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Biomedical subjects

N E Vieira

Publications and source records attributed to N E Vieira.

At least 19 recordsLinked to original sources

Direct vertical electroelution of protein from a PhastSystem band for mass spectrometric identification at the level of a few picomoles.

An electroelution apparatus prototype of a new design was constructed. In that design, the electric field passes vertically through the protein band located on a horizontal (PhastSystem) minigel polymerized on a net of Gel-Fix (Serva). A simple, home-made apparatus allows for electroelution of protein bands at the level of a few picomoles and their identification, after concentration, by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The technique is applicable to one-dimensional (1-D) or two-dimensional (2-D) gels of any size, but has been exemplified only by application to 1-D minigels to demonstrate the lower limits of protein load of the method. When in the course of further development of the prototype it will be combined with a modification to two dimensions of the electroelution mechanism under computer control of the high-performance gel electrophoresis apparatus (formerly of LabIntelligence), the new design appears uniquely qualified for an automated spot elution from 2-D gels under avoidance of gel sectioning.

Electrophoresis, Polyacrylamide Gel↗

Calcium fractional absorption and metabolism assessed using stable isotopes differ between postpartum and never pregnant women.

Determining the fractional absorption (FA) of calcium using the incorporation into urine of stable isotopes given intravenously (IV) and orally has become a routine procedure. We investigated the FA of calcium in two groups of (2-3 mo) postpartum women lactating (LACT) (n = 6) and nonlactating (PPNL) (n = 6), and in never pregnant (NP) women (n = 7). The women consumed a controlled diet containing 30-33 mmol/d calcium (Ca) for 21 d. On d 7 of the controlled diet, the women received 0.05 mmol of 42Ca IV and 0.25 mmol 44Ca orally in milk. Urine samples (24-h) were collected for the next 14 d and morning blood samples were collected from fasting subjects before dosing and at 24 and 48 h after receiving the isotopes. Milk samples from the LACT women were collected from each feeding beginning 24 h before to 72 h after dosing. There were no significant differences in the FA of calcium as measured by stable isotope incorporation into urine (23.8 +/- 2.9%), serum (24.0 +/- 3.4%) or milk (23.6 +/- 3.6%) of LACT women. The fractional calcium absorption measured in urine of the postpartum women (LACT and PPNL, 23.8 +/- 2.9% and 25.0 +/- 3.3%, respectively) did not differ but was greater (P < 0.028) than that of the NP women (17.3 +/- 1.3%). The postpartum LACT and PPNL women had a reduced urinary excretion of calcium (P < 0.01) compared with the NP women. There was a significantly greater incorporation (P < 0.001) by LACT women of the oral isotope dose into milk than into urine. Calcium FA can be determined from incorporation of stable isotopes into breast milk and serum as well as urine.

Absorption↗

Tracking pathology with proteomics: identification of in vivo degradation products of alphaB-crystallin.

Soemmerring's 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 Soemmerring's 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 Soemmerring's rings were generally similar to that of cortical fiber cells of normal human lens with some notable exceptions. Several post-translationally modified forms of alphaB-crystallin(1-175) were identified. Two degradation products, alphaB-crystallin(1-170) and alphaB-crystallin(1-174), each make up 9.5-27% of the total alphaB-crystallin in the Soemmerring's rings and less than 1% in the normal lenses. Other modified forms of alphaB-crystallin are aberrant in the fiber cells of the Soemmerring rings relative to normal lens.

Aged↗

Calcium kinetics in children with osteogenesis imperfecta type III and IV: pre- and post-growth hormone therapy.

Children with osteogenesis imperfecta (OI) type III and type IV were studied using a (42)Ca stable isotope technique. Serum dilution kinetics of (42)Ca were studied pre- and post-growth hormone (GH) treatment in 9 OI III (age range 5-9 years) and 8 OI IV patients (age range 5-12 years). Each subject was studied twice: at baseline and following GH therapy (range 1-1.5 years). Isotopic enrichments of (42)Ca were followed over 7 days using thermal ionization mass spectrometry. A binding site model, which describes reversible and irreversible binding of calcium (Ca) ions to postulated short- and long-term binding sites in bone, was used to analyze the kinetic data. In type III patients, GH treatment (1) increased the fraction of short-term binding sites, theta (0.777 +/- 0.112 versus 0.877 +/- 0.05, respectively; P = 0.034); (2) increased the apparent half-life of a Ca ion attached to the long-term binding site by 76% (P = 0. 009); (3) although not statistically significant (P = 0.098), a trend toward an increased growth rate was observed with increasing change in theta (Deltatheta); (4) patients experienced a 75% increase in growth rate during the first 6 months of treatment. In type IV patients, GH treatment increased the apparent half-life of a Ca ion attached to the long-term binding site by 83% (P = 0.048), however, no trend toward an increased growth rate was observed with increasing Deltatheta in these patients. These significant changes in Ca binding to bone may influence growth in type III patients.

Bone Development↗

Relationship of intestinal calcium absorption to 1,25-dihydroxyvitamin D [1,25(OH)2D] levels in young versus elderly women: evidence for age-related intestinal resistance to 1,25(OH)2D action.

Intestinal calcium absorption decreases with aging, but it is unclear whether this is attributable to an age-related intestinal resistance to 1,25-dihydroxyvitamin D [1,25(OH)2D] action. Thus, we assessed the in vivo dose response of active intestinal calcium absorption to a broad range of circulating 1,25(OH)2D levels in elderly [age (mean +/- SD), 72.5+/-3.0 yr] vs. young women (age, 28.7+/-5.3 yr; n = 20 per group), who were stratified into 5 subgroups: group 1 was given a high calcium intake of 75 mmol/day, suppressing 1,25(OH)2D levels; group 2 was given a normal calcium diet of 15-30 mmol/day, representing basal 1,25(OH)2D levels; group 3 was given a low-calcium diet of 5 mmol/day to stimulate endogenous 1,25(OH)2D production; group 4 was given the low-calcium diet plus 1 microg/day 1,25(OH)2D; and group 5 was given a low-calcium diet plus 2 microg/day 1,25(OH)2D. After 7 days of diet and/or 1,25(OH)2D treatment, fasting fractional calcium absorption (FCA) was assessed by a double-tracer method using stable calcium isotopes. Serum 1,25(OH)2D and vitamin D-binding protein levels were measured concurrently, and the free 1,25(OH)2D index [molar ratio of 1,25(OH)2D to DBP] was calculated. FCA was significantly correlated with the free 1,25(OH)2D index in the young (R = 0.63, P = 0.003) but not in the elderly women (R = 0.27, P = 0.25). Moreover, the slope of the relationship between FCA and free 1,25(OH)2D index (representing intestinal sensitivity to 1,25(OH)2D) was significantly greater in the young (compared with the elderly) women [mean +/- SEM, 0.15+/-0.04 (young) vs. 0.03+/-0.02, elderly, P = 0.03]. Thus, using an experimental design that allowed us to assess FCA over a wide range of 1,25(OH)2D levels, we demonstrate that elderly women have a resistance to 1,25(OH)2D action that may contribute to their negative calcium balance, secondary hyperparathyroidism, and bone loss.

Adult↗

Insulin-like growth factor I and growth hormone (GH) treatment in GH-deficient humans: differential effects on protein, glucose, lipid, and calcium metabolism.

We examined the effects of recombinant human (rh) insulin-like growth factor I (IGF-I) vs. rhGH in a variety of metabolic paths in a group of eight severely GH-deficient young adults using an array of contemporary tools. Protein, glucose, and calcium metabolism were studied using stable labeled tracer infusions of L-[1-13C]leucine, [6,6-2H2]glucose, and 42Ca and 44Ca; substrate oxidation rates were assessed using indirect calorimetry; muscle strength was determined by isokinetic and isometric dynamometry of the anterior quadriceps, as well as growth factors, hormones, glucose, and lipid concentrations in plasma before and after 8 weeks of rhIGF-I (60 microg/kg, sc, twice daily), followed by 4 weeks of washout, then 8 weeks ofrhGH (12.5 microg/kg-day, sc); the treatment order was randomized. In the doses administered, rhIGF-I and rhGH both increased fat-free mass and decreased the percent fat mass, with a more robust decrease in the percent fat mass after rhGH; both were associated with an increase in whole body protein synthesis rates and a decrease in protein oxidation. Neither hormone affected isokinetic or isometric measures of skeletal muscle strength. However, rhGH was more potent than rhIGF-I at increasing lipid oxidation rates and improving plasma lipid profiles. Both hormones increased hepatic glucose output, but rhGH treatment was also associated with decreased carbohydrate oxidation and increased glucose and insulin concentrations, indicating subtle insulin resistance. Neither hormone significantly affected bone calcium fluxes, supporting the concept that these hormones, by themselves, are not pivotal in bone calcium metabolism. In conclusion, rhIGF-I and rhGH share common effects on protein, muscle, and calcium metabolism, yet have divergent effects on lipid and carbohydrate metabolism in the GH-deficient state. These differences may allow for better selection of treatment modalities depending on the choice of desired effects in hypopituitarism.

Adolescent↗

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.

Adolescent↗

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 approximately 1200 mg/day. At baseline (D1), subjects received 42Ca intravenously as well as 44Ca 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 of 42Ca and 44Ca 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.

Adult↗

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 Turner's 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 Turner's 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.

Bone Regeneration↗

Calcium kinetics in glycogen storage disease type 1a.

Glycogen storage disease type 1a (Von Gierke's disease) is one of the more common glycogen storage diseases (GSD). GSD 1a patients can have severe idiopathic osteopenia, often beginning at a young age. Since calcium tracer studies offer a sensitive probe of the bone microenvironment and of calcium deposition, kinetics might be disturbed in patients with GSD 1a. Plasma dilution kinetics obtained using the stable isotope 42Ca are shown in this paper to be quite different between GSD 1a patients and age-matched controls. Comparison of kinetic parameters in these two populations is made using a new binding site model for describing calcium dynamics at the plasma-bone interface. This model describes reversible binding of calcium ions to postulated short-term and long-term sites by a retention probability density function psi (t). Using this analysis, adult GSD subjects exhibited a significant decrease (P = 0.023) in the apparent half-life of a calcium ion on the longer-term site compared with controls. The general theory of calcium tracer dilution kinetics is then discussed in terms of a new model of short-term calcium homeostasis recently proposed by Bronner and Stein [5].

Adolescent↗

Intestinal calcium absorption of women during lactation and after weaning.

We investigated whether intestinal calcium absorption and serum 1,25-dihydroxycholecalciferol (calcitriol) concentrations are higher in women during lactation and after weaning to compensate for calcium lost in breast milk. Measurements were obtained at 4.6 mo postpartum in 24 lactating women and 24 nonlactating women, at 9.6 mo postpartum in 24 lactating women (2.6 mo after complete weaning) and 24 nonlactating women. One-half of the women in each group were randomly assigned to receive 1 g supplemental Ca/d as calcium carbonate. Fractional calcium absorption was measured by using stable isotopic tracers 42Ca and 44Ca. Fractional absorption was 0.32+/-0.02 (+/-SEM) in both lactating and nonlactating women, but was higher in lactating women after weaning (0.37+/-0.02) compared with nonlactating postpartum control subjects (0.31+/-0.02). These effects were independent of calcium intake. Changes in serum calcitriol paralleled changes in fractional absorption. There were no differences in calcitriol concentrations between lactating and nonlactating women, but calcitriol was greater in women after weaning compared with postpartum control subjects. Lactating women who had resumed menses had higher fractional absorption and serum calcitriol than did lactating women who had not. Serum calcium and phosphorus concentrations were greater in lactating compared with nonlactating women; there were no differences between groups after weaning. We conclude that lactation stimulates increases in fractional calcium absorption and serum calcitriol, but the responses are only apparent after weaning or the resumption of menses.

Adult↗

Extraction of serum and urine calcium with ion exchange membrane filters for isotope enrichment determination using thermal ionization mass spectrometry.

The extraction of calcium from water, serum, and urine using Bio-Rex 25 mm ion exchange membrane filters was compared with the oxalate precipitation procedure currently used in our laboratory. Total recoveries of a known quantity of calcium loaded onto the membrane filters for water, serum and urine were as follows: (a) cation exchange filter, 85%, 74%, and 66%; (b) Chelex, 65%, 98%, and 20%; and (c) oxalate precipitation, 93%, 100%, and 96%, respectively. Regression analysis for precipitation versus ion exchange isotope ratio measurements of standards prepared using highly enriched calcium-44 showed slopes of unity. An improvement of automated sample analysis was observed for water and urine calcium samples extracted with ion exchange filters.

Calcium↗

A three-hour measurement to evaluate bone calcium turnover.

It is well established that short-term clearance of an intravenous calcium load in vivo reflects bone uptake. Using results from isotope-dilution experiments with 42Ca, a 3-h test has been developed to measure a quantity, gamma, related to bone accretion. This test is proposed as a useful, clinically applicable measure of bone status. For early times, t, after a bolus of 42Ca, plasma tracer dilution was well approximated by t-gamma, where gamma is related to the fractional rate of loss of tracer, q, from blood into bone (1/q)(dq/dt) = -gamma/t). Gamma was evaluated from kinetic measurements on 91 normal female children, adolescents, and adult women in the age range 4-50 years. For t < or = 3 h, all clearance curves were well fit by a power function. Gamma was found to vary from 0.244 +/- 0.031 for adult premenopausal women (N = 22) to 0.392 +/- 0.056 for prepubertal children (N = 29). Using the Spearman rank-order correlation test, gamma was correlated with bone accretion measured from classic calcium kinetic studies with a correlation coefficient of 0.721, significant at p < 0.005. In those cases in which accretion and resorption remain tightly linked, gamma also provides information on the state of calcium loss from bone. Gamma was evaluated in 14 subjects with bone disease characterised by increased resorption (osteoporosis, Paget's disease) and in 27 subjects with decreased accretion (osteogenesis imperfecta, types I, III, IV; steroid-treated juvenile dermatomyositis). All subjects with Paget's disease and with osteoporosis showed increased gamma, consistent with high bone turnover. The osteoporotic patients furthermore exhibited gamma increasing monotonically by approximately 1% per year after age 55.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

Calcium homeostasis and bone metabolism during pregnancy, lactation, and postweaning: a longitudinal study.

Ten women were followed serially to determine the effect of stages of reproduction on calcium and bone metabolism. The study periods were nonpregnant nonlactating, the end of each trimester of gestation, 3 mo lactation, and postweaning. Comparisons were with nonpregnant nonlactating status for each individual. Fractional calcium absorption (P < 0.0001) and concentrations of 1,25-dihydroxyvitamin D (P < 0.01) were higher in the second and third trimesters. Total urinary calcium was higher during pregnancy and lower postweaning. Parathyroid hormone (PTH) concentrations were higher only postweaning (P < 0.01). Markers of bone turnover increased at the third trimester and during lactation: serum tartrate resistant acid phosphatase and bone specific alkaline phosphatase, and urinary deoxypyridinoline (P < 0.01). Serum procollagen I carboxypeptides increased only in the third trimester (P < 0.01). Bone mineral density by single-photon absorptiometry did not differ by period. We conclude that absorption and urinary excretion of calcium increase during pregnancy whereas bone turnover increases during late pregnancy and lactation; only renal changes consistent with an increase in PTH were seen postweaning.

Absorption↗

Use of stable isotopic tracers in studies of whole body calcium metabolism.

Stable isotopic tracers of calcium have been used to characterize the absorption of dietary calcium and the subsequent distribution of this element through the body. For a group of 7 healthy children, ages 4-14, and 7 children with osteogenesis imperfecta (OI), types I, III and IV, ages 6-17, there were no detectable differences in the fractional absorption of dietary calcium, 0.29+/-0.11 and 0.28+/-0.16, respectively. The total exchangeable pool of calcium was found to be 161+/-52 mg/kg for the healthy children and 95+/-29 mg/kg for the 3 children with Type I OI, 250+/-75 mg/kg for the 3 children with Type III OI and 216 mg/kg for the child with Type IV OI.

Administration, Oral↗

Extraction of magnesium from biological fluids using 8-hydroxyquinoline and cation-exchange chromatography for isotopic enrichment analysis using thermal ionization mass spectrometry.

The use of 8-hydroxyquinoline to precipitate magnesium was evaluated as a method for preparing biological samples for isotopic enrichment analysis using thermal ionization mass spectrometry (TIMS). Standard curves in matrices of water, serum, and urine were prepared using varying amounts of 25Mg. The 25Mg/24Mg isotope ratio was measured by TIMS using a silica gel/phosphoric acid technique. Although the total Mg recovered by precipitation from the matrices varied considerably and was dependent on matrix, recovery was sufficient for isotopic enrichment analysis. Urine samples required cation-exchange chromatography (Bio-Rex AG 50W-X8 filter membrane, Bio-Rad Laboratories) prior to precipitation to remove contaminants which interfered with the thermal ionization process. The observed versus expected 25Mg/24Mg enrichments were evaluated using linear regression analysis: water, y = 0.016 + 1.022x; serum, y = 0.5 + 1.097x; urine, y = -0.004 + 0.943x. This method has proven useful for the isolation of magnesium from the biofluids tested.

Body Fluids↗

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.

Absorptiometry, Photon↗

A non-Markovian model for calcium kinetics in the body.

We present a new generalized compartmental model for calcium kinetics as measured by tracer concentration in blood plasma. The parameter measuring incorporation of calcium in bone discriminates between different levels of physical development in female teenagers and between teenagers and adults.

Adolescent↗