530

Preliminary Communication TABLE I-CLASSIFICATION OF SERA WITH RESPECT TO THE SLOWER

MOVING ALKALINE-PHOSPHATASE COMPONENT IN INDIVIDUALS OF
DIFFERENT BLOOD-GROUPS

SERUM-ALKALINE-PHOSPHATASE AND THE

ABO BLOOD-GROUPS
Arfors et al. 12
reported that the electrophoretic patterns
of serum-alkaline-phosphatase in normal individuals fell
into two groups. Group 1 had a single zone of activity,
while Group 2 had an additional, slower moving zone of
which the intensity varied considerably from serum to
serum. There was a strong association between these
serum groups and certain red-cell antigens. Group-2
sera were found much more commonly in people with
blood-groups 0 and B, and in people whose red cells
were Le(a-)-i.e., mainly ABH secretors.2
We have found that detection of the slower moving was inferior to that obtained with diluted specimens. Other
zones with alkaline-phosphatase activity are occasionally seen
alkaline-phosphatase zone, on which the classification of in normal serum. One of these migrates slightly more slowly
Arfors et al. is based, depends very much on the experi- than the main fast zone, but ahead of the slower zone seen in
mental conditions employed. Increasing the sensitivity the p+ and p++ sera. The other migrates very slowly and
appears just ahead of the origin. When they were observed
these zones were relatively weak, and they were ignored in the
classification discussed above.
Total serum-alkaline-phosphatase was determined by a
modification of the method of Bessey et awl. using p-nitro-
phenylphosphate as substrate. The reaction mixture contained
0-5 ml. 0.02 M p-nitrophenylphosphate disodium salt in
carbonate/bicarbonate buffer (pH 10-3, 1=0-2), 0-4 ml.
0-005 M magnesium chloride in water, and 0-1 ml. serum. This
was incubated for 15 minutes at 375°, and the reaction was
Fig. 1-Electrophoretic patterns of serum-alkaline-phosphatase stopped by the addition of 2 ml. 0-25 N sodium hydroxide. A
classified as po, p+, and p++. blank containing the same quantities of buffered substrate and
of the method causes an increasing proportion of sera to 5. Bessey, O. A., Lowry, O. H., Brock, M. J. J. biol. Chem. 1946, 164, 321.
show the slower moving band, and there seems to be no
clear-cut distinction between sera showing the band and
sera not showing it. In order to examine more closely the
association with the blood-groups we have used a simple
grading procedure for classifying sera with respect to this
particular alkaline-phosphatase component. We now
report our findings with sera from people with different
blood-groups, and relate these findings to the levels of
total alkaline-phosphatase activity in the various sera.
MATERIALS AND METHODS
Serum diluted 1 in 3 with water, and then 50 1. was
was

subjected to vertical starch-gel electrophoresis3 using the

borate-buffer system (pH 8-6) described by Smithies.4 Electro-
phoresis was carried out for 6 hours with a voltage gradient of
5V per cm. in a cold room at +5°C. The alkaline-phosphatase
zones were developed using a reaction mixture containing 50 mg.

x-naphthyl sodium phosphate, 50 mg. Fast Blue RR salt, and

120 mg. magnesium sulphate in 100 ml. 0-06 M borate buffer
(pH 9-7). The gels were incubated in the reaction mixture at
37°C for 1 hour and then washed in a methanol/water/acetic-
acid mixture (5:4:1 by volume).
Sera were classified as pO with only a single band, p+ with an
additional slower moving band which was quite weak, or p++
with a relatively intense slow band which was often as strong
as the fast band and sometimes even stronger. Although the
distinction between p and p-’ is necessarily arbitrary, we
have found that with a standardised electrophoretic and
staining technique, and with the inclusion of appropriate
control sera in each experiment, different observers in our
laboratory have recorded almost the same results. Examples of
patterns classified in this way are shown in fig. 1.
It was found that if po sera were run undiluted a very weak
slow band could often be detected. This was not, however,
used for classification, because the electrophoretic separation
Fig. 2-Distributions of levels of total serum-alkaline-phosphatase
1. Arfors, K. E., Beckman, L., Lundin, L. G. Acta genet. 1963, 13, 89. in sera classified electrophoretically as p", p , and p++. The
2. Arfors, K. E., Beckman, L., Lundin, L. G. ibid. p. 366.
3. Smithies, O. Biochem. J. 1959, 71, 585. numbers tested (n), the means (m), and the standard deviations
4. Smithies, O. ibid. 1955, 61, 629. (S.D.) are given for each class.

TABLE II-SERUM-ALKALINE-PHOSPHATASE LEVELS IN INDIVIDUALS OF
DIFFERENT BLOOD-GROUPS
magnesium chloride was incubated for the same time, and then,
after the addition of 2 ml. 0-25 N sodium hydroxide, 0-1 ml. of
serum was added. Optical densities were read at 410 m[i in a
Unicam spectrophotometer (S.P. 500) and the amount of
p-nitrophenol liberated was determined from a standard curve.
The results were expressed as ,moles of p-nitrophenol liberated
by 1 ml. of serum in one hour.
Sera from 468 volunteer blood-donors were examined. They
were selected so as to include approximately equal numbers of
blood-groups 0, A, B, and AB. The red cells of these donors
were also typed for the Lewis antigen Lea.
RESULTS
The frequency in each blood-group of sera classified
with respect to the slow-moving alkaline phosphatase as
p°, p+ or p++ is given in table I. It is apparent that there
is a much higher incidence of p++ and p+ sera with groups
0 and B than with group A. There is also a strikingly
higher incidence in Le(a-) than in Le(a+) individuals.
The frequencies of the three grades are similar with
blood-groups 0 and B, and with Al and Az. The fre-
quency of p++ is appreciably greater with A2B than with
AIB, but the number of A2B individuals studied is still
rather small.
Fig. 2 gives the distributions of the levels of total alkaline
phosphatase activity observed in sera classified electro-
phoretically as po, p+ or p++. There is considerable
variation in level of activity from serum to serum within
each of these three groups. The average level of activity
in p++ sera is, however, about 30% greater than in p°
sera, and that in p+ sera is about 9% greater than in p°
sera. The differences between the means of the three
distributions are highly significant.
From these results one would expect that the average
level of total serum-alkaline-phosphatase would be greater
with 0 and B than with other blood-groups. That this is
so is shown in table II. In people of group 0 or B whose
red cells are Le(a-), the average level of serum alkaline
phosphatase is 15% higher than that found with groups
A and AB.
DISCUSSION
Two different hypotheses have been put forward about
the nature of the slower moving serum-alkaline-phospha-
tase component. Arfors et al. suggest that it might
represent a complex containing serum-alkaline-phospha-
tase protein and a blood-group substance (possibly
H substance). If this were so one would not necessarily
expect that the average level of total alkaline phosphatase
activity would be greater in sera showing the slow com-
6-9
ponent. The second hypothesis suggests that the two
alkaline-phosphatase zones in normal serum are qualita-
different the
tively enzymes, faster one being derived from
liver and perhaps bone, and the slower one from intestine.
If this were so, and if the amounts varied independently,
then one would expect that sera in which the slower
"
intestinal " zone could be clearly detected would, on the
average, have higher levels of total alkaline-phosphatase
activity than sera in which it was either absent or present
6. Hodson, A. W., Latner, A. L., Raine, L. Clin. chim. Acta, 1962, 7, 255
7. Fishman, W. H., Kreisher, J. H. Ann. N.Y. Acad. Sci. 1963, 103, 951.
8. Cunningham, V. R., Rimer, J. G. Biochem. J. 1963, 89, 50P.
9. Robinson, J. C., Pierce, J. E. Nature, Lond. 1964, 204, 472.
531
in very low concentrations. The present results suggest,
if this second hypothesis is correct, that the higher alkaline-
phosphatase levels of people with blood-group 0 or B
whose red cells are Le(a-)-i.e., who are mainly ABH
secretors-is due to the presence of increased amounts of
the " intestinal " enzyme.
Associations between ABO blood-groups, secretor
status, and certain gastrointestinal disorders have been
extensively studied in recent years, though their cause is
still not understood. Studies of the slow-moving alkaline-
phosphatase component in these conditions will be of
obvious interest, and might possibly provide some insight
into the causal relations involved.
ADDENDUM
10
Beckman has confirmed the association between the
extra alkaline-phosphatase band and secretion of ABH
substances, in a large Brazilian population. Rendel et al.ll
have reported that the level of alkaline-phosphatase activity
is higher in sheep of blood group 0, all of which have an
extra phosphatase band, than in those of group R, which
as a rule do not. K. F. BAMFORD
PH.D. Wales
H. HARRIS
M.D. Cantab.
J. E. LUFFMAN
B.SC. Sheff.
M.R.C. Human Biochemical Genetics
Research Unit and Department of
Biochemistry, King’s College,
E. B. ROBSON
London, W.C.2 PH.D. Lond.
T. E. CLEGHORN
South London Transfusion Centre,
M.D. Sheff.
Sutton, Surrey*
10.
11.
Beckman, L. Acta genet. 1964, 14, 286.
Rendel, J., Aalund, O., Freedland, R. A., Möller, F. Genetics, 1964, 50,
*
973.
Present address: North London Blood Transfusion Centre, Edgware,
Middlesex.
Reviews of Books
Tumours of the Kidney and Ureter
Vol. v Neoplastic Disease at Various Sites. Editor: Sir ERIC
of
RICHES, M.c., M.S., F.R.C.S., emeritus urological surgeon,
Middlesex Hospital. Edinburgh: E. & S. Livingstone. 1964. Pp.
416. 90s.
THE study of tumours of the kidney and ureter has become
of exceptional interest not only because of their clinical be-
haviour but because of the variety of methods now available for
their detection. Although the clinical triad of hasmaturia, local
pain, and swelling continue to dominate the clinical picture,
more early lesions are now being discovered by new radiological
techniques and other means. Much more has become known
about the natural history of the disease, and, from a practical
viewpoint, the prognosis can now be assessed with reasonable
accuracy in terms of pathological grading.
Tumours which develop in duplicated organs permit poten-
tially greater freedom in management. Nevertheless, the
fact that most of them are malignant tends to restrict the
field to ablative surgery and radiotherapy. The indications
for these methods and the speculative role of hormone therapy
in advanced inoperable cases are fully discussed in this
latest contribution to the series of monographs on neoplastic
diseases at various sites. With his team of experts, Sir Eric
Riches is to be congratulated on producing a valuable and well-
illustrated symposium. The sections dealing with diagnosis
and treatment will obviously appeal most strongly to clinicians.
The illustrations demonstrating various radiological techniques
are of high quality, and the analysis of the results of treatment
by surgery and radiotherapy is far-reaching and authoritative.
Other sections, on the statistics of renal tumour, its pathological
features, and its occurrence in childhood, will interest many
readers.