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Serotonin is transformed into melatonin under the control of the light/dark cycle, representing a cornerstone of circadian rhythmicity. Serotonin also undergoes extensive metabolism to produce 5-hydroxyindoleacetic acid (5-HIAA), a biomarker for the diagnosis and monitoring of serotonin secreting neuroendocrine tumors (NETs). While serotonin, melatonin and their metabolites are part of an integrated comprehensive system, human observations about their respective plasma concentrations are still limited. We report here for the first time a multiplex UHPLC-MS/MS assay for the quantification of serotonin, 5-HIAA, 5-hydroxytryptophol (5-HTPL), N-acetyl-serotonin (NAS), Mel, 6-OH-Mel, 5-methoxytryptamine (5-MT), 5-methoxytryptophol (5-MTPL), and 5-methoxyindoleacetic acid (5-MIAA) in human plasma. Analytes were extracted by protein precipitation and solid phase extraction. Plasma concentrations for these analytes were determined in 102 healthy volunteers. The LLOQ of the assay ranges from 2.2 nM for serotonin to 1.0 pM for 6-OH-Mel. This sensitivity enables the quantification of circulating serotonin, 5-HIAA, NAS, Mel, and 5-MIAA, even at their lowest diurnal concentrations. This assay will enable specific, precise and accurate measurement of serotonin, Mel and their metabolites to draw a detailed picture of this complex pineal metabolism, allowing a dynamic understanding of these pathways and providing promising biomarkers and a metabolic signature for serotonin-secreting NETs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Urinary 5-hydroxyindoleacetic acid (5-HIAA), vanillylmandelic (VMA), homovanillic acid (HVA), catecholamines and metanephrines are produced in excess by catecholamine-producing tumors. These biogenic amines are unstable at low or high pH and require hydrochloric acid (HCl) to prevent their degradation. However, HCl addition may result in very low pH causing degradation or deconjugation of several metabolites. This study evaluated the buffering properties of sodium citrate to stabilize all biogenic amines. The metabolite concentrations were measured by LC-MS/MS or by a coulometric assay in 22 urine samples collected native and with HCl or sodium citrate. We studied the effect of pH, time (48 h, four weeks) and storage temperature at 22 °C, 4 °C, and -20 °C. We found that catecholamines degradation was prevented by HCl and citrate and that 5-HIAA was degraded in 5 out of 22 samples collected with HCl. All biogenic amines were efficiently stabilized by citrate for four weeks at 22 °C, except epinephrine (48 h at 4 °C, or four weeks at -20 °C). Sodium citrate did not cause quantification or analytical artefacts concerns. In conclusion, sodium citrate is a non-hazardous alternative to HCl for patients to send unfrozen urine samples to the laboratory which may safely store the sample for four weeks., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Total (i.e. free+sulfated) metanephrines in plasma is a biomarker for the diagnosis of pheochromocytoma/paraganglioma. Sulfated metanephrines must be completely deconjugated by perchloric acid hydrolysis or sulfatase treatment prior to analytical measurement to enable quantification by current techniques. In this report, we compare the yield and efficiency of both methods., Methods: The deconjugation rate of synthetic sulfated metanephrines (normetanephrine (S-NMN), metanephrine (S-MN) and methoxytyramine (S-MT)) spiked in charcoal-stripped plasma was determined by boiling perchloric acid and compared to sulfatase treatment. Total plasma metanephrines (MN, NMN and MT) were also determined in patient samples by both methods., Results: The complete deconjugation of sulfated metanephrines is achieved after 30 min incubation with 0.1M boiling perchloric acid or upon sulfatase treatment. Ten minutes of acid hydrolysis (gold-standard) leads to a 30% underestimation of metanephrine concentrations. The enzyme hydrolysis is time and amount of sulfatase dependent. The rate of hydrolysis is analyte-dependent (MT>>NMN>MN), although it must contain at least 0.8 U/ml of sample. The Deming regression curves comparing acid versus enzyme hydrolysis on patient samples assessed that both methods gave similar unbiased concentrations., Conclusion: Enzyme and acid treatments are equivalent and efficient for removing sulfate from metanephrines as long as the optimal protocol is used for each method. However, the gold standard method for acid hydrolysis at 10 min established more than 20 years ago was not satisfactory regarding the hydrolysis of metanephrines in plasma., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: The clinical manifestations of CMTX have been well described but the natural history has not yet been studied in detail. We studied phenotype variability in a family with a Pro 87 to Leu mutation of the connexin 32 (Cx32) gene., Methods: A total of 32 family members, of which 19 patients were affected, underwent clinical, electrophysiological, and genetic studies., Results: Onset was in the second decade. Clinical features were similar in both sexes when quantitative scores were compared, but more males had a steppage gait and skeletal deformities. All adult patients had a predominant involvement of the thenar muscles. The median values of nerve conduction velocities (NCVs) were not statistically different in men and in women. The correlation coefficients were low between motor NCVs within the same extremities, indicating nonuniform slowing between nerves, the ulnar nerve being the least affected. When disability was rated, a strong correlation was seen in male patients between severity of motor axonal loss and duration of the disease. The main pathological features were axonal loss, clusters of regenerating fibers and paranodal demyelination, the hallmark of a Schwann cell pathology., Conclusions: Our data support the hypothesis that clinical disability in CMTX is caused by loss of large myelinated axons in men. Furthermore, this study shows that the nerves are not uniformly affected in terms of axonal loss. Preventing axonal degeneration and promoting axonal regeneration in the most affected nerves might be the best therapeutic approaches to ameliorate disability in CMTX.