Stabilization of urinary biogenic amines measured in clinical chemistry laboratories.

Urinary 5-hydroxyindoleacetic acid (5-HIAA), vanillylmandelic (VMA), homovanillic acid (HVA), catecholamines and metanephrines are produced in excess by catecholamine-producing tumors. These biogenic amines are unstable at low or high pH and require hydrochloric acid (HCl) to prevent their degradation. However, HCl addition may result in very low pH causing degradation or deconjugation of several metabolites. This study evaluated the buffering properties of sodium citrate to stabilize all biogenic amines. The metabolite concentrations were measured by LC-MS/MS or by a coulometric assay in 22 urine samples collected native and with HCl or sodium citrate. We studied the effect of pH, time (48 h, four weeks) and storage temperature at 22 °C, 4 °C, and -20 °C. We found that catecholamines degradation was prevented by HCl and citrate and that 5-HIAA was degraded in 5 out of 22 samples collected with HCl. All biogenic amines were efficiently stabilized by citrate for four weeks at 22 °C, except epinephrine (48 h at 4 °C, or four weeks at -20 °C). Sodium citrate did not cause quantification or analytical artefacts concerns. In conclusion, sodium citrate is a non-hazardous alternative to HCl for patients to send unfrozen urine samples to the laboratory which may safely store the sample for four weeks.

Quantification of vanillylmandelic acid, homovanillic acid and 5-hydroxyindoleacetic acid in urine using a dilute-and-shoot and ultra-high pressure liquid chromatography tandem mass spectrometry method.

BACKGROUND: Urinary vanillylmandelic acid (VMA) and homovanillic acid (HVA) are biomarkers for the diagnosis and follow-up of neuroblastoma, whereas urinary 5-hydroxyindoleacetic acid (5-HIAA) is used to assess a carcinoid tumor. These analytes are conventionally analyzed in a single run by chromatography (LC) coupled with electrochemical detection (LC-ECD) using commercial kits. A rapid dilute-and-shoot LC tandem mass spectrometry (LC-MS/MS) assay was validated in order to replace the LC-ECD method and therefore improve analytical specificity and throughput. METHODS: Sample preparation was carried out by dilution of the urine sample with a solution containing the deuterated internal standards. The separation was achieved on an ultra-high pressure LC system with MS detection using a triple quadrupole mass spectrometer. The method was validated according to the current EMA and FDA guidelines. RESULTS: The full chromatographic run was achieved in 8 min. The method validation showed excellent linearity (r2>0.999 for all three analytes), precision (CV <15%), negligible matrix effect (recoveries >90%), low carryover (<1%) and LLOQ of 0.25, 0.4 and 0.4 µM for VMA, HVA and 5-HIAA, respectively. Deming fits and Bland-Altman analyses showed no significant differences between the values obtained between the two assays. CONCLUSIONS: The LC-MS/MS method proposed in this study is fast and robust, and the simple sample preparation saves time and avoids the additional costs of dedicated kits used for the LC-ECD assays by switching to LC-MS/MS. Additionally, the near-perfect correlation observed herein between both assays allows the previously established reference ranges to be maintained.

Enzyme and acid deconjugation of plasma sulfated metanephrines.

BACKGROUND: Total (i.e. free+sulfated) metanephrines in plasma is a biomarker for the diagnosis of pheochromocytoma/paraganglioma. Sulfated metanephrines must be completely deconjugated by perchloric acid hydrolysis or sulfatase treatment prior to analytical measurement to enable quantification by current techniques. In this report, we compare the yield and efficiency of both methods. METHODS: The deconjugation rate of synthetic sulfated metanephrines (normetanephrine (S-NMN), metanephrine (S-MN) and methoxytyramine (S-MT)) spiked in charcoal-stripped plasma was determined by boiling perchloric acid and compared to sulfatase treatment. Total plasma metanephrines (MN, NMN and MT) were also determined in patient samples by both methods. RESULTS: The complete deconjugation of sulfated metanephrines is achieved after 30 min incubation with 0.1M boiling perchloric acid or upon sulfatase treatment. Ten minutes of acid hydrolysis (gold-standard) leads to a 30% underestimation of metanephrine concentrations. The enzyme hydrolysis is time and amount of sulfatase dependent. The rate of hydrolysis is analyte-dependent (MT>>NMN>MN), although it must contain at least 0.8 U/ml of sample. The Deming regression curves comparing acid versus enzyme hydrolysis on patient samples assessed that both methods gave similar unbiased concentrations. CONCLUSION: Enzyme and acid treatments are equivalent and efficient for removing sulfate from metanephrines as long as the optimal protocol is used for each method. However, the gold standard method for acid hydrolysis at 10 min established more than 20 years ago was not satisfactory regarding the hydrolysis of metanephrines in plasma.

Synthetic calibrators for the analysis of total metanephrines in urine: revisiting the conditions of hydrolysis.

BACKGROUND: The quantification of total (free+sulfated) metanephrines in urine is recommended to diagnose pheochromocytoma. Urinary metanephrines include metanephrine itself, normetanephrine and methoxytyramine, mainly in the form of sulfate conjugates (60-80%). Their determination requires the hydrolysis of the sulfate ester moiety to allow electrochemical oxidation of the phenolic group. Commercially available urine calibrators and controls contain essentially free, unhydrolysable metanephrines which are not representative of native urines. The lack of appropriate calibrators may lead to uncertainty regarding the completion of the hydrolysis of sulfated metanephrines, resulting in incorrect quantification. METHODS: We used chemically synthesized sulfated metanephrines to establish whether the procedure most frequently recommended for commercial kits (pH 1.0 for 30 min over a boiling water bath) ensures their complete hydrolysis. RESULTS: We found that sulfated metanephrines differ in their optimum pH to obtain complete hydrolysis. Highest yields and minimal variance were established for incubation at pH 0.7-0.9 during 20 min. CONCLUSION: Urinary pH should be carefully controlled to ensure an efficient and reproducible hydrolysis of sulfated metanephrines. Synthetic sulfated metanephrines represent the optimal material for calibrators and proficiency testing to improve inter-laboratory accuracy.

In vitro and in vivo characterization of the activity of telmisartan: an insurmountable angiotensin II receptor antagonist.

In vitro studies have shown that telmisartan is an insurmountable angiotensin II subtype-1 (AT1) receptor antagonist. Herein, the molecular basis of this insurmountable antagonism has been investigated in vitro, and the effect of telmisartan has been compared in vivo with that of irbesartan and candesartan. Association and dissociation kinetics of telmisartan to AT1 receptors have been characterized in vitro on rat vascular smooth muscle cells (RVSMC) expressing solely the AT1 receptor subtype. In a second set of experiments, the antagonistic efficacy of single intravenous doses (0.1, 0.3, and 1 mg/kg) of telmisartan was compared with that of irbesartan (0.3, 1.0, 3.0, and 10.0 mg/kg) and candesartan (0.3 and 1 mg/kg) in conscious, normotensive, male Wistar rats. The results show that the specific binding of [(3)H]telmisartan to the surface of living RVSMC is saturable and increases quickly to reach equilibrium within 1 h. Telmisartan dissociates very slowly from the receptor with a dissociation half-life (t(1/2)) of 75 min, which is comparable with candesartan and almost 5 times slower than angiotensin II (AngII). In vivo, telmisartan blunts the blood pressure response to exogenous AngII dose dependently. The blockade is long lasting and remains significant at 24 h at doses >0.1 mg/kg. Ex vivo assessment of the AT1 receptor blockade using an in vitro AngII receptor binding assay shows similar results. When administered intravenously in rats, telmisartan is 10-fold more potent than irbesartan and comparable to candesartan. Taken together, our in vitro data show that the insurmountable antagonism of telmisartan is due at least in part to its very slow dissociation from AT1 receptors.

Comparative angiotensin II receptor blockade in healthy volunteers: the importance of dosing.

OBJECTIVES: We have reported previously that 80 mg valsartan and 50 mg losartan provide less receptor blockade than 150 mg irbesartan in normotensive subjects. In this study we investigated the importance of drug dosing in mediating these differences by comparing the AT(1)-receptor blockade induced by 3 doses of valsartan with that obtained with 3 other antagonists at given doses. METHODS: Valsartan (80, 160, and 320 mg), 50 mg losartan, 150 mg irbesartan, and 8 mg candesartan were administered to 24 healthy subjects in a randomized, open-label, 3-period crossover study. All doses were given once daily for 8 days. The angiotensin II receptor blockade was assessed with two techniques, the reactive rise in plasma renin activity and an in vitro radioreceptor binding assay that quantified the displacement of angiotensin II by the blocking agents. Measurements were obtained before and 4 and 24 hours after drug intake on days 1 and 8. RESULTS: At 4 and 24 hours, valsartan induced a dose-dependent "blockade" of AT(1) receptors. Compared with other antagonists, 80 mg valsartan and 50 mg losartan had a comparable profile. The 160-mg and 320-mg doses of valsartan blocked AT(1) receptors at 4 hours by 80%, which was similar to the effect of 150 mg irbesartan. At trough, however, the valsartan-induced blockade was slightly less than that obtained with irbesartan. With use of plasma renin activity as a marker of receptor blockade, on day 8, 160 mg valsartan was equivalent to 150 mg irbesartan and 8 mg candesartan. CONCLUSIONS: These results show that the differences in angiotensin II receptor blockade observed with the various AT(1) antagonists are explained mainly by differences in dosing. When 160-mg or 320-mg doses were investigated, the effects of valsartan hardly differed from those obtained with recommended doses of irbesartan and candesartan.

Does protein binding modulate the effect of angiotensin II receptor antagonists?

Introduction Angiotensin II AT 1-receptor antagonists are highly bound to plasma proteins (≥ 99%). With some antagonists, such as DuP-532, the protein binding was such that no efficacy of the drug could be demonstrated clinically. Whether protein binding interferes with the efficacy of other antagonists is not known. We have therefore investigated in vitro how plasma proteins may affect the antagonistic effect of different AT1-receptor antagonists. Methods A radio-receptor binding assay was used to analyse the interaction between proteins and the ability of various angiotensin II (Ang II) antagonists to block AT1-receptors. In addition, the Biacore technology, a new technique which enables the real-time monitoring of binding events between two molecules, was used to evaluate the dissociation rate constants of five AT1-receptor antagonists from human serum albumin. Results The in vitro AT 1-antagonistic effects of different Ang II receptor antagonists were differentially affected by the presence of human plasma, with rightward shifts of the IC50 ranging from one to several orders of magnitude. The importance of the shift correlates with the dissociation rate constants of these drugs from albumin. Our experiments also show that the way that AT1-receptor antagonists bind to proteins differs from one compound to another. These results suggest that the interaction with plasma proteins appears to modulate the efficacy of some Ang II antagonists. Conclusion Although the high binding level of Ang II receptor antagonist to plasma proteins appears to be a feature common to this class of compounds, the kinetics and characteristics of this binding is of great importance. With some antagonists, protein binding interferes markedly with their efficacy to block AT1-receptors.