Diagnosis of Carcinoid Tumors in the Small Intestine by LC-MS/MS Analysis of 5-Hydroxyindoleacetic Acid (5-HIAA) in Serum
Anders Lindgren(1), Magnus Kjellman(2), Henrik Falhammar(2), Linda Touma(1), Britt Lundin(1) Gösta Eggertsen(1) (1) Clinical Chemistry, Karolinska University Laboratory, Stockholm, Sweden (2) Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden
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Anders Lindgren (Presenter) Karolinska University Laboratory
Relevant Financial Disclosures
(within past 24 months)
No relevant financial relationship(s) to disclose.
Abstract
Introduction
Carcinoid tumors are neuroendocrine tumors (NET), mainly occurring in intestines. In most cases the neoplastic cells produce serotonin, which is metabolized to 5-hydroxy-indolacetic acid (5-HIAA). In Sweden 50-100 new cases are discovered annually. For diagnosis, determination of urinary output of 5-HIAA for 24 hours have been used.
Objective
To establish an automated method for quantitation of 5-HIAA in serum using LC-MS/MS.
Patient samples
Serum samples were collected from healthy student volunteers. Patient serum specimens were admitted for analysis to the Clinical Chemical Laboratory, mainly from the Department of Endocrine and Sarcoma Surgery, Karolinska University Hospital. The patients were fasting, and should not have consumed food rich in serotonin 24 hours before drawing the blood samples.
Methods
Sample preparation were carried out by a pipetting robot (Hamilton MicrolabSTARlet). Briefly, to the serum samples were added an internal standard of 3H-labelled 5-HIAA, and proteins were precipitated with acetonitrile. Phospholipids and precipitated proteins were then removed by a ‘phospholipid removal plate’ (Phenomenex), whereafter acetonitrile was evaporated and 0.2% formic acid was added. The amount of 5-HIAA was determined by reversed phase chromatography and tandem mass spectrometry (Waters, Acquity-Xevo TQ).
Results
Total CV of the method was 6.5% at 690 nmol/L and 7,4 at 146 nmol/L The limit of quantification was determined to 25 nmol/L (CV less than 20%). Linearity range from 25 – 3600 nmol/L. Standard addition experiments in serum showed accuracies between -4.8 to to 2.6% and comparison experiments with an external laboratory showed acceptable agreement. The highest values in patient samples were 9,000-10,000 nmol/L, and these patients all had NET tumors with metastatic spreading. Generally, most patients having pathologically elevated serum concentrations also showed increased excretion of urinary 5-HIAA during 24 hours. In cases with only slightly elevated serum levels of 5-HIAA (up to 200 nmol/L), urinary excretion of 5-HIAA usually was not elevated.
Conclusions: Quantitation of 5-HIAA in serum is a useful and reliable method for diagnosis of intestinal carcinoid tumors. The analysis is less laborious than determination of 5-HIAA in urine and more convenient for the patients.
= Discovery stage.
= Translation stage.
= Clinically available.
