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Vitamin D supplementation in tuberculosis patients: A cross-sectional study
Published in Cut Adeya Adella, Stem Cell Oncology, 2018
D.K. Sari, N.K. Arrasyid, R.L. Kusumawati, Y.S. Harahap
We measured 25(OH)D serum concentration by Chemiluminescent Immunoassay (CLIA) technology (Diasorin, Stillwater, MN). Measures were between 4.0 and 150 ng/mL. The lowest value was 4.0 ng/mL, which is based on an inter-assay precision of 3.90% CV. Reference ranges were <20 ng/mL, categorised as deficiency, 20-30 ng/mL (insufficiency), and 30-100 ng/mL (sufficiency) (Holick, 2007). To convert ng/mL to nmol/L, multiply by 2.496. Calcium serum was measured by ADVIA Bayer Assayed Chemistry Controls, with principal procedure: calcium ions form a violet complex with o-cresolphthalein complexone in an alkaline medium. The reaction is measured at 545/658 nm, and normal concentration of calcium was 8.3-10.6 mg/dL.
Methodology
Published in Wilson Harvey, Alan Bennett, Prostaglandins in Bone Resorption, 2020
The alternative method is the measurement of net 40Ca release into the culture medium by either atomic absorption spectrometry or colorimetric analysis. We use this assay in our laboratories, essentially as described by Zanelli et al.16 Following a 24-hr preincubation of 5-day-old mouse calvaria halves, followed by 72-hr exposure to control or test media, the calcium content is measured by automated colorimetric analysis. The color reaction product of calcium with cresolphthalein complexone (phthalein purple) in the presence of 8-hy-droxyquinoline to free protein-bound calcium and combine the magnesium is monitored at 570 nm and expressed as the increase in calcium concentration above that of the culture medium. Zanelli et al. related the difference in calcium release between the paired test and control cultures to their spontaneous resorption rate during the first 24-hr culture period. This reduced the variation in calcium release between the different calvaria. We have recently abandoned the use of paired cultures and randomize the half calvaria between the treatment groups. This has not adversely affected the within-group variance of the assay and has increased the number of treatments possible for a given number of mice. Basal (spontaneous) resorption in control cultures ranges from 0.1 to 0.4 mg/dℓ, and extensive resorption results in increases up to approximately 3.0 mg/dℓ per culture. The choice of cresolphthalein was made as it is a standard procedure in automated analysis of calcium. However, other reagents such as methymol22 have been used effectively for analysis of calcium in culture medium.23 How much faith can we place in these assays? This question has two components, one of statistics and one of interpretation. The statistical side is more easily dealt with and concerns reproducibility. All biological assays are inherently more variable than their chemical counterparts, and this limits their precision. In terms of intra-assay variability, the reported coefficient of variation (SD/mean of the sample, × 100%) ranges from as little as 5% to above 40%, in both isotopic and colorimetric assays. The effects of this variability can be reduced by increasing sample size (e.g., to eight bones per experimental group instead of four or five) and, more importantly, by repetition of the experiments. It is advisable to pay more attention to the size and consistency of the response than to individual probability levels or their absence. However, it is often difficult to assess consistency in the results of resorption studies reported in the literature as individual experiments are usually shown as “representative”.
Enhanced osteogenic activity with boron-doped nanohydroxyapatite-loaded poly(butylene adipate-co-terephthalate) fibrous 3D matrix
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Aysu Arslan, Soner Çakmak, Menemşe Gümüşderelioğlu
Determination of alkaline phosphatase (ALP) activity, collagen and calcium amounts via colorimetric assays. For collagen assay, the scaffolds were first transferred into sterile Eppendorf tubes and they were homogenized in 100 µL distilled water. Then, 100 µL of 12 N HCl was added to each Eppendorf tube and hydrolysis was carried out at 120 °C for 3 h. For calcium analysis, calcium was extracted by using 5% (w/v) trichloroacetic acid under sonication for 30 min. Amounts of ALP, collagen and calcium were determined via colorimetric assay kits (Biovision, Milpitas, CA, USA) using the supernatants obtained as mentioned above. All assays were carried out by following the instructions of the assay kits. In brief, ALP activity was determined due to hydrolysis of p-nitrophenyl phosphate (pNPP) to p-nitrophenol (pNP). UV absorbance of pNP was read via microplate reader (ASYS, Hitech UVM 340 plate reader, UK) at 405 nm. Collagen amount in the ECM was directly proportional to hydroxyproline amount and it was determined via measuring the absorbance of hydroxyproline at 560 nm. Finally, calcium amounts were determined by measuring the absorbance of calcium–cresolphthalein complex at 575 nm. Samples without cells were used as negative controls for ALP, collagen and calcium assays and their absorbances were subtracted from each sample containing cells. ALP activities, collagen and calcium amounts were normalized to ng of DNA. The mineral formation on the scaffolds was also supported by an energy-dispersive X-ray spectroscopy (energy dispersive X-ray [EDX], Xflash 3001 SDD-EDS detector) analysis attached to the SEM.
Contribution of oxidative stress in acute intestinal mucositis induced by 5 fluorouracil (5-FU) and its pro-drug capecitabine in rats
Published in Toxicology Mechanisms and Methods, 2018
Kaïs Rtibi, Slimen Selmi, Dhekra Grami, Mohamed Amri, Hichem Sebai, Lamjed Marzouki
H2O2 level in mucosa of small intestine was performed according to Dingeon et al. (1975). Briefly, the H2O2reacts with p-hydroxybenzoic acid and 4-aminoantipyrine in the presence of peroxidase leading to the formation of quinoneimine that has a pink color detected at 505 nm. The non-haem iron was measured colorimetrically using ferrozine as described by Leardi et al. (1998). Summarily; the iron that was dissociated from the transferrin-iron complex by a solution of guanidine acetate and reduced by ascorbic acid reacts with ferrozine to give a pink complex measured at 562 nm. Calcium (Ca2+) level was measured using a colorimetric method according to Stern and Lewis (1957). In brief, in an alkaline medium, Ca2+ reacts with cresolphthalein leading to a colored complex measurable at 570 nm.
Supplementation of all trans retinoic acid ameliorates ethanol-induced endoplasmic reticulum stress
Published in Archives of Physiology and Biochemistry, 2018
Saritha S. Nair, Syam Das S, Reshma P. Nair, M. Indira
The liver microsomes were isolated in cold as described by Schenkman and Jansson (1999) employing differential ultracentrifugation. This was used for the assays of microsomal NADPH cytochrome P450 Reductase (Dignam and Strobel 1975). Glutathione (GSH) levels were estimated by the method of Patterson and Lazarow (1955). This was also used in the estimation of the lipid peroxidation products. Conjugated dienes (CD) were estimated as described by Recknagel and Ghoshal (1966), hydroperoxides (HP) as described by Mair and Hall (1971), and protein carbonyls (PC) by the method of Abraham and Packer (1993). Protein was estimated by the method of Lowry et al. (1951). Free fatty acids (FFA) in liver homogenate were estimated as given by Falholt et al. (1973), cholesterol by the method of Abell et al. (1952), triglycerides (TG) as given by Van Handel and Zilversmith (1957), and phospholipids as described by Zilversmith and Davis (1950). The microsomal calcium level was estimated by O-cresolphthalein complex one kit procured from Accurex Biomedical Pvt. Ltd. (Bangalore, India; Product code: A-4) as per the instructions of the manufacturer.