Spectrophotometric assay for serum platelet-activating factor acetylhydrolase activity

We developed a spectrophotometric assay for serum platelet-activating factor acetylhydrolase (PAF-AH, EC 3.1.1.47.) activity using a platelet-activating factor (PAF) analogue with a 4-nitrophenyl group as substrate. PAF-AH hydrolyzes the sn-2 position of the substrate ?1-myristoyl-2-(p-nitrophenylsuccinyl)phosphatidylcholine, producing p-nitrophenyl succinate. This liberation was spectrophotometrically monitored and the activity determined from the change in absorption. The assay does not require radioisotopes and is applicable to an automatic analyzer. Utilizing this assay with an automatic analyzer, it is possible to measure the activities of thousands of samples in a few hours with excellent precision (CV 0.5%, n=30) and high correlation (r=0.979, n=100) with the results of a conventional radioisotopic assay. The assay should be particularly useful for clinical diagnostics.     

血清学抗体检测对炎症性肠病的诊断价值

目的 测定抗酿酒酵母菌抗体（ASCA）、抗胰腺腺泡抗体（PAB）、抗小肠杯状细胞抗体（GAB）、抗中性粒细胞胞浆抗体（pANCA）在炎症性肠病（IBD）中的表达,探讨其对溃疡性结肠炎（UC）和克罗恩病（CD）的诊断价值.方法 2006年1月至2011年7月收集天津市人民医院住院患者510例,分成UC组、CD组及对照组,UC组358例,男192例,女166例,平均年龄（45.7±15.8）岁,CD组31例,男20例,女11例,平均年龄（42.8±19.2）岁,对照组121例,男69例,女52例,平均年龄（56.8±13.2）岁.空腹采集静脉血2ml,采用间接免疫荧光法检测血清中IgA型和IgG型ASCA、PAB、GAB和pANCA 4种抗体的表达.结果 ASCA-IgA和IgG 1种以上亚型阳性在CD组、UC组和对照组阳性率分别为12.9％、3.6％、1.7％,CD组与后两组比较差异有统计学意义.PAB各组无一例阳性表达.GAB在UC组和CD组比较差异无统计学意义.pANCA在UC组中阳性率为54.7％,显著高于CD组（3.2％）和对照组（2.5％）,UC组和后两组比较差异有统计学意义.ASCA（＋）诊断CD的敏感性、特异性、阳性和阴性预测值分别为12.9％、96.4％、23.5％、92.7％,pANCA（＋）诊断UC的敏感性、特异性、阳性和阴性预测值分别为54.7％、96.8％、99.5％、15.6％.结论 IBD相关抗体检测的特异性较高,但敏感性低.pANCA对于诊断UC较有价值,同时检测IgA型和IgG型ASCA可提高CD诊断的敏感性,PAB对于诊断IBD意义不大.     

Plasma platelet-activating factor acetylhydrolase activity in critically ill patients

OBJECTIVE: Platelet-activating factor (PAF) is a potent proinflammatory mediator in systemic inflammation and sepsis and is inactivated by the enzyme PAF-acetylhydrolase (PAF-AH). Recently, a large phase III clinical trial using recombinant PAF-AH to treat patients with severe sepsis was performed but failed to reduce 28-day mortality rate. To get more information on the activity of PAF-AH in sepsis, we repeatedly measured its activity in plasma in critically ill patients compared with healthy controls. DESIGN: Retrospective cohort study. SETTING: Intensive care unit. PATIENTS: Two hundred thirty-one patients who were admitted to an operative intensive care unit within 1 yr were enrolled and evaluated daily for American College of Chest Physicians/Society of Critical Care Medicine criteria. PAF-AH activity was measured as the release of [H]-acetate from [H]-acetyl-PAF. INTERVENTIONS: Analysis of plasma samples. MEASUREMENTS AND MAIN RESULTS: At the day of admission, PAF-AH activity of patients was below controls but markedly increased over time. Higher activities were seen in patients with severe sepsis or septic shock compared with those without organ failure. With respect to the clinical outcome, lower values were found in nonsurvivors only as long as they had not developed organ failure. In severe sepsis/septic shock, values of nonsurvivors exceeded those of survivors. PAF-AH activity was positively correlated with plasma levels of inflammatory mediators such as neopterine and tumor necrosis factor-alpha but not with acute phase reactants such as C-reactive protein, interleukin-6, or PCT. In addition, parenteral nutrition with lipid emulsions was seemingly associated with low PAF-AH activity compared with enteral nutrition. CONCLUSION: The data indicate severity- and time-dependent changes in PAF-AH activity and may help to explain the failure of recombinant PAF-AH treatment strategies that were not based on activity measurements.     

CT小肠造影在炎症性肠病中的诊断价值

目的 探讨结肠镜及CT小肠造影(CTE)在炎症性肠病(IBD)诊断中的价值.方法 选取昆明医科大学第一附属医院消化内科2017年9月至2019年9月经内镜及病理检查确诊的IBD患者为研究对象,其中溃疡性结肠炎(UC)患者68例,克罗恩病(CD)患者37例,回顾性分析所有患者的结肠镜及CTE表现.结果 105例患者均完成...     

Diagnostic biomarkers in inflammatory bowel disease

Inflammatory bowel diseases (IBD), which includes ulcerative colitis and Crohn's disease, are chronic intestinal disorders of unknown etiology. The diagnosis of IBD is based upon clinical history, endoscopic, and histological findings; however, accurate diagnosis can still be difficult with these current methodologies. Recent advances in molecular techniques in proteomics and genetic analysis have driven the discovery of novel IBD biomarkers and genetic susceptibility factors that may facilitate the diagnosis of IBD. In the future, biomarkers will play a key role in the diagnosis of IBD and the development of individual courses of treatment.     

Exogenous platelet-activating factor acetylhydrolase reduces mortality in mice with systemic inflammatory response syndrome and sepsis

Current evidence indicates that dysregulation of the host inflammatory response to infectious agents is central to the mortality of patients with sepsis and in those with systemic inflammatory response syndrome. Strategies to block inflammatory mediators, often with complicated outcomes, are currently being investigated as new adjuvant therapies for sepsis. Here, we determined if administration of recombinant platelet-activating factor (rPAF)-acetylhydrolase (rPAF-AH), an enzyme that inactivates PAF and PAF-like lipids, protects mice from inflammatory injury and death after administration of lipopolysaccharide (LPS) or cecal ligation and puncture (CLP). Administration of rPAF-AH increased plasma PAF-AH activity and reduced mortality in both models. Treatment with rPAF-AH increased peritoneal fluid levels of monocyte chemoattractant protein 1/CCL-2 and decreased interleukin 6 and migration inhibitory factor levels after LPS administration or CLP. Administration of a broad-spectrum antibiotic together with rPAF-AH was more protective than single treatment with either of these agents. The combined treatment was associated with reduced interleukin 6 levels in mice subjected to CLP. We observed acute decreases in plasma PAF-AH activity in mice subjected to CLP or challenged with LPS and in human patients with sepsis. We conclude that alterations in the endogenous PAF-AH contribute to the pathophysiology of sepsis and that administration of exogenous rPAF-AH reduces inflammatory injury and mortality in models relevant to the clinical syndrome. Variations in endogenous PAF-AH activity may potentially account for variable responses to exogenous rPAF-AH in previous clinical trials. Serial measurements of plasma PAF-AH activity in murine models demonstrate dynamic regulation of the endogenous enzyme, potentially explaining the variations in human subjects.     

Characterization of serum platelet-activating factor (PAF) acetylhydrolase. Correlation between deficiency of serum PAF acetylhydrolase and respiratory symptoms in asthmatic children

Platelet-activating factor (PAF) acetylhydrolase has been recognized as an enzyme that inactivates PAF. We developed a convenient and reproducible method for determining human serum PAF acetylhydrolase activity. The assay was based on measurement of [14C]acetate produced from 1-O-alkyl-2-[14C]-acetyl-sn-glycero-3-phosphocholine upon precipitation of the complex of radioactive substrate and albumin with TCA. The apparent Km value of PAF acetylhydrolase (near the physiological concentration of serum protein) was 1.5 X 10(-4) M PAF. 32 subjects with serum PAF acetylhydrolase deficiency were found among 816 healthy Japanese adults. The low PAF acetylhydrolase activity in the deficient serum might not be due to the presence of enzyme inhibitor. Both the sensitivity to PAF and the metabolism of PAF in platelets from PAF acetylhydrolase-deficient subjects were almost the same as those of normal subjects. Deficiency in serum PAF acetylhydrolase appeared to be transmitted by autosomal recessive heredity among five Japanese families. Among healthy adults, healthy children, and asthmatic children, who were grouped into five classes on the basis of respiratory symptoms (remission, wheezy, mild, moderate, and severe groups), the probability of PAF acetylhydrolase deficiency was significantly higher in groups with severe symptoms (moderate and severe) (P less than 0.01). These results suggest that deficiency of serum PAF acetylhydrolase might be one of the factors leading to severe respiratory symptoms in asthmatic children.     

粪便钙卫蛋白及血清自身抗体联合检测对炎症性肠病的诊断价值

目的探讨联合检测血清抗胰腺外分泌腺抗体(PAB)、抗酿酒酵母抗体(ASCA)、抗杯状细胞抗体(GAB)、抗中性粒细胞胞浆抗体(PANCA)及粪便钙卫蛋白(FC)在炎症性肠病(IBD)诊断和鉴别诊断中的价值。方法收集确诊的克罗恩病(CD)患者107例及溃疡性结肠炎(UC)患者98例作为IBD组,非IBD肠病疾病患者79例作为疾病对照组,用间接免疫荧光法检测血清PANCA、ASCA、GAB和PAB,用双抗体夹心ELISA法检测FC浓度。对3组血清PANCA、GAB、PAB、ASCA及FC检测的数据进行对比分析。结果 205例IBD患者血清PANCA、GAB、PAB、ASCA的阳性率分别为36.1%、29.8%、38.0%、4.9%;IBD组、CD组与UC组FC浓度均高于疾病对照组,差异有统计学意义(P0.01),CD组与UC组间比较无统计学意义(P0.05)。PANCA在CD和UC组的阳性率分别为8.4%、66.3%;PAB在CD和UC组的阳性率分别为65.4%、8.2%;PAB、PANCA、GAB、ASCA、FC及5项联合在鉴别诊断IBD与非IBD肠道疾病中的敏感性和特异性分别为38.0%、36.1%、29.8%、4.9%、54.1%、63.4%和98.7%、96.2%、94.9%、100%、68.4%、93.7%。5项联合鉴别诊断IBD与非IBD肠道疾病的ROC曲线下面积为0.819;PANCA用于UC鉴别诊断的ROC曲线下面积为0.816;PAB用于CD鉴别诊断的ROC曲线下面积为0.823。结论 GAB为一种IBD相关自身抗体,可用于IBD的辅助诊断;PAB和PANCA分别为CD和UC重要的血清学标记物,对该类疾病诊断具有高度敏感性和特异性;将FC与自身抗体联合对于IBD与非IBD胃肠道疾病的鉴别具有重要意义,但对于区分CD或UC价值不大。     

Serum platelet-activating factor acetylhydrolase (PAF-AH) activity in more than 3000 healthy Japanese

BACKGROUND: A spectrophotometric assay for platelet-activating factor acetylhydrolase (PAF-AH) activity differs from the radioisotopic assay in its value because of a difference in substrate specificity. The spectrophotometric assay is more precise than the radioisotopic assay, providing information that is not clear with the radioisotopic assay. METHODS: We measured the serum PAF-AH activity in 3106 healthy Japanese, utilizing the spectrophotometric assay with an Hitachi 7170 automatic analyzer. We also measured the serum PAF-AH activity in 18 healthy volunteers to investigate the effect of diet and the change in activity in a day and over 6 weeks. Changes were examined at 0 (day 1), 1, 2, 4 and 6 weeks. RESULTS: The mean value for females was significantly lower than that of males at the 5% level and both male and female activity had a tendency to increase with advancing age. It is known that the PAF-AH is primarily associated with LDL in blood and the PAF-AH activity correlated with the total cholesterol (r=0.52, n=126) and the LDL cholesterol (r=0.60, n=126) concentrations. In the diet study, there was no observable effect on activity. No difference in PAF-AH activity was observed between serum and plasma sample types. The serum PAF-AH activity was stable at 7 degrees C for at least 7 days and at -20 degrees C for at least 2 months. CONCLUSIONS: The serum PAF-AH activity in women was lower than in men until the menopausal age was reached. We could use not only fresh fasting serum, but also plasma sample, non-fasting sample and stored sample to estimate the PAF-AH activity.     

炎症性肠病病情活动监测指标的临床价值

目的探讨髓过氧化物酶(MPO)和超氧化物歧化酶(SOD)作为炎症性肠病(IBD)病情活动临床监测指标的价值。方法分别观察了IBD活动组患者15例[其中活动期溃疡性结肠炎(UC)10例,活动期克罗恩病(CD)5例],IBD非活动组患者15例(其中缓解期UC10例,缓解期CD5例)和12例对照组患者的结肠黏膜病理变化,按Oshitani评分标准和d'Haens评分标准进行UC和CD组织学评分,测定结肠黏膜MPO和SOD活性。结果IBD活动组、IBD非活动组病理组织评分均比对照组高(8±5 vs 3±1)、(5±2 vs 3±1)(P<0.01),IBD活动组病理组织评分亦较IBD非活动组高(8±5 vs 5±2)(P<0.01);IBD活动组、IBD非活动组肠黏膜MPO活性均较对照组高[(3.55±0.38)U/g vs(1.52±0.24)U/g、(2.28±0.30)U/g vs(1.52±0.24)U/g](P<0.01),IBD活动组MPO活性较IBD非活动组高[(3.55±0.38)U/g vs(2.28±0.30)U/g](P<0.01);IBD活动组、IBD非活动组肠黏膜SOD活性均较对照组低[(104.58±18.91)U/mg vs(212.44±14.22)U/mg、(170.91±13.57)U/mg vs(212.44±14.22)U/mg](P<0.01),IBD活动组SOD活性较IBD非活动组低[(104.58±18.91)U/mg vs(170.91±13.57)U/mg](P<0.01)。结论MPO活性与IBD病情活动程度呈正相关,SOD活性与IBD病情活动程度呈负相关,两者可作为IBD病情活动的临床监测指标。     

Deficiency of platelet-activating factor acetylhydrolase is a severity factor for asthma

Asthma, a family of airway disorders characterized by airway inflammation, has an increasing incidence worldwide. Platelet-activating factor (PAF) may play a role in the pathophysiology of asthma. Its proinflammatory actions are antagonized by PAF acetylhydrolase. A missense mutation (V279F) in the PAF acetylhydrolase gene results in the complete loss of activity, which occurs in 4% of the Japanese population. We asked if PAF acetylhydrolase deficiency correlates with the incidence and severity of asthma in Japan. We found that the prevalence of PAF acetylhydrolase deficiency is higher in Japanese asthmatics than healthy subjects and that the severity of this syndrome is highest in homozygous-deficient subjects. We conclude that the PAF acetylhydrolase gene is a modulating locus for the severity of asthma.     

钙卫蛋白及抗中性粒细胞胞浆抗体联合检测对炎症性肠病的诊断价值研究

目的 探讨钙卫蛋白与抗中性粒细胞胞浆抗体(ANCA)联合检测对炎症性肠病(IBD)的诊断价值.方法 收集确诊为IBD的患者79例作为IBD组,腹痛、腹泻等排除IBD的患者42例作为疾病对照组,健康体检者34例作为健康对照组.分别检测血液样本中ANCA和粪便样本中钙卫蛋白的水平.结果 79例IBD患者粪便钙卫蛋白浓度为(493.86±204.18)μg/g高于疾病对照组[(71.46±60.51)μg/g]和健康对照组[(36.19±13.46)μg/g].钙卫蛋白在IBD组、疾病对照组和健康对照组中的阳性率分别为57.0%、19.0%、0.0%;ANCA在三组中的阳性率分别为63.3%、4.8%、0.0%.IBD组的钙卫蛋白、ANCA阳性率显著高于其他两组(P＜0.05).钙卫蛋白与ANCA联合检测在IBD组、疾病对照组和健康对照组中的阳性率分别为78.5%、23.8%和0.0%.结论 钙卫蛋白和ANCA联合检测可显著提高IBD的诊断率,为临床IBD的早期诊断和治疗提供可靠依据.     

Advances in the assessment of disease activity in inflammatory bowel disease

Knowledge of the severity and extent of the inflammation in inflammatory bowel diseases provides a means of determining rational therapeutic strategies in affected patients. During the past 3 decades, several clinical, laboratory, and combined indices have been proposed for the assessment of inflammatory bowel disease; refinements in radiologic methods and the availability of endoscopy and biopsy have facilitated the accurate assessment of the extent and severity of the disease. In relapsing conditions such as inflammatory bowel disease, however, the use of such procedures is limited by the radiation exposure or the relatively invasive nature of the technique. In this article, we review the proposed methods and recent advances in assessment of patients with inflammatory bowel disease; we also discuss possible strategies at the time of diagnosis, during recurrence, and in evaluation of the efficacy of drug or dietic therapy.     

Platelet activating factor acetylhydrolase activity in the urine of patients with renal disease

Platelet activating factor has been demonstrated in blood and urine and has a broad range of effects on kidney function. The kidney possesses the enzymes responsible for PAF synthesis as well as the specific acetylhydrolase which deactivates PAF. We used a radioactive assay to measure PAF-acetylhydrolase activity in urine samples obtained from normal individuals and patients with various nephropathies. Activity was detected in the majority of normal urines with a mean + 2 SD = 0.70 nmol/30 min/ml. Activity exceeding this value was measured in the urines of 11 of 14 patients with diabetes mellitus, 14 of 22 with glomerulonephritis, and 5 of 16 with hypertensive renal disease. Further study is necessary to relate urine hydrolase activity to specific functional and structural abnormalities of the kidney.     

血小板活化因子乙酰水解酶基因Val379Ala位点多态性与脓毒症易感性相关性研究

目的 探讨血小板活化因子乙酰水解酶(PAF-AH)基因Val379Ala位点多态性与脓毒症易感性及预后的关系.方法 采用PCR-RFLP技术,分析82例不同程度脓毒症患者及90名健康对照者PAF-AH基因Val379Ala位点的多态性,对含有等位基因多态性的片段进行DNA测序分析.结果 全部样本经PCR-RFLP分析后,脓毒症组82例中,仅1例纯合子Val/Val型,24例杂合子Val/Ala型,57例纯合子Ala/Ala型;对照组90例中,2例Val/Val型,29例Val/Ala型,59例Ala/Ala型.Val379Ala基因型和等位基因频率:脓毒症组与对照组比较差异无统计学意义(P>0.05),存活组、死亡组与对照组比较差异无统计学意义(P>0.05),轻型脓毒症组、严重脓毒症组与对照组比较差异无统计学意义(P>0.05).结论 PAF-AH基因Val379Ala位点多态性与脓毒症易感性、预后及病情严重程度可能不相关.     

Chemiluminescence in inflammatory bowel disease patients: a parameter of inflammatory activity

Background: Reactive oxygen species (ROS) are produced in excess in the inflamed mucosa and peripheral blood of patients with inflammatory bowel disease. These species have emerged as a common pathway of tissue injury in a wide variety of inflammatory and other disease processes. The present study was conducted to assess ROS production and to correlate this with parameters of inflammatory activity. Methods: In 25 patients with Crohn's disease (CD), 20 patients with ulcerative colitis (UC) and 65 age- and sex-matched healthy volunteers ROS production was measured using the whole blood luminol enhanced chemiluminescence assay (LECA). Disease activity was assessed using the Crohn's disease activity index and the Ulcerative Colitis Symptoms Score (UCSS) for CD and UC, respectively. Furthermore, the effect of various scavengers, enzymes and enzyme inhibitors on LECA was studied to assess the contribution of different ROS. Results: LECA was significantly higher in CD and UC patients compared with healthy controls (7.1±4.7 and 9.8±6 vs. 5.2±2.8×10³ counts per minute (cpm), p<0.05 and <0.001). In CD, relative LECA (patient/control) was correlated with the Crohn's disease activity index and C-reactive protein (CRP) (r=0.54, p=0.001 and r=0.51, p=0.01). In UC, CRP but not LECA was correlated with the Ulcerative Colitis Symptoms Score (C-reactive protein: r=0.42, p=0.01). Addition of azide, superoxide dismutase, deferoxamine and dimethylthiourea resulted in a decrease of LECA values. Conclusion: Whole blood LECA is increased in patients with CD and UC. This parameter is correlated with disease activity in CD. The observed chemiluminescence is probably due to generation of superoxide and the hydroxyl radical.     

Neutrophil adherence to bladder microvascular endothelial cells following platelet-activating factor acetylhydrolase inhibition

Interstitial cystitis (IC) is an inflammatory bladder condition of unknown etiology. Tryptase released from elevated numbers of activated mast cells is a proposed mediator of the inflammatory process in IC. We have previously shown that tryptase increases human bladder microvascular endothelial cell (HBMEC) calcium-independent phospholipase A(2) (iPLA(2)) activity, resulting in the production of multiple biologically active phospholipid metabolites, including platelet-activating factor (PAF), that can mediate inflammation. Because the design of selective PLA(2) inhibitors may provide a useful therapeutic strategy to reduce the inflammatory process in IC, we tested several frequently used PLA(2) inhibitors on PAF production in tryptase-stimulated HBMEC. Among the inhibitors tested, methyl arachidonyl fluorophosphonate (MAFP) was found to be a potent inhibitor of PAF-acetylhydrolase activity. Pretreatment of HBMEC with MAFP significantly increased PAF production in both unstimulated and tryptase-stimulated cells. In addition, MAFP pretreatment of tryptase-stimulated HBMEC increased both surface expression of P-selectin and polymorphonuclear leukocyte adherence to the HBMEC monolayer. These effects suggest that MAFP has a proinflammatory effect, irrespective of its ability to inhibit PLA(2).     

Collagenase activity in colonic mucosa during inflammatory bowel disease

A simple test for collagenase activity was performed on colonic mucosa specimens of 35 patients with inflammatory bowel disease, 7 patients with carcinoma of the colon, 3 with benign polyps, and 34 normal subjects. Increased collagenase activity was present in 94.2% of the specimens taken from the inflamed mucosa and 71.4% of those obtained from colonic carcinoma, as compared to 8.8% of the control group.