Kinetic spectrophotometric determination of traces of molybdenum(VI) by its inhibitory effect on the oxidation of 4-hydroxycoumarine by potassium permanganate.
(9/71)The present paper describes a simple, selective and sensitive kinetic method for the determination of trace amounts of molybdenum(VI) based on its inhibitory effect on the reaction oxidation of 4-hydroxycoumarine by KMnO(4) in the presence of hydrochloric acid, at pH 1.75 at 25 degrees C. The rate of the indicator reaction was followed spectrophotometrically by measuring the decrease in the absorbance of KMnO(4) at 525 nm. The development method includes optimization of the reagent concentration and temperature. The calibration graph was linear in the range of concentrations from 20 to 200 ng/cm(3) of molybdenum(VI). The probable relative error was in the interval 3.10 - 10.52% for the concentration range of 200 - 20 ng/cm(3) molybdenum(VI), respectively. The interference effects of the foreign ions were determined to assess the selectivity of the method. The developed method was found to have relatively good selectivity, sensitivity, simplicity and rapidity. The proposed method was applied to the determination of molybdenum(VI) in a particular type of steel and alloy (hastelloy). (+info)
Discovery of a small-molecule HIV-1 integrase inhibitor-binding site.
(10/71)Herein, we report the identification of a unique HIV-1 integrase (IN) inhibitor-binding site using photoaffinity labeling and mass spectrometric analysis. We chemically incorporated a photo-activatable benzophenone moiety into a series of coumarin-containing IN inhibitors. A representative of this series was covalently photo-crosslinked with the IN core domain and subjected to HPLC purification. Fractions were subsequently analyzed by using MALDI-MS and electrospray ionization (ESI)-MS to identify photo-crosslinked products. In this fashion, a single binding site for an inhibitor located within the tryptic peptide (128)AACWWAGIK(136) was identified. Site-directed mutagenesis followed by in vitro inhibition assays resulted in the identification of two specific amino acid residues, C130 and W132, in which substitutions resulted in a marked resistance to the IN inhibitors. Docking studies suggested a specific disruption in functional oligomeric IN complex formation. The combined approach of photo-affinity labeling/MS analysis with site-directed mutagenesis/molecular modeling is a powerful approach for elucidating inhibitor-binding sites of proteins at the atomic level. This approach is especially important for the study of proteins that are not amenable to traditional x-ray crystallography and NMR techniques. This type of structural information can help illuminate processes of inhibitor resistance and thereby facilitate the design of more potent second-generation inhibitors. (+info)
Acute bromadiolone intoxication.
(11/71)A 55-year-old man came to the hospital with a bleeding wound on his tongue. The coating of his tongue was green, and his sputum was red. Because an increased international normalized ratio-value was measured, a blood sample was sent to our laboratory with the suspicion of coumarin intoxication. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis confirmed the poisoning was by bromadiolone, with its maximum serum concentration at 440 microg/L. The analysis of further samples resulted in a calculated elimination half-life of 140 h. The analytical method described was developed for the determination and quantitation of bromadialone using LC-MS. This method is suitable for the simultaneous identification and quantitation of 10 indirect anticoagulants in human serum, which include five superwarfarins (brodifacoum, bromadiolone, difenacoum, difethialone, and flocoumafen) as rodenticides licenced in Germany and five other vitamin K antagonists (acenocoumarol, coumatetralyl, coumachlor, phenprocoumon, and warfarin). The method is based on an acidic (pH 4.2) liquid-liquid extraction followed by LC-ESI-MS analysis. Analytical separation was carried out using an Atlantis C18 column (2.1 x 20 mm, 3 microm). The mobile phase consisted of methanol/0.1% formic acid; the flow rate was 0.6 mL/min, and the time needed for analysis was 5 min. The lower limit of quantitation was 5 microg/L (signal-to-noise > 10). (+info)
Synthesis of the photoaffinity probe 3-(p-azidobenzyl)-4-hydroxycoumarin and identification of the dicoumarol binding site in rat liver NAD(P)H:quinone reductase (EC 18.104.22.168).
(12/71)A photoaffinity analog of 4-hydroxycoumarin containing an azidobenzyl group at the 3-position and, if desired, carbon-14 or tritium radionuclides has been synthesized and characterized. This compound, 3-(p-azidobenzyl)-4-hydroxycoumarin, serves as an effective competitive inhibitor of the dicoumarol-sensitive NAD(P)H:quinone reductase (EC 22.214.171.124; DT-diaphorase) from rat liver, having an apparent inhibition constant of 6.6 x 10(-8) M, a value comparable to that observed for dicoumarol (1.7 x 10(-9) M), significantly lower than for Warfarin (3.5 x 10(-5) M) and well within the range required of an effective photoaffinity reagent. Irradiation of the reductase with ultraviolet light in the presence of the photoprobe resulted in the covalent labeling of up to 10% of the protein. Greater than 99% of the covalent incorporation is precluded by the addition of 15 microM dicoumarol, consistent with the specific labeling of the 4-hydroxycoumarin binding site of this enzyme by this photoaffinity reagent. Further evidence of a high degree of specificity is provided by the isolation and sequence analysis of the peptides covalently modified by this reagent. A single region within the protein was found to be labeled, with threonine 127 and tyrosine 128 being the only amino acid residues that were observed to be modified. These results, for the first time, define a portion of the 4-hydroxycoumarin binding site within a protein that has a well established sensitivity to this type of anticoagulant and, because dicoumarol serves as a competitive inhibitor for pyridine nucleotides in this enzyme, may also define a portion of this unusual pyridine nucleotide binding site. In addition, these results suggest that this reagent may be effective as a highly specific photoaffinity probe in the identification of other proteins that are similarly inhibited by 4-hydroxycoumarin derivatives, such as the microsomal enzymes associated with the vitamin K-dependent carboxylation system. (+info)
Ingestion of superwarfarin leading to coagulopathy: a case report and review of the literature.
(13/71)Superwarfarins are found in many pesticides, including D-con, Prufe I and II, Ramik, Talon-G, Ratak, and Contrac. Ingestion of can lead to significant morbidity and even mortality. Physicians need to consider this diagnosis in any patient presenting with coagulopathy of unclear etiology. We present a patient with superwarfarin-induced coagulopathy and review previous cases in adults in the literature. The patient is a 60-year-old man who presented to our medical center with painless hematuria. Laboratory studies revealed an elevated prothrombin time (PT) (42.5 seconds), partial thromboplastin time (PTT) (64.6 seconds), and international normalized ratio (INR) of 7. Liver-associated enzymes were normal, and complete blood cell count (CBC) showed no evidence of disseminated intravascular coagulation. Subsequent work-up included the absence of an inhibitor by mixing study and deficiencies of vitamin K-dependent coagulation factors. The patient's warfarin level was negative. A brodifacoum level was positive, confirming superwarfarin-induced coagulopathy. The patient is currently doing well with normal coagulation studies after receiving high doses of vitamin K for several weeks. The cause of his exposure to superwarfarin remains uncertain. Physicians need to be cognizant of this unusual cause of coagulopathy in adults. The appropriate diagnostic work-up and unique features of therapy are discussed. (+info)
Fatal brodifacoum poisoning in a pony.
(14/71)Fatal brodifacoum poisoning in a pony is described; this condition has not previously been reported in ponies. Discussion of what factors in the pony's history and treatment may have predisposed to the severity and ultimate death is provided. (+info)
The long-term effects of the rodenticide, brodifacoum, on blood coagulation and vitamin K metabolism in rats.
(15/71)1. The long-term (30 days) effects of a single dose of brodifacoum (0.2 mg kg-1, orally) on blood clotting activity and on liver parameters of the vitamin K cycle were investigated in rats. Maximal effect on blood clotting activity was seen on day one. On day seven blood clotting activity had returned to normal. 2. Liver microsomal vitamin KO reductase activity was maximally suppressed (10% of control activity) on day one, steadily recovered to about 40% on day 15 to remain at that level. The same time course was seen for the number of microsomal warfarin binding sites. 3. The persistent inhibition of the vitamin K cycle was also verified in vivo; following vitamin K administration (10 mg kg-1, i.v.) on day 30, the brodifacoum-treated rats accumulated vitamin KO in the liver. 4. Although clotting factor synthesis was normal, brodifacoum-treated rats were highly sensitive to warfarin. 5. Brodifacoum rapidly accumulated in the liver until the saturation of the microsomal binding site. Brodifacoum binding to the target prevented its elimination from the liver; liver content on day 30 was not different from day 7. 6. The results show (1) an over capacity for the hepatocellular vitamin K cycle, (2) a dissociation of the vitamin K epoxidation and the vitamin K-dependent carboxylation, (3) the 'superwarfarin' rodenticides to be extremely persistent due to their binding to the target. (+info)
Convenient replacement of the hydroxy by an amino group in 4 hydroxycoumarin and 4-hydroxy-6-methyl-2-pyrone under microwave irradiation.
(16/71)The reaction of 4-hydroxycoumarin (1) with some primary amines 2a-h and morpholine (2i) under microwave irradiation occurred without opening of the lactone ring to give N-substituted 4-aminocoumarins 3a-i in excellent yields. Under the same experimental conditions, 4-hydroxy-6-methyl-2-pyrone (4) reacted with benzylamine (2e) or 2-phenyl- ethylamine (2f) to give the corresponding N,N'-disubstituted 4-amino-6-methyl-2-pyridones 5e,f. The main advantages of this procedure are dramatically shortened reaction times, higher amine utilization and considerably improved yields. (+info)