veterinary Pathology and Hygiene, College of Veterinary Medicine, University of Illinois, Urbana, Ill. 61801 (U.S.A.] (Received
January
13, 1975)
Summary The isoenzyme of alkaline phosphatase from normal liver, the corticosteroid induced isoenzyme of alkaline phosphatase from serum and liver and a hepatocellul~ variant isoenzyme of alkaline phosphatase induced by lymphosarcoma have been partially purified and their enzymatic properties compared. The corticosteroid-induced isoenzymes, as well as the isoenzyme induced by neoplasia, appear to have identical properties based on the experiments performed, being distinctly different from the normal hepatic alkaline phosphatase.
Introduction Observations of variant alkaline phosphatase (AP) isoenzymes produced in hepatocellular neoplasia have been reported in humans [l-3], The enzymatic characteristics of these tumor variants have been compared to the placental isoenzyme of AP. Although many similarities have been found between the placental enzyme and the tumor variants, they have been shown to be distinctly different isoenzymes [ 1,2]. The observation of an induced isoenzyme of AP in serum of dogs treated with corticosteroids or suffering from hyperactivity of adrenal cortex has recently been reported [4]. It was suggested that the origin of the induced isoenzyme was the liver. However, this induced isoenzyme has a greater anodal migration than the normal liver AP. Since that report an isoenzyme with identical electrophoretic characteristics has been observed in serum and liver from an animal having lymphosarcoma which had extensively infiltrated the liver. This study was under~ken in the interest of confirming the co~icosteroid induced liver alkaline phosphatase (LAP-CS) as the source of the corticosteroid induced isoenzyme of serum (SAP-CS), as well as confirming the possibility
138
that the variant alkaline phosphatase produced in the liver by lymphosar~oma (LAP-L) might well be identical to the corticosteroid-induced enzymes. Enzymatic properties of the partially purified variant isoenzymes of serum and liver were compared to each other, as well as to the partially purified normal liver alkaline phosphatase (LAP-N).
Materials and Methods Serum was obtained from a dog with hyperactivity of the adrenal cortex. After euthanasia, a portion of the liver was removed from this dog, as well as from five normal dogs, and from a dog with advanced stages of lymphos~~oma metastasized to the liver. All specimens were frozen until use. The condition of each animal was confirmed at necropsy and the subsequent histopathological study. Liver from each source was subjected to butanol extraction using approximately 25 g of tissue, 75 ml of 0.85% saline, and 25 ml of n-butanol. The aqueous extracts of the three hepatic sources, as well as the serum diluted 1 : 1 with saline, were then subjected to ethanol fractionation by adding slowly, with stirring, 99% ethanol chilled to -20°C until the desired percentage of alcohol was reached for precipitation of the isoenzymes. After 15 min the mixture was centrifuged at 1000 X g for 10 min and the precipitate and supernatant were separated. Those fractions which contained either the normal liver AP, the corticosteroid-induced AP or the neoplastic-induced AP redissolved in 0.05 M carbonate/bicarbonate buffer (pH 9.7) resulting in an AP activity 50-100 times greater than that of normal serum. An aliquot of each of these concentrated solutions of AP was subjected to cellulose acetate electrophoresis and stained as previously described [4]. After prolonged incubation to allow maximum staining, each zymogram was evaluated to insure purity of each of the specific isoenzymes in question. Based on the results obtained from cellulose acetate electrophoresis, the authors found that each isoenzyme in question was free from other isoenzymes of AP. AP assays were done in duplicate at 37°C by incubation of a mixture of 0.5 ml of 0.1 M ~~bonate-bic~bonate buffer (pH 9.7), and 0.5 ml of 0.0152 M nitrophenyl phosphate for 5 min prior to adding 0.1 ml of extract. After 15 min 10 ml of 0.02 M NaOH was added and the absorbance read at 410 nm. For those studies involving amino acid inhibition, the amino acid was included in the carbonate-bicarbonate buffer at twice the final concen+ration desired. The effect of pH on each of the four enzyme preparations was examined at pH range of 9.3 to 10.7 using the carbonate-bicarbonate buffer. The Michaelis constant for nitrophenyl phosphate with each of the isoenzymes was determined using a double reciprocal plot in the usual manner. Heat stabilities of the isoenzymes were determined by incubating the extracts at 56°C for times varying from 3 to 18 min. After removal from the 56” C bath, the tubes were rapidly chilled and assayed for AP activity. Inhibitory effects of the amino acids L-glycine, L-phenyl~anine, and L-leucine were determined at concentrations of the amino acids from 0.001 M
139
Minutes Fig. 1. Effects of incubation (a), SAP-CS (‘).
0.1 M. Care was taken pH 9.7.
to
(56”~)
for various
times at 56’C
Prior to AP aSSaY. LAP-N
in each case to adjust
the amino
(*), LAP-CS
(I>). LAP-L
acid solution
to
Results Ethanol fractionation revealed that the normal hepatic isoenzyme is precipitated between 33% and 50% ethanol, whereas the corticosteroid induced
Glycine Fig. 2. Effects SAP-CS (0).
of various
(Log of Cont.) concentrations
of GglYcine
on AP activity.
Lap-N
(0). LAP-CS
(A). LAP-L
(0).
Phenylalanine Fig. 3. Effects of various (“). SAP-CS (0).
(Log of COnC.)
concentrations
of L-phenylalanine
on AP activity.
LAP-N
(a), LAP-CS
(” ), LAP-L
AP and the AP induced by neoplasia are precipitated between 54% and 67% ethanol. The pH optimum of the LAP-N was 10.2, whereas the SAP-CS, LAP-C%, and LAP-L was pH 9.7. Michaelis constants for the LAP-N, LAP-C%, LAP-L, and SAP-CS were 0.87, l-45,1.49, and 1.48, respectively.
Fig. 4. Effects SAP-CS (0).
of various
concentrations
of Gleucine
on AP activity.
LAP-N
(0). LAP-W
(a), LAP-L
(0).
141
Effects of incubation at 56°C prior to AP assays are shown in Fig. 1. The LAP-CS, LAP-L, and SAP-CS have essentially the same heat stability, being subs~ntially more stable than LAP-N. Inhibitory effects of L-glycine on the four enzymes are shown in Fig. 2. LAP-C& LAP-L, and SAP-CS are not inhibited by L-glycine until the concentration of glycine exceeds 0.05 M, whereas some inhibitory effect of L-glycine on LAP-N is seen at 0.001 M L-glycine and this effect increases as the concentration of L-glycine increases. Inhibitory effects of L-phenylal~ine are shown in Fig. 3. At concentrations of L-phenylalanine 0.001 M and 0.005 M the inhibition of the four enzymes is not substantially different; however, at 0.01 M, 0.05 M, and 0.1 M L-phenylalanine the LAP-N is inhibited considerably less than the other three enzymes. Inhibition of the four enzymes by L-leucine is shown in Fig. 4. The degree of inhibition of all four of the enzymes is essentially the same at each concentration of L-leucine.
Discussion From the results presented here it is concluded that the SAP-CS is indeed identical to and derived from the LAP-CS in dogs suffering from hyperactivity of the adrenal cortex or excess treatment with corticosteroids. It is also concluded that based on the biochemical criteria used, the enzyme found in the liver of an animal with lympho~coma is identical to the LAP-CS. That this enzyme is not also a steroid induced enzyme is based on the fact that the animal had no prior history of steroid treatment, showed no clinical signs related to excess production of glucocorticoids and showed no evidence of adrenal cortical hyperplasia on gross observation at necropsy or on the subsequent histopathological study. That the tumor cells themselves produced a steroid cannot be ruled out. The steroid induced alkaline phosphatase, as well as the enzyme produced by the lymphosarcoma, are distinctly different from the normal hepatic enzyme as shown by the fact that they could be distinguished from the normal enzyme in all of the experiments conducted with the exception of the study of the effects of leucine inhibition. In this case all of the AP isoenzymes showed nearly identical properties. Both the steroid-induced AP and the AP produced by the lymphosarcoma are similar to the enzymes previously reported in human hepatocellular cancers [1,3], with respect to the normal liver AP, in that there is an increased anodal migration on electrophoresis, increased heat stability and increased susceptibility to L -phenyl~anine inhibition.
Acknowledgement This study was supported
in part by the Biff Everitt Memorial
Fund.
142
References 1
K.
Higashino,
M.
Hashinotsume,
K.
Kang,
Y.
Takahshi
Chim.
Acta.
and
Y.
Yamamura,
Clin.
67-81 2
B. Jacoby
3
M.L.
4
J.L.
and
Warnock Darner,
K.D.
Bagshawe.
and
R. Reisman,
W.E.
Hoffmann
Clin Clin. and
G.B.
Chim. Long,
Acta, Am.
35 24
(1971)
472-481
(1969)
J. Vet.
5-11
Res..
35
(1974)
1457-1458
Chim.
Acta.
40
(1972)
Clinica Chimica Acta, 62 (1975) 137-142 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7141
A COMPARISON OF CANINE NODAL HEPATIC ALKALINE PHOSPHATASE AND VARIANT ALKALINE PHOSPHATASES AND LIVER
WALTER
E. HOFFMANN
and JOSEPH
OF SERUM
L. DORNER
Department of Veterinary Pathology and Hygiene, College of Veterinary Medicine, University of Illinois, Urbana, Ill. 61801 (U.S.A.] (Received
January
13, 1975)
Summary The isoenzyme of alkaline phosphatase from normal liver, the corticosteroid induced isoenzyme of alkaline phosphatase from serum and liver and a hepatocellul~ variant isoenzyme of alkaline phosphatase induced by lymphosarcoma have been partially purified and their enzymatic properties compared. The corticosteroid-induced isoenzymes, as well as the isoenzyme induced by neoplasia, appear to have identical properties based on the experiments performed, being distinctly different from the normal hepatic alkaline phosphatase.
Introduction Observations of variant alkaline phosphatase (AP) isoenzymes produced in hepatocellular neoplasia have been reported in humans [l-3], The enzymatic characteristics of these tumor variants have been compared to the placental isoenzyme of AP. Although many similarities have been found between the placental enzyme and the tumor variants, they have been shown to be distinctly different isoenzymes [ 1,2]. The observation of an induced isoenzyme of AP in serum of dogs treated with corticosteroids or suffering from hyperactivity of adrenal cortex has recently been reported [4]. It was suggested that the origin of the induced isoenzyme was the liver. However, this induced isoenzyme has a greater anodal migration than the normal liver AP. Since that report an isoenzyme with identical electrophoretic characteristics has been observed in serum and liver from an animal having lymphosarcoma which had extensively infiltrated the liver. This study was under~ken in the interest of confirming the co~icosteroid induced liver alkaline phosphatase (LAP-CS) as the source of the corticosteroid induced isoenzyme of serum (SAP-CS), as well as confirming the possibility
138
that the variant alkaline phosphatase produced in the liver by lymphosar~oma (LAP-L) might well be identical to the corticosteroid-induced enzymes. Enzymatic properties of the partially purified variant isoenzymes of serum and liver were compared to each other, as well as to the partially purified normal liver alkaline phosphatase (LAP-N).
Materials and Methods Serum was obtained from a dog with hyperactivity of the adrenal cortex. After euthanasia, a portion of the liver was removed from this dog, as well as from five normal dogs, and from a dog with advanced stages of lymphos~~oma metastasized to the liver. All specimens were frozen until use. The condition of each animal was confirmed at necropsy and the subsequent histopathological study. Liver from each source was subjected to butanol extraction using approximately 25 g of tissue, 75 ml of 0.85% saline, and 25 ml of n-butanol. The aqueous extracts of the three hepatic sources, as well as the serum diluted 1 : 1 with saline, were then subjected to ethanol fractionation by adding slowly, with stirring, 99% ethanol chilled to -20°C until the desired percentage of alcohol was reached for precipitation of the isoenzymes. After 15 min the mixture was centrifuged at 1000 X g for 10 min and the precipitate and supernatant were separated. Those fractions which contained either the normal liver AP, the corticosteroid-induced AP or the neoplastic-induced AP redissolved in 0.05 M carbonate/bicarbonate buffer (pH 9.7) resulting in an AP activity 50-100 times greater than that of normal serum. An aliquot of each of these concentrated solutions of AP was subjected to cellulose acetate electrophoresis and stained as previously described [4]. After prolonged incubation to allow maximum staining, each zymogram was evaluated to insure purity of each of the specific isoenzymes in question. Based on the results obtained from cellulose acetate electrophoresis, the authors found that each isoenzyme in question was free from other isoenzymes of AP. AP assays were done in duplicate at 37°C by incubation of a mixture of 0.5 ml of 0.1 M ~~bonate-bic~bonate buffer (pH 9.7), and 0.5 ml of 0.0152 M nitrophenyl phosphate for 5 min prior to adding 0.1 ml of extract. After 15 min 10 ml of 0.02 M NaOH was added and the absorbance read at 410 nm. For those studies involving amino acid inhibition, the amino acid was included in the carbonate-bicarbonate buffer at twice the final concen+ration desired. The effect of pH on each of the four enzyme preparations was examined at pH range of 9.3 to 10.7 using the carbonate-bicarbonate buffer. The Michaelis constant for nitrophenyl phosphate with each of the isoenzymes was determined using a double reciprocal plot in the usual manner. Heat stabilities of the isoenzymes were determined by incubating the extracts at 56°C for times varying from 3 to 18 min. After removal from the 56” C bath, the tubes were rapidly chilled and assayed for AP activity. Inhibitory effects of the amino acids L-glycine, L-phenyl~anine, and L-leucine were determined at concentrations of the amino acids from 0.001 M
139
Minutes Fig. 1. Effects of incubation (a), SAP-CS (‘).
0.1 M. Care was taken pH 9.7.
to
(56”~)
for various
times at 56’C
Prior to AP aSSaY. LAP-N
in each case to adjust
the amino
(*), LAP-CS
(I>). LAP-L
acid solution
to
Results Ethanol fractionation revealed that the normal hepatic isoenzyme is precipitated between 33% and 50% ethanol, whereas the corticosteroid induced
Glycine Fig. 2. Effects SAP-CS (0).
of various
(Log of Cont.) concentrations
of GglYcine
on AP activity.
Lap-N
(0). LAP-CS
(A). LAP-L
(0).
Phenylalanine Fig. 3. Effects of various (“). SAP-CS (0).
(Log of COnC.)
concentrations
of L-phenylalanine
on AP activity.
LAP-N
(a), LAP-CS
(” ), LAP-L
AP and the AP induced by neoplasia are precipitated between 54% and 67% ethanol. The pH optimum of the LAP-N was 10.2, whereas the SAP-CS, LAP-C%, and LAP-L was pH 9.7. Michaelis constants for the LAP-N, LAP-C%, LAP-L, and SAP-CS were 0.87, l-45,1.49, and 1.48, respectively.
Fig. 4. Effects SAP-CS (0).
of various
concentrations
of Gleucine
on AP activity.
LAP-N
(0). LAP-W
(a), LAP-L
(0).
141
Effects of incubation at 56°C prior to AP assays are shown in Fig. 1. The LAP-CS, LAP-L, and SAP-CS have essentially the same heat stability, being subs~ntially more stable than LAP-N. Inhibitory effects of L-glycine on the four enzymes are shown in Fig. 2. LAP-C& LAP-L, and SAP-CS are not inhibited by L-glycine until the concentration of glycine exceeds 0.05 M, whereas some inhibitory effect of L-glycine on LAP-N is seen at 0.001 M L-glycine and this effect increases as the concentration of L-glycine increases. Inhibitory effects of L-phenylal~ine are shown in Fig. 3. At concentrations of L-phenylalanine 0.001 M and 0.005 M the inhibition of the four enzymes is not substantially different; however, at 0.01 M, 0.05 M, and 0.1 M L-phenylalanine the LAP-N is inhibited considerably less than the other three enzymes. Inhibition of the four enzymes by L-leucine is shown in Fig. 4. The degree of inhibition of all four of the enzymes is essentially the same at each concentration of L-leucine.
Discussion From the results presented here it is concluded that the SAP-CS is indeed identical to and derived from the LAP-CS in dogs suffering from hyperactivity of the adrenal cortex or excess treatment with corticosteroids. It is also concluded that based on the biochemical criteria used, the enzyme found in the liver of an animal with lympho~coma is identical to the LAP-CS. That this enzyme is not also a steroid induced enzyme is based on the fact that the animal had no prior history of steroid treatment, showed no clinical signs related to excess production of glucocorticoids and showed no evidence of adrenal cortical hyperplasia on gross observation at necropsy or on the subsequent histopathological study. That the tumor cells themselves produced a steroid cannot be ruled out. The steroid induced alkaline phosphatase, as well as the enzyme produced by the lymphosarcoma, are distinctly different from the normal hepatic enzyme as shown by the fact that they could be distinguished from the normal enzyme in all of the experiments conducted with the exception of the study of the effects of leucine inhibition. In this case all of the AP isoenzymes showed nearly identical properties. Both the steroid-induced AP and the AP produced by the lymphosarcoma are similar to the enzymes previously reported in human hepatocellular cancers [1,3], with respect to the normal liver AP, in that there is an increased anodal migration on electrophoresis, increased heat stability and increased susceptibility to L -phenyl~anine inhibition.
Acknowledgement This study was supported
in part by the Biff Everitt Memorial
Fund.
142
References 1
K.
Higashino,
M.
Hashinotsume,
K.
Kang,
Y.
Takahshi
Chim.
Acta.
and
Y.
Yamamura,
Clin.
67-81 2
B. Jacoby
3
M.L.
4
J.L.
and
Warnock Darner,
K.D.
Bagshawe.
and
R. Reisman,
W.E.
Hoffmann
Clin Clin. and
G.B.
Chim. Long,
Acta, Am.
35 24
(1971)
472-481
(1969)
J. Vet.
5-11
Res..
35
(1974)
1457-1458
Chim.
Acta.
40
(1972)
