Ethanol

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Name	Description	Cat.#	Category
QuantiChrom™ Ethanol Assay Kit	Quantitative determination of ethanol by chemical colorimetric (580nm) method. Procedure: 10 min. Kit size: 500 tests. Detection limit: 0.04%. Shelf life: 12 months. Shipping: ambient temp; storage: 2-8°C.	DIET-500	Metabolism
EnzyChrom™ Ethanol Assay Kit	Quantitative determination of ethanol by enzymatic colorimetric (565nm) method. Procedure: 30 min. Kit size: 100 tests. Detection limit: 0.0008%. Shelf life: 6 months. Shipping: dry ice; storage: -20°C.	ECET-100	Blood/urine chemistry; Metabolism

QuantiChromTM Ethanol Assay Kit (DIET-500)

Colorimetric Determination of Ethanol at 580 nm

DESCRIPTION

Alcoholic drinks are among the daily consumed beverages. Studies have shown heavy alcohol consumption may lead to various forms of liver diseases and to increased mortality rates. Quantitative determination of alcohol (ethanol, C2H5OH) finds applications in basic research, drug discovery, clinic studies and winery. Simple, direct and automation-ready procedures for measuring ethanol concentration are very desirable. BioAssay Systems' QuantiChromTM ethanol assay kit is based on an improved dichromate method, in which dichromate is reduced by ethanol to a bluish chromic (Cr3+) product. The intensity of color, measured at 580 nm, is a direct measure of the alcohol concentration in the sample. The optimized formulation substantially reduces interference by substances in the raw samples and exhibits high sensitivity.

APPLICATIONS

Ethanol determination in alcohol containing samples such as beverages (e.g. wine, beer) and yeast cultures. For samples containing less than 0.1% alcohol such as serum or plasma, EnzyChromTM Ethanol Assay Kit (Cat# ECET-100) is recommended.

KEY FEATURES

Sensitive and accurate. Detection range 0.04 – 4% alcohol in 96-well plate assay.

Convenient and high-throughput. The procedure involves adding a single working reagent, incubation for 8 min, adding a Stop Reagent, and reading the optical density. Can be readily automated as a highthroughput 96-well plate assay for thousands of samples per day.

Versatility. Assays can be executed in 96-well plate or cuvet.

KIT CONTENTS (500 tests in 96-well plates)

Reagent A: 50 mL Reagent B: 50 mL

10% TCA: 50 mL Standard: 2 mL 10% (v/v) ethanol

Storage conditions. The kit is shipped at room temperature. Store reagents at room temperature and the ethanol standard at 4°C. Shelf life of at least 6 months (see expiry dates on labels).

Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

PROCEDURES

Procedure using 96-well plate:

1. Prepare 600 μL 2% Premix by mixing 120 μL 10% Standard and 480 μL distilled water. Dilute standard as follows. Transfer 100 μL standards and samples into wells of a clear bottom 96-well plate.

2. Add 100 μL Reagent A quickly using a multi-channel pipettor. Tap plate lightly to mix.

3. Incubate 8 to 30 min at room temperature. The reagent color changes from yellow to visibly bluish in wells 1-4. Add 100 μL Stop Reagent B quickly using a multi-channel pipettor. Tap plate to mix.

4. Read OD at 570-600nm (peak 580nm).

Procedure using cuvette:

1. Prepare 2%, 1%, 0.5% standards and use distilled water as blank control. Transfer 400 μL diluted Standards and 400 μL samples to 1.5-mL centrifuge tubes.

2. Add 400 μL Reagent A quickly to each tube and vortex briefly to mix.

3. Incubate 8 to 30 min at room temperature. Add 400 μL Reagent B quickly and mix briefly.

4. Transfer to cuvettes and read OD at 570-600nm (peak 580nm).

Note: for the cuvette assay, it is recommended that an interval be applied between additions, e.g., add Reagent A to Tube 1 and 1 min later to Tube 2 etc. After the incubation step is completed, add the Stop Reagent B to Tube 1 and 1 min later to Tube 2 etc. This will ensure identical incubation time between tubes.

CALCULATION

Subtract blank OD (water, #8) from the standard OD values and plot the OD against standard alcohol concentrations. Determine sample ethanol concentration from the standard curve.

Conversions: 1% (v/v) ethanol equals 170 mM or 785 mg/dL.

MATERIALS REQUIRED, BUT NOT PROVIDED

Pipeting (multi-channel) devices.

Procedure using 96-well plate: Clear-bottom 96-well plates (e.g. Corning Costar) and plate reader.

Procedure using cuvette: Centrifuge tubes, table centrifuge, cuvets and spectrophotometer.

GENERAL CONSIDERATIONS

1. If sample contains glucose or glycerol, use BioAssay Systems’ specific ethanol assay kit (ECET-100). For samples containing only sugars (e.g. glucose), use Saccharide Removal Kit (DSRK-500) to remove the interferents prior to assay with the DIET-500 Kit.

2. This assay is based on a kinetic reaction. Addition of Reagent A and B (Stop reagent) should be quick and mixing should be brief but thorough.

3. Sample pretreatment. Proteinaceous samples, e.g. plasma, serum, culture media, should be deproteinated by adding 1 vol sample to 2 vol 10% TCA (provided). Pellet for 5 min at 14,000 rpm on a table centrifuge, carefully transfer supernatant for assay (n = 3). Saliva and urine can be analyzed directly (n = 1). For wines, dilute samples to approximately 1 to 2% prior to assay.

LITERATURE

1. Jetter WW (1950). Modified dichromate method for determination of ethyl alcohol in biologic tissue. Am J Clin Pathol. 20:473-475.

2. Pilone GJ (1985). Determination of ethanol in wine by titrimetric and spectrophotometric dichromate methods: collaborative study. J Assoc Off Anal Chem. 68:188-190.

3. Dubowski KM (1980). Alcohol determination in the clinical laboratory. Am J Clin Pathol. 74:747-750.

Related products EnzyChromTM Ethanol Assay Kit (ECET-100)

Saccharide Removal Kit (DSRK-500)

EnzyChromTM Ethanol Assay Kit (ECET-100)

Ultrasensitive Colorimetric Determination of Ethanol at 565 nm

DESCRIPTION

Alcoholic drinks are among the daily consumed beverages. Studies have shown heavy alcohol consumption may lead to various forms of liver diseases and to increased mortality rates. Quantitative determination of alcohol (ethanol, C2H5OH) has applications in basic research, drug discovery, clinic studies and in the alcoholic industry. Simple, direct and automation-ready procedures for measuring ethanol concentration are very desirable. BioAssay Systems' EnzyChromTM ethanol assay kit is based on alcohol dehydrogenase catalyzed oxidation of ethanol, in which the formed NADH is coupled to the formazan (MTT) chromogen. The intensity of the product color, measured at 565 nm, is proportionate to the ethanol concentration in the sample.

APPLICATIONS

Direct Assays: ethanol in serum, plasma, urine and saliva samples.

Pharmacology: effects of drugs on alcohol metabolism.

KEY FEATURES

Sensitive and accurate. Detection limit 0.0008 vol % (140 μM or 8 ppm), linearity up to 0.1% ethanol in 96-well plate assay.

Convenient. The procedure involves adding working reagent, incubating for 30 min and stropping reaction. No 37°C heater is needed.

High-throughput. Can be readily automated as a high-throughput 96- well plate assay for thousands of samples per day.

KIT CONTENTS (100 tests in 96-well plates)

Assay Buffer: 10 mL NAD Solution: 1 mL

MTT Solution: 1.5 mL Enzyme Mix: 120 μL

Stop Reagent: 12 mL Standard: 1.5 mL 1% ethanol

Storage conditions. Store Stop Reagent at room temperature and all other reagents at -20°C. Shelf life of at least 6 months.

PROCEDURES

1. Calibration Curve. Prepare 0.1% alcohol Premix by mixing 25 μL 1% Standard and 225 μL distilled water. Dilute standard as follows. Transfer 10 μL standards into wells of a clear bottom 96-well plate.

Samples: add 10 μL sample per well in separate wells.

IMPORTANT: saliva samples should be diluted 10-fold in PBS prior to assay.

2. Reaction. Spin the Enzyme Mix tube briefly before pipetting. For each well of reaction, prepare Working Reagent by mixing 80 μL Assay Buffer, 1 μL Enzyme Mix, 2.5 μL NAD and 14 μL MTT. Fresh reconstitution is recommended. Add 90 μL Working Reagent per well quickly. Tap plate to mix briefly and thoroughly. Incubate 30 min at room temperature. Add 100 μL Stop Reagent per well. Tap plate to mix.

3. Read optical density at 565 nm (520-600nm).

4. Calculation. Subtract blank (water, #4) from OD values for the standard wells. Plot Standard Curve (DOD vs Standard ethanol concentrations) to determine the slope. Sample ethanol concentration is calculated,

where ODSAMPLE and ODBLANK are the OD565nm values of the sample and blank (water, #4). n is the dilution factor (n = 10 for saliva).

Note: if the sample ethanol concentrations are higher than 0.1%, dilute sample in distill water and repeat this assay. Multiply the results by the dilution factor.

Conversions: 1 vol % ethanol equals 170 mM or 785 mg/dL.

MATERIALS REQUIRED, BUT NOT PROVIDED

Pipeting (multi-channel) devices. Clear-bottom 96-well plates (e.g. Corning Costar) and plate reader.

GENERAL CONSIDERATIONS

1. This assay is based on an enzyme-catalyzed kinetic reaction. Addition of Working Reagent should be quick and mixing should be brief but thorough. Use of multi-channel pipettor is recommended.

2. The following substances interfere and should be avoided in sample preparation: ascorbic acid, SDS (>0.2%), sodium azide, NP-40 (>1%) and Tween-20 (>1%).

PUBLICATIONS

1. Dembele, K. et al (2008). Effects of ethanol on pancreatic beta-cell death: interaction with glucose and fatty acids. Cell Biol Toxicol. 25(2):141-52.

2. Ou XM et al (2010). A novel role for glyceraldehyde-3-phosphate dehydrogenase and monoamine oxidase B cascade in ethanolinduced cellular damage. Biol Psychiatry 67(9):855-63.

3. Tapia H and Morano KA (2010). Hsp90 nuclear accumulation in quiescence is linked to chaperone function and spore development in yeast. Mol Biol Cell. 21(1):63-72.