REPRINTS, COPIES

1.	Phosphate buffer (0.1 M, pH 6.5). Na2HPO4 (4.25 g) and 9.52 g of KH2PO4 were dissolved in water and brought to a final volume of one liter.
2.	Iodine solution (0.02 N). I2(0.252 g) and 5 g KI were dissolved in 5 ml water and diluted to 100 ml.
3.	5 N NaOH
4.	Alkaline sulfite solution. Na2SO3(2. 52 g) was dissolved in 10 ml water and diluted to 100 ml with 5 N NAOH.
5.	Acetic acid (5 N). Glacial acetic acid (28. 5 ml) was diluted to 100 ml with water.

The following steps were performed in carrying out the assay:

1.	The pH of the eluate was adjusted to approximately 6.5 with 5N K2CO3 and made to 50 ml with glass-distilled water. (A sodium base cannot be used to adjust the pH!)
2.	One to 3 ml of sample (0. 2 to 2 microgram doparnine), 0.5 ml buffer, water to give a total volume of 3. 8 ml, and 0. 05 ml iodine solution were added to a test tube.
3.	After 5 minutes, 0.5 ml alkaline sulfite solution was added.
4.	After another 5 minutes, 0.6 ml 5 N acetic acid was added (pH drops to about 5.3).
5.	The tubes were heated for 30 minutes at 45C under standard laboratory lighting conditions (Udenfriend, 1962).
6.	Fluorescence was measured with a spectrofluorometer (9Aminco-Bowman Spectrophotofluorometer, American Instrument Company, Inc., Silver Springs, Maryland 20910.) an activation wavelength of 345 nm and a fluorescence wave-length of 410 nm.

A standard and a reagent blank were run along with the sample. When tissue extracts were analyzed, an internal standard and a tissue blank were run. The internal standard was an aliquot of tissue extract treated as above, except that a known amount of dopamine was added to check for quenching substances that might be present in the banana extract. The tissue blank was an aliquot of tissue extract treated as above, except that 5 N NaOH replaced the alkaline sulfite solution.

Iodine caused the oxidation of dopamine to dopamine quinone. Rearrangement to 5,6-dihydroxyindole was effected by the alkaline sulfite solution. Fluorescence was increased by lowering the pH with acetic acid. Concentration of dopamine in an extract of banana was determined by reference to a standard curve (Figure 2). The standard curve was prepared by plotting relative fluorescence against concentration of dopamine. Concentrations of dopamine of 0.05, 0.10, 0.15, 0.20, 0.30 and 0.40 micrograms per ml were used for the standard curve. A linear relationship was found between relative fluorescence and concentration of dopamine, up to 0. 3 micrograms per ml. Recovery of a known amount of dopamine added to banana prior to extraction was 91 percent.

Updated: Wednesday, June 20, 2007.