Urinary sampling for 5HIAA and metanephrines determination: revisiting the recommendations.

CONTEXT: Biogenic amines such as 5-hydroxy-indole acetic acid (5HIAA) the main metabolite of serotonin or metanephrines (catecholamines metabolites) are used as biomarkers of neuroendocrine tumours. OBJECTIVE: To re-evaluate the recommendations for urinary sampling (preservatives, diet, drugs, etc.) as many of the reported analytical interferences supporting these recommendations are related to obsolete assays. METHODS: Bibliographic analysis of old and modern assays concerning preservation, extraction, assay and interferences. RESULTS: 5HIAA may degrade as soon as urine is excreted. Thus, acids as preservatives (hydrochloric or acetic acid) have to be immediately added. Care should be taken not to decrease the pH under 2. Urine preservative for metanephrine assays is not mandatory. Diets including serotonin-, tryptophan- and dopamine-rich foods have to be avoided depending on the biomarkers investigated (bananas, plantain, nuts, etc.). Tryptophan-rich over-the-counter formulas have to be prohibited when 5HIAA has to be assayed. Acetaminophen may interfere with electrochemical detection depending on high-pressure liquid chromatography (HPLC) parameters. No interference is known with mass spectrometric assays but with the one described for metanephrines determination. Some drugs interfere however with serotonin and catecholamines secretion and/or metabolism (monoamine oxidase inhibitors, serotonin or dopamine recapture inhibitors, etc.). CONCLUSION: Revisited recommendations are provided for the diet, the drugs and the preservatives before HPLC coupled with electrochemical and mass spectrometry assays.

Detection of AMP-activated protein kinase in human sera by immuno-isoelectric focusing.

AMPKalpha is a subunit of AMP-activated protein kinase (AMPK), a heterotrimeric enzyme that works as a fuel sensor activated in response to the depletion of cellular ATP. AMPKalpha is considered as a master switch in regulating glucose and lipid metabolism. Determining its presence in patient sera may help in diagnosing metabolic diseases. Using isoelectric focusing and Western blotting, we were able to detect AMPKalpha in human sera. Using specific antibodies, we showed that the AMPKalpha1 and alpha2 isoforms were apparently present in equal amounts in human sera. To characterize normal and abnormal AMPKalpha patterns, we used an antibody which recognized both isoforms (alpha1 and alpha2) to analyze sera of patients and healthy individuals. We also analyzed sera of HIV patients because several studies suggest that AMPK may play a role in the mechanism of lipodystrophy in HIV patients under antiretroviral therapy. We found that patients with type 2 diabetes or liver diseases presented abnormal AMPK IEF patterns. AMPK was poorly detectable in sera of patients with end-stage liver disease. Abnormal AMPK IEF patterns were more frequent in treated HIV-patients compared to those who are untreated suggesting a possible association between AMPK and the side-effects of antivirals. Our findings highlight the potential of serum AMPK as a new diagnostic biomarker and may help to study the regulation of AMPK activity in tissues.