Alterations of gut microbiota and cytokines in elevated serum diamine oxidase disorder

The present study aimed to explore gut microbiota alterations and host cytokine responses in a population with elevated serum diamine oxidase (DAO) disorder. A total of 53 study participants were included in this study, segregated into 2 groups: subjects with high-level DAO (DAO-H, n = 22) subjects with normal DAO level (DAO-N, n = 31). We investigated the clinical and demographic parameters of study participants. The fecal bacterial communities and serum cytokines in 2 groups were assessed by 16S ribosomal RNA gene sequencing and immunoassay. High-pressure liquid chromatography was used to determine hemoglobin Alc. Flow cytometry was used to find the cytokine level in the blood serum. There is no difference in age, total cholesterol (TCHO), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), hemoglobin Alc, fasting plasma glucose (FPG) and homocysteine between the 2 groups. No significant difference were found in α-diversity between the 2 groups, however, the gut microbiota of subjects in DAO-H were characterized by marked interindividual differences, decreased abundance of Phocaeicola, Lachnospira, Bacteroides, Alistipes, Agathobacter, Lachnospira and Bactetoides and increased abundances of Mediterraneibacter, Blautia, Faecallibacterium, Agathobacter, and Parasutterella. Furthermore, the cytokines were no related to the DAO level in both groups and exhibited no significant differences between DAO-H and DAO-N. This study adds a new dimension to our understanding of the DAO and gut microbiota, and revealed that an increase in the DAO level in the intestinal mucosa could alter the gut microbiota composition, which can cause gut-related complications. Research is needed to extensively evaluate downstream pathways and provide possible protective or treatment measures pertaining to relevant disorders.     

Decreased plasma postheparin diamine oxidase levels in celiac disease

The highest diamine oxidase activity is contained in small-bowel mucosa and, after heparin administration, the enzyme is released by the intestine into the plasma. Previous experimental studies showed that measurement of plasma postheparin diamine oxidase activity is a sensitive test for quantitating the length and severity of small-bowel mucosal injury. On this basis, we measured plasma diamine oxidase activity in celiac disease, a condition characterized by a loss of mature enterocyte mass. Twenty-five untreated celiac patients, 21 celiac patients on a gluten-free diet, 16 patients with small-bowel diseases other than celiac disease (abnormal controls), and 18 healthy controls were studied. Diamine oxidase activity was measured using [¹⁴ C]putrescine as substrate and expressed as units per milliliter of plasma. Basal diamine oxidase levels in controls and patients were too low for significant differences between the groups to be detected. After preliminary experiments in which, on separate occasions, heparin was intravenously administered at doses of 75 and 150 units/kg and in which the second blood sample was taken 10 and 30 min after heparin injection, it was decided to use the 150 unit/kg dose and to measure plasma diamine oxidase activity in the blood sample taken 10 min after heparin stimulation in all the remaining subjects taking part in the study. Postheparin diamine oxidase levels were significantly lower in untreated celiac patients (mean 1.53 units/ml) than in healthy controls (mean 5.85), treated celiac patients (mean 4.82), and abnormal controls (mean 2.62). Except in three patients, no overlap between healthy controls and untreated celiac patients was observed. No significant difference was detected between healthy controls and treated celiac patients. Our results show that plasma postheparin diamine oxidase activity mirrors not only mucosal damage but also mucosal recovery of the small bowel, thus providing a new circulating marker for the status of the human intestinal mucosa. Measurement of plasma diamine oxidase may represent a useful test to screen patients for intestinal biopsy and to follow up the response to gluten-free diet.Part of this work was presented at the meeting of the American Gastroenterological Association in San Francisco, California, on May 19, 1986, and was published in abstract form (Gastroenterology 90:1381, 1986).This work was partly supported by the CNR grants 84.02351.56/115.06909 and 840729.     

Evaluation of diamine oxidase activity (DAO) in the rat intestinal mucosa by measuring expired14CO2 after oral administration of14C-putrescine

This study was performed to investigate whether mucosal diamine oxidase activity could be assessed by measuring expired¹⁴CO₂ after oral administration of¹⁴C-putrescine. Immediately after giving 5 μCi of¹⁴C-putrescine, the¹⁴CO₂ was collected at 1-h intervals for 8 h into a vial containing 1 ml of 10 mM hyamine hydroxide, 2 ml of ethanol, and an appropriate amount of phenolphthalein dye. The expired¹⁴CO₂ caused the color to disappear. The amount of¹⁴CO₂, determined by scintillation counting, reached a maximum 1 h after¹⁴C-putrescine administration, and gradually decreased thereafter. A positive correlation between the mucosal diamine oxidase activity and the maximal expired¹⁴CO₂ value was obtained. There is no doubt that this test can be used to easily detect mucosal DAO activity and avoid the necessity of mucosal biopsy.     

Analysis of a non-synonymous single nucleotide polymorphism of the human diamine oxidase gene (ref. SNP ID: rs1049793) in patients with Crohn's disease

Objective. To analyse the possible influence of a non-synonymous single nucleotide polymorphism (SNP) of the histamine-degrading enzyme diamine oxidase (DAO) on genetic susceptibility to Crohn's disease (CD). Material and methods. In this prospective, case-control study, 210 unrelated Caucasian consecutive CD patients were recruited at the Inflammatory Bowel Disease Unit of a single tertiary centre (Hospital Clínico San Carlos) in Madrid, Spain. A total of 261 healthy volunteers from the same geographic area were also recruited and matched with patients. Both cases and controls were analysed for the presence of a non-synonymous SNP (rs1049793) of DAO using amplification-restriction procedures of the genotype obtained in a blood sample. Results. No significant differences were found in the distribution of carriers of the non-synonymous SNP of DAO between CD patients and controls (OR 1.2 (95% CI 0.9–1.6; p=0.3)). Nor were any differences found between carriers and non-carriers of the non-synonymous SNP in demographic characteristics, phenotypes, complications or treatment of CD. Conclusions. The study of a non-synonymous SNP (rs1049793) of DAO does not seem to be of use in assessing susceptibility to CD, either as a marker of disease activity or as a marker of clinical behaviour in patients with the disease.     

急性肝衰竭大鼠血浆D-乳酸、二胺氧化酶和内毒素的变化及其意义

目的探讨血浆D-乳酸、二胺氧化酶（DAO）和内毒素水平在急性肝衰竭大鼠的变化及其意义。方法选取健康雌性SD大鼠40只,随机分为对照组（15只）和实验组（25只）,采用分光光度法检测血浆中D-乳酸、DAO和内毒素水平,分别用HE染色及电镜观察大鼠回肠的组织形态和超微结构。结果 实验组大鼠血浆D-乳酸、DAO和内毒素水平均明显高于对照组（P〈0.05）;实验组大鼠回肠黏膜明显萎缩,部分绒毛断裂、脱落。结论急性肝衰竭大鼠肠黏膜屏障出现明显障碍。     

延迟快速复苏烧伤犬血浆二胺氧化酶的变化

目的探讨烧伤后延迟复苏情况下,快速补液对伤后早期肠粘膜损伤的影响.方法利用犬40%TBSAⅢ度烫伤模型,32只犬被随机分为烧伤对照组、血定安延迟均匀补液组、血定安延迟快速补液组和血浆延迟快速补液组进行补液.观察伤前、伤后2,6,8,12,24,36和48 h血浆二胺氧化酶、乳酸的变化及小肠病理检查.结果烧伤后各组血浆二胺氧化酶、乳酸逐渐升高,至伤后2 h～6h即显著高于伤前并继续升高,但补液三组上升幅度明显低于烧伤对照组,其中快速补液组烧伤8 h后(快速补液后)均显著低于均匀补液组.血浆快速组与血定安快速组相比二胺氧化酶、乳酸几乎无明显差别.结论在烧伤后延迟复苏情况下,快速补液可以减轻肠粘膜的损伤.     

Evaluation of intestinal mucosal function by measuring expired (14)CO(2) after oral administration of (14)C-putrescine

BACKGROUND: Diamine oxidase (DAO) is the enzyme that degrades putrescine, the key main product of polyamine metabolism, and reflects enterocytic maturity of absorption because diamine oxidase activity is highest in the small intestine. We have already shown that expired (14)CO(2) after oral administration of (14)C-putrescine correlated with intestinal DAO activity. However, the influence of food composition and the mucosal adaptation after intestinal resection have not been elucidated. METHODS: Male Wistar rats were fed normal chow or an elemental diet (ED) for 2 weeks. Resected rats underwent 50% jejunectomy or 50% ilectomy. Expired (14)CO(2) levels, following oral administration of (14)C-putrescine were measured in all rats, and compared with the intestinal DAO activity and other mucosal parameters. RESULTS: In the ED group, the (14)CO(2) levels reached a peak earlier, and values were 2.9-fold higher than in the group fed with normal chow. Mucosal alkaline phosphatase (ALP) and DAO activity in the ED group were also higher than in the group fed normal chow, although the mucosal wet weight was significantly lower in the ED group. In the resection groups, all expired (14)CO(2) values increased during measurement. The peak (14)CO(2) values in the jejunectomy group shifted earlier in the postoperative period. The mucosal DAO activity in both the resection groups was higher than it was in the control group at the fifth and 10th postoperative day. However, there were no differences among the three groups at the 15th postoperative day. CONCLUSIONS: Our studies suggested that expired (14)CO(2) after oral administration of (14)C-putrescine correlates with mucosal DAO activity, and that it also reflects intestinal function.     

Effect of intestinal ischemia on diamine oxidase activity in rat intestinal tissue and blood

This study examines the effect of increasing duration of intestinal ischemia on the mucosal integrity and the release of the enzyme diamine oxidase from the small intestine. Acute ischemia was produced by the occlusion of the superior mesenteric artery, and the subsequent changes in DNA and 125I-albumin content in the lumen were taken as indices of intestinal lesions. Diamine oxidase activity was measured in the intestinal lumen, mucosa, lymph, and serum. Occlusions of the superior mesenteric artery for periods of more than 60 min resulted in significant leakage of 125I-albumin (i.v.) into the lumen. In contrast, luminal DNA content rose significantly after 15 min of ischemia and continued to increase proportionally with the increased duration of the occlusion up to 120 min. Similarly, diamine oxidase activity was augmented in the lumen after 15 min of occlusion and rose sharply as the ischemic period was lengthened up to 60 min, leveling off thereafter. Increases in the diamine oxidase activity were also observed in the intestinal lymph and serum, reaching levels that were 2.6 and 3.6 times that of the control respectively after 60 min of ischemia. These findings suggest that intestinal ischemia reduces the diamine oxidase content in the intestinal mucosa by desquamation of the surface epithelial cells and by releasing the enzyme into the intestinal interstitial fluid, from which at least a portion is transported to the blood via the lymphatics. The early release of diamine oxidase seems to occur before the mucosal barrier is broken.     

肝炎肝硬化患者血浆D-乳酸、二胺氧化酶和内毒素的检测及其临床意义

目的　探讨血D 乳酸、二胺氧化酶 (DAO)和内毒素水平在肝炎肝硬化患者中的变化及其临床意义。方法　将 5 0例肝炎肝硬化患者和 3 0例健康体检者分为试验组和对照组 ,采用分光光度法检测外周血中D 乳酸、DAO和内毒素的活性。结果　肝炎肝硬化患者试验组D 乳酸活性明显高于对照组 (P <0 .0 1) ,试验组 3组间比较差异有显著性 (P <0 .0 1) ,治疗后显著低于治疗前 (P <0 .0 1) ;试验组DAO活性明显高于对照组 ,组间比较Child PughC级组活性明显低于Child PughB级组 (P <0 .0 1) ,治疗后A级组及B级组水平显著低于治疗前 (P <0 .0 5 ) ,C级组水平显著高于治疗前 (P <0 .0 5 ) ;Child PughA级组内毒素活性与对照组比较差异显著性 (P >0 .0 5 ) ,Child PughB、C级组明显高于对照组 (P <0 .0 1) ,治疗后A、B级组水平与治疗前比较差异无显著性 (P >0 .0 5 ) ,C级组水平显著降低 (P <0 .0 1)。相关分析显示 3者水平均相关。结论　血浆D 乳酸、DAO水平是肠粘膜损伤早期诊断的敏感指标 ,内毒素血症是肝硬化患者病情加重的重要因素     

Stimulation of clonal tumor cell growth in vitro by inhibiting the serum polyamine oxidase activity

Summary Several tumors are characterized by elevated levels of polyamines involved in vital cell proliferation processes. Polyamine oxidases (PAO), present in ruminant and particularily in fetal calf serum (FCS), degrade polyamines to polyaminoaldehydes and other products that inhibit cell proliferation. Since most in vitro assays for cloning tumor stem cells use FCS as an essential supplement of the nutrient media, we examined the effects of specifically inhibiting the PAO activity on the clonal growth of leukemic cells and the following normal lymphocytes: the W 25 rat chloroleukemia, the M1 mouse myeloblastic and the L 1210 rat lymphoblastic leukemia, a primary human acute myeloblastic leukemia (AML) and acute lymphocytic leukemia (ALL) as well as normal human PHA-stimulated lymphocytes. In the presence od horse serum, nontoxic doses of the PAO inhibitor 1-hydroxybenzyloxyamine did not affect colony growth of either cell type. However, in the presence of FCS, clonal growth of W 25, ALL, AML, and PHA lymphocytes was significantly stimulated by the enzyme inhibitor. Our data suggest (a) that poor cell proliferation of several tumors in vitro may result from the reaction of polyamines (from cells) and PAO (from serum), (b) that this can be easily tested by means of a specific PAO inhibitor, and (c) that the growth conditions can be optimized by adding nontoxic doses of the enzyme inhibitor or by exchanging FCS for another serum.     

Monoamine oxidase B inhibition reduces gastric mucosal blood flow,basal acid secretion,and cold water restraint-induced gastric mucosal injury in rats

Inhibition of monoamine oxidase B (MAO B) by selective inhibitors pargyline and l-deprenyl increases dopamine (DA) and norepinephrine (NE) concentrations in nucleus accumbens (NACB) and is associated with reduction in cold water restraint-induced gastric mucosal injury, inhibition of basal gastric acid output, and regional gastric mucosal blood flow. Similar effects were not observed with administration of MAO A inhibitors. These observations suggest that activation of central dopamine and norepinephrine receptors, particularly in NACB, are involved in the control of gastric mucosal function.Supported by PHS grant DK 38198 (G.K.).     

Decreased serum paraoxonase-1 activity during intestinal nematode (Nippostrongylus brasiliensis) infection in rats

Reduced paraoxonase-1 (PON1) activity has been observed in a number of pathological conditions; however, little is known about the effects of intestinal nematode infections, such as Nippostrongylus brasiliensis, on paraoxonase activity. We observed a significant reduction in serum paraoxonase and arylesterase activity after N. brasiliensis infection in Wistar rats from Day 6 until Day 12 post-infection (p.i.) for serum paraoxonase and from Day 3 until Day 24 p.i. for arylesterase. In addition, N. brasiliensis infection increased serum concentrations of pro-inflammatory cytokines (interleukin-1, interleukin-6, and tumor necrosis factor-alpha), with maximum concentrations observed on Day 9 p.i. These cytokines are known to inhibit the synthesis of hepatic PON1 mRNA. Thus, the observed reduction in PON1 activity during N. brasiliensis infection is likely associated with inflammatory reactions mounted against the parasites.     

脑外伤大鼠肠运动功能障碍及与血清Ghrelin的关系

目的探讨创伤性脑损伤（TBI）后大鼠肠运动功能异常及其与血清Ghrelin改变的关系。方法雄性Wistar大鼠64只,随机分为两组。TBI组采用改良Feeney自由落体撞击伤法建立TBI模型;对照组只开骨窗,不行落体致伤。两组大鼠分别在致伤后3、6、12、24 h观察血清Ghrelin水平、小肠传输系数及肠黏膜病理改变。结果 TBI后各组大鼠光镜下肠黏膜上皮细胞明显受损,电镜下可见细胞连接较对照组增宽;各时间点的肠道传输系数及血清Ghrelin值均低于对照组,二者呈正相关。结论大鼠在TBI后早期即可出现小肠黏膜损伤和小肠运动功能减低,在此期间血清Ghrelin的变化可能起一定作用。     

慢性间歇低氧大鼠的血压变化及其颈动脉体中还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶的表达

目的 探讨慢性间歇低氧(chronic intermittent hypoxia,CIH)大鼠的血压变化及其颈动脉体中还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶的表达情况,以明确CIH致血压升高的可能机制.方法 清洁级雄性SD大鼠30只按随机数字表法分为CIH组、慢性持续缺氧组及对照组.尾袖法测量大鼠尾动脉收缩压,采用RT-PCR法检测大鼠颈动脉体中NADPH氧化酶各亚基gp91 phox、p22phox及p47phox mRNA的表达,对颈动脉体行免疫组织化学染色,并对p22phox的表达情况行半定量分析.结果 CIH组大鼠的尾动脉收缩压为[(145±11)mm Hg,1 mm Hg=0.133kPa],较CIH组(129±9)mm Hg及对照组(124±7)mm Hg显著升高(F值为19.895,P＜0.01),CIH组gp91phox、p22phox及p47phox mRNA的表达(分别为2.82±0.51、2.74±0.45和2.88±0.47)较慢性持续缺氧组(分别为2.35±0.42、2.25±0.38和2.41±0.43)及对照组(分别为2.23±0.35、2.16±0.30和2.30±0.36)显著升高(F值分别为5.794、6.854和7.163,P＜0.01)免疫组织化学检查结果显示CIH组p22phox蛋白相对表达量(99±12)较其他两组(分别为38±7和34±8)增多.结论 CIH可刺激大鼠颈动脉体中NADPH氧化酶的表达上调并使血压升高,NADPH氧化酶在颈动脉体中的过度表达可能与OSAHS患者发生高血压病存在相关联系.

Abstract：

Objective Chronic intermittent hypoxia (CIH) occurs in patients with obstructive sleep apnea-hyponea syndrome (OSAHS) and has adverse effects on multiple physiological functions. Previous studies have shown that reflexes arising from carotid bodies mediate CIH evoked circulation-respiratory responses, and reactive oxygen species (ROS) play important roles in eliciting systemic responses to CIH.But very little is known about the molecular mechanisms underlying CIH. NADPH oxidase is the most important sources of ROS. In the present study we examined changes of blood pressure and expression of NADPH oxidase in carotid body in rats exposed to intermittent hypoxia. Methods Thirty healthy male SD rats were randomly divided into 3 groups, a CIH group, a chronic continuous hypoxia group and a control group. The systolic blood pressure (SBP) was measured with tail-cuff method. RT-PCR was used to examine mRNA expressions of NADPH oxidase subunit gp91phox, p22phox, p47phox. Immunohistochemistry and semiquantitative analysis of NADPH oxidase subunits p22phox were done in the carotid body sections of all rats. Results Compared with normal group [ ( 124 ± 7 ) mm Hg, 1 mm Hg = 0. 133 kPa ] and chronic continuous hvpoxia group[ (129 ± 9) mm Hg], the SBP in CIH group [( 145 ± 11 ) mm Hg] was significantly higher( F = 19. 895, P ＜0. 01 ＝, and the expression of NADPH oxidase subunits gp91phox,p22phox,p47phox mRNA in CIH group ( 2. 82 ± 0. 51, 2. 74 ± 0. 45, 2. 88 ± 0. 47, respectively ) were significantly higher than those in chronic continuous hypoxia group ( 2. 35 ± 0. 42, 2. 25 ± 0. 38, 2. 41 ±0. 43, respectively)and normal group(2. 23 ±0. 35, 2. 16 ±0. 30, 2. 30 ±0. 36, respectively) ( F =5.794,6. 854, 7. 163, respectively, P ＜ 0. 01 ). The Immunohistochemistry and semiquantitative analysis showed that the expression of NADPH oxidase subunit p22phox in the carotid body in CIH group ( 99 ± 12 ) were more than those in chronic continuous hypoxia and control groups ( 38 ± 7 and 34 ± 8, P ＜ 0. 05 ).Conclusion CIH upregulates the expression of NADPH oxidase in rat carotid body and elevates the rat SBP. These results indicate that NADPH oxidase up-expression is closely associated with OSAHS patients with hypertension.     

Role of food in appearance of growth-stimulating activity in rat proximal intestine after small-bowel resection

The oral intake of food is important for the observed compensatory hyperplasia in the remnant small intestine after resection but the molecular events governing this response are not known. Peptides, of molecular weight 4500 and 1000 daltons, present in the proximal intestine for 96 hr after resection and mitogenic for the intestine have been implicated in the adaptive hyperplasia. In this study, the role of food in the appearance of these peptides was assessed. The results show that after resection, rats nourished intravenously demonstrated neither a significant adaptation nor any of the detectable mucosal mitogens, whereas the rats nourished intragastrically demonstrated both hyperplasia and the mitogenic peptide(s). The association of the hyperplasia with the appearance of the mitogenic peptides in the small intestine suggests that they are important in the mechanism by which food promotes the adaptive hyperplasia.This work was supported by a grant from the Medical Research Council of Canada (MA-3320), and from the Justine Lacoste-Beaubien Foundation. This paper was presented in part at the Annual Meeting of the Canadian Society for Clinical Investigation (1986).     

Histaminase (diamine oxidase) activity in human tumors: an expression of a mature genome.

High histaminase [amine:oxygen oxidoreductase (deaminating) (pyridoxal-containing), EC 1.4.3.6] activity is found in certain human tumors and in the placenta of most mammals. The present study explores the relationship of tumor histaminase to histaminases found in placenta and other human, pig, and rat tissues. The electrophoretic mobility and Michaelis constants for the deamination of histimine and putrescine were identical for histaminases from human placenta and from medullary thyroid carcinoma. An antibody was raised in rabbits against human placental histaminase that was highly purified by a new affinity procedure. In separate studies, using inhibitory concentrations of antibody and a second antibody precipitation technique, identical patterns of immunoreactivity were found for histaminases from human placenta, kidney, medullary thyroid carcinoma, and small cell lung carcinoma; human intestinal histaminase crossreacted well but less strongly than did enzymes from these other tissues. Histaminases from pig kidney, pig intestine, and rat intestine showed no crossreaction; histaminases from rat thymus and adrenal gland showed minimal crossreactivity. The findings suggest that placental histaminase activity is not a unique product of a fetal or trophoblastic genome. The presence of histaminase in malignancies does not appear to be an example of ectopic tumor production of a placental trophoblastic protein.