Bone alkaline phosphatase isoenzyme in renal osteodystrophy.
ABSTRACT Serum total alkaline phosphatase is the most commonly used biochemical marker of bone disease in renal patients, but alkaline phosphatase originates from different organs and sometimes lacks specificity. Bone isoenzyme measurement is considered superior to total alkaline phosphatase for the assessment of bone metabolism. We have studied the value of bone isoenzyme, determined by a new. IRMA (Tandem-R-Ostase), in haemodialysis patients with secondary hyperparathyroidism and renal osteodystrophy. Fifty-six haemodialysis patients were studied. Intact parathyroid hormone (PTH), osteocalcin, total alkaline phosphatase and bone alkaline phosphatase were determined. A transiliac bone biopsy was performed in 20 of the 56 patients after double tetracycline labelling. There was a significant correlation between bone alkaline phosphatase and PTH (r = 0.79, P < 0.001) and between bone and total alkaline phosphatase (r = 0.84, P < 0.001) in all patients. The patients who underwent a bone biopsy showed osteitis fibrosa in 17, mixed lesion in one, adynamic bone disease in one and normal bone in one. Bone alkaline phosphatase showed a significant correlation with static and dynamic histomorphometric indices similar to that obtained with PTH and better than those of total alkaline phosphatase and osteocalcin. It is concluded that bone alkaline phosphatase (ostase) seems to be a useful non-invasive marker of bone metabolism in patients on haemodialysis with high turnover bone disease. More studies are necessary to know its value in low turnover bone disease.
- SourceAvailable from: clinchem.org[show abstract] [hide abstract]
ABSTRACT: This two-site IRMA includes specific monoclonal antibodies for measuring skeletal alkaline phosphatase (B-ALP) in human serum. Assay calibration is based on mass units (micrograms per liter) and was established with purified B-ALP from a human osteosarcoma cell line, SAOS-2. Precision studies demonstrated intra- and interassay CVs of 3-5% and 5-7%, respectively. Relative reactivity studies showed that the assay has a sevenfold preference for detecting B-ALP compared with the liver isoenzyme in serum. The normal reference interval for 478 healthy adults was 5-22 micrograms/L. Method comparison studies showed good correlation between this B-ALP assay (y) and commercially available electrophoretic methods (x) (y = 0.3540x + 20.5, R2 = 0.929) in a pagetic population. Temporal profiles for total ALP, this IRMA B-ALP assay, and B-ALP by electrophoresis in three pagetic patients were parallel. We conclude that this assay demonstrates good analytical performance and would be useful for the clinical assessment of metabolic bone disorders.Clinical Chemistry 06/1994; 40(5):822-8. · 7.15 Impact Factor
- Clinica Chimica Acta 02/1990; 186(2):315-20. · 2.85 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: We measured serum bone alkaline phosphatase (B-ALP) with a new immunoradiometric assay (IRMA) in a large sample of healthy controls comprising 173 women and 180 men, 20-88 yr of age, and in patients with metabolic bone disease. Using serum samples from patients with liver disease and patients with Paget's disease with elevated total alkaline phosphatase (T-ALP) as a source of, respectively, liver and bone isoenzymes, we determined a liver cross-reactivity of the IRMA of 16% that was confirmed by electrophoresis of the circulating alkaline phosphatase isoenzymes. The IRMA was linear for serial sample dilutions, the recovery ranged from 89-110%, and the intra- and interassay variations were below 7% and 9%, respectively. B-ALP increased linearly with age in both sexes, and the mean B-ALP serum levels were not significantly different for women and men (11.3 +/- 4.8 ng/mL for women; 11.0 +/- 4.0 ng/mL for men). The increase in B-ALP after the menopause was significantly higher than that in T-ALP (+77% vs. +24%; P < 0.001). When the values of postmenopausal women were expressed as the SD from the mean of premenopausal women, the mean Z scores were 2.2 +/- 1.8 for B-ALP and 0.9 +/- 1.3 for T-ALP (P < 0.001 between the two). Serum B-ALP was increased from control values in patients with Paget's disease (n = 57; mean, 171.8 +/- 135.6 ng/mL; P < 0.001), in patients with primary hyperparathyroidism (n = 18; mean, 17.2 +/- 5.9 ng/mL; P < 0.001), and in patients with chronic renal failure on hemodialysis (n = 83; mean, 36.6 +/- 35.7 ng/mL; P < 0.001). In patients with Paget's disease, B-ALP was highly correlated with T-ALP (r2 = 0.94; P < 0.001), and the decrease in its serum level was larger than that in T-ALP after treatment with the bisphosphonate pamidronate (-58% vs. -43%; P < 0.03). In patients with various liver diseases, B-ALP was slightly increased, but stayed within the normal range (mean +/- 2 SD) until T-ALP did not exceed 4.5 mu katal/L. We conclude that this new IRMA for B-ALP is reliable, has a low cross-reactivity with the liver isoenzyme, and appears to be more sensitive than T-ALP for the clinical investigation of patients with osteoporosis and other metabolic bone diseases.Journal of Clinical Endocrinology & Metabolism 10/1993; 77(4):1046-53. · 6.43 Impact Factor
Nephrol Dial Transplant (1996) 11 [Suppl 3]: 43-46
Bone alkaline phosphatase isoenzyme in renal osteodystrophy
C. Jarava1, J. R. Armas2, M. Salgueira1 and A. Palma1
'Nephrology and 2Pathology Services, Hospital Universitario Virgen Macarena, 41071 Seville, Spain
Abstract. Serum total alkaline phosphatase is the most
commonly used biochemical marker of bone disease in
renal patients, but alkaline phosphatase originates
from different organs and sometimes lacks specificity.
Bone isoenzyme measurement is considered superior
to total alkaline phosphatase for the assessment of
bone metabolism. We have studied the value of bone
isoenzyme, determined by a new IRMA (Tandem-R-
Ostase), in haemodialysis patients with secondary
hyperparathyroidism and renal osteodystrophy. Fifty-
six haemodialysis patients were studied. Intact para-
thyroid hormone (PTH), osteocalcin, total alkaline
phosphatase and bone alkaline phosphatase were deter-
mined. A transiliac bone biopsy was performed in 20
of the 56 patients after double tetracycline labelling.
There was a significant correlation between bone alkal-
ine phosphatase and PTH (r=0.79, P<0.001) and
between bone and total alkaline phosphatase (r=0.84,
P< 0.001) in all patients. The patients who underwent
a bone biopsy showed osteitis fibrosa in 17, mixed
lesion in one, adynamic bone disease in one and normal
bone in one. Bone alkaline phosphatase showed a
significant correlation with static and dynamic histo-
morphometric indices similar to that obtained with
PTH and better than those of total alkaline phosphat-
ase and osteocalcin. It is concluded that bone alkaline
phosphatase (ostase) seems to be a useful non-invasive
marker of bone metabolism in patients on haemodia-
lysis with high turnover bone disease. More studies are
necessary to know its value in low turnover bone
Key words: bone alkaline phosphatase; renal
Bone histomorphometry is accepted as the best method
for the study of renal osteodystrophy. However bone
biopsy is an invasive procedure accompanied by tech-
nical difficulties in the processing and studying of the
Correspondence and offprint requests to: Dr Alfonso Palma, Servicio
de Nefrologia, Hospital Universitario Virgen Macarena, Avda.
Dr Fedriani, s/n, 41071 Sevilla, Spain.
specimens. This explains why bone biopsy is not widely
performed and why numerous biochemical markers
have been proposed for examining bone disease.
Serum alkaline phosphatase has been used as a
biochemical marker of bone disease for many years.
But total alkaline phosphatase originates from several
organs (liver, bone, intestine, placenta etc.) and its
measurement lacks specificity . Recently a new
method for serum bone alkaline phosphatase measure-
ment has been developed. This method, Tandem-R-
Ostase (Hybritech, San Diego, CA), is a two site
IRMA that involves specific monoclonal antibodies to
measure bone alkaline phosphatase in serum, and has
been previously validated [1-4].
The aims of the present study were: (1) to determine
the correlations between serum bone alkaline phos-
phatase and other biochemical parameters of bone
metabolism in haemodialysis patients; and (2) to
analyse the relationship between bone alkaline phos-
phatase and the histomorphometric parameters in a
group of dialysis patients.
Subjects and methods
The study was carried out in 56 chronic renal failure patients
being treated with chronic haemodialysis. They were 32
males and 34 females, aged 48+15 years (mean + SD) and
on haemodialysis treatment for 71+53 months. They were
dialysed with a standard membrane and had been under
treatment with aluminium hydroxide or calcium carbonate
in different doses. None of the patients had clinical or
biochemical evidence of liver disease. At least half of the
patients were receiving oral calcitriol at a dose not greater
than 1.5ng/week, but they had stopped treatment no less
than 15 days before the study was performed. Immediately
before a dialysis session, blood samples were drawn from
the arterial port for the assay of intact parathyroid hormone
(PTH), osteocalcin, total alkaline phosphatase and bone
Bone biopsy was performed in 20 of the 56 patients.
Transiliac bone biopsy was obtained with a 7 mm Bordier
trephine after double tetracycline labelling which was separ-
ated by an interval of 10 days. Bone samples were fixed in
70% ethanol and then embedded in methylmethacrylate.
Bone sections were stained with Masson-Goldner stain and
the aurintricarboxylic acid method for the identification of
aluminium at the mineral-osteoid interface. For histo-
© 1996 European Dialysis and Transplant Association-European Renal Association
by guest on July 14, 2011
C. Jarava et al.
dynamic evaluation ultraviolet light was used for the identi-
fication of the fluorescent tetracycline labels. The following
histological parameters were measured: osteoblast surface
(ObS/BS, %), osteoclast number per mm2 tissue section
(NOc/TA, mm2), osteoclastic surface (OcS/BS, %), volume
of fibrosis (FbV/TV%), osteoid surface (OS/BS %), osteoid
volume (OV/BV, %) and osteoid seam thickness (OTh, urn).
The dynamic parameters evaluated were: mineral apposi-
tional rate (MAR, urn/day) and bone formation rate
(BFR/BS, |im3/um2/day). Bone biopsies were classified
according to the method of Sherrard et al. [5,6].
Intact PTH was measured by immunoradiometric assay
(Allegro, Nichols Institute, San Juan Capistrano, CA, USA);
the normal range for this assay is 10-65 pg/ml. Serum
osteocalcin was measured by radioimmunoassay (INCSTAR
Corporation, Rochester, Minnesota); the normal range is
5-35 ng/ml for males and 8-55 ng/ml for females. Total
alkaline phosphatase (normal range 30-115 U/l) was deter-
mined by autoanalyser (SMAC, Technicon Instruments
Corp., Tarrytown, New York, USA). Bone alkaline phos-
phatase was measured by an IRMA using two monoclonal
antibodies directed against the human bone isoenzyme and
bone alkaline phosphatase purified from human SAOS-2
osteosarcoma cells as a standard (Ostase, Hybritech, San
Diego, CA, USA). Intra- and interassay coefficients of
variation are less than 7% and 9%, respectively. The assay
has shown a cross-reactivity of only 16% with the circulating
liver isoenyme . The sensitivity of the assay is 0.2 ng/ml.
The normal range for bone alkaline phosphatase is
Statistical analysis included the -evaluation of correlation
matrix, linear regression analysis and analysis of variance.
Data are expressed as the mean ± SD.
Mean humoral values of the patients under study are
shown in Table 1, together with the normal ranges.
Table 2 shows a correlation matrix. Mean values above
the normal range suggest a secondary hyperparathy-
roidism in most of our patients.
Bone alkaline phosphatase correlated significantly
Table 3. Relationship between biochemical indices of bone turnover
and histomorphometric indices of bone metabolism in 20 patients
with the other humoral parameters, with a correlation
coefficient of 0.79 (P< 0.001) with PTH and 0.84
(P<0.001) with total alkaline phosphatase (Table 2).
The histopathological diagnoses in the 20 patients
who had a bone biopsy were: severe osteitis fibrosa in
15, mild osteitis fibrosa in two, mixed lesion in one,
adynamic bone disease in one and normal bone in one.
Aluminium staining was negative in the 20 patients.
Table 3 shows the correlation between the four hum-
oral indices and the histomorphometric parameters.
Humoral markers correlated significantly with most
of the histomorphometric indices. Bone alkaline phos-
phatase and PTH showed the best correlation coeffi-
cients with both static and dynamic parameters.
Figure 1 shows the relationship between bone alkaline
phosphatase and several histomorphometric indices
(ObS/BS, Fb/TV, NOc/mm2, BFR).
Table 1. Mean biochemical values in 56 patients on haemodialysis
38 + 30
513 + 460
573 + 560
SCa, serum calcium; SP, serum phosphate; TAP, total alkaline
phosphatase; BAP, bone alkaline phosphatase; BGP, bone GLA
protein or osteocalcin.
This study analyses the value of serum bone alkaline
phosphatase, determined by a new IRMA (Ostase), as
a marker of bone turnover in haemodialysis patients.
Serum bone alkaline phosphatase is considered super-
ior to the determination of total alkaline phosphatase
activity for assessing bone metabolism . Several
methods have been developed to measure serum bone
alkaline phosphatase, including heat inactivation ,
agarose gel electrophoresis , wheat germ lectin
precipitation  and HPLC . However, these
methods have been considered to be cumbersome and
not always specific and they have not received wide
acceptance [1,3,7,12]. More recently, immunoassays
by guest on July 14, 2011
Bone alkaline phosphatase in renal osteodystrophy
0 10 20 30 40 50 60 70 80 90 100 110 120
N.Oc / mm2
0 10 20 30 40 50 60 70 80 90 100 110 120
0 10 20 30 40 50 60 70 80 90 100 110 120
Fig. 1. Relationship between bone alkaline phosphatase (BAP) and four histological indices: ObS/BS (%) (osteoblastic surface) Fb/TV(%)
(volume of fibrosis) NOc/mm2 (osteoclast number) and BFR (um3/um2/day) (bone formation rate).
with monoclonal antibodies that preferentially distin-
guish between bone and liver isoenzymes have been
developed [2,13,14]. We have used the new inmmunor-
adiometric assay (Tandem-R Ostase)  to measure
the serum bone alkaline phosphatase in our patients.
This assay has been used to study bone metabolism in
patients with Paget disease of bone, postmenopausal
osteoporosis, malignancies, primary hyperparathyroid-
ism, and secondary hyperparathyroidism in patients
with chronic renal failure [1,3,4,15,16]. Van Hoofer al.
compared the results of this IRMA with those of
agarose electrophoresis for a group of patients includ-
ing patients on haemodialysis . They found a good
correlation between the two methods except for low
values of bone alkaline phosphatase and in some
patients with increased concentrations of total alkaline
phosphatase, both due to cross-reactivity of anti-bone
alkaline phosphatase antibodies with liver isoenzyme.
A similar study by Garnero et al. reported a cross-
reactivity of the bone alkaline phosphatase (ostase) for
the liver alkaline phosphase of 16% . They found
that this IRMA is a sensitive method to measure bone
alkaline phosphatase and that, even in patients with
liver disease, can be used to assess bone turnover when
total alkaline phosphatase does not exceed 2.6-fold of
the upper limit of the normal range. Our patients had
no clinical or biochemical evidence of liver disease and
total alkaline phosphatase never exceeded those limits.
In our group of haemodialysis patients serum bone
alkaline phosphatase correlated positively and signi-
ficantly with other biochemical markers of renal osteo-
dystrophy. This is in agreement with other previous
studies that include haemodialysis and CAPD patients
and also predialysis patients [17,18]. Serum bone alkal-
ine phosphatase also showed a significant correlation
to both static and dynamic histomorphometric para-
meters, with r values similar to those of PTH and
greater than those of total alkaline phosphatase and
osteocalcin, as has been previously reported .
Bone alkaline phosphatase is a tetrameric glycopro-
tein found on the cell surface of active osteoblasts
lining the osteoid tissue undergoing the mineralization
process [19,20]. Consequently it is commonly consid-
ered a marker of bone formation . However, in
some pathological states both events, bone formation
and bone resorption, may be coupled and in balance
and the biochemical markers will reflect the overall
rate of bone turnover . This explains why bone
alkahne phosphatase shows a similar correlation to
histomorphometric parameters of formation and
Most of our patients had osteitis fibrosa. As a
by guest on July 14, 2011
consequence our study can only suggest that bone
alkaline phosphatase (ostase) may be a useful marker
of high bone turnover in patients on haemodialysis, as
has been previously reported [16,18]. More studies are
necessary to know the usefulness of this IRMA in
patients with low bone turnover (osteomalacia,
adynamic bone disease).
1. Panigrahi P, Delmas PD, Singer F et al. Characteristics of a
two-site immunoradiometric assay for human skeletal alkaline
phosphatase in serum. Clin Chem 1994; 40: 822-828
2. Hill CS, Wolfert RL. The preparation of monoclonal antibodies
which react preferentially with human bone alkaline phosphatase
and not liver alkaline phosphatase. Clin Chim Ada 1989; 186:
3. Garnero P, Delmas PD. Assessment of the serum levels of bone
alkaline phosphatase with a new Immunoradiometric assay in
patients with metabolic bone disease. J Clin Endocrinol Metab
1993; 77: 1046-1053
4. De la Piedra C, Rapado A, Diaz Martin A, Lopez Gavilanes E,
Gonzalez Parra E. Correlation among levels .of skeletal alkaline
phosphatase (sALP) measured by a new immunoradiometric
assay and intact PTH and other biochemical markers of bone
turnover in patients with chronic renal failure (CRF) (Abst).
Calcif Tissue Int 1993; 52 (suppl): 24
5. Sherrard DJ, Hercz G, Pei Y et al. The spectrum of bone disease
in end-stage renal failure: An evolving disorder. Kidney Int 1993;
6. Hernandez D, Conception MT, Lorenzo V et al. Adynamic
bone disease with negative aluminium staining in predialysis
patients: prevalence and evolution after maintenance dialysis.
Nephrol Dial Transplant 1994; 9: 517-523
7. Deftos LJ. Bone protein and peptide assays in the diagnosis and
management of skeletal disease. Clin Chem 1991; 37: 1143-1148
8. Moss DW, Whitby LG. A simplified heat inactivation method
for investigating alkaline phosphatase isoenymes in serum. Clin
Chim Ada 1975; 61: 63-71
9. Van Hoof VO, Lepoutre LG, Hoylaerts MF, Chevigne R,
DeBroe ME. Improved agarose electrophoretic method for
C. Jarava et al.
separating alkaline phosphatase isoenzymes in serum. Clin Chem
1988; 34: 1857-1862
10. Rosalki SB, Foo AY. Two new methods for separating and
quantifying bone and liver alkaline phosphatase. Clin Chem
1984; 30: 1182-1186
11. Magnusson P, Lofrnan O, Larsson L. Determination of alkaline
phosphatase isoenzymes in serum by high-performance liquid
chromatography with post-column
J Chromatogr 1992; 576: 79-86
12. Stepan JJ, Lachmanova J, Strakova M, Pacovsky V. Serum
osteocalcin, bone alkaline phosphatase isoenyme and plasma
tartrate-resistant add phosphatase in patients on chronic main-
tenance hemodialysis. Bone Min 1987; 3: 177-183
13. Lawson GM, Katzmann JA, Kimlinger TK, O'Brien JF.
Isolation and preliminary characterization of a monoclonal
antibody that interacts preferentially with the liver isoenzyme of
human alkaline phosphatase. Clin Chem 1985; 31: 381-385
14. Seabrook RN, Bailyes EM, Price CP, Siddle K, Lizio JP. The
distinction of bone and liver isoenzymes of alkaline phosphatase
in serum using a monoclonal antibody. Clin Chem Ada 1988;
15. Garnero P, Shih WJ, Gineyts E, Karpf DB, Delmas P.
Comparison of new biochemical markers of bone turnover in
late postmenopausal osteoporotic women in response to alend-
ronato treatment. J Clin Endocrinol Metab 1994; 79: 1693-1700
16. Van Hoof VO, Martin M, Blockx P et al. Immunoradiometric
method and electrophoretic system compared for quantifying
bone alkaline phosphatase in serum. Clin Chem 1995; 41:
17. Garcia Ramon R, Miguel A, Alonso JC, Tajahuerce M, Laporta
P. Estudio comparativo del isoenzima oseo de la fosfatasa
alcalina con otros marcadores de remodelado oseo. Nefrologia
(Madr) 1994; 14: 322-328
18. Lorenzo V, Conception T, Hernandez D et al. Valor predictivo
de la isoenzima osea de la FA (FAO) en el diagnostico histoldg-
ico de la osteodistrofia renal (ODR) (Abst). Nefrologia (Madr)
1994; 14 (suppl 3): 54
19. Howard AD, Berger J, Gerber L, Familletti P, Udenfriend S.
Characterization of the phosphatidylinositol-glycan membrane
anchor of the human placental alkaline phosphatase. Proc Natl
Acad Sci USA 1987; 84: 6055-6059
20. Coen G, Mazzaferro S. Bone metabolism and its assessment in
renal failure. Nephron 1994; 67: 383-401
by guest on July 14, 2011