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Differences of bone alkaline phosphatase isoforms in metastatic bone disease and discrepant effects of clodronate on different skeletal sites indicated by the location of pain.

Patients with breast, lung, and prostate cancer are particularly prone to develop metastatic bone disease. Most of the prostate cancer patients, >60%, will develop skeletal metastases at some stage of the disease; consequently bone pain is a major problem in these patients (1, 2). The recent development of new biochemical markers of bone turnover has generated interest to use these markers for early detection of bone metastases and to assess efficacy and the response to antiresorptive treatment for patients with metastatic bone disease (3-6). Two collagen markers of bone turnover, cross-linked carboxy-terminal telopeptide of type I collagen (ICTP) [5] and carboxy-terminal propeptide of type I procollagen, give some information about the type and activity of bone metastases; moreover, ICTP has been reported to have a prognostic value in prostate cancer (7). Serum alkaline phosphatase (ALP, EC and tartrate-resistant acid phosphatase (EC are increased in prostate cancer patients with bone metastases, and the highest predictive value from a positive bone scintigraphy was obtained with bone ALP (0.88) (8).

ALP is the most frequently used biochemical marker of osteoblastic bone formation. Four human gene loci are encoding for the ALP isoenzymes: "tissue nonspecific", placental, germ cell, and small intestinal locus (9). ALP from the tissue nonspecific locus is expressed in tissues such as bone and liver and constitutes ~95% of the total ALP activity in serum with a ratio of approximately 1:1 during healthy conditions in adults (10). Because bone and liver ALP are encoded by the same gene locus, they are referred to as isoforms of the same isoenzyme. Different carbohydrate side chains or maybe remaining fragments of the in situ cell membrane glycosyl-phosphatidylinositol anchor, or both, yield "tissue specific" structures in the ALP isoforms from this gene locus (11,12). In this study, we used a previously described HPLC method that can detect six different ALP isoforms in serum from healthy adults: one bone/intestinal (B/I), two bone (B1 and B2), and three liver ALP isoforms (L1, L2, and L3) (13,14).

Clodronate has been reported to have multiple effects on human bone, such as reducing bone pain, hypercalcemia, hypercalciuria, formation of new osteolytic lesions, further growth of existing lesions, and development of vertebral fractures in patients with tumor bone disease, and to prevent bone loss (15-20). The pharmacologically active bisphosphonates have indeed proved to be powerful inhibitors of bone resorption when tested in a variety of conditions both in vitro and in vivo; however, the precise mode of action is still not completely elucidated. In vitro studies of bisphosphonates have demonstrated powerful inhibitory effects on the function of existing osteoclasts with minor or conflicting effects on osteoclast recruitment (21-23). It has also been demonstrated that bisphosphonates affect osteoclasts not only directly but also indirectly via effects on cells of the osteoblast family (24, 25). The distribution of clodronate in bone of adult rats and its effects on trabecular and cortical bone has recently been described. The highest activity of [sup.14.C-clodronate] was found in the primary spongiosa of the distal femoral metaphysis and in the cortical bone of the femoral diaphysis (26). The distribution of clodronate in human bone has not, to our knowledge, been reported. The human skeleton is a heterogeneous tissue with a wide morphologic, functional, and metabolic variety in which cortical bone mainly fulfills a mechanical and protective function and the trabecular bone a metabolic function (27). Trabecular bone has ~5-fold more surface area per unit of volume than cortical bone, and a higher rate of metabolic activity and remodeling. Despite all these known differences, reports are sparse on biochemical markers of bone turnover in relation to trabecular and cortical bone. Bone markers are thus far not able to distinguish metabolic events in trabecular from cortical bone (28-30), although some differences have been found (31).

The aim of this randomized, double-blind study (clodronate or placebo) was to examine and compare serum total ALP, bone ALP isoforms, osteocalcin, ICTP, and prostate-specific antigen (PSA) in 45 healthy men with those same markers in 42 patients with primarily or secondarily hormone-refractory prostate cancer with skeletal metastases and who were suffering from persisting pain. We also investigated these markers of bone turnover with respect to the extent of metastases according to Soloway score, I to IV, and their association to different skeletal sites indicated by the localization of pain.

Materials and Methods


The study group was composed of 42 patients (mean age, 71 years; range, 54-86 years) with primarily or secondarily hormone-refractory prostate cancer, histopathologically confirmed evidence of skeletal metastases, and suffering from persisting pain despite analgesic treatment. Patients with other malignant diseases or who had a previous cancer that might interfere in the assessments were not included. No extreme hypercalcemic patients were included, defined as a corrected serum calcium concentration >2.85 mmol/L, corrected for serum albumin concentration. All patients had serum creatinine values <155 [micro]mol/L, indicating no serious deterioration of the glomerular filtration. None of the patients were treated with any other drugs known to affect calcium metabolism within 30 days or palliative radiotherapy within 3 weeks before the start of study treatment. The analgesic effect and safety of clodronate as well as the effect on quality of life compared with placebo in this material is reported elsewhere (32).

Bone metastases were diagnosed by bone scintigraphy after intravenous administration of [sup.99m.Tc-labeled] methylene disphosphonate and x-ray examinations of selected painful areas. Bone scintigraphy was performed on all patients and evaluated at one center (Uppsala) according to Soloway score I to N (33, 34). Pain intensity was assessed using a 10-cm-long graded Visual Analog Scale (35) on day 1 and after 1 month of treatment. The primary pain efficacy variable was the "main pain', which corresponded to mean pain during the last week and recorded at day 1 before treatment. A body picture was also filled in where the pain was localized and characterized as upper (U), lower (L), or upper and lower (UL) body pain. The pelvis and hip were classified as belonging to the lower body.

The control group for osteocalcin, ICTP, and bone ALP isoforms was composed of 45 healthy men (blood donors) with a mean age of 44 years (range, 25-65 years). This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committees of each study center: Danderyd, Eskilstuna, Goteborg, Linkoping, Norrkoping, and Uppsala. Written informed consent was obtained from each patient before participation in the study.


The patients were randomized either to the clodronate (n = 22) or the placebo arm (n = 20). The clodronate (Bonefos, Leiras Oy) group started with intravenous administration, 300 mg/day for 3 days, followed by oral administration of clodronate, 3200 mg/day (1600 mg twice a day) during 4 weeks. The placebo group started with isotonic saline solution for 3 days, followed by placebo capsules during 4 weeks. Thirty-nine of the 42 patients completed the treatment period. One patient in the placebo group stopped treatment because of severe pain, and two patients in the clodronate group ended because of severe diarrhea and a femur fracture, respectively. Samples for determination of serum total ALP, bone and liver ALP isoforms, osteocalcin, ICTP, and PSA were taken before treatment and after 1 month of treatment.


The bone and liver ALP isoforms were separated and quantified by a previously described HPLC method (13,14). A weak anion-exchange column, SynChropak AP300 (250 X 4.6 mm i.d.; MICRA Scientific, Inc.) was used instead of the referred SynChropak AX300. SynChropak AP300 is a modified SynChropak AX300, optimized for bone ALP isoform analysis (10). Serum total ALP activity was measured on a Hitachi 917 analyzer (Boehringer Mannheim GmbH) at 37[degrees]C (36). The relation between the enzymatic activity units and [micro]kat is 1/60, i.e., 1 U/L corresponds to 0.01667 [micro]kat/L. Serum osteocalcin was determined by RIA with a double antibody technique (INCSTAR) (37), and serum ICTP was determined by RIA (Orion Diagnostica) (38). Serum PSA was determined by an IMx immunoassay (Abbott Laboratories) (39).



All calculations were performed with the StatView [R] 4.5 program (Abacus Concepts, Inc.). Nonparametric statistics were used because the distributions were not gaussian according to the Kolmogorov-Smirnov test. The Mann-Whitney test was used to test for differences between the groups and subgroups: healthy men, prostate cancer patients with bone metastases, placebo, clodronate, Soloway score, main pain, and upper and lower pain of the body. To examine differences between the paired observations before treatment and 1 month after treatment, we used the Wilcoxon signed-rank test. To measure the association of linear relationship between variables before treatment and after 1 month, we calculated Kendall's tau rank correlation coefficient. For all statistical tests, a difference was considered significant at P <0.05.


We were able to separate and quantify six ALP isoforms in each serum sample during the entire study period: three bone (B/I, B1, and B2), and three liver ALP isoforms (L1, L2, and L3). ALP isoform chromatographic profiles are presented from one healthy male and one prostate cancer patient with bone metastases (Fig. 1).


Osteocalcin, ICTP, total ALP, and all three bone ALP isoforms, B/I, B1, B2, were increased in prostate cancer patients with bone metastases as compared with healthy men (Table 1). The most apparent increase was observed for B2. Patients and healthy men had a B2 activity corresponding to 75% and 35% of the total ALP activity, respectively. The calculated ratio B1/B2 was significantly decreased because of considerable large B2 isoform activities in the patient group with bone metastases (Table 1). PSA was increased in 37 patients (median, 348 [micro]g/L; range, 2.4-6400 [micro]g/L) compared with age-specific reference interval limits (40). As expected, the correlation coefficients were high between total ALP and all bone ALP isoforms (Table 2). ICTP correlated with all the other biochemical markers at baseline, including PSA (Table 2). We also found a positive correlation between PSA and the bone ALP isoform B2. However, no significant correlation was found between PSA and the bone ALP isoform B1, total ALP, or osteocalcin (Table 2).

The patients were classified according to Soloway score and grouped into three categories: Soloway I (n = 7), Soloway II (n = 19), and Soloway III-IV (n = 11). Markers of bone turnover and PSA increased with the extent of bone metastases indicated by Soloway score from group I to group III-IV. No differences were found for markers of bone turnover and PSA between Soloway groups I and II. However, we found significant differences between Soloway groups II and III-IV for total ALP and the bone ALP isoforms, B1 and B2.

We found significant negative correlations between the primary pain efficacy variable main pain and total ALP, r = -0.22; P <0.05, and between main pain and B2, r = -0.23; P <0.05. The other markers of bone turnover and PSA showed no significant associations. The patients were also classified in terms of body pain according to where the pain was localized. Body pain was characterized as U (n = 11), L (n = 9), and UL (n = 22). No significant differences were found at baseline for markers of bone turnover and PSA between the body pain groups U and L (Table 3). However, a significant difference was found between the body pain groups L and UL for the bone ALP isoform B1 (P <0.05; Table 3).


We found increased concentrations of PSA but no significant differences for the markers of bone turnover in the placebo group (Fig. 2). In the clodronate group, we found increased PSA, ICTP, total ALP, B/I, B1, and B2, whereas no change was observed for osteocalcin (Fig. 2). We found no differences in the placebo group between the body pain groups U and L for PSA and markers of bone turnover. However, after 1 month of clodronate treatment, significantly (P <0.05) higher activities were found for osteocalcin, ICTP, total ALP, B1, and B2 in the body pain group U (n = 6) compared with L (n = 6), despite the small number of patients in each group. No significant associations were observed between the primary pain efficacy variable main pain and the markers of bone turnover or PSA.


The results of the present study demonstrate, as expected, that all the measured markers of bone turnover were higher in the prostate cancer patients with skeletal metastases compared with healthy men. However, we found significant differences between the markers of bone turnover. The largest increase was observed for the bone ALP isoform B2. Prostate cancer patients and healthy men had a B2 activity corresponding to 75% and 35% of the total ALP activity, respectively (P <0.0001). Increased B2 activities have previously been demonstrated for patients with Paget's disease of bone (41). An opposite finding has been observed in growth hormone-deficient adults who have increased B1 activities as compared with healthy adults (10). Other selective differences between the bone ALP isoforms have also been described in disease states such as hypophosphatasia and stress fractures (41). Moreover, we have previously shown that adolescent girls reach higher values than boys of the B1 /B2 ratio in Tanner stage N-V, because of a more rapid decline of the bone ALP isoform B2 compared with B1 after puberty (42). Taken together, we propose that the different bone ALP isoforms reflect different stages in osteoblast differentiation during osteogenesis, where one isoform is presented before the other during the extracellular matrix maturation phase. The bone ALP isoforms may indicate further osteoblast differentiation, i.e., when osteoblasts mature to bone lining cells, or osteocytes, or undergo apoptosis.

Osteogenesis involves three major events: proliferation with collagen synthesis, matrix maturation, and mineralization. In vitro studies have shown that gene expression of collagen type I occurs first, followed by ALP, and then osteocalcin (43). Reflecting the modest increase at baseline of osteocalcin in most of our patients, our results indicate that the bone metastases to a large extent consist of nonmineralized tissue, which also is in agreement with the morphological findings of metastatic bone. The same interpretation can be made in patients with osteomalacia in whom the ALP activities are strongly increased and the osteocalcin concentrations are within reference values or slightly raised (44). Previously described findings of slightly raised concentrations of the carboxy-terminal propeptide of type I procollagen and osteocalcin and grossly increased ALP activities in patients with skeletal metastases (44) indicate that osteoblast differentiation is disturbed during the mineralization phase of osteosclerotic lesions. During unaffected osteoblast differentiation, mineralization down-regulates expression of genes, such as ALP, associated with the matrix maturation period (43). One previously proposed function for ALP is that ALP is necessary for the initiation of mineralization but not for the continuation of mineralization of bone nodules in vitro (45, 46). Osteocalcin, the most abundant osteoblast-specific noncollagenous protein, has been reported to be 21-fold more abundant in cortical than trabecular bone on the basis of bone weight (31). Cortical bone is less metabolically active than trabecular bone; therefore, this could be one of the reasons why osteocalcin sometimes do not respond as well as other markers of bone turnover during certain pharmacological therapies and in different disease groups, such as in chronic renal failure, Paget's disease, multiple myeloma, skeletal metastases, and osteomalacia (44, 47, 48).

The increased concentrations of total ALP, bone ALP isoforms, and ICTP after 1 month of clodronate treatment in metastatic bone disease are in conjunction with other reports (49, 50). However, a decrease in bone formation markers tends to occur 2-3 months later, reflecting the coupling between resorption and formation. The increase in markers of bone formation soon after bisphosphonate treatment has been attributed to increased recruitment of osteoblasts to sites undergoing bone resorption or increased activity of preexisting osteoblasts (51). Either mechanism might suggest the repair of metastatic bone. The development of osteomalacia because of hypocalcemia, hypophosphatemia, and secondary hyperparathyroidism has also been proposed as a reason for increased bone ALP activities during clodronate treatment (49). The increased markers of bone turnover were significantly associated with different skeletal sites indicated by the location of body pain. With respect to the small number of patients in each body pain group, further investigations must be made before any major conclusions can be drawn on the basis of our findings. We suggest, however, that the uptake of clodronate by the skeleton was not uniform and appeared to be dependent in part on bone blood flow, and it was particularly marked at sites of active bone formation and mineralization.


Despite almost exclusively osteosclerotic lesions in prostate cancer, we found increased concentrations of ICTP. This finding could to some extent be explained by coupling of bone formation to resorption induced by growth factors (52). ICTP reflects the degradation of mature type I collagen, i.e., type I collagen cross-linked by pyridinolines or pyrroles. Bone collagen degradation is tightly coupled with bone resorption. However, if the newly formed nonmineralized organic matrix of bone is not mineralized, it is possible that it will be degraded, and such a process is not bone resorption by definition. Thus, the breakdown of the collagenous matrix may give rise to an increased ICTP concentration despite the absence of an obvious bone resorption.

In conclusion, we found that prostate cancer patients with skeletal metastases have grossly increased activities of the bone ALP isoform B2. We propose that the different bone ALP isoforms, B1 and B2, reflect different stages of osteoblast differentiation during osteogenesis, where one isoform is presented before the other during the extracellular matrix maturation phase. The bone ALP isoforms may indicate further osteoblast differentiation, i.e., when osteoblasts mature to bone lining cells, or osteocytes, or undergo apoptosis. After 1 month of clodronate administration, we demonstrated increased concentrations for all markers of bone turnover, except for osteocalcin, which were significantly associated with pain located only in the upper part of the body. Therefore, we suggest that the uptake of clodronate by the skeleton was not uniform during our treatment period. Future investigations, such as determination of the different bone ALP isoforms with respect to trabecular and cortical bone, are necessary to clarify the clinical significance of the site-specific differences found in this study. The bone ALP isoforms B1 and B2 should also be further evaluated concerning their ability for early detection of bone metastases and to assess efficacy and the response to antiresorptive treatment for patients with metastatic bone diseases.

This study was supported by grants from Leiras Oy, Finland; Astra Lakemedel AB, Sweden; and the County Council of 0stergotland (Nos. 94/180 and 95/123). This multicenter study was performed in five Swedish cities. Principal investigators were as follows: Dr. Sten Nilsson and Dr. Peter Strang, Department of Oncology, Uppsala University Hospital, Sweden; Dr. Stephan Brandstedt and Dr. Jan Sehlin, Department of Urology, Danderyd Hospital, Sweden; Dr. Eberhard Varenhorst, Department of Urology, Norrkoping Hospital, Sweden; Dr. Goran Borghede, Department of Oncology, Sahlgrenska University Hospital, Goteborg, Sweden; Dr. Ulf Bandmann, Department of Oncology; and Dr. Leif Borck, Department of Urology, Eskilstuna Hospital, Sweden.


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[1] Bone and Mineral Metabolic Unit, Division of Clinical Chemistry, Department of Biomedicine and Surgery, Linkoping University Hospital, S-581 85 Linkoping, Sweden.

[2] Department of Mathematical Statistics, Royal Institute of Technology, S-100 44 Stockholm, Sweden.

[3] Division of Oncology, Department of Biomedicine and Surgery, Linkoping University Hospital, S-581 85 Linkoping, Sweden.

[4] Clinical Research, Medical Department, Astra Lakemedel AB, 5-151 85 Sodertalje, Sweden.

[5] Nonstandard abbreviations: ICTP, cross-linked carboxy-terminal telopeptide of type I collagen; ALP, alkaline phosphatase; B/I, bone/intestinal alkaline phosphatase; B1, bone 1 alkaline phosphatase; B2, bone 2 alkaline phosphatase; B1/B2 ratio, bone 1 alkaline phosphatase/bone 2 alkaline phosphatase ratio; L1, liver 1 alkaline phosphatase; L2, liver 2 alkaline phosphatase; L3, liver 3 alkaline phosphatase; PSA, prostate-specific antigen; U, upper body; and L, lower body.

* Author for correspondence. Fax 46-13-223240; e-mail Per.Magnusson@

Received January 30, 1998; revision accepted June 2, 1998.
Table 1. Markers of bone turnover in prostate cancer patients with
bone metastases compared with healthy males. (a)

 Biochemical markers Healthy males (e) Patients with bone
 metastases (a)

Total ALP, U/L 156 (102-234) (b) 732 (132-3540) (b,c)
B/I, U/L 4.2 (2.4-9.0) 9.0 (3.0-54) (c)
B1, U/L 23 (11-44) 101 (14-756) (c)
B2, U/L 57 (29-98) 526 (41-2824) (c)
B1/B2 ratio 0.42 (0.23-0.81) 0.20 (0.08-0.57) (c)
Osteocalcin, [micro]g/L 3.0 (0.6-4.3) 4.9 (0.6-15.9) (c)
ICTP, [micro]g/L 3.1 (1.8-4.8) 10.2 (2.5-40.0) (c)

(a) Healthy males, n = 45. Prostate cancer patients with bone
metastases, n = 42.

(b) Values are given as median, with the minimum and maximum values in

(c) P <0.0001, Mann-Whitney test between healthy males and prostate
cancer patients with bone metastases.

Table 2. Correlation coefficients between markers of bone turnover and
PSA in prostate cancer patients with bone metastases. (a)

 Biochemical markers Total ALP B/I B1 B2

B/I 0.67 (b)
B1 0.81 (b) 0.76 (b)
B2 0.96 (b) 0.65 (b) 0.79 (b)
B1/B2 ratio -0.34 (b) -0.07 -0.15 -0.36 (d)
Osteocalcin 0.32 (b) 0.20 0.30 (c) 0.34 (c)
ICTP 0.55 (b) 0.44 (b) 0.50 (b) 0.56 (b)
PSA 0.21 0.17 0.17 0.22 (e)

 Biochemical markers B1/B2 ratio Osteocalcin ICTP

B1/B2 ratio
Osteocalcin -0.20
ICTP -0.29 (c) 0.38 (d)
PSA -0.18 0.17 0.41 (d)

(a) n = 42.

(b-e) Values represent Kendall's tau rank correlation coefficient: (b)
P <0.0001; (c) P <0.01; (d) P <0.001; (e) P <0.05.

Table 3. Markers of bone turnover and PSA in prostate cancer patients
with bone metastases classified according to localization of body
 Localization of body pain

 Biochemical markers U (a) UL

Total ALP, U/L 540 (174-2520) (b) 1068 (192-3540) (b)
B/I, U/L 9.6 (3.6-25) 9.6 (3.0-54)
B1, U/L 99 (26-342) 133 (14-756)
B2, U/L 377 (85-2015) 727 (67-2824)
B1/B2 ratio 0.20 (0.12-0.50) 0.20 (0.08-0.37)
Osteocalcin, [micro]/L 6.3 (0.6-14.2) 5.0 (0.6-15.9)
ICTP, [micro]/L 8.4 (3.3-27.8) 10.9 (2.7-40.0)
PSA, [micro]/L 163 (2.4-4700) 498 (14-6400)

 Localization of body pain

 Biochemical markers L

Total ALP, U/L 354 (132-2940) (b)
B/I, U/L 6.6 (3.0-19)
B1, U/L 49 (22-329) (c)
B2, U/L 238 (41-2395)
B1/B2 ratio 0.20 (0.08-0.57)
Osteocalcin, [micro]/L 3.7 (1.7-9.0)
ICTP, [micro]/L 6.5 (2.5-15.8)
PSA, [micro]/L 66 (7.0-986)

(a) U, n = 11; UL, n = 22; L, n = 9.

(b) Values are given as median, with the minimum and maximum values in

(c) P <0.05, Mann-Whitney test between the body pain group UL compared
with L.
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Title Annotation:Enzymes and Protein Markers
Author:Magnusson, Per; Larsson, Lasse; Englund, Gunnar; Larsson, Brita; Strang, Peter; Selin-Sjogren, Lena
Publication:Clinical Chemistry
Article Type:Clinical report
Date:Aug 1, 1998
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