Analysis of the relationship between MYCN gene and serum NSE and urinary VMA levels and neuroblastoma pathological features and prognosis.

The purpose of this study was to explore the relationship between the MYCN gene, serum neuron-specific enolase (NSE), urinary vanillylmandelic acid (VMA) levels, and neuroblastoma pathological features and prognosis. Ninety-four children with neuroblastoma treated in the hospital were selected to compare the differences in MYCN gene amplification, serum NSE, and urinary VMA levels in children with different clinicopathological features and prognoses. The proportion of children with MYCN gene copy number ≥10 in INSS stage 3-4 was higher than that of children with INSS stage 1-2 (P < 0.05); the proportion of children with MYCN gene copy number ≥10 in high-risk children in the COG risk stratification was higher than that of children with intermediate and low risk (P < 0.05); the serum NSE of children aged >12 months higher than that of children aged ≤12 months (P < 0.05); serum NSE of children with tumors >500 cm3 higher than that of children with tumors ≤500 cm3 (P < 0.05); serum NSE and urinary VMA of children with INSS staging of stages 3-4 were higher than that of children with stages 1 to 2 (P < 0.05); serum NSE and urinary VMA in children with lymph node metastasis were higher than that of children without lymph node metastasis (P < 0.05); serum NSE of children with MYCN gene copy number ≥10 was higher than that of children without lymph node metastasis (P < 0.05); the proportion of children with MYCN gene copy number ≥10 who died, and the percentages of serum NSE and urinary VMA were higher than those of the surviving children (P < 0.05). MYCN gene amplification and serum NSE and urinary VMA levels were related to the age, tumor size, INSS stage, COG stage, lymph node metastasis, and prognosis of the children with neuroblastoma.

Plasma & urinary catecholamines & urinary vanillylmandelic acid levels in patients with generalized vitiligo.

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.

Usefulness of fluorodeoxyglucose positron emission tomography/computed tomography for detection of a neuroblastic nodule in a ganglioneuroblastoma: a case report.

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.

Clinical Metabolomics to Segregate Aromatic Amino Acid Decarboxylase Deficiency From Drug-Induced Metabolite Elevations.

BACKGROUND: Phenotyping technologies featured in the diagnosis of inborn errors of metabolism, such as organic acid, amino acid, and acylcarnitine analyses, recently have been supplemented by broad-scale untargeted metabolomic phenotyping. We investigated the analyte changes associated with aromatic amino acid decarboxylase (AADC) deficiency and dopamine medication treatment. METHODS: Using an untargeted metabolomics platform, we analyzed ethylenediaminetetraacetic acid plasma specimens, and biomarkers were identified by comparing the biochemical profile of individual patient samples to a pediatric-centric population cohort. RESULTS: Elevated 3-methoxytyrosine (average z score 5.88) accompanied by significant decreases of dopamine 3-O-sulfate (-2.77), vanillylmandelate (-2.87), and 3-methoxytyramine sulfate (-1.44) were associated with AADC deficiency in three samples from two patients. In five non-AADC patients treated with carbidopa-levodopa, levels of 3-methoxytyrosine were elevated (7.65); however, the samples from non-AADC patients treated with DOPA-elevating drugs had normal or elevated levels of metabolites downstream of aromatic l-amino acid decarboxylase, including dopamine 3-O-sulfate (2.92), vanillylmandelate (0.33), and 3-methoxytyramine sulfate (5.07). In one example, a plasma metabolomic phenotype pointed to a probable AADC deficiency and prompted the evaluation of whole exome sequencing data, identifying homozygosity for a known pathogenic variant, whereas whole exome analysis in a second patient revealed compound heterozygosity for two variants of unknown significance. CONCLUSIONS: These data demonstrate the power of combining broad-scale genotyping and phenotyping technologies to diagnose inherited neurometabolic disorders and suggest that metabolic phenotyping of plasma can be used to identify AADC deficiency and to distinguish it from non-AADC patients with elevated 3-methoxytyrosine caused by DOPA-raising medications.

A Mercury Toxicity Case Complicated by Hyponatremia and Abnormal Endocrinological Test Results.

Mercury (Hg) poisoning is considered a rare disease by the National Institutes of Health and the diagnosis can present great challenges to clinicians. Children who are exposed to Hg can present with a wide variety of symptoms, including acrodynia, tremor, excessive salivation, and psychiatric symptoms, including insomnia. However, endocrinologic manifestations from Hg exposure are less well known. This is a case report of a 12-year-old boy who presented with body rash, irritability, insomnia, and profuse sweating after returning from a summer camp. The child was initially managed in the outpatient setting, and the investigation was mainly targeted toward infectious etiology, including Rocky Mountain spotted fever and Lyme disease. He was eventually admitted to the hospital with altered mental status and was noted to have hyponatremia with serum sodium of 121 mEq/L. Thyroid studies also revealed elevated free thyroxine levels in the presence of normal triiodothyronine and thyrotropin. The patient developed hypertension and tachycardia, and was found to have elevated 24-hour vanillylmandelic acid and metanephrines. Finally, heavy metal measurements revealed a blood Hg level that was greater than the reference values of 0 to 9 ng/mL. Chelation treatment with 2,3-dimercaptopropane-1-sulfonate was subsequently initiated and over a period of 8 months his symptoms resolved and his thyroid function test returned to normal. This case highlights some of the challenges commonly encountered in identifying Hg exposure. More importantly, it illustrates that exposure to Hg should be considered in children who present with the symptoms and abnormal endocrinologic test results described in this report.

Neuroblastoma.

Neuroblastoma is the most common extracranial solid tumour occurring in childhood and has a diverse clinical presentation and course depending on the tumour biology. Unique features of these neuroendocrine tumours are the early age of onset, the high frequency of metastatic disease at diagnosis and the tendency for spontaneous regression of tumours in infancy. The most malignant tumours have amplification of the MYCN oncogene (encoding a transcription factor), which is usually associated with poor survival, even in localized disease. Although transgenic mouse models have shown that MYCN overexpression can be a tumour-initiating factor, many other cooperating genes and tumour suppressor genes are still under investigation and might also have a role in tumour development. Segmental chromosome alterations are frequent in neuroblastoma and are associated with worse outcome. The rare familial neuroblastomas are usually associated with germline mutations in ALK, which is mutated in 10-15% of primary tumours, and provides a potential therapeutic target. Risk-stratified therapy has facilitated the reduction of therapy for children with low-risk and intermediate-risk disease. Advances in therapy for patients with high-risk disease include intensive induction chemotherapy and myeloablative chemotherapy, followed by the treatment of minimal residual disease using differentiation therapy and immunotherapy; these have improved 5-year overall survival to 50%. Currently, new approaches targeting the noradrenaline transporter, genetic pathways and the tumour microenvironment hold promise for further improvements in survival and long-term quality of life.

Preanalytical validation and reference values for a mass spectrometric assay of serum vanillylmandelic acid for screening of catecholamine secreting neuroendocrine tumors.

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.

Analysis of vanillylmandelic acid and homovanillic acid by UPLC-MS/MS in serum for diagnostic testing for neuroblastoma.

BACKGROUND: Vanillylmandelic acid (VMA) and homovanillic acid (HVA) are typically measured in urine for the diagnosis and monitoring of neuroblastoma, a tumor in children <5 y. A protocol for evaluation of serum VMA and HVA has been utilized at our institution for approximately 25 y, originally validated using high performance liquid chromatography (HPLC) with an electrochemical detector. We recently validated a serum VMA/HVA method by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). METHODS: After solvent extraction and clean up with Ultrafree centrifugal filters, samples were analyzed by UPLC-MS/MS in multiple reaction monitoring mode. RESULTS: The assay was linear between 2 and 1000 ng/ml for VMA and HVA. Within run and run to run CVs were <5% for VMA at all levels, <10% for HVA at high levels, and <20% at low levels. Correlation with the HPLC method was acceptable with a constant bias. The reference interval for VMA by UPLC-MS/MS was determined to be ≤20 ng/ml, and HVA≤30 ng/ml. Original patient data comparing urine to serum showed diagnostic agreement >80% for both VMA and HVA. CONCLUSION: Correlation of VMA and HVA was acceptable after adjustment of reference intervals. Collection of a single serum sample instead of 24-h urine collection saves time and improves accuracy of measurement due to difficulty of collecting a 24-h urine sample in infants and young children. UPLC-MS/MS also offers improved analyte specificity, improved signal to noise, and rapid analysis time.

An autopsy case of bilateral adrenal pheochromocytoma-associated cerebral hemorrhage.

The autopsy findings of a 30-year-old woman who died of cerebral hemorrhage induced by bilateral adrenal pheochromocytoma are presented. The cerebral hemorrhage was shown on the left cerebral hemisphere widely. Her both adrenal glands were severe swelling, and their parenchyma was occupied by a dark red-brown tumorous positive for chromogranin A. The serum catecholamine and their metabolite, vanillylmandelic acid (VMA) levels were markedly high. Furthermore, cardiac hypertrophy and sclerosis of the arteries of various organs had progressed, suggesting an influence of persistent endocrinal hypertension. The measurement of serum VMA level was thought to be valuable for a postmortem diagnosis of pheochromocytoma. Bilateral adrenal pheochromocytoma may have excessively secreted catecholamine and subsequently caused secondary hypertension, leading to cerebral hemorrhage.

Adrenal haemangioblastoma presenting as phaeochromocytoma: a rare manifestation of extraneural hemangioblastoma.

PURPOSE: Adrenal haemangioblastoma presenting clinically as pheochromocytoma is a rare manifestation of extraneural haemangioblastoma. We present an unusual case of von Hippel-Lindau (VHL) disease that had adrenal and cerebellar haemangioblastoma with multiple renal cysts, and a review of the literature. METHODS: Unlike the usual manifestations of secondary polycythemia or increased intracranial pressure and hydrocephalus due to cerebellar lesion, this 36-year-old male presented with hypertension. Investigations revealed right suprarenal mass with raised urinary catecholamines and serum vanillylmandelic acid (VMA) levels, apparently confirming the clinical diagnosis of phaeochromocytoma. RESULTS: Histopathology of the biopsy specimen showed features of haemangioblastoma, which was confirmed by immunohistochemistry using antibodies to neuron specific enolase and aquaporin-1. Based on this, the patient was screened for possible features of VHL, which revealed cerebellar haemangioblastoma and multiple renal cysts with angiomatous lesion. Postoperative follow-up showed normal levels of catecholamines without any symptoms of phaeochromocytoma. CONCLUSIONS: Adrenal haemangioblastoma is a rare entity with only four cases reported in the literature. Surgical removal is the treatment of choice. However, screening for other possible features of VHL, even in the absence of clinical features, is essential to exclude other potential lesions.

Sensitive, rapid and easy analysis of three catecholamine metabolites in human urine and serum by liquid chromatography tandem mass spectrometry.

A sensitive and easy analytical method for catecholamine metabolites including 4-hydroxy-3-methoxyphenylglycol sulfate (HMPG sulfate), vanillylmandelic acid (VMA) and homovanillic acid (HVA) determination was developed based on liquid chromatography-tandem mass spectrometry in a negative multiple reaction monitoring mode. The analytes were rapidly separated on a reversed-phase Waters Xbridge C18 column (150 × 2.1 mm i.d.) with the mobile phase of 15% (v/v) acetonitrile containing 2 mM ammonium formate and 85% (v/v) formic acid solution (0.05%, v/v). Mass spectrometric conditions, such as characteristic fragmentations and quantification ion transitions, both with chromatographic conditions including separation column type and mobile phase composition, were systematically investigated to get optimal sensitivity and specificity. The limits of detection were in the range of 0.03-0.7 ng/mL for the targets. Recovery rates of spiked urine samples with three different concentration levels (low, middle and high) were above 86% with precisions less than 5.7%. For serum analysis, acetonitrile chosen both as protein precipitation reagent and extraction solvent facilitates to reduce matrix effects. Recovery rates of spiked serum sample were in the range of 90.6% to 111.1% for three targets. The intra-day and inter-day precisions were satisfactory less than 8.7%. This proposed method was successfully applied to determine HMPG sulfate, HVA and VMA present in human urine and serum.

Fast analysis of catecholamine metabolites MHPG and VMA in human plasma by HPLC with fluorescence detection and a novel SPE procedure.

A fast and sensitive high-performance liquid chromatographic method has been developed for the determination in human plasma of MHPG (3-methoxy-4-hydroxyphenylethylenglycol) and VMA (vanillyl mandelic acid), the main metabolites of epinephrine and norepinephrine. Analyses were carried out at 325 nm while exciting at 285 nm on a reversed-phase column (Atlantis C18, 150 mm x 4.6 mm I.D., 5 microm) using a mobile phase composed of 2% methanol and 98% aqueous citrate buffer at pH 3.0. A careful solid-phase extraction procedure, based on mixed-mode reversed-phase - strong anion exchange Oasis cartridges (MAX, 30 mg, 1 mL), was developed for the pre-treatment of plasma samples. Extraction yields were satisfactory, always higher than 90%. Calibration curves were linear over the 0.2-40.0 ng mL(-1) concentration range for MHPG and over the 0.5-40.0 ng mL(-1) concentration range for VMA. The method was successfully applied to plasma samples of former drug users undergoing detoxification therapy and subjects "at risk" of developing drug addiction.

The release of sympathetic neurotransmitters is impaired in aged rats after an inflammatory stimulus: a possible link between cytokine production and sympathetic transmission.

Aging results in a general decline in the response to external insults, including acute inflammatory challenges. In young animals, the inflammatory response requires activation of the sympathetic system, including neurotransmitters such as ATP, and catecholamines (epinephrine and norepinephrine). To test whether aging affects activation of this axis, and whether this in turn might affect cytokine release, we administered lipopolysaccharide (LPS) i.p. to adult, middle-aged and aged Fisher 344 rats (6-, 15- and 23-month old, respectively) and evaluated the early (0-12h) serum levels of Neuropeptide-Y (NP-Y), ATP and vanillyl mandelic acid (VMA, as an indirect measurement of catecholamine levels). In addition, we evaluated the association between these factors and serum levels of the cytokines tumor necrosis factor-alpha (TNFalpha) and interleukin-10 (IL-10). Induction of both ATP and NP-Y was markedly reduced in the serum of aged animals, when compared to their younger counterparts, while induction of VMA was not affected by age. In spite of these changes, serum levels of TNFalpha and IL-10 were strongly hyper induced and delayed in aged rats. The results suggest that during aging there is a dysregulation in sympathetic neurotransmitter regulatory mechanisms, and this might play a role in the impairment of the inflammatory response.

Nine cases of childhood adrenal tumour presenting with hypertension and a review of the literature.

AIM: To describe the clinical features, treatment and outcome of children adrenal tumors presenting with hypertension. METHODS: The records of nine children under 16 years of age with adrenal tumours presenting with hypertension were analysed. Details were recorded for family history, clinical presentation, biochemistry, imaging, histology, treatment and outcome. RESULTS: Abdominal mass was palpable only in one patient at diagnosis. Besides hypertension-associated symptoms, Cushing's syndrome was the common presentation form (n = 4). Abdominal computed topography showed adrenal mass in all patients. Tumours were completely resected for each patient. The median tumour weight was 73 g (11-530 g) and the size ranged from 1.5 x 1.5 to 12 x 14 cm2. Pheochromocytoma (n = 2), adrenocortical adenoma (n = 3), adrenocortical carcinoma (n = 1), neuroblastoma (n = 2) and ganglioneuromas (n = 1) were found. In one case, adrenal pheochromocytoma first occurred and non-functioning islet cell tumour successively occurred at pancreas. A better status is common at a median follow-up time of 3.5 years. CONCLUSIONS: Childhood adrenal tumours presented with hypertension showed an atypical course, variable presentation. We report a unique case of adrenal pheochromocytoma followed by the occurrence of non-functioning islet cell tumour. Reversal of hypertension by surgery is crucial. Imaging techniques are important to detect adrenal tumours.

Sympathetic sudomotor disturbance in early type 1 diabetes mellitus is linked to lipid peroxidation.

The present study was performed to determine whether increased lipid peroxidation, as assessed from malondialdehyde (MDA) excretion, is associated with deterioration in peripheral nerve function in early type 1 diabetes mellitus. These parameters were measured annually for 3 years in 36 patients who entered the study less than 2 years after the diagnosis of diabetes. Malondialdehyde excretion was 1.51 +/- 0.20 micromol/g creatinine in the controls, and 2.43 +/- 0.21, 2.39 +/- 0.22, and 1.93 +/- 0.21 micromol/g creatinine at the first, second, and third evaluations, respectively (P < .005). The increased MDA was seen only in the female participants. Malondialdehyde excretion was increased in those with high vs low hemoglobin Alc across all years (P < .05). Malondialdehyde excretion correlated negatively with sudomotor function below the waist. The mean sweat production from the 3 evaluations correlated with mean MDA excretion across all years in the proximal leg (r = -0.42, P < .005) and distal leg (r = -0.40, P < .01). Below the waist, sweating correlated with MDA (r = -0.40, P < .01) as did total sweat (r = -0.38, P < .01). The response amplitudes of the peroneal nerves correlated negatively with MDA excretion (for the mean values at the second 2 evaluations, P < .005, r = -0.45). Tests of sensory function correlated inconsistently with MDA excretion. In summary, lipid peroxidation, as assessed from malondialdehyde excretion, is associated with sudomotor dysfunction in early diabetes.

[Slight hormonal alterations in a patient with a large pheochromocytoma]. / Mínimas alterações hormonais em paciente com grande feocromocitoma.

We present a case report that the patient had symptoms suggesting pheochromocytoma, a large tumor (> 50 g) and a single minimally altered laboratorial test, exemplifying a diagnostic pitfall. A 31 y.o. male patient had two acute abdominal events, the last one accompanied by headache, arterial hypertension, facial flushing, perspiration and cutaneous pallor. In another admission, the patient had sustained arterial hypertension and cardiac arrhythmia. From laboratory analysis, the vanililmandelic acid was slightly modified. Scintigraphy disclosed a large adrenal mass suggesting pheochromocytoma. Histopathology confirmed this hypothesis. This report points out that patients with symptoms suggesting pheochromocytoma, even when plasma catecholamines and urinary metanephrines levels are normal, may harbor large tumors with a high catecholamines turnover or that had undergone hemorrhagic necrosis.

Tumour markers are poor predictors for relapse or progression in neuroblastoma.

The value of the tumour markers vanillylmandelic acid (VMA) and homovanillic acid (HVA) in urine (u) and serum (s), neurone-specific enolase (NSE), and lactate dehydrogenase (LDH) in the early prediction of relapse/progression in neuroblastoma is not known. We analysed the data of neuroblastoma patients who had successfully completed first-line treatment and had laboratory results available from their initial diagnosis and from relapse/progression (n=196). Patients' overall survival from relapse or progression was 21.5+/-4.2% (mean+/-standard deviation). At diagnosis, we found abnormal results in 75% for VMA and/or HVA (s), 92% for VMA and/or HVA (u), 90% for NSE, and 81% for LDH. We found a lower incidence of abnormal results at relapse or progression with 40% for VMA and/or HVA (s), 54% for HVA and/or VMA (u), 61% for NSE, and 48% for LDH. Sensitivity of all markers was higher for metastatic compared with local recurrence. NSE was the best, being able to detect 42% of the localised relapses, 77% of the combined local/metastatic relapses, and 69% of the metastatic recurrences. Relapse or progression in neuroblastoma cannot be detected reliably by monitoring tumour markers alone. Therefore, follow-up of neuroblastoma patients must include clinical assessment and imaging studies.

Sources and significance of plasma levels of catechols and their metabolites in humans.

Human plasma contains several catechols, including the catecholamines norepinephrine, epinephrine, and dopamine, their precursor, L-3,4-dihydroxyphenylalanine (L-DOPA), and their deaminated metabolites, dihydroxyphenylglycol, the main neuronal metabolite of norepinephrine, and dihydroxyphenylacetic acid, a deaminated metabolite of dopamine. Products of metabolism of catechols include 3-methoxytyrosine (from L-DOPA), homovanillic acid and dopamine sulfate (from dopamine), normetanephrine, vanillylmandelic acid, and methoxyhydroxyphenylglycol (from norepinephrine), and metanephrine (from epinephrine). Plasma levels of catechols and their metabolites have related but distinct sources and therefore reflect different functions of catecholamine systems. This article provides an update about plasma levels of catechols and their metabolites and the relevance of those levels to some issues in human health and disease.