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.

Clinical and biochemical assessment of depressive symptoms in patients with Alkaptonuria before and after two years of treatment with nitisinone.

OBJECTIVE: Concerns exist over hypertyrosinaemia that is observed following treatment with nitisinone. It has been suggested that tyrosine may compete with tryptophan for uptake into the central nervous system, and or inhibit tryptophan hydroxylase activity reducing serotonin production. At the National Alkaptonuria (AKU) Centre nitisinone is being used off-licence to treat AKU, and there is uncertainty over whether hypertyrosinaemia may alter mood. Herein results from clinical and biochemical assessments of depression in patients with AKU before and after treatment with nitisinone are presented. PATIENTS AND METHODS: 63 patients were included pre-nitisinone treatment, of these 39 and 32 patients were followed up 12 and 24 months after treatment. All patients had Becks Depression Inventory-II (BDI-II) assessments (scores can range from 0 to 63, the higher the score the more severe the category of depression), and where possible urinary monoamine neurotransmitter metabolites and serum aromatic amino acids were measured as biochemical markers of depression. RESULTS: Mean (±standard deviation) BDI-II scores pre-nitisinone, and after 12 and 24 months were 10.1(9.6); 9.8(10.0) and 10.5(9.9) (p ≥ 0.05, all visits). Paired scores (n = 32), showed a significant increase at 24 months compared to baseline 10.5(9.9) vs. 8.6 (7.8) (p = 0.03). Serum tyrosine increased at least 6-fold following nitisinone (p ≤ 0.0001, all visits), and urinary 3-methoxytyramine (3-MT) increased at 12 and 24 months (p ≤ 0.0001), and 5-hydroxyindole acetic acid (5-HIAA) decreased at 12 months (p = 0.03). CONCLUSIONS: BDI-II scores were significantly higher following 24 months of nitisinone therapy in patients that were followed up, however the majority of these patients remained in the minimal category of depression. Serum tyrosine and urinary 3-MT increased significantly following treatment with nitisinone. In contrast urinary 5-HIAA did not decrease consistently over the same period studied. Together these findings suggest nitisinone does not cause depression despite some observed effects on monoamine neurotransmitter metabolism.

A simple, fast and low-cost turn-on fluorescence method for dopamine detection using in situ reaction.

A simple, fast and low-cost method for dopamine (DA) detection based on turn-on fluorescence using resorcinol is developed. The rapid reaction between resorcinol and DA allows the detection to be performed within 5 min, and the reaction product (azamonardine) with high quantum yield generates strong fluorescence signal for sensitive optical detection. The detection exhibits a high sensitivity to DA with a wide linear range of 10 nM-20 µM and the limit of detection is estimated to be 1.8 nM (S/N = 3). This approach has been successfully applied to determine DA concentrations in human urine samples with satisfactory quantitative recovery of 97.84%-103.50%, which shows great potential in clinical diagnosis.

Spectrophotometric determination of dopamine hydrochloride in pharmaceutical, banana, urine and serum samples by potassium ferricyanide-Fe(III).

In the present work, we developed a simple, sensitive and inexpensive method to determine dopamine hydrochloride using potassium ferricyanide-Fe(III) by spectrophotometry. The results show that Fe(III) is deoxidized to Fe(II) by dopamine hydrochloride at pH 4.0, and then Fe(II) reacts with potassium ferricyanide to form a soluble prussian blue (KFe(III)[Fe(II)(CN)6]). The absorbance of this product was monitored over time using a spectrophotometer at an absorption maximum of 735 nm, and the amount of dopamine hydrochloride could be calculated based on the absorbance. A good linear relationship of the concentration of dopamine hydrochloride versus absorbance was observed, and a linear regression equation of A = 0.022 + 0.16921C (microg mL(-1)) was obtained. Moreover, the apparent molar absorption coefficient for the indirect determination of dopamine hydrochloride was 3.2 x 10(4) L mol(-1) cm(-1). This described method has been used to determine dopamine hydrochloride in pharmaceutical, banana, urine and serum samples with satisfactory results.

Urinary excretion of catecholamines, cortisol and their metabolites in Meishan and large white sows: validation as a non-invasive and integrative assessment of adrenocortical and sympathoadrenal axis activity.

Urinary free corticoids (cortisol and cortisone), catecholamines (norepinephrine or NE, epinephrine or E, dopamine or DA, and their O-methoxylated metabolites) as well as creatinine (Cr) were analysed in 42 spontaneously voided urine samples from Large White (LW, n = 20), Meishan (MS, n = 6), and LW x MS (F1, n = 16) lactating sows. The cortisol concentration in the urine of MS (28.1 pg/micrograms Cr) was five-fold greater than that of LW sows (6.2 pg/micrograms Cr, P < 10(-4)). F1 were intermediate (12.0 pg/micrograms Cr). Mean cortisone concentration was also larger in MS (13.5 pg/micrograms Cr) compared to LW (7.1 pg/micrograms Cr, P < 0.01). Although the differences were less pronounced, the concentrations of the catecholamines were also greater in MS than in LW sows (norepinephrine: 25.4 versus 5.9 pg/micrograms Cr, epinephrine: 8.7 versus 2.8 pg/micrograms Cr and dopamine: 59.2 versus 17.8 pg/micrograms Cr, P < 10(-4)). These results confirmed the hypercortisolism state of MS pigs previously shown by plasma cortisol assay and supported the hypothesis that the sympathetic nervous system is hyperactive in this breed. These urinary investigations may offer possible applications for the assessment of chronic stress.

Assessment of renal dopaminergic system activity during the recovery of renal function in human kidney transplant recipients.

BACKGROUND: The urinary excretion of free dopamine has been used as an index of the renal synthesis of amine. However, it is now well recognized that in the kidney, newly-formed dopamine is significantly inactivated through deamination to 3,4-dihydroxyphenylacetic acid (DOPAC) by monoamine oxidase (MAO). The aim of the present study was to assess the renal dopaminergic system activity during the recovery of renal function in kidney transplant recipients and to assess which parameters are appropriate for the evaluation of renal amine synthesis under these conditions. METHODS: Twenty-four-hour urinary excretion of L-DOPA, dopamine and its metabolites (DOPAC; 3-MT; HVA) were continuously monitored in 19 renal transplant recipients from the first day of surgery until the twelfth day post-transplantation. RESULTS: In 11 patients (Group 1), renal function consistently recovered throughout the study (plasma creatinine levels decreased from 6.2 +/- 0.4 to 2.1 +/- 0.1 mg/dl). Eight patients presented with acute tubular necrosis (Group 2) and minimal renal function was maintained until the twelfth post-operative day. The urinary excretion of L-DOPA did not differ throughout the study between the two groups of patients. In contrast, the 24-h urinary levels of dopamine, DOPAC and HVA were significantly higher throughout the study in patients of Group 1: dopamine (Group 1, 179 +/- 26 to 422 +/- 51 nmol/24 h; Group 2, 25 +/- 3 to 57 +/- 13 nmol/ 24 h), DOPAC (Group 1, 698 +/- 57 to 3487 +/- 414 nmol/ 24 h; Group 2, 158 +/- 22 to 1014 +/- 193 nmol/24 h) and HVA (Group 1, 13,058 +/- 1199 to 20,387 +/- 1559 nmol/ 24 h; Group 2, 4140 +/- 848 to 15,219 +/- 1037 nmol/24 h). CONCLUSIONS: The recovery of renal function in renal transplant recipients is accompanied by an enhanced ability to synthesize dopamine and inactivate it to DOPAC and HVA. It is suggested that the urinary levels of DOPAC may be a useful parameter for the assessment of dopamine formation in renal tissues.

Assessment of renal dopaminergic system activity during cyclosporine A administration in the rat.

1. Administration of cyclosporine A (CsA; 50 mg kg-1 day-1, s.c.) for 14 days produced an increase in both systolic (SBP) and diastolic (DBP) blood pressure by 60 and 25 mmHg, respectively. The urinary excretion of dopamine, DOPAC and HVA was reduced from day 5-6 of CsA administration onwards (dopamine from 19 to 46%, DOPAC from 16 to 48%; HVA from 18 to 42%). In vehicle-treated rats, the urinary excretion of dopamine and DOPAC increased (from 7 to 60%) from day 5 onwards; by contrast, the urinary excretion of HVA was reduced (from 27 to 60%) during the second week. 2. No significant difference was observed between the Vmax and Km values of renal aromatic L-amino acid decarboxylase (AAAD) in rats treated with CsA for 7 and 14 days or with vehicle. 3. Km and Vmax of monoamine oxidase types A and B did not differ significantly between rats treated with CsA for 7 and 14 days or with vehicle. 4. Maximal catechol-O-methyltransferase activity (Vmax) in homogenates of renal tissues obtained from rats treated with CsA for 7 or 14 days was significantly higher than that in vehicle-treated rats; Km (22.3 +/- 1.5 microM) values for COMT did not differ between the three groups of rats. 5. The accumulation of newly-formed dopamine and DOPAC in cortical tissues of rats treated with CsA for 14 days was three to four times higher than in controls. The outflow of both dopamine and DOPAC declined progressively with time and reflected the amine and amine metabolite tissue contents. No significant difference was observed between the DOPAC/dopamine ratios in the perifusate of renal tissues obtained from CsA- and vehicle-treated rats. In addition, no significant differences were observed in k values or in the slope of decline of both DA and DOPAC between experiments performed with CsA and vehicle-treated animals. 6. The Vmax for the saturable component of L-3,4-dihydroxyphenylalanine (L-DOPA) uptake in renal tubules from rats treated with CsA was twice that of vehicle-treated animals. Km in CsA- and vehicle-treated rats did not differ. 7. The decrease in the urinary excretion of sodium and an increase in blood pressure during CsA treatment was accompanied by a reduction in daily urinary excretion of dopamine. This appears to result from a reduction in the amount of L-DOPA made available to the kidney and does not involve changes in tubular AAAD, the availability of dopamine to leave the renal cells and dopamine metabolism. The enhanced ability of the renal tissues of CsA-treated animals to synthesize dopamine, when exogenous L-DOPA is provided, results from an enhanced activity of the uptake process of L-DOPA in renal tubular cells.

Assessment of renal dopaminergic system activity in the nitric oxide-deprived hypertensive rat model.

1. The present paper reports changes in the urinary excretion of dopamine, 5-hydroxytryptamine and amine metabolites in nitric oxide deprived hypertensive rats during long-term administration of NG-nitro-L-arginine methyl ester (L-NAME). Aromatic L-amino acid decarboxylase (AAAD) activity in renal tissues and the ability of newly-formed dopamine to leave the cellular compartment where the synthesis of the amine has occurred were also determined. 2. Twenty four hours after exposure to L-NAME, both systolic (SBP) and diastolic (DBP) blood pressure were increased by 20 mmHg; heart rate was slightly decreased. During the next 13 days both SBP and DBP increased progressively reaching 170 +/- 3 and 116 +/- 3 mmHg, respectively. 3. Baseline urinary excretion of L-DOPA, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT) and homovanillic acid (HVA) during the 4 day period of stabilization averaged 4.4 +/- 0.5, 13.8 +/- 0.3, 37.4 +/- 0.8, 180.0 +/- 2.7 and 206.1 +/- 6.7 nmol day-1, respectively. The urinary excretion of L-DOPA, dopamine and DOPAC, but not that of 3-MT and HVA, were increased from day 6-8 of L-NAME administration onwards (L-DOPA, up to 13.4 +/- 2.1; dopamine, up to 23.0 +/- 1.6; DOPAC, up to 62.8 +/- 3.7 nmol day-1). Baseline daily urinary excretion of 5-hydroxytryptamine and 5-hydroxyindolacetic acid (5-HIAA) averaged 73.5 +/- 1.1 and 241.7 +/- 5.4 nmol day-1, respectively. During the first week of L-NAME administration, the urinary excretion of both 5-hydroxytryptamine and 5-HIAA did not change significantly; however, as was found with dopamine and DOPAC, changes in the urinary excretion of 5-hydroxytryptamine were evident during the second week of L-NAME administration. 4. In experiments performed on homogenates of isolated renal tubules, the decarboxylation of L-DOPA to dopamine was dependent on the concentration of L-DOPA used (10 to 5000 microM) and saturable at 1000 microM. AAAD activity as determined in homogenates (Vmax, in nmol mg-1 protein h-1; Km in microM) was significantly (P < 0.01) higher in rats given L-NAME for 14 days (Vmax = 25 +/- 2; Km = 72 +/- 10) than in control rats (Vmax = 14 +/- 1; Km = 63 +/- 7), rats given L-NAME for 7 days (Vmax = 15 +/- 1; Km = 69 +/- 5) and rats given L-NAME plus L-arginine (Vmax = 13 +/- 1; Km = 60 +/- 3) for 14 days. 5. A considerable amount of the total dopamine formed from added L-DOPA in kidney slices escaped into the incubation medium. The application of the Michaelis-Menten equation to the net transport of newly-formed dopamine allowed the identification of a saturable (carrier-mediated transfer) and a non-saturable component (diffusion). No significant differences in the diffusional rate of transfer(0.14 +/- 0.02 micro mol-1) were observed between the four experimental groups. However, the saturable outward transfer of dopamine (Vmax, in micromol mg-1 protein h-1; Km in microM) was higher in control animals(Vmax= 2.3 +/- 0.2; Km = 568 +/- 67) than that in rats treated with L-NAME for 14 days (Vmax = 0.8 +/- 0.02;Km = 241 +/- 21), but similar to that observed in rats receiving L-NAME plus L-arginine (Vmax= 2.4+/- 0.2; Km= 618 +/- 61); the saturable dopamine outward rate of transfer in rats given L-NAME for 7days (Vmax = 3.9 +/- 0.2; Km = 1006 +/- 32) was higher than in controls.6. In conclusion, the present studies show that the hypertensive response resulting from the long-term administration of L-NAME is accompanied by an increased urinary excretion of dopamine and 5-hydroxytryptamine, which appears to follow an enhanced activity of renal AAAD. The observation that the increased AAAD activity can be reversed by the administration of L-arginine to L-NAME treated rats favours the view that the adaptational response which results in an enhanced AAAD activity probably involves a decrease in the generation of nitric oxide.

In vivo assessment of decarboxylase inhibition or potentiation: urinary dopamine and L-dopa output after L-dopa administration.

In the L-Dopa treated rat, a decreased urinary output of free and conjugated dopamine and an increase in free and conjugated L-Dopa excretion after administration of decarboxylase-inhibiting drugs provide a good in vivo index of Dopa decarboxylase inhibition. With the exception of free dopamine output, which showed an equivocal change, these measurements appear to provide a good yardstick of decarboxylase status in man also. Using this approach, it was not possible to find any evidence of facilitation of decarboxylase action, in L-Dopa-treated parkinsonians given pyridoxine supplements, to account for the ability of this compound to neutralize the beneficial effect of L-Dopa.