A Screen for Spore Wall Permeability Mutants Identifies a Secreted Protease Required for Proper Spore Wall Assembly

The ascospores of Saccharomyces cerevisiae are surrounded by a complex wall that protects the spores from environmental stresses. The outermost layer of the spore wall is composed of a polymer that contains the cross-linked amino acid dityrosine. This dityrosine layer is important for stress resistance of the spore. This work reports that the dityrosine layer acts as a barrier blocking the diffusion of soluble proteins out of the spore wall into the cytoplasm of the ascus. Diffusion of a fluorescent protein out of the spore wall was used as an assay to screen for mutants affecting spore wall permeability. One of the genes identified in this screen, OSW3 (RRT12/YCR045c), encodes a subtilisin-family protease localized to the spore wall. Mutation of the active site serine of Osw3 results in spores with permeable walls, indicating that the catalytic activity of Osw3 is necessary for proper construction of the dityrosine layer. These results indicate that dityrosine promotes stress resistance by acting as a protective shell around the spore. OSW3 and other OSW genes identified in this screen are strong candidates to encode enzymes involved in assembly of this protective dityrosine coat.     

添加谷氨酰胺的肠内营养对急性重症胰腺炎大鼠肠上皮细胞间紧密结合蛋白的影响

目的探讨早期肠内营养中应用谷氨酰胺(glutine,Gln)对急性重症胰腺炎(severe acute pancreatitis,SAP)大鼠肠上皮细胞间紧密结合蛋白的作用。方法:雄性SD大鼠89只,随机分成对照组,SAP+PN(肠外营养)组,SAP+EN(肠内营养)组,SAP+EN+Gln,各组又分为4 d喂养组和7 d喂养组。分别在第4 d、第7 d剖杀大鼠检测各组各项指标。免疫组化法检测小肠组织中occludin蛋白的表达。结果:(1)空肠黏膜蛋白质含量测定:各EN组粘膜蛋白质含量显著高于PN组(p<0.01),EN+Gln组的4 d、7 d分别高于EN组的4 d、7 d(p<0.01)。(2)小肠粘膜occludin蛋白表达及含量测定:PN7 d、4 d组的平均灰度值均显著高于EN、EN+Gln组(p<0.01),EN4 d、7 d组平均灰度均显著高于EN+Gln组(p<0.05)。结论:肠内营养中应用谷氨酰胺能更有效增加occludin蛋白表达,改善肠上皮紧密连接,维护肠上皮屏障完整性。     

AICAR Attenuates Organ Injury and Inflammatory Response after Intestinal Ischemia and Reperfusion

Intestinal ischemia and reperfusion (I/R) is encountered in various clinical conditions and contributes to multiorgan failure and mortality as high as 60% to 80%. Intestinal I/R not only injures the intestine, but affects remote organs such as the lung leading to acute lung injury. The development of novel and effective therapies for intestinal I/R are critical for the improvement of patient outcome. AICAR (5-aminoimidazole-4-carboxyamide ribonucleoside) is a cell-permeable compound that has been shown to possess antiinflammatory effects. The objective is to determine that treatment with AICAR attenuates intestinal I/R injury and subsequent acute lung injury (ALI). Male Sprague Dawley rats (275 to 325 g) underwent intestinal I/R injury with blockage of the superior mesenteric artery for 90 min and subsequent reperfusion. At the initiation of reperfusion, vehicle or AICAR (30 mg/kg BW) was given intravenously (IV) for 30 min. At 4 h after reperfusion, blood and tissues were collected for further analyses. Treatment with AICAR significantly decreased the gut damage score and the water content, indicating improvement in histological integrity. The treatment also attenuated tissue injury and proinflammatory cytokines, and reduced bacterial translocation to the gut. AICAR administration after intestinal I/R maintained lung integrity, attenuated neutrophil chemotaxis and infiltration to the lungs and decreased lung levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6. Inflammatory mediators, lung-inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins, were decreased in the lungs and lung apoptosis was significantly reduced after AICAR treatment. These data indicate that AICAR could be developed as an effective and novel therapeutic for intestinal I/R and subsequent ALI.     

早期肠内营养在重症急性胰腺炎中的应用

目的:探讨早期肠内营养在重症急性胰腺炎治疗中的应用价值。方法:将重症急性胰腺炎患者30例分为实验组和对照组,对照组予以常规治疗,实验组在常规治疗的基础上通过鼻空肠管予以早期肠内营养,记录患者WBC、CRP、血淀粉酶、尿淀粉酶恢复时间、白蛋白变化情况、感染率、病死率、并发症发生率、住院时间、住院费用等。结果:实验组WBC、CRP、血淀粉酶、尿淀粉酶恢复时间较对照组明显低,有显著性差异(P<0.05);实验组血清白蛋白升高较对照组明显高,有显著性差异(P<0.05);实验组感染率、病死率、并发症发生率较对照组明显低,但无显著差别(P>0.05);实验组住院时间、住院费用较对照组低,有显著性差异(P<0.05)。结论:早期肠内营养可以改善急性重症胰腺炎营养状况,缩短病程、减低感染率、病死率、并发症发生率、住院时间及住院费用。     

肠内营养治疗对重症脑卒中患者炎症应激程度的影响及肠道菌群失调的影响因素

摘要 目的：分析肠内营养治疗对重症脑卒中患者炎症应激程度的影响及肠道菌群失调的影响因素。方法：选择自2022年1月至2023年5月收治的82例重症脑卒中患者,分为对照组和观察组,各41例。对照组予以常规治疗,观察组加用肠内营养治疗。比较两组治疗前后的炎症反应指标、应激反应指标、APACHEⅡ评分、GCS评分,使用因素分析发生肠道菌群失调的影响因素。结果：治疗后,与对照组比较：观察组C-反应蛋白（hs-CRP）、白介素-2（IL-2）、IL-6、肿瘤坏死因子-α（TNF-α）、去甲肾上腺素（NE）、皮质醇（COR）及APACHEⅡ评分表达水平低,对氧磷酶-1（PON-1）、谷胱甘肽过氧化（GSH-Px）、GCS评分高（P＜0.05）;经因素分析,内毒素、高血压和糖尿病均是重症脑卒中患者发生肠道菌群失调的独立危险因素（P＜0.05）。结论：肠内营养治疗能有效降低重症脑卒中患者的炎症应激程度,进而增加患者的临床获益,而患者发生肠道菌群失调与高血压、糖尿病和高水平内毒素有关。     

益生菌联合膳食纤维的肠内营养对重型颅脑损伤患者术后营养状况、免疫功能和肠黏膜屏障功能的影响

摘要 目的：观察重型颅脑损伤患者术后经膳食纤维的肠内营养联合益生菌干预后,患者免疫功能、肠黏膜屏障功能以及营养状况的变化。方法：选取2016年6月~2020年5月期间我院收治的重型颅脑损伤患者136例。根据入院顺序奇偶法分为对照组68例和研究组68例,对照组给予膳食纤维的肠内营养干预,研究组在对照组的基础上联合益生菌干预,对比两组格拉斯哥昏迷量表（GCS）评分、营养状况、免疫功能、肠黏膜屏障功能及并发症发生情况。结果：两组干预14 d后白蛋白（ALB）、血红蛋白（Hb）、转铁蛋白（TR）均较干预前升高,且研究组较对照组高（P<0.05）。两组干预14 d后免疫球蛋白G（IgG） 、免疫球蛋白A（IgA）、免疫球蛋白M（IgM）均较干预前升高,且研究组较对照组高（P<0.05）。两组干预14 d后总超氧化物歧化酶（T-SOD）、谷胱甘肽过氧化物酶（GSH-PX）均较干预前升高,且研究组较对照组高（P<0.05）,丙二醛（MDA）较干预前降低,且研究组较对照组低（P<0.05）。两组干预14 d后GCS评分升高,且研究组较对照组高（P<0.05）。研究组的并发症发生率低于对照组（P<0.05）。结论：益生菌联合膳食纤维的肠内营养干预可有效改善重型颅脑损伤术后患者营养状况、免疫功能和肠黏膜屏障功能,同时还可减少并发症发生率,改善患者预后。     

燕麦米汤对严重烧伤患者休克期肠道复苏的影响

目的：研究严重烧伤患者休克期经肠道给予燕麦米汤对肠道复苏的影响。方法：选取烧伤面积≥30%TBSA的患者42例,并随机分为：燕麦米汤组,伤后24h内开始经鼻肠管给予燕麦米汤;对照组,伤后24h内开始经鼻肠管给予50g/L葡萄糖盐水,连续4d,每组21例。在伤后1、2、3、4 d分别检测其血清二氨氧化酶（DAO）值及动脉血乳酸（LAC）含量,观察两组患者腹胀缓解时间、肠鸣音恢复时间等临床指标。结果：两组患者伤后血清二氨氧化酶及动脉血乳酸含量均呈下降趋势,燕麦米汤组血清二氨氧化酶在伤后2、3、4d显著低于对照组（P〈0.01）,而脉血乳酸含量在伤后2、3d显著低于对照组（P〈0.05或0.01）,治疗过程中燕麦米汤组其腹胀缓解时间、肠鸣音恢复时间、排气时间、开始完全肠内营养时间、继发感染例数均优于对照组（P〈0.01）。结论：在严重烧伤休克期尽早鼻饲燕麦米汤能较好地促进肠道复苏。     

燕麦米汤对严重烧伤患者休克期肠道复苏的影响

目的:研究严重烧伤患者休克期经肠道给予燕麦米汤对肠道复苏的影响。方法:选取烧伤面积≥30%TBSA的患者42例,并随机分为:燕麦米汤组,伤后24h内开始经鼻肠管给予燕麦米汤;对照组,伤后24h内开始经鼻肠管给予50g/L葡萄糖盐水,连续4d,每组21例。在伤后1、2、3、4 d分别检测其血清二氨氧化酶(DAO)值及动脉血乳酸(LAC)含量,观察两组患者腹胀缓解时间、肠鸣音恢复时间等临床指标。结果:两组患者伤后血清二氨氧化酶及动脉血乳酸含量均呈下降趋势,燕麦米汤组血清二氨氧化酶在伤后2、3、4d显著低于对照组(P<0.01),而脉血乳酸含量在伤后2、3d显著低于对照组(P<0.05或0.01),治疗过程中燕麦米汤组其腹胀缓解时间、肠鸣音恢复时间、排气时间、开始完全肠内营养时间、继发感染例数均优于对照组(P<0.01)。结论:在严重烧伤休克期尽早鼻饲燕麦米汤能较好地促进肠道复苏。     

Preconditioning Prevents the Inhibition of Na+,K+-ATPase Activity after Brain Ischemia

Application of single transient forebrain ischemia (ISC) in adult Wistar rats, lasting 2 or 10 min, caused inhibition of Na⁺,K⁺-ATPase activity in cytoplasmic membrane fractions of hippocampus and cerebral cortex immediately after the event. In the 2-min ISC group followed by 60 min of reperfusion, the enzyme inhibition was maintained in the cortex, while there was an increase in hippocampal enzyme activity; both effects were over 1 day after the event. However, in the 10-min ISC group enzyme inhibition had been maintained for 7 days in both cerebral structures. Interestingly, ischemic preconditioning (2-min plus 10-min ISC, with a 24-hour interval in between) prevented the inhibitory effect of ischemia/reperfusion on Na⁺,K⁺-ATPase activity observed either after a single insult of 2 min or 10 min ischemia. We suggest that the maintenance of Na⁺,K⁺-ATPase activity afforded by preconditioning be related to cellular neuroprotection.     

Fecal Protease Activity Is Associated with Compositional Alterations in the Intestinal Microbiota

Objective

Intestinal proteases carry out a variety of functions in the gastrointestinal (GI) tract. Studies have reported that elevated enteric proteases in patients with GI disease can alter intestinal physiology, however the origin (human vs. microbial) of elevated proteases in patients with GI disease is unclear.

Aim

The aim of this study was to investigate the association between protease activity and the microbiota in human fecal samples.

Design

In order to capture a wide range of fecal protease (FP) activity stool samples were collected from 30 IBS patients and 24 healthy controls. The intestinal microbiota was characterized using 454 high throughput pyro-sequencing of the 16S rRNA gene. The composition and diversity of microbial communities were determined and compared using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline. FP activity levels were determined using an ELISA-based method. FP activity was ranked and top and bottom quartiles (n=13 per quartile) were identified as having high and low FP activity, respectively.

Results

The overall diversity of the intestinal microbiota displayed significant clustering separation (p = 0.001) between samples with high vs. low FP activity. The Lactobacillales, Lachnospiraceae, and Streptococcaceae groups were positively associated with FP activity across the entire study population, whilst the Ruminococcaceae family and an unclassified Coriobacteriales family were negatively associated with FP activity.

Conclusions

These data demonstrate significant associations between specific intestinal bacterial groups and fecal protease activity and provide a basis for further causative studies investigating the role of enteric microbes and GI diseases.     

A Protease that Participates in Yeast Cell Wall Lysis during Zymolyase Digestion

Zymolyase B decreased the turbidity of a yeast cell wall suspension by about 50% and caused release of peptide-mannan from the cell walls. However cell walls treated with the enzyme still maintained the cell shape. The effect of the enzyme on the cell walls was inhibited by yeast mannan and completely counteracted by treatment of the enzyme with DFP. The activity was not affected by pH, but was considerably reduced by incubation of the enzyme at 55°C for 15 min, a treatment that did not affect the proteolytic activity. Heat-treatment decreased the molecular weight of the enzyme from 29,000 to 22,500 and its sensitivity to yeast mannan. Yeast mannan caused noncompetitive inhibition of the proteolytic activity of the native enzyme and competitive inhibition of that of the heat-treated enzyme. Modification of tryptophan residues of Zymolyase B resulted in decreased sensitivity to yeast mannan and a decrease in the activity of the enzyme on yeast cell walls as well as heat-treatment. On the basis of these results, it is hypothesized that Zymolyase B binds to the cell wall mannans and changes their conformation, making the attached proteins susceptible to proteolysis, and then releases peptide-mannan from the cell walls.     

免疫增强型与普通肠内营养制剂对老年重症肺炎营养状态、肠黏膜屏障功能及T细胞亚群的影响

摘要 目的：观察免疫增强型与普通肠内营养制剂对老年重症肺炎营养状态、肠黏膜屏障功能及T细胞亚群的影响。方法：选择2017年7月~2020年3月期间我院收治的136例老年重症肺炎患者,根据随机数字表法分为研究组（n=68）、对照组（n=68）,对照组患者给予普通肠内营养制剂进行干预,研究组给予免疫增强型肠内营养制剂进行干预,对比两组肠道菌群失调发生率、营养状态、肠黏膜屏障功能、T细胞亚群及并发症发生率。结果：研究组的肠道菌群失调发生率低于对照组（P<0.05）。并发症发生率两组对比无差异（P>0.05）。两组干预10 d后各项营养状态指标：白蛋白（ALB）、前白蛋白（PAB）、血红蛋白（HGB）均较干预前升高,且研究组高于对照组（P<0.05）。干预10 d后两组各项肠黏膜屏障功能指标：内毒素（ET）、二胺氧化酶（DAO）均较干预前降低,且研究组低于对照组（P<0.05）。干预10 d后两组CD3⁺、CD4⁺、CD4⁺/CD8⁺均较干预前升高,且研究组高于对照组（P<0.05）,CD8⁺较干预前降低,且研究组低于对照组（P<0.05）。结论：免疫增强型肠内营养制剂与普通肠内营养制剂相比,安全性相当,但前者对老年重症肺炎患者的营养状态、肠黏膜屏障功能、免疫功能改善效果均更好,且肠道菌群失调发生率更低。     

不同肠内营养液对老年重度AECOPD机械通气患者肠道屏障功能、免疫功能和炎症因子的影响

摘要 目的：探讨不同肠内营养液对老年重度慢性阻塞性肺疾病急性加重期（AECOPD）机械通气患者营养状况、肠道屏障功能、免疫功能和炎症因子的影响。方法：纳入2019年10月-2021年10月在我院住院治疗的老年重度AECOPD患者90例作为研究对象,采用密封信封法随机分为含膳食纤维肠内营养组（FEN组）及不含膳食纤维肠内营养组（NFEN组）各45例。两组患者均接受重度AECOPD规范治疗,并接受机械通气治疗。FEN组给予能全力进行营养支持治疗,NFEN组给予能全素进行营养支持治疗,两组患者均接受持续30 d的肠内营养。比较两组患者治疗前和治疗30 d后营养状况指标血清白蛋白（ALB）、前白蛋白（PA）、血红蛋白（Hb）,肠道屏障功能指标D-乳酸（DLA）、细菌内毒素（BE）、二胺氧化酶（DAO）,T淋巴细胞亚群（CD4⁺、CD8⁺、CD4⁺/CD8⁺）、体液免疫功能指标免疫球蛋白（Ig）G、IgM、IgA,炎症因子降钙素原（PCT）和白介素-6（IL-6）水平的变化,以及两组患者ICU住院天数、机械通气时间和并发症的差异。结果：治疗30 d后,FEN组ALB、PA和Hb水平均显著高于NFEN组（P<0.05）;FEN组DLA、BE和DAO水平均显著低于NFEN组（P<0.05）;FEN组CD4⁺、CD4⁺/CD8⁺以及IgG、IgA水平均显著高于NFEN组（P<0.05）;FEN组PCT、IL-6水平均显著低于NFEN组（P<0.05）。FEN组患者ICU住院天数、机械通气时间及并发症均显著少于NFEN组,差异有统计学意义（P<0.05）。结论：含膳食纤维肠内营养有利于改善老年重度AECOPD机械通气患者营养状况,修复肠道黏膜屏障功能,提高免疫功能,减轻炎症反应,从而改善预后,含膳食纤维的肠内营养支持治疗效果更佳。     

Sex Differences in Ischemia/Reperfusion Injury: The Role of Mitochondrial Permeability Transition

Neurochemical Research - Brain and heart ischemia are among the leading causes of death and disability in both men and women, but there are significant sex differences in the incidence and severity...     

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using "intestinal sacs" are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (P_app)of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.     

Probiotic Bacteria Regulate Intestinal Epithelial Permeability in Experimental Ileitis by a TNF-Dependent Mechanism

Background

We previously showed that the probiotic mixture, VSL#3, prevents the onset of ileitis in SAMP/YitFc (SAMP) mice, and this effect was associated with stimulation of epithelial-derived TNF. The aim of this study was to determine the mechanism(s) of VSL#3-mediated protection on epithelial barrier function and to further investigate the “paradoxical” effects of TNF in preventing SAMP ileitis.

Methods

Permeability was evaluated in SAMP mice prior to the onset of inflammation and during established disease by measuring transepithelial electrical resistance (TEER) on ex vivo-cultured ilea following exposure to VSL#3 conditioned media (CM), TNF or VSL#3-CM + anti-TNF. Tight junction (TJ) proteins were assessed by qRT-PCR, Western blot, and confocal microscopy, and TNFRI/TNFRII expression measured in freshly isolated intestinal epithelial cells (IEC) from SAMP and control AKR mice.

Results

Culture with either VSL#3-CM or TNF resulted in decreased ileal paracellular permeability in pre-inflamed SAMP, but not SAMP with established disease, while addition of anti-TNF abrogated these effects. Modulation of the TJ proteins, claudin-2 and occludin, occurred with a significant decrease in claudin-2 and increase in occludin following stimulation with VSL#3-CM or TNF. TNF protein levels increased in supernatants of SAMP ilea incubated with VSL#3-CM compared to vehicle, while IEC-derived TNFR mRNA expression decreased in young, and was elevated in inflamed, SAMP versus AKR mice.

Conclusions

Our data demonstrate that the previously established efficacy of VSL#3 in preventing SAMP ileitis is due to direct innate and homeostatic effects of TNF on the gut epithelium, modulation of the TJ proteins, claudin-2 and occludin, and overall improvement of intestinal permeability.     

Inhibition of JNK Aggravates the Recovery of Rat Hearts after Global Ischemia: The Role of Mitochondrial JNK

c-Jun N-terminal kinase (JNK), a stress-activated MAPK, is activated during cardiac ischemia-reperfusion (IR). The role of JNK inhibitors in cardioprotection against IR still remains controversial, in part, due to spill-over effects of non-specific inhibitors. In the present study, we sought to examine whether inhibition of JNK by SU3327, a specific JNK inhibitor that inhibits upstream JNK signaling rather than the kinase activity of JNK, improves cardiac function and reduces heart damage during IR. Hearts of male Sprague-Dawley rats perfused by Langendorff were subjected to 25 min of global ischemia followed by 30 min reperfusion in the presence or absence of SU3327. Cardiac function was monitored throughout the perfusion period. Myocardial damage was extrapolated from LDH activity in the coronary effluent. At the end of reperfusion, mitochondria were isolated and used to measure respiration rates and mitochondrial permeability transition pore opening. Protein analysis of mitochondria predictably revealed that SU3327 inhibited JNK phosphorylation. Although SU3327 significantly reduced cell damage during the first minutes of reperfusion, it did not improve cardiac function and, furthermore, reduced the mitochondrial respiratory control index. Interestingly, SU3327 activated the other stress-related MAPK, p38, and greatly increased its translocation to mitochondria. Mitochondrial P-JNK and P-p38 were co-immunoprecipitated with complex III of the electron transfer chain. Thus, JNK plays an essential role in cardiac signaling under both physiological and pathological conditions. Its inhibition by SU3327 during IR aggravates cardiac function. The detrimental effects of JNK inhibition are associated with reciprocal p38 activation and mitochondrial dysfunction.     

肠内营养联合益生菌对直肠恶性肿瘤术后大鼠肠粘膜屏障及血清炎症因子的影响

摘要 目的：研究肠内营养（enteral nutrition, EN）联合益生菌对直肠恶性肿瘤术后大鼠肠粘膜屏障及血清炎症因子的影响。方法：选取32只SPF级雄性SD大鼠,随机分对照组、模型组、EN组和EC组,每组8只。模型组、EN组和EC组建立直肠恶性肿瘤术后模型,EN组灌胃给予肠内营养混悬液,EC组灌胃肠内营养混悬液加益生菌颗粒,对照组和模型组灌胃同体积的生理盐水,四组大鼠均连续灌胃7 d。观察各组大鼠小肠超微结构;检测occludin蛋白、D-乳糖及炎症因子的表达水平。结果：模型组大鼠小肠上皮细胞紧密连接和绒毛结构遭破坏,EN组和EC组与模型组相比有明显改善。与对照组相比,模型组大鼠occludin蛋白和抑炎因子IL-10的表达水平均较低,D-乳糖、血清白介素1β（interleukin-1β, IL-1β）和肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）的表达水平较高（P＜0.05）;与模型组相比,EN组和EC组的occludin蛋白和IL-10的表达水平较高,D-乳糖、IL-1β和TNF-α的表达水平较低（P＜0.05）;与EN组相比,EC组occludin蛋白和IL-10的表达水平较高,D-乳糖、IL-1β和TNF-α的表达水平较低（P＜0.05）。结论：肠内营养联合益生菌可有效改善直肠恶性肿瘤术后大鼠的肠道屏障功能,提高occludin蛋白的表达,降低D-乳糖的表达,降低促炎因子IL-1β和TNF-α的表达同时升高抑炎因子IL-10的表达。     

Liquid Chromatography-Tandem Mass Spectrometry for Analysis of Intestinal Permeability of Loperamide in Physiological Buffer

Analysis of in vitro samples with high salt concentrations represents a major challenge for fast and specific quantification with liquid chromatography-tandem mass spectrometry (LC-MS/MS). To investigate the intestinal permeability of opioids in vitro employing the Ussing chamber technique, we developed and validated a fast, sensitive and selective method based on LC–MS/MS for the determination of loperamide in HEPES-buffered Ringer''s solution. Chromatographic separation was achieved with an Atlantis dC18 column, 2.1 mm×20 mm, 3 µm particle size and a gradient consisting of methanol/0.1% formic acid and ammonium acetate. The flow rate was 0.7 ml/min, and the total run time was 3 min. For quantification, two mass transitions for loperamide and a deuterated internal standard (methadone-d₃) were used. The lower limit of loperamide quantification was 0.2 ng/ml. This new LC-MS/MS method can be used for the detection of loperamide in any experimental setup using HEPES-buffered Ringer''s solution as a matrix compound.