Scoring system for diagnosis and pretreatment risk assessment of neuroblastoma using urinary biomarker combinations.

The urinary catecholamine metabolites, homovanillic acid (HVA) and vanillylmandelic acid (VMA), are used for the adjunctive diagnosis of neuroblastomas. We aimed to develop a scoring system for the diagnosis and pretreatment risk assessment of neuroblastoma, incorporating age and other urinary catecholamine metabolite combinations. Urine samples from 227 controls (227 samples) and 68 patients with neuroblastoma (228 samples) were evaluated. First, the catecholamine metabolites vanillactic acid (VLA) and 3-methoxytyramine sulfate (MTS) were identified as urinary marker candidates through comprehensive analysis using liquid chromatography-mass spectrometry. The concentrations of these marker candidates and conventional markers were then compared among controls, patients, and numerous risk groups to develop a scoring system. Participants were classified into four groups: control, low risk, intermediate risk, and high risk, and the proportional odds model was fitted using the L2-penalized maximum likelihood method, incorporating age on a monthly scale for adjustment. This scoring model using the novel urine catecholamine metabolite combinations, VLA and MTS, had greater area under the curve values than the model using HVA and VMA for diagnosis (0.978 vs. 0.964), pretreatment risk assessment (low and intermediate risk vs. high risk: 0.866 vs. 0.724; low risk vs. intermediate and high risk: 0.871 vs. 0.680), and prognostic factors (MYCN status: 0.741 vs. 0.369, histology: 0.932 vs. 0.747). The new system also had greater accuracy in detecting missing high-risk neuroblastomas, and in predicting the pretreatment risk at the time of screening. The new scoring system employing VLA and MTS has the potential to replace the conventional adjunctive diagnostic method using HVA and VMA.

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.

Optimising urinary catecholamine metabolite diagnostics for neuroblastoma.

INTRODUCTION: The analysis of urinary catecholamine metabolites is a cornerstone of neuroblastoma diagnostics. Currently, there is no consensus regarding the sampling method, and variable combinations of catecholamine metabolites are being used. We investigated if spot urine samples can be reliably used for analysis of a panel of catecholamine metabolites for the diagnosis of neuroblastoma. METHODS: Twenty-four-hour urine or spot urine samples were collected from patients with and without neuroblastoma at diagnosis. Homovanillic acid (HVA), vanillylmandelic acid (VMA), dopamine, 3-methoxytyramine, norepinephrine, normetanephrine, epinephrine and metanephrine were measured by high-performance liquid chromatography coupled with fluorescence detection (HPLC-FD) and/or ultra-performance liquid chromatography coupled with electrospray tandem mass spectrometry (UPLC-MS/MS). RESULTS: Catecholamine metabolite levels were measured in urine samples of 400 neuroblastoma patients (24-hour urine, n = 234; spot urine, n = 166) and 571 controls (all spot urine). Excretion levels of catecholamine metabolites and the diagnostic sensitivity for each metabolite were similar in 24-hour urine and spot urine samples (p > .08 and >.27 for all metabolites). The area under the receiver-operating-characteristic curve (AUC) of the panel containing all eight catecholamine metabolites was significantly higher compared to that of only HVA and VMA (AUC = 0.952 vs. 0.920, p = .02). No differences were observed in metabolite levels between the two analysis methods. CONCLUSION: Catecholamine metabolites in spot urine and 24-hour urine resulted in similar diagnostic sensitivities. The Catecholamine Working Group recommends the implementation of spot urine as standard of care. The panel of eight catecholamine metabolites has superior diagnostic accuracy over VMA and HVA.

Semi-quantitative detection of a vanillactic acid/vanillylmandelic acid ratio in urine is a reliable diagnostic marker for aromatic L-amino acid decarboxylase deficiency.

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.

Likelihood of Diagnosing Neuroblastoma in Isolated Horner Syndrome.

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.

Analytical validation and clinical application of urinary vanillylmandelic acid and homovanillic acid by LC-MS/MS for diagnosis of neuroblastoma.

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.

Specific Urinary Metabolites in Malignant Melanoma.

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 µmol/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.

Methylphenidate increases the urinary excretion of vanillylmandelic acid in rats that is attenuated by buspirone co-administration.

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 (P<0.01) raised urinary VMA excretion in methylphenidate group however significant (P<0.01) 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.

Olive ingestion causing a false suspicion of relapsed neuroblastoma: A case of "oliveblastoma?"

Measurement of the urine catecholamine metabolites homovanillic acid (HVA) and vanillylmandelic acid (VMA) are the standard method for detecting disease recurrence in neuroblastoma. We present a case of abnormal concentrations of catecholamine metabolites that prompted investigations for relapsed neuroblastoma. However, further study revealed that the abnormal biochemistry was likely due to ingestion of olives. Olive ingestion should be considered when interpreting urine HVA and VMA results, and excluded if concentrations are unexpectedly abnormal.

Quantification of vanillylmandelic acid, homovanillic acid and 5-hydroxyindoleacetic acid in urine using a dilute-and-shoot and ultra-high pressure liquid chromatography tandem mass spectrometry method.

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 <15%), negligible matrix effect (recoveries >90%), low carryover (<1%) and LLOQ of 0.25, 0.4 and 0.4 µM for VMA, HVA and 5-HIAA, respectively. Deming fits and Bland-Altman analyses showed no significant differences between the values obtained between the two assays. 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.

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.

Values of vanillylmandelic acid and homovanillic acid in the urine as potential prognostic biomarkers in ischaemic stroke patients.

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.

Corticomedullary mixed tumour resembling a small adrenal gland-involvement of cancer stem cells: case report.

BACKGROUND: Adrenal corticomedullary mixed tumours are very rare. Its mechanism is rarely reported. Here we report the first case of a corticomedullary mixed tumour resembling a "small adrenal gland" with distinct arrangement of the cortical and medullary layers. We further hypothesize regarding the tumorigenic mechanism of this tumour. CASE PRESENTATION: A 58-year man had been diagnosed with diabetes and hypertension for 3 years. His 24-h urine vanillylmandelic acid (VMA) levels were slightly elevated. An abnormal circadian cortisol rhythm was noted, and his cortisol levels were not suppressed by dexamethasone. Abdominal computed tomography (CT) revealed a right adrenal gland lesion (diameter, 30 × 38 mm), while an enhanced CT showed enhancement and hypervascularization. The tumour was positive for adrenocorticotropic hormone, chromogranin A (CGA), and steroidogenic factor-1 (SF-1) on the tumour surface. Acetaldehyde dehydrogenase 1(ALDH1), CD44, CD133, Nestin, Nerve growth factor receptor (NGFR), and Sex determining region y-box 9(SOX9) staining were positive. Although administration of medications for diabetes and hypertension was stopped until surgery was performed, the blood sugar level and blood pressure were maintained after surgery. CONCLUSIONS: This is the first report about a possible mechanism by which cancer stem cells induce adrenal corticomedullary tumours.

Polychlorinated biphenyls and depression: cross-sectional and longitudinal investigation of a dopamine-related Neurochemical path in the German HELPcB surveillance program.

BACKGROUND: Exposure to polychlorinated biphenyls (PCBs) is associated with depressive symptomatology. A cause of depressive symptoms is a disturbance in the neurotransmitter system of dopamine (DA). Animal as well as human studies report that PCBs can influence the DA system. This study examined whether PCB-related depressive symptoms are affected by DA metabolites in humans with high PCB body burden. METHODS: This study is part of the German HELPcB surveillance program (Health Effects in high Level exposure to PCB) for occupationally exposed workers and their relatives. Data was collected from 178 participants on two measurement time points (t1 and t2) with a one-year time lag in between the two time points. PCBs were analyzed in plasma via human biomonitoring and a validated questionnaire was used to identify existence and severity of depressive symptoms. As a surrogate for DA, we measured its metabolites homovanillic acid (HVA) and vanillylmandelic acid (VMA) in urine. Mediation analyses were performed to test whether the association between PCB exposure and severity of depressive symptoms is mediated by urinary concentration of DA metabolites HVA and VMA. The mediation was tested with the SPSS macro MEDIATE. RESULTS: We found a significant mediation over time for lower-chlorinated, higher-chlorinated and dioxin-like PCBs. The positive association between PCB exposure with severity of depressive symptoms was mediated by the main DA metabolite HVA. At t1 a higher exposure with PCBs was associated with lower concentration in urinary HVA. A reduced HVA concentration at t1 was correlated with increased depressive symptoms severity at t2. No meditations were found for VMA. CONCLUSIONS: This work indicates that the association of PCB exposure and an increase of depressive symptoms after one year is mediated by the DA metabolite HVA as a surrogate for DA. These are first steps towards finding an explanation for an underlying neurochemical pathomechanism of PCB-related depressive symptomatology.

Long-term follow-up results of the observation program for neuroblastoma detected at 6-month mass screening.

We conducted an observation program of neuroblastoma in infants, detected by mass screening at 6 months of age; we followed up with them for 15 years. No recurrence was observed after disappearance of tumors, and persistent tumors showed no malignant transformation or metastasis. Histology of the resected tumors showed age-related differentiation.

Serotonergic, noradrenergic and dopaminergic markers are related to cognitive function in adults with 22q11 deletion syndrome.

Patients with 22q11 deletion syndrome (22q11DS) have a high prevalence of psychiatric disorders and intellectual disability. At present the neurobiology underlying psychopathology in 22q11DS is still not understood. In the present study, we analyzed urinary serotonergic, dopaminergic and noradrenergic markers in 67 adults with 22q11DS. Levels of serotonin and the catecholamine metabolite homovanillic acid were significantly lower in the 22q11DS subjects compared to healthy controls. Within the 22q11DS group, levels of dopamine, homovanillic acid, norepinephrine, vanillyl mandelic acid and serotonin positively correlated with Full Scale Intelligence Quotient scores. Our results suggest that cognitive deficits in 22q11DS are associated with abnormal function of several neurotransmitters.

Gender-related effects on urine L-cystine metastability.

Cystinuria is an autosomal recessive disease that causes L-cystine precipitation in urine and nephrolithiasis. Disease severity is highly variable; it is known, however, that cystinuria has a more severe course in males. The aim of this study was to compare L-cystine metastability in first-morning urine collected from 24 normal female and 24 normal male subjects. Samples were buffered at pH 5 and loaded with L-cystine (0.4 and 4 mM final concentration) to calculate the amount remaining in solution after overnight incubation at 4 °C; results were expressed as Z scores reflecting the L-cystine solubility in each sample. In addition, metabolomic analyses were performed to identify candidate compounds that influence L-cystine solubility. L-cystine solubility Z score was +0.44 ± 1.1 and -0.44 ± 0.70 in female and male samples, respectively (p < 0.001). Further analyses showed that the L-cystine solubility was independent from urine concentration but was significantly associated with low urinary excretion of inosine (p = 0.010), vanillylmandelic acid (VMA) (p = 0.015), adenosine (p = 0.029), and guanosine (p = 0.032). In vitro L-cystine precipitation assays confirmed that these molecules induce higher rates of L-cystine precipitation in comparison with their corresponding dideoxy molecules, used as controls. In silico computational and modeling analyses confirmed higher binding energy of these compounds. These data indicate that urinary excretion of nucleosides and VMA may represent important factors that modulate L-cystine solubility and may represent new targets for therapy in cystinuria.

Plasma free metanephrines in the diagnosis of pheochromocytoma: diagnostic accuracy and strategies for Japanese patients.

Measuring the levels of the plasma free metanephrines (PFMs) represents a recently developed and promising test for the diagnosis of pheochromocytoma in the United States and Europe. As this test has not yet been evaluated in Japan, it is necessary to evaluate the diagnostic efficacy of measuring the levels of PFMs compared with the standard measurement of the urinary excretion of metanephrines (uMNs) whose reliability is well established to detect of pheochromocytoma. A total of 101 Japanese subjects clinically suspected of having pheochromocytoma in were included in this study. Subsequently, we prospectively measured the PFMs levels in all patients, compared with those of biochemical markers of the catecholamine secretion and metabolisms in the plasma and urine. All subjects with adrenal tumors underwent tumor excision. Data were available for 84 of the 101 patients, 47 of whom had histopathologically proven pheochromocytoma and 37 were finally diagnosed with non-pheochromocytoma. The results of comparisons in the accuracy of measurement for diagnosis of pheochromocytoma between PFMs and the urinary excretion of metanephrines (uMNs) were 0.980 VS 0.951 for AUC of receiver operatorating characteristic (ROC) curve, 0.957 VS 0.894 for sensitivity, and 0.973 VS 0.946 for specificity, respectively. Although the differences were small, the results of our study definitely demonstrated that measurement of PFMs was not inferior to standard urinary metanephrines (uMNs) measurement, which is established to be the most reliable biochemical method to detect pheochromocytoma. This study clearly shows measuring the PFMs levels to be a reliable and efficient method for diagnosing pheochromocytoma in Japanese patients, as demonstrated in previous reports.

[Clinical manifestations of three neonates with family histories of Menkes disease].

OBJECTIVE: To examine indications for the early diagnosis of Menkes disease (MD). METHODS: We compared clinical examination results of 3 neonate males with family histories of MD who were at risk of being affected (1 infant was affected and 2 were unaffected). RESULTS: Kinky hair, a characteristic shown by MD patients, was observed just after birth in the affected infant; however, this characteristic was extremely mild and easy to miss. In the first month after birth, serum copper level declined over time in the affected infant,while it increased in the unaffected infants. Urine homovanillic acid/vanillylmandelic acid (HVA/VMA) ratio, which is typically high in MD patients, was observed to be 4.9-8.0 (cut-off, 4.0) in the affected infant. In the unaffected infants, the urine HVA/MVA ratio showed a high value of 11.0 during catecholamine administration, but this decreased to below the cut-off value when catecholamine was not administered. CONCLUSIONS: Variations in the serum copper level and urine HVA/VMA ratio over time were considered as useful indicators for the early diagnosis of MD.

[Determination of four biogenic amine metabolites in urine by high-performance liquid chromatography].

OBJECTIVE: To establish a method for simultaneously determining vanilmandelic acid (VMA), 5-hydroxyindoleacetic (5-HIAA), 3, 4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in urine by high-performance liquid chromatography (HPLC). METHODS: After being filtered with a 0.45 µm membrane syringe filter, the urinary samples were injected directly into the HPLC system using a C18 chromatographic column and a fluorescence detector. The excitation and emission wavelengths were chose as 280 nm and 315 nm, respectively, and the urinary samples were carried with a mobile phase of methanol-0.1 mol/L phosphate buffered solution (V/V = 20:80) at a flow rate of 1.0 ml/min and an injection volume of 20 µl. RESULTS: Using the method reported here, the correlation coefficients of VMA, 5-HIAA, DOPAC, and HVA were 0.9999, 0.9998, 0.9997, 0.9999, respectively, over linear ranges of 0-2.5, 0-2.0, 0-2.0, and 0-2.5 µg/ml, the minimum detectable concentrations were 0.006, 0.008, 0.012, and 0.0082 µg/ml, the average precisions were 4.2%, 3.7%, 4.9%, and 3.6%, and the recovery rates were 91%∼102%, 93%∼101%, 94%∼101%, and 89%∼ 102%. CONCLUSION: This determination method is simple, efficient, accurate, and sensitive for the simultaneous detection of VMA, 5-HIAA, DOPAC, and HVA in urine.