Investigation of Metabolite Profile of YM758, a Novel If Channel Inhibitor.

BACKGROUND: YM758 monophosphate is a novel If channel inhibitor that has an inhibitory action for If current and shows a strong and specific activity, selectively lowering the heart rate and decreasing oxygen consumption by heart muscle. OBJECTIVES: The objectives of the current study were to investigate the in vivo metabolic profiles of YM758 in mice, rats, rabbits, dogs, and monkeys and to elucidate the structures of YM758 metabolites. METHODS: Biological samples were analyzed by liquid chromatography hyphenated with a radiometric detection system and liquid chromatography coupled with a mass spectrometer to clarify their metabolic patterns. To elucidate their structures, metabolites were isolated and analyzed by mass spectrometry and nuclear magnetic resonance spectroscopy. RESULTS: Our results from in vivo metabolic profiling in humans and animals indicated there is no significant species difference in the metabolism of YM758, and the metabolic pathways of YM758 are considered to be oxidation, hydration, and demethylation followed by sulfate or glucuronide conjugation.

Analytical challenges: determination of tetrodotoxin in human urine and plasma by LC-MS/MS.

Tetrodotoxin (TTX) is a powerful sodium channel blocker found in puffer fish and some marine animals. Cases of TTX poisoning most often result from puffer fish ingestion. Diagnosis is mainly from patient's signs and symptoms or the detection of TTX in the leftover food. If leftover food is unavailable, the determination of TTX in the patient's urine and/or plasma is essential to confirm the diagnosis. Although various methods for the determination of TTX have been published, most of them are for food tissue samples. Dealing with human urine and blood samples is much more challenging. Unlike in food, the amount of toxin in the urine and blood of a patient is generally extremely low; therefore a very sensitive method is required to detect it. In this regard, mass spectrometry (MS) methods are the best choice. Since TTX is a very polar compound, there will be lack of retention on conventional reverse-phase columns; use of ion pair reagent or hydrophilic interaction liquid chromatography (HILIC) can help solve this problem. The problem of ion suppression is another challenge in analyzing polar compound in biological samples. This review will discuss different MS methods and their pros and cons.

Development and validation of a high-throughput double solid phase extraction-liquid chromatography-tandem mass spectrometry method for the determination of tetrodotoxin in human urine and plasma.

A sensitive analytical method for the determination of tetrodotoxin (TTX) in urine and plasma matrices was developed using double solid phase extraction (C18 and hydrophilic interaction liquid chromatography) and subsequent analysis by HPLC coupled with tandem mass spectrometry. The double SPE sample cleanup efficiently reduced matrix and ion suppression effects. Together with the use of ion pair reagent in the mobile phase, isocratic elution became possible which enabled a shorter analysis time of 5.5 min per sample. The assay results were linear up to 500 ng mL(-1) for urine and 20 ng mL(-1) for plasma. The limit of detection and limit of quantification were 0.13 ng mL(-1) and 2.5 ng mL(-1), respectively, for both biological matrices. Recoveries were in the range of 75-81%. To eliminate the effect of dehydration and variations in urinary output, urinary creatinine-adjustment was made. TTX was quantified in eight urine samples and seven plasma samples from eight patients suspected of having TTX poisoning. TTX was detected in all urine samples, with concentrations ranging from 17.6 to 460.5 ng mL(-1), but was not detected in any of the plasma samples. The creatinine-adjusted TTX concentration in urine (ranging from 7.4 to 41.1 ng µmol(-1) creatinine) correlated well with the degree of poisoning as observed from clinical symptoms.

K+ secretion in the rat kidney: Na+ channel-dependent and -independent mechanisms.

Renal Na(+) and K(+) excretion was measured in rats with varying dietary K(+) intake. The requirement for channel-mediated distal nephron Na(+) reabsorption was assessed by infusing the animals with the K(+)-sparing diuretic amiloride via osmotic minipumps. At infusion rates of 2 nmol/min, the concentration of amiloride in the urine was 38 microM, corresponding to concentrations of 9-23 microM in the distal tubular fluid, sufficient to block >98% of Na(+) transport through apical Na(+) channels (ENaC). With a control K(+) intake (0.6% KCl), amiloride reduced K(+) excretion rates (U(K)V) from 0.85 +/- 0.15 to 0.05 +/- 0.01 micromol/min during the first 2 h of infusion, suggesting that distal nephron K(+) secretion was completely dependent on the activity of Na(+) channels. When K(+) intake was increased by feeding overnight with a diet containing 10% KCl, amiloride reduced U(K)V from 7.5 +/- 0.7 to 1.3 +/- 0.1 micromol/min despite an increased plasma K(+) of 9 mM, again suggesting a major but not exclusive role for the Na(+) channel-dependent pathway of K(+) secretion. The maximal measured rates of amiloride-sensitive K(+) excretion correspond well with estimates based on apical K(+) channel activity in distal nephron segments. However, when the animals were adapted to the high-K(+) diet for 7-9 days, the diuretic decreased U(K)V less, from 6.1 +/- 0.6 to 3.0 +/- 0.8 micromol/min, indicating an increasing fraction of K(+) excretion that was independent of Na(+) channels. This indicates the upregulation of a Na(+) channel-independent mechanism for secreting K(+).

Sample preparation and RPHPLC determination of diuretics in human body fluids.

This article describes reverse phase high-performance liquid chromatography (RPHPLC) methods for determination of diuretics in different human body fluids (whole blood, plasma, serum or urine). Sample preparation procedures, including solid-phase extraction, liquid-liquid extraction, dilution, precipitation as well as automated RPHPLC procedures, are discussed in order to present the advantages and disadvantages of each type of sample preparation. Also, values of analytical recovery of each procedure used for sample preparation are summarized. The most important RPHPLC parameters (detection mode, stationary phase, mobile phase, sensitivity, etc.) are also summarized and discussed.

Acute tetrodotoxin-induced neurotoxicity after ingestion of puffer fish.

This study documents the effects of puffer-fish poisoning on peripheral nerve. Excitability measurements investigated membrane properties of sensory and motor axons in four patients. The median nerve was stimulated at the wrist, with compound muscle potentials recorded from abductor pollicis brevis and compound sensory potentials from digit 2. Stimulus-responses, strength-duration time constant (tau(SD)), threshold electrotonus, and current-threshold relations were recorded. The urine of each patient tested positive for tetrodotoxin. Compared with controls, axons were of higher threshold, compound muscle action potentials and compound sensory nerve action potentials were reduced in amplitude, latency was prolonged, and tau(SD) was reduced. In recovery cycles, refractoriness, superexcitability, and late subexcitability were decreased. Threshold electrotonus of motor axons exhibited distinctive abnormalities with less threshold decline than normal on depolarization and greater threshold increase on hyperpolarization (p < 0.0005 for each patient). The changes in excitability were reproduced in a mathematical model by reducing sodium (Na(+)) permeabilities by a factor of two. This study confirms that the neurotoxic effects of puffer-fish poisoning can be explained by tetrodotoxin blockade of Na(+) channels. It demonstrates the ability of noninvasive nerve excitability studies to detect Na(+) channel blockade in vivo and also the utility of mathematical modeling to aid interpretation of altered excitability properties in disease.