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- Materials and methods
- Supporting Information
Dopamine is a catecholamine that serves as a neurotransmitter in the central and peripheral nervous system. Non-invasive, reliable, and high-throughput techniques for its quantification are needed to assess dysfunctions of the dopaminergic system and monitor therapies. We developed and validated a competitive ELISA for direct determination of dopamine in urine samples. The method provides high specificity, good accuracy, and precision (average inter-assay variation < 12%). The analysis is not affected by general urinary components and structurally related drugs and metabolites. The correlation between ELISA and LC-MS/MS analyses was very good (r = 0.986, n = 28). The reference range was 64–261 μg/g Cr (n = 64). Week-to-week biological variations of second morning urinary dopamine under free-living conditions were 23.9% for within- and 35.5% for between-subject variation (n = 10). The assay is applied in monitoring Parkinson's disease patients under different treatments. Urinary dopamine levels significantly increase in a dose-dependent manner for Parkinson's disease patients under l-DOPA treatment. The present ELISA provides a cost-effective alternative to chromatographic methods to monitor patients receiving dopamine restoring treatment to ensure appropriate dosing and clinical efficacy. The method can be used in pathological research for the assessment of possible peripheral biological markers for disorders related to the dopaminergic system.
The catecholamines (dopamine, norepinephrine, and epinephrine; see Fig. 1), are a class of neurotransmitters and hormones that play a key role in the regulation of many physiological processes in both the central and peripheral nervous system. They are involved in the pathophysiology of many neurological, psychiatric, endocrine, and cardiovascular diseases (Eisenhofer et al. 2004; Goldstein 2010; Tayebati et al. 2011). A number of studies suggest that catecholamines are also key players in the modulation of immune responses (Bergquist et al. 1998; Franco et al. 2007; Haroon et al. 2011).
The dopaminergic system is thought to affect a wide range of behaviors and functions, including cognition, motor activity, pleasure/reward, memory, eating and drinking behaviors, neuroendocrine regulation, and selective attention (Dalley and Everitt 2009). A deficiency in dopamine can cause impairment in these biological functions. In addition, abnormalities in the dopamine system have been associated with movement disorders (Parkinson's disease, Segawa disease), neuropsychiatric disorders (anxiety, depression, attention-deficit hyperactivity disorder (ADHD), drug, and alcohol addiction, suicide), and metabolic diseases (Willner 1983; Naranjo et al. 2001; Kienast and Heinz 2006; Goldstein 2010).
Treatment of dopamine deficiency-related diseases has been monitored by following levels of the major dopamine metabolite homovanillic acid (HVA) in cerebrospinal fluid and serum prolactin (Isaac et al. 2008). Yet, research suggests that the assessment of urinary dopamine might be used as a non-invasive alternative to HVA (Marc et al. 2011). The measurement is relatively easy to obtain, urinary concentrations are higher than those in plasma, and collection procedures are well established and non-invasive. In fact, some studies have evaluated the effects of l-DOPA treatment on urinary dopamine levels in Parkinson's patients (Routh et al. 1971; Dutton et al. 1993; Davidson et al. 2007). It has been demonstrated that after l-DOPA therapy, dopamine levels in the central and peripheral nervous system increase. Furthermore, preliminary evidence suggests that dopaminergic agents have the potential to improve quality of life for patients with major depressive disorder (IsHak et al. 2009; Howland 2012). The measurement of urinary dopamine might be used to monitor patients treated with selective dopamine reuptake inhibitors (SDRIs) in the clinical treatment of ADHD, narcolepsy, fatigue, obesity, mood disorders, anxiety disorders, and drug addiction. The availability of a simple and cost-effective analytical method for urinary dopamine screening would be helpful in assessing the suitability of patient's drug dosing and compliance, in avoiding overdose, and suggesting a change of therapy in case of ineffectiveness.
Several methods have been published on the analysis of dopamine (DA) and related catecholamines in biological fluids. Measurement of catecholamines and their metabolites in plasma and urine are commonly used to aid in the detection and monitoring of neuroblastoma and pheochromocytoma and the evaluation of hypotension or hypertension (Peaston and Weinkove 2004). Traditionally, high performance liquid chromatography (HPLC) combined with electrochemical or fluorescence detection is applied (Peaston and Weinkove 2004; Tsunoda 2006; Pussard et al. 2009). Recently, LC-tandem mass spectrometry is the method of choice because of increased selectivity and sensitivity (Kushnir et al. 2002; de Jong et al. 2010a, 2011; Moriarty et al. 2011). Automated on-line solid phase extraction and subsequent unique MS/MS fragmentation provided the possibility to replace laborious sample preparation, reduce extended LC separation time, and eliminate potential interferences from structurally related metabolites, drugs, and dietary constituents. Despite the technological advances that led to the expansion of the tandem mass methodology in clinical laboratories in the last decade, LC-MS/MS still requires expensive equipment and maintenance, highly qualified personnel, and cannot offer the high throughput provided by immunoassays.
The development and the application of immunoassays for catecholamine detection in biological fluids have been accompanied by difficulties related to their low physiological concentrations, the need of highly specific antibodies and the tendency of the catechol group to be easily oxidized (Knoll and Wisser 1984; Peaston and Weinkove 2004; Kim et al. 2008, 2010). Most of the undertaken approaches are based on the detection of their COMT–metabolites [3-O-methylated catecholamines (metanephrine, normetanephrine, 3-methoxytyramine) and 3-O-methylated acids (homovanilic and vanillylmandelic acid)] which are excreted in higher levels and are more stable. Although some ELISA and radioimmunoassay kits are commercially available, they still have limited clinical use because of complicated and time consuming sample preparation procedures. Free catecholamines are extracted using a cis-diolspecific affinity gel, followed by acetylation and enzymatic conversion (COMT methylation) into their n-acylated metadrenalines prior to immunochemical measurement (Westermann et al. 2002).
We present here the in depth validation of a high-throughput and cost-effective ELISA screening method for direct analysis of urinary dopamine (DA). We also demonstrate its application as a tool for monitoring dopamine concentrations in therapies with dopaminergic agents.
- Top of page
- Materials and methods
- Supporting Information
A competitive ELISA for quantitative determination of dopamine in urine samples was established. The proposed method is very simple to perform and includes specimen neutralization followed by in situ glutaric aldehyde derivatization prior to the immunochemical quantification. The derivatization step with glutaric aldehyde mimics the preparation of the immunogen used to generate the anti-dopamine-specific antibody (Huisman et al. 2010a, b).
In contrast to the traditional approaches used in clinical urinary testing the creatinine normalization is an intrinsic part of the methodology described in this work. The volume of urine used in the analysis is equivalent to 120 μg Cr. In this way, samples are diluted according to their Cr level and the urinary matrix is normalized. This approach reduces matrix interferences and allows direct dopamine measurement in whole urine without the need of sample purification.
The analytical procedure was characterized by excellent precision, good sensitivity, and analytical recovery. The average inter-assay variability of 11.4% (see Table S1) was less than one half of the average within-subject biological variation (CVI = 23.9%) of urinary dopamine (see section ‘Biological variation’) which is an established criteria for desirable performance of laboratory measurements (Fraser et al. 1997). Although the sensitivity of the immunoassay (LoD of 7.3 μg/g Cr) is not as high as those reported for LC methods (Kushnir et al. 2002; Tsunoda 2006; de Jong et al. 2010a, 2011), it is certainly sufficient for the analysis of free dopamine in urine samples. The linearity studies showed that the analytical measurement range of the assay allows the direct quantification of endogenous urinary dopamine in the general population and also in patients under dopamine-modulating treatment where elevated dopamine levels might be expected. Very good correlation (r = 0.986, n = 28) was observed between urinary dopamine concentrations determined by the competitive ELISA and LC-MS/MS.
The present dopamine ELISA is based on the use of a highly specific antibody. The specificity studies suggest that the degree of recognition of the anti-dopamine antibody is directly related to the presence of the catechol (3,4-dihydroxy) group, to the ethyl-amino moiety available for amino coupling with the protein and to the glutaric aldehyde linker. If any of those three epitopes is absent in the analyte, no antibody binding is observed. For example, the lack of 3-hydroxy group in the molecules of tyramine and 3-methoxytyramine and the absence of 4-hydroxy group in the molecule of 3-hydroxy-4-methoxyphenylethylamine (4-methoxytyramine) result in no cross-reactivity with those compounds (see Fig. 1). Furthermore, molecules containing methylated imine group instead of amino group (such as epinephrine) were not detected in the assay because of weaker reactivity of the imine group in the glutaric aldehyde derivatization. Even more, any functional substitution in the ethylene arm (ex. norepinephrine) results in negligible interference.
The high selectivity of the anti-dopamine antibody used in the competitive assay allows for very specific dopamine determination in whole urine samples without sample pre-treatment. Thus, major urinary components do not affect the dopamine measurement (see Table 1). No significant interferences were observed by dopamine precursors, metabolites of the tyrosine pathway, related biogenic amines, dietary supplements, and drugs. The assay is not affected by urinary concentrations of l-DOPA up to 45 mg/L. Considering that ~ 1% of l-DOPA dosage is excreted as a parent compound in Parkinson's therapy (Routh et al. 1971), it can be expected that therapeutic doses up to 4.5 g l-DOPA per day will not interfere with the dopamine measurement. Therefore, the interference free quantification of urinary dopamine makes this ELISA a valuable tool in clinical settings for monitoring dopamine.
One of the aims of this study was to estimate if spot urine collection is an appropriate method to study biological variation of dopamine, to distinguish cause by disease from natural variations. Currently, the 24 h timed urinary collection remains the most common method for evaluation of dopamine excretion despite the practical difficulties in this sampling. However, in a study on Parkinson's patients random urine specimens were used and significant positive correlation with daily dose of l-DOPA was observed (Davidson et al. 2007). Thus, we have compared dopamine levels (expressed as μg of dopamine per g of creatinine) determined in 24 h urine collection and second morning spot urine. The results of this comparative study showed very good correlation (p < 0.01; r = 0.90; n = 24; see Fig. 3). Therefore, spot urine sampling could be considered an alternative collection for dopamine assessment. Furthermore, we observed that the week-to-week variation of dopamine in second morning spot urine collections for 10 individuals is very reproducible. We would like to note that the observed biological variations (within-subject: CVI of 23.9%; between-subject: CVG of 35.5%) and the corresponding individuality index (I of 0.67) for urinary dopamine are comparable to those seen for other types of urinary metabolites widely used in clinical laboratories, such as creatinine, urea, protein, sodium. (Ricós et al. 2012). Studies on day-to-day and within- and between-subject biological variations of catecholamine excretion under free-living conditions and on normal healthy subjects are very limited (Curtin et al. 1996; Souza et al. 1998). Souza et al. reported that the 24 h dopamine excretion during habitual daily activities for normal subjects had within-subject variation of 31% for women (n = 22) and 39% for men (n = 12) and between-subject variation of 42% for women and 39% for men (Souza et al. 1998). The biological variations for second morning urine collection observed by us are lower than the variations reported by Souza et al. for 24 h samples. A second morning urine sample seems to be reliable and more appropriate than 24 h collection for clinical use. The determination of biological variation of urinary DA established here will be of great value when considering patients with possible neuroendocrine disorders and other physiologic and pathologic conditions related to dopaminergic imbalances. Future studies are needed to assess the long-term biological variations in healthy population and in symptomatic patients to improve their diagnosis and treatment. No circadian rhythms were seen in the 28 h excretion pattern of free dopamine for 19 healthy volunteers. Our data are in agreement with earlier findings obtained by HPLC analysis that circadian variations in free dopamine excretion are relatively smaller compared to those found for norepinephrine and epinephrine (Fibiger et al. 1984; Westernik and ten Kate 1986; de Jong et al. 2010b).
The ability of the ELISA method to measure urinary dopamine was demonstrated by the excretion profiles obtained after very low dose (25 mg) of l-DOPA oral administration to 24 apparently healthy volunteers (Fig. 6). Maximum dopamine levels were found 2 h post-dose for most of the individuals similarly to a study reported by Brown and Collery (1981). Although significant inter-individual differences in the pharmacokinetics of l-DOPA has been observed, most of the ingested l-DOPA was completely eliminated as DA in ~ 17 h (data not shown). This suggests that in the case of steady state circulating levels of DA assessment urine sampling should be performed at least 17 h post-dose.
To evaluate whether the present ELISA is useful for detecting variations in DA levels under pathological conditions, we compared the steady state DA concentrations in second morning urine samples collected from 162 Parkinson's disease patients receiving different therapies (see Fig. 7). As demonstrated, long term DA support with DA precursors significantly increased DA concentration in the circulation and as a consequence higher DA levels in the urine were observed: the mean DA levels determined for the PD patients under DA support (group C) were ~ 8-fold higher than those observed for patients not taking anti-PD medications nor DA precursors (group A) and ~ 2-fold higher than those determined for the patients treated with anti-PD drugs other than DA precursors (group B). Interestingly, we have observed that intake of anti-PD medications such as DA agonists, anti-cholinergic drugs, SDRIs and/or MAO inhibitors (group B) resulted in significant but much smaller increase in circulating DA with respect to PD patients that were not taking any anti-PD drugs nor DA support (group A). The relationship between urinary DA levels and the clinical response of the patients remains to be established. Nevertheless, our observations suggest that the DA assay presented here would be a valuable tool in future clinical research on DA support therapies.
Finally, we have demonstrated the clinical utility of the dopamine immunoassay to monitor therapy of PD patients (n = 20) treated with different l-DOPA daily doses (Fig. 8). Pre-treatment (baseline) and under treatment (retest) DA levels were assessed and the relative increase in circulating DA was determined. Dose-dependent changes in urinary dopamine levels in response to l-DOPA administration were observed. We found that daily l-DOPA doses greater than 200 mg/day are necessary to significantly increase urinary dopamine concentrations in PD patients. Similarly, significant increase of urinary dopamine after l-DOPA treatment in a dose-dependent manner was reported in previous PD studies (Routh et al. 1971; Dutton et al. 1993; Davidson et al. 2007). Furthermore, it should be noted that the percent increase in DA levels is not only dose-dependent but also patient dependent. Our results indicate considerable differences in the individual responses to the same dose of l-DOPA. Also, the same % increase in DA levels can be achieved by low, mid or high dose depending on the patient. Both the pharmacokinetics disposition and the pharmacodynamic response to a given dose of dopamine modulators in individual patients may vary widely as a direct consequence of epigenetic differences. It is well known that the individual metabolism of patients may not only differ from one another but may change in the same patient during treatment. Thus, anti-parkisonian treatments lose effectiveness with progression of the disease. These strongly suggest the need of urinary dopamine assessment to determine optimal dosing and patient compliance of l-DOPA treatment. An understanding of the individual's metabolism of l-DOPA is crucial to establish the optimal therapeutic regimen for that agent.
In summary, this study outlines a validated ELISA method for the direct analysis of dopamine in urine samples. We conclude that determining the ratio of dopamine per g Cr in second morning urine samples presents an accurate, convenient, inexpensive, and reliable estimate of dopamine excretion. The method for urinary dopamine presented here is robust, sensitive and very specific and can be used in monitoring dopamine-modulating therapies. Comparing of urinary DA concentrations to a baseline level might be helpful in assessing the suitability of each patient's dosage, assessing patient's compliance, and avoiding overdose. The application of our method in overall clinical assessment still needs to be defined.
The immunoassay described here may provide a viable cost- effective alternative to chromatographic analysis as it offers higher throughput and there is no need of time-consuming and complicated sample pre-treatment. The dopamine ELISA can be run in parallel with other immunoassays for the detection of corresponding metabolites of interest (Huisman et al. 2010b; Nichkova et al. 2012). The method can be further validated as part of a biomarker panel for the determination of patterns of monoamines (norepinephrine, epinephrine, serotonin, etc.) that can be used for monitoring and treatment of neurological disorders.