Bone,
12, 261-263 (1991) Printed in the USA. All rights reserved.
8756-3282191 $3.00 + .OO Copyright 0 1991 Pergamon Press plc
Serum Osteocalcin Levels before and after 1,25 Dihydroxy-Vitamin D Stimulation in a Family with Hypophosphatasia J.D. MACFARLANE,
‘M. FROLICH
and *SE. PAPAPOULOS
Departments of Rheumatology, ‘Clinical Chemistry, and ‘Endocrinology,
University Hospital, L.&en,
The Netherlands
Addressfir correspondence and reprints: J.D. Macfarlane, University Hospital, Department of Rheumatology, Building 1, CZ-Q, P.O. Box 9600, 2300 Rd Leiden, The Netherlands.
Abstract
In the present study we examined the serum concentration of osteocalcin in two subjects with HP and their parents and its response to 1,25(OH),D, stimulation.
Because defective bone mineralization occurs in hypophosphatasia (HP) and the source of bone ahcahne phosphatase is the osteoblast, we investigated another marker of osteoblast activity, namely the production of osteocalcht In an HP family and controls. The mean basal osteocalcht levels ht the two affected young adults and their parents (the apparent heteroxygotes) were 3.4 ng/ml (range 2.m.6) and were not different from the levels in age- and sex-matched controls (3.6 ng/ml, range 2.5-4.6). Furthermore, the ratio of carboxylated to total osteocalcht was normal. The rise in osteocaicin after 1,2%lihydroxycholecalciferol stimulation (2 Fg daily for one week) was slightly greater in the controls than in the HP family. These results support the concept that there is no global defect in osteoblast function in this family with HP. Key Words: Osteoblast-Bone phatase.
Gla-protein-Alkaline
Subjects and Methods The younger subjects are two sisters (A,B), aged 30 and 20 years, with HP. Clinical details have been published elsewhere (Macfarlane et al. 1988). In brief, both have had premature shedding of primary teeth and have almost identical abnormalities in the five biochemical parameters associated with HP, namely serum APase and phosphate, plasma pyridoxal-S-phosphate, and urinary PEA and pyrophosphate. The proband (A) is wheelchair-bound by incapacitating bone and joint pains, and has marked radiographic changes including diffuse intra- and periarticular calcifications (Macfarlane et al. 1986), whereas her sister is symptom-free and has only minimal epiphyseal irregularities at the ends of a few long bones. Their parents (C,D), the presumed obligate heterozygotes, who are clinically and radiologically normal (except for minimal chondrocalcinosis of the knee in the mother, D), have no detectable PEA in the urine, have low-normal serum APase values and show only a mild elevation in urinary pyrophosphate excretion. The parents have a common ancestor six generations back. The controls for A and B comprised four healthy young adult women matched for age and contraceptive pill use. The father C had two age-matched controls, and the control for D was matched for age and menstrual status. All subjects received one week’s treatment with 1,25(OH),D, (Rocaltrol, Roche Lab, Mijdrecht) in a dosage of 0.5 pg orally four times daily.
phos-
Introduction Hypophosphatasia (HP) is a heritable condition characterized by a low serum alkaline phosphatase (APase) activity, an excessive excretion of phosphoethanolamine (PEA) in the urine and a disturbance in bone formation (McKusick 1988; Whyte 1989). Alkaline phosphatase (APase) activity is usually low or absent in bone in the matrix vesicles of osteoblasts, the earliest site of mineralization (Fallon et al. 1984). The majority of reports suggest osteomalacia (Weinstein & Whyte 1981), but the mechanism for this defect is unclear. A point mutation in the tissue nonspecific APase gene has been identified in one case of infantile HP (Weiss et al. 1988). Because APase is produced by the osteoblast, the question is raised whether defective APase activity might be reflected in a more global malfunction of the osteoblast. It is now possible to investigate this further in vivo by measuring serum levels of bone-Gla-protein (osteocalcin). Osteocalcin contains a glutamate whose gamma-carboxylation is vitamin K-dependent. This protein is found exclusively in bone and is produced by the osteoblast; a fraction is released into the serum. The vitamin D metabolite 1,25dihydroxycholecalciferol [1,25(OH),D,] stimulates osteocalcin formation both in vitro from osteoblasts (Price & Baukol 1980) and in vivo (Duda et al. 1986; Zerwekh et al. 1985).
Methods Fasting state blood samples were obtained in the early morning on days 1 (baseline), 5, and 8. The following levels were determined on days 1 and 8: serum calcium, phosphate, albumin, creatinine, APase, osteocalcin, parathyroid hormone, and 1,25(OH),D. Serum 25-hydroxy-vitamin D [25(OH)D] was estimated on day 1. On day 5 only the serum calcium, albumin, and osteocalcin were measured. Calcium, phosphate, creatinine, and APase were measured with a fully automated analyzer (Technicon, Tarrytown, USA). 1,25(OH),D was measured by a radioreceptor assay (Jungen et al. 1981) and 25(OH)D by a competitive protein binding assay (Edelstein et al. 1974). 261
J.D. Macfarlane
Table I. Serum variables results in hypophosphatasia kindred before and during one week’s treatment with 1,25(OH),D Variable (normal range) APase (15-60 U/l) Phosphate (0.81-1.45 mmol/l) Calcium (2.25-2.55 mmol/l) Osteocalcin (1.6-6.7 ng/ml) 25(OH)D (19-126 nmoV1) 1,25(OH),D (40-140 pmol/l) PTH (
12, 261-263 (1991) Printed in the USA. All rights reserved.
8756-3282191 $3.00 + .OO Copyright 0 1991 Pergamon Press plc
Serum Osteocalcin Levels before and after 1,25 Dihydroxy-Vitamin D Stimulation in a Family with Hypophosphatasia J.D. MACFARLANE,
‘M. FROLICH
and *SE. PAPAPOULOS
Departments of Rheumatology, ‘Clinical Chemistry, and ‘Endocrinology,
University Hospital, L.&en,
The Netherlands
Addressfir correspondence and reprints: J.D. Macfarlane, University Hospital, Department of Rheumatology, Building 1, CZ-Q, P.O. Box 9600, 2300 Rd Leiden, The Netherlands.
Abstract
In the present study we examined the serum concentration of osteocalcin in two subjects with HP and their parents and its response to 1,25(OH),D, stimulation.
Because defective bone mineralization occurs in hypophosphatasia (HP) and the source of bone ahcahne phosphatase is the osteoblast, we investigated another marker of osteoblast activity, namely the production of osteocalcht In an HP family and controls. The mean basal osteocalcht levels ht the two affected young adults and their parents (the apparent heteroxygotes) were 3.4 ng/ml (range 2.m.6) and were not different from the levels in age- and sex-matched controls (3.6 ng/ml, range 2.5-4.6). Furthermore, the ratio of carboxylated to total osteocalcht was normal. The rise in osteocaicin after 1,2%lihydroxycholecalciferol stimulation (2 Fg daily for one week) was slightly greater in the controls than in the HP family. These results support the concept that there is no global defect in osteoblast function in this family with HP. Key Words: Osteoblast-Bone phatase.
Gla-protein-Alkaline
Subjects and Methods The younger subjects are two sisters (A,B), aged 30 and 20 years, with HP. Clinical details have been published elsewhere (Macfarlane et al. 1988). In brief, both have had premature shedding of primary teeth and have almost identical abnormalities in the five biochemical parameters associated with HP, namely serum APase and phosphate, plasma pyridoxal-S-phosphate, and urinary PEA and pyrophosphate. The proband (A) is wheelchair-bound by incapacitating bone and joint pains, and has marked radiographic changes including diffuse intra- and periarticular calcifications (Macfarlane et al. 1986), whereas her sister is symptom-free and has only minimal epiphyseal irregularities at the ends of a few long bones. Their parents (C,D), the presumed obligate heterozygotes, who are clinically and radiologically normal (except for minimal chondrocalcinosis of the knee in the mother, D), have no detectable PEA in the urine, have low-normal serum APase values and show only a mild elevation in urinary pyrophosphate excretion. The parents have a common ancestor six generations back. The controls for A and B comprised four healthy young adult women matched for age and contraceptive pill use. The father C had two age-matched controls, and the control for D was matched for age and menstrual status. All subjects received one week’s treatment with 1,25(OH),D, (Rocaltrol, Roche Lab, Mijdrecht) in a dosage of 0.5 pg orally four times daily.
phos-
Introduction Hypophosphatasia (HP) is a heritable condition characterized by a low serum alkaline phosphatase (APase) activity, an excessive excretion of phosphoethanolamine (PEA) in the urine and a disturbance in bone formation (McKusick 1988; Whyte 1989). Alkaline phosphatase (APase) activity is usually low or absent in bone in the matrix vesicles of osteoblasts, the earliest site of mineralization (Fallon et al. 1984). The majority of reports suggest osteomalacia (Weinstein & Whyte 1981), but the mechanism for this defect is unclear. A point mutation in the tissue nonspecific APase gene has been identified in one case of infantile HP (Weiss et al. 1988). Because APase is produced by the osteoblast, the question is raised whether defective APase activity might be reflected in a more global malfunction of the osteoblast. It is now possible to investigate this further in vivo by measuring serum levels of bone-Gla-protein (osteocalcin). Osteocalcin contains a glutamate whose gamma-carboxylation is vitamin K-dependent. This protein is found exclusively in bone and is produced by the osteoblast; a fraction is released into the serum. The vitamin D metabolite 1,25dihydroxycholecalciferol [1,25(OH),D,] stimulates osteocalcin formation both in vitro from osteoblasts (Price & Baukol 1980) and in vivo (Duda et al. 1986; Zerwekh et al. 1985).
Methods Fasting state blood samples were obtained in the early morning on days 1 (baseline), 5, and 8. The following levels were determined on days 1 and 8: serum calcium, phosphate, albumin, creatinine, APase, osteocalcin, parathyroid hormone, and 1,25(OH),D. Serum 25-hydroxy-vitamin D [25(OH)D] was estimated on day 1. On day 5 only the serum calcium, albumin, and osteocalcin were measured. Calcium, phosphate, creatinine, and APase were measured with a fully automated analyzer (Technicon, Tarrytown, USA). 1,25(OH),D was measured by a radioreceptor assay (Jungen et al. 1981) and 25(OH)D by a competitive protein binding assay (Edelstein et al. 1974). 261
J.D. Macfarlane
Table I. Serum variables results in hypophosphatasia kindred before and during one week’s treatment with 1,25(OH),D Variable (normal range) APase (15-60 U/l) Phosphate (0.81-1.45 mmol/l) Calcium (2.25-2.55 mmol/l) Osteocalcin (1.6-6.7 ng/ml) 25(OH)D (19-126 nmoV1) 1,25(OH),D (40-140 pmol/l) PTH (
