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Serum alkaline phosphatase of intestinal origin in patients with cancer and with cirrhosis of the liver
298
CLINICA
SERUM WITH
ALKALINE CANCER
W. H. FISHMAN,
PHOSPHATASE
AND WITH
NORMA
OF INTESTINAL
CIRRHOSIS
I. INGLIS
OF THE
ORIGIN
CHIMICA
ACTA
IN PATIENTS
LIVER*
AND M. J. KRANT
Departmentof Pathology (Oncology) and Medicine, Tufts University School of Medicine and the
Cancer Research Department, New England Center Hospital and the Medical (Oncology) Lemuel Shattuck Hospital, Boston. Mass. (U.S.A) (Received
October
Service,
3rd, 1964)
SUMMARY
A study has been completed on the L-phenylalanine-sensitive alkaline phosphatase in sera of normal subjects, of cancer patients and of patients with cirrhosis of the liver. It has been previously established that only intestinal alkaline phosphatase is inhibited by L- and not D-phenylalanine. In normal subjects, some 40% of the serum alkaline phosphatase is composed of enzyme contributed to by the intestine. The upper value for this component was found to be 3.00 units. Patients with cancer evidence a lower proportion than normal of the contribution of intestinal alkaline phosphatase to the total serum value. The elevated total alkaline phosphatase values encountered in such patients must represent contribution to the serum level from tissues other than intestine. Subjects with cirrhosis of the liver show a normal average but a wide range of contributions (IO-80%) of the intestinal component. In IO out of 33 patients with cirrhosis of theliver, theintestinal alkaline phosphatase exceeded 3.4 units, and was 8.3 units in one individual. In 8 of these IO patients, portal hypertension was obviously present. It is now possible to divide patients with liver disease exhibiting an abnormally elevated alkaline phosphatase into those which can be explained by a high L-phenylalanine-sensitive alkaline phosphatase and into those which cannot. It is further necessary to recognize the quantitative importance of the contribution of the intestinal source to the total alkaline phosphatase value in health and disease, in general.
INTRODUCTION
Hitherto an elevated value for serum alkaline phosphatase has been considered to originate from bone or from liver, and in some cases (of metastatic cancer) from both. It has now been discovered
that in certain patients
with liver disease, abnormal
* Aided by grants at the Tufts-New England Medical Center from the American Cancer Society, Inc., New York (P-106, P-107) and from the National Cancer Institute, National Institutes of Health, Bethesda, Md. (CS-9734, CA-07538-or), and at the Shattuck Hospital, from the National Cancer Institute, (CA o7Igo-oISRC). Clin. Chim. Acta, IZ (1965) 298-303
SERUM ALKALINE
PHOSPHATASE
IN CANCER
299
amounts of alkaline phosphatase with the properties of the intestinal enzyme exist in the circulation. At times, this component accounts for the elevated serum total alkaline phosphatase. The biogenesis, transport, excretion and physiologic and pathologic mechanisms which regulate levels in the circulation of intestinal alkaline phosphatase now become relevant in the interpretation of abnormal values of the serum alkaline phosphatase. Earlier work from this laboratory rl, 2had demonstrated the clinical advantage in studies of prostatic cancer of using the organ-specific inhibitor, L-tartrate, for measuring serum acid phosphatase of prostatic origin. Encouraged by this experience, the authors made a systematic attempt to find organ-specific reagents for alkaline phosphatase 3. Amongst the most promising compounds was L-phenylalanine which proved to be a stereospecific, moderately good inhibitor of rat intestinal alkaline phosphatase. Of unique interest is the recent finding that this amino acid inhibits close to 80% of the enzyme activity of human intestinal alkaline phosphatase but does not appreciably alter alkaline phosphatase activity of liver or bone4. More direct evidence now exists which favors the concept of a distinct alkaline phosphatase protein specifically sensitive to L-phenylalanine. It is possible to identify the slower moving of two serum alkaline phosphatases which migrate during electrophoresis on starch gel as L-phenylalanine-sensitiveb. It travels to the same position reached by purified human intestinal alkaline phosphatase. Also, this band, in the region of the haptoglobins, is intensified in electrophoresed sera taken from subjects with a high intestinal alkaline phosphatase fraction6. Moreover, human intestinal cells growing in tissue culture produce only L-phenylalanine-sensitive alkaline phosphatase7. Finally, this enzyme is located in the striated border of the intestinal epithelium of the rat. The fact that this remarkable organ-specific inhibitor is endowed with stereospecificity favors the quantitative assay of intestinal alkaline phosphatase. Thus, the composition of control and test enzyme digests are exactly the same (substrate, buffer, serum, phenylalanine) except that the test digest contains 0.005 M L-phenylalanine and the control digest 0.005 M n-phenylalanine. Thus, the only difference between test and control digests is the configuration of the amino acid, phenylalanine. The assay has been capable of measuring satisfactorily known amounts of human intestinal alkaline phosphatase added to serum4 and has been employed in the present study. It is also the basis of the histochemical localization of L-phenylalanine-sensitive intestinal alkaline phosphatase7. A survey of sera directed towards locating disorders associated with abnormal values for intestinal alkaline phosphatase is in progress. However, the finding of elevated values in liver diseases was unexpected and is of such unique interest as to merit its separate publication at this time. EXPERIMENTAL
Method A measurement is made of the ability of serum to hydrolyze phenylphosphate (0.009 M) in the absence of phenylalanine and in the presence separately of 0.005 M n-phenylalanine and of 0.005 M L-phenylalanine. Three fresh buffered substrate solutions are prepared and are adjusted to PH Clin. Chim. Acta, IZ (1965) 29%303
w.
300
H.
FISHMAN
et
al.
9.8 (glass electrode).
These are Solution I, 0.009 M phenylphosp~~ate in 0.02 M veronal buffer 9.8; Solution II, same as Solution I but containing 0.005 M L-phenyllanine (final concentration) and Solution III, same as Solution I but containing 0.005 M D-phenylalanine*. Digests are prepared as indicated in Table I ; all are incubated for I h, and the reaction stopped by adding 2.0 ml of 1.5 M formaldehyde. Color development of the liberated phenol is then completed exactly as described by TABLE
I
Tubes 5, 6 and 7 are substrate reagent blanks used to set the colorimcter at roof,; transmittance for Solns. I, II and 111 respectively. The subsequent readings of tubes I, 3 and 4 when substituted into a previously established calibration curve yield the ,ng quantities (X, S’, X”) of total phenol found in the digests. The value for Y (tube z) is subtracted from S, X’, X” to yield the number of ,lg due directly to the hydrolytic action of the phosphatase on the substrate. From previous works. TT~!{,inhibition by L-phenylalanine is experienced by intestinal alkaline phosphatsse. C-B Calculation**
C = ratio of intestinal~total alkaline phosphatase = K.
:
77 Ii s roe x T = I units of intestinal alkaline phosphatasc, units of total alkaline phosphatase.
where ‘I represents the
Stolbach et al.“, and the amount of phenol is obtained from the conventionally prepared phenol calibration curve. The micrograms of phenol are divided by 5 to convert them to Shinowara values in order to make possible ready comparisons between present and previous data 3~?. The factor of 5.0 was established by performing alkaline ~hosphatase assays on twenty sera by both the Shinowarag and the phenylphosphate procedure described here and computing their numerical ratio. In order to test the reproducibility of the enzyme assay, sera were divided into two portions, coded and submitted to the NECH laboratory for blinded assay. Variability of duplicate aliquots of the same specimen is small, averaging in the normal individual, 3.78% total, and 8.56%, intestinal alkaline phosphatase. In the cirrhotic population, the corresponding averages for duplicate variability are 2.98oj, (total) and 15.9% (intestinal). It should be noted that the smaller numbers in the intestinal component mean a smaller denominator and as a-result alargervariability can be expected where the same experimental error applies to both total and intestinal alkaline phosphatase. In addition, intestinal and total alkaline phosphatase measurements have been made on several individuals over a period of time and these show consistency in the * The D- and r=phenylalanine were purchased from CALBIOCHEM, 3625 Medford St., Los Angeles, California. ** The dilution factor (5.0) and the numerical ratio (5.0) of the Shinowara and phenylphosphatc procedure cancel each other and are not indicated in the formula. C&z. Chim. Acta,
12
($965)
298-303
SERUM ALKALINE PHOSPHATASE IN CANCER
301
total alkaline phosphatase and its intestinal fraction (Table II). Finally, is made in Fig. I between ambulatory and hospitalized controls. TABLE
II
SEVERhL
VALUES
FOR
TOTAL
AND
INTESTINAL
ALKALINE
PHOSPHATASE
IN
a comparison
INDIVIDUAL
HEALTHY
SUBJECTS
Subject
Alkaline phosphatase*
Date
W.H.F.
3/5/62 10/20/62 rr/3/62 i r I7162 1213164
Intestinal
2.45 3.68
0.73 1.52 1.0
I.91
2.83 2.93
I.09
4.29 4.36
I.50
12/14/62 S/10/62 s/14/62 s/15/62 S/16/62 ro/4/62 1117163
4.3 3.6 3.2 3.4 4.08 4.43
1.85
2.96
s/6/62
2.4 3.5
0.72 0.98
J.S.F.
I I
J.H.K.
Total
N.I.
/2g/62
6113163
1.13
I.Oj
1.83 1.20 I.31 1.81
* Shinowara units per IOO ml of fresh serum.
TABLE ALKhLINE
III PHOSPHATASE*
OF
NORMAL
SUBJECTS
AND
OF
PATIENTS
WITH
CANCER
AND
LIVER
CIRRHOSIS
Summaw
A’*.
Patient
Mean _______ ~ Total
Int.
Standard
IIT
Total
x
x IO0
TJUP Normals* * Cancer a) Lung b) Breast c) Other 3. Liver cirrhosis I
2.
39.3 21.4 23.8
36 64 17
3.02 6.79 4.86
I.34
16
5.92
0.98
18.9
31 33
8.35 6.07
1.66 259
21.7 44.5
1.21
1.13
Median
Deviation Int.
1.168
0.651
6.40
1.28
4.45
I.95
Total
IIT
Int.
II.22
8.52
21.3
2.72 4.75
0.92
1.02
39.2 20.8
4.50
I.75
37.5
“t” test Alkaline
phosphatase
Comparison Group
Total Total Total Intestine Intestine Intestine
Normal Normal Cancer Normal Normal Cancer
I/T x 100
100
__of the means OS.
of
t
P
Group Cancer Liver cirrhosis Liver cirrhosis Cancer Liver cirrhosis Liver cirrhosis
3.77 4.18 0.58 0.59 4.18 3.79
* Shinowara units per IOO ml of serum preserved by freezing. ** 14 females, average age 34.0 years: 22 males, average age 27.2 years. Clin. Chim.
Acta,
12
(1965) 298-303
W. H. FISHMAN et al.
302
Clinical material The control group consisted of 36 subjects (physicians, hospital personnel, students) who were employed at the Lemuel Shattuck Hospital. The cancer group of 64 patients constituted a hospitalized population with advanced disease, and under several forms of treatment. The cirrhosis of the liver group numbered 33. RESULTS AND DISCUSSION
The frequency distribution curves for-the groups appear in Fig. 2 and the means, standard statistical
treatment
in Table
control, cancer and liver cirrhosis deviations, median distribution and
III. IO
NORMALS
5
0
TOTAL HOSP. Patknk
INTESTINAL
7.0 r
;2 5.0 z 3 4.0 f
f
:,
5.0 -
.
m . . .
2.0 -
0%
1.0-
’
.
3.0 r
.
. .
. x *I*
Amb. Nomwk
E=,H:?c
. 6.0 -
HOSP. Patients
Amb. Normok
2.5 -
i
10
. 2.0 -
.;.
,.5-
&Q ._. . :.p
2-f .
1.00.5 -
: .
_:* *.. . .. .*.
o-
o-
:i . l @ yp
13 0.’ .
Fig. I. Values of alkaline phosphatase in hospitalized and in ambulatory subjects. The difference of intestinalfraction betweenpatients and ambulant subjects is highly significant. Fig. 2. Frequency distributionof total (blank) and intestinal (shaded) alkaline phosphatase in control, cancer and liver cirrhosis groups.
UNITS/100
It is evident that on the average, 40% (range, zo-65%) of the total alkaline phosphatase represents a contribution from the intestine in normal individuals. This percentage is reduced in the cancer group to 21.4% (range 8.7-35.6%), but not in cirrhosis of the liver 44.5% (range 9.9-82%). An examination of the data of subjects with elevated total alkaline phosphatase (over 4.0 Shinowara units), reveals several in the control series with values of intestinal component up to 3.00 units. However, many more with cirrhosis of the liver exhibit high intestinal fractions (nearly one-third of the group), while the cancer group, by contrast, shows such values very infrequently. Clin. Chim. Acta,
IZ
(1965) 298-303
SERUM
ALKALINE
PHOSPHATASE
303
IN CANCER
A correlation was attempted between various clinical and laboratory features of the limited number of patients with cirrhosis and the presence of an elevated intestinal alkaline phosphatase value. The presence or absence of a shunt operation, hyperbilirubinemia, abnormal liver function tests, hemoglobin value below IO g %, high or low protein diet, elevated BUN or neomycin therapy did not appear to correlate significantly with enzyme values. However, there was a strong suggestion that portal hypertension was relatively common in those cases with elevated enzyme levels. The examination of this suggestion and its possible significance is the subject of further studies. What also arrests one’s attention uals whose total alkaline phosphatase
is the existence of presumably healthyindividis slightly abnormal and in whom this eleva-
tion can be explained by an increment in the intestinal component.Whether or not this is a genetic trait remains to be established. Also, it is remarkable that the hospitalized subjects register significantly lower values than ambulatory ones (Fig. I). These hospitalized subjects were selected because of the absence of disease states known to be associated with elevated serum total alkaline phosphatase. This group tended to be in bed more, and had limited exercise. With regard to cancer patients, it is widely accepted that an elevation in serum alkaline phosphatase is often associated with the appearance of metastases to the liver. It is interesting that in the patients studied, this event is not the consequence of an elevated intestinal alkaline phosphatase. Mention should also be made of elevated intestinal alkaline phosphatase encountered in congestive heart disease in the absence of liver disease. This latter finding (unpublished results), together with the suggestive relationship of portal hypertension to serum intestinal alkaline phosphatase, serve to direct our attention to a hemodynamic mechanism as an important factor in explaining elevated values of the intestinal component. ACKNOWLEDGEMENTS
We thank Dr. T. C. Chalmers for his interest and criticism of the manuscript and we are grateful to Dr. H. Muench and Miss R. Klintz for their statistical treatment of the data. Dr. James Patterson, Dr. Franz Ingelfinger and Dr. I. Levitan are also thanked for their cooperation in supplying blood specimens for the survey of diseases possibly related to intestinal alkaline phosphatase. Mr. Sidney Green has contributed to the development of the enzyme assay procedure.
REFERENCES
I 2 3 4 5 6 7 8 9
W. W. W. W. W. J. K. L. G.
H. FISHMAN AND F. LERNER, J. Biol. Chem., rzoo (1953) 89. H. FISHMAN, C. D. BONNER AND F. HOMBURGER. Nezer En.&. 1. Med., 255 (1956) 925. H. FISHMAN; S. GREEN AND N. I. INGLIS, Biochim. Biophys. Acta, 62 (I&‘) 363’. H. FISHMAN, S. GREEN AND N. I. INGLIS, Nature, 198 (1963) 685. H. FISHMAN AND J. H. KREISHER, Ann. N.Y. Acad. Sci., 103 (1963) Art. 2, 951. H. KREISHER, V. CLOSE AND W. H. FISHMAN, C&z. Chim. Acta, II (1965) 122. WATANABE AND W. H. FISHMAN, J. Histochem. Cytochem., IZ (1964) 252. STOLBACH, J, S. NISSELBAUM AND W. H. FISHMAN, Am. J. Clin. Pathol., 29 (1958) 379. Y. SHINOWARA, L. M. JONES AND H. L. REINHART, J. Biol. Chem., 142 (1942) 921. Clin. Chim.
Acta,
12 (1965)
298-303
CLINICA
SERUM WITH
ALKALINE CANCER
W. H. FISHMAN,
PHOSPHATASE
AND WITH
NORMA
OF INTESTINAL
CIRRHOSIS
I. INGLIS
OF THE
ORIGIN
CHIMICA
ACTA
IN PATIENTS
LIVER*
AND M. J. KRANT
Departmentof Pathology (Oncology) and Medicine, Tufts University School of Medicine and the
Cancer Research Department, New England Center Hospital and the Medical (Oncology) Lemuel Shattuck Hospital, Boston. Mass. (U.S.A) (Received
October
Service,
3rd, 1964)
SUMMARY
A study has been completed on the L-phenylalanine-sensitive alkaline phosphatase in sera of normal subjects, of cancer patients and of patients with cirrhosis of the liver. It has been previously established that only intestinal alkaline phosphatase is inhibited by L- and not D-phenylalanine. In normal subjects, some 40% of the serum alkaline phosphatase is composed of enzyme contributed to by the intestine. The upper value for this component was found to be 3.00 units. Patients with cancer evidence a lower proportion than normal of the contribution of intestinal alkaline phosphatase to the total serum value. The elevated total alkaline phosphatase values encountered in such patients must represent contribution to the serum level from tissues other than intestine. Subjects with cirrhosis of the liver show a normal average but a wide range of contributions (IO-80%) of the intestinal component. In IO out of 33 patients with cirrhosis of theliver, theintestinal alkaline phosphatase exceeded 3.4 units, and was 8.3 units in one individual. In 8 of these IO patients, portal hypertension was obviously present. It is now possible to divide patients with liver disease exhibiting an abnormally elevated alkaline phosphatase into those which can be explained by a high L-phenylalanine-sensitive alkaline phosphatase and into those which cannot. It is further necessary to recognize the quantitative importance of the contribution of the intestinal source to the total alkaline phosphatase value in health and disease, in general.
INTRODUCTION
Hitherto an elevated value for serum alkaline phosphatase has been considered to originate from bone or from liver, and in some cases (of metastatic cancer) from both. It has now been discovered
that in certain patients
with liver disease, abnormal
* Aided by grants at the Tufts-New England Medical Center from the American Cancer Society, Inc., New York (P-106, P-107) and from the National Cancer Institute, National Institutes of Health, Bethesda, Md. (CS-9734, CA-07538-or), and at the Shattuck Hospital, from the National Cancer Institute, (CA o7Igo-oISRC). Clin. Chim. Acta, IZ (1965) 298-303
SERUM ALKALINE
PHOSPHATASE
IN CANCER
299
amounts of alkaline phosphatase with the properties of the intestinal enzyme exist in the circulation. At times, this component accounts for the elevated serum total alkaline phosphatase. The biogenesis, transport, excretion and physiologic and pathologic mechanisms which regulate levels in the circulation of intestinal alkaline phosphatase now become relevant in the interpretation of abnormal values of the serum alkaline phosphatase. Earlier work from this laboratory rl, 2had demonstrated the clinical advantage in studies of prostatic cancer of using the organ-specific inhibitor, L-tartrate, for measuring serum acid phosphatase of prostatic origin. Encouraged by this experience, the authors made a systematic attempt to find organ-specific reagents for alkaline phosphatase 3. Amongst the most promising compounds was L-phenylalanine which proved to be a stereospecific, moderately good inhibitor of rat intestinal alkaline phosphatase. Of unique interest is the recent finding that this amino acid inhibits close to 80% of the enzyme activity of human intestinal alkaline phosphatase but does not appreciably alter alkaline phosphatase activity of liver or bone4. More direct evidence now exists which favors the concept of a distinct alkaline phosphatase protein specifically sensitive to L-phenylalanine. It is possible to identify the slower moving of two serum alkaline phosphatases which migrate during electrophoresis on starch gel as L-phenylalanine-sensitiveb. It travels to the same position reached by purified human intestinal alkaline phosphatase. Also, this band, in the region of the haptoglobins, is intensified in electrophoresed sera taken from subjects with a high intestinal alkaline phosphatase fraction6. Moreover, human intestinal cells growing in tissue culture produce only L-phenylalanine-sensitive alkaline phosphatase7. Finally, this enzyme is located in the striated border of the intestinal epithelium of the rat. The fact that this remarkable organ-specific inhibitor is endowed with stereospecificity favors the quantitative assay of intestinal alkaline phosphatase. Thus, the composition of control and test enzyme digests are exactly the same (substrate, buffer, serum, phenylalanine) except that the test digest contains 0.005 M L-phenylalanine and the control digest 0.005 M n-phenylalanine. Thus, the only difference between test and control digests is the configuration of the amino acid, phenylalanine. The assay has been capable of measuring satisfactorily known amounts of human intestinal alkaline phosphatase added to serum4 and has been employed in the present study. It is also the basis of the histochemical localization of L-phenylalanine-sensitive intestinal alkaline phosphatase7. A survey of sera directed towards locating disorders associated with abnormal values for intestinal alkaline phosphatase is in progress. However, the finding of elevated values in liver diseases was unexpected and is of such unique interest as to merit its separate publication at this time. EXPERIMENTAL
Method A measurement is made of the ability of serum to hydrolyze phenylphosphate (0.009 M) in the absence of phenylalanine and in the presence separately of 0.005 M n-phenylalanine and of 0.005 M L-phenylalanine. Three fresh buffered substrate solutions are prepared and are adjusted to PH Clin. Chim. Acta, IZ (1965) 29%303
w.
300
H.
FISHMAN
et
al.
9.8 (glass electrode).
These are Solution I, 0.009 M phenylphosp~~ate in 0.02 M veronal buffer 9.8; Solution II, same as Solution I but containing 0.005 M L-phenyllanine (final concentration) and Solution III, same as Solution I but containing 0.005 M D-phenylalanine*. Digests are prepared as indicated in Table I ; all are incubated for I h, and the reaction stopped by adding 2.0 ml of 1.5 M formaldehyde. Color development of the liberated phenol is then completed exactly as described by TABLE
I
Tubes 5, 6 and 7 are substrate reagent blanks used to set the colorimcter at roof,; transmittance for Solns. I, II and 111 respectively. The subsequent readings of tubes I, 3 and 4 when substituted into a previously established calibration curve yield the ,ng quantities (X, S’, X”) of total phenol found in the digests. The value for Y (tube z) is subtracted from S, X’, X” to yield the number of ,lg due directly to the hydrolytic action of the phosphatase on the substrate. From previous works. TT~!{,inhibition by L-phenylalanine is experienced by intestinal alkaline phosphatsse. C-B Calculation**
C = ratio of intestinal~total alkaline phosphatase = K.
:
77 Ii s roe x T = I units of intestinal alkaline phosphatasc, units of total alkaline phosphatase.
where ‘I represents the
Stolbach et al.“, and the amount of phenol is obtained from the conventionally prepared phenol calibration curve. The micrograms of phenol are divided by 5 to convert them to Shinowara values in order to make possible ready comparisons between present and previous data 3~?. The factor of 5.0 was established by performing alkaline ~hosphatase assays on twenty sera by both the Shinowarag and the phenylphosphate procedure described here and computing their numerical ratio. In order to test the reproducibility of the enzyme assay, sera were divided into two portions, coded and submitted to the NECH laboratory for blinded assay. Variability of duplicate aliquots of the same specimen is small, averaging in the normal individual, 3.78% total, and 8.56%, intestinal alkaline phosphatase. In the cirrhotic population, the corresponding averages for duplicate variability are 2.98oj, (total) and 15.9% (intestinal). It should be noted that the smaller numbers in the intestinal component mean a smaller denominator and as a-result alargervariability can be expected where the same experimental error applies to both total and intestinal alkaline phosphatase. In addition, intestinal and total alkaline phosphatase measurements have been made on several individuals over a period of time and these show consistency in the * The D- and r=phenylalanine were purchased from CALBIOCHEM, 3625 Medford St., Los Angeles, California. ** The dilution factor (5.0) and the numerical ratio (5.0) of the Shinowara and phenylphosphatc procedure cancel each other and are not indicated in the formula. C&z. Chim. Acta,
12
($965)
298-303
SERUM ALKALINE PHOSPHATASE IN CANCER
301
total alkaline phosphatase and its intestinal fraction (Table II). Finally, is made in Fig. I between ambulatory and hospitalized controls. TABLE
II
SEVERhL
VALUES
FOR
TOTAL
AND
INTESTINAL
ALKALINE
PHOSPHATASE
IN
a comparison
INDIVIDUAL
HEALTHY
SUBJECTS
Subject
Alkaline phosphatase*
Date
W.H.F.
3/5/62 10/20/62 rr/3/62 i r I7162 1213164
Intestinal
2.45 3.68
0.73 1.52 1.0
I.91
2.83 2.93
I.09
4.29 4.36
I.50
12/14/62 S/10/62 s/14/62 s/15/62 S/16/62 ro/4/62 1117163
4.3 3.6 3.2 3.4 4.08 4.43
1.85
2.96
s/6/62
2.4 3.5
0.72 0.98
J.S.F.
I I
J.H.K.
Total
N.I.
/2g/62
6113163
1.13
I.Oj
1.83 1.20 I.31 1.81
* Shinowara units per IOO ml of fresh serum.
TABLE ALKhLINE
III PHOSPHATASE*
OF
NORMAL
SUBJECTS
AND
OF
PATIENTS
WITH
CANCER
AND
LIVER
CIRRHOSIS
Summaw
A’*.
Patient
Mean _______ ~ Total
Int.
Standard
IIT
Total
x
x IO0
TJUP Normals* * Cancer a) Lung b) Breast c) Other 3. Liver cirrhosis I
2.
39.3 21.4 23.8
36 64 17
3.02 6.79 4.86
I.34
16
5.92
0.98
18.9
31 33
8.35 6.07
1.66 259
21.7 44.5
1.21
1.13
Median
Deviation Int.
1.168
0.651
6.40
1.28
4.45
I.95
Total
IIT
Int.
II.22
8.52
21.3
2.72 4.75
0.92
1.02
39.2 20.8
4.50
I.75
37.5
“t” test Alkaline
phosphatase
Comparison Group
Total Total Total Intestine Intestine Intestine
Normal Normal Cancer Normal Normal Cancer
I/T x 100
100
__of the means OS.
of
t
P
Group Cancer Liver cirrhosis Liver cirrhosis Cancer Liver cirrhosis Liver cirrhosis
3.77 4.18 0.58 0.59 4.18 3.79
* Shinowara units per IOO ml of serum preserved by freezing. ** 14 females, average age 34.0 years: 22 males, average age 27.2 years. Clin. Chim.
Acta,
12
(1965) 298-303
W. H. FISHMAN et al.
302
Clinical material The control group consisted of 36 subjects (physicians, hospital personnel, students) who were employed at the Lemuel Shattuck Hospital. The cancer group of 64 patients constituted a hospitalized population with advanced disease, and under several forms of treatment. The cirrhosis of the liver group numbered 33. RESULTS AND DISCUSSION
The frequency distribution curves for-the groups appear in Fig. 2 and the means, standard statistical
treatment
in Table
control, cancer and liver cirrhosis deviations, median distribution and
III. IO
NORMALS
5
0
TOTAL HOSP. Patknk
INTESTINAL
7.0 r
;2 5.0 z 3 4.0 f
f
:,
5.0 -
.
m . . .
2.0 -
0%
1.0-
’
.
3.0 r
.
. .
. x *I*
Amb. Nomwk
E=,H:?c
. 6.0 -
HOSP. Patients
Amb. Normok
2.5 -
i
10
. 2.0 -
.;.
,.5-
&Q ._. . :.p
2-f .
1.00.5 -
: .
_:* *.. . .. .*.
o-
o-
:i . l @ yp
13 0.’ .
Fig. I. Values of alkaline phosphatase in hospitalized and in ambulatory subjects. The difference of intestinalfraction betweenpatients and ambulant subjects is highly significant. Fig. 2. Frequency distributionof total (blank) and intestinal (shaded) alkaline phosphatase in control, cancer and liver cirrhosis groups.
UNITS/100
It is evident that on the average, 40% (range, zo-65%) of the total alkaline phosphatase represents a contribution from the intestine in normal individuals. This percentage is reduced in the cancer group to 21.4% (range 8.7-35.6%), but not in cirrhosis of the liver 44.5% (range 9.9-82%). An examination of the data of subjects with elevated total alkaline phosphatase (over 4.0 Shinowara units), reveals several in the control series with values of intestinal component up to 3.00 units. However, many more with cirrhosis of the liver exhibit high intestinal fractions (nearly one-third of the group), while the cancer group, by contrast, shows such values very infrequently. Clin. Chim. Acta,
IZ
(1965) 298-303
SERUM
ALKALINE
PHOSPHATASE
303
IN CANCER
A correlation was attempted between various clinical and laboratory features of the limited number of patients with cirrhosis and the presence of an elevated intestinal alkaline phosphatase value. The presence or absence of a shunt operation, hyperbilirubinemia, abnormal liver function tests, hemoglobin value below IO g %, high or low protein diet, elevated BUN or neomycin therapy did not appear to correlate significantly with enzyme values. However, there was a strong suggestion that portal hypertension was relatively common in those cases with elevated enzyme levels. The examination of this suggestion and its possible significance is the subject of further studies. What also arrests one’s attention uals whose total alkaline phosphatase
is the existence of presumably healthyindividis slightly abnormal and in whom this eleva-
tion can be explained by an increment in the intestinal component.Whether or not this is a genetic trait remains to be established. Also, it is remarkable that the hospitalized subjects register significantly lower values than ambulatory ones (Fig. I). These hospitalized subjects were selected because of the absence of disease states known to be associated with elevated serum total alkaline phosphatase. This group tended to be in bed more, and had limited exercise. With regard to cancer patients, it is widely accepted that an elevation in serum alkaline phosphatase is often associated with the appearance of metastases to the liver. It is interesting that in the patients studied, this event is not the consequence of an elevated intestinal alkaline phosphatase. Mention should also be made of elevated intestinal alkaline phosphatase encountered in congestive heart disease in the absence of liver disease. This latter finding (unpublished results), together with the suggestive relationship of portal hypertension to serum intestinal alkaline phosphatase, serve to direct our attention to a hemodynamic mechanism as an important factor in explaining elevated values of the intestinal component. ACKNOWLEDGEMENTS
We thank Dr. T. C. Chalmers for his interest and criticism of the manuscript and we are grateful to Dr. H. Muench and Miss R. Klintz for their statistical treatment of the data. Dr. James Patterson, Dr. Franz Ingelfinger and Dr. I. Levitan are also thanked for their cooperation in supplying blood specimens for the survey of diseases possibly related to intestinal alkaline phosphatase. Mr. Sidney Green has contributed to the development of the enzyme assay procedure.
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