Free Metadrenalines Research Articles

A review of the management of positive biochemical screening for phaeochromocytoma and paraganglioma: a salutary tale.

Phaeochromocytomas (PC) and paragangliomas (PGL) are rare neuroendocrine tumours of chromaffin cells. Diagnosis depends on biochemical evidence of excessive production of catecholamines. This is straightforward when test results are orders of magnitude above the concentrations expected in healthy individuals and those with essential hypertension. Equivocal results pose a management dilemma. We reviewed biochemical screens that were positive and the ensuing management for PC/PGL at our institution. The objective was to inform the development of a standardised approach to investigation and clinical follow-up. All records of positive biochemical screening for PC/PGL were extracted from the laboratory information system between January 2004 and June 2012. Clinical notes of patients with positive results were reviewed. A total of 2749 biochemical screens were performed during the evaluation period. Of these, 106 (3.9%) performed on 82 patients were positive. Chart review determined that 12/82 patients had histologically confirmed PC/PG. Of the 70 patients remaining, the most common indication for biochemical screening was hypertension and the medical subspecialty most frequently requesting the test was Endocrinology. The primary team carried out repeat testing on 35/70 (50%) patients and in 29 results normalised. Notably, 35/70 (50%) patients did not have any follow-up of positive test results. This study highlights the necessity for a standardised diagnostic protocol for PC/PGL. We suggest that appropriate follow-up of borderline-elevated results should first include repeat biochemical testing. This should be performed under standardised pre-analytical conditions and where possible off all potentially interfering medications, measuring plasma free metadrenalines.

Urinary free (unconjugated) metadrenalines in different hereditary forms of catecholamine-secreting phaeochromocytoma/paraganglioma

Catecholamine-producing neuroendocrine tumours are found in chromaffin cells of the adrenal medulla (phaeochromocytoma) or extra-adrenal paraganglia (paraganglioma), known collectively as PPGLs. In approximately a quarter or more of cases of PPGL, these rare tumours arise as a result of germline mutations of several tumour susceptibility genes. At the Crosshouse laboratory, urine tests include free metadrenalines (fMAs) (also known as free metanephrines) which demonstrate superior sensitivity over that obtained by urinary vanillyl mandelic acid, catecholamines or plasma catecholamines in the diagnosis of PPGL. This retrospective audit was to determine if urinary fMAs offered discrimination among the hereditary forms of PPGL. Retrospective biochemical and genetic data were gathered from 1997 to 2011. The identified urine specimens were those obtained at the time of first diagnosis or recurrence of PPGL. Results of catecholamines and metabolites were standardized as multiples of their respective relevant upper reference limits (URLs). Results were available for 29 affected patients (15 females and 14 males), median age 26 (range 9-63) years, comprising three mutation groups: succinate dehydrogenase subunit B or D ([SDHB/D] 16 patients), multiple endocrine neoplasia type 2 ([MEN 2] 6 patients) and von Hippel-Lindau disease ([VHL] 7 patients). The parent catecholamines exhibited increased values for noradrenaline (NA) and/or adrenaline (AD) for 25/29 (86.2%) patients. Either or both free normetadrenaline (fNMA) and fMA were elevated in 29/29 (100%) patients. The ratio of the multiples of URL for fMA/fNMA displayed a clearer separation of MEN 2 patients from those with SDHB/D or VHL than did the equivalent AD/NA ratio.

Improved plasma free metadrenaline analysis requires mixed mode cation exchange solid-phase extraction prior to liquid chromatography tandem mass spectrometry detection

The investigation and effective management of phaeochromocytoma involves biochemical measurement of either conjugated total urine or plasma free metadrenalines. Current analytical methods include enzyme-linked immunosorbent assays, high-performance liquid chromatography (HPLC) with electrochemical detection (ECD) or liquid chromatography tandem mass spectrometry (LCMS/MS). Since the first two methods are either extremely laborious, necessitate low sample run numbers, result in slow turnaround times or are subject to analytical interference, a robust, routine clinical method is not achievable. We established a novel sample preparation method to measure plasma free metadrenalines using LCMS/MS. Three different solid-phase extraction (SPE) methods were compared: hydrophilic-lipophilic balance sorbent (HLB), weak cation exchange (WCX) and mixed mode cation exchange (MCX) and their ability to remove interfering compounds prior to LCMS/MS analysis. Maximum recovery of plasma free metadrenaline and plasma free normetadrenaline were achieved by positively charging compounds prior to SPE application. Compared with HLB and WCX cartridges, MCX extraction resulted in chromatography without co-eluting interference with superior assay precision and accuracy. Additionally, samples that could not be quantified because of interference using HPLC/ECD could be readily assayed using this new method. The use of the MCX SPE method with LCMS/MS detection provides an improved assay to measure plasma free metadrenalines in comparison to many available alternative methods.

Age-related medical decision limits for urinary free (unconjugated) metadrenalines, catecholamines and metabolites in random urine specimens from children

Neuroblastoma is the most common extracranial solid tumour in childhood (8% of all childhood cancers), the most frequently diagnosed in infancy, and has one of the highest death rates, while chromaffin tumours rarely present in childhood. Both tumour types produce catecholamines and their metabolites. It is difficult to produce reference ranges for tests in children, and currently, no age-related medical decision limits for free metadrenalines (free metanephrines) in random urine specimens exist in the paediatric literature. Results of vanillylmandelic acid (VMA), 5-hydroxyindoleacetic acid, homovanillic acid (HVA), noradrenaline (NA), adrenaline, dopamine (DA), free normetadrenaline (fNMA), free metadrenaline and free 3-methoxytyramine (f3MT) in 1658 random urines obtained from infants, children and young adults were measured by high performance liquid chromatography with electrochemical detection. Specimens were excluded from consideration if obtained from the following categories, i.e. (a) harbouring neuroblastic, chromaffin, carcinoid or other tumours or malignancies; (b) medical conditions having known association with excess catecholamine excretion; (c) patients administered catecholamine or paracetamol; (d) overly dilute urine; and (e) manifesting outlying values following visual inspection. There remained 872 specimens that were grouped into seven age ranges (<1; 1 or 2; 3 or 4; 5-7; 8-10; 11-13; 14-19 y) for which medical decision limits were determined for each analyte. There was no significant difference between the results for boys or girls. In 55 patients harbouring neuroblastic tumours, HVA (54/55), f3MT (14/16), VMA (45/53) and DA (43/53) were the most frequently elevated analytes at time of diagnosis. In 11 patients presenting in childhood with chromaffin tumours, fNMA (11/11) followed by NA (10/11) were the most frequently elevated. Discussion The likely reasons for outlying or missing values, together with the reasons for variation in the distinctive biochemical patterns of analytes exhibited in individuals harbouring either neuroblastic or chromaffin tumours are discussed.

Measurement of urinary metadrenaline and normetadrenaline by liquid chromatography tandem mass spectrometry for the diagnosis of phaeochromocytoma

Measurement of metadrenalines has been recommended in the investigation of phaeochromocytoma. Urinary assays remain the most common; however, drug interference is still one of the main challenges for analytical systems. We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of total urinary normetadrenaline and total urinary metadrenaline, which does not require extraction procedures prior to analysis. Total urinary normetadrenaline and total urinary metadrenaline were individually quantified by electrospray ionization in the multiple-reaction monitoring mode. Deuterated internal standards were used and an acid hydrolysis step was used to convert conjugated metabolites into free metadrenalines. Chromatographic separation was achieved using a Phenomenex Luna 3 micro PFP column followed by analysis on an API 3200 LC-MS/MS. Linearity was exhibited across the calibration range for both normetadrenaline (r = 1, P < 0.0001) and metadrenaline (r = 1, P < 0.0001) with the limit of quantification of 0.05 and 0.02 micromol/L, respectively. Intra-assay imprecision for both normetadrenaline and metadrenaline was less than 5.5% with % coefficient of variations of less than 4%. Inter-assay imprecision was less than 13%. Neither noradrenaline or adrenaline interfere with the assay as determined by the spiking of samples with high concentrations of noradrenaline or adrenaline (P > 0.05). Acceptable analytical performance was seen with comparison to a high-performance liquid chromatography method and on External Quality Assessment returns. An analytically simple and sensitive method has been developed and evaluated for the analysis of total urinary normetadrenaline and total urinary metadrenaline which is now in routine use.

Parkinson's disease: the effect of L-dopa therapy on urinary free catecholamines and metabolites

L-dopa is an important antiparkinsonian drug. It is a precursor of dopamine and the other catecholamines. Potentially, administration of L-dopa could lead to increased urinary excretion of catecholamines and their metabolites to abnormal amounts. The current study aimed to determine these excretions in patients with Parkinson's disease (PD) receiving L-dopa compared with suitable controls. This is the first assessment of the effect of exogenous administration of L-dopa on urinary free metadrenalines. Using one-way analysis of variance (ANOVA), urine catecholamines and metabolites, expressed as mmol per mole creatinine, were compared in: patients with PD who were receiving L-dopa; patients with PD but not receiving L-dopa; and patients without PD who were being investigated for the presence of phaechromocytoma but were found not to have the disease. Significantly higher values for urinary dopamine, homovanillic acid, free normetadrenaline and free metadrenaline were found in patients with PD receiving L-dopa compared with the other two control groups. In all the patients with PD, these four analytes were significantly correlated with daily dose of L-dopa. L-dopa therapy can result in production of false positives for urinary excretion of dopamine, homovanillic acid, free normetadrenaline or free metadrenaline and thereby decrease the diagnostic value of these measurements in identifying phaeochromocytoma and related tumours.

Paracetamol-associated interference in an HPLC-ECD assay for urinary free metadrenalines and catecholamines.

Some high-performance liquid chromatography techniques with electrochemical detection for urinary catecholamines and their metabolites can be subject to interference from drugs and their metabolites. Prime amongst these interferences are those due to paracetamol ingestion. The prevalence of paracetamol contamination was determined by measuring the drug in all patient specimens submitted for catecholamine analysis over a 2-month period. These findings were then related to the proportion of unreportable results at each range of specimen paracetamol concentration. The apparent results from a small representative sample of the paracetamol-positive specimens are illustrated. Approximately one-third of urine specimens were found to contain paracetamol. Low-level contamination can produce apparent patterns of results which may easily be confused with those found in predominantly adrenaline-, or metadrenaline-secreting phaeochromocytomas. Despite the fact that significantly less than 5% of an oral dose of paracetamol is excreted as the free drug, its analysis provides a good surrogate marker for the likelihood of spurious results. It is useful to know prior to urinary free metadrenaline or catecholamine analysis which urine specimens are likely to contain potentially interfering paracetamol metabolites.

Plasma metadrenalines: do they provide useful information about sympatho-adrenal function and catecholamine metabolism?

1. The clinical utility of plasma metadrenalines for examination of sympatho-adrenal function and catecholamine metabolism was assessed from plasma measurements of these metabolites in a number of clinical conditions (hypertension, cardiac failure, bilateral adrenalectomy and X-chromosomal deletions of the gene for monoamine oxidase), and before and during activation of sympathetic outflow or infusions of noradrenaline and adrenaline. 2. Plasma concentrations of normetadrenaline were less than 25% of those of noradrenaline, concentrations of metadrenaline and adrenaline were similar and those of sulphate-conjugated metadrenalines were 20- to 30-fold higher than free metadrenalines. Hypertensive patients had elevated plasma concentrations of adrenaline, noradrenaline and conjugated but not free metadrenalines. Cardiac failure patients had 2- to 4-fold increases in plasma noradrenaline and free and conjugated normetadrenaline. Adrenalectomy resulted in undetectable plasma concentrations of adrenaline, 91-97% decreases in free and conjugated metadrenaline and a 40% decrease in normetadrenaline relative to noradrenaline. Patients with X-chromosomal deletions of the gene for monoamine oxidase had 6- and 16-fold increases in plasma free and conjugated normetadrenaline and 2- and 4-fold increases in free and conjugated metadrenaline. 3. Infusion of catecholamines increased plasma concentrations of free metadrenalines by less than 6% of increases in precursor amines, indicating that most plasma normetadrenaline (84%) and metadrenaline (90%) is derived from metabolism of catecholamines before their entry into the circulation. Considerable O-methylation of catecholamines within the adrenals explains why sympatho-adrenal activation resulted in smaller proportional increases in plasma metadrenalines than catecholamines. 4. Plasma metadrenalines provide supplementary information about sympatho-adrenal activity to that provided by catecholamines, but are more useful for examination of the extraneuronal inactivation of catecholamines, particularly detection of neurochemical phenotypes in genetic disorders of catecholamine metabolism. Significant formation of metadrenalines within chromaffin tissue explains why measurements of plasma metadrenalines provide an extraordinarily sensitive method for diagnosis of phaeochromocytoma.

Metabolic products of adrenaline (epinephrine) during long-term constant rate intravenous infusion in the human

Four subjects were infused with 0.063 μg/kg/min of dl-adrenaline-2- 14C (10 μc) for 8 hr. Urine was collected at 2-hr intervals for 12 hr and then at the end of 18 and 24 hr. The urinary metabolites were separated and quantitated by combination column and filter paper chromatography. The radioactivity recovered in the urine reached a constant level 4 hr after the beginning of the infusion and continued at a constant level for 4 hr. The primary metabolites of adrenaline, i.e. metradrenaline and 3,4-dihydroxymandelic acid (DOMA), were recovered in constant amounts after 2 hr, whereas the secondary metabolites, 3-methoxy-4-hydroxymandelic acid (MOMA) and metadrenaline sulfate, were not recovered in constant amounts until after 4 hr. During this period of constant urinary radioactivity, the infusion rate was 1.25 μc/hr, DOMA was recovered in the urine at 0.018 μc/hr, free metadrenaline at 0.031 μc/hr, metradrenaline sulfate at 0.30 μc/hr and MOMA at 0.24 μc/hr. By extrapolating these results in terms of the normal endogenous rate of adrenaline secretion (860 μc/24 hr), it can be calculated from Table 1 that on the order of 100–300 μg/hr of MOMA normally occurs in the urine as the result of adrenaline metabolism. Since the normal urinary output of MOMA is 3–5 mg/24 hr, this then means that most of the MOMA is derived from endogenous noradrenaline.