Cells were cultured in SD medium at 30 °C with gentle shaking at 200 rpm for 24 h. The cultured cells were then washed twice with distilled water. After washing, the cells were inoculated to a 200-mL Erlenmeyer flask containing 50 mL of growth test medium consisting of 6.7 g/L yeast nitrogen base without amino acids (Difco Laboratories), 1.46 g/L Yeast Synthetic Drop-out Media Supplements (Y1501, Sigma-Aldrich, Burlington, USA), 76 mg/L uracil, and 20 g/L xylose. Cells were inoculated at an optical density of OD600 = 0.1 and cultured at 30 °C with gentle shaking at 200 rpm. Cell density was measured using a UV–VIS spectrophotometer (uVmini-1240, Shimadzu Corp. Kyoto, Japan).
Uvmini 1240
Manufactured by Shimadzu
Sourced in Japan, Germany, China, United States, Italy, Australia, United Kingdom, Ireland, Brazil, Canada
About the product
The UVmini-1240 is a compact and lightweight ultraviolet-visible (UV-Vis) spectrophotometer designed for routine analysis. It provides accurate absorbance measurements across the UV-Vis wavelength range.
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The Shimadzu UVmini 1240 spectrophotometer has been discontinued and is no longer available from Shimadzu or their authorized distributors. However, replacement parts such as halogen and deuterium lamps are still available through third-party suppliers.
For those seeking a current model, Shimadzu recommends the UV-1900i UV-Vis Spectrophotometer as a modern alternative to the discontinued UVmini 1240.
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882 protocols using «uvmini 1240»
1
Yeast Growth Protocol in Xylose Medium
2
Quantification of Heme Pathway Metabolites
The optical density at 600 nm (OD600) was detected by a spectrophotometer (UVmini-1240, Shimadzu Corporation, Kyoto, Japan) to reflect cell growth. The concentrations of ALA, PBG, UP, CP, PPIX, and heme were analyzed using HPLC (Shimadzu, Kyoto, Japan) equipped with a ZORBAX Eclipse XDB-C18 (5 μm, 4.6 × 250 mm, Agilent, Santa Clara, CA, USA).
For the analysis of ALA and PBG, the method was based on that described by Park et al. through the derivatization of amino acids with OPA [52 (link)]. Mobile phase A contained 3.01 g sodium acetate in 1 L water with 200 μL triethylamine and 5 mL tetrahydrofuran (pH 7.2); mobile phase B contained 3.01 g sodium acetate in 200 mL water (pH 7.2), 400 mL methanol, and 400 mL acetonitrile. The parameters were set as follows: a flow rate of 0.8 mL/min; the mobile phase B ratio was 22% during the first 5 min, 30% at 29 min, 100% at 31.5 min, maintained at 100% to 35 min, 8% at 35.5 min, and maintained at 8% to 40 min; column temperature of 40 °C and monitored absorption wavelengths of 338 nm.
For the analysis of UP and CP, mobile phase A was 1M ammonium acetate aqueous solution (pH 5.15) with 8% acetonitrile; mobile phase B was methanol with 8% acetonitrile and 10% water. The parameters were set as follows: a flow rate of 0.8 mL/min; the mobile phase B ratio was 0 during the first 8 min, 65% at 38 min, maintained at 65% to 48 min, 0 at 49 min, and maintained at 0 to 55 min; column temperature of 40 °C and monitored absorption wavelengths of 404 nm.
For the analysis of heme and PPIX, mobile phase A was water with 0.1% trifluoroacetic acid; mobile phase B was methanol with 0.1% trifluoroacetic acid. The parameters were set as follows: a flow rate of 0.8 mL/min; a mobile phase B ratio of 30% for 1 min, 100% at 20 min, 100% at 35 min, and 30% at 37 min; column temperature of 40 °C and monitored absorption wavelengths of 400 nm.
For the analysis of ALA, heme, and PPIX by HPLC-MS, mobile phase A was 10 mM ammonium formate containing 0.1% formic acid and mobile phase B was acetonitrile containing 0.1% formic acid. First, 1 μL of samples were loaded onto an Agilent ZORBAX Eclipse Plus C18 column (2.1 × 50 mm, 1.8 μm), separated using Agilent 1290 Infinity II LC system, and quantified by an Agilent 6495C triple quadrupole mass spectrometer (Agilent, Santa Clara, CA, USA) equipped with an electrospray ionization (ESI) interface in the positive ion mode. The parameters were set as follows: a flow rate of 0.4 mL/min; mobile phase B ratio of 30% for 0.1 min, 40% at 0.4 min, 55% at 3 min, 100% at 4 min, 30% at 4.8 min, and maintained at 30% to 5.2 min. Mass transition, m/z 616.2 → 557.2 was selected to monitor heme, m/z 132.2 → 86.1 was selected to monitor ALA, and m/z 563.3 → 429.2 was selected to monitor PPIX. The capillary voltage was set at 2.5 kV. Nitrogen was used as the drying gas at a flow rate of 14 L/min at 200 °C. The nebulizer pressure was set at 24 psi.
For the analysis of ALA and PBG, the method was based on that described by Park et al. through the derivatization of amino acids with OPA [52 (link)]. Mobile phase A contained 3.01 g sodium acetate in 1 L water with 200 μL triethylamine and 5 mL tetrahydrofuran (pH 7.2); mobile phase B contained 3.01 g sodium acetate in 200 mL water (pH 7.2), 400 mL methanol, and 400 mL acetonitrile. The parameters were set as follows: a flow rate of 0.8 mL/min; the mobile phase B ratio was 22% during the first 5 min, 30% at 29 min, 100% at 31.5 min, maintained at 100% to 35 min, 8% at 35.5 min, and maintained at 8% to 40 min; column temperature of 40 °C and monitored absorption wavelengths of 338 nm.
For the analysis of UP and CP, mobile phase A was 1M ammonium acetate aqueous solution (pH 5.15) with 8% acetonitrile; mobile phase B was methanol with 8% acetonitrile and 10% water. The parameters were set as follows: a flow rate of 0.8 mL/min; the mobile phase B ratio was 0 during the first 8 min, 65% at 38 min, maintained at 65% to 48 min, 0 at 49 min, and maintained at 0 to 55 min; column temperature of 40 °C and monitored absorption wavelengths of 404 nm.
For the analysis of heme and PPIX, mobile phase A was water with 0.1% trifluoroacetic acid; mobile phase B was methanol with 0.1% trifluoroacetic acid. The parameters were set as follows: a flow rate of 0.8 mL/min; a mobile phase B ratio of 30% for 1 min, 100% at 20 min, 100% at 35 min, and 30% at 37 min; column temperature of 40 °C and monitored absorption wavelengths of 400 nm.
For the analysis of ALA, heme, and PPIX by HPLC-MS, mobile phase A was 10 mM ammonium formate containing 0.1% formic acid and mobile phase B was acetonitrile containing 0.1% formic acid. First, 1 μL of samples were loaded onto an Agilent ZORBAX Eclipse Plus C18 column (2.1 × 50 mm, 1.8 μm), separated using Agilent 1290 Infinity II LC system, and quantified by an Agilent 6495C triple quadrupole mass spectrometer (Agilent, Santa Clara, CA, USA) equipped with an electrospray ionization (ESI) interface in the positive ion mode. The parameters were set as follows: a flow rate of 0.4 mL/min; mobile phase B ratio of 30% for 0.1 min, 40% at 0.4 min, 55% at 3 min, 100% at 4 min, 30% at 4.8 min, and maintained at 30% to 5.2 min. Mass transition, m/z 616.2 → 557.2 was selected to monitor heme, m/z 132.2 → 86.1 was selected to monitor ALA, and m/z 563.3 → 429.2 was selected to monitor PPIX. The capillary voltage was set at 2.5 kV. Nitrogen was used as the drying gas at a flow rate of 14 L/min at 200 °C. The nebulizer pressure was set at 24 psi.
3
Protein Quantification via Bradford Assay
The protein content was assessed spectrophotometrically using the Bradford method [22 (link)] on a UV–vis spectrophotometer (UVmini-1240; Shimadzu, Kyoto, Japan), with bovine serum albumin as the standard.
4
Quantifying Phenolic Content in Purslane
Total phenol quantities of purslane samples were displayed using Folin–Ciocalteu reagent based on a previous study [22 (link)]. A total of 2.5 mL of Folin–Ciocalteu reagent and 2 mL of Na2CO3 solution were mixed onto 0.5 mL of extract. The total phenolic contents of the samples kept in the dark at room temperature for 2 h were determined at 725 nm wavelength in a spectrophotometer (Shimadzu, UV mini 1240, Kyoto, Japan). Findings were stated as mg GAE/100 g.
5
Adaptive Laboratory Evolution of C. glutamicum
Initially, the aecD gene disruptant of C. glutamicum was cultured in a modified M9 medium containing 1.5 g L−1 L-cysteine hydrochloride monohydrate (Cys·HCl·H2O) for 24 h. The culture was then diluted with the fresh modified M9 medium containing the same concentration of Cys·HCl·H2O to achieve the optical density at 660 nm (OD660) of 0.1, and the cells were then cultured for 24 h; OD660 values were measured using a spectrophotometer UVmini-1240 (Shimadzu Corporation, Kyoto, Japan). The serial transfer process was repeated until the specific growth rate increased. After that, the concentration of Cys·HCl·H2O in the M9 medium was increased from 1.5 to 2 g L−1 and the serial transfer process was repeated. The Cys·HCl·H2O concentration was stepwise increased to 2.5, 3, and 4 g L−1. After the serial transfer, the previous culture was mixed with glycerol (15% (v/v), final concentration) and then stored at − 80 °C. Finally, the ALE experiments were continued until the specific growth rate of the cultured cells increased and became stable in the presence of 4 g L−1 Cys·HCl·H2O. In the present study, five series of cultures for ALE, designated ALE1, ALE2, ALE3, ALE4, and ALE5, were independently performed.
Top 5 protocols citing «uvmini 1240»
1
Soil Enzyme Activities in Intercropping Systems
Soil samples were collected to 20 cm depth using an auger after harvest to minimize damage to the plots in 2011 and 2012. Three replicate cores were collected from intercropping strips of maize rows and associated crops and mixed together to give composite samples and the same method was used in the monoculture plots. The composite samples were stored in plastic bags, air-dried and sieved through a 2.0 mm mesh and plant residues and roots were removed by hand prior to chemical analysis. Soil samples were also collected and stored at 4°C prior to analysis for soil enzyme activities.
Soil OM was determined by wet oxidation using the acidified dichromate method [39] . Soil total N was measured after Kjeldahl digestion according to standard protocols (SKD-800, Shanghai, Peiou Corporation). Soil Olsen P was determined using standard procedures [40] by colorimetry (Uvmini-1240, Shimadzu Corporation). Soil exchangeable K was extracted using 1 mol L−1 ammonium acetate solution buffered at pH 7 and determined by flame photometry (M410, Sherwood Corporation, UK). Soil CEC was measured by the sodium saturation method [41] . Soil pH was measured in soil suspension with deionized-distilled water (2.5∶1, w/v) (pHS-3C, SPSIC Corporation).
Soil urease activity was determined by the method described by Guan (1986) [42] with minor modification. Five grams of fresh soil were placed in a 50-mL volumetric flask together with 1.0 mL toluene. Fifteen minutes later, 5 mL 10% urea solution and 10 mL citrate buffer (pH 6.7) were added. The flask was shaken and then placed in an incubator at 37±0.1°C for 24 h. After incubation, deionized water at 38°C was added to a volume of 50 mL. The suspension was filtered. To a 50-mL volumetric flask were added 1.0 mL of the filtrate with 9 mL deionized water, 4 mL sodium phenate solution and 3 mL sodium hypochlorite solution. Twenty minutes later, deionized water was added up to a volume of 50 mL. Finally, urease activity was determined colorimetrically at 578 nm and expressed as mg NH3-N (g soil)−1 (24 h)−1.
Soil nitrate reductase activity was determined by a colorimetric method [42] with modification. Triplicate 5 g soil samples were incubated with 4 ml of 2,4-dinitrophenol solution 1 ml potassium nitrate solution and 5 ml distilled water at 25°C for 24 h. A similar set up was prepared for the control. The control sample was incubated at −20°C for 24 h. After incubation, 10 ml 4 M KCl solution was added to all the soil samples including the control. This was shaken for 30 minutes and filtered. To 5 ml of the filtrate, 3 ml of NH4Cl buffer (pH 8.5) and 2 ml of color reagent were added. This was kept for 15 minutes for color development. Optical density was was determined in a spectrophotometer against the blank at 520 nm. The enzyme activity was expressed as micrograms of NO−2–N per gram daily
Soil sucrase activity was determined by the method described by Guan (1986) with modification. Five grams of fresh soil were placed in a 50-mL Erlenmeyer flask together with 15 mL 8% of sucrose solution, 5 mL phosphate buffer (pH 5.5) and 1 ml of toluene. The flask was shaken and then placed in an incubator at 37.0±0.1°C for 24 h. After incubation, the sample was filtered through a quantitative filter paper. Then, 1 mL of the filtrate and 3 mL salicylic acid were taken to a 50-mL volumetric flask and heated for 5 min at 100°C in a water bath. After heating, the flask was cooled for 3 min with flowing tap water and deionized water was added to make up to 50 mL, and sucrase activity was measured colorimetrically at 508 nm (U-2800, Japan). Sucrase activity is expressed as mg glucose g soil−1 (24 h)−1.
Soil acid phosphatase activity was determined using p-nitrophenyl phosphate disodium (PNPP) as substrate [43] . On the basis of a modified universal buffer stock solution, the pH for the acid phosphatase analysis was adjusted to 6.5 with HCl. The pNP released by phosphatase was determined colorimetrically at 400 nm. Enzyme activity was expressed as micrograms of p-nitrophenol produced per gram of soil.
Soil OM was determined by wet oxidation using the acidified dichromate method [39] . Soil total N was measured after Kjeldahl digestion according to standard protocols (SKD-800, Shanghai, Peiou Corporation). Soil Olsen P was determined using standard procedures [40] by colorimetry (Uvmini-1240, Shimadzu Corporation). Soil exchangeable K was extracted using 1 mol L−1 ammonium acetate solution buffered at pH 7 and determined by flame photometry (M410, Sherwood Corporation, UK). Soil CEC was measured by the sodium saturation method [41] . Soil pH was measured in soil suspension with deionized-distilled water (2.5∶1, w/v) (pHS-3C, SPSIC Corporation).
Soil urease activity was determined by the method described by Guan (1986) [42] with minor modification. Five grams of fresh soil were placed in a 50-mL volumetric flask together with 1.0 mL toluene. Fifteen minutes later, 5 mL 10% urea solution and 10 mL citrate buffer (pH 6.7) were added. The flask was shaken and then placed in an incubator at 37±0.1°C for 24 h. After incubation, deionized water at 38°C was added to a volume of 50 mL. The suspension was filtered. To a 50-mL volumetric flask were added 1.0 mL of the filtrate with 9 mL deionized water, 4 mL sodium phenate solution and 3 mL sodium hypochlorite solution. Twenty minutes later, deionized water was added up to a volume of 50 mL. Finally, urease activity was determined colorimetrically at 578 nm and expressed as mg NH3-N (g soil)−1 (24 h)−1.
Soil nitrate reductase activity was determined by a colorimetric method [42] with modification. Triplicate 5 g soil samples were incubated with 4 ml of 2,4-dinitrophenol solution 1 ml potassium nitrate solution and 5 ml distilled water at 25°C for 24 h. A similar set up was prepared for the control. The control sample was incubated at −20°C for 24 h. After incubation, 10 ml 4 M KCl solution was added to all the soil samples including the control. This was shaken for 30 minutes and filtered. To 5 ml of the filtrate, 3 ml of NH4Cl buffer (pH 8.5) and 2 ml of color reagent were added. This was kept for 15 minutes for color development. Optical density was was determined in a spectrophotometer against the blank at 520 nm. The enzyme activity was expressed as micrograms of NO−2–N per gram daily
Soil sucrase activity was determined by the method described by Guan (1986) with modification. Five grams of fresh soil were placed in a 50-mL Erlenmeyer flask together with 15 mL 8% of sucrose solution, 5 mL phosphate buffer (pH 5.5) and 1 ml of toluene. The flask was shaken and then placed in an incubator at 37.0±0.1°C for 24 h. After incubation, the sample was filtered through a quantitative filter paper. Then, 1 mL of the filtrate and 3 mL salicylic acid were taken to a 50-mL volumetric flask and heated for 5 min at 100°C in a water bath. After heating, the flask was cooled for 3 min with flowing tap water and deionized water was added to make up to 50 mL, and sucrase activity was measured colorimetrically at 508 nm (U-2800, Japan). Sucrase activity is expressed as mg glucose g soil−1 (24 h)−1.
Soil acid phosphatase activity was determined using p-nitrophenyl phosphate disodium (PNPP) as substrate [43] . On the basis of a modified universal buffer stock solution, the pH for the acid phosphatase analysis was adjusted to 6.5 with HCl. The pNP released by phosphatase was determined colorimetrically at 400 nm. Enzyme activity was expressed as micrograms of p-nitrophenol produced per gram of soil.
2
Physiological Effects of Fertilization Treatments
The effects of fertilization treatments were assessed by periodical height measurements of whole plants during the vegetative season and evaluation of their biomass yield and energy value in the autumn. Assessments of the physiological activity of plants (gas exchange, index of chlorophyll content, enzyme activity) were carried out on fully developed leaves situated under the top of plants. In each experimental variant, one leaf from each of 10 plants was taken for the evaluation of gas exchange and enzyme activity. The material was collected in the third week of July in a temperature range of 25–30 °C, in sunshine and air humidity of 50–60%.
The height of the plants was measured at monthly intervals throughout the growing season [17 (link)]. The weights of fresh green biomass, and dry (dried at 130 °C for 3 days) were assessed in November on the basis of 5 plants taken from each experimental variant. The data presented were calculated for one plant as an average for the treatment [17 (link)]. Assessments of gas exchange (net photosynthesis, transpiration, stomatal conductance, and intercellular CO2 concentration) were performed using a TPS-2 -Portable Photosynthesis System (PP Systems, Amesbury, MA, USA) [17 (link),20 (link)].
An index of the chlorophyll content in leaves was estimated using a SPAD-502 chlorophyll meter (Konica Minolta, Osaka, Japan) [17 (link)]. The activities of acid (pH 6) (EC 3.1.3.2) and alkaline (pH 7.5) (EC 3.1.3.1) phosphorylases (U g−1 (FM) min−1) in leaves and RNase (EC 3.1.27.5) (U g−1(FM) min−1) were studied using the methods demonstrated by Knypl and Kabzinska [50 (link)]. The activity of total dehydrogenases (EC 1.1.1.-) was measured using the procedure presented by Górnik and Grzesik [20 (link),51 (link)] with a spectrophotometer (UVmini-1240, Shimadzu, Japan) for formazan determination at a wavelength of 480 nm.
The height of the plants was measured at monthly intervals throughout the growing season [17 (link)]. The weights of fresh green biomass, and dry (dried at 130 °C for 3 days) were assessed in November on the basis of 5 plants taken from each experimental variant. The data presented were calculated for one plant as an average for the treatment [17 (link)]. Assessments of gas exchange (net photosynthesis, transpiration, stomatal conductance, and intercellular CO2 concentration) were performed using a TPS-2 -Portable Photosynthesis System (PP Systems, Amesbury, MA, USA) [17 (link),20 (link)].
An index of the chlorophyll content in leaves was estimated using a SPAD-502 chlorophyll meter (Konica Minolta, Osaka, Japan) [17 (link)]. The activities of acid (pH 6) (EC 3.1.3.2) and alkaline (pH 7.5) (EC 3.1.3.1) phosphorylases (U g−1 (FM) min−1) in leaves and RNase (EC 3.1.27.5) (U g−1(FM) min−1) were studied using the methods demonstrated by Knypl and Kabzinska [50 (link)]. The activity of total dehydrogenases (EC 1.1.1.-) was measured using the procedure presented by Górnik and Grzesik [20 (link),51 (link)] with a spectrophotometer (UVmini-1240, Shimadzu, Japan) for formazan determination at a wavelength of 480 nm.
3
Quantification of Total Phenolic Content
Total phenolic content was determined according to the Folin-Ciocalteu method.[27 ] Gallic acid was used as standard and total phenolic content expressed as mg of gallic acid equivalents (GAE) per gram.[16 (link)] For preparation of gallic acid stock solution, in a 100 ml volumetric flask, 500 mg of dry gallic acid in 10 ml of ethanol 96% was dissolved and diluted to volume with water. For preparation of calibration curve, 0, 1, 2, 3, 5 and 10 ml of the phenol stock solution were added into 100 ml volumetric flasks, and then diluted to volume with water. These solutions will have phenol concentrations of 0, 50, 100, 150, 250 and 500 mg/L gallic acid.[27 ] For preparation of sample solution, Jaft powder of Q. brantii and fruit powder of C. sativum (10 g) were added to 250 ml boiling water and extracted under continuous stirring for 10 min. Then the extracts were filtrated and concentrated using rotary evaporator. The concentrated extracts were lyophilized by freeze dryer (Snijders Tilburg 2040, Holland) and dry extracts were obtained.[16 (link)28 (link)] 100 mg dry extract of plants is dissolved in 15 ml of hot water and filtrated.[11 ]
Folin-Ciocalteu test: Briefly, 1.58 ml water was added to each calibration solution, sample and blank (20 μl) and then 100 μl of Folin-Ciocalteu reagent was added and well mixed. After 30 seconds, 300 μl of 20% sodium carbonate solution was added and the sample tubes were left at room temperature for 2 h. The absorbance of each solution was determined at 765 nm against the blank with a UV-VIS spectrophotometer (UVmini 1240, Shimadzu) and plot absorbance vs. concentration. For C. sativum, 20 μl was added as for the calibration solutions, but in the case of Q. brantii, the sample was diluted by 10 first, then 20 μl was added.[27 ]
Folin-Ciocalteu test: Briefly, 1.58 ml water was added to each calibration solution, sample and blank (20 μl) and then 100 μl of Folin-Ciocalteu reagent was added and well mixed. After 30 seconds, 300 μl of 20% sodium carbonate solution was added and the sample tubes were left at room temperature for 2 h. The absorbance of each solution was determined at 765 nm against the blank with a UV-VIS spectrophotometer (UVmini 1240, Shimadzu) and plot absorbance vs. concentration. For C. sativum, 20 μl was added as for the calibration solutions, but in the case of Q. brantii, the sample was diluted by 10 first, then 20 μl was added.[27 ]
4
Prevalence of G6PD Deficiency in Myanmar
Between December 2014 and March 2015, 1000 adult healthy volunteers were randomly recruited among persons who were attending the Out Patient Department of Township Medical Centers of Ahlone and Insein Townships in Yangon Region, Myanmar. After signing informed consent, volunteers had a clinical examination and a 2ml venous blood sample was collected in EDTA tubes. Samples were transported refrigerated to the Department of Medical Research (DMR) Biochemistry laboratory and analyzed for complete blood count and G6PD phenotypes within 6 hours.
Malaria RDT (SD Malaria Ag P.f/P.v, Standard Diagnostic, Korea) was performed as per manufacturer instructions using 5μl of whole blood.
The Complete Blood Count (CBC) was performed using a hematology analyzer (pocH-100i, Sysmex, USA). The CBC included white blood cells (WBC) total and 3-part differential count, red blood cells number (RBC), red blood cells size (MCV), haemoglobin content (MCH and MCHC), total haemoglobin concentration (HGB), haematocrit (HCT) and platelets count (PCT). Quality controls were run every day before analysis of samples. Anaemia was defined by Hb ≤11.5g/dL [18 (link)].
Hemoglobin typing (Hb typing) was performed using IsoElectric Focusing (IEF) electrophoresis according to protocol from Gianazza and colleagues [19 (link)]. The technique allows for detection of abnormal structural hemoglobins (such as HbE, HbC and HbS). Reticulocyte count on 1000 red blood cells was performed using New Methylene Blue staining. Both Hb typing and reticulocyte count were performed at the Pathology laboratory of DMR.
The G6PD spectrophotometric assay (G-6-PDH quantitative kit, code345-B, Trinity Biotech, Ireland) was performed in duplicate using 10μL of whole blood per replicate; instructions from supplier were followed for reagents preparation. A UV spectrophotometer (UV mini-1240, SHIMADZU, Japan) with electronically controlled temperature compartment was used to detect the absorbance at 340 nm during 10 minutes at 30°C. G6PD activity was calculated as IU/gHb and IU/RBC using the results of the complete blood count on the same blood. Normal, intermediate and deficient G6PD controls (code G6888, G5029 and G5888) were run in double at the beginning of each analysis day; their assessed activity was compared to reference activity and concordance between replicates was analyzed in terms of coefficient of variance (CV). Training on the spectrophotometric assay was performed both in SMRU and at DMR Biochemistry laboratory.
The G6PD fluorescent spot test (FST) (code 203-A, Trinity Biotech, Ireland) was performed using 5μL of blood mixed with 100μL kit reagents. After 10 minutes of incubation at room temperature, a 15μL aliquot was spotted on filter paper and allowed to air dry. The spots were then visualized under UV light; spots that showed fluorescence were classified as normal, spots that failed to show fluorescence were classified as deficient. A normal and a deficient control sample were analyzed along with each batch of samples.
The CareStart test (AccessBio, USA) was performed according to the manufacturer’s instructions: 2 μL of blood were placed in the device and the buffer added immediately; after 10 minutes the reading window was inspected for development of color. Tests showing a pink color were classified as normal, tests showing very faint or no color were classified as deficient, tests that showed remaining blood in the reading window were considered invalid and repeated. Remarks about the uniformity of color in the reading window were collected.
Both G6PD qualitative tests were read by two laboratory technicians unaware of the sprectrophotometric results. If the results were discordant a third reading was performed by a third laboratory technician. Although great effort was put in the initial training and in obtaining only a binary result (deficient or normal) in some cases it was not possible to decide either way and an “Intermediate” result was reported.
Laboratory temperature and humidity were recorded every day at the beginning of sample processing. Temperature range was 24.1–27.1°C and humidity range was 41.1–58.1%.
Malaria RDT (SD Malaria Ag P.f/P.v, Standard Diagnostic, Korea) was performed as per manufacturer instructions using 5μl of whole blood.
The Complete Blood Count (CBC) was performed using a hematology analyzer (pocH-100i, Sysmex, USA). The CBC included white blood cells (WBC) total and 3-part differential count, red blood cells number (RBC), red blood cells size (MCV), haemoglobin content (MCH and MCHC), total haemoglobin concentration (HGB), haematocrit (HCT) and platelets count (PCT). Quality controls were run every day before analysis of samples. Anaemia was defined by Hb ≤11.5g/dL [18 (link)].
Hemoglobin typing (Hb typing) was performed using IsoElectric Focusing (IEF) electrophoresis according to protocol from Gianazza and colleagues [19 (link)]. The technique allows for detection of abnormal structural hemoglobins (such as HbE, HbC and HbS). Reticulocyte count on 1000 red blood cells was performed using New Methylene Blue staining. Both Hb typing and reticulocyte count were performed at the Pathology laboratory of DMR.
The G6PD spectrophotometric assay (G-6-PDH quantitative kit, code345-B, Trinity Biotech, Ireland) was performed in duplicate using 10μL of whole blood per replicate; instructions from supplier were followed for reagents preparation. A UV spectrophotometer (UV mini-1240, SHIMADZU, Japan) with electronically controlled temperature compartment was used to detect the absorbance at 340 nm during 10 minutes at 30°C. G6PD activity was calculated as IU/gHb and IU/RBC using the results of the complete blood count on the same blood. Normal, intermediate and deficient G6PD controls (code G6888, G5029 and G5888) were run in double at the beginning of each analysis day; their assessed activity was compared to reference activity and concordance between replicates was analyzed in terms of coefficient of variance (CV). Training on the spectrophotometric assay was performed both in SMRU and at DMR Biochemistry laboratory.
The G6PD fluorescent spot test (FST) (code 203-A, Trinity Biotech, Ireland) was performed using 5μL of blood mixed with 100μL kit reagents. After 10 minutes of incubation at room temperature, a 15μL aliquot was spotted on filter paper and allowed to air dry. The spots were then visualized under UV light; spots that showed fluorescence were classified as normal, spots that failed to show fluorescence were classified as deficient. A normal and a deficient control sample were analyzed along with each batch of samples.
The CareStart test (AccessBio, USA) was performed according to the manufacturer’s instructions: 2 μL of blood were placed in the device and the buffer added immediately; after 10 minutes the reading window was inspected for development of color. Tests showing a pink color were classified as normal, tests showing very faint or no color were classified as deficient, tests that showed remaining blood in the reading window were considered invalid and repeated. Remarks about the uniformity of color in the reading window were collected.
Both G6PD qualitative tests were read by two laboratory technicians unaware of the sprectrophotometric results. If the results were discordant a third reading was performed by a third laboratory technician. Although great effort was put in the initial training and in obtaining only a binary result (deficient or normal) in some cases it was not possible to decide either way and an “Intermediate” result was reported.
Laboratory temperature and humidity were recorded every day at the beginning of sample processing. Temperature range was 24.1–27.1°C and humidity range was 41.1–58.1%.
5
DPPH Free Radical Scavenging Assay
The free radical scavenging activity of the plant extract (EECA) was carried out in terms of hydrogen donating or radical-scavenging ability using the stable radical DPPH (1,1 Diphenyl-1-picrylhydrazyl) by the method described by Braca et al. [13 (link)]. Briefly, 0.1 mL of extract at various concentrations (3.125–100 μg/mL) was mixed with 3 mL of freshly prepared DPPH solution (0.004%) in methanol. The mixture was homogenized and incubated for 30 min at room temperature in the dark. The absorbance was measured at 517 nm in a spectrophotometer (UVmini-1240, Shimadzu, Japan). Ascorbic acid was used as the antioxidant standards; methanol used as a blank; methanol plus DPPH solution used as a negative control (Ac). The degree of decolorization of DPPH from purple to yellow indicated the scavenging efficiency of the extract. All experiment was conducted in triplicates. The percentage of free radical-scavenging activity was expressed with the following formula: Scavenging effect (%) = [(Ac − As)/Ac] × 100. Where Ac is the absorbance of the control, and As is the absorbance of the sample (ascorbic acid or extract). The IC50 (half-maximal inhibitory concentration) was calculated graphically using a calibration curve in the linear range by plotting the extract concentration versus the corresponding scavenging effect.
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