Lethal Injection of a Castor Bean Extract: Ricinine Quantification as a Marker for Ricin Exposure Using a Validated LC-MS/MS Method.

Ricin is a highly toxic agent derived from the castor bean plant (Ricinus communis). Poisoning occurs commonly by oral ingestion of the beans. Injection of ricin is believed to be more lethal. Ricin is a large glycosylated protein difficult to detect in clinical samples. Instead, ricinine, a small alkaloid found in the same beans, is used as surrogate marker for ricin exposure. We describe a simple LC-MS/MS method for the detection of ricinine in serum, blood and urine, validated according to EMA guidelines and successfully applied to patient samples of a suicidal death after injection of a castor bean extract. A 26-year-old man self-presented to the emergency department with severe abdominal cramps and nausea after injection of a castor bean extract. Due to rapid deterioration of his hemodynamic function despite early aggressive fluid resuscitation, he was transferred to ICU. Abdominal cramps worsened and a fulminant diarrhea developed, resulting in hypovolemic shock and cardiorespiratory collapse. Despite full supportive therapy, the patient died approximately 10 hours after injection due to multiple organ failure. Ricinine was quantified by LC-MS/MS after LLE with diethyl ether using ricinine-D3 as internal standard. Six hours after injection, ricinine concentrations in serum and blood were 16.5 and 12.9 ng/mL, respectively, which decreased to 12.4 and 10.6 ng/mL, 4 hours later. The urinary concentration was 81.1 ng/mL 7 hours after injection, which amply exceeded the levels previously reported in similar cases with lethal outcome. Concentrations of ricinine, compatible with a lethal exposure to castor beans, were detected in serum, blood and urine. Ricinine was also found in bile and liver tissue.

Supplementation with resveratrol as Polygonum cuspidatum Sieb. et Zucc. extract induces changes in the excretion of urinary markers associated to aging in rats.

An UPLC-HR-MS metabolomics approach was used to study the effects of a 49-days oral supplementation with Polygonum cuspidatum extract in healthy rats. Multivariate analysis allowed to observe significant differences in the excretion of several markers between treated animals and control group. Among the others, the amounts of N-methyl-2-pyridone-5-carboxamide (2PY) and phenylacetylglycine (PAG) were reduced in the treated group compared to control. These compounds have been previously considered as markers of aging. Furthermore, the excretion of 3-hydroxysebacic acid and 4,6-dihydroxyquinoline was also changed following supplementation, although not significantly. Despite the relatively short time of treatment (7 weeks), the significant changes in the urinary levels of aging markers observed at day 49 suggests a potential role of this type of studies as a new approach in the evaluation of the anti-aging effects of plant extracts.

Oral Glucose Tolerance and Tryptophan Metabolism in Non-Obese and Non-Insulin-Dependent Diabetic Goto-Kakizaki Rats Fed High-Tryptophan Diets.

We investigated oral glucose tolerance and tryptophan (Trp) metabolism in non-obese and non-insulin-dependent diabetic Goto-Kakizaki (GK) rats fed high-Trp diets. Five-week-old male Wistar and GK rats were fed a 20% casein diet (control diet) or the same diet supplemented with 1%, 2%, 3%, or 5% Trp for 58 d. Oral glucose tolerance tests were performed on Days 14 and 28 of the experimental period. Urine as well as livers and blood were collected on the last day of the experiment. The glucose concentration and the amount of Trp metabolites were measured. On Day 14 of the experiment, the incremental blood glucose concentrations integrated over a period of 2 h (ΔAUC0-2h) of blood glucose in rats fed the 3% and 5% Trp diets had decreased by 13% and 18%, respectively, compared with that of the control-GK rats. However, no significant differences were found in the rats fed +1% or +2% Trp diets compared with control-GK rats. On Day 28, there were no significant differences found in the ΔAUC0-2h of blood glucose levels in any group including the control-GK group. On the last day, the concentrations of plasma glucose, total cholesterol, and triglyceride did not show differences in any group. There were no specific phenomena observed in the metabolism of Trp in GK rats even when fed an excess of Trp, compared with that of Wistar rats. Oral Trp administration and its continuous use may not improve blood glucose levels in type 2 diabetic rats.

Urinary Excretion of Niacin Metabolites in Humans After Coffee Consumption.

SCOPE: Coffee is a major natural source of niacin in the human diet, as it is formed during coffee roasting from the alkaloid trigonelline. The intention of our study was to monitor the urinary excretion of niacin metabolites after coffee consumption under controlled diet. METHODS AND RESULTS: We performed a 4-day human intervention study on the excretion of major niacin metabolites in the urine of volunteers after ingestion of 500 mL regular coffee containing 34.8 µmol nicotinic acid (NA) and 0.58 µmol nicotinamide (NAM). In addition to NA and NAM, the metabolites N1 -methylnicotinamide (NMNAM), N1 -methyl-2-pyridone-5-carboxamide (2-Py), and nicotinuric acid (NUA) were identified and quantified in the collected urine samples by stable isotope dilution analysis (SIVA) using HPLC-ESI-MS/MS. Rapid urinary excretion was observed for the main metabolites (NA, NAM, NMNAM, and 2-Py), with tmax values within the first hour after ingestion. NUA appeared in traces even more rapidly. In sum, 972 nmol h-1 of NA, NAM, NMNAM, and 2-Py were excreted within 12 h after coffee consumption, corresponding to 6% of the ingested NA and NAM. CONCLUSION: The results indicate regular coffee consumption to be a source of niacin in human diet.

Comparison of the effects of nicotinic acid and nicotinamide degradation on plasma betaine and choline levels.

AIM: The present study was to compare the effects of nicotinic acid and nicotinamide on the plasma methyl donors, choline and betaine. METHODS: Thirty adult subjects were randomly divided into three groups of equal size, and orally received purified water (C group), nicotinic acid (300 mg, NA group) or nicotinamide (300 mg, NM group). Plasma nicotinamide, N1-methylnicotinamide, homocysteine, betaine and choline levels before and 1.5-h and 3-h post-dosing, plasma normetanephrine and metanephrine concentrations at 3-h post-dosing, and the urinary excretion of N1-methyl-2-pyridone-5-carboxamide during the test period were examined. RESULTS: The level of 3-h plasma nicotinamide, N1-methylnicotinamide, homocysteine, the urinary excretion of N1-methyl-2-pyridone-5-carboxamide and pulse pressure (PP) in the NM group was 221%, 3972%, 61%, 1728% and 21.2% higher than that of the control group (P < 0.01, except homocysteine and PP P < 0.05), while the 3-h plasma betaine, normetanephrine and metanephrine level in the NM group was 24.4%, 9.4% and 11.7% lower (P < 0.05, except betaine P < 0.01), without significant difference in choline levels. Similar but less pronounced changes were observed in the NA group, with a lower level of 3-h plasma N1-methylnicotinamide (1.90 ± 0.20 µmol/l vs. 3.62 ± 0.27 µmol/l, P < 0.01) and homocysteine (12.85 ± 1.39 µmol/l vs. 18.08 ± 1.02 µmol/l, P < 0.05) but a higher level of betaine (27.44 ± 0.71 µmol/l vs. 23.52 ± 0.61 µmol/l, P < 0.05) than that of the NM group. CONCLUSION: The degradation of nicotinamide consumes more betaine than that of nicotinic acid at identical doses. This difference should be taken into consideration in niacin fortification.

Comparison of pharmacokinetics and urinary iron excretion of two single doses of deferiprone in ß-thalassemia/hemoglobin E patients.

Dose-related pharmacokinetics and urinary iron excretion (UIE) of an orally active iron chelator, deferiprone (L1), was investigated in 12 severe ß-thalassemia/hemoglobin E patients. The patients received two single doses of 25 and 50 mg/kg with a 2-week washout period. Deferiprone was rapidly absorbed and reached maximum concentration (C(max)) within 1 h after administration. Pharmacokinetic parameters including C(max) and area under concentration time curve from time zero to infinity (AUC(0-∞)) as well as urinary excretion of non-conjugated and glucuronide-conjugated deferiprone (L1 and L1-G) increased proportionally with the dose of deferiprone. A constant ratio of AUC(0-∞) of L1-G to L1 and a percentage of urinary excretion of L1-G indicated that increasing the dosage does not influence deferiprone biotransformation. Longer terminal elimination half-lifeand higher volume of distribution of L1 were observed with the high dose and correlated with deferiprone-chelated iron in serum. Unexpectedly, UIE did not show a linear relationship with the increased dose of deferiprone. The correlation between UIE and creatinine clearance suggested the possibility of L1-iron complex redistribution in patients with renal impairment treated with high-dose deferiprone.

A comparative study on mimosine, 3,4-dihydroxy pyridone (3,4-DHP) and 2,3-dihydroxy pyridone (2,3-DHP), purine derivatives (PD) excretion in the urine, thyroid hormone and blood metabolites profiles of Thai swamp buffalo (Bubalus bubalis) and Murrah buffalo (Bubalus bubalis).

Four Thai swamp buffaloes (SB) and four Murrah buffaloes (MB) fed a based diet of fresh ruzi grass (Bachiaria ruziziensis) with an increased proportion of fresh leucaena leaves. Intake of nutrients in animals increased when ruzi grass was mixed with leucaena. Digestibility of nutrients were the highest in SB and MB fed diets containing 25% and 50% of leucaena, respectively, and nitrogen (N) balances in both animal breeds were varied among diets. The regression equation coefficient of mimosine + DHP in the urine was twice as high in SB than in MB. Urinary purine derivatives excretion rate in SB was higher than that in MB. Plasma triiodothyronine and thyroxine declined in both animal breeds fed a diet containing >1 g mimosine intake/kg BW(0.75)/day. Plasma urea-N was the lowest in SB, but the highest in MB when fed a diet containing 84% of leucaena. Plasma ß-HBA in SB have declined when diets contained >50% of leucaena but that in MB was not affected by any diet. In conclusion, the effect of leucaena in diet upon buffalo breeds depends on the proportion of leucaena in the diet, mimosine contents and condensed tannins components.

Pharmacokinetic and pharmacodynamic interaction for a binary mixture of chlorpyrifos and diazinon in the rat.

Chlorpyrifos (CPF) and diazinon (DZN) are two commonly used organophosphorus (OP) insecticides and a potential exists for concurrent exposures. The primary neurotoxic effects from OP pesticide exposures result from the inhibition of acetylcholinesterase (AChE). The pharmacokinetic and pharmacodynamic impact of acute binary exposures of rats to CPF and DZN was evaluated in this study. Rats were orally administered CPF, DZN, or a CPF/DZN mixture (0, 15, 30, or 60 mg/kg) and blood (plasma and RBC), and brain were collected at 0, 3, 6, 12, and 24 h postdosing, urine was also collected at 24 h. Chlorpyrifos, DZN, and their respective metabolites, 3,5,6-trichloro-2-pyridinol (TCP) and 2-isopropyl-4-methyl-6-hydroxypyrimidine (IMHP), were quantified in blood and/or urine and cholinesterase (ChE) inhibition was measured in brain, RBC, and plasma. Coexposure to CPF/DZN at the low dose of 15/15 mg/kg did not alter the pharmacokinetics of CPF, DZN, or their metabolites in blood. A high binary dose of 60/60 mg/kg increased the C(max) and AUC and decreased the clearance for both parent compounds, likely due to competition between CPF and DZN for CYP450 metabolism. At lower doses, most likely to be encountered in occupational or environmental exposures, the pharmacokinetics were linear. A dose-dependent inhibition of ChE was noted in tissues for both the single and coexposures, and the extent of inhibition was plasma > RBC > or = brain. The overall relative potency for ChE inhibition was CPF/DZN > CPF > DZN. A comparison of the ChE response at the low binary dose (15/15 mg/kg), where there were no apparent pharmacokinetic interactions, suggested that the overall ChE response was additive. These experiments represent important data concerning the potential pharmacokinetic and pharmacodynamic interactions for pesticide mixtures and will provide needed insight for assessing the potential cumulative risk associated with occupational or environmental exposures to these insecticides.

Effects of dose and timing of calcium supplementation on bone resorption in early menopausal women.

Bone resorption follows a circadian rhythm that peaks at night, reflecting the circadian rhythm of serum parathyroid hormone. Our previous studies in early postmenopausal women have established that 1000 mg of calcium given at 9 p. m. reduced bone resorption markers overnight, but not during the day. In contrast, 1000 mg given as a divided dose (500 mg doses at 9 a. m. and 9 p. m. each) reduced bone resorption markers during the day, but not during the night. We have now evaluated the effect of 1500 mg of calcium given as a divided dose of 500 mg in the morning and 1000 mg in the evening on bone resorption. We studied 26 healthy women (median age 56 years) whose menopause was less than five years before. On two days, urine was collected from 9 a. m. to 9 p. m. (day collection), and from 9 p. m. to 9 a. m. (night collection); a further fasting (spot) urine sample was obtained at 9 a. m. at the end of the night collection. On the second day, 500 mg of calcium in the carbonate form was taken at 9 a. m. (at the start of the collection) and a further 1000 mg at 9 p. m. (at the start of the second night collection). Calcium supplementation decreased urinary deoxypyridinoline (DPyr/Cr) during the day (p = 0.08) and night (p < 0.05), as well as urinary pyridinoline (Pyr/Cr) both by day (p < 0.05) and night (p < 0.001). There were also decreases in urine hydroxyproline. We conclude that the acute administration of 500 mg of calcium in the morning and 1000 mg in the evening to early postmenopausal women suppresses bone resorption markers during both the day and night.

Drought associated poisoning of cattle in South Texas by the high quality forage legume Leucaena leucocephala.

Approximately 80 head of yearling cattle grazing on 680 acres exhibited signs of Leucaena leucocephala toxicosis, which was confirmed in 3 animals by detection of 3-hydroxy-4 (IH)-pyridone, the metabolite of the poisonous principle mimosine, in their urine. The animals had grazed leucaena almost exclusively due to lack of alternative forage resulting from drought conditions. Toxicosis from this otherwise high quality forage would likely not have occurred had animals consumed lower amounts of leucaena and could probably have been prevented, as it has been elsewhere, had the animals been colonized with Synergistes jonesii, a beneficial ruminal bacterium capable of degrading the toxic metabolites.