Bile acid profiles in urine of patients with liver diseases

Abstract. Quantitative gas chromatography-mass spectrometry was used to study the metabolic profiles of unconjugated, conjugated and sulphated bile acids in urine of patients with intermittent intrahepatic cholestasis of unknown aetiology, cirrhosis of the liver, primary biliary cirrhosis, viral and toxic hepatitis and extrahepatic cholestasis. A large number of bile acids was present which can broadly be classified into four groups: cholic and chenodeoxycholic acids constituted between 49·4% and 77·9% of the total bile acids (mean values of the groups); deoxycholic and other 3,12-disubstituted bile acids between 1·3% and 12·3%, monohydroxy bile acids between 6·7% and 14·4% and bile acids hydroxylated at C-1 or C-6 between 4·6% and 14·6%. The high proportion of bile acids from the latter group, and the presence of tetrahydroxylated bile acids, clearly distinguished hepatic disease from the normal state. The metabolic profiles were very variable and there were few consistent differences between the groups of diseases studied. Norcholic acid constituted a significantly higher percentage of the total bile acids in cirrhotic patients (6·2 ± 6·8%) than in non-cirrhotic patients (1·3±1·8%, P<0·001). With this exception, no profile was specific for any type of intra- or extra-hepatic cholestasis. The excretion rates of the major l-hydroxylated bile acids were positively correlated to each other. The same was true for the major 6-hydroxylated bile acids. This may indicate that cholic, chenodeoxycholic and deoxycholic acids act as substrates for common 1- and 6-hydroxylating enzymes. Possibly the taurine conjugates are preferred substrates since 1-hydroxylated bile acids and hyocholic acid were found mainly in this fraction. A positive correlation between the excretion of sulphated 3β-hydroxy-5-cholenoic acid and 3β,12α-dihydroxy-5-cholenoic acid indicates a direct metabolic relationship between these compounds. Confirming previous data, a high proportion of bile acids was sulphated. The degree of sulphation increased with decreasing number of hydroxyl groups, reaching 100% for the monohydroxy and most of the dihydroxy acids. Tetrahydroxycholanoates were not sulphated, and sulphation of trihydroxycholanoates was positively correlated to the renal bile acid excretion rate. Bile from patients with intermittent intrahepatic cholestasis did not contain the tetrahydroxylated bile acids present in urine. Hyocholic acid was a very minor, mainly taurine conjugated, bile acid. Monohydroxy bile acids were usually below the detection limit. These data do not support the hypothesis that lithocholic acid participates in the initiation or perpetuation of intermittent intrahepatic cholestasis of unknown aetiology.     

气相色谱-质谱联用法检测粪便中短链脂肪酸

摘要：目的：建立气相色谱-质谱联用快速检测粪便中短链脂肪酸的方法,并探讨将其应用于肠道疾病辅助诊断的可能性。 方法：分别取健康C57BL/6小鼠、SD大鼠和成年人志愿者粪便,以及用硫酸葡聚糖溶液诱导的肠炎模型C57BL/6小鼠的粪便;用气相色谱-质谱联用法检测上述标本中短链脂肪酸的种类和相对丰度。 结果：所有标本均检测出多种短链脂肪酸,其种类主要有乙酸、丙酸、丁酸和戊酸。在生理条件下,人与鼠短链脂肪酸的种类和相对丰度比都十分相似。肠炎可使粪便中除乙酸外的其他短链脂肪酸的相对丰度下降,其中,尤以丁酸下降最为明显。 结论：气相色谱-质谱联用法可快速检测粪便中的短链脂肪酸,并有可能应用于肠炎等肠道疾病的辅助诊断。     

衍生化气相色谱法测定冠心病患者血清中游离脂肪酸的方法学研究

目的建立同时测定冠心病患者血清中11种游离脂肪酸(FFA)含量的测定方法。方法采用N,O-双(三甲基硅烷基)三氟乙酰胺(BSTFA)对11种FFA进行硅烷化反应,并利用气相色谱质谱联用技术(GC/MS)同时测定冠心病患者血清冻干粉中11种FFA的含量。结果 11种FFA色谱峰完全分离,峰形良好,血清中杂质对测定不产生干扰。在测定浓度范围内线性关系良好,11种FFA的回收率均75%,日内、日间精密度和重复性实验的相对标准偏差(RSD)均10%。结论该方法具有操作简单、灵敏度高、准确性好、线性范围宽等优点,可应用于临床实际血清样品中FFA的分析检测。     

Urinary excretion of bile alcohols in normal children and patients with α1-antitrypsin deficiency during development of liver disease

Abstract. Healthy infants and children were found to excrete bile alcohol glucuronides in urine. Following isolation and hydrolysis, the bile alcohols were estimated by capillary gas-liquid chromatography. The daily urinary excretion of the major compound, 27-nor-5 β -cholestane-3α,7α,12α,24ξ,25ξ-pentol (a C₂₆ bile alcohol), ranged from 0·1 to 1·1 μmol/24 h per m² body surface area for healthy infants and children. Two groups of patients with α₁-antitrypsin deficiency (phenotype PiZ) were also studied during infancy and childhood, and biochemical liver function tests and liver morphology were compared to the excretion of bile alcohols. The highest excretion of the C₂₆ bile alcohol in urine was found in patients with α₁-antitrypsin deficiency and juvenile cirrhosis (2·1–8·4 μmol 24 h^-1 m^-2) regardless of preceding neonatal cholestasis. Patients with α₁-antitrypsin deficiency, neonatal cholestasis and subsequent fibrosis or normal liver morphology excreted bile alcohols within the normal range. The C₂₆ bile alochol constituted an average of 36% of the total bile alcohols in forty-three urine samples. This percentage was about the same in the three groups studied. The findings suggest that determination of urinary bile alcohols may be a valuable non-invasive diagnostic tool for patients with or at risk of developing liver cirrhosis.     

吹扫捕集-气相色谱-质谱联用法测定生活饮用水中的二氯甲烷

目的建立生活饮用水中二氯甲烷的吹扫捕集-气相色谱-质谱联用测定方法。方法采用吹扫捕集富集水中二氯甲烷,解吸后用气相色谱-质谱测定,选择离子扫描方式下用标准曲线法进行定量分析。结果该方法操作简便,检出限低(0.005ng/mL),回收率均大于97%,相对标准偏差小于5%。结论该方法适合生活饮用水中的二氯甲烷的测定。     

Kinetics of primary bile acids in patients after orthotopic liver transplantation

Administration of cyclosporin A (CsA) may induce cholestasis, and this effect has been attributed to impaired hepatocellular uptake, transport, secretion and intestinal absorption of bile acids. Disturbances of the enterohepatic circulation may affect metabolism of bile acids. To test whether liver transplantation and treatment with CsA alters pool sizes or synthesis and turnover rates, we determined kinetics of primary bile acids in patients after orthotopic liver transplantation on CsA. Two male and four female patients were studied 6–20 months after transplantation. They had no overt signs of cholestasis, graft dysfunction or rejection. Kinetics of cholic acid (CA) and chenodeoxycholic acid (CDCA) were simultaneously determined after oral administration of [24-¹³C]-CA and [24-¹³C]-CDCA on the basis of isotope dilution in a single pool of bile acids. Ten healthy volunteers served as controls. After orthotopic liver transplantation, pool sizes, fractional turnover rates and synthesis rates of both primary bile acids, CA and CDCA were not significantly different from control subjects. In spite of the known interference of CsA with the enterohepatic circulation of bile acids, in the majority of patients after orthotopic liver transplantation without cholestasis, graft dysfunction or rejection, treatment with CsA does not disturb kinetics of primary bile acids.     

Capillary gas chromatographic determinations of urinary bile acids and bile alcohols in CTX patients proving the ineffectivity of ursodeoxycholic acid treatment

Urine samples and serum samples of a patient with cerebrotendinous xanthomatosis (CTX) were investigated by means of capillary gas chromatography, both before and during oral treatment with ursodeoxycholic acid (UDCA), and the results compared with those obtained during chenodeoxycholic acid (CDCA) therapy. The predominantly excreted bile alcohol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol and two abnormal bile acids, i.e. 23-norcholic acid and 23-hydroxycholic acid were determined. In addition, the serum cholestanol/cholesterol ratio was determined. Whereas previous experiments demonstrated that the urinary excretion of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol and the abnormal bile acids decreased within a few weeks during CDCA therapy, the present study shows that their urinary excretions remain essentially the same during UDCA treatment. In contrast to the decrease in the serum cholestanol/cholesterol ratio during CDCA therapy, this ratio remains essentially the same during UDCA therapy. It is therefore concluded that, in contrast to CDCA therapy, UDCA treatment is not effective in the treatment of CTX.     

Accurate enzymatic measurement of fecal bile acids in patients with malabsorption

Quantitation of fecal bile acid excretion can help elucidate the cause of diarrhea or steatorrhea. Fecal bile acids can be measured with gas chromatography-mass spectrometry, but this is time-consuming, expensive, and not available for clinical use. Relatively simple enzymatic methods have been described for the measurement of fecal 3alpha-hydroxy bile acids, but these have not been validated in patients with gastrointestinal disease. We found that an enzymatic method yielded falsely low results in patients with malabsorption syndromes for two reasons: First, the preliminary hydrolysis step did not completely deconjugate bile acids, precluding their extraction into diethyl ether for enzymatic assay. Second, long-chain fatty acids inhibited 3alpha-hydroxysteroid dehydrogenase activity. By increasing the duration of hydrolysis and the concentration of enzyme, we developed a simple, accurate, and reproducible method for measuring fecal 3alpha-hydroxy bile acids that agreed well with values obtained with the use of gas chromatography-mass spectrometry (R =.95), both in normal subjects and in patients with malabsorption syndromes.     

Pharmacokinetics of flunitrazepam following single dose oral administration in liver disease patients compared with healthy volunteers

The pharmacokinetic behaviour of flunitrazepam and its main active metabolite, N-desmethyl flunitrazepam, was investigated in 12 patients with liver disease (cirrhosis or hepatitis) compared to 6 healthy volunteers. A gas-liquid chromatographic method allowing for simultaneous determination of flunitrazepam and N-desmethyl flunitrazepam in plasma samples was developed. The accuracy and the precision near the quantification limit of ca. 1 ng/ml were better than 5%. Plasma levels of flunitrazepam were not significantly altered by hepatic failure, whereas plasma levels of N-desmethyl flunitrazepam were lower in patients than in healthy subjects. Pharmacokinetic parameters did not differ significantly between healthy subjects and liver disease patients: the oral clearance was 3.5 +/- 0.8, 3.5 +/- 1.9 and 4.0 +/- 1.2 ml/min/kg, respectively in healthy subjects, patients with hepatitis and patients with cirrhosis. The apparent elimination half-life was 22 +/- 5 h in healthy subjects, 25 +/- 10 h in patients with hepatitis and 20 +/- 6 h in patients with cirrhosis. However, the expected increase of the drug free fraction during liver disease could decrease the therapeutic and toxic ranges of flunitrazepam in these patients.     

Recent advances of liquid chromatography–(tandem) mass spectrometry in clinical and forensic toxicology

Liquid chromatography (LC) coupled to mass spectrometry (MS) or tandem mass spectrometry (MS/MS) has become increasingly important in clinical and forensic toxicology as well as doping control and is now a robust and reliable technique for routine analysis in these fields. In recent years, methods for LC–MS(/MS)-based systematic toxicological analysis using triple quadrupole or ion trap instruments have been considerably improved and a new screening approach based on high-resolution MS analysis using benchtop time-of-flight MS instruments has been developed. Moreover, many applications for so-called multi-target screening and/or quantification of drugs, poisons, and or their metabolites in various biomatrices have been published. The present paper will provide an overview and discuss these recent developments focusing on the literature published after 2006.     

健康体检人群体重指数与血脂水平及非酒精性脂肪肝关系的研究

目的探讨体重指数与血脂水平及非酒精性脂肪肝(non-alcoholic fatty liver disease, NAFLD)的关系。 方法对2012年1月至2013年1月于安徽省立医院参加健康体检,病史及饮酒史资料完整的10000名体检者的人体质量指数(BMI)与其相关临床指标进行统计分析。按照BMI将人群分组,不同体重指数人群间血脂水平及NAFLD检出率差异采用χ²检验。 结果(1)所有体检人群中超重及肥胖者5996例,占60%,正常体重者3829例,占38.3%;(2)超重和肥胖在不同年龄组人群中的构成比差异有统计学意义(χ²＝161.733,P＝0.000;(3)除高密度脂蛋白(HDL-C)外,超重组和肥胖组的甘油三脂(TG)、总胆固醇(TC)、低密度脂蛋白(LDL-C)水平均明显高于体重正常组;(4)NAFLD和高脂血症的发生率随BMI水平升高而逐渐增加;(5)性别(男性)、BMI、TG、TC为NAFLD发生的独立危险因素,HDL-C为保护因素;(6)ROC曲线分析BMI预测NAFLD发生的最佳切点为(男性：24.85或24.95;女性24.95)。 结论超重及肥胖是发生NAFLD和高脂血症的危险因素,应加强对体重的干预,以改善脂代谢和降低NAFLD的发生。     

生物检材中复方芬太尼的气相色谱和气相色谱/质谱检测

目的：建立生物检材中复方芬太尼的气相色谱（GC）和气相色谱/质谱检测方法（GC/MS法）。方法：检材经10%氢氧化钠调pH=10,乙醚萃取,气/质联用法定性,气相色谱内标法定量检测生物检材中复方芬太尼。结果：GC/MS法分析芬太尼的选择离子m/z为245,146;异丙嗪的选择离子m/z为284,72。GC检测的回归方程、线性检测范围、相关系数、回收率、最低检出浓度分别为：心血中芬太尼为Y=23.376X＋0.516 8（μg/mL）,（0.1~220）μg/mL,0.985,（98.5±3.5）%,0.1μg/mL,异丙嗪为Y=16.537X＋0.158 4（μg/mL）,（0.12~200）μg/mL,0.974,（97.50±2.0）%,0.12μg/mL;肝组织中芬太尼为Y=82.236X＋0.413 4（μg/mL）,（0.1~220）μg/g,0.975,（95.6±0.75）%,0.1μg/g,异丙嗪为Y=40.437X＋0.134 2（μg/mL）,（0.12~200）μg/g,0.964,（94.8±0.15）%,0.12μg/g。染毒家兔心血及心、肝、脾、肺、肾和脑中芬太尼的含量依次为：（24.39±6.43）μg/mL及（84.96±1.03）,（73.82±1.73）,（55.74±2.07）,（42.64±1.38）,（35.94±2.87）,（10.15±7.05）μg/g,异丙嗪的含量依次为：（146.69±2.43）μg/mL及（204.96±13.03）,（213.32±1.73）,（115.74±3.07）,（95.64±5.38）,（195.34±2.87）,（170.15±1.05）μg/g。结论：生物检材中GC/MS法选择性好,定性准确,GC检测简便,快速,灵敏,定量结果准确,可用于芬太尼麻醉意外中毒的临床快速检验诊断和芬太尼滥用中毒死亡案件的法医学鉴定。     

稳定同位素iTRAQ标记联合液相色谱-串联质谱法定量分析药物性肝损伤患者血清氨基酸的变化

目的探索药物性肝损伤(DILI)患者治疗的不同阶段血清氨基酸水平的变化差异,探讨其临床意义及可用于DILI早期诊断及疗效评价的氨基酸类生物标志物。方法收集21例健康志愿者(健康组)及24例DILI患者不同阶段(初诊、治疗、转归)的血清标本,采用稳定同位素iTRAQ标记方法联合液相色谱串联质谱技术检测血清氨基酸进行定量并分析其差异。结果在血清中共定量氨基酸28种,与健康组比较,DILI患者共有14种氨基酸浓度在诊疗不同阶段发生显著性改变;其中异亮氨酸、亮氨酸、苯丙氨酸及丝氨酸的浓度在初诊时显著升高,随治疗进程降低,逐渐接近于正常水平。结论 4种氨基酸中,苯丙氨酸与DILI的相关性更高,可能为DILI早期诊断及治疗效果评价的潜在生物标志物。     

Urinary bile acids in late pregnancy and in recurrent cholestasis of pregnancy

The metabolic profiles of urinary bile acids in pregnant women in the last trimester and patients with recurrent intrahepatic cholestasis of pregnancy (RCP) were studied. Following separation according to mode of conjugation, about thirty different bile acids were quantitatively analysed by gas chromatography-mass spectrometry. In all patients the sulphate fraction comprised 50--90% of the total bile acids. In RCP a shift from glycine to taurine conjugation was noted to together with a slight relative increase in sulphation. A ten- to hundred-fold increase in cholic and chenodeoxycholic acids was seen in RCP, the increase being mainly in the sulphate fraction. Tetrahydroxylated bile acids, tentatively regarded as 1- and 6-hydroxylated products of cholic acid, were quantitatively important in patients with RCP. The relative amounts of the secondary bile acids, deoxycholic and lithocholic acids, decreased with increasing cholestasis. Metabolites hydroxylated at C-6 were common, and the excretion of hydroxylated at C-6 were common, and the excretion of hyocholic acid was positively correlated to that of chenodeoxycholic acid. An increase in the excretion of 5 alpha-configurated bile acids in RCP was noted. A positive correlation between the excretion of 3 beta-hydroxy-5-cholenoic acid and 3 beta,12 alpha-dihydroxy-5-cholenoic acid indicates a metabolic relationship between the two compounds. Because of the relatively small amounts of lithocholic and 3 beta-hydroxy-5-cholenoic acids in patients with RCP, these compounds do not seem to be of pathogenetic importance in this type of cholestasis.     

225例肝病患者自身抗体检测结果分析

目的：探讨各种肝病患者中自身抗体检测在自身免疫性肝病（autoimmune liver disease, ALD）包括自身免疫性肝炎（autoimmune hepatitis, AIH）、原发性胆汁性肝硬化（primary biliary cirrhosis, PBC）及原发性硬化性胆管炎（primary sclerosing cholangitis, PSC）中的临床意义。方法选择我院2013年1月-2014年2月消化科、肝胆外科、移植外科等收治的各种肝病患者225例,分为ALD患者组和其他肝病患者组。用间接免疫荧光法检测225份临床血清标本中抗核抗体（antinuclear antibody, ANA）、抗平滑肌抗体（antismooth muscle antibody, SMA）和抗线粒体抗体（anti-mitochondrial antibody, AMA）,用欧蒙印迹法检测抗肝肾微粒体抗体（liver kidney microsomal, LKM）、抗肝细胞溶质抗原Ⅰ型抗体（liver cytosol-1, LC-1）、抗可溶性肝抗原抗体/抗肝胰抗体（anti-soluble liver antigen antibody/liver pancreas antigen, SLA/LP）、AMAⅡ型（AMA-M2）并查阅临床资料,对检测结果作出分析。结果 ALD 患者组ANA阳性率为77．78％,SMA阳性率为7．41％,AMA阳性率为55．56％,LKM 阳性率为7．41％,AMA-M2阳性率为48．15％,SLA/LP 阳性率为7．41％,LC-1阳性率为3．70％。其他肝病患者组 ANA 阳性率为26．26％,SMA 阳性率为4．55％,AMA 阳性率为11．11％,LKM 阳性率为0．51％,AMA-M2阳性率为5．05％,SLA/LP阳性率为2．53％,LC-1阳性率为1．52％。ALD患者组自身抗体阳性率与其他肝病患者组相比较,ANA、AMA、LKM、AMA-M2差异均有统计学意义（P均＜0．05）。结论 ALD患者自身抗体检出率较高,为临床医生鉴别病毒性肝炎、ALD及其他肝病提供可靠依据。     

2型糖尿病患者非酒精性脂肪肝与尿白蛋白排泄率的关系

目的 探讨合并非酒精性脂肪肝(NAFLD)的2型糖尿病(T2DM)患者尿白蛋白排泄率(UAER)的变化特点及其相关性.方法 T2DM患者228例,根据是否合并NAFLD分为糖尿病合并NAFLD组(135例)和糖尿病无NAFLD组(93例),另选年龄、性别匹配的40例健康受试者为对照组,比较两组患者体重指数(BMI)、空腹血糖(FPG)、血脂、空腹胰岛素(FINS)、空腹C肽、胰岛素抵抗指数(HOMA-IR)、24h UAER的差异,用卡方检验分析两组间正常、微量和大量UAER的分布差异,进一步以T2DM合并NAFLD为应变量,以各临床生化指标为自变量,用单因素和多因素Logistic回归分析方法分析T2DM合并NAFLD的危险因素.结果 糖尿病合并NAFLD组BMI、甘油三酯(TG)、FINS和空腹C肽、HOMI-IR、UAER高于非NAFLD组,差异有统计学意义(P均<0.05).卡方检验显示,糖尿病合并NAFLD患者微量白蛋白尿和大量白蛋白尿发生率高于无NAFLD组,差异有统计学意义(χ2=23.905,P=0.001).Logistic回归分析显示,BMI(OR=4.66,P=0.001)、TG(OR=8.46,P=0.000)及UAER(OR=3.73,P=0.003)升高是T2DM合并NAFLD的危险因素.结论 T2DM患者中NAFLD与BMI、TG及UAER的升高密切相关,T2DM合并NAFLD患者糖尿病肾病的发病率显著高于无合并NAFLD患者.     

肝病患者血清胆碱酯酶检测临床意义分析

目的探讨肝病患者检测血清胆碱酯酶(ChE)的临床意义。方法检测肝病患者112例(实验组)和健康者106例(对照组)血清ChE活性。结果各肝病患者组与对照组检测结果比较差异有统计学意义(P<0.05)。结论肝病患者血清ChE活性低于健康者,并随肝病病情加重而逐渐下降;血清ChE活性能较好反映肝脏合成功能,准确反映肝病患者病情严重程度。     

人工肝治疗终末期肝病肝衰竭的终末期肝病模型预后分析

目的　通过观察应用终末期肝病模型 (MELD)对人工肝治疗终末期肝病预后的预测作用 ,探讨其在临床的应用价值及人工肝支持系统在终末期肝病治疗中的效果。方法　 43例患者随机分为治疗组与对照组 ,对其进行MELD评分 ,并对治疗组进行人工肝后评分 ,观察 3个月后的死亡率。结果　治疗组中 4例治疗前MELD评分大于 40分者 ,3个月死亡率为 10 0 %,17例治疗前评分 30～ 40分者 3个月死亡率为 5 3%,对照组中 6例MELD评分大于 40分者 ,3个月死亡率为 10 0 %,16例治疗前评分 30～ 40分者 3个月死亡率为87%,与治疗组相比P <0 .0 5。结论　MELD评分可以引入临床推广应用 ,人工肝治疗MELD评分在 30～ 40分的终末期肝病是安全有效的。