Biomedical subjects
J C Netelenbos
Publications and source records attributed to J C Netelenbos.
Intestinal absorption of strontium chloride in healthy volunteers: pharmacokinetics and reproducibility.
1. The absorption kinetics of orally administered strontium chloride and its reproducibility were investigated in healthy volunteers after administering strontium either under fasting conditions (study I, n = 8) or in combination with a standardized meal (study II, n = 8). Each subject received strontium orally at day 0, 14, and 28 and intravenously at day 42. The study was performed as part of a project in which a simple clinical test for measuring intestinal calcium absorption is being developed, based on the use of stable strontium as a marker. 2. Plasma strontium concentration-time curves were analysed by noncompartment analysis and a four compartment disposition model. Within a volunteer each oral curve was fitted simultaneously with the intravenous curve, by which means a two segment model for absorption was revealed. 3. Mean absolute bioavailability of strontium was 25% without a meal and 19% with a meal, whereas the intraindividual variation was 24% and 20%, respectively. 4. Various limited sampling absorption parameters were determined in order to select a potential test parameter for measuring intestinal calcium absorption using strontium as a marker. Fractional absorption at 4 h (Fc240), obtained after co-ingestion of strontium with a meal, appeared to be the best test parameter, because it represented bioavailability well (r = 0.90).
Intra-individual variation in bone resorption markers in urine.
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Growth hormone induces tyrosine phosphorylation of annexin I in rat osteosarcoma cells.
GH induces phosphorylation of a number of cellular proteins, of which several have now been identified, such as mitogen-activated protein kinase, insulin receptor substrate-1, and members of the JAK kinase and STAT families of proteins. However, other phosphorylated proteins remain unidentified. Growth factors and cytokines, including epidermal growth factor, insulin, pp60v-scr, and angiotensin II, induce a rapid phosphorylation of annexin I, a 35-kDa member of the annexin family of Ca2+ and phospholipid-binding proteins. The osteoblast-like rat osteosarcoma cell-line UMR-106.01, in which GH acts as a mitogen via a high affinity GH receptor, was used as a model for GH-induced protein phosphorylation. It is demonstrated by immunoblotting and immunoprecipitation techniques that GH induces the phosphorylation of annexin I on tyrosine residues. This phosphorylation is dose and time dependent. Induction of annexin I phosphorylation is delayed compared with that of JAK2. These results identify annexin I as a protein that becomes tyrosine phosphorylated under the influence of GH and show that phosphorylation of annexin I is a general phenomenon that follows activation of a cell by hormones or cytokines.
Growth hormone binds to a single high affinity receptor site on mouse osteoblasts: modulation by retinoic acid and cell differentiation.
Growth hormone (GH) exerts direct differentiative and proliferative effects on osteoblasts. We studied 125I-labeled human (h) GH binding to primary mouse osteoblasts derived from collagenase-treated 18-day fetal mouse calvaria. Scatchard analysis of the data revealed a single class of high affinity GH receptors (apparent Ka = 5.74 x 10(9) M-1) with 2200 sites per cell. Affinity crosslinking and SDS-PAGE electrophoresis showed two bands with apparent molecular masses of 120 and 70 kDa. Mouse osteoblasts express GH receptor mRNA with gene transcripts of 4.2 and 1.2 kb, at levels which reach approximately 1/6 of those in mouse liver and 1/3 of those in mouse muscle. Two populations of undifferentiated and diffentiated osteoblasts, obtained by sequential collagenase digestion of mouse calvaria, were used to study the relationship between osteoblastic phenotype and GH receptor expression. Although the affinity of the receptors in undifferentiated and differentiated cells was the same, the capacity was significantly higher (1.45 +/- 1.0% vs 2.39 +/- 0.9%, P = 0.03) in differentiated cells. This stresses the specific importance of the osteoblast as a target cell for GH. The differentiating potential of the vitamin A derivative retinoic acid was subsequently used experimentally to induce differentiation in the cells. Retinoic acid increased 125I-hGH binding to preosteoblasts (153%, P = 0.02). Together, these data demonstrate the presence of a high affinity GH receptor in mouse osteoblasts which is related to differentiation.
Immunoadsorption assay for bone alkaline phosphatase compared with wheat germ agglutinin precipitation assay in serum from (pre)pubertal girls.
The performance characteristics of two bone alkaline phosphatase (ALP; EC 3.1.3.1) assays, a wheat germ agglutinin (WGA) precipitation assay and a new immunoadsorption assay (IAA), were compared. The within- and between-run imprecision of the IAA (3.6-4.2% and 3.6-7.7%) was comparable with that of the WGA assay. The mean cross-reactivity with liver ALP appeared to be 4% in the WGA assay and 11% in the IAA. The reference ranges in a group of 155 healthy Caucasian (pre)pubertal schoolgirls were: 149-401 U/L (total ALP, 30 degrees C), 105-349 U/L (bone ALP, 30 degrees C, WGA assay), and 58-205 U/L (bone ALP, 25 degrees C, IAA). Comparison of the WGA assay (x) with the IAA (y) demonstrated a correlation coefficient of 0.95 [Deming regression equation: y = (0.56 +/- 0.01)x + (2.0 +/- 1.5); Sy[symbol: see text]x = 5.3 U/L]. Correlation studies of the WGA assay and the IAA results with total ALP demonstrated r = 0.98 and 0.96, respectively.
Growth hormone receptor activity is stimulated by insulin-like growth factor binding protein 5 in rat osteosarcoma cells.
Osteoblast-like UMR-106.01 rat osteosarcoma cells express high affinity growth hormone (GH) receptors (GHRs). Because osteoblasts secrete insulin-like growth factor binding protein-5 (IGFBP-5), we evaluated whether it also modulates GH binding and GHR expression in UMR cells. Human recombinant intact IGFBP-5 stimulated 125I-hGH binding in a dose-dependent manner (dose range 300-3000 ng/ml), inducing an increase to 193.6 +/- 2.1% of control binding at 3000 ng/ml (P < 0.001). Carboxy-truncated IGFBP-5 also stimulated GH binding but with less potency (125 +/- 2.7% of control at 3000 ng/ml, P < 0.01). GHRs identified by chemical crosslinking of 125I-hGH to cell monolayers increased after treatment with IGFBP-5 and decreased in response to insulin-like growth factor-I (IGF-I). GHR mRNA levels, as quantitated by a solution hybridization RNAse protection assay, increased up to 3 to 7-fold in a time-dependent manner by intact IGFBP-5 but not by carboxy-truncated IGFBP-5. An antiserum to IGFBP-5 reduced basal GH binding to 56.7 +/- 4.3% of control value at a concentration of 0.5% (P < 0.001), showing that IGFBP-5 produced by the cells is a strong regulator of GH binding. IGFBP-5 antiserum also decreased GH binding to 85.9 +/- 0.9% of IGFBP-5 stimulated value (P < 0.001), showing the specificity of IGFBP-5 stimulation. To determine whether the GHR upregulation was physiologically significant, cell proliferation was evaluated after coincubation of IGFBP-5 with low, non-stimulatory concentrations of GH. IGFBP-5 (1000 ng/ml) induced cell proliferation to 116.2 +/- 3.2% of control levels, and coincubation with hGH at 10 ng/ml induced an increase to 133.3 +/- 0.1% of control levels. We conclude that exogenous and endogenous IGFBP-5 upregulate GHR mRNA levels and GH binding and this interaction potentiates GH-stimulated mitogenesis in osteoblastic cells.
Measurement of intestinal calcium absorption by using stable strontium.
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Insulin-like growth factor binding proteins-2 and -3 stimulate growth hormone receptor binding and mitogenesis in rat osteosarcoma cells.
GH exerts its biological actions on osteoblasts through a specific high affinity receptor expressed on these cells. GH receptor binding is positively modulated by a number of factors, including retinoic acid and dexamethasone, whereas fetal calf serum strongly decreases the binding. To identify responsible factors in serum, components of serum, the insulin-like growth factors (IGFs)-I and -II, and IGF binding proteins (IGFBPs)-2 and -3 were tested for a possible negative modulatory role. IGF-I and -II decreased [125I]hGH binding at an optimal concentration of 30 ng/ml for IGF-I and 100 ng/ml IGF-II, reducing the binding to 51% and 55%, respectively, of control values. A stimulation of [125I]hGH binding was observed with IGFBP-2 as well as IGFBP-3, inducing an increase to 148% and 151% of control binding at an optimal concentration of 3000 ng/ml for both peptides. The effects of all peptides were dependent on the incubation time, being significantly increased after 8 h of incubation and reaching the full effect thereafter. The effects were declined at 24 h compared with 16 h for IGFBP-2 and -3 but not for IGF-I and -II. Coincubation of the cells with IGF-I and -II and IGFBP-2 and -3 neutralized the effects of the factors alone. In conclusion, these results show that IGF-I and -II on the one hand and IGFBP-2 and -3 on the other hand exert opposite actions on [125I]hGH binding, IGFBP-2 and -3 exerting probably an IGF-independent effect. Further, IGF-I and -II decreased GH receptor messenger RNA (mRNA) levels, as quantified by a solution hybridization ribonuclease protection assay, from 8.65 +/- 1.78 attomoles (amol)/microgram DNA (control) to 2.4 +/- 0.68 and 2.16 +/- 0.92 amol/microgram DNA, respectively. IGFBP-2 increased GH receptor mRNA levels from 5.26 +/- 1.17 (control) to 13.19 +/- 3.48. Incubation with IGFBP-3 did not result in stimulation of GH receptor mRNA levels (8.59 +/- 2.91 amol/microgram DNA). This shows that the mechanism of regulation of the GH receptor is, except for IGFBP-3, at least in part on the mRNA level. Lastly, IGFBP-2 and IGFBP-3 are mitogenic for UMR-106.01 rat osteosarcoma cells, inducing an increase in cell number to 125% and 142% of control cell counts after 48 h of incubation with 1000 ng/ml IGFBP-2 and -3, whereas IGF-I, IGF-II and Long R3 IGF-I did not stimulate proliferation. IGFBP-2 and -3 potentiate hGH induced mitogenesis at low hGH concentrations of both factors, whereas at higher concentrations no such effect is observed.(ABSTRACT TRUNCATED AT 400 WORDS)
[Osteoporosis and jaw bone reduction].
The relationship between osteoporosis and residual ridge reduction has been investigated in many studies. As a result it can be concluded that it is not easy to measure the extent of osteoporosis of the jaws. Several studies have shown that individuals with general signs of osteoporosis do not necessary have an osteoporotic mandible or maxilla. The same applies to the relationship between osteoporosis and residual ridge reduction in edentulous patients and osteoporosis and bone loss in patients with periodontal diseases. Taken into account the positive effect of healthy food intake and functional loading in the prevention and treatment of osteoporosis, it can be expected that they also influence the bone quality of the jaws. Preservation of strategic teeth or roots under the overdenture as well as the insertion of implants have a positive effect on the bone of the jaws.
Intestinal strontium absorption: from bioavailability to validation of a simple test representative for intestinal calcium absorption.
Calcium absorption tests have rarely been validated for being representative for absolute bioavailability (true absorption) or for intraindividual variation. Therefore, we investigated the reproducibility of the absolute bioavailability of strontium chloride, a marker for intestinal calcium absorption, in healthy male volunteers (n = 8) by measuring the area under the plasma strontium concentration-time curve after oral and intravenous administration of strontium. Subsequently, we selected a simple test variable as being representative of absolute bioavailability. The mean absolute bioavailability (+/- SD) was 25% +/- 7%. The best test variable appeared to be the fractional absorption at 240 min (Fc240) after oral intake, which demonstrated the highest correlation with absolute bioavailability (r = 0.66). The intraindividual variations of the data for this variable and for the absolute bioavailability are similar to those described for various absorption tests based on the use of calcium isotopes. Thus, the Fc240 of strontium offers the potential of a simple clinical test for use as a measure of intestinal calcium absorption and its modulation.
Sensitive method for analysis of strontium in human and animal plasma by graphite furnace atomic absorption spectrophotometry.
For long-term pharmacokinetic studies in humans as well as in experimental animals, an analysis of strontium (Sr) with a low detection limit and high sensitivity is necessary. The data presented here describe the optimization of the furnace program and sample handling for measuring Sr in plasma by using graphite furnace atomic absorption spectrophotometry. The method was validated and applied to studies on the pharmacokinetics of SrCl2 in humans and rats. Calibration curves were linear up to 57.1 nmol/L. The limit of detection and lower limit of quantification were 0.21 and 0.57 nmol/L, respectively. Reciprocal sensitivity was 0.53 nmol/L Sr at A = 0.0044. The intraassay precision was 2.2%, 1.5%, and 1.1% (n = 6) at 4.57, 22.7, and 49.2 nmol/L Sr, respectively. At these concentrations, the interassay precision and recovery were 0.7%, 1.5%, and 1.8% and 100.4%, 99.1%, and 100.6% (n = 12), respectively. The endogenous Sr concentration in human plasma samples was 0.27 +/- 0.07 mumol/L (n = 18). Pharmacokinetic studies in a human volunteer and in rats demonstrated that this procedure was suited for measuring low Sr concentrations and was applicable to small sample volumes.
Long-term effect of calcium supplementation on bone loss in perimenopausal women.
We observed in a controlled 2 year longitudinal trial in 248 perimenopausal women that a daily calcium supplement of either 1000 or 2000 mg Ca2+ significantly reduced lumbar bone loss and bone turnover in the first year of calcium supplementation. In the second supplementation year the rate of lumbar bone loss in the treated subjects was not significantly different from that in the control group, although two of the three biochemical parameters of bone turnover remained decreased throughout the study. To quantify further the long-term effect of calcium supplementation, we extended the study for another year in 214 women. In the women of the control group who were menstruating until the last year of the trial, the mean change in lumbar bone mineral density after 3 years was -3.2% of the initial value versus 1.6% in the calcium-supplemented groups (p < 0.01). The decrease in lumbar bone loss in these supplemented premenopausal and early perimenopausal women remained statistically significant in the second and third years of supplementation. In the women who stopped menstruating before or during the study, the long-term reduction in lumbar bone loss was not significant (mean difference between control and treatment groups < 0.6% points after 3 years). The decrease in metacarpal cortical thickness (MCT) in the treated subjects during 3 years was on average -3.0% of the initial value in the control versus -2.0% in the supplemented subjects (P < 0.01). The effect of calcium supplementation on MCT was not significantly related to the menopausal status of the subjects.(ABSTRACT TRUNCATED AT 250 WORDS)
Topically applied low-dose calcitriol has no calciotropic effect in patients with stable plaque psoriasis.
BACKGROUND: Topical calcitriol, a potent inhibitor of cell proliferation and inducer of terminal cell differentiation, can clear psoriasis. However, possible side effects on calcium and bone metabolism from transdermal absorption have not been evaluated. OBJECTIVE: The calciotropic effects of low-dose calcitriol (3 micrograms/gm) ointment, applied twice daily for 6 weeks, were investigated. METHODS: A double-blind study was carried out in 18 patients with chronic stable plaque-type psoriasis, of whom nine were treated with calcitriol (3 micrograms/gm) and nine with betamethasone dipropionate (500 micrograms/gm). The main end points were calcitriol plasma concentrations, intestinal calcium absorption, and bone turnover. RESULTS: Serum alkaline phosphatase concentrations increased slightly (p < 0.02) and intestinal calcium absorption decreased slightly (p < 0.01) in the calcitriol-treated group. However, the alterations were too small to have any clinical relevance. CONCLUSION: Low-dose calcitriol, topically applied for 6 weeks on a maximal body surface area of 30%, can be considered as safe regarding calcium and bone metabolism.
Dexamethasone increases and serum decreases growth hormone receptor binding to UMR-106.01 rat osteosarcoma cells.
Dexamethasone (DEX) is known to exert major effects on functions of osteoblast-like cells. We investigated its action on the regulation of GH receptors in the osteoblast-like osteosarcoma cells UMR-106.01. DEX stimulated [125I]human GH (hGH) binding to UMR-106.01 cells. This effect was dose dependent and significant in a concentration range of 10(-8)-10(-6) M. The maximum effect was an increase of 42 +/- 1.4% (n = 3; mean +/- SE) above control, P < 0.01, at 10(-7) M DEX. Time dependence of this stimulation was observed, with a peak between the 12th and the 16th h of incubation, an effect being still detectable at 48 h. Cycloheximide decreased [125I]hGH binding and completely abolished the stimulating effect of DEX, suggesting that modulation of [125I]hGH binding by DEX is fully dependent on protein synthesis. Addition of fetal calf serum (FCS) resulted in a dose-dependent decrease of [125I]hGH binding to 24 +/- 2% of control (n = 3; mean +/- SE), P < 0.001, without interfering with the stimulatory effect of DEX, the ratio of DEX vs. control being higher with increasing FCS doses. Taken together, these results suggest the existence of different pathways for the regulation of GH receptor binding to UMR-106.01 cells, including a stimulatory one at the pretranslational level for DEX and an inhibitory one for (growth) factors present in FCS.
Estrogen replacement therapy continuously combined with four different dosages of dydrogesterone: effect on calcium and lipid metabolism.
Lipid and bone metabolism was studied in 165 healthy postmenopausal women treated with 2 mg 17 beta-estradiol continuously combined with one of four doses (2.5, 5, 10, or 15 mg) of dydrogesterone in a double blind randomized study design. Fasting blood and urine samples were drawn at baseline and after 3 and 6 months of treatment. Bone remodeling was significantly reduced in all four treatment groups, as indicated by the decrease in serum corrected calcium, phosphate, and alkaline phosphatase and the urinary calcium/creatinine ratio. A dose response of dydrogesterone on these indices was not found. With all four dosages of dydrogesterone, lipid profile (total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol, and apolipoproteins) improved significantly; however, this was less pronounced with the highest dydrogesterone dose. Our data suggest that continuously applied dydrogesterone in combined hormone replacement therapy does not annihilate the beneficial effects on bone remodeling and lipid metabolism induced by estrogens.
One-hour test for estimating intestinal absorption of calcium by using stable strontium as a marker.
Intestinal absorption of calcium is a relevant marker in a broad spectrum of diseases; however, its determination in clinical practice is difficult. Our aim was to design a simple test for this based on stable strontium (88Sr). The correlation between the intestinal absorption of simultaneously administered 45Ca and 88Sr was investigated in patients with various disorders of the bone and calcium metabolism. The area under the curve for the period 0-60 min after dosing (AUC0-60 in mmol.L-1.min), being a representative measure of intestinal absorption, showed a close correlation between both elements (r = 0.90, P < 0.001). Moreover, the measure of agreement in classifying the patients as low, intermediate, or high absorbers was high (kappa = 0.80). We conclude that a test based on measuring AUC0-60 of strontium is a fast and inexpensive way to obtain reliable information about the level of intestinal calcium absorption.