Metabonomic evaluation of Schaedler altered microflora rats

Previously, we identified two distinct metabonomic phenotypes in Sprague-Dawley rats sourced from two different rooms (colonies) in the Charles River, Raleigh facility [Robosky, L. C., Wells, D. F., Egnash, L. A., Manning, M. L., Reily, M. D., and Robertson, D. G. (2005) Metabonomic identification of two distinct phenotypes in Sprague-Dawley (Crl:CD(SD)) rats. Toxicol. Sci. 87, 277-284]. On the basis of literature reports and cohabitation experiments, we concluded that the differing phenotypes were due to different gut flora populations. One hypothesis explaining this phenomenon was attributed to the practice of initiating new colonies with rats derived from foundation colonies that had limited gut floral populations, the Charles River altered Schaedler flora (CRASF) rats. We hypothesized that the lack of differentiation of CRASF rats to the full complement of microflora was responsible for the altered phenotype characterized by increased urinary chlorogenic acid metabolites and decreased hippurate (CA rats) as opposed to the prevalent phenotype characterized by the inverse ratio of these metabolites (HIP rats). Upon receipt, it was confirmed that the CRASF rats exhibited a metabonomic profile similar to CA rats that remained constant while animals were housed individually in a dedicated animal room. However, exposure of CRASF rats to HIP rats, or their bedding, led to a relatively rapid but variable rate of reversion to the historic HIP type metabolic profile. On the basis of the results, we conclude that CRASF rats have a unique metabolic profile due to their limited gut flora constitution. If rigorous isolation procedures are not employed, the CRASF phenotype will eventually differentiate into the more typical HIP phenotype with a time course that may be quite variable. Given the marked metabolic heterogeneity between the phenotypes, this work highlights the importance of monitoring rat metabolic profiles.     

Communication Regarding Metabonomic Identification of Two Distinct Phenotypes in Sprague-Dawley (Crl:CD(SD)) Rats

In a recent publication, we reported the identification andcharacterization of two distinct phenotypes in Sprague-Dawleyrats obtained from the Charles River Raleigh facility (Roboskyet al., 2005). The phenotypes were designated as HIP (high hippuricacid level in urine) and CA (high chlorogenic     

建立大鼠原位肠-肝灌流模型评价绿原酸的代谢

目的：建立大鼠原位肠-肝灌流模型，运用该模型研究绿原酸在大鼠肠、肝中的代谢转化。方法：采用液相色谱质谱联用（HPLC-MS）法测定大鼠原位肠-肝灌流模型灌流液中绿原酸及其代谢产物咖啡酸、阿魏酸和马尿酸。结果：绿原酸十二指肠给药后，灌流液中主要活性代谢产物为阿魏酸，同时在灌流液中也检测到少量活性代谢产物咖啡酸和大量代谢终产物马尿酸。结论：大鼠原位肠-肝灌流模型适用于绿原酸生物转化和代谢动力学的研究；绿原酸在肠中存在广泛的代谢。     

Detection of carboxylic acids and inhibition of hippuric acid formation in rats treated with 3-butene-1,2-diol, a major metabolite of 1,3-butadiene.

Epidemiological studies have indicated that 1,3-butadiene exposure is associated with an increased risk of leukemia. In human liver microsomes, 1,3-butadiene is rapidly oxidized to butadiene monoxide, which can then be hydrolyzed to 3-butene-1,2-diol (BDD). In this study, BDD and several potential metabolites were characterized in the urine of male B6C3F1 mice and Sprague-Dawley rats after BDD administration (i.p.). Rats given 1420 micromol kg(-1) BDD excreted significantly greater amounts of BDD relative to rats administered 710 micromol kg(-1) BDD. Rats administered 1420 or 2840 micromol kg(-1) BDD excreted significantly greater amounts of BDD per kilogram of body weight than mice given an equivalent dose. Trace amounts of 1-hydroxy-2-butanone and the carboxylic acid metabolites, crotonic acid, propionic acid, and 2-ketobutyric acid, were detected in mouse and rat urine after BDD administration. Because of the identification of the carboxylic acid metabolites and because of the known ability of carboxylic acids to conjugate coenzyme A, which is critical for hippuric acid formation, the effect of BDD treatment on hippuric acid concentrations was investigated. Rats given 1420 or 2272 micromol kg(-1) BDD had significantly elevated ratios of benzoic acid to hippuric acid in the urine after treatment compared with control urine. However, this effect was not observed in mice administered 1420 or 2840 micromol kg(-1) BDD. Collectively, the results demonstrate species differences in the urinary excretion of BDD and show that BDD administration in rats inhibits hippuric acid formation. The detection of 1-hydroxy-2-butanone and the carboxylic acids also provides insight regarding pathways of BDD metabolism in vivo.     

基于GC-TOF/MS技术分析肾性高血压大鼠血清代谢组学变化

目的基于气相色谱-质谱(GC-TOF/MS)技术分析两肾一夹(2K1C)肾性高血压大鼠血清代谢物的变化情况。方法 20只健康雄性SD大鼠按照随机数字表法分为假手术(Sham)组和2K1C组,每组10只。比较建模前及建模4周后2组大鼠体质量及尾动脉收缩压变化。采用GC-TOF/MS检测血清代谢产物的表达水平,正交偏最小二乘法-判别分析(OPLS-DA)筛选出差异表达代谢物,并对筛选出的差异表达代谢物进行聚类分析及KEGG通路分析。结果 4周后2K1C组共7只大鼠建模成功。与Sham组相比,2K1C组共筛选到14种差异表达代谢物,其中,花生四烯酸(arachidonic acid)、马尿酸(hippuric acid)、七烷酸(heptadecanoic acid)、吲哚乳酸酯(indolelactate)、木糖(xylose)、顺-巨头鲸鱼酸(cis-gondoic acid)、富马酸(fumaric acid)、胞苷-磷酸(cytidine-monophosphate)、乳酰胺(lactamide)、2-羟基-3-异丙基丁二酸(2-hydroxy-3-isopropylbutanedi...     

Dose-dependent pharmacokinetics of benzoic acid following oral administration of sodium benzoate to humans

Summary Plasma concentration-time data for benzoic and hippuric acids and urinary excretion-time data for hippuric acid were analyzed simultaneously after oral doses of 40, 80 or 160 mg/kg sodium benzoate administered at least one week apart to 6 healthy subjects.The mean AUCs of benzoic acid after the doses of 80 and 160 mg/kg of sodium benzoate were 3.7- and 12.0-times greater, respectively, than after 40 mg/kg. However, the mean AUC of hippuric acid was roughly proportional to the benzoate doses. The observed data were explained by a one-compartment model with first-order rate absorption and Michaelis-Menten elimination of benzoic acid, together with a one-compartment model with first-order elimination for hippuric acid.Although the maximum rate of biotransformation of benzoic acid to hippuric acid varied between 17.2 and 28.8 mg·kg^–1·h^–1 among the six individuals, the mean value (23.0 mg·kg^–1·h^–1) was fairly close to that provided by daily maximum dose (0.5 g·kg^–1·day^–1) recommended in the treatment of hyperammonaemia in patients with inborn errors of ureagenesis.The individual maximum rate of metabolism can be estimated from the urinary excretion rate of hippuric acid 1.5 to 3 h after the single oral dose of 80 to 160 mg·kg^–1 sodium benzoate. The justification of this concept requires further studies in patients with inborn errors of urea synthesis.This study was supported in part by a grant-in-aid from the Ministry of Human Health and Welfare, Tokyo, Japan     

Pharmacokinetic aspects of the mode of action of EGYT-475, a new antidepressant agent

EGYT-475 (N-benzyl-piperazine-picolinyl fumarate; Trelibet) metabolism was compared in rats, dogs and man. In the rat 7 urinary metabolites of EGYT-475 were found, and 4 were identified as: N-picolinyl-piperazine (EGYT-1354) (30%), picolinic acid (28.5%), hippuric acid (53%) and N-benzylpiperazine (EGYPT-2760) (3.7%). The results show that debenzylation is the main route of EGYT-475 metabolism in the rat. By this route EGYT-2760, the active EGYT-475 metabolite, is further metabolized. In the dog the main metabolic pathway is hydrolysis, and because of that the formed EGYT-2760 is not metabolized further and its urinary content exceeds 50%. In man, similarly as in the rat, debenzylation is the preferred route of EGYT-475 metabolism. These results explain much higher toxicity of EGYT-475 in dogs than in rats and man.     

Species differences in the metabolism of 3-phenoxybenzoic acid

The metabolism of 3-phenoxybenzoic acid (3PBA) (10 mg/kg, ip) has been studied in ten mammalian and one avian species in comparison with that of benzoic acid. 3PBA exhibits wide species diversity in its metabolism, unlike benzoic acid, of which benzoylglycine (hippuric acid) is the major urinary metabolite in all species studied. With 3PBA, glycine conjugation is the major route of metabolism in three species (sheep, cat, and gerbil), whereas in the mouse the taurine conjugate is the principal metabolite. The ferret eliminates similar amounts of each of these metabolites, whereas the glycylvaline dipeptide conjugate is the major metabolite isolated from the excreta of the mallard duck. Conversely, glucuronic acid conjugates of 3PBA and its 4'-hydroxy derivative (4'HO3PBA) are the major urinary metabolites in the marmoset, rabbit, guinea pig, and hamster; the rat appears unique in eliminating the O-sulfate conjugate of 4'HO3PBA as the principal urinary metabolite. In most cases, where amino acid conjugates are the major excretory products, the proportions of hydroxylated metabolites present are minimal. The pattern of metabolism of 3PBA does not significantly vary with dose (0.1-100 mg/kg) or route (ip and po) in the sheep, gerbil, or mouse. When administered 4'HO3PBA, the gerbil and mouse eliminate principally glucuronide and sulfate conjugates rather than amino acid conjugates, which are only minor components (less than 10% of the dose) in each case. This implies that hydroxylation is a primary metabolic event in determining the eventual fate of 3PBA in many species.     

Accumulation and turnover of metabolites of toluene and xylene in nasal mucosa and olfactory bulb in the mouse

Autoradiography of male mice following inhalation of the radioactively labelled solvents, toluene, xylene, and styrene, revealed an accumulation of non-volatile metabolites in the nasal mucosa and olfactory bulb of the brain. Since no accumulation occurred after benzene inhalation, it was assumed that the activity represented aromatic acids, which are known metabolites of these solvents. This was supported by the finding that also radioactive benzoic acid (main metabolite of toluene) and salicylic acid accumulated in the olfactory bulb. High-performance liquid chromatography revealed that after toluene inhalation (for 1 hr), nasal mucosa and olfactory bulb contained mainly benzoic acid, with a strong accumulation in relation to blood plasma, and considerably less of its glycine conjugate, hippuric acid. After xylene inhalation, on the other hand, methyl hippuric acid dominated over the non-conjugated metabolite, toluic acid. The results indicate a specific, possibly axonal flow-mediated transport of aromatic acids from the nasal mucosa to the olfactory lobe of the brain. The toxicological significance of these results remains to be studied.     

Some observations on the urinary excretion of glycine conjugates by laboratory animals

1. An isotope dilution assay has been developed for measuring the amount of conjugated glycine present in urine. The average daily excretion of conjugated glycine by male and female mouse, rat, guinea-pig and rabbit has been determined.

2. The glycine conjugates excreted by the four species have been identified. All species excreted only benzoylglycine and phenylacetylglycine.

3. The metabolism of [carboxy-¹⁴C]benzoic acid and [1-¹⁴C]phenylacetic acid has been investigated in the Sprague-Dawley rat. When administered over a range of concn. from 10 μg/kg to 1?g/kg, benzoic acid is converted to hippuric acid while phenylacetic acid is converted to phenylacetylglycine and phenylacetylglutamine.

4. Neither the rate of excretion nor the composition of the urinary metabolites arising from each acid is changed when low doses of one acid are co-administered with a high dose of the other.

5. The origin of the conjugated benzoic and phenylacetic acids is discussed.     

基于靶向代谢组学的腹膜透析患者血清氨基酸水平探究

目的:分析腹膜透析(PD)终末期肾病(ESKD)患者血清氨基酸谱,探究其氨基酸代谢失衡状况,为氨基酸腹膜透析液的科学合理调整提供参考。方法:回顾性分析2015年1月-2015年6月某院肾内科147例终末期肾病行腹膜透析管植入术患者,与147例同期进行体格检查且未见异常的健康志愿者的完整临床资料。运用超高效液相色谱-串联质谱(UHPLC-MS/MS)法定量检测血清样本中24种氨基酸含量,结合多变量统计学分析筛选腹膜透析患者与健康志愿者的差异代谢物(t检验、变量投影重要性准则)。运用R软件对差异代谢物和实验室检测指标进行相关性分析,探究差异代谢物的生物学功能,并实现PD患者血清氨基酸谱系统聚类。结果:腹膜透析患者与健康志愿者的实验室检测指标与血清氨基酸含量多数存在统计学差异。血清氨基酸定量检测结果的多变量统计分析显示:腹膜透析患者与健康志愿者的氨基酸谱得分图呈现良好的分离趋势。进一步差异代谢物筛选表明,丙氨酸(P<0.001)、马尿酸(P<0.001)等在腹膜透析患者血清中显著升高,而牛磺酸(P<0.001)显著降低,是主要的差异氨基酸。相关性研究表明,不同类型氨基酸(芳香族氨基酸、支链氨基酸等)与实验室检测指标的相关性不同,马尿酸与血肌酐呈现中等强度的相关性(ρ=0.58,P=0.03)。结论:运用UHPLC-MS/MS能够快速、有效识别腹膜透析患者血清氨基酸代谢状况。马尿酸、牛磺酸、丙氨酸3种氨基酸的变化可为氨基酸腹膜透析液的临床合理优化提供参考。     

Arachidonic acid metabolism in glucocorticoid-induced hypertension

1. Products of metabolism of arachidonic acid, such as 20-hydroxyeicosatetraenoic acid (20-HETE), thromboxane A(2) (TXA(2)) and prostaglandin I(2) (PGI(2)), regulate vascular tone. Among them, 20-HETE is a potent constrictor in small arteries that also has natriuretic properties. The present study investigated changes in urinary concentrations of 20-HETE and metabolites of TXA(2) and PGI(2) in glucocorticoid-hypertension in rats, a sodium-independent model. 2. Male Sprague-Dawley rats were treated with saline, adrenocorticotrophic hormone (ACTH; 0.2 mg/kg) or dexamethasone (20 microg/kg) by daily s.c. injection for 12 days. Systolic blood pressure (SBP) was measured using the tail-cuff method. Metabolic cages were used for 24 h urine collection. Thymus weight and urinary concentrations of 20-HETE, TXA(2) and PGI(2) were determined. 3. In the present study, SBP was increased by both ACTH (from 102 +/- 2 to 134 +/- 7 mmHg; n = 10; P < 0.01) and dexamethasone (from 106 +/- 5 to 122 +/- 4 mmHg; n = 10; P < 0.01). Thymus weight, a marker for glucocorticoid activity, was significantly decreased by both ACTH and dexamethasone (56 +/- 9 and 76 +/- 5 mg/100 g bodyweight, respectively; n = 10; P' < 0.01) compared with the saline control (151 +/- 5 mg/100 g bodyweight; n = 20). Urinary 20-HETE excretion was increased by ACTH (501 +/- 115 pmol/g creatinine; n = 10; P' < 0.05) but not by dexamethasone (126 +/- 13 pmol/g creatinine; n = 10) compared with the saline control (219 +/- 54 pmol/g creatinine; n = 20). Neither ACTH nor dexamethasone affected urinary excretion of TXB(2) or PGI(2) compared with the saline control. 4. In conclusion, ACTH but not dexamethasone increased urinary 20-HETE excretion in male Sprague-Dawley rats. Urinary concentrations of the metabolites TXB(2) and PGI(2) were unchanged in both models of glucocorticoid-hypertension. The vasoconstrictor 20-HETE may play a role in the genesis of ACTH-induced hypertension.     

The effect of prolonged sodium phenobarbitone treatment on hepatic xenobiotic metabolism and the urinary excretion of metabolites of the d-glucuronic acid pathway in the rat

Male Sprague-Dawley rats were fed a 0.1 % (w/w) sodium phenobarbitone (PB) diet for periods of 4, 12 and 24 weeks. Phenobarbitone treatment resulted in a marked increase in liver size which was accompanied by the induction of several parameters of hepatic xenobiotic metabolism including mixed function oxidase enzyme activities, UDP-glucuronyltransferase, cytochrome P450 and the microsomal protein content. In addition, treatment with the barbiturate led to the stimulation of the urinary excretion of d-glucaric acid, l-gulonic acid, free d-glucuronic acid, l-ascorbic acid and xylitol. The levels of stimulation of both the hepatic and urinary parameters remained relatively constant throughout the period of treatment. On cessation of PB treatment the parameters of hepatic xenobiotic metabolism reverted to control levels at a faster rate than the excretion of the d-glucuronic acid metabolites. The results demonstrate a correlation between the urinary excretion of metabolites of the d-glucuronic acid pathway and hepatic xenobiotic metabolizing enzyme activities during the prolonged administration of PB.     

牛蒡皮提取物对高脂血症大鼠血脂及抗氧化作用的研究

目的观察牛蒡皮提取物对实验性高脂血症大鼠血脂代谢的影响及抗氧化作用。方法喂饲法建立高脂血症大鼠模型,牛蒡皮提取物(以绿原酸计)60,120mg/(kg.bw),按牛蒡皮计为12,24g/(kg.bw)。给大鼠连续灌胃4周,测定血清甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-c)含量,血清超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性,红细胞丙二醛(MDA)含量。结果牛蒡皮提取物能明显降低TG、MDA,使动脉硬化指数(AI)降低,HDL-c升高,并能提高高脂血症大鼠血清SOD、GSH-Px活性。结论牛蒡皮提取物能调节高脂血症大鼠血脂代谢及纠正自由基代谢紊乱,发挥抗动脉硬化作用。     

vMAlteration of bile acid metabolism in pseudo germ-free rats

To characterize the impact of gut microbiota on host bile acid metabolism, we investigated the metabolic profiles of oxysterols and bile acids (BAs) in a conventional rat model (SD) (n=5) and its pseudo germ-free (GF) equivalent (n=5). GF rats were developed by the oral administration of bacitracin, neomycin and streptomycin (200 mg/kg, each) twice a day for 6 days. Urinary levels of oxysterols and bile acid metabolites were quantified using gas chromatography-mass spectrometry (GC-MS). The activity levels of enzymes involved in the bile acid metabolic pathway were determined through urinary concentration ratio between product to precursor. Cholic acid (CA) and ??-/??-muricholic acid (??-/??-MCA) were significantly elevated at pseudo germ-free condition. An increase of hydroxylase (cholesterol 7??-hydroxylase, oxysterol 7??-hydroxylase and cytochrome P450 scc) and a significant decrease of 7??-dehydroxylase were observed. The urinary concentration ratio of primary bile acids, a marker for hepatotoxicity, increased in pseudo germfree conditions. Therefore, it was found that gut microbiota could play a significant role in the bile acids homeostasis and metabolism.     

Comparative metabolism of bis(2-methoxyethyl)ether in isolated rat hepatocytes and in the intact rat: Effects of ethanol on in vitro metabolism

The metabolism of the reproductive and developmental toxicant bis(2-methoxyethyl)ether (diglyme) was studied in isolated rat hepatocytes and in the intact rat. Male Sprague-Dawley rats (190–220 g) were used in both studies. Hepatocytes, isolated by a two-step in situ collagenase perfusion of the liver, were cultured as monolayers and incubated with [¹⁴C]diglyme at 1, 10, 30, and 50 M for up to 48 h. For the in vivo study, rats were given single oral doses of [¹⁴C]diglyme at 5.1 mmol/kg body wt, and urine was collected for up to 96 h. Radioactive compounds in the culture medium or in the urine were separated by high performance liquid chromatography and quantified with an in-line radioactivity monitor. Metabolites were identified by comparison of their chromatographic retention times and their mass spectra with those of authentic compounds. The principal metabolite from hepatocytes and in the urine was (2-methoxyethoxy)acetic acid (MEAA). This metabolite accounted for approximately 36% of the radioactivity in the 48-h culture medium and about 67% of the administered dose in the 48-h urine. Other prominent metabolites common to both systems included 2-(2-methoxyethoxy)ethanol, methoxyacetic acid (MAA), 2-methoxyethanol, and diglycolic acid. The diglyme metabolite profiles from urine and from hepatocytes were qualitatively similar, demonstrating that, in the rat, hepatocytes serve as a good model system for predicting the urinary metabolites of diglyme. Moreover, MEAA was shown to be the metabolite best suited for use as a short-term biological marker of exposure to diglyme. Pretreatment of rats with ethanol resulted in a marked increase in the overall in vitro metabolism of diglyme. The major metabolic pathways for diglyme involve O-demethylation and cleavage of the central ether bond, and it is the latter pathway that leads to the formation of MAA, the metabolite associated with the reproductive and developmental toxicity of diglyme. The amounts of MAA formed in hepatocytes from ethanol-pretreated rats ranged from two to four times those formed in hepatocytes from untreated rats.Mention of Company or product names is not to be considered an endorsement by the National Institute for Occupational Safety and Health.     

Measuring inhalant abuse among homeless youth in southern Brazil

In order to measure urinary level of hippuric acid among Brazilian homeless youth suspected of having inhaled toluene in previous hours, urine samples were voluntarily collected from the youth around two hours after they arrived at an "open-school" in Porto Alegre, Brazil. Hippuric acid levels were determined by gas chromatography in urine specimens of 50 homeless children and adolescents. In 86% of the urine specimens, hippuric acid levels were within the range of 3.6 g/g creatinine and 46.1 g/g creatinine. Very high urinary hippuric acid level may indicate voluntary toluene abuse. A cut-off urinary hippuric acid level value needs to be established for toluene self-exposure diagnosis.     

Bioavailability, metabolism, and renal excretion of benzoic acid in the channel catfish (Ictalurus punctatus)

The pharmacokinetics and metabolism of the model compound benzoic acid were examined after intravascular (iv) and po administration at 10 mg/kg in the channel catfish (Ictalurus punctatus). A two-compartment pharmacokinetic model best described the plasma disposition of parent benzoic acid after iv dosing. The following pharmacokinetic values were estimated: elimination half-life, 5.9 hr; total body clearance, 61 ml/hr/kg; and volume of distribution (steady-state), 369 ml/kg. Plasma protein binding of [14C]benzoic acid was 18%. Benzoic acid was rapidly and extensively absorbed after po administration; absorption half-life was 0.8 hr and bioavailability was 95%. Renal excretion was the primary route of elimination of benzoic acid and metabolites. More than 80% of the iv-administered 14C was recovered in the urine in 24 hr. Unchanged benzoic acid comprised more than 90% of the urinary radiolabel. The major urinary metabolite was benzoyltaurine, which comprised 6-7% of the excreted 14C. Channel catfish were qualitatively similar to other teleost fishes in the formation of the taurine conjugate of benzoic acid. In contrast, the primary mammalian metabolite is the glycine conjugate, hippuric acid.     

The urotoxic effects of N,N'-dimethylaminopropionitrile. 2. In vivo and in vitro metabolism

The urotoxicity and metabolism of N,N'-dimethylaminopropionitrile (DMAPN) were investigated in male Sprague-Dawley rats. Animals treated with 525 mg DMAPN/kg or equimolar doses of commercially available potential DMAPN metabolites showed varying levels of urinary retention. About 44% of the administered dose of DMAPN was excreted unchanged in 5 days. beta-Aminopropionitrile and cyanoacetic acid were identified as urinary metabolites. The urinary excretion of cyanoacetic acid was nonlinearly proportional to the volume of urine retained in the bladders. In vitro, the metabolism of DMAPN to cyanide, formaldehyde, and cyanoacetic acid was localized mostly in the microsomal fraction of liver, kidney, and urinary bladders. This reaction required NADPH and oxygen for maximal activity. Metabolism of DMAPN was increased in hepatic microsomes obtained from phenobarbital-treated rats (220% of control) and decreased following CoCl1 treatments (73% of controls). Addition of SKF 525-A to the incubation mixtures inhibited the metabolism of DMAPN to formaldehyde (47-64% of controls). Addition of sulfhydryl compounds (glutathione and cysteine) to the incubation mixtures did not affect the rate of these reactions. These findings indicate that DMAPN is primarily metabolized via a cytochrome P450-dependent mixed-function oxidase system and that the urotoxic effects of DMAPN may be related to this metabolism.     

Effects of chlorogenic acid and its metabolites on the sleep-wakefulness cycle in rats

The effect of chlorogenic acid on the sleep-wakefulness cycle in rats was investigated in comparison with those of caffeic acid (the metabolite of chlorogenic acid) and dihydrocaffeic acid (the metabolite of caffeic acid). A significant prolongation of sleep latency was observed with chlorogenic acid and caffeic acid at a dose of 500 and 200 mg/kg, respectively. On the other hand, no remarkable effects were observed with dihydrocaffeic acid even at a dose of 500 mg/kg. Caffeine caused a significant increase in sleep latency and waking time and decrease in non-rapid eye movement sleep time at a dose of 10 mg/kg. In contrast, chlorogenic acid and its metabolites had no significant effects on each sleep state. From these results, it may be concluded that chrologenic acid caused a mild arousal effect compared with that of caffeine, and the effect of chlorogenic acid may have occurred through its metabolite caffeic acid.