Nancy E. Vieira

Measurement of True Calcium Absorption in Premature Infants Using Intravenous 46Ca and Oral 44Ca

ABSTRACT: We have developed a method for measuring true fractional calcium absorption (α) in premature infants using two stable isotopes of calcium and tested it in seven studies in seven infants (birth weight 1543 ± 65 g, gestation 32.8 ± 7 wk). A total of 7.5 μg/kg 46Ca was given as a single intravenous bolus. Immediately thereafter 1.25 mg/kg of 44CA was given in a single gavage feeding of standard infant formula (Enfamil). A metabolic isolette was used to obtain 4-h collections of urine for 24 h total. 46Ca and 44Ca were measured in urine by thermal ionization mass spectroscopy and expressed as the ratio to naturally occurring 48Ca. The differences in the 46Ca/48Ca and 44Ca/48Ca ratios from natural levels (Δ% excess 46Ca and Δ% excess 44Ca) were calculated. Percent absorption (α) equals a constant times cumulative Δ% excess 44Ca/A% excess 46Ca. The calculation of α is independent of urine volume or concentration. The Δ% excess 46Ca, showed the expected multiexponential decline as a function of time, and Δ% excess 44Ca usually peaked during a 4− to 8-h urine collection. Calculations of α using increasingly long sampling times showed that a plateau had been reached by 12 h. α values calculated after 16–24 h in the seven infants at 2 wk of age were 41, 48, 45, 46, 25, 55, and 51%. Repeat studies at 3 wk of age were 46, 60, and 54%. These values are somewhat higher than net percent calcium absorption values reported for standard formula and thus appear very appropriate. This methodology will be very valuable in studying factors that may affect true calcium absorption in premature infants.

Stable Isotopic Measurement of Endogenous Fecal Calcium Excretion in Children

Using a stable isotopic technique in which 42Ca was administered via a bolus injection, we measured endogenous fecal calcium excretion, Vf, in five healthy children, aged 3-14 years. The Vf averaged 1.4 +/- 0.4 mg/kg/day, and was lower than urinary Ca excretion (Vu) in four of the five children. These results for Vf are consistent with previously reported results for Vf in healthy adults and much lower than those reported in premature infants. These results may be useful in understanding developmental changes in Ca metabolism and in interpreting dual tracer Ca isotope studies in children.

Calcium Kinetics in the Hyperprostaglandin E Syndrome

ABSTRACT: Metabolic investigations, including the use of stable isotopes of calcium, were used to study calcium kinetics in three children with the hyperprostaglandin E syndrome. The studies were performed both during indomethacin treatment and in the absence of therapy. Off therapy, each child had hypercalciuria (mean urinary calcium excretion 0.478 mM/kg/d), hyperprostaglandinuria, and elevated serum calcitriol concentration. All had diminished bone density and were euparathyroid. Indomethacin treatment was associated with a marked reduction in serum calcitriol concentration, as well as decreased prostaglandin E excretion. Mean urinary calcium excretion fell to 0.135 mM/kg/d. The stable isotope studies defined two components to the hypercalciuria of this disease: an indomethacin-sensitive dietary contribution and a relatively indomethacin-resistant bone resorptive element. Bone densitometry confirmed the presence of the resorptive element by demonstrating skeletal demineralization.

Human calcium metabolism including bone resorption measured with 41Ca tracer

Accelerator mass spectrometry is so sensitive to small quantities of 41Ca that it might be used as a tracer in the study of human calcium kinetics to generate unique kinds of data. In contrast with the use of other Ca isotopic tracers, 41Ca tracer can be so administered that the tracer movements between the various body pools achieve a quasi steady state. Resorbing bone may thus be directly measured. We have tested such a protocol against a conventional stable isotope experiment with good agreement.

Estrogen Therapy Enhances Calcium Absorption and Retention and Diminishes Bone Turnover in Young Girls With Turner's Syndrome: A Calcium Kinetic Study

Using stable tracers of calcium, we have previously shown a significant increase in calcium absorption and retention in prepubertal boys treated with exogenous testosterone. To investigate the effects of estrogen replacement on measures of calcium absorption, retention, and bone turnover, we studied a group of seven hypogonadal girls with Turners syndrome (mean +/- SE age, 12.5 +/- 0.7 years). At baseline, 42Ca intravenously (IV) and 44Ca orally were administered, and blood and urine samples were collected for approximately 130 hours. Estrogen therapy was begun as oral ethinyl estradiol (4 or 20 micrograms/d) or intramuscular depot estradiol given over 4 weeks, after which an identical study was repeated. Analysis of calcium enrichment in blood and urine was performed using mass spectrometry methods. After estrogen therapy, there was a significant increase in calcium absorption ([Va] P = .03) and total calcium retention ([Vbal] P = .04), similar to the effects of testosterone in boys. Bone accretion (Vo+) decreased after estrogen therapy (P = .004), as did resorption ([Vo-] P = .004). The overall rate of whole-body calcium turnover (Vt) was significantly decreased after estrogen administration (P = .04). These findings were opposite of those observed in prepubertal boys treated with testosterone. The contribution of bone resorption to whole-body turnover (E) also decreased after estrogen therapy (P = .05). These changes were associated with increased levels of 1,25-dihydroxyvitamin D after therapy with estrogens (P = .05). We conclude that estrogen supplementation is significantly anabolic for calcium metabolism by markedly increasing calcium absorption and retention and diminishing the estimated whole-body calcium turnover in girls with severe hypogonadism and Turners syndrome. Further studies assessing the dietary calcium and/or vitamin D intake and bone mineral density of hypogonadal girls whose estrogen replacement is intentionally delayed will further define the need for calcium or vitamin D supplements in the peripubertal years in this condition.

Influence of age on effects of endogenous 1,25-dihydroxyvitamin D on calcium absorption in normal women

Recent reports of increases in serum 1,25-dihydroxyvitamin D [1,25(OH2)D] concentration with aging despite no changes or decreases in calcium absorption suggest that elderly women have intestinal resistance to vitamin D action. Thus, in 15 young adult (30±1 year) and 15 elderly (74±1 year) women (mean±SE), we assessed the responsiveness of intestinal calcium absorption to increases in circulating 1,25(OH)2D induced by 4 days of an experimental diet (150 mg calcium and 1600 mg phosphorus daily). True fractional calcium absorption (FCA) (44Ca mixed with food and 42Ca given intravenously, then their ratio in urine measured by mass spectrometry) was determined. Baseline serum intact parathyroid hormone (PTH) concentration was higher in the older women (P=0.01) whereas serum 1,25(OH)2D concentration and true FCA were similar. In both groups, serum 1,25(OH)2D concentrations increased (P<0.002) on the experimental diet. After 4 days on the diet, serum 1,25(OH)2D increased over baseline by 30.5 and 35.6% and, despite these increases, true FCA was 40±3 versus 40±4%/24 hours (NS between groups) in the young and elderly women, respectively. These data suggest that either elderly women have normal intestinal responsiveness to vitamin D or that the resistance to it is too mild to be detected by these methods.

Profound Hypogonadism Has Significant Negative Effects on Calcium Balance in Males: A Calcium Kinetic Study

The impact of estrogen deficiency on bone has been extensively studied in the female; however, the effects of androgen deficiency on calcium fluxes in males have been less well characterized. We investigated the effect of short‐term, severe androgen deficiency on measures of calcium absorption and kinetics as well as on markers of bone turnover in males. To accomplish this, 11 healthy male volunteers were recruited (mean age 23.3 ± 0.5 years [SEM], body mass index 25.3 ± 0.8 kg/m2). They consumed a weight maintenance diet for at least 3 days prior to admission to our Research Unit, with a calcium intake of ∼1200 mg/day. At baseline (D1), subjects received42Ca intravenously as well as44Ca PO mixed with milk or juice. A 29‐h urine collection was begun and blood samples collected at frequent intervals for the measurement of the isotopic enrichment of42Ca and44Ca using thermal ionization mass spectrometry. Twice daily urine samples were collected for 5 days after the administration of the isotopes. A gonadotropin‐releasing hormone agonist (Lupron) was given after D1, again 3 weeks later, and studies repeated identically 4 weeks (D2, n = 6) and 10 weeks from baseline (D3, n = 7) (two subjects completed three studies). Testosterone concentrations were markedly suppressed on both D2 and D3 (−95%, p < 0.006), whereas there were no detectable changes in growth hormone and insulin‐like growth factor‐1 concentrations. Urinary calcium excretion increased significantly after 4 weeks (43%, p = 0.0007) and 10 weeks (73%, p = 0.003) of sustained hypogonadism. Using a multicompartmental kinetic model, the contribution of oral calcium to the urinary losses was decreased by D3 (−41%, p = 0.01), yet the contribution of bone calcium to urine losses increased by 10 weeks (+11%, p = 0.01). There was a 21% decrease in bone calcium deposition (Vo+) by D3 (p < 0.05) with no significant change in bone resorption rates (Vo−). There was a significant correlation between the decrease in testosterone concentration and the increase in urinary calcium excretion, especially at 10 weeks (R2 = 0.84, p = 0.004). These kinetic changes were accompanied by a decrease in osteocalcin concentrations on D2, with improvements by D3. Urinary N telopeptide, a measure of bone resorption, also increased during the studies. In summary, profound hypogonadism in young males is associated with marked increases in urinary calcium losses, with a greater contribution of bone calcium to those losses and decreased kinetic markers of bone calcium deposition. We conclude that even short‐term, severe deficiency in gonadal steroids can have profound negative effects on calcium and bone metabolism in males.

Compartmental analysis of calcium metabolism in very-low-birth-weight infants.

ABSTRACT: The calcium metabolism of 13 very-low-birth-weight infants fed a high-calcium diet was evaluated by means of stable isotope kinetic and balance studies. The studies used orally and i.v. administered stable isotopes, and the kinetic data were evaluated with the aid of a sequential, three-compartment model. The infants (postmenstrual age 33 ± 1 wk, weight 1.34 ± 0.03 kg) had higher bone calcium deposition rates (160 ± 7 mg·kg−1.d−1 or 4.00 ± 0.18 mmol·kg−1·d−1) than those previously reported for either older children or adults. Furthermore, when analyzed as a function of net calcium absorption, bone calcium deposition rates increased markedly and significantly as net calcium absorption increased (r = 0.70, p < 0.01), whereas in older individuals, bone calcium deposition is a relatively invariant function of absorption. A relatively smaller response of bone calcium removal to calcium absorption was found for the very-low-birth-weight infants in this study (r = −0.39, p = 0.18), whereas in adults, bone calcium removal constitutes the major regulatory response. It is suggested that the calcium kinetic results in the very-low-birth-weight infants reflect the high rate of bone growth typical of the third trimester of gestation.

Effect of growth hormone treatment on calcium kinetics in patients with osteogenesis Imperfecta Type III and IV

Using a dual stable isotope technique, the effect of growth hormone (GH) on whole body calcium (Ca) metabolism was studied in children (ages 5-14 years) with type III (n = 9) and IV (n = 8) osteogenesis imperfecta. Each subject was studied twice: at baseline and following a GH (0.1-0.2 U/kg per day) treatment period of 1-1.5 years. Subjects were given 42Ca intravenously and 44Ca orally. The sera and urine 42Ca and 44Ca isotopic enrichments were followed over 7 days using thermal ionization mass spectrometry. The SAAM program was used to fit a three-compartment model to the tracer data. No significant differences were observed between: (1) children with type III and IV disease; or (2) baseline studies of boys and girls within each disease type. However, GH treatment significantly increased: (1) the exchangeable calcium pool (EP) in type III patients (2086 vs. 4422 mg/day, p = 0.02); and (2) the parameter associated with bone calcium accretion in type IV patients (Vo+: 973 vs. 1560 mg/day,p = 0.03) with boys responding with a significantly greater increase than girls (p = 0.008). Although not statistically significant, a trend toward an increase in Vo+ in type III patients and in EP in type IV was observed following treatment. Our observations imply that more Ca was available for bone mineralization following GH treatment in these subjects.

Tracking pathology with proteomics: Identification ofin vivo degradation products of αB-crystallin

Soemmerrings ring is one form of “after cataract” that can occur following cataract surgery. The ring structure is formed by adherence of the anterior lens capsule to the posterior lens capsule. Epithelial cells remaining after surgery differentiate into lens fiber cells but the resulting tissue mass does not remain transparent. The protein in normal lens and in Soemmerrings rings from four individuals was analyzed using two‐dimensional (2‐D) gel electrophoresis, matrix assisted laser desorption/ionization‐time of‐flight‐mass spectrometry (MALDI‐TOF‐MS) and image analysis with Phoretix software. The 2‐D protein patterns of the Soemmerrings rings were generally similar to that of cortical fiber cells of normal human lens with some notable exceptions. Several post‐translationally modified forms of αB‐crystallin(1—175) were identified. Two degradation products, αB‐crystallin(1—170) and αB‐crystallin(1—174), each make up 9.5—27% of the total αB‐crystallin in the Soemmerrings rings and less than 1% in the normal lenses. Other modified forms of αB‐crystallin are aberrant in the fiber cells of the Soemmerring rings relative to normal lens.