Genetic deletion of Sost or pharmacological inhibition of sclerostin prevent multiple myeloma-induced bone disease without affecting tumor growth.

Multiple myeloma (MM) causes lytic bone lesions due to increased bone resorption and concomitant marked suppression of bone formation. Sclerostin (Scl), an osteocyte-derived inhibitor of Wnt/ß-catenin signaling, is elevated in MM patient sera and increased in osteocytes in MM-bearing mice. We show here that genetic deletion of Sost, the gene encoding Scl, prevented MM-induced bone disease in an immune-deficient mouse model of early MM, and that administration of anti-Scl antibody (Scl-Ab) increased bone mass and decreases osteolysis in immune-competent mice with established MM. Sost/Scl inhibition increased osteoblast numbers, stimulated new bone formation and decreased osteoclast number in MM-colonized bone. Further, Sost/Scl inhibition did not affect tumor growth in vivo or anti-myeloma drug efficacy in vitro. These results identify the osteocyte as a major contributor to the deleterious effects of MM in bone and osteocyte-derived Scl as a promising target for the treatment of established MM-induced bone disease. Further, Scl did not interfere with efficacy of chemotherapy for MM, suggesting that combined treatment with anti-myeloma drugs and Scl-Ab should effectively control MM growth and bone disease, providing new avenues to effectively control MM and bone disease in patients with active MM.

Liquid chromatography with multi-channel electrochemical detection for the determination of epigallocatechin gallate in rat plasma utilizing an automated blood sampling device.

A liquid chromatography method with multi-channel electrochemical detection was developed for the determination of epigallocatechin gallate (EGCG) in rat plasma. After administration of EGCG, blood samples were periodically collected by Culex (an automated blood sampling robot). EGCG was extracted from 50 microl of diluted blood (blood and saline at a ratio of 1:1) with ethyl acetate. Chromatographic separation was achieved within 10 min using a C8 (150x4.6 mm) 5 microm column with a mobile phase containing 20 mM sodium monochloroacetate, pH 2.8 and 12% acetonitrile at a flow-rate of 1.2 ml/min. A four-channel detector with glassy carbon electrodes was used with applied potentials of +700, 600, 500, 400 mV vs. Ag/AgCl. The limit of detection was 2 ng/ml at a signal-to-noise ratio of 3:1 and the limit of quantitation was 5 ng/ml. The calibration curve was linear over the range of 5-800 ng/ml. The intra- and inter-assay precisions were in the range of 1.3-4.5% and 2.2-4.4%, respectively. Using this method it was possible to determine plasma concentration following a single dose of EGCG to rats with good accuracy and precision. Thus the pharmacokinetic properties of EGCG in rats can be examined for intravenous, intraperitoneal and oral dosing.

In vivo microdialysis and reverse phase ion pair liquid chromatography/tandem mass spectrometry for the determination and identification of acetylcholine and related compounds in rat brain.

A method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed for the determination of basal acetylcholine (ACh) in microdialysate from the striatum of freely moving rats. A microdialysis probe was surgically implanted into the striatum of the rats and Ringer's solution was used as the perfusion medium at a flow rate of 2 microL per minute. The samples were then analyzed off-line by LC/MS/MS experiments. The separation of ACh and choline (Ch) was carried out using reverse phase ion pair liquid chromatography with heptafluorobutyric acid as a volatile ion pairing reagent. Analytes were detected by electrospray ionization tandem mass spectrometry in the positive ion mode. The detection limit for ACh was 1.4 fmol on column, which is at least three times lower than previously reported. Three quaternary ammonium compounds in the rat brain microdialysate were also identified by tandem mass spectrometry experiments in which the unknown mass spectra were compared with standard reference compounds. These compounds were identified as carnitine, acetylcarnitine and (3-carboxypropyl)trimethylammonium. This is the first known report of the compound (3-carboxypropyl)trimethylammonium being found in rat brain.

Liquid chromatography with multichannel electrochemical detection for the determination of trans-resveratrol in rat blood utilizing an automated blood sampling device.

A sensitive and selective multichannel liquid chromatography-electrochemistry method was developed for the determination of the natural product trans-resveratrol (resveratrol) in rat blood. After administration of resveratrol, blood samples were periodically collected by an automated blood sampling device. Resveratrol was extracted from 150 microl of diluted blood (blood and saline at a ratio of 1:1) with acetonitrile containing 1% of trichloroacetic acid. Chromatographic separation was achieved within 12 min using a C18 (100x2.0 mm) 3 microm column with a mobile phase containing 20 mM sodium acetate, 0.5 mM EDTA, pH 4.5 and 21% acetonitrile at a flow-rate of 0.4 ml/min. A multichannel detector with glassy carbon electrodes was used, which can control up to four working electrodes simultaneously with applied potentials of +800, 700, 600, 500 mV vs. Ag/AgCl. The limit of detection was 2 ng/ml at a signal-to-noise ratio of 3:1 and the limit of quantitation was 4 ng/ml. The linearity of the calibration curve was obtained over the analytical range of 5-1000 ng/ml. The intra- and interassay precision was in the range of 2.5-4.4% and 1.2-4.3%, respectively. Using this method it was possible to quantify blood concentration following a single dose of resveratrol to rats with good accuracy and precision. Thus the pharmacokinetic properties of resveratrol in rats can be examined for intraperitoneal, oral and intravenous dosing.

Ultrafiltrate and microdialysis DL probe in vitro recoveries: electrolytes and metabolites.

UF ultrafiltration and DL microdialysis probes are well-suited for sampling interstitial concentrations of ions and metabolites in peripheral tissue. The first step in utilization of membrane sampling techniques is to determine the recovery characteristics of the probes in vitro.

Histochemical demonstration of endothelial superoxide and hydrogen peroxide generation in ischaemic and reoxygenated rat tissues.

OBJECTIVE: The aims were to test and evaluate two novel and independent histochemical methods for detecting the initial postischaemic burst of superoxide and hydrogen peroxide in buffer perfused rat tissues during reflow after 60 min warm ischaemia. METHODS: The first is a high manganese/diaminobenzidine technique, in which superoxide oxidises Mn2+ to Mn3+, which in turn oxidises diaminobenzidine to form amber coloured polymers, observable by light microscopy. The second is a high iron/diaminobenzidine technique, in which hydrogen peroxide oxidises diethylenetriaminepenta-acetate chelated Fe2+ to form intermediate species, which in turn oxidise diaminobenzidine similarly to Mn3+. Various isolated organs of the rat were rendered ischaemic for 60 min, and reperfused with oxygen or air equilibrated buffers containing diaminobenzidine and either Mn2+ or Fe2+. Tissues were fixed by perfusion with Trump's solution and processed for light microscopy. RESULTS: Both manganese and iron methods consistently showed the appearance of reaction product on the luminal surfaces of arterial, capillary, and venular endothelial cells in lung, heart, and intestine of the rat during the first 2 to 3 min of reoxygenation after ischaemia. The histochemical reactions were nearly absent in non-manganese-treated and non-iron-treated controls. Superoxide dismutase strongly inhibited Mn2+/diaminobenzidine reaction product formation and catalase strongly inhibited Fe2+/diaminobenzidine reaction product formation, when tested in specially perfused lung preparations in which these specific antioxidant enzymes were concentrated. CONCLUSIONS: These histochemical techniques provide direct, visual evidence that a burst of reactive oxygen species is generated in postischaemic rat tissues. The Mn2+/diaminobenzidine and Fe2+/diaminobenzidine techniques permit investigation of the endothelium derived reactive oxygen by simple laboratory procedures available to almost any investigator at low marginal cost. The endothelial oxidants so revealed may be of pathophysiological significance in a variety of cardiovascular disorders.

Endothelial superoxide production in the isolated rat heart during early reperfusion after ischemia. A histochemical study.

This paper describes a histochemical study of superoxide generation in buffer-perfused, isolated rat hearts during the first 2 minutes of reperfusion after 60 minutes of warm ischemia. Superoxide radical production was demonstrated by a modification of Karnovsky's manganese/diaminobenzidine technique, in which superoxide oxidizes Mn++ to Mn ions, which in turn oxidize diaminobenzidine to form amber, osmiophilic polymers, observable by light or electron microscopy. Isolated hearts were rendered ischemic, reperfused with oxygen equilibrated buffer containing Mn++ and diaminobenzidine, fixed by perfusion with Trump's solution, and processed for light and electron microscopy. The method consistently demonstrated evidence of superoxide generation near the luminal surfaces of arterial, capillary, and venular endothelial cells during the first 2 minutes of reoxygenation after ischemia. The histochemical reaction was absent or markedly reduced in non-manganese-treated or nonischemic hearts, as well as in hearts perfused with calcium-free or oxygen-free buffers. The histochemical differences were statistically significant on quantitative morphometric analysis. These results provide direct, visual evidence of the existence and endothelial localization of a burst of superoxide radicals in intact, postischemic myocardium and suggest the pathophysiologic importance of calcium-dependent endothelial cell activation in the initiation of reperfusion injury.

Endothelial superoxide production in buffer perfused rat lungs, demonstrated by a new histochemical technique.

This paper describes a new approach to the histochemical demonstration of superoxide generation by pulmonary vascular endothelial cells using a supravital high manganese/diamine technique, in which nascent superoxide radicals induce formation of amber, osmiophilic polymers of diaminobenzidine (DAB), detectable by light or electron microscopy. Superoxide oxidizes Mn2+ ions to the Mn3+ valence state. In turn trivalent manganese readily initiates formation of the polymerized DAB reaction product. Isolated rat lungs were perfused in situ with bloodless, buffered high manganese/DAB salt solution via the pulmonary artery. The aortic root was ligated to minimize outflow from the left heart, so that perfusate shunted across pulmonary capillary endothelium, to fill the alveolar spaces and drain via the trachea. Lungs were perfused for 3 min with oxygen equilibrated buffer, with or without 60 min prior warm anoxia, induced by initial perfusion with argon sparged buffer. After aldehyde fixation and tissue processing DAB reaction product was detected on the inner, luminal surface of the vascular endothelium by both light and electron microscopy. Bronchi and epithelial cells never stained positively. The histochemical reaction was absent or markedly reduced in non-manganese treated or superoxide dismutase treated lungs, as well as in lungs perfused with calcium free buffer. The histochemical reaction was not prevented by the xanthine oxidase inhibitors allopurinol or methylene blue. The high manganese/diamine technique provides direct visual evidence of a calcium dependent mechanism by which pulmonary vascular endothelial cells can generate superoxide radicals.

Effect of calcium on blood pressure, platelet aggregation and erythrocyte sodium transport in Dahl salt-sensitive rats.

Forty 11-week-old Dahl salt-sensitive rats were divided into four groups matched for blood pressure and weight. Group I was given a sodium-deficient diet, group II a sodium-enriched diet, group III a sodium-enriched diet plus a calcium supplement and group IV a sodium-enriched diet plus nitrendipine, a calcium antagonist. For the first 18 weeks, when the sodium-enriched diet contained 2.6% sodium, there were no differences in blood pressure between the groups; the sodium content was then increased to 8%, and the diets continued for 12 more weeks. At 41 weeks old, the rats in group II had significantly (P less than 0.05) higher systolic blood pressures than the other groups. Erythrocytes from the rats on the low-sodium diet had significantly (P less than 0.025) lower intracellular sodium (3.9 +/- 0.4 mmol/l) while cells from the rats given nitrendipine had significantly (P less than 0.005) higher intracellular sodium (13.3 +/- 0.8 mmol/l) than those from the rats on a high-salt diet (7.4 +/- 1.4 mmol/l). Nitrendipine caused significant (P less than 0.05) decreases in both ouabain-sensitive and furosemide-sensitive sodium efflux. Platelet aggregation in response to 2 mumol/l adenosine diphosphate was not significantly affected by the nitrendipine. The evidence that nitrendipine markedly affects sodium transport supports the hypothesis that an interaction of calcium and sodium may be involved in blood pressure control.