Multi-residue Determination of Polar Veterinary Drugs in Livestock and Fishery Products by Liquid Chromatography/Tandem Mass Spectrometry.

Residues of 37 polar veterinary drugs belonging to six families (quinolones, tetracyclines, macrolides, lincosamides, sulfonamides, and others) in livestock and fishery products were determined using a validated LC-MS/MS method. There were two key points in sample preparation. First, extraction was performed with two solutions of different polarity. Highly polar compounds were initially extracted with Na2EDTA-McIlvaine's buffer (pH 7.0). Medium polar compounds were then extracted from the same samples with acetonitrile containing 0.1% formic acid. Secondly, cleanup was performed using a single SPE polymer cartridge. The first extracted solution was applied to the cartridge. Highly polar compounds were retained on the cartridge. Then, the second extracted solution was applied to the same cartridge. Both highly and medium polar compounds were eluted from the cartridge. This method satisfied the guideline criteria for 37 out of 37 drugs in swine muscle, chicken muscle, bovine muscle, prawn, salmon trout, red sea bream, milk, and honey; 35 out of 37 in egg; and 34 out of 37 in flounder. The LOQ ranged from 0.1 to 5 µg/kg. Residues were detected in 24 out of 110 samples and analyzed using the validated method.

Single-laboratory validation study of rapid analysis method for multi-class veterinary drugs in milk, fish and shellfish by LC-MS/MS.

A method of rapid analysis of multi-class residual veterinary drugs in milk, fish and shellfish was validated in accordance with Japanese guidelines for the validation of analytical methods for residual agricultural chemicals in food. Using LC-MS/MS, 43 multi-class veterinary drugs, including sulfonamides, quinolones, coccidiostats and antiparasites, could be analyzed in one injection. Analytes were extracted from samples with two kinds of solvent, acetonitrile containing 1 vol% formic acid and anhydrous acetonitrile, and salted out with 4.0 g of magnesium sulfate, 1.5 g of trisodium citrate and 2.0 g of sodium chloride. This method was assessed by performing recovery tests in retail milk and 4 kinds of fresh cultured fish and shellfish (salmon, tiger shrimp, red sea bream and bastard halibut) spiked with the 43 target analytes at the levels of 10 and 100 µg/kg. Using this method, 40 out of 43 drugs satisfied the guideline criteria in milk, 37 drugs in salmon, 42 drugs in tiger shrimp, 41 drugs in red sea bream and 39 drugs in bastard halibut.

Development and validation of rapid analysis method for multi-class veterinary drugs in livestock products by LC-MS/MS.

A method for rapid analysis of multi-class residual veterinary drugs in livestock products was developed and validated in accordance with the Japanese guideline for pesticides. Using LC-MS/MS, 43 multi-class veterinary drugs, including sulfonamides, quinolones, coccidiostats and antiparasites, could be analyzed simultaneously in only 18 minutes. The extraction process was developed by modifying the QuEChERS approach to provide faster and less expensive extraction. The samples were extracted by using two kinds of solvent, acetonitrile and acetonitrile including 1 vol% formic acid, and salted out with magnesium sulfate, trisodium citrate and sodium chloride. Using these two extractants, 40 out of 43 drugs satisfied the guideline criteria in bovine muscle and swine muscle, 39 drugs were found in chicken muscle, and 37 drugs were found in eggs. The limit of quantification was less than the MRL for all analytes.

Rapid determination of nitroimidazole residues in honey by liquid chromatography/tandem mass spectrometry.

We developed a rapid and efficient means of determining residues of four nitroimidazoles-i.e., dimetridazole, ipronidazole, metronidazole, and ronidazole-and three hydrophilic metabolites- i.e., 2-hydroxymethyl-1-methyl-5-nitroimidazole, 1 -methyl-2-(2'-hydroxyisopropyl)-5-nitroimidazole, and 1-(2-hydroxyethyl)-2-hydroxymethyl-nitroimidazole--in honey. We applied a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) procedure improved to suit a nitroimidazole analysis, which is fast (approximately 30 min) and uses less organic solvent. The procedure involves initial single-phase extraction of 5 g of honey with acetonitrile containing 1% acetic acid, followed by liquid-liquid partitioning involving the addition of 5 g sodium chloride, 1.5 g trisodium citrate dihydrate, and 4 g magnesium sulfate. Moreover, matrix from honey was reduced by an SPE method with an alumina-N cartridge. The samples were analyzed using LC/MS/MS. Chromatographic separation of these nitroimidazoles and metabolites was performed in the gradient mode on a pentafluorophenylpropyl-bonded silica column (150x2.0 mm, 3 pm particle size) at 40 degrees C. The mobile phase consisted of a 0.01% acetic acid solution and acetonitrile, and the flow rate was 0.2 mL/min. The method was validated using honey spiked with these nitroimidazoles from 0.1 to 0.5 microg/kg. The overall recovery of the seven nitroimidazoles ranged from 76.1 to 98.5%; intra- and interassay CV values were <9.5 and <14.2%, respectively. The LOQ ranged from 0.1 to 0.5 microg/kg. LC/MS/MS coupled with the QuEChERS method showed good potential as a method for determining nitroimidazole residues in honey.

Screening assay of residual antibiotics in livestock samples by LC-MS/MS.

A LC-MS/MS screening assay of multi-class antibiotics was developed for 19 residual antibiotics in livestock samples. Sample preparation employed the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) approach using 0.5% formic acid in acetonitrile-methanol (8 : 2), with salting-out using magnesium sulfate, trisodium citrate and sodium chloride. Recovery values from 5 different livestock samples ranged from 45.5 to 121.6%, and the RSDs were under 18% at two concentration levels. The limit of quantification values of 19 analytes were under 10 µg/kg in all livestock samples, and the procedure can detect almost all analytes under the MRL. Screening capability was confirmed by employing spiked samples. This new screening assay for residual antibiotics in livestock samples is expected to be useful for routine laboratory tests.

Age-associated reduction of stimulatory effect of ghrelin on food intake in mice.

Aging is associated with a progressive decrease in appetite and food intake. We focused on the age-associated changes of the stimulatory effect of the appetite-regulating peptide, ghrelin. Food intake and the concentrations of acyl ghrelin and desacyl ghrelin in the plasma and in the stomach were measured with and without overnight fasting in young and old mice. Moreover, the food intake in response to the intraperitoneal administration of graded doses of acyl ghrelin was compared between young and old mice. Fasting drives food intake in young mice, but not in old mice. The concentrations of acyl ghrelin and desacyl ghrelin in the plasma and in the stomach were higher in the old mice than in the young mice. Food intake did not increase in old mice when stimulated by the administration of 1-3 nmol of acyl ghrelin, which could produce a significant increase in food intake in young mice. In conclusion, food intake did not increase in old mice after either overnight fasting or the administration of acyl ghrelin. The release and synthesis of ghrelin seem to be rather higher in old mice compared to young mice. These increases might be the results of compensation for the decline of receptor (and/or post-receptor) functions.

Administration of ursodeoxycholate failed to prevent sludge and/or gallstone formation in cholecystokinin-1(A) receptor-deficient mice.

Gallstone disease is one of the most prevalent digestive diseases. The frequency of gallstone disease is about 10% in middle-age persons and 20% in aged persons. Gallbladder dysmotility and stasis of bile flow promote sludge and/or gallstone formation. Gallbladder contraction depends on cholecystokinin (CCK) via CCK-1 receptors (R)s. Previously, we raised CCK-1R deficient (-/-) mice and observed sludge and/or gallstone formation in more than 30% at 12-24 months of age. As ursodeoxycholate (UDCA) is commonly used for patients with gallstone disease, we expected that continuous administration of UDCA could prevent sludge and/or gallstone formation in CCK- 1R(-/-) mice. A diet containing 0.1% UDCA was administered in age-matched CCK-1R(-/-) and wild-type male and female mice for 8 months. Administration of UDCA decreased the frequency of sludge and/or gallstone formation compared with the control (CRF-1) diet (39%→26% in male, 35%→25% in female mice); however, these effects did not attain a level of statistical significance. Although the body weight was significantly higher in UDCA-fed than CRF-1-fed male mice regardless of genotypes, the plasma lipid concentrations did not differ between the two diets. In conclusion, administration of UDCA was less effective than expected at preventing sludge and/or gallstone formation in CCK-1R(-/-) mice.

Rapid determination of fumagillin residues in honey by liquid chromatography-tandem mass spectrometry using the QuEChERS method.

A new, rapid, and efficient method for determining the fumagillin residues in honey was developed. The samples extracted were analyzed using LC/MS/MS. Chromatographic separation of fumagillin was performed in gradient mode on a C8 column (100 x 2.0 mm, 5 microm) at 40 degrees C. The mobile phase consisted of a mixture of 2 mM ammonium formate-0.01% formic acid solution and methanol; the flow rate was set to 0.2 mL/min. Under these conditions, it was possible to measure fumagillin and its isomers as a single peak. The sample preparation procedure used is based on the QuEChERS (quick, easy, cheap, effective, rugged, and safe) method, which is fast (approximately 30 min) and uses less organic solvent. The fumagillin was extracted with acetonitrile containing 0.1% formic acid, then purified using a solid-phase extraction method with an Oasis mixed-mode weak anion-exchange cartridge. The overall recovery of fumagillin ranged from 88.1 to 99.4%; the intra- and interassay CVs were <4.5% and <4.9%, respectively. The LOQ was 0.1 microg/kg. LC/MS/MS coupled with the QuEChERS method showed strong potential as a method for determining fumagillin residues in honey.

[Determination of nequinate and buquinolate in livestock products using liquid chromatography-tandem mass spectrometry].

We studied the simultaneous determination of nequinate and buquinolate, which are used as feed additives to prevent coccidiosis, by means of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The sample was extracted with acetonitrile, then loaded onto an HLB mini-column with 20% methanol. After clean-up with 20% methanol, the analytes were eluted with acetonitrile-methanol (1 : 1). The coccidiostats in the purified samples were determined using ESI-MRM mode LC-MS/MS with a sample matrix calibration curve. Mean recoveries of nequinate and buquinolate from 8 kinds of livestocks samples (chicken muscle, chicken liver, chicken heart, swine muscle, swine heart, cattle muscle, sheep muscle, egg) were in the range of 89.5% to 108.6%, and the relative standard deviation values were <20% (n=10) at the levels of 0.01 µg/g and 0.05 µg/g, respectively. The limits of quantification of these compounds were 0.001 µg/g in each sample.

Determination of dimetridazole, metronidazole and ronidazole in salmon and honey by liquid chromatography coupled with tandem mass spectrometry.

A liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed to determine the residues of dimetridazole (DMZ), metronidazole (MNZ) and ronidazole (RNZ) in salmon and honey. These compounds were extracted with ethyl acetate from samples and cleaned up using a silica solid phase extraction (SPE) cartridge. These compounds were determined by reversed-phase LC using a C18 column with distilled water-methanol as the mobile phase, and MS detection in the positive mode by applying selected reaction monitoring (SRM). DMZ-d(3), MNZ-(13)C(2),(15)N(2) and RNZ-d(3) were used as internal standards. The method was validated in salmon and honey spiked with these compounds at 0.4-2 µg/kg, and average recoveries were in the range of 91.2-107.0%. Repeatability was 1.7-17.1% and intermediate precision was less than 20%. The detection limits of DMZ, MNZ and RNZ in salmon and honey were 0.05-0.2 µg/kg. The method was applied to 3 salmon and 20 honey samples. The concentrations of these compounds in all samples were lower than the detection limits established by the Ministry of Health, Labour and Welfare in Japan.

Rapid determination of fluoroquinolone residues in honey by a microbiological screening method and liquid chromatography.

A rapid and efficient method was developed for the simultaneous determination of seven fluoroquinolone (FQ) residues: norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, orbifloxacin, sarafloxacin, and difloxacin in honey. The samples were first screened with a microbiological method by using test plates made from metal-free purified agar seeded with Bacillus subtilis BGA. When a sample was found to contain FQ residues by using the microbiological method, it was analyzed by LC with fluorescence detection (LC/FL). FQs were extracted with Na2EDTA-McIlvaine buffer and purified by a dual SPE method in which a cation-exchange cartridge was connected to an anion-exchange cartridge. The overall recoveries of the seven FQs ranged from 70.0 to 92.1%. The intra-assay and interassay CVs were < or = 7.8 and < or = 5.1%, respectively. For the microbiological method, the LOD values ranged from 2 to 9 microg/kg. For LC/FL, the LOQ values ranged from 2 to 7 microg/kg. The developed method was used to analyze 70 honey samples. In 14 samples in which the microbiological method detected the presence of FQ residues, norfloxacin, ciprofloxacin, and enrofloxacin were identified by LC/FL.

Determination of bithionol, bromophen, nitroxynil, oxyclozanide, and tribromsalan in milk with liquid chromatography coupled with tandem mass spectrometry.

LC/MS/MS was developed to determine the residues of bithionol (BTN), bromofen (BMF), nitroxynil (NTX), oxyclozanide (OCZ), and tribromsalan (TBS) in milk. Samples were extracted with ethyl acetate and cleaned up by liquid-liquid separation with acetonitrile and n-hexane. The compounds were determined by RP-LC using a C18 column with 0.1% formic acid-methanol. Mass spectral acquisition was performed in the negative mode by applying selected-reaction monitoring. The method was validated in milk spiked with these compounds at 5-600 microg/kg; average recoveries were in the range 83.8-97.1%, with RSD values of 1.4-8.0%. The interassay RSDs were less than 11%. The LODs of these compounds in milk were 0.1 microg/kg. The method was applied to 24 raw milk samples. The concentration of these compounds in all samples was lower than the Japanese maximum residue limits. The method is rapid, sensitive, and specific for monitoring residues of BTN, BMF, NTX, OCZ, and TBS in milk.

Stimulatory effect of N-methyltyramine, a congener of beer, on pancreatic secretion in conscious rats.

BACKGROUND: Alcoholic beverages stimulate gastric acid secretion and increase the appetite. Although ingested ethanol stimulates pancreatic secretion, alcoholic beverages contain several congeners. N-methyltyramine (NMT) was isolated from beer as a factor in stimulating gastric acid secretion. In this study, we examined NMT to determine whether the congener stimulated pancreatic secretion in conscious rats. METHODS: Cannulae were inserted into male Wistar rats to separately drain bile and pancreatic secretions: 2 duodenal cannulae, a gastric cannula, and an external jugular vein cannula. The rats were placed in modified Bollman-type restraint cages. After a 4-day recovery period, experiments were conducted on unanesthetized rats. Different concentrations of NMT (5, 25, and 50 microg/kg) solutions were infused into the stomach. To examine the mechanism, the effects of the proton pump inhibitor, cholecystokinin (CCK-BR) antagonist (YM022), CCK-AR antagonist (CR1505), and atropine were administered prior to the NMT (25 microg/kg) infusion. The effect of intravenous infusion of NMT (7.5 microg/kg) was then determined. Moreover, dispersed acini were prepared, and the effect of different concentrations of NMT on amylase release was determined. RESULTS: Intragastric administration of NMT significantly increased pancreatic exocrine secretion in a dose-dependent manner. Atropine eliminated the stimulatory effect of NMT, but the infusion of the proton pump inhibitor, YM022, and CR1505 did not. Intravenous infusion of NMT did not affect pancreatic secretion, and NMT did not stimulate amylase release in vitro. CONCLUSIONS: N-methyltyramine stimulates pancreatic secretion via the cholinergic gastro-pancreatic reflex. The NMT content in beer was 2 mg/l, so that if a person weighing 60 kg consumes a 750 ml of beer, 25 microg/kg NMT will be ingested. Therefore, the stimulatory effect of beer on pancreatic secretion was produced not only by ethanol but also by the congener, NMT.

Gastric acid secretion in cholecystokinin-1 receptor, -2 receptor, and -1, -2 receptor gene knockout mice.

Gastrin is important for stimulating acid secretion as well as differentiating gastric mucosal cells via cholecystokinin-2 receptors (CCK-2Rs). In turn, CCK acts preferably via CCK-1R to release somatostatin, and somatostatin has been postulated to exhibit a tonic inhibition of gastrin bioactivity. The present study was designed to examine the hypothesis that CCK-1R and 2R may act in opposite directions in gastric acid secretion. Having generated CCK-1R(-/-), 2R(-/-), and 1R(-/-)2R(-/-) mice, we examined the regulation of gastric acid secretion in four genotypes including wild-type mice. Parietal cells possess histamine receptors, muscarinic receptors, and CCK-2Rs. Since histamine increases cAMP and carbachol increases calcium, the responses of gastric acid secretion to graded doses of histamine, carbachol, and a combination of histamine + carbachol were determined. The sensitivity to histamine did not differ among the four genotypes, while the maximal acid secretion was lower in CCK-2R(-/-) mice than in wild-type mice. In addition, sensitivity to carbachol was impaired in mice without CCK-2R. The interaction of histamine and carbachol was conserved in all genotypes. In conclusion, CCK-2R is necessary to respond to carbachol as well as to produce the maximal acid secretion, while the role of CCK-1R in acid secretion is less important.

Age-associated gallstone formation in male and female CCK-1(A) receptor-deficient mice.

BACKGROUND: Gallbladder dysmotility accelerates cholelithiasis. In turn, gallbladder dysmotility can occur secondary to inflammation and excess cholesterol accumulation in gallbladder smooth muscle. METHODS: The present study was designed to determine how much gallbladder dysmotility contributes to gallstone formation as a primary cause and whether a sex difference exists in gallstone formation by comparing cholecystokinin-1 receptor gene-deficient [CCK-1R(-/-)] male and female mice. RESULTS: No sludge or gallstone formation was observed in mice at 6 months of age. The frequency of sludge and gallstone formation in mice at 12 and 24 months of age was slightly higher in female CCK-1R(-/-) mice than in males, but the difference was not significant. CONCLUSIONS: Gallbladder dysmotility may have accelerated sludge and gallstone formation, but its contribution was limited. A 12-month period was required to produce gallstones, and after the mice reached 12 months of age, further ageing did not increase the frequency of gallstones. The effect of sex did not reach a significant level.

Susceptibility to obesity and gallbladder stasis produced by a protein- and fat-enriched diet in male mice compared with female mice.

BACKGROUND: The frequency of Japanese subjects over 20 years old with metabolic syndrome is 45.6% in men but just 16.7% in women. The reason why Japanese male subjects are more susceptible to metabolic syndrome than women is unknown. One possibility is the higher frequency of Japanese male subjects (40-70 years old) who had a drinking habit (67%), while that of female subjects was only 25%. In addition, daily fat intake was markedly increased in Japanese subjects (from 9% to 25%), and cholesterol cholelithiasis is one of the most rapidly increasing digestive diseases during the past 50 years. The object of this study is to examine whether a potential sex-related risk factor exists in the manifestation of metabolic syndrome as well as gallstone formation. METHODS: Gallbladder dysmotility accerelates gallstone formation and gallbladder contraction depends on cholecystokinin (CCK) and its receptor (CCK-1R). We developed CCK-1R gene knockout (-/-) mice. The effects of the fat- and protein- enriched diet OA-2 on body weight, hyperlipidemia, and frequencies of sludge and gallstone formation were examined, and compared between wild-type and CCK-1R(-/-) male and female mice. The OA-2 diet contains slightly higher protein and fat (7.9 % fat and 27.6 % protein) compared with a standard diet (CRF-1) (5.6 % fat and 22.6 % protein), but their total energies are similar. After weaning, CRF-1 was provided until 3 months of age in all animals. Administration of an OA-2 diet was started when age-matched CCK-1R(-/-) and wild-type male and female mice reached maturity, at 3 months of age. Administration of CRF-1 was continued in the rest of the animals. Mice were sacrificed by guillotine at 6 and 12 months of age and the blood was collected to measure plasma levels of triglyceride and cholesterol. The gallbladder was removed and classified as normal (clear gallbladder), clouded (sludge formation), and/or containing gallstone formations. RESULTS: As long as CRF-1 was provided, the frequency of sludge and/or gallstone formation in CCK-1R(-/-) male mice was 3 of 8 (35%) and 4 of 9 (45%) in females at 12 months of age, whereas no gallstone formation was observed at 6 months of age. On the other hand, male mice fed OA-2 increased their body weight and plasma lipid concentrations, compared with those fed CRF-1 regardless of genotype. Under the OA-2 diet, sludge and gallstone formation was observed at 6 months of age, not only in CCK-1R(-/-) male mice but also in wild-type male mice. In contrast, parameters in female mice did not differ between the two diets. CONCLUSION: Male mice were more susceptible to protein- and fat-enriched diet-induced obesity than female mice, and hyper-nutritional status accelerated sludge and gallstone formation in male mice.

Decreased hydrogen-potassium-activated ATPase (H+-K+-ATPase) expression and gastric acid secretory capacity in aged mice.

Gastric acid secretion in response to chemical stimulation and to mechanical stimulation was investigated in adult and old mice. The protein expression of a proton pump (H(+)-K(+)-ATPase), a marker of parietal cell function, was determined by Western blotting. Acid secretion was stimulated by histamine (500 and 1000 microg/kg) or carbachol (10 and 20 microg/kg). To investigate the response to mechanical stimulation, the stomach was distended by an intragastric injection of isotonic saline (0.5, 1.0, 1.5, and 2.0 ml). Administration of two doses of histamine produced a dose-dependent increase in acid secretion in adult mice, whereas a higher dose of histamine failed to produce a further increase in old mice. Gastric acid secretion, whether produced by carbachol or mechanical stimulation, did not differ between the two age groups. The protein expression of H(+)-K(+)-ATPase was significantly lower in old mice than in adult. Insofar as histamine increases acid secretion via the cyclic AMP (cAMP) pathway in parietal cells, while carbachol and gastric distention do so via the calcium signaling pathway, the cAMP signaling pathway may be more susceptible to aging than the calcium signaling pathway. The decrease in the secretory capacity of acid secretion in the old mice may be partly attributable to a decrease in parietal cell function, as shown by decrease in H(+)-K(+)-ATPase protein expression.

Lack of ghrelin secretion in response to fasting in cholecystokinin-A (-1), -B (-2) receptor-deficient mice.

Cholecystokinin receptors (CCK-Rs) have been classified into two subtypes: CCK-AR (1R) and -BR (2R). We generated CCK-AR(-/-), CCK-BR(-/-), and CCK-AR(-/-)BR(-/-) mice and found that the gastric emptying of a liquid meal was increased in CCK-BR(-/-) and AR(-/-)BR(-/-) mice, compared with wild-type and CCK-AR(-/-) mice. Given that enhanced gastric emptying leads to eating, food intake after overnight fasting was examined, as was the effect of CCK-8S on food intake. Male mice 6-8 months of age were deprived of food for 16 h with free access to water, after which they were injected intraperitoneally (0.1 ml/mouse) with either vehicle or CCK-8 (0.3, 1.0, or 3.0 nmol/mouse), and their food intake was monitored for 4 h. CCK-8S inhibited food intake in wild-type and CCK-BR(-/-) mice, but not in CCK-AR(-/-) or AR(-/-)BR(-/-) mice. Unexpectedly, we observed a lower food intake in CCK-AR(-/-)BR (-/-) mice treated with vehicle than in mice of the other genotypes. To examine the mechanism of decrease in food intake in CCK-AR(-/-)BR(-/-) mice, the involvement of ghrelin was determined in wild-type and CCK-AR(-/-)BR(-/-) mice. Fasting plasma ghrelin levels were significantly lower in CCK-AR (-/-)BR(-/-) mice than in wild-type mice, and no increase in response to fasting was observed in CCK-AR(-/-)BR(-/-) mice. An administration of acyl-ghrelin produced a small increase in food intake in CCK-AR(-/-)BR(-/-) mice, but not to the levels of wild-type mice. In conclusion, CCK-AR(-/-)BR(-/-) mice showed lower food intake as well as lower response to exogenous ghrelin, and a lower plasma ghrelin level after fasting, though which receptor is more important is unknown.

Acute inhibition of lymphatic cholesterol transport in rat intestine by SCH 58053.

To clarify the pharmacological mechanism of ezetimibe, SCH 58053, an analog of ezetimibe, was intraduodenally administered to lymph-fistula rats, and its effect on lymphatic lipid transport in the intestine was monitored. SCH 58053, 5.0 mg/kg body weight, was administered one hour before a 5-hour infusion of a lipid emulsion containing 40 micromol/h of triolein and 2.74 micromol/h of cholesterol (Experiment 1) or co-administered at 5.0 mg/kg body weight/h with the lipid emulsion for 4 hours to rats that had been infused with the lipid emulsion alone for 3 hours (Experiment 2). SCH 58053 administration significantly inhibited lymphatic cholesterol transport, but not triglyceride transport, in both groups compared to control rats that did not receive SCH 58053. The ratio of free cholesterol to total cholesterol in the lymph of the treated rats was unchanged compared to the control rats. Thus, the results showed that SCH 58053 is a potent, rapid, and selective inhibitor of lymphatic cholesterol transport in the intestine.

Disturbance of response to acute thermal pain in naturally occurring cholecystokinin-a receptor gene knockout Otsuka Long-Evans Tokushima Fatty (OLETF) rats.

Otsuka Long-Evans Tokushima Fatty (OLETF) rats lack cholecystokinin-A receptor (CCK-AR) because of a genetic abnormality. We observed that body temperature homeostasis in response to changes in ambient temperature was deteriorated in OLETF rats, while the functions of the signal outputs from the hypothalamus to effectors were not impaired. Deteriorated homeostasis was also seen in CCK-AR deficient (-/-) mice. In the present study, we examined whether the sensory pathway involved in transmitting signals about temperature from the skin to the brain was impaired in OLETF rats. To elucidate the involvement of CCK-AR function, we conducted the same experiment in CCK-AR(-/-) mice. Responses to thermal pain were assessed using the Hargreaves' plantar test apparatus. Shortening of withdrawal latency was observed in OLETF rats compared to control rats, indicating thermal hyperalgesia. Behavioral responses following paw withdrawal were disturbed in OLETF rats. The 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid contents in the hippocampus and frontal cortex of OLETF rats were significantly higher than in those of the controls. CCK-AR(-/-) mice did not show any differences from wild-type mice. In conclusion, OLETF rats showed thermal hyperalgesia and disturbed responses to thermal pain, and an alteration of 5-HT function might have a role in this disturbance.