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Name	Description	Cat.#	Category
EnzyChrom™ Phospholipid Assay Kit	Quantitative determination of phospholipid by colorimetric (570nm) or fluorimetric (530/585nm) methods. Procedure: 20 min. Kit size: 100 tests. Detection limit: colorimetric assay 3 µM, fluorimetric assay 0.6 µM. Shelf life: 3 months. Shipping: on ice; storage: 4, -20°C.	EPLP-100	Blood/urine chemistry; Metabolism

EnzyChromTM Phospholipid Assay Kit (EPLP-100)

Quantitative Colorimetric/Fluorimetric Phospholipid Determination

DESCRIPTION

PHOSPHOLIPIDS are a class of lipids which constitute a major component of cell membranes and play important roles in signal transduction. Most phospholipids contain one diglyceride, a phosphate group, and one choline. BioAssay Systems' method provides a simple, direct and high-throughput assay for measuring choline-containing phospholipids in biological samples. In this assay, phospholipids (such as lecithin, lysolecithin and sphingomyelin) are enzymatically hydrolyzed to choline which is determined using choline oxidase and a H2O2 specific dye. The optical density of the pink colored product at 570nm or fluorescence intensity (530/585 nm) is directly proportional to the phospholipid concentration in the sample.

KEY FEATURES

Sensitive. Use 20 μL samples. Linear detection range: colorimetric assay 3 - 200 μM, fluorimetric assay 0.6 - 20 μM phospholipid.

APPLICATIONS

Assays: phospholipid in biological samples such as serum and non- EDTA plasma.

Drug Discovery/Pharmacology: effects of drugs on choline-containing phospholipid metabolism.

KIT CONTENTS

Assay Buffer: 10 mL PLD Enzyme: 120 μL

Enzyme Mix: 120 μL Dye Reagent: 120 μL

Standard: 400 μL 2 mM phosphatidylcholin e

Storage conditions. The kit is shipped on ice. Store all components at -20°C. Shelf life of three months after receipt.

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.

COLORIMETRIC ASSAY

Liquid samples such as serum and plasma can be assayed directly. Solid samples can be homogenized in the assay buffer.

Note: SH-containing reagents (e.g. b–mercaptoethanol, dithiothreitol, > 5 μM), sodium azide, EDTA, and sodium dodecyl sulfate are known to interfere in this assay and should be avoided in sample preparation.

1. Equilibrate all components to room temperature. Briefly centrifuge the tubes before opening. Keep thawed tubes on ice during assay.

2. Standards: mix 24 μL 2 mM Standard with 216 μL dH2O (final 200 μM). Dilute standard in dH2O as follows.

Transfer 20 μL diluted standards into separate wells of a clear flatbottom 96-well plate.

Samples: transfer 20 μL of each sample into separate wells of the plate.

Note: if a sample is known to contain choline, prepare an extra sample blank well with 20 μL of the sample.

3. Color reaction. Prepare enough Working Reagent by mixing, for each well, 85 μL Assay Buffer, 1 μL PLD Enzyme, 1 μL Enzyme Mix and 1 μL Dye Reagent. Add 80 μL Working Reagent to each well. For samples that contain choline, prepare a blank control reagent with no PLD Enzyme (i.e., 85 μL Assay Buffer, 1 μL Enzyme Mix and 1 μL Dye Reagent). Add 80 μL of the Control Reagent to the Sample Blank well.

Tap plate to mix. Incubate 15 min at room temperature.

Note: if precipitation occurs with certain samples, carry out the reaction in centrifuge tubes. After the 15 min incubation, centrifuge 5 min at 14,000 rpm. Transfer the supernatant into the wells for OD reading.

4. Read optical density at 570nm (550-585nm).

FLUORIMETRIC ASSAY

The fluorimetriC assay procedure is similar to the colorimetric procedure except that (1) 0, 6, 12 and 20 μM phospholipid standards and (2) a black 96-well plate are used. Read fluorescence intensity at lex = 530 nm and l em = 585 nm.

Note: if the calculated phospholipid concentration of a sample is higher than 200 μM in the Colorimetric Assay or 20 μM in the Fluorimetric Assay, dilute sample in 0.5% Triton X-100 and repeat the assay. Multiply result by the dilution factor n.

CALCULATION

Subtract blank value (#4) from the standard values and plot the DOD or DF against standard concentrations. Determine the slope and calculate the phospholipid concentration of Sample,

RSAMPLE and RBLANK are optical density or fluorescence intensity readings of the Sample and H2O Blank (or Sample Blank if sample contains choline), respectively. n is the sample dilution factor.

MATERIALS REQUIRED, BUT NOT PROVIDED

Pipetting devices, centrifuge tubes, clear flat-bottom uncoated 96-well plates, optical density plate reader; black flat-bottom uncoated 96-well plates, fluorescence plate reader.

LITERATURE

1. Nie, Y. et al. (1993). A micro enzymic method for determination of choline-containing phospholipids in serum and high density lipoproteins. Lipids. 28:949-951.

2. Grohganz, H. et al. (2003). Quantification of various phosphatidylcholines in liposomes by enzymatic assay. AAPS PharmSciTech. 4(4):E63.

3. Hojjati, MR, Jiang XC (2006). Rapid, specific, and sensitive measurements of plasma sphingomyelin and phosphatidylcholine. J Lipid Res. 47:673-676.