Variability of PDYN and OPRK1 genes in four argentinian populations and its genetic association with clinical variables related to acute postsurgical pain / Variabilidad de los genes PDYN y OPRK1 en cuatro poblaciones argentinas y su asociación con variables clínicas relacionadas al dolor agudo post-quirúrgico

ABSTRACT Several population studies showed an association between variation in pain sensitivity and genetic polymorphisms located in Prodynorphin (PDYN) and Kappa Opioid Receptor (OPRK1) human genes. We analysed polymorphisms of these two genes to characterise their variation in Argentinian populations, as well as to evaluate their association with acute pain sensitivity. We studied 11 genetic markers in individuals from four locations in Argentina (Ciudad Autónoma de Buenos Aires, La Plata, Resistencia, and Misión Nueva Pompeya), calculated the population parameters, and evaluated the possible association among pain sensitivity, clinical, and genetic variables through a Generalised Estimating Equation model. High linkage disequilibrium was observed in the four populations for both genes, and significant differences were found among frequencies of Argentinian populations and those from other continents reported in the 1000 Genomes Project. Four PDYN gene polymorphisms from 3´ untranslated region and exon 4 showed association with acute pain sensitivity. One genotype of each of these polymorphisms was associated with a higher pain sensitivity, probably related with the activation of the N-methyl-D-aspartate (NMDA) receptors. We found a strong association with acute pain for the following clinical variables: 1) time after surgery, 2) intravenous klosidol supplied every 8 h, and 3) type of incision. Our results highlight the importance of a regional study of genetic variants which influence pain sensitivity and analgesic response.

RESUMEN La asociación entre la sensibilidad al dolor y los polimorfismos que presentan los genes humanos de prodinorfina (PDYN) y receptor opioide kappa (OPRK1) se ha evidenciado en distintos estudios poblacionales. Con el objetivo de caracterizar la variación de estos genes y evaluar su asociación con dolor agudo en la población argentina, analizamos 11 polimorfismos en individuos provenientes de cuatro localidades argentinas (Ciudad Autónoma de Buenos Aires, La Plata, Resistencia, y Misión Nueva Pompeya). Calculamos los parámetros poblacionales y evaluamos la posible asociación entre sensibilidad al dolor, variables clínicas y variables genéticas a través de un modelo de ecuación generalizada de estimación. Se observó alto desequilibrio de ligamiento para ambos genes en las cuatro poblaciones analizadas, y se encontraron diferencias significativas entre las frecuencias de poblaciones argentinas y las reportadas en el Proyecto 1000 Genomes para poblaciones de otros continentes. Cuatro polimorfismos de la región 3´UTR y el exón 4 de PDYN mostraron asociación con la sensibilidad al dolor agudo. En cada uno de estos polimorfismos, un genotipo resultó asociado con alta sensibilidad al dolor, probablemente en relación con la activación de receptores N-metil-D-aspartato (NMDA). Encontramos una fuerte asociación con dolor agudo para las siguientes variables clínicas: 1) tiempo post-cirugía, 2) administración intravenosa de klosidol cada 8 h, y 3) tipo de incisión. Nuestros resultados resaltan la importancia de realizar estudios regionales de variables genéticas que influyen en la sensibilidad al dolor y la respuesta analgésica.

Increased calcium uptake and improved trabecular bone properties in intestinal alkaline phosphatase knockout mice.

Previous studies have demonstrated a negative correlation between intestinal alkaline phosphatase (IAP) activity and calcium (Ca) absorption in the gut, as IAP acts as a protective mechanism inhibiting high Ca entry into enterocytes, preventing Ca overload. Here we evaluated Ca absorption and bone properties in knockout mice (KO) completely devoid of duodenal IAP (Akp3 -/- mice). Female C57BL/6 control mice (WT, n = 7) and KO mice (n = 10) were used to determine Ca absorption in vivo and by in situ isolated duodenal loops followed by histomorphometric analysis of duodenal villi and crypts. Bone mineral density, morphometry, histomorphometry and trabecular connectivity and biomechanical properties were measured on bones. We observed mild atrophy of the villi with lower absorption surface and a significantly higher Ca uptake in KO mice. While no changes were seen in cortical bone, we found better trabecular connectivity and biomechanical properties in the femurs of KO mice compared to WT mice. Our data indicate that IAP KO mice display higher intestinal Ca uptake, which over time appears to correlate with a positive effect on the biomechanical properties of trabecular bone.

Low-Level Fluoride Exposure Increases Insulin Sensitivity in Experimental Diabetes.

The effect of chronic fluoride (F) exposure from the drinking water on parameters related to glucose homeostasis was investigated. Wistar rats were randomly distributed into 2 groups (diabetic [D] and nondiabetic [ND]; n = 54 each). In D, diabetes was induced with streptozotocin. Each group was further divided into 3 subgroups (0, 10, or 50 mgF/L in drinking water). After 22 days of treatment, plasma and liver samples were collected. No alterations in glycemia, insulinemia, K(ITT), and HOMA2-IR (homeostasis model assessment 2 of insulin resistance) were seen for ND. F-exposure of D rats led to significantly lower insulinemia, without alterations in glycemia (increased %S). Proteomic analysis detected 19, 39, and 16 proteins differentially expressed for the comparisons D0 vs. D10, D0 vs. D50, and D10 vs. D50, respectively. Gene Ontology with the most significant terms in the comparisons D0 vs. D10, D0 vs. D50, and D50 vs. D10 were organic acid metabolic process and carboxylic acid metabolic process, organic acid metabolic process, and cellular ketone metabolic process. Analysis of subnetworks revealed that proteins with fold changes interacted with GLUT4 in comparison D0 vs. D10. Among these proteins, ERj3p was present in D10. Upregulation of this protein in the presence of F might help to explain the higher %S found in these animals. These data suggest that fluoride might enhance glucose homeostasis in diabetes and identify specific biological mechanisms that merit future studies.

Sequential treatment with monofluorophosphate and zoledronic acid in osteoporotic rats.

OBJECTIVE: Osteoporosis is the consequence of an imbalance in bone remodeling caused by excessive resorption or inappropriate bone formation. This paper proposes a sequential treatment with monofluorophosphate (MFP) and zoledronic acid (Z), together with changes in the calcium content in the diet. METHOD: Seven-week-old female Sprague Dawley rats were divided into five groups (n = 21 per group): (1) sham-operated rats (Sham); (2) ovariectomized (OVX) rats fed with a normal calcium diet (OVX); (3) OVX rats fed with a normal calcium diet and treated sequentially with monofluorophosphate and zoledronic acid (OVX.G1); (4) OVX rats sequentially fed with a low calcium diet and then a high calcium diet, without treatment (OVX.G2); (5): OVX rats fed with a low calcium diet and then a high calcium diet, treated sequentially with monofluorophosphate and zoledronic acid (OVX.G3). RESULTS: After 150 days, the OVX.G3 group showed a similar bone volume to that of the Sham group due to an increase in trabecular number. Dual X-ray absorptiometry bone analysis showed an increase of 9.8% compared with OVX rats. Additionally, an increase in the fracture load at the cortical bone and higher fracture load, ultimate load and stiffness in the compression test were found. CONCLUSION: The sequential treatment with monofluorophosphate and zoledronic acid increases trabecular bone mass, bone mineral density and bone strength.

Effect of the treatment with alpha-macroglobulin on the development of experimental pancreatitis.

UNLABELLED: Alpha-macroglobulins are proteinase inhibitors. Monofluorophosphate increases alpha-macroglobulin levels in plasma, inducing a higher survival rate and lower pancreatic damage in rats with pancreatitis. The aim of this study was to evaluate the effect of alpha-macroglobulin on the development of pancreatitis. Pancreatitis was surgically induced in Sprague-Dawley rats divided into groups of 16 rats each and -subjected to the following intravenous treatments for 3 days: CONTROLS: pancreatitis without treatment, Enriched plasma: pancreatitis+-alpha-macroglobulin-enriched plasma, Normal plasma: pancreatitis+plasma with normal levels of alpha-macroglobulin, Saline Solution: pancreatitis+saline solution, Purified alpha-macroglobulin: pancreatitis+purified alpha-macroglobulin. After 14 days pancreatic damage was assessed using a score that measures: edema, fibrin, neutrophils, mononuclear leukocytes, necrosis, vascular congestion, thrombosis, hemorrhage and fibrosis. Pancreatic damage decreased and the percentage of animals with pancreatitis was lower in enriched-plasma and purified alpha-macroglobulin groups. We conclude that the intravenous administration of alpha-macroglobulins causes a reduction in the histological damage produced by pancreatitis.

Mathematical model for the homeostasis of alpha-macroglobulins in the rat.

Alpha-macroglobulins (AM) are proteins that inactivate proteinases. Sodium monofluorophosphate (MFP) binds to AM and transiently changes AM plasma levels. As a consequence MFP is useful to modify AM homeostasis. A mathematical model to study the homeostasis of AM is proposed in this paper. The model describes changes in plasma concentration of AM, MFP concentration in the gastrointestinal tract, MFP plasma concentration, plasma concentration of AMMFP and includes rate constants of the processes involved in AM homeostasis. Estimation of the rate constants values was achieved using experimental and mathematical resources. The homeostasis of AM after an oral dose of 80 µmol of MFP was analyzed with a simulation tool. Experimental conditions that modify the homeostasis of AM had been simulated and validated using specific drugs that change some parameter of the system. The mathematical model describes accurately the behavior of the biological model. The results allow concluding that the simplifications made did not underestimate the main processes involved in the homeostasis and, also that the assumptions made were correct.

Bone morphometry and differences in bone fluorine containing compounds in rats treated with NaF and MFP.

Sodium fluoride (NaF) and sodium monofluorophosphate (MFP) are drugs used to increase bone mass. They have been considered equivalent but the results of the treatments were not always coincident. Most studies have been carried out in osteoporotic women or ovariectomized rats pointing to the result in bone mass rather than at the mechanism of action. Convincing evidence indicates that pharmacokinetic of NaF is different from MFP. While only fluoride is found in bones and plasma of rats treated with NaF, in MFP-treated rats, there are also fluorine (F) bound to plasma alpha-macroglobulin and bone covalently bound F. A significant increase in bone mass of rats was observed after 30 days of treatment with NaF and MFP in young rats. This increase in bone mass correlates with the increase in number and thickness of trabeculas in cancellous bone. In the femur of MFP-treated rats, there was an increase in the inertia momentum of the diaphysis without changes in bone width. In addition, bone F content of MFP-treated animals is twice of the content of NaF-treated rats. This difference is the consequence of bone covalently bound F, which is absent in NaF-treated rats. In addition, alpha-macroglobulin was detected in noncollagenous bone matrix of MFP-treated rats. Although F in feces and plasma did not differ among treatments, the urinary excretion of F was lower in MFP than in NaF-treated rats, which is consistent with the higher bone F content.

Effect of disodium monofluorphosphate on plasma and blood viscosity in the rat.

Alpha macroglobulins (AM) are plasma proteins whose main function is to inactivate proteinases, protecting the tissues from the action of these enzymes. AM have influence on plasma viscosity (PV) and binds monofluorophosphate (MFP), which disturbs its homeostasis. The aim of this work was to evaluate whether the administration with MFP could modify blood viscosity. AM levels (micromol/l), PV (mPa.s), viscosity of red blood cells suspensions in NaCl 9 g/l (VES) and in autologue plasma (VEP) were measured in fifty-day old rats after a single dose of 80 micromol MFP or after 30 days of treatment with 80 micromol of MFP. Relative viscosity (RV) was calculated as the ratio VEP/PV. AM and PV increased significantly after 30 min of an oral dose of MFP. Controls (n=6), AM: 19.65+/-0.85, PV: 1.39+/-0.01, treated (n=6), AM: 22.88+/-0.75 (p<0.05), PV: 1.76+/-0.14 (p<0.05). After 30 days of treatment with MFP, AM and PV increased significantly. Controls (n=6), AM: 10.76+/-1.33, PV: 1.19+/-0.04, treated (n=6), AM: 17.66+/-1.27 (p<0.05), PV: 1.38+/-0.03 (p<0.05). The treatment with MFP modifies neither the VEP nor the RV. These results would indicate that AM and/or MFP did not interact with erythrocyte membrane and did not modify erythrocyte deformability.

Intestinal absorption of fluoride at high luminal concentration of fluoride.

The paper reports that high fluoride concentrations in the intestinal lumen hinders the absorption of this anion. This conclusion was verified with three different experimental models. Pharmacokinetic experiments done in human volunteers revealed that the bioavailability of fluoride from sodium fluoride (NaF, CAS 7681-49-4) enteric coated tablets was 33% of that of plain (immediate release) tablets. The latter findings were confirmed in rats receiving 1 ml of NaF solutions (40, 80 or 160 mmol/l) by gavage. The greatest AUC (area under the curve of fluoremia as a function of time) was obtained with an oral dose of 80 mumol of NaF. This parameter was significantly greater (p < 0.01) with 80 mumol than with 40 mumol NaF, but similar to that observed with 160 mumol. Fecal fluoride excretions (in the 24 h following a single dose of NaF) and the bone fluoride contents (found at the end of 30 days of treatment with 40, 80 or 160 mumol NaF/day), agreed with the AUC values. The rate of fluoride absorption (v, mumol/10 min) through the intestinal wall was investigated with perfused, isolated rat duodenum in vivo. Fluoride absorption increased between 0 and 10 mmol/l luminal fluoride and decreased with higher concentrations. Oxygen consumption of duodenal-tissue decreased exponentially between zero (1.12 mumol O2 min-1 g-1) and 10 mmol/l fluoride (0.45 mumol O2 min-1 g-1).

Measurement of total and diffusible serum fluoride.

This article describes a technique for the measurement of total and diffusible F content of serum, at clinical significant concentrations of F (1-10 microM). The proposed procedure avoids the interference of unknown serum components with the ion-specific electrode. Sample F is concentrated fivefold through distillation of hydrofluoric acid (Taves' method). Ionic fluoride is presented to the electrode in a simple solution at concentrations within the linear response of the electrode. Average recoveries of F from serum or its ultrafiltrate were 96+/-7% (21%) and 97+/-12% (53%) (mean+/-SEM [CV]), respectively. With four replicates of each sample, the technique produce within-run standard deviations of 0.6 microM and 2.2 microM at 1 and 10 microM F, respectively. Total precision assessment gave standard deviations of 0.6 microM and 2.6 microM at 1 and 10 microM F, respectively. The fasting serum F levels of normal climacteric women, 45 to 65 years, showed an asymmetric distribution. The data obtained started at the detection limit of the technique (0.1 mM). The 75 percentile was 1.85 microM for total and 0.5 microM for diffusible F. In patients (n = 25) treated with NaF (30 mg F/day) the fasting levels of total serum F (4.5 +/-1.7 microM) did not differ from those of diffusible F (4.2+/-1.5 microM). In patients (n = 50) treated with sodium monofluorophosphate (15 mg F/day) the fasting levels of total and diffusible serum F were 6.5+/-1.7 microM and 0.5+/-0.03 microM, respectively. In conclusion, this paper establishes the presence of two fractions of serum fluorine: diffusible and nondiffusible (or protein bound) and describes a technique for their clinical estimation. In untreated subjects and in patients receiving NaF, the former fraction contains ionic fluoride. In patients treated with MFP, diffusible serum fluorine is composed by ionic fluoride and low molecular weight, peptide-bound, acid-labile fluorine.

Metabolism of the complex monofluorophosphate-alpha 2-macroglobulin in the rat.

Sodium monofluorophosphate (MFP) is a drug used in the treatment of primary osteoporosis. Following the intake of MFP, a small fraction of the drug is absorbed intact and forms a complex with alpha 2-macroglobulin (MFP-alpha 2M) inactivating the antiproteasic activity of the globulin. The complex has been shown to occur in the serum of rats and human being. This paper reports data on the metabolism of this complex in the rat. In vitro experiments showed that liver and bone tissue remove MFP-alpha 2M from the incubation medium. When the experiments were pursued beyond the time needed to reduce the complex concentration to very low levels, fluorine (F) reappears in the medium in two forms: bound to low molecular weight macromolecule/s (2,200 +/- 600 Da) and as ionic F. Concentrations of these F fractions increase while that of the complex decreases as a function of time. In vitro, uptake of the complex by liver or bone tissue was not affected by the presence of colchicine or methylamine. These drugs, however, inhibited intracellular metabolism of the complex, as indicated by the impairment of the return of F species to the extracellular space and the increase in F content of the tissue. The cellular receptors responsible for the uptake of the complex in liver and bone are insensitive to low concentration of calcium and inhibited by polyinosinic acid[5']. These features characterize the "scavenger" receptor, one of the two receptor types known to remove inactive alpha 2M from the circulation. Injection of polyinosinic acid [5'] to living rats also hindered the disappearance of the complex from serum. It is concluded that the metabolism of the MFP-alpha 2M complex involves binding to receptors, uptake by cells, lysosomal degradation and return of F bound to low molecular weight macromolecule/s to the extracellular space. It is assumed, however, that inorganic F is the final product of lysosomal hydrolysis of the protein moiety.

In postmenopausal osteoporosis the bone increasing effect of monofluorophosphate is not dependent on serum fluoride.

According to previous pharmacokinetic studies the bioavailability of fluorine (F) from sodium monofluorophosphate (MFP) doubles that of sodium fluoride (NaF). This paper reports a study designed to verify whether the vertebral bone mass increasing effect of NaF (30 mg F/day) was comparable to that of MFP (15 mg F/day), given for 18 months to osteoporotic postmenopausal women. The BMD of lumbar vertebrae of both groups showed significant increases (MFP: 60 +/- 15 mg/cm2, NaF: and 71 +/- 12 mg/cm2) over basal levels (P < 0.001). The difference between treatments was not significant (P = 0.532). The serum levels of ionic F (the mitogenic species on osteoblasts) were not related to the above mentioned effects. In NaF-treated patients, the fasting levels of total serum F increased significantly (6.7 +/- 0.9 microM vs. Basal: 2.0 +/- 0.8 microM; P < 0.001). This phenomenon was accounted for by ionic fluoride that increased over 20-fold (6.5 +/- 1.9 microM vs. Basal: 0.3 +/- 0.04 microM). In MFP-treated patients the fasting serum levels of total (7.0 +/- 0.7 microM vs. Basal: 2.2 +/- 0.9 M) and diffusible F (0.5 +/- 0.02 microM vs. Basal 0.2 +/- 0.02 microM) increased significantly (P < 0.001). The increase in the non diffusible F fraction is accounted for by protein-bound F, probably by the complexes formed between MFP and alpha 2-macroglobulin and C3. Serum diffusible F was formed by two fractions: ionic F and F bound to low molecular weight macromolecule/s (2,200 +/- 600 Da), in approximately equal amounts. The general information afforded by the present observations support the hypothesis that ionic F is released progressively during the metabolism of MFP bound to alpha 2-macroglobulin and C3. These phenomena explain why comparable effects to those obtained with 30 mg F/d of NaF could by obtained with one half the dose of MFP.

Binding of monofluorophosphate to alpha2-macroglobulin and C3.

After administering an oral dose of monofluorophosphate (MFP) to human beings or rats, a fraction of the drug appears in plasma that is bound to proteins, establishing a previously undetected compartment of nondiffusible fluoride. This article documents experiments performed in vitro, describing the binding of MFP to two plasma globulins: alpha2-macroglobulin and C3 (a beta-globulin). MFP binds irreversibly to these proteins through a stable bond. MFP binds to purified alpha2-macroglobulin or to C3 with a molar ratio MFP: protein close to unity. MFP binding reduces significantly the biological activity of these proteins, which share in common a macrocyclic 4-residue ring thiolactone (Cys-Gly-Glu-Glu). The binding site of MFP is as yet unknown. Protein-bound MFP appeared in the plasma of volunteers during the 5-7 hours following intake. Peak concentration of protein-bound MFP and maximal reduction of alpha2-macroglobulin activity was observed 2 hours after intake. Clearance of protein-bound MFP coincided with the return of alpha2-macroglobulin to basal levels.

[Changes in bone mass and in glucose homeostasis in subjects with high spontaneous fluoride intake]. / Modificaciones en la masa osea y en la homeostasis de la glucosa en residentes de la zona de Bahía Blanca con alta ingesta espontanea de flúor.

This paper reports metabolic data of 24 women and two men, 44-66 years old, ex-residents in an area of endemic fluorosis close to Bahía Blanca city. Fasting fluoremias of these subjects (0.5 to 9.2 microM) and daily urinary fluoride excretion (> 60 mumoles/day) are characteristics of zones with endemic fluorosis. Bone mineral density (BMD) at the lumbar spine (L2-4 1330 +/- 41 mg/cm2) and femoral neck (1045 +/- 10 mg/cm2) were significantly above average of normal subjects of the same age and sex. A significant correlation was observed between the daily excretion of fluoride and BMD L2-4 (r = 0.43, P < 0.05). The Area Under the Curve of insulin during a standard glucose tolerance test showed an inverse relationship with fluoremia. This observation coincides with experiments published elsewhere indicating that fluoride intake at concentrations 5 microM or greater, inhibits the secretion of insulin.

Bioavailability of fluoride administered as sodium fluoride or sodium monofluorophosphate to human volunteers.

This paper reports a reassessment of the bioavailability of fluoride from monofluorophosphate (MFP, CAS 10163-15-2). It was prompted by recent work from this laboratory reporting that, following an oral dose of MFP, a fraction of the drug appears in plasma bound to globulins forming a previously undetected compartment of non-diffusible fluoride. The presence of protein-bound MFP in plasma after the intake of this drug hinders its straightforward comparison with NaF (CAS 7681-49-4). After an oral of NaF, all plasma fluoride is diffusible. After intake of MFP, on the other hand, plasma contains diffusible fluoride and protein-bound fluoride during the 6-8 h following intake. The area under the curve of total plasma fluoride for MFP (1540 +/- 117 mumol.min/l) doubles that of NaF (811 +/- 52 mumol.min/l p < 0.001). On this basis, in agreement with findings previously reported for the rat, it is concluded that the bioavailability of fluoride for MFP doubles that of NaF.

Comparative study of the effect of sodium fluoride and sodium monofluorophosphate on glucose homeostasis in the rat.

The effect of acute and chronic administration of sodium fluoride (NaF) or sodium monofluorophosphate (MFP) on the glucose homeostasis of the rat are compared. The oral administration of a single dose of 40 mumol/100 g b.w. of either compound produced similar increases in plasma glucose (up to 1.8 g/l) and diffusible fluoride (up to 130 mumol/l). In long-term experiments (three months of duration), treatment with NaF (a 5 mmol/l solution as the water supply) produced, in the first month of experiment, abnormal glucose tolerance tests and increased plasma diffusible fluoride levels (range: 2-12 mumol/l). Treatment with MFP, on the other hand, did not affect glucose homeostasis; plasma diffusible fluoride was always below 2 mumol/l. The results of these experiments indicate that glucose homeostasis is affected when plasma diffusible fluoride exceeds 5 mumol/l. The basal and glucose-stimulated insulin secretion of isolated Langerhans rat islets (incubated with solutions containing 2, 5, 10 and 20 mumol/l NaF) was significantly inhibited by 5 to 20 mumol/l fluoride. No effect was observed under similar conditions with MFP at concentrations of 2, 5, 10, 20 and 50 mumol/l.

Pharmacokinetic differences between sodium fluoride and sodium monofluorophosphate and comparative bone mass increasing activity of both compounds in the rat.

After the administration of an oral dose of 80 mumol/l of NaF (CAS 7681-49-4) to rats, the area under the curve of total plasma fluoride equals 10,200 mumol.min/l. After an oral dose of 80 mumol of monofluorophosphate (MFP/CAS 10,163-15-2), two forms of fluoride appear in plasma: protein-bound MFP and diffusible fluoride. The areas under the curve of total (protein-bound + diffusible) and diffusible fluoride equal 22,200 and 8,850 mumol.min/l, respectively. The activity of MFP for increasing the bone mass of the rat was assessed with NaF as the standard. The animals were treated chronically for 100 days since weaning with food ad libitum and 5 mmol/l NaF, 5 or 2.5 mmol/l MFP solutions as the water supply. The effect obtained with 2.5 mmol/l MFP was similar to that produced by 5 mmol/l NaF, indicating a potency ratio MFP is twice as active as NaF.

Gastric and intestinal absorption of monofluorophosphate and fluoride in the rat.

This paper reports data indicating that, in the rat, sodium monofluorophosphate (MFP, CAS 10163-15-2) is absorbed without hydrolysis through the stomach. This phenomenon and the fact that MFP binds to plasma globulins produce a prompt increase in protein-bound MFP concentration, significantly greater than the increase in plasma ultrafilterable fluoride. Intestinal absorption of MFP was investigated with the isolated duodenal loop in situ. The drug was hydrolyzed to fluoride at the intestinal lumen with a rate of approx. 3 times greater than that for fluoride absorption. After filling the duodenal loop with a MFP solution, ultrafiltrable fluoride accounts for the increase in plasma fluoride concentration, with no evidence of protein-bound MFP. This report also includes analytical techniques for the measurement of MFP in test solutions and in plasma.

Bone mass increase and glucose tolerance in rats chronically treated with sodium fluoride.

Female rats were treated with fluoride for 100 days (between 21 and 121 days of age) replacing the water supply with a 5 mM NaF solution. Bone mass was assessed by destructive physical and chemical measurements on the whole skeleton, that gave an overall view not reported previously. Bone mass (dry, fat-free weight of the skeleton/100 g of body weight) increased 7% (P less than 0.001) with respect to control animals. This phenomenon was equally evident in the head, the axial and the appendicular skeleton. Fluoride treatment did not affect the ratio ashes/organic matrix. Treated animals showed a subtle disturbance of glucose tolerance as shown by glucose tolerance tests. The disturbance was manifest as high plasma and soft tissue levels of fluoride during the period of bone mass increase. Glucose tolerance was normalized when the maximum bone mass was achieved and plasma and soft tissue fluoride returned to control levels.

Estimation of bone turnover in climacteric women by the whole body retention of fluoride.

This paper reports the measurement of whole body retention using fluoride (WBRF) as an estimator of skeletal turnover in a group of climacteric women that received an oral dose of 700 mumol of sodium fluoride. WBRF is defined as 100(1-(urinary fluoride/fluoride load)). WBRF was significantly correlated with whole body retention of 99m-Tc-methylene-diphosphonate, the serum levels of the bone alkaline phosphatase and the urinary excretion of hydroxyproline. WBRF values ranged from 20% to 95% and were affected by calcium intake and the urinary calcium excretion. In normal subjects with high turnover, the measurement of serum alkaline phosphatase activity and/or urinary hydroxyproline excretion helps to distinguish these cases from patients with metabolic bone diseases due to metastases, Paget disease, etc. The fact that the fraction of fluoride not incorporated into bone is not further metabolized plus the accuracy, preciseness and rapidity of fluoride measurements in urine are the main advantages of this technique.