Acta Pndiatr Scand 79: 36-40, 1990
High Total and Free 1,25-Dihydroxyvitamin D Concentrations in Serum of Premature Infants R. SCHILLING, E HASCHKE, C. SCHATTEN, M. SCHMID, W. WOLOSZCZUK, I. STEFFAN and E. SCHUSTER From the Department of Paediatrics, the Ludwig-Boltzmann-Institute of Clinical Endocrinologx the Institute of Analytical Chemistry, and the Institute of Medical Computer Sciences, University of Vienna, Austria
ABSTRACT. Schilling, R., Haschke, F., Schatten, C., Schmid, M., Woloszczuk, W., Steffan, 1. and Schuster, E. (Department of Paediatrics, Ludwig-Boltzmann-Institute of Clinical Endocrinology, Institute of Analytical Chemistry, Institute of Medical Computer Sciences, University of Vienna, Austria). High total and free 1,25-dihydroxyvitamin D concentrations in serum of premature infants. Acta Paediatr Scand 79: 36, 1990. We investigated the relationship between serum total and free 1,25-dihydroxyvitamin D (1,25-OH2D)and the biochemical regulation of 1,25-OH2Dproduction in premature infants. We measured 1,25-OH2D, vitamin D binding protein and related biochemical parameters and calculated the free 1,25-OH2Dindex in serum of 17 premature infants (birthweight 8101700 g; gestational age 31-36 weeks) on two different occasions defhed by body weight (Study A 179-1850 g, Study B: 2 100-2200 9). Dietary calcium (Ca) intake was 1,sor 2,6 mmol/kg/d,-phosphorus (P) intake 1,7 mmol/kg/d and vitamin D intake lo00 IU/d. Biochemical results were similar in infants with different Ca intakes and all were within reference ranges. Concentrations of vitamin D binding protein (Study A 0.15f0.03 g/l, Study B 0.14k0.03 g/l; i f S D ) were lower, concentrations of 1,25(OH)*D(Study A 180f67 pmolh, Study B 216f53 pmol/l) were higher, and consequently the free 1,25-OH2Dindex (Study A 6.6k2.7, Study B 8.8f2.6) was 4 to 6 times higher than in previously studied term infants. 1,25-0H2D and the free 1,25-OH2Dindex increased significantly with age and were not COP related with serum P or parathyroid hormone. The data indicate that in premature infants with normal biochemical parameters of Ca and P metabolism elevated concentrations of 1,25-OH2Ds i g w an increased fraction of free 1,25-OH2Dand that increased production of 1,25-OH2Dis not due to hypophosphatemia or hyperparathyroidism. Kcy words: vitamin D, 1,25-dihydnaryvitamin D, free 1,25-dihydnoxyvitarnin D, vitamin D binding protein, prematun infants.
1,25-dihydroxyvitamin D (1 ,25-OH2D) is the biologically most active metabolite of vitamin D. Its main function is stimulation of intestinal calcium (Ca) and phosphorus (P) absorption (1). Its synthesis from 25-hydroxyvitamin D (25-OHD) by the kidneys is stimulated by parathyroid hormone (PTH) and by hypophosphatemia. In normal adults, serum concentration of 1,25-OH2D is closely related to the concentration of its carrier protein, the vitamin D binding protein (DBP) (2). In this way the small free fraction of serum 1,25-OH2D, which is assumed to be the biologically active fraction, is kept constant. Even at very low gestational age healthy premature infants seem to have adequate ability to synthesize 1,25-OH2D: serum 1,25-OH2D concentrations at birth are comparable to reference values for healthy children and adults, increase significantly during the first few days of life (3, 4, 5) and are far above reference values between 3 and 12 weeks of life (5, 6). Concentrations of DBP in cord blood of premature infants are below reference values for term infants and adults, but increase to comparable levels by 40-42 weeks postconceptional age (7). In view of these findings it could be assumed that the free fraction of 1,25-OH2Dis considerably larger in premature than in term infants or in adults. However, no data on concentration of free 1,25-OH2D and its regulation in premature infants are available. Considering the high Ca and P requirements of premature infants for skeletal mineralisation and growth
Acta Paediatr Scand 79
Vitamin
D
metabolism in premature infants
(8), we speculated that elevated 1,25-OH2D concentrations might be due to inadequate dietary Ca and P intake and a concomitant increase of serum PTH or a decrease of serum P. Our study therefore was designed to investigate the relationship between serum total and free 1,25-OH2D concentrations and the biochemical regulation of 1,25-OH2D production in premature infants at different postnatal ages (PNA) and postconceptional ages (PCA) while receiving different dietary Ca intakes.
SUBJECTS AND METHODS Seventeen premature infants (7 females, 10 males) with a mean birthweight of 1395 g (range 810-1 700 g) and a mean gestational age of 33 weeks (range 31-36 weeks) who were admitted to the neonatal intensive care unit participated in the study. Written informed consent was obtained from all parents prior to the study. The infants were free of major congenital anomalies and entered the study when they were able to be maintained on enteral feedings of pooled pasteurised human milk given via nasogastric tube. Infants were randomly assigned to receive a supplement of either glucose-l-phosphate (4.5 mmol PI1 milk; Group P, n=8) or of glucose-l-phosphate and Ca-gluconate (same P,plus 7 mmol Call milk; Group Ca-P, n=9). An oral supplement of vitamin D3 (1 OOO I.U.) was given daily from 7 days of age. Venous blood was obtained when body weight reached 1750-1 850 g (Study A) and again at 2 1002200 g (Study B). Blood was centrifuged and the serum frozen immediatedly and stored at -20°C until analysis. From the day the infants entered the study until the day of Study B milk intake was recorded daily and body weight was determined daily to the nearest 10 g using infant scales. Mean daily Ca and P intakes during the study period were calculated from the milk volume consumed and the Ca and P content of milk (mean Ca and P content of pooled human milk determined on 7 different occasions-Ca 8.0 mmolll, P 5.0 mrnolfl-plus the amount of Ca and P from the supplements). Gestational age was determined by the clinical score of Petrussa (9). Ca concentration in human milk was determined by atomic absorption spectrometry (Perkin Elmer 303) after dry ashing at 450°C and P concentration was measured by a colorimetric method (10). Serum Ca, P and alkaline phosphatase activity (AP) were measured by routine colorimetric methods using a selective autoanalyser (Greiner G 400, Langenthal, Switzerland). Immunoreactive parathyroid hormone (iFTH) in serum was measured by a radioimmunoassay with mid-regional specifcity (Sorin Biomedica, Italy). 25-OHD was measured by a competitive protein binding assay as described previously (11). 1,25-OH2D was measured by a radioreceptor assay after extraction and purification of samples on C18 and silica cartridges as described by Reinhardt et al. (12) using a kit (Immuno Nuclear, Stillwater Mn., USA). In our laboratory the sensitivity of the assay is 7 pg/ml and intra- and interassay variances are 10.4% and 11.8%. respectively. DBP was measured by radial immunodiffusion (GC-Globulin Partigen", Behringwerke, FRG). Free 1,25-OH2D was estimated by calculating the ratio of the molar concentrations of 1,25-OH2D and DBP [free 1,25-OH2D index, FDI; (2, 13)]. Reference values for 1,25-OH2D, DBP and the FDI were obtained in healthy term infants aged 6-12 months. Data for infants receiving the different supplements were compared by the r-test. Data from Studies A and B were compared by the paired f-test. Correlations between variables were tested by linear regression analysis.
RESULTS Group P and Group Ca-P did not differ significantly in age and weight at birth nor in age and weight on study days A or B. For both groups combined, postnatal age on the day of Study A averaged 30 days (range 14-70 days) and postconceptional age averaged 37 weeks (range 34-41 weeks). On the day of Study B the respective ages were 43 days (range 28-84 days) and 39 weeks (range 36-43 weeks). Mean daily Ca intake during the study period was significantly (p
High Total and Free 1,25-Dihydroxyvitamin D Concentrations in Serum of Premature Infants R. SCHILLING, E HASCHKE, C. SCHATTEN, M. SCHMID, W. WOLOSZCZUK, I. STEFFAN and E. SCHUSTER From the Department of Paediatrics, the Ludwig-Boltzmann-Institute of Clinical Endocrinologx the Institute of Analytical Chemistry, and the Institute of Medical Computer Sciences, University of Vienna, Austria
ABSTRACT. Schilling, R., Haschke, F., Schatten, C., Schmid, M., Woloszczuk, W., Steffan, 1. and Schuster, E. (Department of Paediatrics, Ludwig-Boltzmann-Institute of Clinical Endocrinology, Institute of Analytical Chemistry, Institute of Medical Computer Sciences, University of Vienna, Austria). High total and free 1,25-dihydroxyvitamin D concentrations in serum of premature infants. Acta Paediatr Scand 79: 36, 1990. We investigated the relationship between serum total and free 1,25-dihydroxyvitamin D (1,25-OH2D)and the biochemical regulation of 1,25-OH2Dproduction in premature infants. We measured 1,25-OH2D, vitamin D binding protein and related biochemical parameters and calculated the free 1,25-OH2Dindex in serum of 17 premature infants (birthweight 8101700 g; gestational age 31-36 weeks) on two different occasions defhed by body weight (Study A 179-1850 g, Study B: 2 100-2200 9). Dietary calcium (Ca) intake was 1,sor 2,6 mmol/kg/d,-phosphorus (P) intake 1,7 mmol/kg/d and vitamin D intake lo00 IU/d. Biochemical results were similar in infants with different Ca intakes and all were within reference ranges. Concentrations of vitamin D binding protein (Study A 0.15f0.03 g/l, Study B 0.14k0.03 g/l; i f S D ) were lower, concentrations of 1,25(OH)*D(Study A 180f67 pmolh, Study B 216f53 pmol/l) were higher, and consequently the free 1,25-OH2Dindex (Study A 6.6k2.7, Study B 8.8f2.6) was 4 to 6 times higher than in previously studied term infants. 1,25-0H2D and the free 1,25-OH2Dindex increased significantly with age and were not COP related with serum P or parathyroid hormone. The data indicate that in premature infants with normal biochemical parameters of Ca and P metabolism elevated concentrations of 1,25-OH2Ds i g w an increased fraction of free 1,25-OH2Dand that increased production of 1,25-OH2Dis not due to hypophosphatemia or hyperparathyroidism. Kcy words: vitamin D, 1,25-dihydnaryvitamin D, free 1,25-dihydnoxyvitarnin D, vitamin D binding protein, prematun infants.
1,25-dihydroxyvitamin D (1 ,25-OH2D) is the biologically most active metabolite of vitamin D. Its main function is stimulation of intestinal calcium (Ca) and phosphorus (P) absorption (1). Its synthesis from 25-hydroxyvitamin D (25-OHD) by the kidneys is stimulated by parathyroid hormone (PTH) and by hypophosphatemia. In normal adults, serum concentration of 1,25-OH2D is closely related to the concentration of its carrier protein, the vitamin D binding protein (DBP) (2). In this way the small free fraction of serum 1,25-OH2D, which is assumed to be the biologically active fraction, is kept constant. Even at very low gestational age healthy premature infants seem to have adequate ability to synthesize 1,25-OH2D: serum 1,25-OH2D concentrations at birth are comparable to reference values for healthy children and adults, increase significantly during the first few days of life (3, 4, 5) and are far above reference values between 3 and 12 weeks of life (5, 6). Concentrations of DBP in cord blood of premature infants are below reference values for term infants and adults, but increase to comparable levels by 40-42 weeks postconceptional age (7). In view of these findings it could be assumed that the free fraction of 1,25-OH2Dis considerably larger in premature than in term infants or in adults. However, no data on concentration of free 1,25-OH2D and its regulation in premature infants are available. Considering the high Ca and P requirements of premature infants for skeletal mineralisation and growth
Acta Paediatr Scand 79
Vitamin
D
metabolism in premature infants
(8), we speculated that elevated 1,25-OH2D concentrations might be due to inadequate dietary Ca and P intake and a concomitant increase of serum PTH or a decrease of serum P. Our study therefore was designed to investigate the relationship between serum total and free 1,25-OH2D concentrations and the biochemical regulation of 1,25-OH2D production in premature infants at different postnatal ages (PNA) and postconceptional ages (PCA) while receiving different dietary Ca intakes.
SUBJECTS AND METHODS Seventeen premature infants (7 females, 10 males) with a mean birthweight of 1395 g (range 810-1 700 g) and a mean gestational age of 33 weeks (range 31-36 weeks) who were admitted to the neonatal intensive care unit participated in the study. Written informed consent was obtained from all parents prior to the study. The infants were free of major congenital anomalies and entered the study when they were able to be maintained on enteral feedings of pooled pasteurised human milk given via nasogastric tube. Infants were randomly assigned to receive a supplement of either glucose-l-phosphate (4.5 mmol PI1 milk; Group P, n=8) or of glucose-l-phosphate and Ca-gluconate (same P,plus 7 mmol Call milk; Group Ca-P, n=9). An oral supplement of vitamin D3 (1 OOO I.U.) was given daily from 7 days of age. Venous blood was obtained when body weight reached 1750-1 850 g (Study A) and again at 2 1002200 g (Study B). Blood was centrifuged and the serum frozen immediatedly and stored at -20°C until analysis. From the day the infants entered the study until the day of Study B milk intake was recorded daily and body weight was determined daily to the nearest 10 g using infant scales. Mean daily Ca and P intakes during the study period were calculated from the milk volume consumed and the Ca and P content of milk (mean Ca and P content of pooled human milk determined on 7 different occasions-Ca 8.0 mmolll, P 5.0 mrnolfl-plus the amount of Ca and P from the supplements). Gestational age was determined by the clinical score of Petrussa (9). Ca concentration in human milk was determined by atomic absorption spectrometry (Perkin Elmer 303) after dry ashing at 450°C and P concentration was measured by a colorimetric method (10). Serum Ca, P and alkaline phosphatase activity (AP) were measured by routine colorimetric methods using a selective autoanalyser (Greiner G 400, Langenthal, Switzerland). Immunoreactive parathyroid hormone (iFTH) in serum was measured by a radioimmunoassay with mid-regional specifcity (Sorin Biomedica, Italy). 25-OHD was measured by a competitive protein binding assay as described previously (11). 1,25-OH2D was measured by a radioreceptor assay after extraction and purification of samples on C18 and silica cartridges as described by Reinhardt et al. (12) using a kit (Immuno Nuclear, Stillwater Mn., USA). In our laboratory the sensitivity of the assay is 7 pg/ml and intra- and interassay variances are 10.4% and 11.8%. respectively. DBP was measured by radial immunodiffusion (GC-Globulin Partigen", Behringwerke, FRG). Free 1,25-OH2D was estimated by calculating the ratio of the molar concentrations of 1,25-OH2D and DBP [free 1,25-OH2D index, FDI; (2, 13)]. Reference values for 1,25-OH2D, DBP and the FDI were obtained in healthy term infants aged 6-12 months. Data for infants receiving the different supplements were compared by the r-test. Data from Studies A and B were compared by the paired f-test. Correlations between variables were tested by linear regression analysis.
RESULTS Group P and Group Ca-P did not differ significantly in age and weight at birth nor in age and weight on study days A or B. For both groups combined, postnatal age on the day of Study A averaged 30 days (range 14-70 days) and postconceptional age averaged 37 weeks (range 34-41 weeks). On the day of Study B the respective ages were 43 days (range 28-84 days) and 39 weeks (range 36-43 weeks). Mean daily Ca intake during the study period was significantly (p
