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Pediatric Endocrinology |
Department of Pediatrics, Okayama University Medical School (Hit.T., S.K., T.O., T.M., Hir.T., Y.S.) Okayama 700; and Diagnostic Development, SRL, Inc. (S.H., S.Y.), Tokyo 163–08, Japan
Address all correspondence and requests for reprints to: Susumu Kanzaki, Department of Pediatrics, Okayama University Medical School, 2–5-1, Shikata-cho, Okayama 700 Japan.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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These results show that B-ALP levels are a useful marker for bone formation because B-ALP levels increased when the growth rate accelerated. Serum B-ALP is a potential predictor of the effectiveness of GH therapy, because the serum level after 3 months of GH therapy reflects the outcome of 1 yr of GH therapy.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In human serum, tissue-nonspecific (liver, bone, and kidney), intestinal, and placental ALP isoforms have been identified (5, 6, 7, 8). These sources can contribute to total ALP activity, making clinical interpretation difficult without fractionation of these ALP isoforms (9). Although separating the intestinal and placental ALPs is relatively easy, it is much more difficult to distinguish between B-ALP and liver ALP because these two isoforms are the products of a single gene and differ only with respect to posttranslational glycosylation (5, 6, 7, 8, 10). The discrimination between these two isoforms is necessary for reliable clinical use.
Traditional methods of differentiating the B-ALP from liver ALP include conventional agarose gel electrophoresis (4, 11, 12, 13), heat and chemical inactivation (14, 15, 16, 17), wheat germ agglutinin precipitation (12, 18, 19, 20, 21), and wheat germ agglutinin-high performance liquid chromatography (22, 23). Unfortunately, none of these methods satisfies the requirements for routine application as a bone formation index such as sensitivity, specificity, ease of application, and reliability.
Recently, an immunoradiometric assay (IRMA) for human B-ALP was developed (24). To verify that the serum level of B-ALP is a specific marker for bone formation, we investigated age-related changes in serum B-ALP levels using this sensitive B-ALP IRMA. Moreover, to examine the effect of GH on skeletal metabolism, we measured serum B-ALP levels in GH-deficient children because they showed significant bone growth during GH therapy.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We studied 363 healthy children (207 males, age 0–18 yr and 156 females, age 0–16 yr) whose height, weight, and annual height gain were normal for their age. No children were receiving medication at the time of blood sampling. In 62 males and 39 females, the stages of puberty were estimated according to Marshall and Tanner (25, 26). In 62 males, the pubertal stage was also determined by testicular volume (prepuberty, < 4 mL; puberty, 4–12 mL; and adult stage, > 12 mL) (27).
Patients with GH deficiency
The GH treatment group consisted of 20 GH-deficient Japanese children, 15 males and 5 females, age 5–14 yr. The diagnosis of GH deficiency was based on their short stature (<2.0 SD below the mean height of age- and sex-matched children), low annual height gain (<5.0 cm/yr) and their failure to raise serum GH levels above 10 µg/L after at least two provocative tests (28). All subjects were diagnosed as having idiopathic GH deficiency, and none of them showed clinical or laboratory evidence of other pituitary hormone deficiencies. GH treatment for these patients was authorized by The Foundation for Growth Science in Japan.
Circadian variation in serum B-ALP
Circadian variation in serum B-ALP levels were studied in 16 normal children (11 males and 5 females, age 2–14 yr). Blood samples were collected from each child at 0600, 0800, 1200, 1600, 1800, and 2400 h.
Experimental protocol
After informed consent was obtained from the parents of all subjects, blood samples were obtained from the nonfasting normal children and the GH-deficient patients between 1000–1100 h, i.e. about 12 h after GH injection.
All GH-deficient patients were treated with recombinant human GH \[0.5 U (0.17 mg)/kg per week\] given subcutaneously at night (2100–2200 h) 6–7 days/week. The annual growth rate was calculated from the height increase observed after 1 yr of GH treatment. Blood samples were collected from GH-deficient patients before and 1, 2, 3, 6, 9, and 12 months after the initiation of GH therapy.
All blood samples were promptly centrifuged, and serum was separated and stored at -70 C until assayed.
Measurements of serum B-ALP levels and total ALP (T-ALP) activities
Serum B-ALP levels were measured with an IRMA (Tandem-R Ostase
kit, Hybritech, San Diego, CA). This assay is a solid-phase, two-site
IRMA (21, 24, 29, 30, 31, 32). Briefly, the assay was carried out as follows;
100 µL of the serum samples or the B-ALP standard solution and a
radiolabeled monoclonal detector antibody (100 µL) were mixed in a
plastic tube. Subsequently, plastic beads coated with a monoclonal
capture antibody were added. After overnight incubation at 2–8 C, the
beads were washed three times to remove the unbound labeled antibody.
The radioactivity bound to the solid phase was counted in a 
-counter
for 1 min. The intra- and interassay coefficients of variation (CVs)
were less than 6.7% and less than 8.1%, respectively. The sensitivity
was 2.0 µg/L, and the analytical recovery rate was 103–112% of the
expected theoretical value. The percentage cross-reactivity of this
IRMA for liver ALP isoenzyme was 11–16% (21, 29).
Serum T-ALP activity was measured with a rate assay using disodium p-nitrophenylphosphate and N-methyl-D-glucamine as the substrate and the buffer, respectively, at pH 10.5 and 25 C (Sun test ALP-N, Sanko Junyaku Co., Ltd. Tokyo, Japan). The sensitivity of this test was 2 U/L. The intra- and interassay CVs were approximately 3% and 2.3%, respectively.
Measurements of serum levels of carboxy-terminal propeptide of type I procollagen (PICP), osteocalcin, and insulin-like growth factor-I (IGF-I)
Serum PICP levels were analyzed with a RIA kit (Orion Diagnostica, Espoo, Finland) (33). The sensitivity of this test was 1.2 µg/L. The intra- and interassay CVs were approximately 3% and 5%, respectively. Serum levels of osteocalcin were measured by IRMA (BGP IRMA kit, Mitsubishi Chemicals Inc., Tokyo, Japan) (34). The sensitivity of this test was 1.0 µg/L, and the intraassay CV was approximately 6.3–6.4%.
IGF-I concentrations were determined by IRMA (somatomedin-C·II Ciba Corning, Ciba Corning Diagnostica, Tokyo, Japan). The sensitivity of this test was 0.3 µg/L. The intra- and interassay CVs were approximately 1.8–8.1% and 3.3–6.4%, respectively (35).
Bone mineral density (BMD) measurement
In 19 GH-deficient patients, the BMD of the lumbar spine (L2 to L4) was measured by dual energy x-ray absorptiometry (Hologic QDR-1000, Hologic Inc., Waltham, MA) before and after 1 yr of GH therapy. The SD score for BMD was calculated using standard data from normal Japanese children (36).
Statistical analysis
Results are given as the mean \ SD. The Mann-Whitney U-test was used to compare groups. The relationships between the different variables were calculated by linear regression analysis. A P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Serum B-ALP levels were measured at different times of the day. The CVs for each child ranged from 2.10–9.66%. No significant circadian variation was observed in the serum B-ALP levels.
Serum B-ALP levels in normal children
Age-related changes in serum B-ALP levels in normal children are
shown in Fig. 1
. In males, the serum B-ALP levels were
extremely high in infants and gradually decreased with age. They began
to rise again at age 9–10 yr and peaked at 13–14 yr. In females, the
serum B-ALP levels were also high in infants and gradually decreased
with age. They began to increase at age 7–8 yr, peaked at 11–12 yr,
and then declined again. Therefore, in both males and females, elevated
serum B-ALP levels were observed during infancy and puberty. The
pattern of this age-related change in serum B-ALP levels was similar to
the shape of the standard height velocity curve for healthy Japanese
children (37).
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The serum B-ALP level in normal males was significantly higher in
midpuberty (testicular volume: 4–12 mL) than either in prepuberty or
in the adult stage (Fig. 2A). The highest serum B-ALP
level was also observed at the stage 3 of breast development in females
and at the stage 3 of pubic hair development in both sexes (Fig. 2, B
and C).
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Serum levels of B-ALP, PICP, and osteocalcin were measured in the
same serum samples from 242 normal children. Serum levels of B-ALP in
normal children showed significant positive correlations with those of
PICP and osteocalcin (r = 0.45, P < 0.0001 for
PICP; r = 0.43, P < 0.0001 for osteocalcin) (Fig. 3).
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The SD scores of serum B-ALP levels in 20 GH-deficient patients before GH therapy ranged from -1.60–1.16 (mean = -0.60). Therefore, there was a considerable overlap between values obtained from patients, and those obtained from healthy subjects. There were no apparent correlations between the serum B-ALP levels and height velocity in GH-deficient patients before GH therapy (data not shown).
With the beginning of GH therapy, serum B-ALP levels increased
significantly (Fig. 4). After 3 months of GH treatment,
a 26% increase (P < 0.005) in serum B-ALP levels was
observed. The maximal B-ALP level was seen after 6 months of GH
therapy, and serum B-ALP levels remained high during GH therapy. The
percent increase in the serum levels of B-ALP 2 and 3 months after the
beginning of GH treatment were significantly higher than those for
T-ALP (P < 0.005). The percent increase in serum B-ALP
levels showed a significant positive correlation with the percent
increase in serum IGF-I levels (r = 0.43, P <
0.0001).
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The percent increase in serum B-ALP level after 3 months of GH
treatment correlated with the GH-induced improvement of the height
SD score after 1 yr of GH therapy [height SD
scores after 1 yr of therapy minus that at the beginning of therapy
(
height SD score); r = 0.531 and P
< 0.05] (Fig. 5). It was also positively correlated
with the GH-induced improvement in the height velocity SD
score during 1 yr of GH therapy [height velocity SD scores
after 1 yr of GH therapy minus that before therapy ( height velocity
SD score); r = 0.608 and P < 0.01]
(Fig. 5). In addition, the increment of SD scores in serum
B-ALP level after 1 yr of GH treatment [B-ALP SD scores
after 1 yr of therapy minus that at the beginning of therapy ( B-ALP
SD score)] significantly correlated with the GH-induced
increment of SD scores in BMD after 1 yr of GH therapy
[BMD SD scores after 1 yr of therapy minus that at the
beginning of therapy ( BMD SD score)] (r = 0.663,
P < 0.005) (Fig. 6).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Although previous analyses demonstrated that serum B-ALP levels were
relatively high in childhood, particularly during puberty (40),
age-related changes in serum B-ALP levels during childhood have not
been studied thoroughly. In this study, we found that serum B-ALP
levels in normal children of both sexes were higher at all ages than in
adults (adult male: 11.0 \ 4.0 µg/L, female: 11.3 \ 4.8
µg/L, mean \ SD) (29). Peak serum B-ALP levels were
observed during infancy as well as during puberty. Therefore, the
age-related changes in serum B-ALP levels closely resembled the height
velocity curves for Japanese children (37). The elevation of serum
B-ALP level during puberty is consistent with previous reports that the
serum levels of other bone formation markers, such as osteocalcin and
PICP, increased during puberty (41, 42). Furthermore, our data
demonstrated that the highest serum B-ALP level was observed in
midpuberty (Fig. 2
). Consistent with our data, Blumsohn et
al. (43) reported that several bone metabolic markers including
B-ALP attained the highest levels during midpuberty. It has been
reported that maximal annual height gain is observed at stages 3 and 4
of pubertal development (25, 26). Moreover, serum B-ALP levels are
significantly correlated with those of the established bone formation
markers, osteocalcin and PICP, in normal children. Therefore, these
results strongly suggest that B-ALP is a good candidate for a bone
formation marker during childhood.
In this study, we found considerable overlap in serum B-ALP levels between GH-deficient children before GH therapy and age- and sex-matched controls. This result concurs with previous studies investigating serum levels of other bone formation markers such as PICP, osteocalcin, and the procollagen type III N-terminal propeptide in GH-deficient children (41, 42, 44, 45, 46, 47, 48). The inability to differentiate GH-deficient children from the normal controls by B-ALP levels can be attributed in part to the fact that bone formation is regulated not only by GH, but also by other hormones such as thyroid hormone and some growth factors (49).
Recently, several reports revealed that serum levels of bone formation
markers increased with the beginning of GH administration (41, 42, 44, 46, 50). In our study, a 26% increase of serum B-ALP levels was
observed 3 months after beginning GH treatment, and the level remained
high during GH therapy. We have previously reported that the serum PICP
and intact molecular osteocalcin levels increased 30% and 120%,
respectively, 3 months after GH treatment began (41, 42). The size of
the GH-induced increase in serum B-ALP levels is almost the same as
that for PICP, but is lower than that for osteocalcin, even though the
study populations were different. Moreover, the improvement of BMD
SD score during 12 months of GH therapy was positively
correlated with the improvement of B-ALP SD score after 1
yr of GH treatment (Fig. 6). These findings support the hypothesis that
B-ALP in serum is a useful marker for bone formation, because serum
B-ALP levels increased when the bone growth rate was accelerated.
In the management of patients receiving GH therapy, early determination
of the long-term growth response is important. Measurement of IGF-I has
been advocated as a tool to assess the likelihood of a growth response
to GH therapy (51). However, serum IGF-I levels have not correlated
well with growth velocity, because IGF-I is synthesized not only in
bone but in many other tissues (51). It is noteworthy that the percent
increase in B-ALP after 3 months of GH treatment was positively
correlated with GH-induced improvements of both the height
SD score and the height velocity SD score
during 1 yr of GH treatment (Fig. 5). Therefore, serum levels of B-ALP
after 3 months of GH treatment may be able to predict the growth
response to 1 yr of GH treatment. Of course, there is sufficient
scatter of the data so that measurements would be useful for research
studies (with pools of patients), but their applicability to the
individual may be limited. We previously demonstrated that osteocalcin
in serum after 1 month of GH therapy was correlated with the height
gain during 1 yr of GH therapy in GH-deficient children (41). We also
reported that the percent increase in serum PICP level after 1 month of
GH treatment was related to the increase in height velocity after 1 yr
of treatment (42). However, the use of these two bone formation markers
has some problems, i.e. PICP in the serum is secreted from
both bone and fibroblasts (52), and osteocalcin has been reported to
have wide circadian variations (38). Crofton et al. (45)
recently reported that B-ALP was the best early predictor of a height
velocity response to GH treatment. For theses reasons, B-ALP in the
serum currently is the best candidate for predicting the efficacy of GH
therapy.
In conclusion, accelerated bone formation is reflected by high levels of serum B-ALP in children, particularly during infancy and puberty, a period in which significant bone growth occurs. Moreover, B-ALP appears to be a useful marker for predicting growth responses to long-term GH therapy.
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Acknowledgments |
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Footnotes |
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Received September 30, 1996.
Revised March 20, 1997.
Accepted April 1, 1997.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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) children as a function of age. Serum B-ALP levels were
increased during infancy and puberty. Females had their peak serum
B-ALP levels during puberty about 2 yr earlier than males. Values are
group mean ± SD.
