Search

This study aimed to confirm the independent risk factors for recurrence-free survival (RFS) in intermediate and high-risk neuroblastoma (NB) patients and set up an effective nomogram model for predicting the recurrence of NB. A total of 212 children with intermediate- and high-risk neuroblastoma, who had ever achieved complete remission (CR) or very good partial remission (VGPR) after standardized treatment in this hospital, were chosen as study objects. After retrospective analysis of the clinical data, Cox regression model was used to explore the factors related to the recurrence of neuroblastoma, to determine the variables to construct the Nomogram. The consistency index would predict the accuracy of this nomogram. RFS rate in 1-year, 3-year, 5-year, and 10-year was 0.811, 0.662, 0.639, and 0.604, respectively. Children with MYCN amplification had a higher neuron-specific enolase (NSE) value (P = 0.031) at the initial diagnosis than MYCN non-amplification. The univariate analysis predicted that increased vanillylmandelic acid (VMA) and NSE value and dehydrogenase (LDH) 1000 U/L were important adverse factors for the recurrence of NB. Multivariate analysis demonstrated that age at diagnosis, tumor localization, MYCN state, histologic subtype, and tumor capsule were significantly associated with RFS (all P values 0.05). Nomograms were established for predicting the recurrence of NB according to the Cox regression analysis. Internal verification by the Bootstrap method showed that the prediction of the nomogram's consistency index (C-index) was 0.824 (P = 0.023). Conclusion: Age at diagnosis, tumor localization, MYCN state, histologic category, and tumor capsule were independent risk factors for the recurrence of NB. The nomogram model could accurately predict the recurrence of children with neuroblastoma. What is Known: • The prognoses of neuroblastoma (NB) could vary greatly due to the high heterogeneity, the 5-year survival rate of low-risk NB exceeded 90%, while the 5-year survival rate of children in the intermediate and high-risk groups was not satisfactory.. What is New: • Increased vanillylmandelic acid (VMA) and neuron-specific enolase (NSE) value, and lactate dehydrogenase (LDH)1000U/L were important adverse factors for the recurrence of NB. • NSE value was more valuable for predicting NB recurrence.

Manjak aktivnosti dekarboksilaze aromatskih L-aminokiselina rijedak je autosomno-recesivni poremećaj koji dovodi do teškog deficita kateholamina i serotonina. Klinička slika uključuje različite stupnjeve neuroloških oštećenja, uključujući motoričke i neuromotoričke simptome. Uspješnost liječenja ovisi o pravovremenom postavljanju dijagnoze i početku terapije. Postavljanje dijagnoze manjka aktivnosti AADC temelji se na određivanju koncentracije neurotransmitera u likvoru i molekularnoj dijagnostici zbog čega se bolesnici izlažu invazivnom postupku uzimanja uzoraka za analizu. Vanillaktat (VLA), vanilmandelična kiselina (VMA) i njihov omjer VLA/VMA mogu se analizirati u sklopu rutinske analize organskih kiselina u urinu i smatraju se potencijalnim neinvazivnim biokemijskim biljezima u postavljanju dijagnoze AADC. Cilj ovog diplomskog rada bio je odrediti referentne intervale za VLA, VMA i njihova omjera VLA/VMA u urinu retrospektivnom analizom organskih kiselina bolesnika suspektnih na neurometaboličke poremećaje. 219 obrađenih bolesnika podijeljeno je u tri dobne skupine, a uzorci su analizirani inhouse metodom ekstrakcije organskih kiselina na sustavu za plinsku kromatografiju s masenom spektrometrijom. Kao dodatni marker korišten je N-acetiltirozin. Određivanje referentnih intervala VLA, VMA i VLA/VMA u urinu u sklopu rutinske analize organskih kiselina može pomoći u diferencijalnoj dijagnostici neurometaboličkih bolesti. Aromatic L-amino acid decarboxylase deficiency is rare autosomal recessive disorder that leads to severe deficit of catecholamine and serotonin. The clinical picture includes varying degree of neurological damage, including motor and neuromotor symptoms. Treatment outcomes depends on the timepoint when therapy started. Key diagnostic test for AADC confirmation are measurement neurotransmiters in cerebrospinal fluid and genetic testing. However, to perform this testing, patients are exposed to intensive lumbar puncture procedure. Determination of vanillactic acid (VLA), vanillylmandelic acid (VMA) and VLA/VMA ratio as part of organic acid analysis in urine is considered a reliable approach using non-invasive biomarkers in the diagnosis of AADC. The aim of this thesis was to determine reference intervals of VLA, VMA and VLA/VMA ratio in urine. Retrospective analysis of urinary organic acids obtained from 219 patients suspected for neurometabolic disorders were included in this study. Patients were divided into groups depending on age. Organic acids in urine were measured using in-house method by gas-chromatography mass spectrometry. As additional biomarker, N-acetyltyrosine was used. Determination of reference intervals of VLA, VMA and VLA/VMA ratio as a part of organic acids analysis in urine may help to differentiate neurometabolic patients with possible AADC deficiency from other neurometabolic disorders.

In 2004, the Japanese government halted nationwide mass screening for neuroblastoma in 6-month-old infants as it led to overdiagnosis of localized tumors with favorable prognoses and failed to reduce neuroblastoma-related mortality. However, a new mass screening program for neuroblastoma in 18-month-old infants (18MS) was conducted in the Osaka prefecture. We assessed the efficacy of the 18MS in screening unfavorable cases. Public health centers in Osaka prefecture, excluding the Osaka city area, provided test kits to the guardians of infants who received a check-up at 18 months of age between 2004 and 2017. For patients whose standardized urinary levels of vanillylmandelic acid or homovanillic acid exceeded the threshold, they were further examined and treated in two specific hospitals Osaka University Hospital and Osaka Women’s and Children’s Hospital. Screening-positive patients with and without neuroblastoma were retrospectively reviewed. Among 142,423 children screened during the 18MS, 85 tested positive, and 14 were diagnosed with neuroblastoma. Twelve patients were classified as very low risk, while 2 were classified as high risk, based on the International Neuroblastoma Risk Group risk classification. The 18MS did not screen unfavorable cases with neuroblastoma efficiently, although few participants benefited from it.

Neuroblastoma and other neural crest tumors can be characterized by the increased production and excretion of catecholamines and their metabolites. Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are important catecholamine metabolites that can be measured to provide relatively rapid laboratory diagnosis and clinical follow-up of neuroblastoma. We present a procedure to quantify HVA and VMA in urine samples which have been diluted to a creatinine concentration of 2 mg/dL. Diluted samples are spiked with deuterated internal standards, acidified, and extracted with an organic solvent. A bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) and pyridine mixture is added to the dried extract to create trimethylsilyl derivatives of HVA and VMA. The derivatized compounds are measured using gas chromatography-mass spectrometry (GC/MS).

Background Aromatic L-amino acid decarboxylase (AADC) deficiency is a primary neurotransmitter defect of the biosynthesis of catecholamines and serotonin. The phenotype consists of varying degrees of neurological impairment, including motor and non-motor symptoms. Treatment outcomes correlate with the time point of diagnosis and treatment initiation; therefore, reliable diagnostic markers are necessary. Increased vanillactic acid (VLA) concentrations in the analysis of organic acids in urine have been reported in AADC deficiency. However, this elevation is often subtle and easily missed. In this study, we evaluate the semi-quantitative determination of VLA and vanillylmandelic acid (VMA) concentrations and establish the ratio of a VLA/VMA as a novel diagnostic marker for AADC deficiency. Methods Urine samples obtained from 10,095 non-AADC deficient controls and 14 confirmed AADC deficient patients were used for organic acid analysis by liquid-liquid extraction of the acidified samples and gas chromatographic-mass spectrometric separation after trimethylsilylation. The semi-quantitative determination of VLA and VMA concentrations and the calculation of a VLA/VMA ratio were evaluated as a diagnostic marker for AADC deficiency. Results The mean VLA and VMA concentrations in 10,095 non-AADCD samples was 0.3 mmol/mol creatinine (SD = 1.18, range 0–57.79) and 5.59 mmol/mol creatinine (SD = 3.87, range 0.04–60.62), respectively. The mean concentration of VLA in 14 patient-derived samples was 10.24 mmol/mol creatinine, (SD = 11.58, range = 0.37–33.06) and 0.45 mmol/mol creatinine for VMA (SD = 0.29, range 0.11–1.27). The mean VLA/VMA ratio in non-AADC controls was 0.07 (SD = 0.37, range 0.0–23.24), whereas AADC deficient patients revealed a mean VLA/VMA ratio of 23.16 (SD = 22.83, range 0.97–74.1). The VLA/VMA ratio thus allows a reliable identification of patients with AADC deficiency, especially in the young age cohort as it decreases with age. To take this into account, age-adjusted thresholds have been developed. Conclusion Determination of individual concentrations of VLA and VMA in urine does not allow a reliable diagnosis of AADC deficiency. In this study, we could demonstrate that a semi-quantitative analysis of organic acids in urine allows the formation of metabolite ratios and that the VLA/VMA ratio is a reliable, easily accessible, new parameter for the diagnosis of AADC deficiency.

The GC-MS quantification of biogenic monoamines (BMAs), together with their acidic metabolites (ACMEs), in a single step, is presented here for the first time. This novel principle is based on the exceptional reactivity of the hexamethyldisilazane (HMDS) and perfluorocarboxylic acid (PFCA) couples [1,2], resulting in the simultaneous trimethylsilylation and acylation of BMAs and ACMEs. For this basic study, tyramine (TYR), 3-methoxytyramine (3-MeTYR), dopamine (DA), epinephrine (EP), normetanephrine (NORMNE), norepinephrine (NOREP), tryptamine (T), 3,4-dihydroxyphenylalanine (L-DOPA), 5-methoxytryptamine (5-MeT), serotonin (ST), and their ACMEs, such as homovanillic acid (HVA), vanillylmandelic acid (VMA), and 5-hydroxyindoleacetic acid (5-HIAA) were selected. These three ACMEs were derived from 3-MeTYR, NORMNE and ST, respectively. The mass fragmentation properties of the fully derivatized products proved to be of stoichiometric distribution. Informative high masses were obtained: such as the molecular ions [ M ] + = and/or their [M-CH3]+ alternatives. The exceptions were EP and NOREP which decomposed to the same specific, abundant mass of m/z 355 representing the C7H3-tri-OTMS ions formed by the loss of their nitrogen-containing moieties. The general rule of this new principle was confirmed by using trifluoroacetic acid (TFA), pentafluoropropionic acid (PFPA), or heptafluorobutyric acid (HFBA) with HMDS in parallel tests. In all three cases, derivatives of close retention properties in a stoichiometric manner were obtained. On the basis of the optimum separation characteristics between the BMA-ACME pairs, the HMDS & PFPA couple was preferred as the reagent of choice. Method validation was carried out, both with model solutions and in the presence of the urine matrices (without any preliminary extraction). Analytical performance characteristics for the model solutions like repeatability (RSD% 3.88–6.4), linearity (R2 0.991–0.999) and limit of quantitation (LOQ 8.8–103 ng/mL) were determined. Analytical performance characteristics for urine matrices were calculated by using the standard addition method applying the urine of a healthy volunteer and also analyzing urines of patients diagnosed with neurological diseases.

A new method for the simultaneous determination of two tumour biomarkers, homovanillic (HVA) and vanillylmandelic acid (VMA), using flow injection analysis (FIA) with amperometric detection (AD) at a commercially available boron doped diamond electrode (BDDE) was developed. It was found that this method is suitable for the determination of HVA (in the presence of VMA) and VMA (in the presence of HVA) in optimum medium of Britton-Robinson buffer (0.04 mol L−1, pH 3.0). Calibration dependences consist of two linear parts for both biomarkers, the first one being in the concentration range from 1 to 10 μmol L−1 and the second one from 10 to 100 μmol L−1 (with obtained LODs 0.44 μmol L−1 for HVA and 0.34 μmol L−1 for VMA, respectively). To minimize any negative effects related to the passivation of the working electrode, suitable cleaning pulses (+2.4 V for 30 s) were imposed on the working electrode after each measurement. An attempt to use FIA with multiple pulse amperometric detection to determine both analytes in one run was not successful. Changing potentials in short intervals in multiple pulse detection probably results in mutual interaction of analytes and/or products of their electrochemical oxidation, thus preventing the application of this approach.

BACKGROUND Paediatric neuroblastoma is a relatively common type of malignant tumour originating from neural crest tissues. Early diagnosis and the performance of specific therapeutic strategies can increase the survival rate and improve the prognosis of children with neuroblastoma. METHODS A total of 86 children with neuroblastoma were recruited in this research, and 50 healthy children aged 1-12 years were also selected as controls. Twenty-four-hour urine vanillylmandelic acid (VMA) was evaluated by high-performance liquid chromatography. Serum carbohydrate antigen 125 CA125 and neuron-specific enolase (NSE) levels were evaluated by electrochemiluminescence in Cobas E411 autoanalyser. RESULTS The serum CA125, NSE and 24-hour urine VMA levels of children with stage III-IVs neuroblastoma were significantly higher than those of children with clinical stages I-II; the serum CA125, NSE and 24-hour urine VMA levels of children in the effective treatment group were significantly lower than those in the treatment-ineffective group. The serum CA125 generated sensitivity and specificity of 71.88% and 59.26%, combined with an AUC (area under the curve) of 0.7049. The serum NSE generated sensitivity and specificity of 68.75% and 81.48%, combined with an AUC of 0.7407. The 24-hour urine VMA generated sensitivity and specificity of 90.63% and 59.26%, combined with an AUC of 0.7986. CONCLUSION In conclusion, serum CA125, NSE and 24-hour urine VMA levels before treatment could assess the condition of children with neuroblastoma and predict the effect of treatment.

Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are end-stage metabolites of catecholamine and are clinical biomarkers for the diagnosis of neuroblastoma. For the first time in Korea, we implemented and validated a liquid chromatography tandem mass spectrometry (LC–MS/MS) assay to measure urinary concentrations of HVA and VMA according to Clinical and Laboratory Standards Institute guidelines. Our LC–MS/MS assay with minimal sample preparation was validated for linearity, lower limit of detection (LOD), lower limit of quantification (LLOQ), precision, accuracy, extraction recovery, carryover, matrix effect, and method comparison. A total of 1209 measurements was performed to measure HVA and VMA in spot urine between October 2019 and September 2020. The relationship between the two urinary markers, HVA and VMA, was analyzed and exhibited high agreement (89.1% agreement, kappa’s k = 0.6) and a strong correlation (Pearson’s r = 0.73). To our knowledge, this is the first study to utilize LC–MS/MS for simultaneous quantitation of spot urinary HVA and VMA and analyze the clinical application of both markers on a large scale for neuroblastoma patients.

Background Neuroblastoma screening aims to reduce neuroblastoma-related mortality. A controlled trial showed no reduction in stage 4 disease incidence and preliminary mortality data. This article presents epidemiologic and clinical data 20 years after cessation of the screening program. Methods The patients with detected disease in the screening area were compared with the clinically diagnosed patients in the control area and in the prestudy and poststudy cohorts. All statistical tests were 2-sided. Results The cumulative incidence for children aged 1 to 6 years in the birth study cohorts (1994-1999) in the screening arm was 13.4 cases per 100 000 births (95% confidence interval [CI] = 12.2 to 14.6) based on 61.2% of screening participants and 38.8% of nonparticipants. Screening participants had a cumulative incidence of 15.7 (95% CI = 14.0 to 17.4) per 100 000 births. The cumulative incidence in the contemporary control cohort was 9.3 (95% CI = 8.2 to 10.3) per 100 000 births, 7.6 (95% CI = 6.8 to 8.4) in the prestudy cohort, and 8.1 (95% CI = 7.4 to 8.9) in the poststudy cohort from 2000 to 2004 (P Conclusions Neuroblastoma screening at 1 year of age reduced neither stage 4 incidence nor neuroblastoma mortality and was affected by overdiagnosis, leading to unnecessary treatment. A few screening-detected stage 4 cases represent a biologically interesting subgroup but do not change the recommendation to close the “catecholamine-based neuroblastoma screening book.”

Introduction Pheochromocytoma and paraganglioma are uncommon tumours whose best known symptoms include high blood pressure, palpitations, headache, and sweating. Clinical identification is not easy, however, and requires biochemical tests that allow for early diagnosis, including measurement of metanephrines levels. The aim of this study was to assess the diagnostic performance of plasma free metanephrines (PMETs) and to verify the transferability of the reference values used. Methods PMETs levels were measured by liquid chromatography coupled to tandem mass spectrometry. Other biochemical tests evaluated (plasma catecholamine, urine metanephrine, catecholamine and vanilmandelic acid levels) were performed by liquid chromatography with electrochemical detection. Requests of these tests from 01/09/2015 to 31/10/2017 were reviewed, and both the reference values (document EP28-A3c) and the parameters of biological variation (Fraser method) for PMETs were estimated. Results The study sample consisted of 1279 patients (61.3% females) aged 0–90 years, including 19 with pheochromocytoma/paraganglioma. Tests requested included: PMETs (n = 662), catecholamines (n = 589), metanephrines (n = 586), and vanilmandelic acid (n = 513) in urine, and plasma catecholamines (n = 228). Tests with higher sensitivity were urinary fractionated metanephrines (91.7%) and PMETs (82.4%). When performance was compared in patients with both tests (n = 243), they detected the same number of tumours (90.9%), but PMETs showed greater specificity (93.5% vs. 88.8%). Plasma normetanephrine levels showed a significant association with age (rho = 0.19, p Conclusion PMETs and urinary fractionated metanephrines are the biochemical tests with better performance in diagnosis of pheochromocytomas/paragangliomas.

Neuroblastoma (NB) is the only pediatric tumor that is screened for nationwide by detecting the urinary levels of homovanillic acid and/or vanillylmandelic acid; however, whether NB screening reduces the mortality rate has not been established. This review compared the incidence and mortality rates among data from international mass screening for NB, as well as an analysis of differences in age of screening, detection methods, and diagnostic biomarkers. A well-designed trial exploring possible benefits and hazards is warranted prior to resuming mass screening for NB.

Pheochromocytoma diagnosis in dogs is challenging because biochemical tests are not always available. In humans, urinary vanillylmandelic acid (VMA) is part of a pheochromocytoma biochemical diagnostic profile, whereas its diagnostic accuracy is currently unknown in dogs with pheochromocytoma. Prospectively, VMA was determined by HPLC and expressed as the ratio with respect to urinary creatinine (VMA:C). The diagnostic accuracy of the VMA:C ratio was evaluated by analyzing the receiver operating characteristic (ROC) curve in 10 healthy dogs, 8 dogs with pituitary-dependent hypercortisolism, 8 dogs with adrenal-dependent hypercortisolism, and 7 dogs with pheochromocytoma. The pheochromocytoma diagnosis was confirmed by histology and immunohistochemistry in all tumors. The VMA:C ratio was significantly higher in dogs with pheochromocytoma (158 [53.4 to 230.8] × 10-3) than in dogs with adrenal-dependent hypercortisolism (48.1 [24.3 to 144.9] × 10-3; P 58.2 × 10-3 for pheochromocytoma diagnosis, a sensitivity of 85.7% and a specificity of 88.4% were obtained. Nevertheless, when using a cut-off ratio of 4 times the median VMA:C ratio determined in healthy dogs, there was no overlap (100% specificity). The area under the ROC curve indicated that the VMA:C ratio test could be used to discriminate between dogs with and without pheochromocytoma, what leads to the conclusion that it is useful for pheochromocytoma diagnosis in dogs.

A novel method towards spectrophotometric determination of catecholamines and their metabolites differing in their functional groups has been developed. This method is based on a change in morphology of silver triangular nanoplates upon the action of cateсholamines and their metabolites, which is manifested by the decrease of the nanoparticle local surface plasmon resonance (LSPR) band intensity or its shift to the short-wavelength region of the spectrum. The shift value of the LSPR band or the change of its intensity increases with increasing concentration of catecholamines or their metabolites, which is proposed for their spectrophotometric determination. The limits of detection of catecholamines and their metabolites under selected conditions increase in the series homovanillic acid

Detecting circulating tumor cells (CTCs) has proven valuable for evaluating the prognosis of cancer patients and for studying the mechanisms of treatment resistance. Owing to the lack of universal and specific tumor markers for neuroblastoma (NB), in this prospective study, we adopted an EpCAM-independent method to detect CTCs in the peripheral blood of NB patients. We used an EpCAM-independent assay to delete leukocytes and to enrich the CTCs. CTCs were identified by immunostaining of CD45, DAPI and DNA fluorescence in situ hybridization (FISH) of the centromere of chromosome 8 probe (CEP8). Cells that were DAPI+/CD45-/CEP8 ≥ 3 were considered CTCs. We collected peripheral blood from 28 NB patients as well as clinical and follow-up data. The number of CTCs among the different risk groups were significantly different (p = .0208, Kruskal-Wallis test). Patients with metastasis had more CTCs than those without metastasis (p .0001, Mann-Whitney test). Patients with ≥3 CTCs per 4 ml of peripheral blood had an increased likelihood of having metastasis (sensitivity, 88.89%; specificity, 78.59%), and patients with ≥10 CTCs per 4 ml of peripheral blood had poorer overall survival. The EpCAM-independent assay along with immunostaining-FISH (i-FISH) described here can detect CTCs in patients with NB at a high sensitivity and may have clinical value for prognosis evaluation and diagnosing metastasis when imaging data are ambiguous.

Background/purpose The prognosis of patients with intermediate-risk neuroblastoma is favorable; therefore, a reduction therapy is desired. However, the long-term prognosis of those with residual tumor is unclear. The aim of this study was to clarify the necessity of residual tumor resection. Methods We retrospectively reviewed the records of patients diagnosed with intermediate-risk neuroblastoma who either were treated by chemotherapy only (nonresection group; n=16), or received postchemotherapy tumor resection (resection group; n=9). Results In the nonresection group, tumor size decreased in 14 patients; 5 had no detectable local tumor at the end of the follow-up period. Tumor size increased in 2 patients 1.5–2.5years postchemotherapy. Both patients received additional treatment and survived. All patients survived during the median follow-up time of 127months. In the resection group, 5 patients received complete resections and 4 patients received nearly complete resections. All patients survived during the median follow-up time of 84months. In 8 out of 9 resected tumors, regression or maturation was pathologically induced by chemotherapy-only treatment. Conclusion Patients with intermediate-risk neuroblastoma with or without postchemotherapy residual tumor resection had an excellent long-term outcome. The tumor pathology with intermediate-risk neuroblastoma might be susceptible to change to regression or maturation by chemotherapy. Level of evidence IV

Background Ganglioneuroblastoma, nodular is defined as a composite tumor of biologically distinct clones. The peripheral neuroblastic tumors in this category are characterized by the presence of grossly visible neuroblastoma nodules coexisting with ganglioneuroblastoma, intermixed, or with ganglioneuroma. Making a correct diagnosis of ganglioneuroblastoma, nodular is often difficult by biopsy or partial tumor resection, because the neuroblastic nodule could be hidden and not sampled for pathological examination. Case presentation We report a case of a Japanese boy aged 3 years, 8 months, with an unresectable abdominal tumor and elevated vanillylmandelic acid and homovanillic acid levels. The initial biopsy was ganglioneuroma. However, after the second biopsy from a hidden neuroblastoma nodule that was clearly highlighted by fluorodeoxyglucose positron emission tomography/computed tomography, we reached the diagnosis of ganglioneuroblastoma, nodular. Because the nodule demonstrated neuroblastoma, differentiating subtype, with a low mitosis-karyorrhexis index (favorable histology) and nonamplified MYCN, the boy was treated according to the intermediate-risk protocol and is now alive and well 4 years after the diagnosis. Conclusions This case illustrates the critical role of fluorodeoxyglucose positron emission tomography/computed tomography for detecting a neuroblastoma nodule in a ganglioneuroblastoma.

Background & objectives: Vitiligo is an acquired skin disease characterized by depigmented areas of the skin. Increased release of catecholamines from autonomic nerve endings in microenvironment of melanocytes in affected skin might be involved in the aetiopathogenesis of vitiligo. Levels of catecholamines are considered as being related to onset or worsening of the disease. Therefore, in this study, the role of catecholamines was evaluated in mapping disease stability and outcome of vitiligo patients undergoing melanocyte transfer. Methods: In this study, circulatory and urinary levels of catecholamine (CA) and vanillylmandelic acid (VMA) were determined in 45 individuals (30 vitiligo patients and 15 healthy controls) using ELISA. Results: A significant increase for plasma and urinary catecholamines along with VMA was observed as compared to healthy controls. When the pre- and post-intervention levels were analyzed in responders and non-responders, respectively, only dopamine showed significant decline in urine, rest of the molecules in plasma as well as urine showed non-significant decline except VMA which showed insignificant increase. Interpretation & conclusions: Levels of plasma/urinary epinephrine, and plasma dopamine, could not be established as biomarkers for disease stability or successful outcome of autologous melanocyte transfer in generalized vitiligo patients. However, dopamine (urine) might be of help in determining the stability in patients with generalized vitiligo undergoing melanocyte transfer. Further studies need to be done on a large sample of patients to confirm our findings.

Chronic kidney disease (CKD) results in decreased bone strength. Serotonin (5-HT) is one of the critical regulators of bone health, fulfilling distinct functions depending on its synthesis site: brain-derived serotonin (BDS) favors osteoblast proliferation, whereas gut-derived serotonin (GDS) inhibits it. We assessed the role of BDS and peripheral leptin in the regulation of bone metabolism and strength in young rats with 5/6 nephrectomy. BDS synthesis was accelerated during CKD progression. Decreased peripheral leptin in CKD rats was inversely related to BDS content in the hypothalamus, brainstem and frontal cortex. Serotonin in these brain regions affected bone strength and metabolism in the studied animals. The direct effect of circulating leptin on bone was not shown in uremia. At the molecular level, there was an inverse association between elevated GDS and the expression of cAMP responsive element-binding protein (Creb) gene in bone of CKD animals. In contrast, increased expression of activating transcription factor 4 (Atf4) was shown, which was associated with GDS-dependent transcription factor 1 (Foxo1), clock gene - Cry-1, cell cycle genes: c-Myc, cyclins, and osteoblast differentiation genes. These results identified a previously unknown molecular pathway, by which elevated GDS can shift in Foxo1 target genes from Creb to Atf4-dependent response, disrupting the leptin-BDS - dependent gene pathway in the bone of uremic rats. Thus, in the condition of CKD the effect of BDS and GDS on bone metabolism and strength can't be distinguished.

Background: Suitable biomarkers that have prognostic values are one of the key points of interest in ischaemic stroke. Increased sympathetic nervous system activity in ischaemic stroke causes multiple local and systemic effects that can be detrimental to the outcome. The mechanism of action is increased secretion and activity of catecholamines, whose end metabolic products are vanillylmandelic acid and homovanilic acid. Aim of our study was to determine whether these compounds can be used as potential prognostic biomarkers in ischaemic stroke, as a unique insight into the activity of the sympathetic nervous system. Methods: Urine samples of 96 patients with ischaemic stroke and transitory ischaemic attacks were analysed. Values of vanillylmandelic and homovanillic acids in urine were tested using liquid chromatography on the first and third day post-stroke. Severity of stroke was determined using the NIHSS scale, while functional outcome was determined using the Modified Rankin Scale. Results: Values of vanillylmandelic and homovanillic acids positively correlated with functional outcome of ischaemic stroke. Favorable outcomes correlated with decreased values, on contrary to increased values, which were associated with unfavourable outcomes. Conclusion: Determining the values of these compounds in the urine is an easily available prognostic tool for the ischaemic stroke outcome, while also influencing potential therapeutic changes

Background Neuroblastomas are pediatric tumors characterized by overproduction of catecholamines. The catecholamine metabolites, vanillylmandelic acid (VMA) and homovanillic acid (HVA), are used in clinical evaluation of neuroblastoma. Tandem mass spectrometry (LC-MS/MS) is an effective analytical method for measurement of VMA and HVA in urine. Methods Dilute-and-shoot sample preparation was performed in a 96-well format using a liquid handler. Chromatographic separation was achieved using a reverse phase column; detection was accomplished by triple quadrupole mass spectrometry with electrospray ionization in positive mode. Data were acquired by multiple reaction monitoring. Two transitions, quantifier and qualifier, were monitored for each analyte and its stable isotope-labeled internal standard. Analytical specificity studies were performed. Results Injection-to-injection time was 4 min. The method was validated for linearity, limit of quantification, imprecision, accuracy, and interference. Linearity was 0.5–100 mg/l for both analytes. Within-run, between-day, and total imprecision were 1.0–4.1% for VMA and 0.8–3.8% for HVA. The method correlated well with our established HPLC method. Interferences precluding quantitation of VMA in 3% of specimens were reduced significantly (to 0.1% of specimens) using a modified LC gradient to reanalyze affected samples. Conclusions A simple, robust, economical, fast LC-MS/MS method was developed and validated for measurement of urinary VMA and HVA.

Wastewater-based epidemiology is a growing research field which provides valuable information on community drug use and chemical exposure. One parameter critical to estimations of drug use is the catchment area population. A population biomarker could be used to provide this information. This study evaluated the analytical suitability of three endogenous biomarkers of human activity: the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) which has previously been proposed and two further candidates, the catecholamine metabolites vanillylmandelic acid (VMA) and homovanillic acid (HVA). An analytical method involving derivatization was developed and validated for two candidates, 5-HIAA and HVA by liquid chromatography - mass spectrometry. The best performance was obtained for VMA as the underivatized analyte. The derivatized extracts produced a 100 times better sensitivity. The three neurotransmitter metabolites were evaluated as population biomarkers in wastewater samples. All were stable in sample, not lost upon filtration and showed stable inter-day mass loads over seven days for a metropolitan wastewater treatment plant. When applied to a small community during a festival period, mass loads of both HVA and VMA reflected the increase in the catchment population, whilst 5-HIAA proved to be more variable. Refereed/Peer-reviewed

Methylphenidate is a psychostimulant used for the treatment of (ADHD) attention deficit hyperactivity syndrome in children and adults. After chronic administration it is known to produce behavioral disorders including anxiety. Previous studies demonstrated that co-administration of buspirone can reduce behavioral and cognitive adverse effects produced by methylphenidate. The aim of the present study is to measure the levels vanillylmandelic acid (VMA) excretion in urine following prolong administration of methylphenidate, buspirone and their combination. Samples of urine for the estimation of the urinary VMA excretion were collected from treated and control male Wistar rats. We found significant (P0.01) raised urinary VMA excretion in methylphenidate group however significant (P0.01) reduction in VMA levels were seen after buspirone co-administration. Excretion of VMA in urine would allow the monitoring of sympatho-adrenomedullary system activity. This study could be helpful to increase the clinical use of methylphenidate in the treatment of different disoders.

Background and objectives: Melanin, which has a confirmed role in melanoma cell behaviour, is formed in the process of melanogenesis and is synthesized from tryptophan, L-tyrosine and their metabolites. All these metabolites are easily detectable by chromatography in urine. Materials and Methods: Urine samples of 133 individuals (82 malignant melanoma patients and 51 healthy controls) were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC). The diagnosis of malignant melanoma was confirmed histologically. Results: Chromatograms of melanoma patients showed increased levels of 5,6-dihydroxyindole-2-carboxylic acid, vanilmandelic acid, homovanilic acid, tryptophan, 5-hydroxyindole-3-acetic acid, and indoxyl sulphate compared to healthy controls. Concentration of indoxyl sulphate, homovanilic acid and tryptophan were significantly increased even in the low clinical stage 0 of the disease (indoxyl sulphate, homovanilic acid and tryptophan in patients with clinical stage 0 vs. controls expressed as medium/ interquartile range in &micro, mol/mmol creatinine: 28.37/15.30 vs. 5.00/6.91, 47.97/33.08 vs. 7.33/21.25, and 16.38/15.98 vs. 3.46/6.22, respectively). Conclusions: HPLC detection of metabolites of L-tyrosine and tryptophan in the urine of melanoma patients may play a significant role in diagnostics as well as a therapeutic strategy of melanoma cancer.

BACKGROUND The need for an extensive evaluation for neuroblastoma in children with Horner syndrome is controversial. METHODS A retrospective study design was used. The cohort included 47 children with anisocoria who were diagnosed with Horner syndrome and 135 children with neuroblastoma evaluated at a pediatric medical center between 2007 and 2015. To detect neuroblastoma, patients with Horner syndrome underwent brain and cervical MRI, abdominal ultrasound, and/or measurement of urinary vanillylmandelic acid (VMA). The neuroblastoma group was evaluated for signs/symptoms of Horner syndrome at the time of diagnosis. RESULTS Seven patients with Horner syndrome were lost to follow-up, and the findings of the remaining 40 were categorized according to the age of the patient. Horner syndrome most frequently was idiopathic (58%), and in only 1 patient did the discovery of neuroblastoma precede the appearance of Horner syndrome. In the 21 patients aged 1-18 years, Horner syndrome was acquired in 15 patients and congenital in 6. The most common etiology was trauma (62%). Imaging was performed in 14 patients and VMA testing in 13. Neuroblastoma was diagnosed in 5 patients; in none was it related to Horner syndrome. In the 135 patients with neuroblastoma, most of the tumors were diagnosed at Stage 4 (60%) or Stage 3 (30%) with 53% originating in the abdomen. In one patient (0.74%) with signs/symptoms of Horner syndrome at diagnosis of neuroblastoma, the tumor had been identified prenatally and the diagnosis confirmed by imaging postnatally. CONCLUSIONS The absence of occult neuroblastoma in children with Horner syndrome and of signs/symptoms of Horner syndrome in the children diagnosed with neuroblastoma suggests that Horner syndrome might not be as frequent a cause of neuroblastoma as previously thought. We recommend that full investigation for neuroblastoma be reserved for suspicious cases associated with additional systemic signs or symptoms.

Exploring innovative technologies to precisely quantify biomolecules is crucial but remains a great challenge for disease diagnosis. Unfortunately, the humoral concentrations of most biotargets generally vary within rather limited scopes between normal and pathological states, while most literature-reported biosensors can detect large spans of targets concentrations, but are less sensitive to small concentration changes, which consequently make them mostly unsatisfactory or even unreliable in distinguishing positives from negatives. Herein, a novel strategy of precisely quantifying the small concentration changes of a certain biotarget by editing the dynamic ranges and sensitivities of a lanthanide-based metal-organic framework (Eu-ZnMOF) biosensor is reported. By elaborately tailoring the biosensor's structure and surface areas, the tunable Eu-ZnMOF is developed with remarkably enhanced response slope within the "optimized useful detection window," enabling it to serve as a powerful signal amplifier (87.2-fold increase) for discriminating the small concentration variation of urinary vanillylmandelic acid (an early pathological signature of pheochromocytoma) within only three times between healthy and diseased subjects. This study provides a facile approach to edit the biosensors' performances through structure engineering, and exhibits promising perspectives for future clinical application in the non-invasive and accurate diagnosis of severe diseases.

Vanillylmandelic acid (VMA) and homovanillic acid (HVA) are clinical biomarkers for diagnosis of neuroblastoma (NB), which commonly occurs in the childhood. Development and application of a robust LC-MS/MS method for fast determination of these biomarkers for optimal laboratory testing of NB is essential in clinical laboratories. In present study, we developed and validated a simple liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quick clinical testing of VMA and HVA for diagnosis of NB. The method was validated according to the current CLSI C62-A and FDA guidelines. The age-adjusted pediatric reference intervals and diagnostic performance were evaluated in both 24 h urine and random urine. Injection-to-injection time was 3.5 min. Inter- and intra-assay coefficients of variation (CVs) were ≤3.88%. The lower limit of quantification and the limit of detection were 0.50 and 0.25 μmol/L for both VMA and HVA. Recoveries of VMA and HVA were in the ranges of 85-109% and 86-100% with CVs ≤5.76%. This method was free from significant matrix effect, carryover and interference. The establishment of age-adjusted pediatric reference intervals by this LC-MS/MS method was favorable for the improvement in diagnostic performance, which was crucial for correct interpretation of test results from children in both 24 h and random urine.

Vanillylmandelic acid (VMA), an important metabolite of catecholamines that is routinely screened as tumor marker, was investigated by the various spectroscopic techniques (IR, Raman, UV-Vis, antioxidant decolorization assay and NMR). Structures optimized by the employment of five common functionals (M05-2X, M06-2X, B3LYP, CAM-B3LYP, B3LYP-D3) were compared with the crystallographic data. The M05-2X functional reproduced the most reliable experimental bond lengths and angles (correlation coefficient >0.999). The importance of intramolecular hydrogen bonds for structural stability was discussed and quantified by the NBO analysis. The most prominent bands in vibrational spectrum were analyzed and compared to the experimental data. The positions of the carbon and hydrogen atoms in NMR spectra were well reproduced. The differences in UV-Vis spectrum were investigated by adding the explicit solvent and by performing NBO and QTAIM analyses. The discrepancy in the two spectra of about 50 nm could be explained by the solvent effect on carboxyl group. The most probable antioxidant activity mechanism was discussed for VMA and its carboxylate anion. The Molecular Docking study with the C - reactive protein additionally proved that variety of functional groups present in VMA and its anion allowed strong hydrogen and hydrophobic interactions.

Background: Urinary vanillylmandelic acid (VMA) and homovanillic acid (HVA) are biomarkers for the diagnosis and follow-up of neuroblastoma, whereas urinary 5-hydroxyindoleacetic acid (5-HIAA) is used to assess a carcinoid tumor. These analytes are conventionally analyzed in a single run by chromatography (LC) coupled with electrochemical detection (LC-ECD) using commercial kits. A rapid dilute-and-shoot LC tandem mass spectrometry (LC-MS/MS) assay was validated in order to replace the LC-ECD method and therefore improve analytical specificity and throughput. Methods: Sample preparation was carried out by dilution of the urine sample with a solution containing the deuterated internal standards. The separation was achieved on an ultra-high pressure LC system with MS detection using a triple quadrupole mass spectrometer. The method was validated according to the current EMA and FDA guidelines. Results: The full chromatographic run was achieved in 8 min. The method validation showed excellent linearity (r2>0.999 for all three analytes), precision (CV 90%), low carryover ( Conclusions: The LC-MS/MS method proposed in this study is fast and robust, and the simple sample preparation saves time and avoids the additional costs of dedicated kits used for the LC-ECD assays by switching to LC-MS/MS. Additionally, the near-perfect correlation observed herein between both assays allows the previously established reference ranges to be maintained.

Background Urinary vanillylmandelic acid (VMA) is used to diagnose and monitor catecholamine secreting neuroendocrine tumors (NETs). We developed and validated a new liquid chromatography tandem mass spectrometry (LC–MS/MS) assay for determination of serum VMA. Methods We used serum samples from healthy volunteers (n = 314) and patients suspected for NET (n = 36). Deuterated VMA as an internal standard was added to samples before solid phase extraction (SPE) and LC–MS/MS analysis. We studied the effects of sample storage, sampling device and a meal on serum VMA and metanephrine concentrations. Diurnal variation and age-dependent reference intervals were established. The diagnostic performance was compared with a urinary HPLC assay for VMA and metanephrines and a serum metanephrine LC–MS/MS assay. Results Serum VMA is stable at least for one day at + 4 °C, seven days at room temperature and 98 days at − 20 °C. Type of sampling device was not critical, but elevated serum VMA occurs after a meal (p = 0.031). Serum VMA increased with age. Therefore, we suggest clinical cut-off values of 62 nmol/L, 80 nmol/L and 108 nmol/L for age groups 18–50 yrs, 51–70 yrs and > 70 yrs, respectively. Comparison between a urinary VMA HPLC assay and serum VMA LC–MS/MS assay showed good correlation. Conclusions Our LC–MS/MS assay is fast and sensitive and suits well for use in a clinical laboratory. Compared to 24-h urine collection our serum assay enables well controlled sampling and convenient preanalytical steps.

BACKGROUND The large animal model of arterial hypertension is very valuable to test the antihypertensive drugs and devices. We characterized a novel swine model of spontaneous hypertension and investigated its response to renal denervation (RDN). METHODS The blood pressure (BP), levels of plasma catecholamines and urine vanillylmandelic acid, and the protein expressions of angiotensin-converting enzyme (ACE) and angiotensin II type 1 (AT1), and type 2 (AT2) receptors in the rostral ventrolateral medulla (RVLM) were compared between domestic pigs and Guizhou mini-pigs. Twelve-month-old Guizhou mini-pigs were divided into sham (n = 7) and ablation (n = 7) groups. The mini-pigs in ablation group received bilateral percutaneous RDN with a saline-irrigated Sniper ablation catheter. Three months after the procedure, the BP was measured and the histology of renal nerves and arteries was analyzed. RESULTS The mini-pigs spontaneously developed hypertension by the age of 6 months and the BP (162.2 ± 11.4/111.8 ± 9.2mm Hg) was significantly higher than age-matched domestic pigs (137.5 ± 1.9/80.2 ± 4.1mm Hg, P < 0.05). The levels of plasma catecholamines and urine vanillylmandelic acid were higher in mini-pigs than domestic pigs. The expressions of ACE and AT1 were increased, but the AT2 was decreased, in RVLM from mini-pigs compared with domestic pigs. Three months after the procedure, the BP was sharply reduced in ablation group (113.8 ± 14.4/79.4 ± 11.7 mm Hg) compared with sham group (192.4 ± 10.5/141.2 ± 5.9 mm Hg, P < 0.01). Renal nerves were substantially destroyed, while renal arteries and function were not significantly affected by ablation. CONCLUSIONS The Guizhou mini-pig is a novel spontaneous hypertensive animal model with sympathetic hyperactivity and responds well to RDN.

Since neurotransmitters (NTs) and their metabolites are involved in some major neurological disorders, there is a demand for suitable analytical techniques allowing highly sensitive determination of NTs in biological samples. We developed and validated a derivatization method for the simultaneous determination of NTs and their metabolites in rat brain homogenates and microdialysates of the prefrontal cortex and hippocampus using high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The NTs and their metabolites were derivatized with dansyl chloride under optimal conditions and separated by an Ultimate XB-C8 column. The method was used for the quantification of monoamine NTs like dopamine (DA), norepinephrine (NE) and serotonin (5-HT), and their metabolites including 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), vanilmandelic acid (VMA), 4-hydroxy-3-methoxyphenylglycol (MHPG), and 5-hydroxyindole-3-acetic acid (5-HIAA). Amino acid neurotransmitters like tryptophan (TRP), kynurenine (KYN), kynurenic acid (KA) and glutamate (GLU), glutamine (GLN) and γ-aminobutyric acid (GABA) were also taken into account. This method exhibited good linearity for all of the analytes with regression coefficients higher than 0.99. The intra-day and inter-day precision were no greater than 16%. The lower limit of quantification (LLOQ) values for NTs ranged from 0.4 to 9.7 pmol mL−1. The mean recovery ranged from 90.3% to 115.0%. The described LC-MS/MS method was successfully applied for quantification of the NTs and their metabolites in the brain homogenates and microdialysates of rats with good precision and accuracy.

Background: Menkes disease is a lethal disorder associated with copper metabolism. Although early treatment with copper-histidine injections can improve outcomes, early diagnosis is difficult because the clinical features of Menkes disease are subtle or do not manifest in affected neonates. Previous report stated that the low activity of dopamine β-hydroxylase, a copper-dependent enzyme, leads to increases in the urine homovanillic acid/vanillylmandelic acid (HVA/VMA) ratios in patients with Menkes disease, and indicated that a urine HVA/VMA ratio cut-off value of >4 is useful in screening for Menkes disease. Methods : We examined the standard values of the urine HVA/VMA ratio in unaffected neonates and assessed its use as a screening parameter for Menkes disease among neonates. In total, 112 neonates, aged between 1 and 6days, were enrolled in the study and were classified into 2 groups based on their urine HVA/VMA ratios: high (>4) and low (⩽4). Results : Multivariate logistic analysis revealed that mechanical ventilation was an independent risk factor for a high urine HVA/VMA ratio (odds ratio: 21.94; 95% confidence interval: 2.82–247.03; p =0.004). The mean urine HVA/VMA ratio was 2.47±0.67 among 92 neonates who did not receive mechanical ventilation. Conclusion : This study established standard values for the urine HVA/VMA ratio in newborn babies that could be useful in screening for Menkes disease among neonates.