11 research outputs found
High-Throughput and Sensitive Quantitation of Plasma Catecholamines by Ultraperformance Liquid Chromatography–Tandem Mass Spectrometry Using a Solid Phase Microwell Extraction Plate
Plasma
catecholamines provide a reliable biomarker of sympathetic activity.
The low circulating concentrations of catecholamines and analytical
interferences require tedious sample preparation and long chromatographic
runs to ensure their accurate quantification by HPLC with electrochemical
detection. Published or commercially available methods relying on
solid phase extraction technology lack sensitivity or require derivatization
of catecholamine by hazardous reagents prior to tandem mass spectrometry
(MS) analysis. Here, we manufactured a novel 96-well microplate device
specifically designed to extract plasma catecholamines prior to their
quantification by a new and highly sensitive ultraperformance liquid
chromatography–tandem mass spectrometry (UPLC–MS/MS)
method. Processing time, which included sample purification on activated
aluminum oxide and elution, is less than 1 h per 96-well microplate.
The UPLC–MS/MS analysis run time is 2.0 min per sample. This
UPLC–MS/MS method does not require a derivatization step, reduces
the turnaround time by 10-fold compared to conventional methods used
for routine application, and allows catecholamine quantification in
reduced plasma sample volumes (50–250 μL, e.g., from
children and mice)
DataSheet_1_Low number of neurosecretory vesicles in neuroblastoma impairs massive catecholamine release and prevents hypertension.pdf
IntroductionNeuroblastoma (NB) is a pediatric cancer of the developing sympathetic nervous system. It produces and releases metanephrines, which are used as biomarkers for diagnosis in plasma and urine. However, plasma catecholamine concentrations remain generally normal in children with NB. Thus, unlike pheochromocytoma and paraganglioma (PHEO/PGL), two other non-epithelial neuroendocrine tumors, hypertension is not part of the usual clinical picture of patients with NB. This suggests that the mode of production and secretion of catecholamines and metanephrines in NB is different from that in PHEO/PGL, but little is known about these discrepancies. Here we aim to provide a detailed comparison of the biosynthesis, metabolism and storage of catecholamines and metanephrines between patients with NB and PHEO.MethodCatecholamines and metanephrines were quantified in NB and PHEO/PGL patients from plasma and tumor tissues by ultra-high pressure liquid chromatography tandem mass spectrometry. Electron microscopy was used to quantify neurosecretory vesicles within cells derived from PHEO tumor biopsies, NB-PDX and NB cell lines. Chromaffin markers were detected by qPCR, IHC and/or immunoblotting.ResultsPlasma levels of metanephrines were comparable between NB and PHEO patients, while catecholamines were 3.5-fold lower in NB vs PHEO affected individuals. However, we observed that intratumoral concentrations of metanephrines and catecholamines measured in NB were several orders of magnitude lower than in PHEO. Cellular and molecular analyses revealed that NB cell lines, primary cells dissociated from human tumor biopsies as well as cells from patient-derived xenograft tumors (NB-PDX) stored a very low amount of intracellular catecholamines, and contained only rare neurosecretory vesicles relative to PHEO cells. In addition, primary NB expressed reduced levels of numerous chromaffin markers, as compared to PHEO/PGL, except catechol O-methyltransferase and monoamine oxidase A. Furthermore, functional assays through induction of chromaffin differentiation of the IMR32 NB cell line with Bt2cAMP led to an increase of neurosecretory vesicles able to secrete catecholamines after KCl or nicotine stimulation.ConclusionThe low amount of neurosecretory vesicles in NB cytoplasm prevents catecholamine storage and lead to their rapid transformation by catechol O-methyltransferase into metanephrines that diffuse in blood. Hence, in contrast to PHEO/PGL, catecholamines are not secreted massively in the blood, which explains why systemic hypertension is not observed in most patients with NB.</p
mRNA and protein quantification of TH, DBH and PNMT in PGL <i>vs</i>. PHEO.
<p>(A) Whisker-box plot of mRNA relative ratio from 11 PGL <i>vs</i>. 50 PHEO (reference). TH: fold change to reference (FC) 0.38, Standard error (SE) 0.08–1.94, 95% confidence Interval (CI) 0.003–13.37; PNMT: FC 0.095, SE 0.003–2.35, CI 0–74.06, DBH: not statistically significant (ns). Reporter genes included the following: EEFIA1, GAPDH and TBP. ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05. (B) Quantification of the signal obtained by western blot for 35 PHEO (filled circles) and 12 PGL (open circles). Results are given as QL (corresponding band pixel values)-BG (background pixel value), and calculated values were divided by the β-actin signal value and reported on the y-axis. Geographic mean and standard deviation for each group are reported in supporting information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125426#pone.0125426.s003" target="_blank">S3 Table</a>). ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05, ns; not statistically significant.</p
TH, DBH and PNMT protein expression in tumors.
<p>(A) Western blot of sample tissues from NorAd PHEO (A, n = 14), mixed PHEO (B, n = 21) and PGL (n = 12). Volumes loaded were 10μl of a 20% w/v extract. E and NE values corresponding to the samples analysed by western blot are given in nanomoles/gram of tissue, lower quantification limit was set at 1nmole/gram. (B) Quantification of the signal obtained by western blot. Results are given as QL (corresponding band pixel values)-BG (background pixel value), and calculated values are divided by the β-actin signal value and reported on the y-axis. Geographic mean and standard deviation for each group are reported in supporting information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125426#pone.0125426.s006" target="_blank">S6 Table</a>). ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05, ns; not statistically significant.</p
Dexamethasone effects on primary tumor cells.
<p>(A) Whisker-box plot of PNMT mRNA relative ratio from PGL cells (tumors P16, P30, P35 and P42) incubated with <i>vs</i>. without dexamethasone 1μM for 24 hrs (reference). Reporter genes included the following: EEFIA1, GAPDH and TBP, ns; not statistically different. (B) Whisker-box plot of TH, DBH and PNMT mRNA relative ratio from PHEO cells (tumors P23, P36, P44, P45, P52, P53, P56, P61 and P62) incubated with <i>vs</i>. without dexamethasone 1μM for 24 hrs (reference). TH: FC 2.8, SE 1.28–7.55, CI 0.68–17.38, reporter genes included the following: EEFIA1, GAPDH and TBP. ** <i>P</i><0.01. (C) Western blot of PHEO cell incubated with (+) or without (-) dexamethasone (Dexa.) 1μM for 24 hrs.</p
Flowchart of the patients and tumor samples included in the study.
<p>For CAT and MNs quantification in tissue 63 patients were enrolled and 66 tumors were collected (two patients had bilateral tumors (P21 and P43) and one had both a PHEO and a PGL (P25)*, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125426#pone.0125426.t001" target="_blank">Table 1</a>. Tumors consisted of 53 PHEO and 13 PGL. Quantification of CAT in plasma was performed on 72 patients from 50 PHEO and 23 PGL patients, (including P25 with both a PHEO and a PGL). The plasma CAT analysis was performed on the same patient recruited for tissue CAT and MNs study with the following exceptions: two plasma were not available (P14 and P27) and two tumors were bilaterally excised at the same time (P43 and P44), finally, 10 additional plasma from PGL patients were added in the study without tissue sample available. For MNs quantification in plasma, sample P62 was not available (instead of P27 for CAT). Three tissue samples for PHEO and two for PGL were stored in perchloric acid, not compatible for mRNA extraction for qPCR assay**. 15 PHEO tissue sample were no longer available for WB assays***.</p
mRNA and protein quantification of TH, DBH and PNMT in PGL <i>vs</i>. mixed and NorAD PHEO.
<p>(A) Whisker-box plot of mRNA relative ratio from PGL (n = 11) <i>vs</i>. mixed PHEO (n = 28) (reference). TH: reaction efficiency (RE) 0.99, fold change to reference (FC) 0.29, Standard error (SE) 0.04–1.65, 95% confidence Interval (CI) 0.003–48.29; DBH: not statistically significant (ns); PNMT: RE 0.98, FC 0.024, SE 0.001–0.41, CI 0–4.82. Reporter genes included the following: EEFIA1, RE 0.99; GAPDH, RE 1.04, TBP, RE 0.97. ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05, (B) Ratio from PGL (n = 11) <i>vs</i>. NorAd PHEO (n = 22) (reference). (C) Ratio from NorAd PHEO (n = 22) <i>vs</i>. mixed PHEO (n = 28) (reference) PNMT: FC 0.07, SE 0.005–0.93, CI 0.001–10.99.</p
PNMT expression in NorAd PHEO <i>vs</i>. mixed tumors.
<p>(A) Whisker-box plot of PNMT mRNA relative ratio from NorAd tumors (including PHEO and PGL, n = 29) <i>vs</i>. mixed tumor (PHEO and PGL, n = 32, reference). Fold change to reference (FC) 0.06, Standard error (SE) 0.003–0.29, 95% confidence Interval (CI) 0–22.36; Reporter genes included the following: EEFIA1, GAPDH and TBP. (B) Quantification of the signal obtained by western blot in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125426#pone.0125426.g006" target="_blank">Fig 6</a> for PNMT signal in mixed and NorAd tumor (independently of PHEO or PGL). Results are given as QL (corresponding band pixel values)-BG (background pixel value) and calculated value is divided by the β-actin signal value and reported on the y-axis. Geo. mean 0.64 <i>vs</i>. 0.18, standard deviation: 0.7 and 0.14 for respectively mixed and NorAd tumor.</p
Quantification of E, NE, MN and NMN in plasma and tumor tissue of PGL, mixed and NorAd tumors.
<p>(A) Quantification of epinephrine (E), norepinephrine (NE), total CAT (E+NE) and NE/E ratio from 30 mixed PHEO (Mix, open diamonds); 23 noradrenergic PHEO (NorAd, filled diamonds) and 13 PGL (open circles) samples. Results are given in nanomoles per gram of tissue and represented on a logarithmic scale. Geographic mean and standard deviation for each group are reported in supporting information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125426#pone.0125426.s004" target="_blank">S4 Table</a>). (B) Same as in A, quantification of CAT in plasma for 28 mixed PHEO, 22 NorAd PHEO and 23 PGL. (C) Quantification of MN and NMN sum of MNs (MN+NMN) and NMN/MN ratio from 30 mixed PHEO (Mix, open diamonds); 23 NorAd PHEO (filled diamonds) and 13 PGL (open circles) samples. (D) Same as in C, quantification of MNs in plasma for 28 mixed PHEO, 22 NorAd PHEO and 23 PGL. ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05, ns: not statistically significant. For CAT and MNs in tumor tissue, minimal values were set at 0.1 nmol/g representing limit of quantification.</p
Patients and tumors characteristics.
<p>Localisation: PHEO were found either in the left (L) or right (R) adrenal gland. PGL were excised from the following location: abd: abdominal; 1: paraortal right; 2: paraortal left; 3: Glomus vagal right; 4: retroperitonal left, paraortal; 5: abdominal, paraortal. NT: non-tested, Sp*: sporadic, RET, SDHB, -D and VHL negative for mutation</p><p>Sp**: RET and SDHB negative. SP<sup>1</sup>: SDHA, -B, -C, -D, MAX, VHL negative. SP<sup>2</sup>: SDHB, -D, RET, VHL, Men1 negative, SP<sup>3</sup>: SDHB, -C, -D, RET, negative, SP<sup>4</sup>: VHL negative, SP<sup>5</sup>: SDHB, -C, -D, Men1, RET negative. For CAT and metabolites in plasma, upper reference limits are (in nmol/l); NE (norepinephrine): 6.55, E (epinephrine): 1.23, free NMN (normetanephrine): 1.39, free MN (metanephrine): 0.85, P65 was later shown not to be a PHEO and do not appear in this table</p><p>Patients and tumors characteristics.</p
