Quantitative Colorimetric/Fluorimetric Determination of Ammonia

AMMONIA (NH3) or its ion form ammonium (NH4+) is an important source of nitrogen for living systems. It is synthesized through amino acid metabolism and is toxic when present at high concentrations. In the liver, ammonia is converted to urea through the urea cycle. Elevated levels of ammonia in the blood (hyperammonemia) have been found in liver dysfunction (cirrhosis), while hypoammonemia has been associated with defects in the urea cycle enzymes (e.g. ornithine transcarbamylase). Simple, direct and automation-ready procedures for measuring NH3 are popular in research and drug discovery. BioAssay Systems' ammonia assay is designed to directly measure NH3 and NH4+. In this assay, NADH is converted to NAD+ in the presence of NH3, ketoglutarate and glutamate dehydrogenase. The decrease in optical density at 340 nm or fluorescence intensity at lem/ex = 450/360 nm is directly proportionate to the NH3concentration in the sample.

Homogeneous and simple procedure. Simple “mix-and-measure” procedure allows reliable quantitation of NH3 within 30 minutes.

Ketogutarate: 120 μL Standard: 400 μL 20 mM NH4Cl

NADH Reagent: 1 mL

Storage conditions. The kit is shipped chilled. Store all components at -20°C. Shelf life of at least 3 months.

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.

Sample preparation: solid samples can be extracted by homogenization in distilled water (dH2O) and filtered, centrifuged or, if necessary,deproteinized to remove any undissolved material. Samples should be clean and colorless with pH adjusted to 7 - 8.Serum and plasma samples can be assayed directly. Cell culture mediashould be diluted 5-10 fold in dH2O prior to assay.

1. Standards and Samples. Equilibrate all components to room temperature. Briefly centrifuge tubes before opening. Prepare a 1000 μM NH3 Standard Premix by mixing 15 μL of the 20 mM Standard and 285μL dH2O. Dilute Standard as follows.

Transfer 20 μL standards into separate wells of a clear, flat-bottom 96-well plate.

Transfer 20 μL of each sample into two separate wells, one serving as a sample blank well (RBLANK) and one as a sample well (RSAMPLE).

2. Enzyme Reaction. For each standard and sample well, prepare Working Reagent by mixing 180 μL Assay Buffer, 1 μL Enzyme, 8 μL

NADH Reagent and 1 μL Ketoglutarate. Add 180 μL Working Reagent to the four Standards and the Sample Wells. Prepare blank control reagent by mixing 180 μL Assay Buffer, 8 μLNADH Reagent and 1 μL Ketoglutarate (No Enzyme). Add 180 μL Blank control reagent only to the Sample Blank Wells. Tap plate to mix. Incubate 30 min at room temperature.

3. Read OD340nm.

The fluorimetric procedure is the same as for the colorimetric assay, except that a black, flat-bottom 96-well plate is used. After incubation for 30 min at room temperature, read fluorescence intensity at lex = 350-360 nm and lem = 450 nm.

Subtract the standard values from the blank value (#4) and plot the DOD or DF against standard concentrations. Determine the slope and calculate the NH3 concentration of Sample,

RSAMPLE and RBLANK are optical density or fluorescence intensity readings of the Sample and Sample Blank, respectively. n is the sample dilution factor.

Note: if the calculated NH3 concentration is higher than 1000 μM, dilute sample in dH2O and repeat assay. Multiply result by the dilution factor n.

Conversions: 1000 μM NH3 equals 1.7 mg/dL or 17 ppm.

Pipeting devices, and clear flat-bottom 96-well plates and optical density plate reader for colorimetric assays; black flat-bottom 96-well plate and fluorescence intensity plate reader for fluorimetric assays.

1. Bruce, A.W. et al. (1978). Two-point determination of plasma ammonia with the centrifugal analyzer. Clin Chem. 24:782-787.

2. Mondzac, A. et al. (1965). An enzymatic determination of ammonia in biological fluids. J Lab Clin Med. 66:526-531.