Nanoparticulate System Based on Calcium-Crosslinked Carbomer Retards Percutaneous Drug Permeation: New Insight Into Skin Barrier Functions.

BACKGROUND: The stratum corneum poses a formidable barrier for dermal and transdermal delivery of drugs. Besides the stratum corneum barrier, the viable epidermis poses another challenge to pharmaceutical formulators. A drug is probably transdermally permeable if it rapidly crosses the epidermal secondary barrier, while stimulation of lamellar body secretion from granular cells and intracellular release of Ca++ from endoplasmic reticulum (ER) result in retardation. OBJECTIVE: To evaluate the skin permeability of lidocaine HCl loaded in nanoparticles made of carbomer calcified with calcium gluconate, while figuring out the physiological mechanism that regulates the Ca++ related skin barrier function. METHODS: Lidocaine hydrochloride was loaded in a nanoparticulate system based on calcified carbomer, fabricated by using a water-in-oil microemulsion as a precursor. In vitro release and percutaneous permeation testing were carried out to compare between calcified and non-calcified nanoparticles. In addition, comparison was also made between calcified nanoparticles using carbomer gels prepared at two pH values and at two different ratios of Ca++/carbomer. RESULTS: A unique structure of the calcified nanoparticles has been proposed, in which the carbomer nanoparticles are partially coated by gluconate ions through hydrogen bonding and partially through ionic interactions with calcium ions. Although the in vitro release data showed no difference between non-calcified and calcified carbomer nanoparticles, a calcium-related phenomenon of skin retardation has been revealed. CONCLUSIONS: It has been proposed that stimulation of lamellar body secretion from granular cells and Ca++ release from ER, which is elicited by the calcium gluconate-coated nanoparticles, result in dermal retardation of lidocaine.

Effect of feeding calcium gluconate embedded in a hydrogenated fat matrix on feed intake, gastrointestinal fermentation and morphology, intestinal brush border enzyme activity and blood metabolites in growing lambs.

Gluconate salts have been identified as a butyrate precursor when fed to non-ruminant species and may increase the butyrate concentration in the large intestine supporting gastrointestinal health and development. The objective of this study was to evaluate the dose response of hydrogenated fat-embedded calcium gluconate (HFCG) on performance and gastrointestinal tract (GIT) development in growing lambs. Thirty-two wether lambs were used in a randomized complete block design and assigned to 1 of 4 treatments differing in the inclusion of HFCG: 0.0% (CON), 0.075% (LOW), 0.30% (MED), and 0.60% of the diet (HIGH). Lambs were allocated into individual pens and fed ad libitum with feed delivered twice daily. Feed intake was recorded daily, and body weight (BW) was assessed at the beginning and the end of the 29-d period. Blood was sampled on day 21, prior to feeding and 6 h post-feeding to evaluate changes in ß-hydroxybutyrate, glucose, and insulin concentrations. Total fecal collection was conducted during days 25 to 28 to assess apparent total tract digestibility. On day 29, lambs were slaughtered, and the entire GIT was separated by region to enable sampling of tissue and digesta. Data were analyzed to assess linear, quadratic, and cubic effects of HFCG dose. Final BW, average daily gain, and dry matter intake decreased linearly (P ≤ 0.02) with increasing HFCG. Increasing inclusion of HFCG linearly decreased (P = 0.01) the thickness of the stratum corneum in ruminal papillae but did not affect other strata (P ≥ 0.34). Omasal digesta weight linearly decreased (P = 0.01) as the concentration of HFCG increased and abomasal digesta weight was cubically affected (P = 0.03) the increasing dose of HFCG. Short-chain fatty acid concentration in the cecum was cubically affected (P < 0.01) with increasing dose of HFCG where low dose had the greatest concentration. Moreover, increasing the dietary supply of HFCG linearly increased the proportion of acetate (P = 0.04) in the cecum and linearly decreased the proportion of propionate in the digesta of both the cecum (P < 0.01) and colon (P = 0.01). Colon crypt depth was quadratically (P = 0.03) affected with the increasing dose of HFCG, where lambs fed MED had greatest crypt depth. We conclude that feeding HFCG to growing lambs did not increase butyrate concentration in the large intestine and consequently does not increase the absorptive surface area of the whole tract, the size of the GIT, or the functionality of the intestine.

Gluconate salts have been reported to be metabolized by microbes in the gastrointestinal tract to yield butyrate. Butyrate has shown potential to enhance functionality of the gastrointestinal tract by increasing the absorptive surface area, enzyme activity, and the barrier function. This study evaluated the inclusion of four levels of hydrogenated fat-embedded Ca-gluconate (HFCG; 0.0%, 0.075%, 0.30%, and 0.60% of the diet) designed to increase the production of butyrate in the large intestine. Thirty-two wether lambs were fed for 28 d, slaughtered, and eviscerated to allow complete evaluation of the gastrointestinal tract and its contents. Growth and dry matter intake decreased linearly with increasing dose of HFCG. Dose of HFCG cubically affected short-chain fatty acid concentration in the cecum with increased concentrations at the 0.075% dose. Moreover, increasing dose of HFCG linearly increased the proportion of acetate and linearly decreased the proportion of propionate in the cecum without altering the proportion of butyrate. Thus, the supplementation of HFCG did not increase butyrate concentration in the large intestine and did not enhance gastrointestinal tract function.

Postruminal infusion of calcium gluconate increases milk fat production and alters fecal volatile fatty acid profile in lactating dairy cows.

Gluconic acid is a carboxylic acid naturally occurring in plants and honey. In nonruminant animals, gluconic acid has been shown to increase gastrointestinal butyrate concentrations and improve growth performance, but a ruminant application remains undescribed. This experiment examined the effects of postruminal calcium gluconate (CaG) on milk production, fecal volatile fatty acid concentrations, and plasma metabolite concentrations in lactating dairy cows. Six rumen cannulated multiparous Holstein cows (60 ± 6 d in milk) were randomly assigned to 6 treatment sequences within a 6 × 6 Latin square design in which each experimental period consisted of 5 d of continuous postruminal infusion followed by a 2 d wash-out period. Test treatments included a negative control (CON; 0.90% NaCl wt/vol), positive control (Na-butyrate, 135 g/d), and 4 doses of CaG (44, 93, 140, and 187 g/d). Cows received a total mixed ration (31% corn silage, 28% alfalfa silage, 5% hay, 36% concentrate) with dry matter intake fixed (25.3 ± 1.7 kg/d) throughout the experiment. On d 5 of each infusion period, samples of milk, feces, and blood were collected from each animal. Calcium gluconate treatments increased milk fat concentration, and a tendency was observed for increased milk fat yield and energy-corrected milk yield above levels achieved by CON, with maximal treatment responses of 4.43% (CON 3.81%), 2.089 kg/d (CON 1.760 kg/d), and 51.8 kg/d (CON 47.1 kg/d), respectively. Concentrations of iso-butyric acid in feces were greater in cows infused with CaG (13.3 µmol/g) treatments compared with CON (9.7 µmol/g). Arterial concentrations of glucose and nonesterified fatty acids were lower (glucose: CaG 2.98 mmol/L, CON 3.29 mmol/L and nonesterified fatty acids: CaG 0.130 mmol/L vs. 0.148 mmol/L) and ß-hydroxybutyrate higher (CaG 1.703 vs. CON 0.812) in cows infused with CaG than CON. Together, these results suggest that postruminal infusion of CaG may alter metabolic mechanisms to support a milk fat production response.

Calcium gluconate as cross-linker improves survival and shelf life of encapsulated and dried Metarhizium brunneum and Saccharomyces cerevisiae for the application as biological control agents.

Calcium chloride (CC) is the most common cross-linker for the encapsulation of biocontrol microorganisms in alginate beads. The aim of this study was to evaluate if calcium gluconate (CG) can replace CC as cross-linker and at the same time improve viability after drying and rehydration, hygroscopic properties, shelf life and nutrient supply. Hence, the biocontrol fungi Metarhizium brunneum and Saccharomyces cerevisiae were encapsulated in Ca-alginate beads supplemented with starch. Beads were dried and maximum survival was found in beads cross-linked with CG. Beads prepared with CG showed lower hygroscopic properties, but a higher shelf life for encapsulated fungi. Moreover, we demonstrated that gluconate has a nutritive effect on encapsulated fungi, leading to increased mycelium growth of M. brunneum and to enhanced CO2 release from beads containing Saccharomyces cerevisiae. The application of CG as cross-linker will pave the way towards increasing drying survival and shelf life of various, especially drying-sensitive microbes.

Fabrication and characterization of the nano-composite of whey protein hydrolysate chelated with calcium.

The nano-composites of whey protein hydrolysate (WPH) chelated with calcium were fabricated in aqueous solution at 30 °C for 20 min, with the ratio of hydrolysate to calcium 15 : 1 (w/w). UV scanning spectroscopy, fluorescent spectroscopy, Fourier transform infrared spectroscopy, dynamic light scattering and atomic force microscopy were applied to characterize the structure of the WPH-calcium chelate. The nano-composites showed the successful incorporation of calcium into the WPH, indicating the interaction between calcium and WPH. The chelation of calcium ions to WPH caused molecular folding and aggregation which led to the formation of a WPH-calcium chelate of nanoparticle size, and the principal sites of calcium-binding corresponded to the carboxyl groups and carbonyl groups of WPH. The WPH-calcium chelate demonstrated excellent stability and absorbability under both acidic and basic conditions, which was beneficial for calcium absorption in the gastrointestinal tract of the human body. Moreover, the calcium absorption of the WPH-calcium chelate on Caco-2 cells was significantly higher than those of calcium gluconate and CaCl2 in vitro, suggesting the possible increase in calcium bioavailability. The findings suggest that the WPH-calcium chelate has the potential in making dietary supplements for improving bone health of the human body.

A single-dose study of denosumab in patients with various degrees of renal impairment.

This 16-week study evaluated pharmacokinetics and pharmacodynamics of denosumab in 55 subjects with renal function ranging from normal to dialysis-dependent kidney failure. Participants received a single 60-mg subcutaneous dose of denosumab. Kidney function groups were based on calculations using the Cockcroft-Gault equation and U.S. Food and Drug Administration (FDA) guidance in place when the study was designed. Renal function did not have a significant effect on denosumab pharmacokinetics or pharmacodynamics. These findings suggest denosumab dose adjustment based on glomerular filtration rate is not required. Rapid decreases in serum C-telopeptide in all groups were sustained throughout the study. The most common adverse events were hypocalcemia (15%), pain in extremity (15%), and nausea (11%). Most adverse events were mild to moderate in severity. Calcium and vitamin D supplementation was not initially required by the study protocol, but was added during the trial. No subject who received adequate calcium and vitamin D supplementation became hypocalcemic. Seven subjects had nadir serum calcium concentrations between 7.5 and <8.0 mg/dL (1.9 and <2.0 mmol/L), and 5 subjects (4 with advanced renal disease) had nadir serum calcium <7.5 mg/dL (<1.9 mmol/L). Two subjects (1 symptomatic, 1 asymptomatic) were hospitalized for intravenous calcium gluconate treatment. At the recommended dose, denosumab is a useful therapeutic option for patients with impaired renal function. Supplementation of calcium and vitamin D is strongly recommended when patients initiate denosumab therapy, particularly in patients with reduced renal function.

The pivotal role of intracellular calcium in oxaliplatin-induced inhibition of neurite outgrowth but not cell death in differentiated PC12 cells.

The antineoplastic efficacy of oxaliplatin, a widely used anticancer drug, is restricted by its adverse effects such as peripheral neuropathy. Infusing a combination of calcium gluconate and magnesium sulfate (Ca/Mg) suppresses the acute neurotoxic side effects of oxaliplatin, although the mechanism is unclear. To elucidate the molecular mechanisms of oxaliplatin-induced neurotoxicity and the effects of Ca/Mg against this toxicity, we examined the effect of Ca/Mg on oxaliplatin-induced inhibition of neurite outgrowth in PC12 cells, a commonly used neuronal cell model. Oxaliplatin and oxalate suppressed nerve growth factor (NGF)-induced neurite outgrowth and reduced the NGF-mediated increase in the intracellular calcium concentration [Ca(2+)](i). A calcium-chelating agent, BAPTA/AM, also exhibited similar inhibitory effects on neurite outgrowth and [Ca(2+)](i). The addition of Ca/Mg attenuated these inhibitions induced by oxaliplatin and oxalate. The NGF-induced upregulation of growth-associated protein-43 (GAP-43) was suppressed by oxaliplatin and oxalate. Oxaliplatin, but not oxalate, suppressed NGF-stimulated extracellular signal-regulated kinase activation, and this inhibition was not affected by Ca/Mg. Ca/Mg did not modify the oxaliplatin-induced loss of cell viability or apoptosis in PC12 or HCT-116 cells, a human colorectal cancer cell line. These results suggest that the inhibition of neurite outgrowth but not tumor cell death induced by oxaliplatin is partly associated with reductions in [Ca(2+)](i) and GAP-43 expression, and this inhibition was suppressed by the addition of Ca/Mg. Therefore, it may be assumed that Ca/Mg is useful for protecting against oxaliplatin-induced neurotoxicity without reducing the antitumor activity of oxaliplatin.

A model for calcium permeation into small intestine.

An in vitro model was used to simulate the intestinal permeation of calcium ions depending on the type of salt (carbonate, fumarate, citrate, or gluconate), its concentration (1.0, 2.5, 5.0, or 10 mM/l), and pH (1.3, 4.2, 6.2, or 7.5). To simulate the conditions for calcium permeation in a patient in a fasting state, the solutions were placed in contact with segments of small intestine of pig: stomach, duodenum, jejunum, and ileum. The percent permeation, its rate, and half-time were measured in each case. In all cases, the maximum permeation was seen at 1 mM concentration, depending on pH: 100% for carbonate at pH 1.3; 82% for fumarate, pH 6.2; 79.5% for citrate at pH 4.2, and 81% for gluconate at pH 7.4. The maximum rate of permeation (% h(-1)) was also observed at 1 mM: 2.16 for carbonate at pH 1.3, 0.29 for fumarate at pH 6.2, 0.26 for citrate at pH 4.2, and 0.28 for gluconate at pH 7.4. The shortest half-time permeation (t (1/2), h) for 1 mM solutions depended also on pH (in parentheses): carbonate 0.3 (1.3), fumarate 2.4 (6.2), citrate 2.6 (4.2), and gluconate 2.5 (7.4). The results suggest that calcium carbonate and citrate can be recommended to patients with normal gastric acidity and hyperacidity while fumarate and gluconate to patients with hypoacidity.

Sorbitol production using recombinant Zymomonas mobilis strain.

A recombinant Zymomonas mobilis strain harboring the plasmid pHW20a-gfo for over-expression of glucose-fructose oxidoreductase (GFOR) was constructed. The specific activity of GFOR enzyme in the new recombinant strain was at least two folds greater than that in the wild strain. The maximum GFOR activity achieved in terms of the volumetric, and the cellular were 2.59 U ml(-1), and 0.70 U mg(-1), respectively, in the batch cultures. A significant improvement of the bioconversion process for the production of sorbitol and gluconic acid from glucose and fructose was made using divalent metal ions which drastically reduced the ethanol yield and significantly increased the yield of target product. Among several divalent metal ions evaluated, Zn(2+) was found to be most effective by inhibiting the Entner-Doudoroff pathway enzymes. The yield of the byproduct ethanol was reduced from 16.7 to 1.8 gl(-1) and the sorbitol yield was increased to almost 100% from 89%. The Ca(2+) enhanced the sorbitol yield and the formation of calcium gluconate salt made the separation of gluconate from the reaction system easier.

Application of 2-factorial design on the enhanced production of calcium gluconate by a mutant strain of Aspergillus niger.

Sixty-eight isolates of the filamentous fungus Aspergillus niger were examined for calcium gluconate production under submerged culture conditions in 500-ml Erlenmeyer flasks. The isolate Chem-15 was selected for improvement through ultraviolet (UV) light-induced mutagenesis. Among viable mutants, strain 32 exhibited the best gluconate productivity, and it was subjected to N-methyl N-nitro N-nitroso guanidine (NG) treatment. Mutant strain NG-7 gave the highest gluconate production (86.48g/L) which varied significantly (p0.05) from that of the wild type. The mutant was cultured overnight and plated on 5-fluorocytosine-PDA medium. Gluconate productivity was increased by 35% when the process parameters, incubation period (72h), initial pH (6.5), glucose as carbon source (15%), inoculum size (1.875x10(6)CFU/ml) and corn steep liquor (CSL) as nitrogen source (0.5%) were optimized using a 2-factorial Plackett-Burman design. Maximal glucose oxidase activity (28U/ml/min) was achieved at the optimal fermentation conditions with 26.5g/L DCM. The model terms were highly significant thus suggesting the potential commercial utility of the mutant (HS, df=3 approximately 0.0182).

A comparative study on several models of experimental renal calcium oxalate stones formation in rats.

In order to compare the effects of several experimental renal calcium oxalate stones formation models in rats and to find a simple and convenient model with significant effect of calcium oxalate crystals deposition in the kidney, several rat models of renal calcium oxalate stones formation were induced by some crystal-inducing drugs (CID) including ethylene glycol (EG), ammonium chloride (AC), vitamin D(3)[1alpha(OH)VitD(3), alfacalcidol], calcium gluconate, ammonium oxalate, gentamicin sulfate, L-hydroxyproline. The rats were fed with drugs given singly or unitedly. At the end of experiment, 24-h urines were collected and the serum creatinine (Cr), blood urea nitrogen (BUN), the extents of calcium oxalate crystal deposition in the renal tissue, urinary calcium and oxalate excretion were measured. The serum Cr levels in the stone-forming groups were significantly higher than those in the control group except for the group EG+L-hydroxyproline, group calcium gluconate and group oxalate. Blood BUN concentration was significantly higher in rats fed with CID than that in control group except for group EG+L-hydroxyproline and group ammonium oxalate plus calcium gluconate. In the group of rats administered with EG plus Vitamin D(3), the deposition of calcium oxalate crystal in the renal tissue and urinary calcium excretion were significantly greater than other model groups. The effect of the model induced by EG plus AC was similar to that in the group induced by EG plus Vitamin D(3). EG plus Vitamin D(3) or EG plus AC could stably and significantly induced the rat model of renal calcium oxalate stones formation.

Bioconversion of waste office paper to gluconic acid in a turbine blade reactor by the filamentous fungus Aspergillus niger.

Gluconic acid production was investigated using an enzymatic hydrolysate of waste office automation paper in a culture of Aspergillus niger. In repeated batch cultures using flasks, saccharified solution medium (SM) did not show any inhibitory effects on gluconic acid production compared to glucose medium (GM). The average gluconic acid yields were 92% (SM) and 80% (GM). In repeated batch cultures using SM in a turbine blade reactor (TBR), the gluconic acid yields were 60% (SM) and 67% (GM) with 80-100 g/l of gluconic acid. When pure oxygen was supplied the production rate increased to four times higher than when supplying air. Remarkable differences in the morphology of A. niger and dry cell weight between SM and GM were observed. The difference in morphology may have caused a reduction of oxygen transfer, resulting in a decrease in gluconic acid production rate in SM.

Enhanced excitability of hippocampal mossy fibers and CA3 neurons under dietary zinc deficiency.

On the basis of the evidence that susceptibility to kainate-induced seizures is enhanced by zinc deficiency and that glutamate concentrations in hippocampal extracellular fluid are excessively increased during seizures, excitability of hippocampal mossy fibers and CA3 neurons was examined using hippocampal slices, which were prepare from mice fed a zinc-deficient diet for 4 weeks. The spatio-temporal dynamics of zinc and calcium was monitored using their indicators, membrane-impermeable ZnAF-2 and membrane-permeable fura-2 AM, respectively. When the molecular layer of dentate gyrus was stimulated with 100mM KCl for 1s, the increased percentages of extracellular zinc in the stratum lucidum and CA3 pyramidal cell layer were higher in zinc-deficient mice than in the control mice, implying that glutamate release from the mossy fibers of the dentate granular cells is enhanced by zinc deficiency. Judging from the increased percentages, however, the amount of zinc released was estimated to be less in zinc-deficient mice. On the other hand, the basal calcium concentrations in the stratum lucidum and CA3 pyramidal cell layer detected with fura-2 were higher in zinc-deficient mice than in the control mice, indicating that hippocampal calcium homeostasis is affected by zinc deficiency. Furthermore, the increased percentage of intracellular calcium in the stratum lucidum by stimulation with high K+ was enhanced by the zinc deficiency. The alteration of hippocampal calcium homeostasis seems to enhance excitability of dentate granular cells in zinc deficiency, following by an enhanced excitability of postsynaptic structures in CA3 neurons.

Comparison of low and high dose of vitamin D treatment in nutritional vitamin D deficiency rickets.

In this study, we compared three different therapy modes (150,000 IU, 300,000 IU, and 600,000 IU vitamin D p.o.) in infants with nutritional vitamin D deficiency rickets (VDR). Our purpose was to determine the most effective dosage of vitamin D with least side effects for treating VDR. The study included 56 patients, 3-36 months of age, with nutritional VDR and 20 age-matched control infants. In all infants, serum calcium, phosphorus, alkaline phosphatase, magnesium, serum 25-hydroxycholecalciferol, plasma intact parathormone levels and urinary Ca/creatine ratio were determined. Of 56 patients, 52 were able to be followed long-term. These patients were reexamined on the 3rd day, 7-10th day, and 25-30th day after treatment. On the 30th day post-treatment, we did not find any difference between the doses in the improvement of rickets. However, hypercalcemia was present in eight infants who had been administered 300,000 IU (two infants) and 600,000 IU (six infants) of vitamin D. In conclusion, our findings showed that 150,000 IU or 300,000 IU of vitamin D was adequate in the treatment of VDR, but 600,000 IU of vitamin D may carry the risk of hypercalcemia.

Substituted glycals as probes of glycosidase mechanisms.

D-Glucal and a series of substituted derivatives have been tested as substrates, inhibitors and inactivators of the Agrobacterium faecalis beta-glucosidase in order to probe structure/function relationships in this enzyme. D-Glucal is shown to be a substrate (kcat = 2.3 min-1, Km = 0.85 mM) undergoing hydration with stereospecific protonation from the alpha-face to yield 2-deoxy-beta-D-glucose. 1-Methyl-D-glucal surprisingly serves as only a poor substrate (kcat = 0.056 min-1, Km = 57 mM), also undergoing protonation from the alpha-face. 2-Fluoro-D-glucal, however is completely inert, as a result of inductive destabilisation of the oxocarbenium ion-like transition state for protonation, and functions only as a relatively weak (Ki = 24 mM) inhibitor. Similar behaviour was seen with almond beta-glucosidase and yeast alpha-glucosidase and for the interaction of 2-fluoro-D-galactal with Escherichia coli beta-galactosidase. A series of of alpha, beta-unsaturated glucal derivatives was also synthesised and tested as potential substrates, inhibitors or inactivators of A. faecalis beta-glucosidase. Of these only 1-nitro-D-glucal functioned as a time dependent, irreversible inactivator (ki = 0.011 min-1, Ki = 5.5 mM), presumably acting as a Michael acceptor. Electrospray mass spectrometric analysis revealed multiple labeling of the enzyme by this inactivator, lessening its usefulness as an affinity label. Less reactive Michael acceptor glycals which might have been more specific (1-cyano-, 2-cyano-, 1-carboxylic acid, 1-carboxylic acid methyl ester) unfortunately did not function as inactivators or substrates, only as relatively weak reversible inhibitors (Ki = 3-96 mM).

Development of a mutant strain of Aspergillus niger and optimization of some physical factors for improved calcium gluconate production.

In the course of mutation studies of Aspergillus niger strain AB with ethylene imine (1:4000), a mutant A. niger AB 501, produced greater amount of calcium gluconate in the culture broth (88.0 g/lit) as against the parent strain (36.0 g/lit) by the surface culture method of fermentation. This mutant was then exposed to UV-rays and a mutant, A.niger AB 1801, was found to produce high calcium gluconate in the culture broth (120 g/lit). The optimum cultural conditions for the production of calcium gluconate by A.niger AB 1801 were pH, 6.5; period of incubation, 9 days; volume of medium in 1 litre flask, 150 ml; temperature, 30 degrees C, volume of inoculum, 7.5 ml of cell suspension containing 2.6 x 10(7) spores and age of inoculum, 6 days old spores of A. niger AB 1801. The maximum yield of calcium gluconate to the above conditions was 168 g/lit. The cultural conditions that support maximum cultural growth did not, however, give optimal yield of calcium gluconate because after having yielded the maximum of calcium gluconate the growth of organism continued to increase further.

Efficient phosphate binding using a combination of gluconolactate and carbonate calcium salts.

Although renal-failure-related hyperphosphataemia can be corrected by various phosphate binders, there remains a need for safer and more efficient formulations to precipitate phosphate. This work describes both a theoretical approach and a phosphate precipitation test in order to design efficient binding calcium salts formulations. The results show that the combination of a soluble calcium salt (the gluconolactate) and a proton-consuming calcium salt (the carbonate) can precipitate phosphate effectively. Furthermore, the theoretical computations correlate well with the ability of the salt to bind phosphate in vitro.

Isotopic exchange plus substrate and inhibition kinetics of D-xylose isomerase do not support a proton-transfer mechanism.

The D-xylose isomerase of Streptomyces olivochromogenes is a Mg2+- or Mn(2+)-dependent enzyme that catalyzes the aldose-ketose isomerization of xylose to xylulose or of glucose to fructose. Proton exchange into water during enzyme-catalyzed isomerization of C-2 tritiated glucose at 15, 25 and 55 degrees C shows < 0.6% exchange (the loss of one proton in every billion turnovers). High concentrations of guanidine hydrochloride and extremes of pH had no effect on the amount of exchange detected. Such a low percentage of exchange is inconsistent with a proton-transfer mechanism as the main kinetic pathway for isomerization. 19F NMR experiments showed no release of fluoride after incubation of the enzyme for 4 weeks with 800 mM 3-deoxy-3-fluoroglucose or 3-deoxy-3-fluoroallose (both are competitive inhibitors with Ki values of 600 mM). This result is also inconsistent with a proton-transfer mechanism. A hydride-shift mechanism following ring opening has been proposed for the isomerization. Enzyme-catalyzed ring opening was directly measured by demonstrating H2S release upon reaction of xylose isomerase with 1-thioglucose. D-Xylose isomerase-catalyzed interconversion of glucose to fructose exhibited linear Arrhenius behavior with an activation energy of 14 kcal/mol from 0 to 50 degrees C. No change in rate-determining step occurs over this temperature range. 13C NMR experiments with glucose show that enzyme-bound magnesium or manganese does not interact specifically with any one site on the sugar. These results are consistent with nonproductive binding modes for the substrate glucose in addition to productive binding.

Transdermal estrogen replacement does not increase calcitonin secretory reserve in postmenopausal women.

The effect of transdermal estrogen replacement on ionized calcium and calcitonin levels was examined in 15 postmenopausal women. Following baseline measurement of calcitonin and ionized calcium in the fasting state, the effect of calcium infusion on calcitonin levels was studied. Estrogen replacement resulted in a fall in baseline ionized calcium, however, the rate of rise of calcium was the same before and after estrogen administration. Thus the time at which a particular calcium concentration was attained was later after the commencement of the calcium infusion following estrogen replacement. Although there was no detectable difference in baseline calcitonin concentrations (pre-estrogen, 2.4 +/- 0.4; post-estrogen, 2.1 +/- 0.4 pmol/l), following estrogen replacement the time at which a particular calcitonin concentration was attained was later after the commencement of the calcium infusion, reflecting the slower attainment of a particular calcium concentration (p = 0.014 by ANOVA). Analysis of total calcitonin production by area under the curve, however, did not show a significant difference before and after estrogen replacement (643 +/- 184 and 407 +/- 115 pmol.l-1.100 min-1, respectively). When the calcitonin response to calcium infusion was compared at the same calcium concentration, estrogen status had no effect on the relationship. We conclude that transdermal estrogen replacement has no effect on calcitonin secretory reserve in postmenopausal women and does not alter the relationship between elevated calcium and calcitonin levels. We cannot exclude an effect of estrogen on baseline calcitonin levels as the calcium concentration was lower but the calcitonin levels not different.