结合胆红素和非结合胆红素的直接测定在高胆红素血症中的应用

目的评价结合胆红素（CBil）和非结合胆红素（UBil）的直接测定在胆汁瘀积性黄疸（CJ）、肝细胞性黄疸（HJ）和新生儿溶血性黄疽（NHJ）中的价值以及δ胆红素／总胆红素（δBil／TBil）比值对CJ患者手术疗效判断的意义。方法用Vitros 250型干式生化仪及配套干片测定18例CJ患者和20例HJ患者血浆TBil、CBil和UBil,计算δBil;13例CJ患者在行胆总管切开引流术后3d、5d、7d复测。同法测定22例NHJ患儿血浆CBil和UBil,计算新生儿总胆红素（NBil）。结果CJ组CBil／TBil比值与HJ组比较差异有统计学意义（P=0．000）,而UBil／TBil比值、δBil和δBil／TBil比值2组间差异均无统计学意义（P=0．241、0．067、0．090）。6例经胆总管切开引流术治疗后排泄良好的CJ患者的TBil、CBil和UBil在术后第3d起逐渐下降,δBil缓慢下降,δBil／TBil比值由术前的0．16～0．44升至0．52～0．78。7例治疗后排泄不良的CJ患者其TBil、CBil和UBil在术后5d～7d缓慢减低,δBil变化甚微,δBil／TBil在0．20～0．46范围内波动。NHJ患儿血浆NBil、UBil及UBil／NBil比值均明显增高。结论CBil和UBil的直接测定及δBil／TBil比值对CJ、HJ和NHJ的诊断和／或鉴别诊断以及CJ患者手术疗效的判断均有很好的应用价值。     

Excretion of conjugated bilirubin in the isolated perfused rat kidney

1. Renal mechanisms of conjugated bilirubin excretion have been studied in isolated rat kidneys perfused with a protein-free dextran medium, containing conjugated bilirubin isolated from human bile. 2. In nine perfused kidneys with a low glomerular filtration rate (GFR) (less than 0.5 ml/min) and depressed tubular function, there was a significant linear correlation between conjugated bilirubin clearance and GFR (r = 0.97). 3. In contrast, nine kidneys with a normal GFR (greater than 0.8 ml/min) and good tubular function exhibited substantial tubular reabsorption of filtered conjugated bilirubin (mean 74%). Reabsorption was proportional to the filtered conjugated bilirubin load and a tubular transport maximum was not observed even at high concentrations (144 mumol/1). 4. The fractional reabsorption of bilirubin was unchanged by the addition of sodium aminohippurate to the medium. Perfusion with an albumin medium (10 g/1) resulted in a tenfold reduction in conjugated bilirubin clearance. 5. These observations indicate that non-protein-bound conjugated bilirubin is freely filtered by the glomeruli and then largely reabsorbed in the tubules. Evidence of tubular secretion was not obtained. 6. Chromatographic separation of bilirubin conjugates showed that the proportion of di- to mono-conjugates in the urine was greater than in the perfusate. Whether this incicated further conjugation by the kidney of the monoconjugates or differential clearance of the conjugates was not established.     

血清结合胆红素的酶法测定

目的 建立用胆红素氧化酶（ＢＯＤ）作为催化剂，选择性地测定血清结合胆红素（ＣＢ）的新方法。方法 测定条件为：０．１ｍｏｌ／Ｌ甘氨酸缓冲液、ｐＨ１０．０，ＢＯＤ活性０．５～０．８Ｕ／反应管，反应时间２分钟。结果 线性至少可达到２２０μｍｏｌ／Ｌ；精密度为批内ＣＶ１．７１～３．９８％、批间ＣＶ８．０～９．７２％；正常参考值范围为０～３．１４μｍｏｌ／Ｌ；与偶氮法所测结果相关性较好。结论 本文所建立的Ｃ     

酶法测定结合胆红素的方法学评价

目的评价胆红素酶法测定血清结合胆红素（CBil）,并与干化学法作比较。方法在pH值5．5、120mmoL／L苯二甲酸氧钾缓冲液中,胆红索氧化酶（BOD）选择性地氧化CBil,引起450nm处吸光度（A）值下降,求得CBil浓度。加入氟化钠（NaF）和N-乙酰半胱氨酸（NAC）,抑制BOD对未结合胆红素（UBil）和δ-胆红素（δBil）的氧化,消除其干扰。结果BOD法测定的线性范同达230μmol／L,批内变异系数（CV）为4．1％（低值）、1．4％（高值）,批问CV为11．65％（低值）、10．26％（商值）,平均回收率为97．8％。BOD法与干化学法比较,Y干化学法=1．0160X酶法＋1．6031,r=0．9907。结论该法能有效地抑制BOD对UBil和δBil的氧化,特异性的检测CBil,并可用于自动牛化分析仪。     

Evaluation of a kinetic method for simultaneous determination of conjugated and unconjugated bilirubin.

This paper describes a fast kinetic method for the simultaneous determination of unconjugated and conjugated bilirugin in the same reaction solution. A stopped-flow mixing system with a stabilized photometer and small computer is used to mix sample and reagent rapidly and to record 250 data points during a 700-ms reaction time, and a regression program is used to resolve these kinetic data into the concentrations of unconjugated and conjugated bilirubin. Data are reported for synthetic single and two-component samples and for serum samples. Kinetic results for synthetic mixtures and serum samples are compared with results obtained by a conventional two-step procedure. Regression equations show good linearity between kinetically determined absorbance changes and concentration, good agreement between taken and found values for total, unconjugated, and conjugated bilirubin for synthetic samples in human serum albumin, and a good correlation between kinetic and equilibrium results for these species in sera. Regression slopes for kinetic vs. equilibrium assay results for total, unconjugated, and conjugated bilirubins in sera were 1.01 +/- 0.05, 1.04 +/- 0.03 and 0.91 +/- 0.04, respectively, with intercepts of 6.6,--2.7, and 3.8 micromol/liter, and standard errors of estimate of 28, 14, and 20 micromol/liter. These data reflect uncertainties in both the kinetic and equilibrium methods.     

酶法测定结合胆红素的方法学评价

目的评价胆红素酶法测定血清结合胆红素(CB il),并与干化学法作比较。方法在pH值5.5、120mmol/L苯二甲酸氢钾缓冲液中,胆红素氧化酶(BOD)选择性地氧化CB il,引起450 nm处吸光度(A)值下降,求得CB il浓度。加入氟化钠(NaF)和N-乙酰半胱氨酸(NAC),抑制BOD对未结合胆红素(UB il)和δ-胆红素(δB il)的氧化,消除其干扰。结果BOD法测定的线性范围达230μmol/L,批内变异系数(CV)为4.1%(低值)、1.4%(高值),批间CV为11.65%(低值)、10.26%(高值),平均回收率为97.8%。BOD法与干化学法比较,Y干化学法=1.016 0X酶法 1.603 1,r=0.990 7。结论该法能有效地抑制BOD对UB il和δB il的氧化,特异性的检测CB il,并可用于自动生化分析仪。     

Detection of bilirubin conjugates in faeces of germfree rats

The presence of bilirubin conjugates in faeces from germfree (GF) and conventional (CONV) rats was tested after using affinity chromatography and Porapak Q chromatography as clean-up procedures. The bilirubin conjugates were detected as their ethyl anthranilate azopigments after separation by thin layer chromatography (tlc). Azopigments prepared from bile of GF and CONV rats served as a reference material for the tlc analysis. After tlc of the faecal azopigment preparations it was concluded that GF rat faeces contains bilirubin conjugates, while faeces from CONV rats is devoid of conjugated bilirubin. The findings suggest that the main bilirubin conjugates present in faeces from GF rats are of the glucuronic acid type.     

Proportion of conjugated bilirubin in bile in relation to hepatic bilirubin UDP-glucuronyltransferase activity

To elucidate the diagnostic relevance of biliary conjugated bilirubin, biliary bilirubin from normal volunteers (NV), patients with Gilbert's syndrome (GS) and Crigler-Najjar syndrome type II (C-N II), and from various rat strains was fractionated. Biliary bilirubin diglucuronide (BDG) was present at lower levels, and bilirubin monoglucuronide (BMG) and unconjugated bilirubin were present at higher levels in GS and C-N II compared with NV, which is consistent with decreased hepatic bilirubin UDP-glucuronyltransferase activity (BGTA). The level of biliary BDG was higher in Wistar-Kyoto rats and lower in heterozygous (Jj) Gunn rats than in SD and Wistar rats. The hepatic BGTA level in heterozygous (Jj) Gunn rats was decreased to 60% of that in Wistar rats, in accordance with decreased biliary BDG. On the other hand, BGTA in Wistar-Kyoto rats whose biliary BDG level was high, was not different from that of Wistar and SD rats. Thus, a correlation between BGTA and biliary bilirubin fractions may not exist on some occasions.     

结合胆红素酶法测定的一种新方法

目的 建立一种特异性更高的酶法血清结合胆红素(CB)测定的新方法。方法 在pH5．5、有氟化钠(NaF)和N—乙酰半肮氨酸(NAC)的存在下，胆红素氧化酶(BOD)选择性氧化CB，引起450nm吸光度下降，计算CB的浓度。结果 批内精密度，n＝20，-↑x＝17．65μmol／L，s＝1．15，CV＝6．52％和-↑x＝301．49μmol/L，s＝1．01，CV＝0．33％；批间精密度，n＝20，-↑x＝31．50μmol／L，s＝3．12，CV＝9．90％和-↑x＝184．12μmol/L，s＝5．01，CV＝2．72％；线性范围至少可达320μmol／L；选择性抑制物的最适浓度，NaF2．5mmol/L，NAC2．5mmol/L；与重氮法的相关性，Y(酶法)＝0．839X(重氮法)—7．965，r＝0．9690。结论 该研究建立的反应条件，能有效地抑制BOD对游离胆红素的“非特异性”氧化，提高CB测定的准确性。     

Enzymatic assay for conjugated bilirubin (Bc) in serum using bilirubin oxidase (BOD).

We described an automated enzymatic assay for conjugated bilirubin (Bc) in serum using the Iatro D-Bil kit, with bilirubin oxidase (EC 1.3.3.5 BOD) from Myrothecium species. The specificity of the enzyme in the Iatro D-Bil kit was examined by analyzing unconjugated bilirubin (Bu), delta bilirubin (Bdelta), and Bc with high-performance liquid chromatography (HPLC), before and after the enzymatic reaction using BOD. The within-assay coefficients of variation (CV) of this method were 0.58 to 5.00% (n = 20) at 1.4 to 155.8 micromol/L. Day-to-day Cvs ranged from 1.61 to 7.14% at 1.2 to 182.1 micromol/L. The analytical recovery was 96 to 101%. The presence of ascorbic acid, reduced glutathione, L-cysteine, uric acid, urea, creatinine, glucose, lipemic material, anticoagulants, hemoglobin, or human serum albumin did not affect this assay system. The correlation coefficient between values obtained with the Iatro D-Bil kit (y) and HPLC method as reference for conjugated fractions (x) was; r = 0.983, y = 0.952x + 8.851 micromol/L, Sy/x = 11.97 (n = 56). We studied serum Bc levels, not including Bu and Bdelta, in patients with hepatic diseases or autoimmune hemolytic anemia. Levels of Bc obtained by the proposed method changed more rapidly than did those of direct bilirubin (D-Bil) obtained by diazo-dye method during the course of the diseases.     

血清结合胆红素酶法测定在自动生化分析仪上的应用

目的　建立酶法测定血清结合胆红素的全自动分析方法 ,并在肝胆疾病患者中临床应用。方法　通过试剂的合理组合 ,分析参数和操作程序的设定 ,建立结合胆红素 (CB)的全自动分析方法 ,同时对总胆红素进行自动化酶法测定。结果　酶法CB测定 :精密度 ,批内n =2 0 , x =6 .4 8μmol/L ,s=0 .11,CV =1.7%和n =2 0 , x =77.9μmol/L ,s=0 .4 2 ,CV =0 .5 3% ;批间n =10 , x =9.4 6 μmol/L ,s=0 .17,CV =1.83%和n =10 , x =5 9.7μmol/L ,s=1.4 3,CV =2 .39% ;线性范围至少可达 340 μmol/L ;比对实验 :与Beckman重氮法试剂 (在BeckmanCX7型生化分析仪上 )比较 ,Y(酶法 ) =0 .75 9X1- 2 .82、r =0 .991,与IagnosticumRT重氮法试剂 (日立 76 0 0 0 2 0型分析仪 )比较 ,Y =0 .84 1X2 - 4 .39、r=0 .96 3。临床研究显示 :与重氮法直接胆红素值相比 ,本法测定CB值能够更灵敏地反映黄疸患者的胆红素分泌功能。结论　建立的CB全自动分析方法具有良好的分析特性 ,可在临床推广应用。