Measurement of plasma 5-hydroxyindoleacetic acid in carcinoid disease: an alternative to 24-h urine collections?

Abstract

The biochemical investigation of carcinoid disease has relied on measurement of the daily excretion of 5-hydroxyindoleacetic acid (5-HIAA) in urine.1 Collection of 24-h urine has considerable disadvantages for the patient. Inconvenience, embarrassment, strict dietary requirements and the need for hazardous preservatives make this test unpopular, and signi®cant errors in collection may be made. The measurement of 5-HIAA in plasma would offer a more convenient alternative. However, the value of plasma 5-HIAA measurements in carcinoid disease has not been evaluated. Highperformance liquid chromatographic (HPLC) methods for measuring plasma 5-HIAA have been described previously, but applied to the investigation of patients with psychiatric and neurological disorders. 2 We describe a simple, isocratic HPLC method with¯uorimetric detection following ether extraction of 5-HIAA from plasma. This method was used to determine a reference range for plasma 5-HIAA in normal individuals and to investigate patients with known carcinoid disease. The effect of serotonin-containing foods on plasma 5-HIAA was also evaluated. MATERIALS AND METHODSOne hundred microlitres of internal standard (3´4 mmol/L 5-hydroxyindole-2-carboxylic acid) was added to 0´5 mL of heparinized plasma and diluted with 1 mL of water. The solution was acidi®ed with 25 mL of 50% glacial acetic acid and saturated with 1 g of sodium chloride. Extraction was carried out by shaking with 5 mL of diethylether for 2 min. After centrifugation at 2000 g for 5 min the ether layer was decanted into a glass tube and evaporated to dryness under nitrogen at 398C. The residue was reconstituted with 350 mL of mobile phase 5-HIAA (0´1 mol/L ammonium acetate, pH 4´55, containing 7% acetonitrile). 5-HIAA and the internal standard were separated isocratically on a 4´66250 mm C 18 , 5 mm Symmetry column (Waters, Watford, Herts, UK) using the mobile phase at a¯ow rate of 1 mL/min at ambient temperature. Peaks were detected using a Perkin Elmer LS5 spectro¯uorimeter (Perkin Elmer, Beacons®eld, Bucks, UK) at an excitation wavelength of 280 nm and emission wavelength of 345 nm. The internal standard peak eluted at 6´7 min and the 5-HIAA at 12´5 min. Lateeluting¯uorescent compounds were removed from the column by incorporating an acetonitrile wash for 1 min between sample injections. The method was calibrated using normal pooled plasma spiked with 5-HIAA concentrations of up to 1000 nmol/L. Samples with 5-HIAA greater than 1000 nmol/L were diluted in pooled plasma and re-analysed.Urine 5-HIAA was measured by HPLC using the method described by Skrinska and Hahn, 3 but substituting methanol in the mobile phase with acetonitrile. Heparinized blood samples were collected from 40 normal, fasting adult volunteers (14 men, 26 women) and 11 patients with carcinoid disease. Seven pairs of normal volunteers had both fasting and 2-h blood samples taken after ingestion of the following serotonin-containing food: plums, tomatoes, kiwi fruit, avocadoes, walnuts, pineapples and bananas.Plasma 5-HIA...

The importance of the measurement of circulating markers in patients with neuroendocrine tumours of the pancreas and gut.

“…Patients with hindgut carcinoid do not excrete these products. An assay for serum 5HIAA has been described which shows similar specificity, sensitivity and diagnostic potential to the urinary assay but has the advantage of being more acceptable and convenient for the patient (Degg et al 2002). A wide range of peptides that are found in the circulation of patients with carcinoid tumours have been described (Table 2) (Ö berg et al 1999).…”

Measurement of plasma 5-hydroxyindole acetic acid by liquid chromatography tandem mass spectrometry—Comparison with HPLC methodology

“…The plasma 5-HIAA HPLC method in routine use at Leeds Teaching Hospitals has been described in detail previously [12]. Briefly, 100 L of internal standard (5-hydroxyindole-2-carboxylic acid) was added to 500 L of heparinised plasma and diluted with 1 mL of water.…”

“…The plasma 5-HIAA method described by Degg et al [12] and currently in routine use at Leeds Teaching Hospitals, U.K. employs HPLC with native fluorescence detection and although the method shows good reliability and precision, the sample preparation procedure requires relatively large sample volumes, uses hazardous and volatile chemicals and has a run time of 17 min [12]. By employing LC-MS/MS and a simpler sample preparation procedure, we aimed to cut both the sample preparation and run time considerably, while improving on some analytical attributes of the HPLC method.…”

Circulating Tumor Markers in Patients with Neuroendocrine Tumors – a Clinical Perspective

“…Plasma 5-HIAA has been suggested as a useful test for patients with NETs [76,[79][80][81]. It suffers some of the same limitations as serotonin and 24-h urine collection of 5-HIAA.…”