Oxalic

Standards of cyanidin (Cy)-, pelargonidin (Pel)- and their 3-O-glucoside (glc), Cy-rutinoside (Cy-rut), Cy-3,5-di-glucoside (Cy-di-glc), Cy-sambubioside (Cy-sam), and Cy-3-O-sophoroside (Cy-sop) were purchased from Polyphenols Laboratory (Sandnes, Norway). Potassium chloride, hydrochloric acid, methanol, acetonitrile, acetone, Folin reagent, phosphoric and trifluoroacetic acid (TFA) were from Merck (Darmstadt, Germany). Ascorbic acid, 4-dimethylamino-cinnamaldehyde (DMAC), ammonium acetate, acetic acid, glucose, fructose, saccharose, citric, isocitric, succinic, malic, fumaric, oxalic, tartaric, cis-aconitic and quinic acid were provided by Sigma-Aldrich (St. Louis, MO, USA). Extrasynthese (Genay, France) supplied catechin (CAT), epicatechin (EC), procyanidin C1 (PC1), procyanidin A2 (PA2) and punicalagin. Water was obtained from the Arium pro apparatus (Sartorius, Milan, Italy). The freeze-dried raspberries (Rubus idaeus) were a gift from FutureCeuticals (Momence, IL, USA).

The strain KPS-11 was inoculated in 100 mL of Pikovskaya’s broth in 500 mL flasks in triplicate and incubated in an orbital shaker at 150 rpm at 28 ± 2°C for up to 10 days (240 h). Twenty micro liter of bacterial culture from each flask was harvested at 5, 7, and 10 days post inoculation, centrifuged at 13,000 × g for 10 min and cell-free supernatant was collected. Phosphate solubilization was determined through Phospho-molybdate blue color method using spectrophotometer (λ = 882 nm) as described by Murphy and Riley (1962) (link). For HPLC analysis, the cell-free supernatant was filtered through 0.2 μm nylon filters (Millipore, USA) and 20 μL was injected to HPLC equipped with Turbochrom software (Perkin Elmer, USA) and C-18 column at a flow rate of 0.6 mL min^-1 using 30:1:70 (v/v/v) methanol: acetic acid: water as mobile phase. Signals were detected at 210 nm. The organic acids gluconic, malic, lactic, oxalic, tartaric, and ascorbic acid (Sigma–Aldrich) were used as standard.

Sugars and organic acids were identified and quantified according to Hernández [15 (link)], with some modifications. Approximately 1 g of sample was diluted in 5 mL of phosphate buffer (pH 7.8), homogenized by Ultra-TurraxTM (IKA L004640, Staufen, Germany) for 1 min, and centrifuged at 15,000× g for 10 min. Finally, samples were filtered through a 0.45 μm Millipore filter. For the determination of the content of sugars and organic acids on samples, an HPLC (high-performance liquid chromatograph) Hewlett-Packard series 1100 (Hewlett-Packard, Wilmington, DE, USA) was used. The elution buffer consisted of 0.1% phosphoric acid with a flow rate of 0.5 mL/min.
Sugars and organic acids were isolated using a Supelco column (Supelcogel TM C-610H column 30 cm × 7.8 mm, Supelco, Inc., Bellefonte, PA, USA) and a precolumn Supelguard (5 cm × 4.6 mm; Supelco), and the absorbance was measured at 210 nm using a diode-array detector (DAD). Standards of sugars (glucose, fructose, sucrose, raffinose, maltitol, and sorbitol) and organic acids (oxalic, citric, tartaric, malic, quinic, shikimic, succinic and fumaric) were obtained from Sigma (Poole, UK). Calibration curves were used for the quantification of sugars and organic acids, showing good linearity (R² = 0.999). Results for both organic acids and sugars were expressed as concentrations g/L of dry weight (dw).

Acetonitrile, formic acid, methanol, DPPH (1,1-diphenyl-2 picrylhydrazyl radical), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), TPTZ (2,4,6-tri(2-pyridyl)-s-triazine), acetic acid, phloroglucinol, arbutin, caffeic acid, betulinic, oleanolic and ursolic acid, fructose, glucose, sorbitol and sucrose, and malic, oxalic, citric, shikimic, succinic, and fumaric acids were purchased from Sigma-Aldrich (Steinheim, Germany). Quercetin 3-O-glucoside, kaempferol 3-O-glucoside, cyanidin 3-O-glucoside, isorhamnetin 3-O-glucoside, apigenin 7-O-glucoside, chlorogenic, quinic, p-coumaric and ferulic acids, (+)-catechin, and procyanidin B2 were purchased from Extrasynthese (Lyon, France). Sodium hydroxide (CAS 1310-73-2), iodine (CAS 7553-56-2), and potassium iodide (CAS 7681-11-0) were purchased from CHEMPUR (Piekary Slaskie, Poland).

100mg/ml of mineralized and demineralized bone particulate <125μm and 125–750μm from 3 donors were stirred at 750RPM combined with either 1M or stock lactic acid, phosphoric acid, oxalic, hydrochloric acid, acetic acid, sulfuric acid, formic acid, citric acid, or trifluoroacetic acid (all from SigmaAldrich, St. Louis, MO) at either 4°C, room temperature (~21°C), or 37°C. Samples were taken a 2hrs, 8hrs, 16hrs, and every 24hrs for 2 weeks for microscopic evaluation. The solubility of BCP, HA, and CPP (Sigma-Aldrich, St. Louis, MO) was also tested in each acid at 100mg/ml and imaged under normal and polarized light. Samples were examined for the appearance of crystals by an Olympus BX41 microscope with a polarizer. CPPD crystals from arthrocentesis were imaged as positive controls.

Standards of flavonoids (scutellarin, baicalin, baicalein, wogonin, wogonoside, luteolin, chrysin), verbascoside, sugars (glucose, fructose, xylose, galactose, rhamnose, sucrose and cellobiose) and organic acids (quinic, malic, ascorbic, succinic, citric, α-keto-glutaric, oxalic and tartaric), methanol and acetonitrile (HPLC gradient grade) were purchased from Sigma-Aldrich (Darmstadt, Germany). Water was obtained from Milipore Milli-Q Gradient water purification system (Barnstead, US).

The organic acids used were of analytical standard grade. Caproic and valeric acids were obtained from Fluka (Steinheim, Germany); succinic acid from Lancaster (Morecambe, UK); 95% formic, thiocyanic, nitric, pyruvic and ascorbic acids, as well as monohydrate citric acid, plus oxalic, propionic, butyric, 85% ortho-phosphoric acids from Sigma-Aldrich (Saint-Quentin-Fallavier, France) and 80% D,L-lactic acid from Avantor Performance Materials Poland S.A. (Gliwice, Poland). A 36–38% hydrochloric acid was delivered by Baker Analyzed and methylhydroxyethylcellulose (M-HEC) by HERCULES (Prague, Czech Republic), β-alanine by Merck (Darmstadt, Germany) and L-histidine by Serva (Heidelberg, Germany); 99.5% acetic acid was obtained from Chempur (Piekary Śląskie, Poland). Deionized water of 18 MΩcm was obtained from Milli Ro & Q water purification system (Merck-Millipore, Billerica, MA, USA).