8756-3282191 $3.00 + .OO Copyright 0 1991 Pergamon Press plc
Bone, 12, pp. 4346; (1991) Printed in the USA. All rights reserved.
Vitamin D Metabolism and Serum Binding Proteins in Anorexia Nervosa J. M. OLMOS, J. A. RIANCHO, J. GONZALEZ-MACfAS
J. A. AMADO,
J. FREIJANES,
Departamento de Medicina Interna, ‘Servicio de Psiquiatrta, Hospital “Marqub
J. MENJ%DEZ-ARANGO’
and
de Valdecilla, ” Universidad de Cantabria, Santander, Spain
Address for correspondence and reprints: J. Gonzalez-Macias, Departamento de Medicina Intema, Hospital “Maquts
de Valdecilla,” Santander,
Spain.
anorectic women. However, Aarskog et al. (1986), reported a reduction in 1,25(OH),D and Fonseca et al. (1988), observed a diminished concentration of 250HD in anorectic patients. This work was undertaken to go further into this subject, by studying vitamin D metabolites and serum transport proteins, as well as other parameters of mineral metabolism, in a group of patients with anorexia nervosa.
Abstract Serum vitamin D metaboiites and other parameters of mineral metabolism were measured in 12 patients with anorexia nervosa. Serum concentrations of calcium, phosphate, aIbumitt, alkaline phosphatase, parathyroid hormone, calcitonin, osteocalcin, and 24-hours calcium excretion were nomud. Serum 25-hydroxyvitamin D (250HD) concentration was similar in patients and normal subjects, whereas 1,25dihydroxyvitamin D (1,25(OH),D) levels were signitIcantly reduced in patients (62 f 17 vs 82 f 17 pmoUI); p < 0.05). The concentration of vitamin D-binding protein (DBP) in patients was normal, but serum binding capacity (Nmax) was diminished in anorectic patients (2.05 f 0.50 vs 2.53 f 0.51 p,moi/I; p < 0.05). The diminished serum binding capacity, in spite of normal concentrations of albumin and DBP, refiects the presence of qualitative rather than quantitative defects in serum transport proteins. Since the reduction in 1,25(OH),D and serum binding capacity was quantitatively similar, it is likely that free 1,25(OH),D levels would be normal.
Material and Methods We studied 12 women aged 14 to 24 years (mean -+: SD: 17 t 3 yrs). All met the criteria for the diagnosis of AN (Feighner et al. 1972). and the mean duration of the disease was 18 months (6-46 mo.). At the time of the study, all had secondary amenorrhea and their weight losses were 20% to 46% of their original body weight. Fasting venous blood samples were obtained at 09.00 h. Serum calcium, phosphate, albumin, creatinine, alkaline phosphatase, and 24 hours-urine calcium and creatinine were measured by standard automatic methods. Serum calcium was corrected for albumin concentration. Serum estradiol (Zer Science Based Industries, Jerusalem, Israel), calcitonin, parathyroid hormone C-terminal (PTHc), and osteocalcin were measured by radioimmunoassay (INCSTAR, Stillwater, USA). Vitamin D metabolites were determined as previously reported (Riancho et al. 1988). Briefly, 2 ml of serum were extracted with acetonitrile and then chromatographied on Sep-Pak Cl8 cartridges (Waters Assoc. Milford, CT, USA), followed by high pressure liquid chromatography (HPLC). The 250HD region eluting from HPLC was collected and quantified by competitive binding assay with rat serum. The 1,25(OH),D collected from HPLC was measured by radioassay with calf thymus receptor (INCSTAR). The interassay coefficients of variation (CV) were 10% and 14% for 250HD and 1,25(OH),D, respectively. Normal ranges are 12.5-125 pmoY1 and 40-137 pmol/l for 25 OHD and 1,25(OH)2D, respectively. The serum concentration of DBP was measured by radial immunodiffusion (Bouillon et al. 1977) using rabbit immunoglobulin to human Gc globulin (Dakopatts, Glostrup, Denmark). On each plate, a standard curve consisting of 5 dilutions of pure human DBP (Sigma, St Louis, USA) was run. After incubation at room temperature for 48 h, the diameter of the sharp ring-shaped precipitates was measured to obtain the concentration of DBP in each sample. CV was 8%. In addition,
Key Words: Anorexia nervosa-25hydroxyvitamin D- 1,25dihydroxyvitamin D-Vitamin D-binding protein-Serum binding capacity. Introduction Anorexia nervosa (AN) is a psychiatric disorder of unknown origin characterized by aberrant eating behavior, fear of becoming fat, and a desire to lose weight (Herzog et al. 1985). Malnutrition, amenorrhea, and estrogen deficiency are among the sequelae of this self-induced starvation (Aro et al. 1986; Gold et al. 1986). The association between several conditions of estrogen deficiency and osteoporosis is well documented (Cann et al. 1987; Ciccarelli et al. 1988; Drinkwater et al. 1984; Riggs & Melton 1986). A reduction in bone mass, which may result in pathological fractures, has also been reported in patients with AN (Baum et al. 1987; Kaplan et al. 1986; Rigotti et al. 1984; Szmukler et al. 1985; Treasure et al. 1987). Nevertheless, the underlying pathogenetic mechanisms have not been completely elucidated. In particular, conflicting results have been reported regarding vitamin D metabolism. Rigotti et al. (1984). found no abnormalities either in 25-hydroxyvitamin D (250HD) or in 1,25-dihydroxyvitamin D (1,25(OH),D) levels in 18 adult 43
J. M. Olmos et al.: Vitamin D and anorexia nervosa
44
Table I. Biochemical features of the patients.
Parameter
Table II. Vitamin D status in patients with anorexia nervosa and controls.
Patients (mean 2 SD)
Normal range Parameter
Calcium (mg/dl)
Phosphate(mg/dl) Creatinine (mgidlf Albumin (gidl) Alk. Phosphatase (U/L) Urine calcium (mg/24 h) U C&U Creat ratio (mgimg) PTHc (ngfml) Calcitonin (ng/ml) Osteocalcin (ng/ml)
9.3 4.3 0.9 4.2 149 98 0.20 0.4 47 4.6
zt -c -t rt It rt t c -t +
0.3 0.5 0.1 0.5 45 45 0.13 0.2 11 2.9
8.5-10.4 2.5-4.5 0.4-1.2 3.5-5.2 76300 95-200 < 0.22
Bone, 12, pp. 4346; (1991) Printed in the USA. All rights reserved.
Vitamin D Metabolism and Serum Binding Proteins in Anorexia Nervosa J. M. OLMOS, J. A. RIANCHO, J. GONZALEZ-MACfAS
J. A. AMADO,
J. FREIJANES,
Departamento de Medicina Interna, ‘Servicio de Psiquiatrta, Hospital “Marqub
J. MENJ%DEZ-ARANGO’
and
de Valdecilla, ” Universidad de Cantabria, Santander, Spain
Address for correspondence and reprints: J. Gonzalez-Macias, Departamento de Medicina Intema, Hospital “Maquts
de Valdecilla,” Santander,
Spain.
anorectic women. However, Aarskog et al. (1986), reported a reduction in 1,25(OH),D and Fonseca et al. (1988), observed a diminished concentration of 250HD in anorectic patients. This work was undertaken to go further into this subject, by studying vitamin D metabolites and serum transport proteins, as well as other parameters of mineral metabolism, in a group of patients with anorexia nervosa.
Abstract Serum vitamin D metaboiites and other parameters of mineral metabolism were measured in 12 patients with anorexia nervosa. Serum concentrations of calcium, phosphate, aIbumitt, alkaline phosphatase, parathyroid hormone, calcitonin, osteocalcin, and 24-hours calcium excretion were nomud. Serum 25-hydroxyvitamin D (250HD) concentration was similar in patients and normal subjects, whereas 1,25dihydroxyvitamin D (1,25(OH),D) levels were signitIcantly reduced in patients (62 f 17 vs 82 f 17 pmoUI); p < 0.05). The concentration of vitamin D-binding protein (DBP) in patients was normal, but serum binding capacity (Nmax) was diminished in anorectic patients (2.05 f 0.50 vs 2.53 f 0.51 p,moi/I; p < 0.05). The diminished serum binding capacity, in spite of normal concentrations of albumin and DBP, refiects the presence of qualitative rather than quantitative defects in serum transport proteins. Since the reduction in 1,25(OH),D and serum binding capacity was quantitatively similar, it is likely that free 1,25(OH),D levels would be normal.
Material and Methods We studied 12 women aged 14 to 24 years (mean -+: SD: 17 t 3 yrs). All met the criteria for the diagnosis of AN (Feighner et al. 1972). and the mean duration of the disease was 18 months (6-46 mo.). At the time of the study, all had secondary amenorrhea and their weight losses were 20% to 46% of their original body weight. Fasting venous blood samples were obtained at 09.00 h. Serum calcium, phosphate, albumin, creatinine, alkaline phosphatase, and 24 hours-urine calcium and creatinine were measured by standard automatic methods. Serum calcium was corrected for albumin concentration. Serum estradiol (Zer Science Based Industries, Jerusalem, Israel), calcitonin, parathyroid hormone C-terminal (PTHc), and osteocalcin were measured by radioimmunoassay (INCSTAR, Stillwater, USA). Vitamin D metabolites were determined as previously reported (Riancho et al. 1988). Briefly, 2 ml of serum were extracted with acetonitrile and then chromatographied on Sep-Pak Cl8 cartridges (Waters Assoc. Milford, CT, USA), followed by high pressure liquid chromatography (HPLC). The 250HD region eluting from HPLC was collected and quantified by competitive binding assay with rat serum. The 1,25(OH),D collected from HPLC was measured by radioassay with calf thymus receptor (INCSTAR). The interassay coefficients of variation (CV) were 10% and 14% for 250HD and 1,25(OH),D, respectively. Normal ranges are 12.5-125 pmoY1 and 40-137 pmol/l for 25 OHD and 1,25(OH)2D, respectively. The serum concentration of DBP was measured by radial immunodiffusion (Bouillon et al. 1977) using rabbit immunoglobulin to human Gc globulin (Dakopatts, Glostrup, Denmark). On each plate, a standard curve consisting of 5 dilutions of pure human DBP (Sigma, St Louis, USA) was run. After incubation at room temperature for 48 h, the diameter of the sharp ring-shaped precipitates was measured to obtain the concentration of DBP in each sample. CV was 8%. In addition,
Key Words: Anorexia nervosa-25hydroxyvitamin D- 1,25dihydroxyvitamin D-Vitamin D-binding protein-Serum binding capacity. Introduction Anorexia nervosa (AN) is a psychiatric disorder of unknown origin characterized by aberrant eating behavior, fear of becoming fat, and a desire to lose weight (Herzog et al. 1985). Malnutrition, amenorrhea, and estrogen deficiency are among the sequelae of this self-induced starvation (Aro et al. 1986; Gold et al. 1986). The association between several conditions of estrogen deficiency and osteoporosis is well documented (Cann et al. 1987; Ciccarelli et al. 1988; Drinkwater et al. 1984; Riggs & Melton 1986). A reduction in bone mass, which may result in pathological fractures, has also been reported in patients with AN (Baum et al. 1987; Kaplan et al. 1986; Rigotti et al. 1984; Szmukler et al. 1985; Treasure et al. 1987). Nevertheless, the underlying pathogenetic mechanisms have not been completely elucidated. In particular, conflicting results have been reported regarding vitamin D metabolism. Rigotti et al. (1984). found no abnormalities either in 25-hydroxyvitamin D (250HD) or in 1,25-dihydroxyvitamin D (1,25(OH),D) levels in 18 adult 43
J. M. Olmos et al.: Vitamin D and anorexia nervosa
44
Table I. Biochemical features of the patients.
Parameter
Table II. Vitamin D status in patients with anorexia nervosa and controls.
Patients (mean 2 SD)
Normal range Parameter
Calcium (mg/dl)
Phosphate(mg/dl) Creatinine (mgidlf Albumin (gidl) Alk. Phosphatase (U/L) Urine calcium (mg/24 h) U C&U Creat ratio (mgimg) PTHc (ngfml) Calcitonin (ng/ml) Osteocalcin (ng/ml)
9.3 4.3 0.9 4.2 149 98 0.20 0.4 47 4.6
zt -c -t rt It rt t c -t +
0.3 0.5 0.1 0.5 45 45 0.13 0.2 11 2.9
8.5-10.4 2.5-4.5 0.4-1.2 3.5-5.2 76300 95-200 < 0.22
