Upsala J Med Sci 88: 127-139, 1983 

Perspectives on Serum Acid Phosphatase in Prostatic Disease 
An evaluation of two methods 

S .  Dedorson,' A .  Fritjofsson,' B .  J. Norlen' ,and G .  Ronquist' 
From the Departments of Urology' and Clinical Chemistry,* 

University Hospital, Uppsala, Sweden 

ABSTRACT 
Acid phosphatase in serum was measured in 116 patients with 

prostatic disease, benign in 59 and malignant in 57 cases. 
Comparisons were made between radioimmunoassay (RIA) and an 
enzymatic method. The correlation coefficient between the 
respective values was 0.96 in patients with untreated prostatic 
cancer, indicating that no significant difference between results 
with the two methods was to be expected. The correlation 
coefficient between RIA values and cancer stage was 0.48, and 
between catalytic activity and cancer stage it was 0.50. The 
validy of the two methods consequently was equal. RIA, however, 
was the more sensitive method, giving elevated values in 10 of 11 
patients with untreated stage I11 or stage IV prostatic cancer, as 
compared with only 4 of the same 11 in the enzymatic assay. This 
seeming paradox most probably was attributable to differing 
intrinsic properties of the methods when the upper limits of 
normal range were established. Neither RIA nor enzymatic analysis 
discriminated early prostatic cancer (stages I and 11) from benign 
lesions. 

INTRODUCTION 
Measurement of acid phosphatase in serum has been employed 

for more than 40 years to detect and monitor malignant 
prostatic disease ( 8 ) .  The prostate gland is the main, but 
not the sole source of this enzyme in man. Research on enzymatic 
assay of acid phosphatase has therefore been focused on making the 
procedure more prostate-specific. Refinements in substrates and 
inhibitors for the reaction, however, have not so far led to 
absolute organ specificity. 

9-838572 127 



The enzymatic activity of the acid phosphatase molecule is 
sensitive to pH, with optimum at pH 5.5. The molecule 
dissociates into its two subunits at pH 2.0 or less and also 
at pH 7 . 4  or higher. The dissociation results in inactivation 
of the enzyme, and the two subunits immediately begin to 
aggregate ( 4 ) .  Inherent difficulties thus limit the 
usefulness of enzymatic assay of acid phosphatase. 

A competitive-binding, 'labelled antigen' technique for 
isotopic assay of PAP (prostatic acid phosphatase) was 
therefore evolved as an alternative procedure (6). Such assay 
offered improvements with regard to prostate-specificity, 
stability, sensitivity and precision (7). Despite controversy 
on particular aspects of radioimmunoassay for PAP, reports 
have continued to emphasize the advantages of this principle 
over the enzymatic technique (5,9,13). 

The aims of the investigation here presented were to 
measure PAP concentrations in sera from patients with benign 
or malignant prostatic disease and to compare results from 
radioimmunoassay (RIA) with those from a conventional 
enzymatic procedure for determining PAP activity. 

PATIENTS AND METHODS 

Radioimmunoassay 
All radioimmunoassays of PAP were run in duplicate, using 

0.1 ml serum samples and a commercially available RIA kit 
(GammaDabR, Clinical Assays, Travenol Laboratories, 
Cambridge, Mass., USA) in accordance with manufacturer's 
instructions. After incubation and centrifugation, however, 
it was found necessary to remove traces of supernatant fluid 
in the tubes, using filter paper, before making counts of the 
sedimented radioactive material in the tubes. 

128 



Enzymatic assay 
The catalytic activity of serum acid phosphatase was 

measured as recommended by the Committee on Enzymes of the 
Scandinavian Society for Clinical Chemistry and Clinical 
Physiology using paranitrophenyl phosphate as substrate with 
I,(+)-tartrate as inhibitor. "he upper limit of the reference 
range €or this method is 58 nkat' 1-'. 
Patients 

The studies were made on 1 1 6  patients consecutively 
referred to this department of urology because of prostatic 
disorder. Prostatic cancer was found in 57 of the men and 
benign prostatic disease in 59. Table I surveys the numbers 
of patients and their mean age distribution according to 
diagnosis. 

Table 1 Distribution of patients according to age and 
diagnosis 

Diagnosis No of Mean age 
- patients (years ) 

Prostatic cancer 57 72.6 

Stage I 
Stage I1 
Stage I11 
Stage IV 

Benign prostatic disease 

Hyperplasia 
Prostatitis 
Other benign process 

3 

1 5  

23 

16 

59 

39 

1 3  

7 

79.0 

74.4 

73.3 

68.7 

63.3 

6 8 . 1  

50.8 

5 9 . 1  

129 



In all cases of prostatic cancer the diagnosis was 
confirmed by transrectal aspiration biopsy for cytological 
study. Intravenous pyelography and X-ray examination of the 
lungs were also performed. Bone scan was done in all but two 
patients of this group (1 with stage I1 and 1 with stage IV 
tumour), with skeletal roentgenography when indicated. Since 
PAP- concentration was the issue under study, it was not 
included in the clinical staging of malignancy, which 
otherwise was in accordance with recommendations by the 
Veterans Administration Cooperative Urological Research Group 
(12): Stage I thus implied clinically unsuspected cancer in a 
transurethral resection specimen, stage I1 a palpable nodule 
confined to the prostate gland, stage I11 periprostatic 
growth of the tumour and stage IV known distant metastases, 
irrespective of PAP level in the serum and findings at rectal 
examination. 

Of the 5 7  patients with prostatic cancer, 20 were 
untreated at the time of the study. The treatment in the 
other 3 7  patients varied - orchiectomy, 
oestrogen/chemotherapy (estramustin, EstracytR) or 
irradiation, alone, in combinations or in succession. The 
duration of therapy and the response were highly variable at 
the time of the investigation. 

In the group of 59 patients with benign prostatic 
disorders, histological or cytological examination was done 
only if surgery was performed or if the clinical findings 
aroused suspicion of malignancy. 

Statistical methods 
A Hewlett-Packard 9820 A calculator, Model 20 with 

standard programmes was used for registering data and for the 
statistical analyses. 

The reliability of RIA was measured as the correlation 
coefficient between the duplicate sets of values (n/set = 
116). The validity of the respective methods was assessed in 
the patients with untreated prostatic cancer (n=20) as the 
correlation between cancer stage and PAP concentration 
according to RIA or to the enzymatic assay (Fig. 3 ) .  The 
validity was additionally calculated as correlation between 

130 



results with the two methods in the 59 patients with benign 
prostatic disease (Fig.l), in the total 57 with prostatic 
cancer (Fig.2), and in the 20 patients with untreated 
prostatic cancer (Fig.3). For calculation of correlation 
coefficients and testing of significance, we used the methods 
described by Snedecor & Cochran (10). 

The sensitivity of the method was judged from the 
proportion of elevated readings among the patients with 
prostatic cancer. The specificity was calculated as the 
proportion of non-elevated readings in patients with benign 
prostatic disease. The significance of differences in 
sensitivity and specificity was calculated with the z test 
(one-tailed, with correction for continuity) as described by 
Snedecor & Cochran (10). 

4.0 

3.2 
c 

L 

c"u 
n 
6 
c#z 
.- - 1.6 a a a 
I 

0.8 

0 

-0- 

0 
0 

I 

I 
I 
I 
I 

+ 1.27 

1 -  I I 1 -  

12 24 36 48 
S-PAP ( E n d  nkat . 1-l 

1 
60 

Fig.1 Correlation between values from enzymatic analysis 
(Enz.)and from radioimmunoassay (RIA) of serum 
phosphatase (S-PAP) in patients with benign prostatic 
disease. 

Regression line 
Upper limit of normal range - - - _ _ _  

131 



RESULTS 
To determine the upper normal limit for RIA results, PAP 

was measured in serum sampled from 58 male blood donors. "he 
mean value was 1.20 p g  - l-l, S . D .  0.80. The upper 
limit of the reference range therefore was set at 2.80 pg 
1-l. 
positive correlation (r=0.20) with the RIA values in the 
patients with benign prostatic disorders, despite the 
relatively narrow range of readings in that group (Fig 1). 
The correlation between the two variables was appreciably 
better among the 57 patients with malignant disease (Fig 2), 
and it was particularly high (0.96) in the 20 patients with 
untreated prostatic cancer (Fig 3 ) .  

The values from enzymatic analysis showed a weak 

'"1 12 
0 0 

0 

/ 

I 
I I 1 1 1 

0 40 80 120 160 200 
S-PAP (Enz.) nkat e 1 - l  

Fig 2.CorrelatLon between Enz. and RIA values in all patients 
with prostatic cancer. 

Regression line 
Upper limit of normal range - - - - - -  

132 



The c o r r e l a t i o n  between t h e  d u p l i c a t e  r u n s  o f  R I A  
( r = 0 . 9 9 7 )  e x p r e s s e d  h i g h  r e l i a b i l i t y  o f  t h e  method. The s t a g e  

of c a n c e r ,  moreover, showed s i g n i f i c a n t l y  (p<O.O5) p o s i t i v e  
c o r r e t a t i o n  w i t h  r e s u l t s  from e n z y m a t i c  a n a l y s i s  ( r = O .  5 0 )  and 
R I A  v a l u e s  ( r = O .  4 8 ) .  Between t h e  t w o  methods, t h e r e f o r e ,  no 

d i f f e r e n c e  i n  v a l i d i t y  w a s  d e t e c t a b l e .  
R I A  g a v e  e l e v a t e d  v a l u e s  i n  4 o f  59 p a t i e n t s  w i t h  b e n i g n  

p r o s t a t i c  d i s e a s e  ( F i g  1, Table 2 ) .  The enzyme t e s t s  showed 
no e l e v a t e d  v a l u e s  i n  t h e  same g r o u p .  The s p e c i f i c i t y ,  i . e .  
t h e  a b i l i t y  t o  r e c o g n i z e  b e n i g n  n a t u r e  of p r o s t a t i c  l e s i o n s ,  
t h u s  w a s  lower w i t h  R I A  ( 0 . 9 3 )  t h a n  w i t h  e n z y m a t i c  a s s a y  
(1.00), t h o u g h  t h e  d i f f e r e n c e  w a s  n o t  s t a t i s t i c a l l y  
s i g n i f i c a n t .  

0 

n = 20 
r = 0.96 
P < 0.001 
y = 0.10~ +0.23 

2 ‘ a  - - - - I - -  ---- - - - - - _ - - - - - _ _  
I 
I 
l 0 
I 

I I I I 1 
40 80 120 160 200 

S-PAP (Enz.) nkat . I-’ 

Fig 3 .  C o r r e l a t i o n  between Enz. and R I A  v a l u e s  i n  p a t i e n t s  
w i t h  u n t r e a t e d  p r o s t a t i c  c a n c e r .  

R e g r e s s i o n  l i n e  

Upper l i m i t  o f  normal r a n g e  - - - - -  

133 



u 
-4 
.tJ 

.tJ 
la 
0 
k a 
C 
-4 

14 

I 
m 

m 

2 
W 
0 

h 
m 
v) 
(0 
m 

N 

a, 
rl 
P 
P 
m 

PI 
2 
5 
& 
a, 
m 

h 

r- 

m 
rl 

m 
r- 
(u 
rl 

a\ 

m 
d 

+I 

(u 

\o 

0 

* 
+I 

r- 
m 

h 
a, 
U 
c 
m u 
0 
-4 
.tJ 
m 
.tJ 

& 
nl 
$ 

d r -  
r l m  

m a l o  
+ I  + I  + I  

m u m  
r l d m  
r - m m  
rim+ 

. . .  
+I +I + I  . . .  

H 
H 

a 
c 

0 

cr 

m 
rl 
rl 

m 
r- 
rl 

+ I  

d 

rl 

P 

4 

+I 

m 
Ln 

0 
.4 
.tJ 
m 
.tJ 
0 
0 
k 

c v )  
m l d  
-4 a, 
c v )  
a, -4 
m a  

0 

0 

cr 
co 
+ I  m 
m 
rl 

Ln 

134 



The means from R I A  and from enzymatic assay were almost 
the same in the patients with stage I or 11 prostatic cancer 
as in those with benign prostatic disease. The proportion of 
patients with values above the upper limits of normal was 
also similar (Table 2). Both parameters, however, distinguished 
cancer stages I11 and I V  from the benign conditions, especially 
when R I A  was used. Thus, elevated values were given by R I A  in 9 of 
the 23 patients with stage I11 cancer, but by the enzyme method in 
only 3 patients. The corresponding figures in stage IV cancer were 
9 and 4 of 16 patients. 

The sensitivity, i.e. the ability to recognize prostatic 
disease as malignant, was 0 . 3 3  with R I A  and 0.14 with the 
enzymatic assay when all 57 cancer patients were included in the 
analysis. This difference was significant (p<O.Ol) and was 
explained solely by the superior sensitivity of R I A  in tumours of 
stage I11 or I V .  

The enhanced sensitivity of R I A  was still more evident in 
untreated prostatic cancer (Table 3 ) .  Of the 11 patients with 
stage I11 or stage I V  tumour, 10 showed elevated values in 
R I A ,  but only 4 in enzymatic assay. 

Table 3 .  Radioirmolnoassay (RIA) and enzyme assay of S-PAP in untreated 
prostatic cancer 

Values above 

RIA enzyme assay 
No of Serum PAP upper range limit 

Diagmsis patients RIA E n z y z ~  assay - 
[pg-~-') (nkat-1-1) 

11 4 

1 0  

6 2  

4 2  

+ 
+ 
+ + 

+ 

P r o s t a t i c  cancer 20 5.6-7.4 51.8?68.5 

Stages I and I1 9 2.1-2.0 19.9+12.1 
Stage I11 7 6.9-6.8 57.4-51.6 
Stage IV 4 11.2212.6 113.5-125.9 

9t -838572 135 



DISCUSSION 
RIA measures antigen determinants of the analyzed enzyme, 

whereas the various methods of enzymatic analysis reflect 
catalytic activity. When RIA was introduced for determination 
of prostatic acid phosphatase in serum it was hoped that this 
assay, based on an antibody against a purified isoenzyme, 
would increase diagnostic acuity. Such specificity should 
contribute to early detection of prostatic cancer and also to 
the more appropriate tumour staging that is essential for 
optimum choice of treatment and for accurate monitoring of 
tumour response. These hopes remain widely held, although it 
is generally recognized that no known isoenzyme of acid 
phosphatase is tumour-specific or even tumour-associated. 

The present study showed good correlation between results 
with RIA and those with the enzymatic method of determining 
serum acid phosphatase in patients with prostatic cancer, 
particularly before treatment had been instituted. A priori, 
therefore, no clear difference was to be expected between the 
two methods in the continued investigations. Their validity, 
accordingly, proved to be similar. In sensitivity, on the 
other hand, the methods clearly differed. The most probable 
explanation of this apparent paradox was intrinsic difference 
in the properties of the methods when the upper normal limits 
were determined. If RIA is a completely organ-specific test, 
the upper limit of its normal range should be 'absolute'.The 
catalytic activity in the enzymatic analysis, however, is 
assumed not to derive exclusively from the prostate. Acid 
phosphatases from other organs may contribute to the results 
of the analysis, and such extra-prostatic 'contaminating 
activities' may diminish with advancing age. The mean age was 
higher in the cancer patients than in those with benign 
prostatic disease. Intergroup comparisons of RIA and 
enzymatic analysis data (Table 2) suggest that the 
extraprostatic contribution to the latter values could have 
been inversely related to age. Use of an age-matched 
reference group might well have eliminated this discrepancy. 

The false positive RIA results occurred only in patients 
with benign prostatic hyperplasia. The 10 per cent of results 

136 



above u p p e r  n o r m a l  l i m i t  i n  t h i s  g r o u p  i s  close t o  t h e  f i g u r e  
r e p o r t e d  b y  F o t i  e t  a l .  ( 6 ) .  I n  d e a l i n g  w i t h  t h e  problem o f  
f a l s e  p o s i t i v e  r e s u l t s ,  these w r i t e r s  i n t r o d u c e d  +4 S .  D .  as 
u p p e r  n o r m a l  l i m i t ,  w h i c h  r e d u c e d  t h e  p r o p o r t i o n  t o  5 per 

c e n t  b u t  decreased t h e  s e n s i t i v i t y  o f  t h e  a s s a y  i n  s t a g e  I 

c a n c e r .  
I n  o u r  s e r i e s  the t w o  a s s a y  t e c h n i q u e s  d i d  n o t  d i f f e r  i n  

r e g a r d  t o  d e t e c t i o n  o f  e a r l y  c a n c e r .  Among t h e  t o t a l  18 
p a t i e n t s  w i t h  s t a g e  I or s t a g e  I1 c a n c e r ,  o n l y  o n e  showed an 
e l e v a t e d  R I A  v a l u e  (1 o f  the 9 w i t h  u n t r e a t e d  e a r l y  c a n c e r ) .  
W e  t h e r e f o r e  c o n c l u d e  t h a t  n e i t h e r  a s s a y  i s  u s e f u l  as a 
s c r e e n i n g  p r o c e d u r e  f o r  e a r l y  d e t e c t i o n  o f  p r o s t a t i c  
c a r c i n o m a .  C o n t r a d i c t o r y  f i n d i n g s  were r e p o r t e d  b y  o t h e r  
a u t h o r s  ( 2 , 3 , 6 , 1 1 , 1 3 ) .  On t h e  other h a n d ,  B r u c e  e t  a1 (1) f o u n d  
e l e v a t e d  R I A  v a l u e s  i n  o n l y  8 p e r  c e n t  o f  p a t i e n t s  w i t h  s t a g e  I or 
11 p r o s t a t i c  c a n c e r .  

Between i n t r a c a p s u l a r  and e x t r a c a p s u l a r  c a n c e r ,  however, 
R I A  d i s c r i m i n a t e d  r e a s o n a b l y  w e l l  i n  o u r  s t u d y .  Thus i n  s t a g e  
I11 or I V ,  R I A  g a v e  e l e v a t e d  v a l u e s  i n  1 0  o f  11 u n t r e a t e d  
p a t i e n t s ,  w h i l e  t h e  e n z y m a t i c  a n a l y s i s  showed o n l y  4 w i t h  
s u c h  v a l u e s .  R I A  t h e r e f o r e  appears t o  be u s e f u l  f o r  the 
s t a g i n g  of p r o s t a t i c  c a n c e r .  

S t a n d a r d i z e d  methods a r e  e s s e n t i a l  when PAP d e t e r m i n a t i o n  
i s  u s e d  i n  t h e  c l a s s i f i c a t i o n  of p r o s t a t i c  disease. E x t e n d e d  
s t u d i e s  o f  l a r g e  series and r e p e a t  a n a l y s e s  i n  i n d i v i d u a l  
p a t i e n t s ,  both R I A  and measurement o f  c a t a l y t i c  a c t i v i t y ,  a r e  
r e q u i r e d  t o  c l a r i f y  i f  the t w o  methods c o n t i n u e  t o  r e f l e c t  
t h e  same c i r c u m s t a n c e s  w i t h  r e g a r d  t o  tumour p r o g r e s s i o n ,  

t h e r a p e u t i c  r e s p o n s e  and p r o g n o s i s .  

137 



REFERENCES 
1. Bruce, A.W., Mahan, D.E., Morales, A., Clark, A.F. & 

Belville, W.D. : An objective look at acid phosphatase 
determinations: A comparison of a biochemical and 
immunological methods. Rr J Urol 51(3), 213-217,1979. 

2. Carroll, B.J: Radioimmunoassay of prostatic acid phosphatase 
in carcinoma of the prostate. N Engl J Med 298(16), 912, 
1978. 

3. Cooper, J.F. & Foti, A.G.: A radioimmunoassay for prostatic 
acid phosphatase. Natol Cancer Inst Monogr 49, 235-237, 1978. 

4. Derechin, M., Rustum, Y.M. & Barnard, E.A. : Acid 
phosphomonesterase of human prostate. Molecular weight, 
dissociation and chemical composition. Biochim Biophys Acta 
250, 143-154, 1971. 

5. Dewers, L.M.: Prostatic acid phosphatase: Assay Comparisons. 
Laboratory Management 19, 8-16, 1981. 

6. Foti, A.G., Cooper, J.F., Herschman, H. & Malvaez, R.R.: 
Detection of prostatic cancer by solidphase radioimmunoassay 
of serum prostatic acid phosphatase. N Engl J Med 297(25), 
1357-1361, 1977. 

7. Griffiths, J.C.: Prostate-specific acid 
phosphatase:reevaluation of radioimmunoassay in diagnosing 
prostatic disease. Clin Chem 26, 433-437, 1980. 

8. Gutman, E.B., Sproul, E.E. & Gutman, A.B.: Significance of 
increased phosphatase activity of bone at the site of 
osteoplastic metastases secondary to carcinoma of the 
prostate gland. Am J Cancer 28, 485-495, 1936. 

9. Lundholm, G.R., Stirton, S . ,  Liedtke, R.J. & Batjer, J.D.: 
Prostatic acid phosphatase by radioimmunoassay. Sensitivity 
compared with enzymatic assay. JAMA 244(18), 2071-2073, 
1980. 

10. Snedecor, G.W. & Cochran, W.G.: Statistical Methods. Sixth 
Edition. The Iowa State University Press. Ames, Iowa, U.S.A., 
1967. 

11. Wajzman, Z . ,  Chu, T.M., Saroff, J., Slack, N. & Murphy, G.P.: 
Two new, direct and specific methods of acid phosphatase 
determination. Urology 13(1), 8-11, 1979. 

Group. Cancer of the prostate: treatment comparisons. J Urol 
12. The Veterans Administration Cooperative Urological Research 

98, 516-522, 1967. 

138 



13. Vihko, P., Sajanti, E., JZnne, O., Peltonen, L. & Vihko, R.: 
Serum prostate-specific acid phosphatase: development and 
validation of a specific radioimmunoassay. Clin Chem 24(11), 
1915-1919, 1978. 

Address for reprints : 
Gunnar Ronquist, M.D. 
Department of Clinical Chemistry, University Hospital 
s-751 85 Uppsala 

10-838572 139