Detection of polyamines by a new enzymatic differential assay. (4). Fundamental study on a new enzymatic differential assay of blood

The enzymatic method for isolation and determination of urinary polyamines was modified to measure the polyamines in the blood. High recovery rates of polyamine in blood by enzymatic hydrolyzation were obtained, namely, 101.9 +/- 4.4% for diamine, 96.0 +/- 5.4% for spermidine and 104.1 +/- 3.3% for spermine. Furthermore excellent linearity was demonstrated. Within-run precision of polyamine in blood was excellent, namely, in C.V., 1.15% for Reaction 1, 2.11% for Reaction 2 and 2.79% for Reaction 3. This method was compared with high pressure liquid chromatography (HPLC), and a close correlation was demonstrated for all the fractions: diamine r = 0.8824, y = 1.367x+0.0417 (n = 15); spermidine r = 0.9878, y = 0.806x+5.218 (n = 15); spermine r = 0.9764, y = 1.068x-0.9195 (n = 15).     

Analytical and clinical evaluation of the Bio-Rad HPLC kit for measurement of type I collagen cross links

 The measurement of hydroxylysylpyridinoline (PYD) and lysylpyridinoline (DPD), the degradation products of type I collagen, by manual HPLC assay posed practical difficulties. The present study was undertaken to evaluate the first commercially available HPLC kit, which provides a convenient substitute for cumbersome classical HPLC methods. The HPLC procedure is based on an improved sample clean-up chromatography, convenient ready-to-use HPLC reagents, and quicker isocratic elution of PYR and DPD on reverse-phase analytical column. The analytical parameters assessed include sensitivity, within- and between-assay variation, method comparison, recoveries, and interference. Clinical evaluation included discriminatory power of PYD and DPD and response to treatment of osteoporosis patients with Alendronate. DPD and PYD concentrations showed linear (r ² > 0.99) response between 10–400 pmol/ml and 75–4000 pmol/ml, respectively. The average within-assay imprecision, over a range of clinically relevant cross-links concentrations, was CV < 7% (n = 30). The total imprecision (n = 35 days), by ANOVA, for PYD and DPD was CV < 7.5% and CV < 10%, respectively. Average spike recovery was 95.4% ± 6.5%. Comparison with the historical HPLC method exhibited a close correlation (r values between 0.87 and 0.91, P < 0.0001). Creatinine-corrected DPD in postmenopausal (Z score = 2.4, P < 0.05, n = 17) and osteoporotic (Z score = 3.0, P < 0.01, n = 29) women were 44% and 64% higher, respectively, compared to premenopausal samples (n = 15). Similarly, PYD concentration was 26% and 54% higher in postmenopausal and postmenopausal osteoporotic women, respectively. There was a 47% (P < 0.001) decrease in DPD concentration (n = 16), and a 30% decrease in PYD concentration after 90 days of treatment of osteoporotic patients with Alendronate. DPD concentration correlated with N-telopeptide with an r value of 0.69 (n = 67, P < 0.0001). The reported kit method is substantially simpler and precise than the manual method. DPD concentrations determined by the current method reaffirm the clinical value in identifying increased bone resorption in pathological conditions and monitoring response to antiresorptive therapy. Received: June 28, 2002 / Accepted: November 6, 2002 Offprint requests to: D.J. Baylink     

大鼠梗阻性黄疽脑内多胺代谢紊乱的实验研究

目的 研究梗阻性黄疽大鼠脑组织多胺代谢变化及临床意义.方法 雄性Wistar大鼠26只,随机分为假手术组(SO组)和胆总管结扎组(BDL组).术后9d门静脉抽血测量血清总胆红素(TBil)水平;取大鼠脑组织,高效液相色谱(HPLC)法测定腐胺、精眯和精胺的含量;比色法测定脑组织多胺氧化酶(PAO)活性水平.结果 BDL...     

Comparison of specific radioimmunoassays for cyclosporine

This study compares two recently introduced radioimmunoassay kits involving specific monoclonal antibodies to cyclosporine. One kit (Sandimmun) involved 3H-labeled CsA (3H-CsA) as tracer and the other (CY-CLO-Trac-SP) involved a 125I-labeled conjugated derivative of CsA. The kits were nearly equivalent in method performance characteristics. They produced superimposed standard curves and equivalent values to transplanted patient samples. Concentrations of CsA determined by either kit were apparently equivalent to values measured by high-performance liquid chromatography, suggesting that the specific monoclonal antibodies used with the kits detect in trough blood mainly native CsA. The 125I-labeled CsA, when compared with the 3H-CsA alternative, increased the sensitivity and precision, decreased the turnaround time, and provided a technically efficient and conveniently capable method of replacing HPLC for measuring native CsA.     

Value of urinary polyamines as noninvasive markers of cardiac allograft rejection in the dog

A noninvasive marker of cardiac allograft rejection would be useful clinically. Lymphocyte proliferation and organ rejection may cause changes in urinary polyamine excretion. To test this hypothesis, cervical heterotopic heart transplantations were performed in a group of 6 nonimmunosuppressed dogs and in a group of 9 dogs treated with cyclosporine (N = 3) or cyclosporine and steroids (N = 6). A group (N = 3) having a sham operation was also studied. Serial biopsies of the transplanted hearts were performed. Urinary polyamine levels were measured daily by high-pressure liquid chromatography of urine specimens. Between 2 and 4 days after transplantation, the transplanted hearts of all animals without immunosuppression demonstrated histological rejection. An early increase in putrescine levels and in total urinary polyamine levels was observed in this group. In the treated groups, histological rejection appeared from the second to the eighth day after transplantation. Each episode of rejection occurred from 1 day to 4 days after a significant increase in urinary polyamine levels compared with the preoperative baseline level (p less than 0.01). In contrast, polyamine excretion in 3 dogs after sham operations remained unchanged. Thus, urinary excretion of polyamines increases before the appearance of histological rejection; this suggests that changes in urinary polyamine levels may be a useful marker of cardiac allograft rejection.     

鸟氨酸脱羧酶和S-腺苷甲硫氨酸脱羧酶双反义腺病毒对肺癌增殖和侵袭抑制作用的研究

目的探讨重组鸟氨酸脱羧酶和S-腺苷甲硫氨酸脱羧酶双反义腺病毒载体（Ad-ODC-AdoMetDCas）介导的鸟氨酸脱羧酶（ODC）和S-腺苷甲硫氨酸脱羧酶（AdoMetDC）反义RNA对肺癌细胞多胺合成、增殖和侵袭的抑制作用。方法采用活细胞计数法观察Ad-ODC-AdoMetDCas对肺癌A-549细胞生长增殖的影响,采用Western印迹法和高效液相色谱分别检测腺病毒对肺癌A-549细胞中ODC和AdoMetDC蛋白表达以及细胞内多胺含量,采用活细胞计数法和流式细胞仪分别检测腺病毒介导的鸟氨酸脱羧酶反义RNA和Ad-ODC-AdoMetDCas对A-549细胞增殖和细胞周期分布的影响,应用Matrigel侵袭实验分析腺病毒对肺癌细胞侵袭活性的改变。结果当感染效率为50时,腺病毒对A-549细胞的感染率约75％。Ad-ODC-AdoMetDCas可明显抑制A-549细胞ODC和AdoMetDC的表达。A-549细胞感染Ad-ODC-AdoMetDCas后,细胞内多胺含量明显降低。感染Ad-ODC-AdoMetDCas的A-549细胞停滞在G1期。Ad-ODC-AdoMetDCas可显著降低A-549细胞的体外侵袭能力。结论ODC和AdoMetDC双反义腺病毒能够显著抑制肺癌A-549细胞的增殖和侵袭。     

Urine polyamine in patients with malignant urological diseases using a polyamine-test enzyme kit

Using a polyamine-test enzyme kit, the urine polyamine concentration was determined in 74 patients with malignant urological disease (12 with renal cell cancer, 13 with pelvic-ureter cancer, 24 with bladder cancer and 25 with prostate cancer), 7 patients with BPH, 20 patients with benign urological disease and 20 normal subjects. The urine polyamine level was significantly elevated in all the patients with any malignant urological disease compared to normal subjects. It was also significantly high in the patients with BPH. Defining the mean +/- 3SD (= 50 mumole/g Cr.) of 20 normal subjects as an upper limit, slightly higher levels not exceeding 100 mumol/g Cr. were frequently observed in the patients with BPH or with benign urological disease. Setting the upper limit at 100 mumole/g Cr., the positive rate amounted to 33% (low stage 17%) in renal cell cancer, 23% (low stage 14%) in pelvic ureter cancer, 13% (low stage 0%) in bladder cancer and 4% (low stage 0%) in prostate cancer. The positive rate was low especially in low stage cases.     

Detection of urinary polyamine by a new enzymatic differential assay. (I). Fundamental study on a new enzymatic differential assay

A new enzymatic method for determining urinary polyamine concentration by fractionation of the urinary acetyl conjugate into free polyamines with acylpolyamine amido-hydrolase and quantification using two kinds of amine-oxidase of different substrate specificity was examined. High recovery rates of urinary polyamine by enzymatic hydrolyzation were obtained, namely, 95 +/- 4% for diamine, 95 +/- 1% for spermidine and 99 +/- 2% for spermine. Furthermore, excellent linearity was demonstrated with up to 150 mumole/l diamine, up to 75 mumole/l spermidine and up to 50 mumole/l spermine. Although urinary polyamine concentration varied diurnally even after correction of urinary creatinine, day-to-day variation disappeared. In 24-hour pooled urine and voluntary urine, diamine, spermidine and spermine correlated relatively well. Urinary leukocytes and erythrocytes exerted no influence on urinary polyamine.     

Inhibition of bone resorption by alpha-difluoromethylornithine may not be mediated by polyamine depletion

We have examined the effect of alpha-difluoromethylornithine (DFMO) on bone polyamine content and parathyroid hormone (PTH)- and calcitriol-stimulated bone resorption in cultures of neonatal mouse calvaria. Polyamine content in bone homogenates was determined by reverse-phase paired-ion HPLC. Treatment with 5 mM DFMO for 48 h reduced putrescine from 0.4 nmol/bone to nondetectable levels, slightly decreased spermidine, and did not affect spermine. Bone resorption elicited by 48 h of treatment with PTH or calcitriol was inhibited by concentrations of DFMO greater than or equal to 5 mM added 48 h prior to hormone. This observation supported the concept that polyamines may play a role in bone resorption. However, other observations cast uncertainty on this conclusion. Measurement of calvarial polyamine content at 2 h intervals revealed no increase in endogenous polyamines for up to 10.5 h after calcitriol addition. Although addition of putrescine restored bone polyamine content, exogenous polyamines failed to reverse the inhibitory effects of DFMO on calcitriol-stimulated resorption. These results suggest that a mechanism other than depletion of polyamines could be contributing to the inhibitory effect of DFMO on resorption.     

Difluoromethylornithine inhibits urogastrone stimulation of neomucosal growth

Urogastrone (UG) stimulates the growth of intestinal neomucosa on patched intestinal defects. This effect may be dependent on increased polyamine biosynthesis. The aim of this study was to determine if difluoromethylornithine (DFMO), a specific inhibitor of polyamine synthesis, would inhibit urogastrone stimulation of neomucosal growth. Twenty-four New Zealand white rabbits (2.1-2.9 kg) had 2 x 5-cm ileal defects patched with adjacent cecal serosal surface. Group I (n = 6) served as controls. Group II (n = 6) received UG 1.5 micrograms/kg/hr subcutaneously. Group III (n = 6) took 2% DFMO orally. Group IV (n = 6) received the same doses of UG and DFMO simultaneously. Animals were sacrificed 7 days after patching to assess neomucosal growth. Urogastrone infusion resulted in significantly greater neomucosal coverage (54 +/- 4%) and neomucosal surface area (236 +/- 18 mm2) in the Group II animals. Neomucosal coverage and contraction of the intestinal defects and neomucosal surface area were similar in the other three groups. Crypt cell production rate (15.5 +/- 2.0 cells/hr) and disaccharidase activity were also significantly greater in Group II than in the other groups. DFMO alone (Group III) resulted in a significantly lower ornithine decarboxylase (ODC) activity, polyamine content, and crypt cell production rate. Group IV animals had lower ODC activity but not lower polyamine content or crypt cell production rate. UG resulted in a significant increase in neomucosal growth. DFMO prevented this stimulatory effect and inhibited ornithine decarboxylase activity. The stimulation of neomucosal growth by UG is dependent, at least in part, on increased polyamine biosynthesis.     

优化和验证人精浆miRNAs提纯方法

目的优化精浆miRNAs提纯方法,为后续实验奠定基础。方法收集国家卫生计生委科研所生殖健康服务中心正常精液样本5例,非梗阻性无精子症(NOA)患者(经北京大学第三医院检查确诊)精液样本22例。根据不同RNA提取方法分为4组:Trizol LS组、蛋白酶K+Trizol LS组、miRNeasy Micro kit组及Trizol LS+miRNeasy Micro kit联合使用组(改良组)。比较4组提纯NOA患者精浆RNA的OD260/280,使用Agilent2100生物分析仪检测改良法提纯精浆miRNAs的纯度和质量,采用实时定量PCR方法比较4组精浆miR-106b的溶解曲线,并比较精液参数正常组(n=5)和NOA患者组(n=10)精液样本中的miR-106b的表达量。结果改良组提纯精浆RNA的OD260/280为(1.90±0.03)显著高于其他3组(P0.05);改良组提纯精浆miRNAs的完整数合格,色谱图位置特异性明显,峰值清晰;4组miR-106b的溶解曲线中,改良组曲线符合标准;NOA患者精浆中miR-106b的表达丰度显著低于参数正常组(P0.01)。结论Trizol LS与miRNeasy Micro kit联合使用提纯的精浆miRNAs质量好,能满足后续实时定量PCR、深度测序等实验要求。小样本分析发现NOA患者精浆miR-106b含量显著低于精液参数正常组,有待大样本验证其是否能作为临床诊断NOA的分子指标。     

Assessment of the efficiency of endovesical laser treatment of urinary bladder tumors on the basis of polyamine content measured in the eluent.

Authors describe a new analytical method for monitoring the treatment of vesicular tumors. Chromatographic analysis of the polyamine content of the eluent allows differential diagnostic judgement of the malignity of the laser treated tumorous tissue. The practical applicability of the method is demonstrated and evaluated on the basis of the analysis of 88 clinical cases.     

Urine levels of polyamine in patients with or without cancer

The urine levels of polyamine (total amount of putrescine, spermidine and cadaverine) were measured in patients with or without cancer by simple enzymatic assay method. In 148 control healthy adults, the urine levels of polyamine were 23.1 +/- 7.1 mumole/g. creatinine, whereas among 52 patients with benign diseases, the level in only 5 patients was slightly higher than normal level. The polyamine levels in 170 patients with cancer was 46.1 +/- 50.6 mumole/g. creatinine, which was about 2-times higher than normal level. In the patients with cancer of the stomach or the colon and rectum, the increase in polyamine level appeared to be correlated with the clinical stage of tumor. Following successful surgical resection of cancer, the polyamine level increased in one week transitorily after operation but gradually decreased to normal level within 5 weeks. Whereas following unsuccessful surgical resection of cancer, the polyamine value maintained high levels. The evidence suggests that the measurement of the urine level of polyamine is useful for the diagnosis of cancer or the clinical stage of tumor, and it will be helpful in the evaluation of therapeutic effects and prognosis.     

Polyamines in breast cancer

Polyamine levels (putrescine, spermidine and spermine) in breast cancers (n = 54) were measured as a potential guide to prognosis. Values (expressed as nmol per 100 mg tumour) ranged from: 0.9 to 4.5 for putrescine, 4.2 to 29.8 for spermidine and 5.6 to 39.7 for spermine concentration. Increased intracellular polyamine levels were positively correlated with factors known adversely to affect survival after mastectomy, namely histological grade III and oestrogen-receptor negative status. Advanced T4 tumours and medullary-type carcinomas also contained high polyamine levels. Tumour size and node status did not affect polyamine levels in primary tumours. Tumours that recurred within 2 years of mastectomy had significantly higher levels of spermidine and spermine than those that did not. Breast cancer polyamine levels are a biological marker of tumour aggressiveness and can be used as a prognostic indicator of early tumour recurrence that is independent of node status.     

A modification of the estimation of urinary oxalate using a Sigma kit

The Sigma kit for estimating urinary oxalate is an enzymatic procedure. However, some errors were encountered using the standard assay system of the kit. Firstly, an overestimate of oxalate may arise from the oxidation of ascorbate during the alkaline wash stage of the extraction of oxalate from urine. Secondly, an underestimate of oxalate may occur because of incomplete extraction of oxalate. A modified assay system for measurement of urinary oxalate using the kit is reported. The following points were modified: urine was diluted two-fold with 0.2 M EDTA and and 0.2 M citrate buffer (pH 3.0), oxalate from urine was extracted with 0.06 N sodium hydroxide to prevent the overestimation by the oxidation of ascorbate, and a plate mixer and addition of a small magnet to the vial were used in the steps of both absorption and extraction of oxalate to keep the accuracy of the estimation. The linearity of standard curve, reproducibility and recovery rates of the modified method were studied, and good results were obtained (linearity; r = 0.999, CV of reproducibility; 5.3%, recovery rate; 101% (300 microM) and 103% (600 microM). A good correlation was seen between the modified Sigma method and high performance liquid chromatography (r = 0.991).     

两种精浆酸性磷酸酶检测方法的比较与评价

目的:对检测精浆酸性磷酸酶(ACP)活性的常规方法(磷酸苯二钠法)和试剂盒方法进行比较,并探讨试剂盒方法取代常规方法用于常规检测的可能性。方法:79份不育男性的精浆,分别用常规方法和试剂盒方法检测其ACP活性。取1份精浆标本用于两种方法的批内检测,另取2份标本用于两种方法的批间检测。随机留取10份精浆标本,稀释后立即或放置30 m in后检测ACP活性。另随机留取10份精浆标本,同时由2位技术人员分别用常规方法检测ACP活性。结果:试剂盒方法所测ACP活性与常规方法显著相关(r=0.745,P=0.000)。试剂盒方法与常规方法相比,两者批内CV(13.72%;10.66%)比较接近,而批间CV(13.8%和15.49%;24.43%和21.04%)明显较低。精浆稀释后放置30 m in,无论是常规方法还是试剂盒方法,所测结果都显著降低(P<0.05)。而2位技术人员用常规方法所测精浆ACP活性无显著区别(P=0.165)。结论:试剂盒方法检测精浆ACP活性优于常规方法,可以取代常规方法。     

A new screening method for cystinuria. A simple and safe screening kit.

The new kit (Urocystin) presented here utilizes the dark brown coloration which a neutral aqueous solution of cystine develops rapidly upon addition of nickel ion and sodium hydrosulfite (Na2S2O4). What is needed is pouring 4 ml of the urine sample into the kit, and the kit is able to judge without fail any urine sample with a cystine concentration of 50 microgram/ml or more as positive. No pretreatment of the sample is necessary. The kit contains no hazardous reagents at all. The kit has thus turned screening of cystinuria into an extremely simple affair.     

Repair of long-term venous catheters

The insertion of long-term Silastic catheters into the vena cava for home parenteral nutrition, antibiotic administration, and chemotherapy is increasing; however, one of the often described complications of these catheters is breakage of the external segment of the catheter. A new device as well as a new method for repair of the catheter has been described. Clinical testing of the catheters repaired with this device by our nutritional support service has demonstrated no leakage or occlusion at the catheter repair site. We believe the catheter repair kit described is simpler to use than the repair kit currently on the market.     

Effect of eflorithine on intestinal regeneration

Patching intestinal defects with adjacent serosal surfaces results in the growth of new intestinal mucosa. Since polyamine biosynthesis is associated with cellular growth and differentiation, it may be important in this regenerative process. Our aim was to determine the effect of eflorithine (difluoromethylornithine), a specific inhibitor of polyamine synthesis, on intestinal regeneration. Forty-eight New Zealand white rabbits had 2 x 5-cm ileal defects patched with adjacent cecal serosal surface. One half of the animals took 2% eflorithine in drinking water postoperatively. Six animals in each group were killed 7, 14, 21, and 28 days after patching. There was no significant difference in neomucosal growth at any time. Villous height, disaccharidase activity, and crypt cell production were significantly lower in the eflorithine-treated animals. Eflorithine-treated animals had significantly lower ornithine decarboxylase activity and polyamine levels. Despite the inhibitory effect of eflorithine on polyamine synthesis and proliferative activity, epithelialization and contraction of the patched defect were not affected. These findings suggest that polyamine synthesis is important in proliferation and differentiation of cells in the neomucosa but does not influence cell migration in intestinal regeneration.     

High-performance liquid chromatography versus immunoassay for the measurement of sirolimus: comparison of two methods

Sirolimus is an immunosuppressive agent administered as prophylaxis of acute rejection to patients after kidney transplantation. Therapeutic drug monitoring (TDM) of whole blood is an important part of immunosuppressive therapy. At present, two methods of measuring drug concentrations are available: the reference method is high-performance liquid chromatography (HPLC) with ultraviolet (UV) or mass spectrometry (MS) detection and a second technique is the IMx sirolimus assay, which is an enzyme immunoassay using microparticles coated with anti-sirolimus antibodies (MEIA). The objective of this study was to compare the two methods. We examined a group of 42 patients receiving sirolimus after kidney transplantation. Blood was taken during routine ambulatory visits. The drug concentration in blood was performed at the same time by the two methods. To compare the methods, a statistical analysis was performed, yielding: r-value = 0.939 (r(2)); slope = 1.04; intercept = +0.38. The mean concentration of sirolimus was higher in the immunoassay than in the HPLC method namely, 9.7 +/- 6.4 ng/mL versus 8.9 +/- 5.8 ng/mL respectively. The HPLC method showed high sensitivity and specificity, but it was time consuming and labor intensive. The MEIA method is burdened with a high risk of methodologic error, due to its lack of specificity caused by cross-reactions with drug metabolites. We concluded that HPLC with its high sensitivity and analytical specificity is still the reference method; however, MEIA may be a fast method for use in clinical practice.