Abuse-related and abuse-limiting effects of methcathinone and the synthetic "bath salts" cathinone analogs methylenedioxypyrovalerone (MDPV), methylone and mephedrone on intracranial self-stimulation in rats.

RATIONALE: Abuse of synthetic cathinones, popularized as "bath salts," has increased dramatically in the USA since their debut in 2010. Preclinical behavioral studies may clarify determinants of the abuse-related effects produced by these compounds. OBJECTIVES: This study examined behavioral effects of (±)-methcathinone, (±)-3,4-methylenedioxypyrovalerone (MDPV), (±)-3,4-methylenedioxymethcathinone (methylone), and (±)-4-methylmethcathinone (mephedrone) in rats using intracranial self-stimulation (ICSS). METHODS: Male Sprague-Dawley rats (n = 18) with electrodes targeting the medial forebrain bundle responded for multiple frequencies of brain stimulation and were tested in two phases. First, dose-effect curves for methcathinone (0.1-1.0 mg/kg), MDPV (0.32-3.2 mg/kg), methylone (1.0-10 mg/kg), and mephedrone (1.0-10 mg/kg) were determined. Second, time courses were determined for effects produced by the highest dose of each compound. RESULTS: Methcathinone produced dose- and time-dependent facilitation of ICSS. MDPV, methylone, and mephedrone produced dose- and time-dependent increases in low rates of ICSS maintained by low brain stimulation frequencies, but also produced abuse-limiting depression of high ICSS rates maintained by high brain stimulation frequencies. Efficacies to facilitate ICSS were methcathinone ≥ MDPV ≥ methylone > mephedrone. Methcathinone was the most potent compound, and MDPV was the longest acting compound. CONCLUSIONS: All compounds facilitated ICSS at some doses and pretreatment times, which is consistent with abuse liability for each of these compounds. However, efficacies of compounds to facilitate ICSS varied, with methcathinone displaying the highest efficacy and mephedrone displaying the lowest efficacy to facilitate ICSS.

Serotonin and norepinephrine transporters: possible relationship between oligomeric structure and channel modes of conduction.

In the past several years there has been significant progress made on the biophysics of neurotransmitter transporters, leading to the proposal of new models of substrate and ion permeation across membranes. Questions arising from these studies are as follows: How are substrate uptake and substrate-induced current related? Where and how does substrate-ion coupling occur? What is the functional significance of the coupled and uncoupled currents? Because of a long-standing interest and collaboration, and because of their importance for normal function and disease, the authors have focused on the properties of human norepinephrine and serotonin transporters, using other clones and mutations as specific needs arise. It has been know for decades that hNETs (human norepinephrine transporters) clear NE+ (norepinephrine) following its release in peripheral sympathetic and central noradrenergic synapses. Neuronal activity influences NE+ uptake, so one is also interested in the acute regulation of hNET. To study these problems, hNET-expressing cells have been developed that are suitable for patch clamp, radioligand uptake, biochemistry, and transiently expressed clones for structure-function analysis, and new protocols have been designed combining patch-clamp, microamperometry, Ca2+ imaging, and native catecholamine transporter preparations to study transporters in whole cells and isolated patches. Using these methods, Na-dependent, NE+-induced hNET currents that are blocked by cocaine and antidepressants, channel modes of NE+ conduction, voltage-dependent uptake coupled to NE+-induced ion channel activity, PKC (phosphokinase C) regulation of NE+ uptake, and transporter modulation by [Ca2+]i have all been discovered. There is also provocative new data on other transporters in this family, such as Li/Na mole fraction experiments in the Drosophila serotonin transporters and sided enkephalin block in proline transporters. These studies have led one to postulate the existence of a narrow pore within transporters through which the substrate (NE+ or serotonin, 5HT+) and other ions (principally Na+) pass. It is hypothesized that the pore resides in an oligomeric structure and that separate gene products of hNET or hSERT (human serotonin transporters) come together to form a channel.

Bioavailability and pharmacokinetics of intravenously and orally administered allopurinol in healthy beagles.

OBJECTIVES: To determine bioavailability and pharmacokinetic parameters for allopurinol and its active metabolite, oxypurinol. ANIMALS: 6 healthy, reproductively intact female Beagles, 4.9 to 5.2 years old, and weighing 9.5 to 11.5 kg. PROCEDURE: In the first part of the study, allopurinol was administered IV at a dosage of 10 mg/kg of body weight to 3 dogs and 5 mg/kg to 3 dogs; the sequence was then reversed. In the second part of the study, allopurinol was administered orally at a dosage of 15 mg/kg to 3 dogs and 7.5 mg/kg to 3 dogs; the sequence was then reversed. In the third part of the study, allopurinol was administered IV (10 mg/kg), orally (15 mg/kg) with food, and orally (15 mg/kg) without food. Plasma samples were obtained at timed intervals, and concentrations of allopurinol and oxypurinol were determined. RESULTS: Maximal plasma allopurinol concentration and area under plasma allopurinol and oxypurinol concentration-time curves were 2 times greater when dogs were given 10 mg of allopurinol/kg IV, compared with 5 mg/kg, and when dogs were given 15 mg of allopurinol/kg orally, compared with 7.5 mg/kg. Allopurinol elimination half-life, time to reach maximal plasma oxypurinol concentration, and oxypurinol elimination half-life were significantly greater when dogs received 10 mg of allopurinol/kg IV, compared with 5 mg/kg, and when dogs received 15 mg of allopurinol/kg orally, compared with 7.5 mg/kg. CONCLUSIONS: Elimination of allopurinol is dependent on nonlinear enzyme kinetics. The bioavailability of allopurinol, and pharmacokinetic parameters of allopurinol and oxypurinol after oral administration of allopurinol, are not affected by administration with food. CLINICAL RELEVANCE: A dose threshold exists beyond which additional allopurinol would not substantially further inhibit xanthine oxidase activity. Oral administration of > 15 mg of allopurinol/kg to dogs would not be expected to result in greater reduction of plasma and urine uric acid concentrations. Also, allopurinol may be administered to dogs for dissolution or prevention of urate uroliths without regard to time of feeding.

Influence of two diets on pharmacokinetic parameters of allopurinol and oxypurinol in healthy beagles.

OBJECTIVES: To determine whether diet influences the metabolism of IV administered allopurinol in healthy dogs. ANIMALS: 6 healthy female Beagles, 4.9 to 5.2 years old and weighing 9.6 to 11.5 kg. PROCEDURES: Allopurinol was administered IV (10 mg/kg) while dogs consumed a 10.4% protein (dry weight), casein-based diet or a 31.4% (dry weight), meat-based diet. After each dose, plasma samples were obtained at timed intervals, and concentrations of allopurinol and its active metabolite, oxypurinol, were determined by high-performance liquid chromatography. An iterative, nonlinear regression analytical program was used to determine the weighted least-squares, best-fit curves for plasma allopurinol and oxypurinol concentration-time data. From these data, pharmacokinetic parameters were calculated. RESULTS: Pharmacokinetic parameters for allopurinol and oxypurinol were not different when comparing the effect of diet. CONCLUSION: There is no influence of diet on pharmacokinetic parameters of allopurinol or oxypurinol. CLINICAL RELEVANCE: In contrast to observations in human beings, allopurinol metabolism is not influenced by diet. Therefore, formation of xanthine-containing calculi in dogs consuming a high-protein diet and receiving allopurinol is probably not attributable to alteration of allopurinol metabolism.

Effects of time and dilution on concentration of xanthine in frozen urine and plasma of dogs.

OBJECTIVE: To evaluate the effects of dilution on stability of xanthine in canine urine stored at -20 C, and to evaluate the effects of storage at -20 C on stability of xanthine in canine plasma. ANIMALS: 6 reproductively intact female Beagles, 3.9 to 4.2 years old and weighing 8.5 to 10.1 kg. PROCEDURE: Dogs were fed a 31.4% protein (dry weight), meat-based diet for 21 days, and administered allopurinol (15 mg/kg of body weight, q 12 h) during days 14 to 21; urine and plasma samples were obtained on day 22. Urine samples were preserved undiluted or diluted, and divided into 1-ml aliquots for storage at -20 C for 1 to 12 weeks. Plasma samples were divided into 1-ml aliquots for storage at -20 C for 1 to 12 weeks. Urine and plasma xanthine concentrations were measured on day of collection (baseline) and after 1, 2, 4, 6, 9, and 12 weeks. RESULTS: Dilution of urine samples did not have a significant effect on consistency of xanthine concentration measured for up to 12 weeks of storage. Although xanthine concentration did not differ significantly between undiluted and diluted urine samples, average xanthine concentration measured in diluted samples was consistently higher, compared with that in undiluted samples. Compared with baseline values, plasma xanthine concentration was significantly lower at 6, 9, and 12 weeks of storage. CONCLUSIONS: Measurement of xanthine concentration is reproducible in undiluted or diluted urine samples for up to 12 weeks, although dilution may provide better results. Measurement of plasma xanthine concentration is reproducible in samples stored for up to 4 weeks. CLINICAL RELEVANCE: To ensure reproducibility of measurements of xanthine concentration in urine samples collected from dogs that are affected with urate uroliths and receiving allopurinol, urine should be diluted 1:20 with deionized water. These measurements may be useful for monitoring dogs that are receiving allopurinol for dissolution or prevention of urate uroliths.

Epidural administration of bupivacaine, morphine, or their combination for postoperative analgesia in dogs.

OBJECTIVE: To compare the analgesic effects of epidural administration of morphine (MOR), bupivacaine hydrochloride (BUP), their combination (COM), and 0.9% sterile NaCl solution (SAL) in dogs undergoing hind limb orthopedic surgeries. DESIGN: Blinded, randomized clinical trial. ANIMALS: 41 healthy dogs admitted for elective orthopedic surgeries involving the pelvis or hind limbs. PROCEDURE: Analgesic and control agents were administered postoperatively prior to recovery from isoflurane anesthesia. Ten dogs received MOR, 0.1 mg/kg of body weight; 10 received BUP, 0.5%, 1 ml/10-cm distance from the occipital protuberance to the lumbosacral space; 11 received COM; and 10 received SAL epidurally. Dogs were monitored for 24 hours after epidural injection for pain score, heart and respiratory rates, blood pressure, time to required administration of supplemental analgesic agent, total number of supplemental doses of analgesic agent required, and plasma concentrations of cortisol, MOR, and BUP. RESULTS: Pain scores were significantly lower in dogs in the COM and BUP groups than in dogs in the SAL group. Pain scores also were significantly lower in dogs in the COM group than in dogs in the MOR group. Time to required administration of supplemental analgesic agent was longer for dogs in the COM group than for dogs in the MOR and SAL groups. Total number of supplemental doses of analgesic agent required was lower for dogs in the BUP and COM groups than for dogs in the SAL group. CLINICAL IMPLICATIONS: Postoperative epidural administration of COM or BUP alone provides longer-lasting analgesia, compared with MOR or SAL.

Effects of various methods of preservation on the stability of uric acid in frozen canine urine.

OBJECTIVE: To evaluate the effects of dilution and alkalinization, separately and together, on the stability of uric acid in canine urine stored at -20 C. DESIGN: Prospective-controlled study. ANIMALS: 5 dogs with confirmed ammonium urate uroliths, 6 Beagles, and 6 mixed-breed dogs. PROCEDURE: Dogs were fed a 31.4% protein (dry weight), meat-based diet for 21 days, and urine samples were collected on day 22. Urine samples were preserved, using combinations of dilution and alkalinization, and divided into 1-ml aliquots for storage at -20 C for 1 to 12 weeks. Urine uric acid concentrations were measured, using high-performance liquid chromatography, on day of collection (baseline), and after 1, 2, 4, 8, and 12 weeks. RESULTS: Alkalinization did not have a significant effect on reproducibility of measurements of uric acid concentrations in urine; however, dilution did have a significant effect. Compared with baseline, uric acid concentrations in urine samples collected from dogs with ammonium urate uroliths and Beagles and diluted 1:10 or 1:20 with deionized water were not different after storage for 1 to 12 weeks. Uric acid concentrations in urine samples collected from mixed-breed dogs did not differ from baseline values during the 12-week storage period whether samples were undiluted or were diluted 1:10 or 1:20 with deionized water. CONCLUSIONS: Measurements of uric acid concentration are most reproducible in canine urine samples stored at -20 C for 1 to 12 weeks when samples are diluted 1:20 with deionized water. CLINICAL RELEVANCE: To ensure reproducibility of measurements of uric acid concentration in urine samples collected from dogs affected with urate uroliths, urine should be diluted 1:20 with deionized water. Alkalinization is not necessary, and is not recommended because of the additional step in processing and its potential to interfere with measurement of other urinary analytes.

Influence of four diets on uric acid metabolism and endogenous acid production in healthy beagles.

OBJECTIVE: To evaluate the influence of 3 diets used to dissolve or prevent ammonium urate uroliths in dogs, and a diet formulated for growth, on 24-hour excretions of uric acid, ammonia, net acid, titratable acid, bicarbonate, and creatinine; 24-hour urine volumes; pH values of 24-hour urine samples; plasma uric acid concentration; serum creatinine concentration; and endogenous creatinine clearance values. DESIGN: Randomized block. ANIMALS: Six reproductively intact female Beagles, 3.9 to 4.2 years old, weighing 8.5 to 11.1 kg. PROCEDURES: Four diets were evaluated for their ability to dissolve magnesium ammonium phosphate hexahydrate (struvite) uroliths (diet S); to minimize uric acid excretion (diet U); to minimize clinical signs associated with renal failure (diet K); and to promote growth in pups (diet P). Each diet was fed for 14 days; then 24-hour urine samples were collected. An adult maintenance diet was fed during a 7-day washout period. RESULTS: Consumption of diet U was associated with lowest plasma uric acid concentration, lowest 24-hour urinary uric acid, ammonia, titratable acid, and net acid excretions, lowest endogenous creatinine clearance values, highest 24-hour urinary bicarbonate excretion and urine pH values, and highest 24-hour urine volumes. Consumption of diet P was associated with opposite results; results of consumption of diets S and K were intermediate between those for diets U and P. CONCLUSION: Consumption of diet U by healthy Beagles is associated with reduced magnitude of urinary excretion of uric acid and ammonia, with alkaluria, and with polyuria, which may be beneficial in the management of ammonium urate uroliths in dogs. CLINICAL RELEVANCE: Results support use of diet U for management of ammonium urate urolithiasis in dogs.

Influence of two amounts of dietary casein on uric acid, sodium urate, and ammonium urate urinary activity product ratios of healthy beagles.

Casein has been used as a protein source in diets designed to dissolve canine ammonium urate uroliths and to prevent their recurrence, because it contains fewer purine precursors than do many other sources of protein. However, an important question is whether reduced quantities of dietary casein have any benefit in modifying saturation of urine with urates. To answer this question, activity product ratios of uric acid, sodium urate, and ammonium urate were determined in 24-hour urine samples produced by 6 healthy Beagles during periods of consumption of a 10.4% protein, casein-based (10.4% casein) diet and a 20.8% protein, casein-based (20.8%casein) diet. Significantly lower 24-hour urinary excretions of ammonia and phosphorus were observed when dogs consumed the 10.4% casein diet. These results suggest that use of the 10.4% casein diet in protocols designed for dissolution and prevention of uric acid, sodium urate, and ammonium urate uroliths in dogs may be beneficial.

Influence of allopurinol and two diets on 24-hour urinary excretions of uric acid, xanthine, and ammonia by healthy dogs.

Hyperxanthinuria and xanthine uroliths have been recognized with increased frequency in dogs with ammonium urate uroliths that had been given allopurinol. We hypothesized that dietary modification might reduce the magnitude of uric acid and xanthine excretion in urine of dogs given allopurinol. To test this hypothesis, excretion of metabolites, volume, and pH were determined in 24-hour urine samples produced by 6 healthy Beagles during periods of allopurinol administration (15 mg/kg of body weight, PO, q 12 h) and consumption of 2 special purpose diets: a 10.4% protein (dry matter), casein-based diet and a 31.4% protein (dry matter), meat-based diet. Significantly lower values of uric acid (P = 0.004), xanthine (P = 0.003), ammonia (P = 0.0002), net acid (P = 0.0001), titratable acid (P = 0.0002), and creatinine (P = 0.01) excreted during a 24-hour period were detected when dogs consumed the casein-based diet and were given allopurinol, compared with the 24-hour period when the same dogs consumed the meat-based diet and were given allopurinol. For the same 24-hour period, urine pH values, urine volumes, and urine bicarbonate values were significantly (P = 0.0004, P = 0.04, and P = 0.002, respectively) higher during the period when the dogs were fed the casein-based diet and given allopurinol than when they were fed the meat-based diet and given allopurinol. Endogenous creatinine clearance was significantly (P = 0.006) lower when dogs were fed the casein-based diet and given allopurinol than when they were fed the meat-based diet and given allopurinol.(ABSTRACT TRUNCATED AT 250 WORDS)

Diet effect on activity product ratios of uric acid, sodium urate, and ammonium urate in urine formed by healthy beagles.

Urine activity product ratios of uric acid, sodium urate, and ammonium urate and urinary excretion of metabolites were determined in 24-hour samples produced by 6 healthy Beagles during periods of consumption of a low-protein, casein-based diet (diet A) and a high-protein, meat-based diet (diet B). Comparison of effects of diet A with those of diet B revealed: significantly lower activity product ratios of uric acid (P = 0.025), sodium urate (P = 0.045), and ammonium urate (P = 0.0045); significantly lower 24-hour urinary excretion of uric acid (P = 0.002), ammonia (P = 0.0002), sodium (P = 0.01), calcium (P = 0.005), phosphorus (P = 0.0003), magnesium (P = 0.01), and oxalic acid (P = 0.004); significantly (P = 0.0001) higher 24-hour urine pH; and significantly (P = 0.01) lower endogenous creatinine clearance. These results suggest that consumption of diet A minimizes changes in urine that predispose dogs to uric acid, sodium urate, and ammonium urate urolithiasis.

Influence of four diets containing approximately 11% protein (dry weight) on uric acid, sodium urate, and ammonium urate urine activity product ratios of healthy beagles.

Urine activity product ratios of uric acid (APRua), sodium urate (APRna), and ammonium urate (APRau), and urinary excretion of 10 metabolites were determined in 24-hour urine samples produced by 6 healthy Beagles during periods of consumption of 4 diets containing approximately 11% protein (dry weight) and various protein sources: a 72% moisture, casein-based diet; a 10% moisture, egg-based diet; a 72% moisture, chicken-based diet; and a 71% moisture, chicken-based, liver-flavored diet. Significantly (P < 0.05) higher APRua, APRna, and APRau were observed when dogs consumed the egg-based diet, compared with the other 3 diets; there were no differences in these ratios among the other 3 diets. Twenty-four-hour urinary excretions of chloride, potassium, phosphorus, and oxalic acid were significantly (P < 0.05) higher when dogs consumed the egg-based diet. Twenty-four-hour urinary excretions of sodium were significantly (P < 0.05) higher when dogs consumed the egg-based diet, compared with the casein-based diet and the chicken-based, liver-flavored diet, but were not significantly different between the egg-based diet and chicken-based diet. Twenty-four-hour urine volume was similar when dogs consumed the 4 diets. Twenty-four-hour endogenous creatinine clearance was significantly (P < 0.05) lower when dogs consumed the casein-based diet; there were no differences among the other 3 diets. Although consumption of all diets was associated with production of alkaline urine, the 24-hour urine pH was significantly (P < 0.05) higher when dogs consumed the egg-based diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular physiology of norepinephrine and serotonin transporters.

Cocaine- and antidepressant-sensitive norepinephrine and serotonin transporters (NETs and SERTs) are closely related members of the Na+/Cl- transporter gene family, whose other members include transporters for inhibitory amino acid transmitters, neuromodulators, osmolytes and nutrients. Availability of cloned NET and SERT cDNAs has permitted rapid progress in the definition of cellular sites of gene expression, the generation of transporter-specific antibodies suitable for biosynthetic and localization studies, the examination of structure-function relationships in heterologous expression systems and a biophysical analysis of transporter function. In situ hybridization and immunocytochemical studies indicate a primary expression of NET and SERT genes in brain by noradrenergic and serotonergic neurons, respectively. Both NET and SERT are synthesized as glycoproteins, with multiple glycosylation states apparent for SERT proteins in the brain and periphery. N-glycosylation of NET and SERT appears to be essential for transporter assembly and surface expression, but not for antagonist binding affinity. Homology cloning efforts have revealed novel NET and SERT homologs in nonmammalian species that are of potential value in the delineation of the precise sites for substrate and antagonist recognition, including a Drosophila melanogaster SERT with NET-like pharmacology. Electrophysiological recording of human NETs and SERTs stably expressed in HEK-293 cells reveals that both transporters move charge across the plasma membrane following the addition of substrates; these currents can be blocked by NET-and SERT-selective antagonists as well as by cocaine.

Plasma ferulenol concentration and activity of clotting factors in sheep with Ferula communis variety brevifolia intoxication.

Dynamics of plasma ferulenol concentration and its effect on the vitamin K-dependent coagulation factors, prothrombin time (PT), and activated partial thromboplastin time (APTT) were determined in 4 sheep intoxicated individually with 600 g of powdered Ferula communis variety brevifolia (FCb) given in 8 doses at intervals of 6 hours. Ferulenol was detected in the plasma of all sheep at initial blood sample collection, 6 hours after the first dose of approximately 75 g of FCb was placed in the rumen. The last observed peak of approximately 20 micrograms/ml was detected at about 12 hours after the last of 8 doses, and the mean concentration then decreased to < 1 microgram/ml during the next 70 hours. Maximal concentration of ferulenol and time for plasma clearance varied with individual sheep. The PT increased steadily to a maximum of 6 times normal about 70 hours after the last peak plasma ferulenol concentration and about 80 hours after FCb administration was stopped. The PT then returned to almost normal (ratio of 1.12) from the maximum (ratio of 6.12) within approximately 5 days. The APTT results generally paralleled the PT results, but the change was not as marked. Maximal PT and APTT ratios were animal-dependent and not always related to plasma ferulenol concentration. The activity of all the vitamin K-dependent coagulation factors was depressed, but the variations were unique to each factor. Factor V, a vitamin K-independent coagulation factor actually had a brief period of increased plasma activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Reliability of single urine and serum samples for estimation of 24-hour urinary uric acid excretion in six healthy beagles.

Urine uric acid-to-urine creatinine ratios (UUA:UC), urine uric acid concentrations, urine uric acid concentrations corrected for glomerular filtration rate, and urinary uric acid fractional excretions were compared with 24-hour urinary uric acid excretions measured in 6 healthy adult female Beagles. Comparisons, using correlation analysis, were made when dogs consumed a 10.4% protein (dry weight), casein-based diet and a 31.4% protein (dry weight), meat-based diet. The UUA:UC, urine uric acid concentrations corrected for glomerular filtration rate, and urinary uric acid fractional excretions were not reliable estimates of 24-hour urinary uric acid excretions during consumption of either diet. Urine uric acid concentrations in samples collected 2, 4, 6, and 24 hours after initiation of collection correlated with 24-hour urinary uric acid excretions when dogs consumed the casein-based diet; correlation was not found at any time interval when dogs consumed the meat-based diet. Therefore, determination of 24-hour urinary uric acid excretion is recommended because UUA:UC are unreliable.

Simultaneous determination of eight anticoagulant rodenticides in blood serum and liver.

A liquid chromatographic method was developed for the analysis of indandione and 4-hydroxycoumarin anticoagulant rodenticides in blood serum and liver. The method enabled the measurement of serum and liver concentrations of eight anticoagulant rodenticides: brodifacoum, bromadiolone, chlorophacinone, coumafuryl, coumatetralyl, diphacinone, difenacoum, and warfarin. Anticoagulants were extracted from serum and liver with acetonitrile. Extracts were applied to solid-phase extraction columns, which contained mixed packings. Column eluates were evaporated to dryness, reconstituted, and subjected to reversed-phase liquid chromatography. Hydroxycoumarins were detected by fluorescence at an excitation wavelength of 318 nm and an emission wavelength of 390 nm. Indandiones were detected by UV absorption at 285 nm. Extraction efficiencies of greater than 75% for serum and greater than 69% for liver were obtained. The within-run precision (CV) ranged from 2.4 to 8.6% for serum and 2.6 to 8.7% for liver. The between-run precision (CV) ranged from 1.5 to 12.2% for serum and from 2.1 to 11.8% for liver. Hydroxycoumarin rodenticides were detected at 1 ng/mL of serum and 1 ng/g of liver. Indandiones were detected at 10 ng/mL of serum and 10 ng/g of liver.

Elevated 25-hydroxy and normal 1,25-dihydroxy cholecalciferol serum concentrations in a successfully-treated case of vitamin D3 toxicosis in a dog.

A 4-y old, 27 kg spayed female German Shepherd dog was observed to ingest one 1-oz package of a rodenticide containing cholecalciferol. An initial serum calcium concentration of 15.7 mg/dl was successfully reduced to normal during 10 d using calcitonin and prednisolone. During that time, the serum 25-hydroxy and 1,25-dihydroxy cholecalciferol concentrations ranged from 637 to 315 ng/ml (normal 32 +/- 6 ng/ml) and 64 to 29 pg/ml (normal 34 +/- 19 pg/ml), respectively. Serum mid-molecule parathyroid hormone concentrations (76 to 97 pcmol/L) were within the normal range (85-140 pcmol/L). These data indicate that hypercalcemia seen in dogs following field exposures to cholecalciferol-containing rodenticides may be associated with elevated 25-hydroxy rather than 1,25-dihydroxy cholecalciferol. Consequently, serum 25-hydroxy cholecalciferol concentrations may be the most conclusive method for diagnosing hypervitaminosis D3 toxicosis in the live dog.

Relationship between the iron regulated outer membrane proteins and the outer membrane proteins of in vivo grown Pasteurella multocida.

The SDS-PAGE patterns of the outer membrane protein (OMP) extracts of Pasteurella multocida strain P1059, grown under iron-restricted, iron-replete and in vivo conditions, were examined. The results showed that the iron-regulated outer membrane proteins (IROMPs) with molecular masses of 76 kDa, 84 kDa, and 94 kDa were expressed by bacteria grown in iron-restricted media. They were also expressed by in vivo grown P. multocida. Convalescent-phase sera, obtained from turkeys which had survived pasteurellosis, contained antibodies that reacted intensly with th three IROMPs. This indicated that these proteins were expressed in vivo. Bacteria expressing the IROMPs showed greater binding to Congo Red when compared to cells not expressing IROMPs. Cells expressing the IROMPs or its OMP extracts grown in iron-restricted media also showed greater binding to 59Fe-pasteurella siderophore (multocidin) when compared to bacteria or its extracts not expressing IROMPs. Convalescent-phase sera, which contained antibodies against the IROMPs, blocked this specific 59Fe-multocidin binding to IROMPs. Autoradiography was used to determine which of these IROMPs functioned as a receptor for the iron-multocidin complex. The results suggested that these three IROMPs have specific epitopes for binding to the iron multocidin complex.

Multicomponent determination of 4-hydroxycoumarin anticoagulant rodenticides in blood serum by liquid chromatography with fluorescence detection.

A sensitive liquid chromatographic method was developed for the analysis of 4-hydroxycoumarin anticoagulant rodenticides in blood serum. The method can simultaneously measure the serum levels of five anticoagulant rodenticides: brodifacoum, bromadiolone, coumatetralyl, difenacoum, and warfarin. Serum proteins are precipitated with acetonitrile and the supernatant is mixed with ethyl ether. The organic phase is separated, evaporated to dryness, and the residue subjected to chromatographic analysis. The anticoagulants are separated by reversed-phase gradient chromatography with fluorescence detection at an excitation wavelength of 318 nm and emission wavelength of 390 nm. Extraction efficiencies of 68.1 to 98.2% were obtained. The within-run precision (CV) ranged from 2.19 to 3.79% and the between-run precision (CV) from 3.72 to 9.57%. The anticoagulants can be quantitated at serum levels of 10 to 20 ng/mL.

Superwarfarin ingestion and detection.

Because of the emergence of warfarin resistance, new potent long-acting anticoagulants are now readily available in several over-the-counter rodenticide products. The availability of these "superwarfarin" compounds has led to accidental and purposeful human ingestions, one of which has resulted in a death. We summarize the prior case reports and report a second death. In addition, we report the availability of an assay to detect the presence of brodifacoum (a superwarfarin compound) in human plasma and tissues.