5-HIAA-For-in-vitro-test

							5-HIAA                                                                                                              For in vitro test

Column Kit
Chromatographic-colorimetric determination of serotonin and 5-hydroxy-3-indole acetic acid in urine

PRINCIPLE
The final products of tryptophane metabolism are adsorbed on a chromatographic gel and selectively eluted. Serotonin (5HT), 5-
hydroxy-triptophane (5HTP) and 5-hydroxy-3-indole acetic acid (5HIAA) form a colored complex with 1-nitrose-2-naphthol.

Recovery:           98 %  2
V.C.:               3%
Response:           linear up to 50 mg/l

REFERENCE VALUES

5-HT + 5-HTP:      0 - 5 mg/24 h
5-HIAA:            2 - 10 mg/24 h

SAMPLE
24-hour urine.

REAGENTS COMPOSITION
1-     Hydrochloric acid                         0.1         mole/l
2-     Ammonium hydroxyde                        0.02        mole/l
A-     Sulfuric acid                             1           mole/l
B-     1-Nitrose-2-naphthol                      6           mmole/l
C-     Sodium nitrite                            0.72        mole/l
D-     1,2 dichloroethane
E-     Serotonin Standard                        0.046       mg/ml
       5H-CK Chromatographic columns

SOLUTIONS

D           Keep Reagent D tightly capped.
E           Store Reagent E at +4°C to +10°C.

PREPARATION OF THE SAMPLE
Collect the 24-hour urine in bottles containing 2-3 ml conc. hydrochloric acid. Check the pH; it should be under 6. Stable for 3-4
days at room temperature.

TEST PROCEDURE

Wavelength:         540 nm
Temperature:        room temperature
Centrifuge 3-4 ml urine.

Pipet as following into a chromatographic column (Note 1):
 Reagent 1                     2.0 ml 
 Urine                         2.0 ml 
 Reagent 1                     5.0 ml 
 Reagent 1                     4.0 ml  eluate I
 Reagent 2                     1.0 ml 
 Reagent 2                     2.0 ml  eluate II
 = discard the eluate
 = collect the eluate

Pipet the following into centrifuge-tubes:
                            Sample                Standard                Blank
                 5HT + 5HTP          5HIAA
 Eluate I            2.0 ml            ---            ---                   ---
 Eluate II             ---           2.0 ml           ---                   ---
 Reagent E             ---             ---          0.5 ml                  ---
 Reagent 2             ---             ---          1.5 ml                2.0 ml


                      Mascia Brunelli S.p.A. – Viale Monza, 272 – 20128 Milano – Tel. 02/25209-1 – Fax 02/2576428
 Reagent A          1.0 ml            1.0 ml          1.0 ml              1.0 ml
 Reagent B          1.0 ml            1.0 ml          1.0 ml              1.0 ml
 Reagent C          1 drop            1 drop          1 drop              1 drop
 Mix and let stand for 10 minutes;   then pipet as follows:
 Reagent D          3.0 ml            3.0 ml          3.0 ml              3.0 ml

Mix for 10 seconds, centrifuge for 2 minutes at 3000 rpm and read the optical density (O.D.) of the supernatant at 540 nm (510-570)
against the Blank (Note 3).

CALCULATION

I)      5HT + 5HTP:
         O.D. Sample
         x 10                  = mg of serotonin/l of urine
        O.D. Standard

II)     5HIAA:
         O.D. Sample
         x 5                   = mg 5-HIAA/l of urine
        O.D. Standard

In calculating 24-hour excretion, multiply the reading obtained by urine volume (in liters).

NOTES
1. Before using the chromatographic columns, shake them vigorously to bring the gel back to suspension. Uncap each column,
   snap off bottom tips with fingers and let the liquid flow into the column.
2. 5-HIAA values of 10 to 100 mg/24 hours can be obtained in cases of thrombosis and hemorrhage; they are higher than 100
   mg/24 hours (up to 2000 mg) in cases of argent affinoma or malignant carcinoids patients, values can be slightly higher than
   normal. This determination is useful in preliminary testing of suspected cases; no false positive values have been noted as yet.
3. If the upper phase is not clear after centrifugation, transfer it to a centrifuge tube containing a spatula-tipfull of anhydrous
   sodium sulfate, mix thoroughly and let deposit.

WARNINGS
1. Aspirin, phenacetine, acetanilide, chloropromazine are positive interferents as are drugs such as reserpine which release
   serotonin. On the other hand, the phenothiazines inhibit the reaction. None of these drugs should be administered for 72
   hours before urine collection. Bananas should also be banned from the diet for the same period of time because they contain
   large quantities of tryptophane, which is metabolized into 5-HTP and 5-HIAA.
2. Determine both Eluate I and Eluate II each time the test is performed. Even though 5-HIAA increases considerably in most
   cases of metastasing tumors, there are times when only 5-HT and 5-HTP increase. The clinical significance is equivalent to an
   increase in 5-HIAA.

REFERENCES
1. S. Udenfriend, E. Titus e H. Weissabach, J. Biol. Chem. 216, 449 (1955) Mod.

                                                                                            COD. 340340000                40 Tests




                      Mascia Brunelli S.p.A. – Viale Monza, 272 – 20128 Milano – Tel. 02/25209-1 – Fax 02/2576428