Urinary Homovanillic Acid Research Articles

Determination of homovanillic acid in urine by stable isotope dilution and electrospray tandem mass spectrometry

We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of homovanillic acid (HVA), a biochemical marker for catecholamine and neurotransmitter metabolism. Urine specimens are spiked with 5 μg of a stable-isotope labeled internal standard, 13C 6 18O-HVA, and prepared by automated solid phase extraction. Residues were dissolved in acetonitrile: 0.05% aqueous acetic acid and analyzed by MS/MS in the selected reaction monitoring mode (HVA: m/ z 181 to m/ z 137; 13C 6 18O-HVA: m/ z 189 to m/ z 145) after separation using a Discovery RP Amide C16 column. Consecutive calibrations ( n=7) between 0.52 and 16.7 mg/l exhibited consistent linearity and reproducibility. At a urine concentration of 0.51 mg/l, the signal-to-noise ratio for HVA was 21:1. Inter- and intra-assay CVs ranged from 0.3% to 11.4%, at mean concentrations ranging 1.8 to 22.7 mg/l. Recovery of HVA added to urine ranged between 94.7% and 105% (1.25 mg/l added), 92.0% and 102% (5.0 mg/l), and 96.0% and 104% (10 mg/l). LC-MS/MS is well suited to replace an HPLC method for routine HVA determination, by providing positive identification, faster turn around time, virtually no repeat analyses and a 44% reduction of personnel necessary to perform the testing.

Natural course of neuroblastoma detected by mass screening: s 5-year prospective study at a single institution.

To describe various favorable courses of neuroblastoma (NBL) detected by mass screening and to present our observation program as a temporary treatment option, to be used until a final decision is made regarding the mass screening program for 6-month-old infants. Between October 1993 and November 1999, 26 of 51 patients with NBL detected by mass screening were enrolled in our observation program. The criteria for observation included urinary vanillylmandelic acid (VMA) and homovanillic acid (HVA) levels less than 50 microg/mg creatinine, smaller tumor size (< 5.0 cm), preoperative status, and granted informed consent. Patients were divided into four groups according to changes in urinary VMA and HVA values and tumor size. Patients who no longer fulfilled criteria underwent surgery. The observation period ranged from 4 to 73 months. Urinary VMA and HVA levels decreased in 19 of 26 patients, often by age 16 months. Eighteen patients had regressing tumors, and in 10 of these cases, the tumor was undetectable or barely detectable by imaging techniques. Four patients younger than 12 months had increased tumor marker levels and tumor volume, histologically reflecting neuroblastic proliferation. The remaining three patients, all older than 18 months, had varied tumor marker levels but increased tumor volume, histologically reflecting an increase in Schwann cells. No upgrading of tumor stage or unfavorable biologic factor was noted in any patient. None of our patients showed evidence of transition from favorable to unfavorable prognosis, a finding that points to a reduction in the significance of screening as a public health measure. Until results of ongoing screening trials involving older patients have been evaluated, the observation program can be used as a temporary measure to avoid, with little risk, unnecessary surgical intervention.

Simultaneous HPLC of twelve monoamines and metabolites shows neuroblastoma cell line releases HVA and HIAA.

Neuroblastoma is a solid tumor occurring usually in children less than 5 years old. It has been difficult to distinguish neuroblastoma from other childhood tumors through morphological diagnosis. Urine homovanillic acid (HVA), which is a metabolite of dopamine, has been proposed as a diagnostic index. Although increased levels of a serotonin metabolite, 5-hydroxyindole-3-acetic acid (HIAA), have also been observed in urine samples of the patients, they were largely attributed to dietary amines. By using an HPLC system with electrochemical detection, which can simultaneously assay 12 monoamines and metabolites, we showed that HVA and HIAA are two of the most prominent monoamine metabolites in the medium after a neuroblastoma cell line (IMR-32) was cultured for 3 days. Moreover, we found that the levels of HVA and HIAA in the media are proportional to the cell densities. These results suggest that the levels of HVA and HIAA in tissue culture media, or in urine from patients whose dietary amines are well controlled, may provide a valuable diagnostic index for neuroblastoma.

新生児頚部原発神経芽細胞腫例

A neonatal boy presented with a painless, smooth, round mass in the left side of his neck at birth. CT, MRI, and echo examinations revealed a solid tumor with calcification occupying the left upper neck. The levels of serum neuron specific enolase (NSE), urine homovanillic acid (HVA), and vanillylmandelic acid (VMA) were elevated, indicating a diagnosis of neuroblastoma. Surgical resection was performed 23 days later because of airway obstruction due to tumor progression. The resected tumor was 43×35×32mm and was diagnosed as a rosette-fibrillary type of neuroblastoma by histopathological analysis. The tumor was of a favorable histological grade and was classified as stage 2A according to the International Neuroblastoma Staging System. After surgery, the patient received systemic chemotherapy of a regimen including cyclophosphamide and vincristine. He is alive and disease free 4 years after diagnosis.

Low platelet-poor plasma concentrations of serotonin in patients with combat-related posttraumatic stress disorder

Background: Combat-related posttraumatic stress disorder (CR-PTSD) is associated with a dysregulation of various neurotransmitter systems. Methods: We assessed levels of platelet-poor plasma (PPP) norepinephrine (NE), and serotonin (5-HT), and 24-hour urinary excretion of NE, dopamine (DA), and homovanillic acid (HVA) in 17 male outpatients with untreated chronic CR-PTSD (age, 33.1 ± 7.4 years) and 10 normal control subjects (age, 35.8 ± 2.7 years). Results: Compared with the control subjects, the PTSD patients showed significantly lower PPP 5-HT levels, elevated PPP NE levels, and significantly higher mean 24-hour urinary excretion of all three catecholamines (NE, DA, and HVA). The 24-hour urinary HVA values of the CR-PTSD patients correlated significantly and positively with the total Impact of Event Scale scores and the avoidance symptoms cluster scores, and the PPP 5-HT levels correlated negatively with the Hamilton Anxiety Rating Scale scores. The PPP NE/5-HT ratio was significantly higher in the study group than in the control subjects. Conclusions: We believe this combined enhanced noradrenergic activity and diminished 5-HT activity may be relevant to the neurobiology of CR-PTSD.

Screening infants for neuroblastoma does not reduce the incidence of poor-prognosis disease

The Quebec Neuroblastoma Screening Project (QNSP) was initiated to determine whether mass screening would reduce mortality from this tumor in a large cohort of infants. A total of 476,603 children were born in the province of Quebec (PQ) from May 1, 1989 through April 30, 1994 and were eligible for urinary homovanillic acid (HVA) and vanillylmandelic acid (VMA) determination at 3 weeks and 6 months of age. Infants with positive screening were referred to one of four pediatric cancer centers in Quebec for uniform evaluation and treatment. Standardized incidence ratios (SIRs) were calculated for PQ (screened cohort) and for two prospective population-based (nonscreened) controls: the state of Minnesota (MN) and the province of Ontario (ONT). In this study, 91% of the PQ cohort (n = 425,816) was screened at 3 weeks and 74% at 6 months (n = 349,706). Through July 31, 1997 with a follow-up of the cohort for 39–99 months, 128 cases of neuroblastoma were diagnosed in the screened cohort: 45 were detected by screening, 20 were detected clinically before 3-week screening, and 62 were detected clinically having normal screens (58) or never screened (4). Three of 128 cohort patients moved outside PQ and were omitted from the calculations; 63 cases of neuroblastoma would have been expected in PQ during the study period, while 125 were observed (SIR = 1.98, 95% confidence interval [CI[ = 1.64–2.34, p < 0.001). In the controls from MN and ONT, 54 and 92 cases of neuroblastoma have been detected, respectively, compared with 44 and 99 expected (SIR = 1.24 and 0.93). SIR for PQ by age at diagnosis showed a markedly increased incidence at less than 1 year of age, with no reduction in incidence in older age. Limiting analysis to only patients diagnosed at older than 1 year of age with advanced-stage disease, 25 cases were clinically detected in PQ versus 27 expected. Data from the two control groups showed no significant change in stage of disease in children younger than or older than 1 year of age. Preliminary mortality data from a subset of the PQ, ONT, and MN cohorts showed standardized mortality ratios (SMRs) of 1.12, 0.72, and 1.01, respectively. Screening for neuroblastoma markedly increases the incidence in infants younger than 1 year of age, without decreasing the incidence of unfavorable advanced-stage disease in older children. Preliminary mortality data is reported. However, it is unlikely that screening for neuroblastoma will reduce mortality from this disease. Med. Pediatr. Oncol. 31:450–454, 1998. © 1998 Wiley-Liss, Inc.

Development of an enzyme-linked immunosorbent assay with monoclonal antibody for quantification of homovanillic in human urine samples

A monoclonal antibody to homovanillic acid (HVA) was prepared by synthesis of a HVA-protein conjugate (HVA-ovalbumin) as an immunogen, immunization of mice, and the subsequent hybridization technique. Monoclonal antibodies were screened on the basis of sensitivity, specificity, and accuracy. An indirect ELISA was developed for quantification of HVA in human urine. The assay was characterized and shown to have high specificity, with cross-reactivities to vanillylmandelic acid and normetanephrine at 0.18% and <0.1%, respectively. The assay coefficients of variation were <10% within the working range of 0.5-40 mg/L. Initial results from testing urine samples of patients with neuroblastoma and other diseases were validated by HPLC, suggesting that this ELISA method is a reliable and convenient system for quantification of HVA in urine and can be used in the mass screening of neuroblastoma in infants.

Urinary homovanillic acid (HVA) and vanillymandelic acid (VMA) in workers exposed to manganese dust

The neurotoxicity of manganese (Mn) is well known, however, the neurochemical effect caused by this metal is less well investigated. In this study, urinary homovanillic acid (HVA) and vanillymandelic acid (VMA), two end products of catecholamine metabolism, were measured in 39 workers chronically exposed to Mn in a manganese smelting plant. The average duration of Mn exposure was 17.4 yr. Nineteen nonexposed workers were also studied. Concentrations of Mn in serum (MnS) and in urine (MnU) were measured by Zeeman graphite furnace atomic absorption spectrophotometry (ZAAS), and HVA and VMA determined by high performance liquid chromatography (HPLC). For Mn-exposed workers, the concentration of MnS was nearly 2.8 times (1.61 +/- 0.16 mg/L vs 0.56 +/- 0.16 mg/L) and MnU about 4.5 times higher (7.62 +/- 0.17 mg/L vs 1.69 +/- 0.16 mg/L) than the nonexposed. Although the geometric mean concentration of HVA in exposed workers was similar to that of the nonexposed (3.09 +/- 1.39 mg/g cre. vs 2.99 +/- 1.40 mg/g cre.), the VMA concentration was significantly higher (3.02 +/- 1.43 mg/g cre. vs 2.49 +/- 1.58 mg/g cre., p = 0.033). Multiple regression analysis showed that although there were no correlations between any of these parameters with the duration of exposure to Mn, both HVA and VMA showed significant correlations with increase in MnS and MnU. These data provide evidence that exposure to Mn was associated with measurable increase in catecholamine metabolites. This finding is compatible with recent observations in laboratory animals that Mn interferes with neurochemical metabolism.

Determination of homovanillic acid and vanillylmandelic acid in neuroblastoma screening by stable isotope dilution GC-MS.

A method for the quantitative determination of homovanillic acid (HVA) and vanillylmandelic acid (VMA), two metabolites of catecholamines, is presented. The assay is based on gas chromatography/electron impact mass spectrometry. The preparation of 13C-labeled VMA from [13C6]vanillin is described. Together with purchased deuterated HVA the 13C-labeled VMA is used as an internal standard in stable isotope dilution GC/MS. The method involves elution from soaked filter-papers, determination of creatinine content, extraction of HVA and VMA from eluted and urinary samples and derivatization to the di-and tri(trimethylsilyl) derivatives, respectively. The detection limits were found to be 4.0 pg for HVA and 0.8 pg for VMA. The method was applied to the routine determination of urinary HVA and VMA in a range from 5 to 100 ng HVA and VMA per microgram creatinine. The lower limits of pathological concentrations are set at 35 ng micrograms-1 creatinine for HVA and to 20 ng micrograms-1 creatinine for VMA, which are in close correlation with the values from other methods, but with the main advantage of reducing the amount of questionable or elevated results from 6.7% (high-performance liquid chromatography (HPLC) alone) to 0.9% (HPLC and GC/MS).

Increased Urinary Hva Levels in Neuroblastoma Screens Related to Diet, Not Tumor

At present, urinary vanillylmandelic acid (VMA) and/or homovanillic acid (HVA), metabolites of catecholamines, are the most sensitive diagnostic markers of neuroblastoma. They can be measured by the high-performance liquid chromatographic method, which has been used to screen for neuroblastoma in infants in Japan. From 6486 urine samples of 18-month-old children, 103 samples showed high HVA levels over the cutoff point for neuroblastoma, and most of them also showed markedly high levels of 5-hydroxyindoleacetic acid (5-HIAA), a metabolite of serotonin. One of the causes of high HVA excretion might be due to the ingestion of banana, common weaning food in early childhood. Our study showed that it is better to restrict banana ingestion within 24 hours before taking a urine sample for diagnosis of neuroblastoma and that 5-HIAA is a good marker of a pseudopositive result due to banana ingestion.

A study of the potential confounding effects of diet, caffeine, nicotine and lorazepam on the stability of plasma and urinary homovanillic acid levels in patients with schizophrenia

Ten men inpatients who met DSM-III-R criteria for schizophrenia participated. On five occasions at least one week apart, each subject had an intravenous line placed at 0730 after an overnight fast. On each occasion blood samples were drawn at 0800 and hourly thereafter through 1200 noon for measurement of plasma homovanillic acid (HVA). Total four-hour urine collections were obtained for measurement of urinary HVA. Subjects received five experimental conditions, in randomized sequence: no intervention, smoking one cigarette per hour, drinking one caffeinated cola per hour, lorazepam 2 mg IV push, or a high monoamine meal. Baseline (0800) plasma HVA measures showed only minor intrinsic variability. The average standard deviation in baseline plasma HVA over five occasions of measurement was low relative to the changes in HVA produced during treatment with antipsychotic medications. The high monoamine meal significantly elevated plasma HVA, with a similar trend for urinary HVA. Neither caffeine, nicotine, nor lorazepam significantly affected plasma or urinary HVA.

Plasma levels of chromogranin A are directly proportional to tumour burden in neuroblastoma.

A novel animal experimental model involving the human, poorly differentiated, and adrenergic neuroblastoma cell line SH-SY5Y xenotransplanted to subcutaneous tissue of 13 nude rats (WAG rnu/rnu) was used to investigate the usefulness of six proposed neuroblastoma markers. It was shown that the plasma concentrations of human chromogranin A (CgA) as measured by RIA were directly proportional to tumour volume (r = 0.83, P < 0.001). To rule out possible liberation of CgA by tumour cell lysis, the CgA degradation product pancreastatin was also measured in plasma by a specific RIA, but was not detectable. Plasma neurone-specific enolase (NSE) was elevated in tumour-bearing animals (P < 0.01), but did not correlate with tumour volume (r = 0.49, P > 0.05). Urine homovanillic acid (HVA), detected by HPLC, was elevated in tumour-bearing animals (P < 0.01), but did not correlate with tumour volume (r = -0.32, P > 0.05). Urine vanillyl mandelic acid was not detectable. Urine dopamine was found in low concentrations that did not correlate with tumour volume. In summary, although plasma NSE and urinary HVA were elevated in tumour-bearing animals only plasma CgA correlated with tumour burden. This makes CgA a promising biochemical marker for neuroblastomas.

Dopamine turnover in schizophrenia before and after haloperidol withdrawal CSF, plasma, and urine studies

The dopamine hypotheses of schizophrenia and antipsychotic drug action suggest that the dopamine metabolite homovanillic acid (HVA) should change with drug withdrawal and change in clinical state. We designed a study of cerebrospinal fluid (CSF), plasma, and urinary HVA on and off haloperidol to examine the effects of drug withdrawal. CSF and plasma HVA samples were obtained in 72 healthy schizophrenic (DSM-III-R) males (age: 36 +/- 7.4 years), before and after haloperidol withdrawal, which was after 6 weeks on placebo or sooner if they met specific criteria for relapse. We collected three 24-hour urine samples in 34 of these patients. In addition, CSF HVA was obtained in 24 well-screened age-matched male normal controls. HVA was measured with high-pressure liquid chromatography (HPLC). CSF HVA decreased significantly after drug withdrawal, particularly in those who met relapse criteria; drug-free CSF HVA levels were not significantly different from those of normals. Plasma HVA increased significantly after haloperidol withdrawal in relapsing patients, but not in clinically stable patients. Urinary HVA excretion decreased after withdrawal with decreased HVA clearance. We conclude that haloperidol withdrawal had a strong effect on dopamine turnover, whereas the patient's clinical state had only a weak central effect, without affecting total body production of HVA. Conceivably, dopamine involvement in schizophrenia reflects the failure of the homeostatic mechanisms that allow for integration of different functional brain components as needed.

Urinary creatinine adjusted reference ranges for homovanillic and vanillylmandelic acid in children and adults

The relationship between urinary homovanillic acid (HVA), vanillylmandelic acid (VMA) and creatinine is investigated and reference ranges are constructed for HVA and VMA. A total of 769 urine samples were obtained from children and adults. The ratios HVA/creatinine and VMA/creatinine were highly correlated with creatinine concentration; hence, reference to creatinine was found to be an inappropriate adjustment for urinary dilution. An alternative method based upon linear regression is proposed. After allowing for differing levels of creatinine, HVA and VMA were found to be significantly correlated with age, height and weight for individuals aged less than 20 years. In those aged over 20 years, HVA was significantly correlated with weight and VMA with age. Differences between sexes were found in the levels of HVA and VMA, the exception being VMA in the over 20 age group. Reference ranges were constructed for HVA and VMA which are variously dependent upon creatinine, age, sex and weight.

3,4-dihydroxyphenylalanine (DOPA) decarboxylase deficiency and resultant high levels of plasma DOPA and dopamine in unfavorable neuroblastoma.

Neuroblastoma (NB) is a tumor which arises from neural crest cells. In the developing neural crest cells, the induction of 3,4-dihydroxyphenylalanine (DOPA) decarboxylase is more delayed than that of tyrosine hydroxylase and dopamine-beta-hydroxylase. If NB cells are arrested in an early stage of neural crest development, the induction of DOPA decarboxylase is insufficient and the accumulation and secretion of DOPA can be caused. The biochemically immature phenotype is thought to represent the undifferentiated characteristics of the cells and might correlate with the grade of malignancy. To investigate whether the hypothesis is clinically applicable or not, we have measured plasma DOPA, dopamine and urinary catecholamine metabolites in NB patients. The levels of plasma DOPA, dopamine, urinary homovanillic acid (HVA) and vanillactic acid (VLA) were significantly higher in patients with unfavorable NBs and the higher plasma DOPA level was significantly associated with the patients' age (> 1 year old), tumor stage (III, IV) and DNA diploidy. Serial determination of plasma DOPA was a good monitor of the disease course. These results are compatible with the hypothesis on DOPA decarboxylase deficiency and DOPA secretion in undifferentiated, unfavorable NBs. In conclusion, the plasma DOPA can be used to predict patients' prognosis as well as to follow up patients with NB.

Influence of renal clearance on peripheral homovanillic acid measurements in healthy subjects and schizophrenic patients

In order to investigate the effect of renal clearance on plasma homovanillic acid (HVA) concentrations, we examined plasma concentration and urinary excretion of HVA and creatinine per 4 h over 24 h in eight male schizophrenic patients and eight healthy male subjects. Renal clearances of HVA and of creatinine were determined per 4 h period. No significant differences were found between groups in the total 24 h excretion of either HVA or creatinine. Although differences between groups in plasma HVA concentrations and urinary HVA excretion per 4 h did not reach statistical significance, the renal clearance of HVA was significantly lower in the patient group than in the control group. There was no difference between groups in creatinine clearance. Diurnal changes were seen in the renal clearance of HVA and creatinine in both groups. Renal HVA clearance decreased from 23.00–07.00 h, with a coincident decrease in urinary HVA excretion and an increase in plasma HVA concentration. We provide evidence that renal clearance is an important determinant of plasma HVA concentration, and should be considered when interpreting plasma HVA data.

Urinary excretion of homovanillic acid in workers exposed to manganese.

Homovanillic acid, an end product of dopamine catabolism, and manganese (Mn) were measured in the urine of 68 male workers exposed to Mn-containing dust in a dry alkaline battery plant or an Mn oxide and salt producing plant, and in 35 control male subjects. The geometric mean of the airborne concentration of inhalable (total) dust amounted to 0.95 and 1.37 mg/m3 in the dry alkaline battery plant and the Mn oxide and salt producing plant, respectively. In the latter, a higher prevalence of increased values of urinary homovanillic acid concentration was found. In the total population, there was a low but statistically significant positive correlation between the concentration of homovanillic acid and Mn in urine (r = 0.20, P = 0.04) but there was no significant correlation between the level of homovanillic acid in urine and Mn in airborne dust or duration of exposure. This observation might be compatible with the stimulation of dopamine turnover in the brain, which has been observed in the early phase of Mn intoxication in animals. However, the large variability in urinary homovanillic acid excretion in control subjects precludes the use of this biological indicator to detect early interference of Mn with the dopaminergic system.

Creatinine related reference ranges for urinary homovanillic acid and vanillylmandelic acid at 6 months of age.

The relationship between homovanillic acid (HVA), vanillylmandelic acid (VMA), and creatinine in the urine of 6 month old babies has been studied and reference ranges in the form of centiles constructed for HVA and VMA against creatinine. Over 10,000 urine samples were collected from babies in four health districts in the north of England. HVA and VMA concentration, either independently or when divided by creatinine concentration, were dependent upon the absolute concentration of creatinine in the sample. After adjustment for creatinine significant differences in the mean concentration of HVA were found between sexes. No such differences were found for VMA. HVA and VMA were also found to be age dependent. Centiles were constructed using a procedure which makes no distributional assumptions about the data. The net effect of utilising these centiles was to increase the predictive value of a positive screening test from 20% to 40% without any increase in the false negative rate.

Catecholamines in Autistic Disorder: Effects of Amisulpride and Bromocriptine in a Controlled Crossover Study

ABSTRACT The biochemical effects on catecholaminergic systems of the dopamine antagonist amisulpride and the dopamine agonist bromocriptine were evaluated in a double-blind, randomized, crossover study in children with autistic disorder. Plasma levels of dopamine, norepinephrine, and epinephrine; urinary concentrations of homovanillic acid (HVA), vanillyImandelic acid (VMA) and 3-methoxy-4-hydroxyphenylethylene glycol (MHPG); and plasma prolactin were measured. At doses of amisulpride and bromocriptine that had the expected opposing effects on plasma prolactin, the drugs' effects on the catecholaminergic systems were similar. Both agents unexpectedly lowered urinary HVA (total, free, and sulfated) although only amisulpride decreased the HVA levels significantly. This paradoxical decrease in HVA levels suggests that both dopamine agonists and antagonists could act on autoreceptors or presynaptic dopaminergic receptors in the central nervous system. There was no significant action of either drug on plasma epinephrine, urinary VMA, or urinary MHPG, suggesting that neither drug altered norepinephrine or epinephrine systems; however, a weak increase of plasma norepinephrine occurred after amisulpride treatment, consistent with effects observed with other neuroleptics. Neither agent altered plasma dopamine, suggesting that peripheral dopamine metabolism was unchanged. The clinical effects of amisulpride and bromocriptine have been reported to be unexpectedly complementary. The complementary clinical effects of a dopamine agonist and dopamine antagonist might speculatively be related to their similar action on dopamine autoreceptors, leading to a correction of the dopaminergic hyperactivity that has been postulated in autistic children.

Cystic neuroblastoma

In the newborn, cystic masses of the adrenal gland are unusual findings and most are secondary to hemorrhage. We present a patient with a clinical history typical for adrenal hemorrhage who was found to have a thick walled cystic adrenal mass on both physical and ultrasound examination. During evaluation the mass decreased in size, but vanillylmandelic acid (VMA) and homovanillic acid (HVA) levels were elevated. Laparotomy and left adrenalectomy confirmed the diagnosis of a cystic neuroblastoma. Adrenal cyst, adrenal abscess, and cystic neuroblastoma are all rare entities in the newborn. We recommend that all cystic masses of the adrenal be evaluated by urinary VMA and HVA and that the possibility of cystic neuroblastoma be kept in mind when an adrenal cystic mass is followed nonoperatively.