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.

A reliable and simple method for the assay of neuroendocrine tumor markers in human urine by solid-phase microextraction-gas chromatography-triple quadrupole mass spectrometry.

Homovanillic acid (HVA), vanylmandelic acid (VMA), and 5-hydroxyindoleacetic acid (5-HIAA) are the metabolites of some catecholamines such as epinephrine, nor-epinephrine, dopamine and serotonin and their quantification is used in the diagnosis and management of patients with neurocrine tumors. A novel approach in the assay of these biomarkers in human urine samples by solid phase microextraction (SPME) combined with gas chromatography-triple quadrupole mass spectrometry (GC-QqQ-MS) is presented. A preliminary derivatization with ethyl chloroformate/ethanol was used and the corresponding derivatives were then extracted by SPME in immersion mode. The performance of five SPME fibers and three chloroformates were evaluated in univariate mode and the best results were obtained using the polyacrylate fiber and ethyl chloroformate. The variables affecting the efficiency of SPME analysis were optimized by the multivariate approach of "Experimental design" and, in particular, a central composite design (CCD) was applied. The optimum working conditions in terms of response values were achieved by performing analysis at room temperature with addition of NaCl (9.5%) and with an extraction time of 25.8 min. Identification and quantification of analytes were carried out by using a gas chromatography-triple quadrupole mass spectrometry (GC-QqQ MS) system in multiple reaction monitoring (MRM) acquisition. An evaluation of all analytical parameters shows that the proposed method provides satisfactory results. Very good linearities were, in fact, achieved in the tested calibration ranges with correlation coefficient values >0.99 for all the analytes and accuracies and RSDs calculated for between-run and tested at concentrations of 1, 10, and 80 mg L(-1) were ranging from 91.3% to 106.6%, and from 0.5 to 8.9%, respectively. Moreover, the LOD values obtained can be considered very satisfactory (1.3, 0.046 and 24.3 µg L(-1) for HVA, VMA and 5-HIAA, respectively). The developed protocol represents, therefore, a simple, rapid and selective tool for assaying these acidic biomarkers in urine samples for neuroendocrine cancer diagnosis.

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.

Non-invasive assessment of cardioregulatory autonomic functions in children with epilepsy.

OBJECTIVES: We aimed to evaluate the interictal cardiovascular autonomic functions in pediatric patients with idiopathic epilepsy, both partial and generalized. MATERIALS AND METHODS: The study included 25 patients with idiopathic epilepsy and 50 control subjects. Patients underwent five standardized clinical cardiovascular reflex autonomic tests [resting heart rate (HR), HR response to deep breathing and to Valsalva maneuver, the 30:15 ratio of HR response to standing, and blood pressure response to standing], as well as a 12 lead surface electrocardiogram. Heart rate variability (HRV) was tested via 24-h Holter monitoring and the time domain parameters (SDNN, PNN50, rMSDD) were assessed. Excretion of vanillyl mandelic acid and metanephrine was measured in 24-h urine collection. RESULTS: Clinical reflex autonomic tests showed mild dysfunction in 8%, moderate dysfunction in 44% and severe dysfunction in 4% of patients. The HRV parameter, SDNN, was reduced in all age groups, while rMSDD and PNN50 were reduced only in the older age group. Metanephrine levels were significantly reduced in the patients group. Patients with uncontrolled epilepsy had a significantly higher frequency of autonomic dysfunction as assessed by clinical scoring. CONCLUSION: Cardiac autonomic dysfunction is not uncommon in pediatric patients with epilepsy. Altered cardiovascular regulation seems to be related to the epilepsy itself rather than to the characteristics of the disorder.

Job strain does not relate to morning level vanilmandelic acid in Japanese civil servants.

Epidemiologic and clinical studies have related acute and, less frequently, chronic life stress to cardiovascular diseases. In addition, animal models suggest that chronic psychological stress could cause atherosclerosis, probably by increasing sympathetic activation. In this cross-sectional study we evaluated the association between job strain, as one of the markers of workplace stress, and the urinary excretion of Vanilmandelic acid (VMA) upon awakening as a sympathoadrenal activity marker in the morning. Subjects were 936 male and 823 female civil servants working in departments related to the municipality of a city in Toyama Prefecture, Japan, in the spring of 2001. VMA was measured by high-performance liquid chromatography. We found that there was an age dependent increase in the level of VMA and females had higher VMA levels than males. Males who were current smokers had significantly lower VMA levels than nonsmokers after adjusting for age. Job strain level did not relate to VMA concentration in urine after adjusting for age and smoking status both in men and women. In addition, working and sleeping hours as predictor variables were also not associated with urinary VMA levels upon awakening in the morning. In conclusion, it seems that job strain does not independently relate to the sympathoadrenal activity, but the interaction between job strain and other variables such as personal characteristics and environmental factors and their relation with sympathoadrenal activity should further be explored.

Assessment of cold induced alterations in catecholamine turnover of lean and glutamate-treated obese rats.

Excretion of norepinephrine (NE) and vanillylmandelic acid (VMA) in urine as well as NE-turnover in tissues from lean and glutamate-treated obese rats were determined in warm and cold environment. NE-and VMA-excretion in urine was elevated by cold exposure, indicating an activation of the sympathetic nervous system in animals of both groups. Organspecific NE-turnover responds with higher sensitivity to cold in obese rats but without complete compensation in brown adipose tissue. Urinary NE- and VMA- excretion as well as NE-turnover in organs confirmed that cold exposure activates the sympathetic nervous system. Measurement of NE-turnover in tissues gives organspecific information regarding alterations in sympathetic activity during cold exposure, whereas excretion of NE and VMA in urine is a summarizing measure for the whole body turnover only.

Direct assay of 3-methoxy-4-hydroxy-phenylglycol (MHPG) in human saliva; a new approach in the assessment of noradrenergic function.

The present paper offers a simple, reliable and specific method for the evaluation of salivary level of conjugated MHPG in 16 children (5-16 years) selected as control group. In the chosen group, we established the urinary level of NA: 17.64 +/- 1.38 micrograms/24 h, of A: 5.87 +/- 0.44 micrograms/24 h, of MHPG: 1.62 +/- 0.14 mg/24 h, of VMA: 1.54 +/- 10.10 mg/24 h. In all patients we assayed salivary MHPG: 57 +/- 5.25 mg/ml; in only 6 patients we also determined salivary NA: 2.21 +/- 0.11 micrograms/l and A: 0.25 +/- 0.03 micrograms/l. The MHPG in saliva correlates with urinary MHPG but doesn't correlate with NA in saliva or in urine. The assay of salivary MHPG may represent a non-invasive procedure of evaluation of noradrenergic tonus in children.

Energy expenditure during carbohydrate overfeeding in obese and nonobese adolescents.

Basal metabolic rate (BMR), thermic effect of food (TEF), and total daily energy expenditure (TDEE) were measured in six nonobese and seven obese adolescents during periods of weight maintenance and 2 wk of carbohydrate overfeeding. BMR and TEF were measured by indirect calorimetry and TDEE by the doubly labeled water method. Fasting blood levels of insulin, thyroid, and norepinephrine (NE) and urinary excretion of NE and 4-hydroxy-3-methoxymandelic acid (VMA) were measured before and after overfeeding. Energy intake was 1.61 X BMR during maintenance and 2.45 X BMR during overfeeding. BMR increased comparably in both groups during overfeeding (obese, 7.9 +/- 1.2%, nonobese, 8.6 +/- 1.9%). TEF was similar and did not change significantly during overfeeding (maintenance: obese, 9.4 +/- 0.6%; nonobese, 9.8 +/- 0.4% vs. overfeeding: obese, 8.6 +/- 0.3%; nonobese, 9.2 +/- 0.7%). TDEE did not differ significantly between obese and nonobese. Increases in insulin and 3,5,3'-triiodothyronine levels were significant but did not differ between the two groups. Plasma NE and urinary excretion of VMA and NE did not increase during overfeeding. The thermogenic response to food or to overfeeding does not appear reduced in obese adolescents, nor does facultative thermogenesis appear to be a significant factor in weight maintenance in adolescents.

Determination of urinary homovanillic and vanillylmandelic acids from dried filter paper samples: assessment of potential methods for neuroblastoma screening.

Urinary homovanillic (HVA) and vanillylmandelic (VMA) acids were analyzed on 200 random urine samples from patients with neuroblastoma and controls, after the samples had been dried onto absorbent filter paper. The acids were determined quantitatively by gas chromatography (GC) and qualitatively by thin layer chromatography (TLC). The results were analyzed for correlation between liquid urine samples and urine dried on filter paper and between TLC and GC methods. A high overall correlation for HVA and VMA (99%) was found between liquid and dried filter samples analyzed by GC. The correlations were more significant for samples with elevated levels of these acids than for those with normal levels. Normalization of the results to the urinary creatinine concentration (UCr) is indicated due to variations in urine concentration. Results from TLC analysis showed a false positive rate of 3.5% and a false negative rate of 0.5% compared to GC analysis. This work suggests that a combination of a sensitive TLC method with a rapid quantitative GC method would be suitable for mass neuroblastoma screening in infants.

Feasibility of chemical screening of urine for neuroblastoma case finding in infancy in Quebec.

Neuroblastoma is the most common fatal solid tumour of childhood. Studies in Japan suggest that screening urine at 6 months for tumour-derived metabolites greatly improves early case finding and prognosis. The incidence rate of neuroblastoma in Quebec is at least 1 per 10,330 live births, higher than that of all other diseases responding to early treatment except congenital hypothyroidism screened for in the Quebec Network of Genetic Medicine. The feasibility of chemical screening of urine for elevated levels of homovanillic acid and vanillylmandelic acid in Quebec was assessed. The cost-effectiveness of screening 100,000 infants per year would be high (cost-benefit ratio 2.4), with a net saving of about $280,000 and eight lives per year. The estimated cost of adding neuroblastoma screening to the existing urine metabolite screening program is $70,700. The apparent sensitivity of the proposed test is 0.859 and the rate of false-positive results about 0.1%, both acceptable values. The attitude of potential participants toward the present urine screening program and the addition of a "tumour test" was positive. The results indicate that a pilot study of neuroblastoma screening in Quebec could be undertaken.

Assessment of the diurnal variations in urinary homovanillic and vanillylmandelic acid excretion for the diagnosis and follow-up of patients with neuroblastoma.

The diurnal variation of urinary homovanillic acid (HVA) and vanillylmandelic acid (VMA) was studied in neuroblastoma patients and in a control group. Urinary HVA and VMA levels in four sequential 6-hour urine collections within a 24-hour period were compared. HVA and VMA levels were expressed in microgram/mg of urinary creatinine (UCr) and in mg/6h specimens. No statistically significant variations between the four time intervals were found when expressed in microgram/mg UCr or mg/6h. The small variations that exist in the excretion of HVA and VMA during different periods of the day are due to variations in renal excretion rather than variations in production. The results from this study indicate that a random urine sample should be as good as a 24-hour collection for diagnosis and follow-up of neural crest tumors.

Assessment of norepinephrine secretion and production.

A noninvasive method is described for assessing the disposition of NE produced by peripheral sympathetic neurons. Total NE production is estimated from integrated NE metabolite excretion. The rate at which NE enters the bloodstream (apparent NE secretion) is estimated by isotope dilution analysis. The rate at which NE is metabolized locally is calculated by subtracting apparent NE secretion from NE production. In 10 normal volunteers, basal NE production by the peripheral sympathetic nervous system was 11.1 +/- 1.0 nmol/m2/min. Apparent NE secretion, by contrast, was only 2.4 +/- 0.18 nmol/m2/min. Our data thus demonstrate that in normal individuals under basal conditions, at least 78% of the NE produced by the sympathetic nervous system undergoes local metabolism and that no more than 22% of NE diffuses into the circulation.

Mass screening of neuroblastoma in infancy.

A vanillylmandelic acid spot test for screening infants for neuroblastoma was developed in cooperation with 11 health centers in Kyoto, Japan. In this system, the parent sends a filter paper spotted with the infant's urine at 6 or 7 months of age. From 78,331 infants screened during the six years from July 1973 to September 1979, four asymptomatic infants with neuroblastoma were discovered. Three of them have already completed their treatments and have recovered. The cost of screening 13,000 infants per year was $7,500. One false-negative test was encountered, as were a number of false-positive tests.

Socialization and the human physiologic response to crowding.

Cross-sectional data were analyzed for a possible relationship between household densities and physiologic alteration, based on socialization experiences with siblings in an earlier home environment. The measure of household density was persons-per-room and the measure of physiologic alteration was urinary vanillylmandelic acid. The results show an interaction between number-of-sibs and number-of-younger-sibs, with a statistically significant positive correlation between household densities and VMA values for subjects with fewer total sibs and no younger sibs, while a negative correlation was observed for subjects with one or more younger sibs. One possible interpretation of these results is that the physiologic response to crowding in humans is dependent at least in part on the earlier socialization experiences of the individual.

PIP: The relationship between crowding, the individual's socialization, and physiologic alteration was studied in 283 females, mostly white, who completed detailed interview forms and provided a urine specimen for vanillymandelic acid (VMA) analysis. VMA is a urinary catecholamine metabolite and indicator of sympathetic adrenal medullary activity. Significantly fewer than expected of those with incomes below $5000 a year supplied specimens so the sample is biased toward those with higher incomes. Household densities ranged as high as 2.2 persons per room, but most were 1 or less per room with mean density of .61. The overall partial correlation between VMA values and household density, controlling for age, is -.040. This, however, breaks down into 2 distinct groups. Those with 0-1 siblings had a strong positive correlation while those with larger numbers of sibs had a weaker negative correlation (p less than .025). Even greater difference is found when VMA density is based on number of younger sibs. Overall partial correlation for respondents with no younger sibs is strongly positive (p less than .01) while those with more showed weaker correlations. 2 explanations are offered. Those who had not experienced crowding as a youngster would have more difficulty developing coping strategies for crowded conditions, or an individual reacts with stress whenever current living conditions are substantially different from those experienced as a child.