Microsoft word - 3636 5-hiaa ing.doc

5 – HIAA
Chromatographic – Colorimetric Determination of 5-Hydroxy-3-Indolacetic Acid (5-HIAA)
Accurately mix each collected eluate, keeping them separate.
COLORIMETRIC REACTION
40 tests REF 3636
INTENDED USE
Kit for quantitative in vitro determination of 5-Hydroxy-3-Indoleacetic Acid PRINCIPLE
The final products of tryptophane metabolism are adsorbed on a neutral resin. After the washing of interfering substances, serotonin (5HT), 5-hydroxy-triptophane (5HTP) and 5-hydroxy-3-indolacetic acid (5-HIAA) are eluted together. The concentration of these metabolites is Mix very carefully and wait for 10 minutes at room temperature. photometrical determined by the products formed in the reaction with REAGENTS AND COLUMNS
Shake on vortex for 10 seconds and centrifuge for 2 minutes at 3000
Kit components:
rpm. Read the absorbencies of the sample (As) and the standard (Astd), at 540 nm (510-570 nm) against blank. REAGENT 1 Hydrochloric acid
REAGENT 2 Hydrated ammonium
CALCULATION
*REAGENT 3 Diluted sulphuric acid
*REAGENT 4 1-nitrose-2-naphthol
*REAGENT 5 Sodium nitrite
*REAGENT 6 1,2-dichloroethane
5HT + 5HTP

Serotonin (mg/L) = (As/ Astd) x 12.5
STANDARD Serotonin 20 mg/L
To calculate the 24 hour excretion, multiply the obtained values by the COLUMNS Chromatographic colums
(*) Dangerous reagents are marked by an asterisk. Refer to MSDS. STABILITY: stored at 2-8°C, sealed reagents and columns, are stable up REFERENCE VALUES

5-HIAA: 2 - 10 mg/24 hours
EQUIPMENT REQUIRED BUT NOT SUPPLIED
Spectrophotometer or filter photometer (540 nm), centrifuge.
1. Aspirin, phenacetine, acetanilide, chloropromazine give positive
interference, same as drugs like reserpine which causes serotonin
24 hour urine.
release. Phenothiazine derivates inhibit the reaction. Do not Collect the 24 hour urine in a container with 2-3 ml of concentrated administer any of these drugs for 72 hours before urine collection. hydrochloric acid. Make sure the pH value is lower than 6. Mix the urine, For the same period of time, ban bananas, walnuts and tomatoes from the diet as their high concentration of tryptophane may give STABILITY: about 5 days at 2-8°C with pH lower than 6. 2. If the supernatant is not completely clear after centrifuge, transfer it
MANUAL ASSAY PROCEDURE
into another centrifuge test-tube containing a spatula-tip of anhydrous sodium sulfate, mix thoroughly and centrifuge or let 3. In case of thrombosis and hemorrhage, 5-HIAA values ranged
among 10 and 100 mg/24 hours may result; they are higher than 100 mg/24 hours (up to 2000 mg) in cases of argentaffinoma or malignant carcinoids. Patients with non-metastasing tumours may have normal values. In cases of pulmonary carcinomas, values are lightly increased. This determination is very useful in preliminary tests of suspected cases; no false positive values have been PREPARATION OF THE COLUMN

Let the columns reach room temperature before use. Shake and turn the
4. Define ELUATE I and ELUATE II each time. 5-HIAA considerably
columns upside down to obtain a complete resin re-suspension and leave increases in most cases of metastasing tumours, while sometimes them for a few minutes in vertical position to allow the resin to sediment only 5HT and 5HTP considerably increase. In such cases the test again. Take the upper cap off and snap the bottom tip off. Let the liquid result will always be positive and values higher than normal values. REFERENCE
CHROMATOGRAPHIC SEPARATION
1. 1. B.L. Goodwin, C.R.J. Ruthven, M.W. Weg and M. Sandler - Clin.
Let the reagents reach room temperature before use.
Gently pipette, washing any eventual urine remains on the column
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