Arylsulfatase A activity of urine in patients with various genitourinary tract disorders

Arylsulfatase A activity was measured in urine from patients with various genitourinary tract disorders such as bladder tumor and inflammation. No significant difference in enzyme activity was found between normal and affected urine on the basis of either the volume or protein content of urine, a finding which differed from previous results. However, it was demonstrated that urine from affected patients was more labile to heat treatment in comparison with the control. Upon pretreatment of urine arylsulfatase A at 62.5 degrees C for 10 min, an average of 57% of the original activity was lost in samples from patients with bladder tumor, 58% in those with testicular tumor and 62% in cystitis and urethritis, respectively, while the enzyme activity in the control urine lost only 27% with similar heat treatment. These results were statistically significant with p < 0.001. The arylsulfatase A from patients with advanced bladder tumors demonstrated the presence of a variant form (pI 5.3) which was not detected in normal urine. This variant of arylsulfatase A was also demonstrated in nephrostomy urine from patients with congenital obstruction of the pelvi-ureteric junction.     

Radioimmunoassay for arylsulfatase A in urine

Purified arylsulfatase A (EC 3.1.6.1) from human urine was radioiodinated under conditions that caused no significant loss of antigenic activity. We used this labeled arylsulfatase A (specific radioactivity 4-7.5 Ci/g) together with nonlabeled enzyme and rabbit antiserum produced against homogeneous enzyme to develop a radioimmunoassay for arylsulfatase A in urine. A solid-phase, second-antibody technique (Immunobead Second Antibody; Bio-Rad Laboratories) was used to separate free enzyme from antigen-antibody complexes. The working range of the assay was 0.1-4.0 ng of enzyme; within- and between-assay CVs were around 10%, and the analytical recovery was 105.5% (SD 7.7%). The lower limit of detection was 0.08 ng of arysulfatase A per assay, substantially less than that of typical activity-based assays. Over a wide range of urinary arylsulfatase A activities, results by this method agreed well (r = 0.99) with those obtained by activity assays. We measured the enzyme in urines of 59 healthy volunteers and 92 patients with different diseases, including a group of colorectal cancer cases, to determine whether this could serve as a reliable marker for cancer of the colon; however, urinary excretion of arylsulfatase A by most patients with colon cancer was within normal limits.     

Clinical significance of a variant form of urinary arylsulfatase A

A variant form of urinary arylsulfatase A which was not detected in normal voided urine was demonstrated in the urine obtained directly from the renal-pelvis [Ishibashi, T. et al.: Biochim. Biophys. Acta, 616, 218-227 (1980)]. The variant form was not observed in urine collected directly from the ureter during operations for uretero-cutaneostomy or ileal conduit. However, the urine from intubated uretero-cutaneostomy patients collected near the pelvi-ureteric junction showed the presence of the variant form, further suggesting the origin specificity of the variant. This arylsulfatase was not demonstrated in the voided urine from patients with non-urologic malignant disorders such as uterine endometrial, uterine cervical, rectal, pancreas head and gastric carcinomas, in spite of its appearance in high levels in the urine from patients with advanced bladder cancer.     

Identification by proteomic analysis of calreticulin as a marker for bladder cancer and evaluation of the diagnostic accuracy of its detection in urine

BACKGROUND: New methods for detection of bladder cancer are needed because cystoscopy is both invasive and expensive and urine cytology has low sensitivity. We screened proteins as tumor markers for bladder cancer by proteomic analysis of cancerous and healthy tissues and investigated the diagnostic accuracy of one such marker in urine. METHODS: Three specimens of bladder cancer and healthy urothelium, respectively, were used for proteome differential display using narrow-pH-range two-dimensional electrophoresis. To evaluate the presence of calreticulin (CRT) as detected by Western blotting, we obtained 22 cancerous and 10 noncancerous surgical specimens from transurethral resection or radical cystectomy. To evaluate urinary CRT, we collected 70 and 181 urine samples from patients with and without bladder cancer, respectively. Anti-CRT COOH-terminus antibody was used to detect CRT in tissue and urine. RESULTS: Proteomic analysis revealed increased CRT (55 kDa; pI 4.3) in cancer tissue. Quantitative Western blot analysis showed that CRT was increased in cancer tissue (P = 0.0003). Urinary CRT had a sensitivity of 73% (95% confidence interval, 62-83%) at a specificity of 86% (80-91%) for bladder cancer in the samples tested. CONCLUSIONS: Proteomic analysis is useful in searching for candidate proteins as biomarkers and led to the identification of urinary CRT. The diagnostic accuracy of urinary CRT for bladder cancer appears comparable to that of Food and Drug Administration-cleared urinary markers, but further studies are needed to determine its diagnostic role.     

Arylsulfatase A in urine of patients with urothelial tumors

A significant increase in urine arylsulfatase A activity (p less than 0.01) was found in patients with urothelial tumors. Arylsulfatase A activity was 1.36 +/- 1.10 U/24-h urine in control specimens, 1.90 +/- 1.66 U/24-h urine in various genitourinary tract disorders, and 3.90 +/- 1.98 U/24-h urine in transitional cell carcinoma specimens. Surgical treatment of the neoplastic patients lowered the arylsulfatase A activity found in urine to within reference values. The arylsulfatase A excreted by patients with these tumors was highly sensitive to thermal inactivation while the enzyme activity in the control urines was less affected by the heat treatment. The time course of the arylsulfatase A reaction with 4-nitrocatechol sulfate was not linear in normal individuals, while it was linear in 90% of patients with urothelial tumors. This difference in the kinetic pattern of the enzyme could be used to increase the diagnostic specificity of the determination.     

Determination of diagnostic and prognostic values of urinary interleukin-8, tumor necrosis factor-alpha, and leukocyte arylsulfatase-A activity in patients with bladder cancer

OBJECTIVES: This study was planned to evaluate the feasibility of the assay of leukocyte arylsulfatase-A (AS-A) activity, and some urinary cytokine levels (tumor necrosis factor-alpha [TNF-alpha] and interleukin-8 [IL-8]), as noninvasive diagnostic tools in different stages of bladder cancer patients. DESIGN AND METHODS: Blood and urine samples of 79 subjects were analyzed, including 28 healthy volunteers, 27 patients with superficial bladder cancer (SBC), and 24 patients with muscle invasive bladder cancer (MIBC). RESULTS: In SBC patients, the mean leukocyte AS-A activity was slightly higher (11.4%) than healthy subjects without reaching statistical significance. On the other hand, the enzyme activity in MIBC patients was significantly higher than those of controls (38.9%) and SBC patients (18.3%). Urinary TNF-alpha levels in both cancer groups were not significantly different from the control group. Urinary IL-8 levels of MIBC patients were significantly increased when compared with the levels of SBC patients and healthy subjects (P < 0.001). CONCLUSIONS: Based on our results, it may be concluded that measurement of leukocyte AS-A activity is not a sufficiently reliable noninvasive diagnostic test in distinguishing early stage bladder cancer from healthy subjects as well as detecting disease progression. Whereas measurement of urinary IL-8 may be valuable as a noninvasive diagnostic and prognostic test especially in patients with advanced bladder cancer. It also appears that complementary biochemical information may be obtained about the prognosis of the disease by monitoring urinary IL-8 profile. However, further confirmatory clinical trials about the prognostic value of the measurement of urinary IL-8 are needed.     

Performance characteristics of multiple urinary tumor markers and sample collection techniques in the detection of transitional cell carcinoma of the bladder

BACKGROUND: The optimal management of bladder cancer requires early detection of both primary tumor as well as recurrences. This study compared the methodologies of the following tumor markers and diagnostic tools: telomerase, bladder tumor-associated analytes (BTA stat), nuclear matrix protein 22 (NMP22), the hemoglobin (Hb) dipstick and chemiluminometric red cell assays, and determined their respective sensitivity and specificity in detection of bladder cancer along with urine cytology. METHODS: Different sample collection techniques were used including bladder tissue from radical cystectomy or transurethral resection, voided and cystoscopically collected urine and bladder washes. Results of these techniques were compared for some of these markers. RESULTS: The first study utilizing cystoscopically collected urine and bladder washes, the sensitivity of telomerase in detecting bladder cancer was 92% and 87%, respectively, whereas for bladder tissue it was 93%. The sensitivities of cytology, NMP22 and original BTA, however, were 62%, 54% and 49%, respectively. In the second study using first single voided urine from patients with bladder cancer and non-bladder cancer controls, the greatest sensitivity (overall 77%) and specificity (overall 98%) was exhibited with telomerase. The combined sensitivity of telomerase with that of either the hemoglobin dipstick or chemiluminometric red cell assays, showed that the overall sensitivity was increased to 89% and specificity to 98%. CONCLUSIONS: The sensitivity and specificity of telomerase activity in conjunction with chemiluminometric red cell or hemoglobin dipstick assay may be considered as an alternative to urinary cytology. It can also supplement cystoscopy for monitoring recurrence of bladder cancer.     

膀胱癌新标志物UPK3A的临床应用研究

目的前瞻性评价人Uroplakin 3A（UPK3A）诊断膀胱癌的作用,探索更好的非侵入性的诊断膀胱癌的方法。方法收集34例健康志愿者、46例良性泌尿外科疾病患者和112例膀胱癌患者的尿液。酶联免疫吸附试验定量检测尿液中UPK3A水平,核基质蛋白22（NMP22）检测试剂盒测定NMP22,同时进行尿脱落细胞学检测。结果膀胱癌患者尿液UPK3A水平明显高于健康志愿者和良性泌尿外科疾病患者,差异有统计学意义（P〈0.01）。UPK3A的受试者工作特征（ROC）曲线显示了一个0.911的良好曲线下面积。以OD0.0685作为临界值可以获得一个较适宜的敏感度（83%）和特异度（84%）的组合。UPK3A、NMP22和尿脱落细胞学诊断膀胱癌的敏感度分别为83%、58%和64%;特异度分别为84%、75%和82%。结论膀胱癌患者尿液UPK3A水平较高,UPK3A可作为诊断膀胱癌的一个敏感的标志物,但还需要大规模的多中心研究进行验证。     

Allelic deletion fingerprinting of urine cell sediments in bladder cancer.

BACKGROUND: Bladder cancer shows frequent nonrandom allelic deletion at various chromosomal regions. Genotypic detection methods could potentially identify patients at risk for recurrent progressive disease. In this study, we examined allelic deletion at specific chromosomal loci in tumor tissue and urine cell sediment samples using a microsatellite-based protocol. Although both allelic deletion and microsatellite instability have been reported in primary bladder cancer, microsatellite instability was not specifically examined in this study. We report a pilot study of 40 patients with bladder cancer in which allelic deletion in tumor tissue and urine cell sediment was compared with conventional urine cytology results. METHODS AND RESULTS: Forty tumors were analyzed using a set of microsatellite primers from chromosomes 3, 4, 8, 11, 14, and 17 to construct allelic deletion fingerprints. Cy5.5-labeled PCR products were analyzed using the OpenGene System and GeneObjects software. Eighty-eight percent of tumors showed allelic deletion. In urine cell sediments, the tumor detection rate was 80% compared with 50% for routine urine cytology. The allelic deletion fingerprinting (ADF) procedure identified 69% of incipient tumors, cases initially classified as normal by routine urine cytology. CONCLUSION: ADF analysis provides a reliable noninvasive method for the detection and monitoring of recurrent cancer in urine cell sediment samples from patients with bladder cancer.     

尿液膀胱肿瘤抗原水平检测在膀胱癌诊断中的意义

目的 　评估尿液膀胱肿瘤抗原（BTA）水平在膀胱癌诊断中的价值。方法　收集98 例膀胱癌患者、57 例非膀 胱癌患者以及68 例健康体检者晨尿标本,利用化学发光分析仪进行各样本中BTA 水平的检测。通过ROC 曲线下面积 计算,分析BTA 对膀胱癌的诊断灵敏度、特异度及最佳临界值。结果　膀胱癌患者尿液中BTA 水平[486.56（66.27, 2 013.81）] U/ml 明显高于健康对照组[25.71（10.40,53.39）] U/ml 及非膀胱癌组[55.47（26.55,296.24）] U/ml,差异 具有统计学意义（χ2=64.172,P <0.01）;BTA 诊断膀胱癌的ROC 曲线下面积为0.816,与参考线下面积相比,差异具 有统计学意义（P <0.01）,BTA 诊断膀胱癌的最佳临界值为84.38U/ml,诊断敏感度为70.8%,特异度为93.5%。结论　 尿液BTA 的检测在膀胱癌的诊断筛查中具有较高的临床价值,可用于膀胱癌的早期筛查。     

Treatment of bladder cancer cells in vitro and in vivo with 2-5A antisense telomerase RNA

Bladder cancer is the most common malignant tumor of the urinary tract. Novel treatment approaches are essential because of the failure of current treatment options to cure a high percentage of patients. Telomerase, a ribonucleoprotein, is detected in almost all bladder cancer, but not in normal bladder tissues. Therefore, telomerase is expected to be a very promising candidate for targeted therapy of bladder cancer. In this study, we synthesized a 19-mer antisense oligonucleotide against the RNA component of human telomerase (hTR) linked to a 2-5A molecule (2-5A-anti-hTR) and investigated its antitumor effect against bladder cancer cells. The 2-5A antisense strategy relies on the recruitment and activation of RNase L at the site of targeted RNA sequence. Here we demonstrate that treatment with 2-5A-anti-hTR reduced the viability of seven bladder cancer cell lines (UM-UC-2, UM-UC-3, UM-UC-6, UM-UC-9, UM-UC-14, RT4 and T24) expressing telomerase activity to 21-55% within 4 days. The cytotoxicity was mainly due to induction of caspase-dependent apoptosis. In contrast, normal fibroblast WI38 cells lacking telomerase activity were resistant to the treatment. Furthermore, treatment of subcutaneous UM-UC-2 tumors in nude mice with 2-5A-anti-hTR significantly suppressed the tumor growth through induction of apoptosis (P < 0.001). These findings may offer a strong support to the feasibility of the 2-5A-anti-hTR treatment for human bladder cancer.     

尿膀胱癌抗原、透明质酸和存活素联合检测对膀胱癌的诊断运用探讨

目的探讨在对膀胱癌的诊断过程中,尿膀胱癌抗原、透明质酸和存活素的应用价值。方法膀胱癌患者74例,泌尿系统良性疾病患者20例,健康志愿者标本10例分别行膀胱癌抗原(UBC)、透明质酸(HA)、存活素(Sur-vivin)和尿脱落细胞学(UC)检查。结果 UBC、HA、Survivin和尿脱落细胞学的检测有利于膀胱癌的诊断,且UBC、HA、Survivin三者的有效性优于尿脱落细胞学的检测。UBC、HA、Survivin在检测膀胱癌的过程中,三者的敏感性和准确性高于尿脱落细胞学。UBC、HA、Survivin在肿瘤的各分级(G1、G2、G3)和各分期(pTa、pT1、≥pT2)中的敏感性明显高于尿脱落细胞学。结论尿膀胱癌抗原、透明质酸和存活素在诊断膀胱癌过程中应用价值优于尿脱落细胞学。     

A modified radioimmunoassay for arylsulfatase A in human serum and urine

A radioimmunoassay was developed for the determination of arylsulfatase A (EC 3.1.6.1) in human serum and urine. An isoenzyme of arylsulfatase A purified from human urine was used as a standard antigen. The enzyme was radioiodinated with 125I using the Chloramine T method and was stable for about 4 wk. Antibody-bound enzyme was separated from free enzyme by means of a double antibody technique in the presence of polyethylene glycol (PEG). The working range of the method was 0.15-5.0 ng of arylsulfatase A per assay. The within-assay CV was about 8% for both biological fluids and the between-assay CV for serum was 14.1%. Analytical recoveries were 93.2 +/- 9.1% and 97.8 +/- 5.5% for serum and urine, respectively, and the sensitivity was 0.040 ng of arylsulfatase per assay. Serum samples of 50 healthy blood donors were assayed to establish the normal serum level of immunoreactive enzyme, which was found to be 8.3 ng/ml +/- 1.8 ng/ml of serum. Storage of frozen serum was shown to have no significant effect on results obtained using this RIA.     

Arginase isoforms in human colorectal cancer

Arginase (EC 3.5.3.1) activity was determined in 54 colorectal tissues obtained from patients with primary colorectal adenocarcinoma as well as in serum of 45 patients and 65 healthy individuals. In patients, the preoperative values of the mean serum arginase activity and the activity in colorectal tumors were much higher than in serum of healthy subjects and control tissues. Two isoforms of arginase, anionic and cationic, were identified in colorectal tissues (normal and cancerogenous), and only one, the cationic form, in serum. These arginases were different from the main human liver cationic arginase (pI 9.3). The anionic colorectal arginase was identical with the human liver anionic isoform (pI 7.7), and the cationic arginase from colorectal tissues and blood serum with the human kidney cationic enzyme (pI 8.9). The total activity and the level of protein of the cationic arginase in colorectal cancer was higher than in control tissue, and it was also higher in serum of patients with colorectal cancer than in healthy subjects. Thus, it can be concluded that the increased arginase activity in blood serum and colorectal cancer in studied patients was due to the raised level of the cationic arginase and this isoform seems to be a discriminating parameter for assessing the presence of colorectal cancer.     

Comparison of cytokeratin 20 RNA and angiogenin in voided urine samples as diagnostic tools for bladder carcinoma

BACKGROUND: We evaluated the diagnostic efficacy of urinary angiogenin (ANG) and cytokeratin 20 (CK-20) mRNA in comparison with voided urine cytology in the detection of bladder cancer patients. OBJECTIVES AND METHODS: A total of 97 Egyptian patients provided a single voided urine sample for ANG, CK-20 and cytology before cystoscopy. Of the 97 cases, 63 were histologically diagnosed as bladder cancer; 33 with transitional cell carcinoma (TCC) and 30 with squamous cell carcinoma (SCC), whereas the remaining 34 had benign urological disorders. A group of 46 healthy volunteers were also included in this study. Voided urine was centrifuged and the supernatant was used for estimation of ANG by EIA and confirmed by Western blotting (WB). The urine sediment was used for cytology and RNA extraction. CK-20 RNA was detected by RT-PCR. RESULTS: The best cutoff value for ANG was calculated by a ROC curve as 322.7 ng/mg protein. The median urinary ANG level in bladder carcinoma, benign urological disorders and healthy volunteer groups was: 802.7, 425 and 33 pg/mg protein, respectively. The positivity rate for urinary CK-20 mRNA of the control, benign and malignant groups was 0%, 2.9% and 82.3%, respectively (P = 0.000); while the rates for ANG were 11.6%, 54.8% and 75.4%, respectively (P = 0.000). There was no significant difference in positivity rates of CK-20 and ANG with respect to sex, smoking, schistosomiasis, urine cytology, tumor grade, tumor stage, hematuria or pus cells. The overall sensitivity and specificity were 71.4% and 90% for voided urine cytology, 75.4% and 70.3% for ANG, and 82.3% and 98.8% for CK-20. Combined sensitivity of voided urine cytology with ANG and CK-20 together (98.2%) was higher than either the combined sensitivity of voided urine cytology with ANG (96.5%) or with CK-20 (91.6%) or than that of the biomarker alone. We demonstrated significant positive correlation between CK-20 positivity with age (P = 0.043) and nodal involvement (P = 0.037); however, there was no significant correlation between CK-20 and ANG with the other clinicopathological parameters. CONCLUSIONS: Our data indicate that CK-20 and ANG in voided urine had higher sensitivities compared to voided urine cytology. However, when specificity was considered, CK-20 alone had superior sensitivity and specificity compared to ANG and voided urine cytology.     

磁共振扩散加权成像在膀胱病变鉴别诊断中的应用价值

目的：探讨磁共振扩散加权成像（DWI）和表观扩散系数（ADC）在膀胱病变鉴别诊断中的应用价值。方法：以53例膀胱癌及12例良性膀胱病变作为病例组,11例正常膀胱壁作为对照组。分析病例组及对照组的DWI并测量病变及正常膀胱壁的ADC值。结果：所有53例膀胱癌表现为扩散受限。12例良性病变中,2例轻度扩散受限,余10例无扩散受限。DWI上检出膀胱癌的敏感度为100%,特异度为83.3%,准确度为96.9%。膀胱癌的平均ADC值为（0.76±0.17）×10-3 mm2/s,显著低于良性病变的（1.21±0.20）×10-3 mm2/s,并显著低于对照组的（1.55±0.15）×10-3 mm2/s（F=89.04,P<0.001）。结论：DWI和ADC在膀胱良恶性病变的鉴别诊断中具有较好的应用价值。     

Lipid peroxidation and antioxidant enzyme activities in cancerous bladder tissue and their relation with bacterial infection: a controlled clinical study

It is well known that antioxidants and reactive oxygen species play an important role in carcinogenesis. In this sudy, we attempted to evaluate antioxidant enzyme activities and lipid peroxidation levels in cancerous bladder tissue and to determine their relationship with bacterial infection. Bacterial culture was made from all urine samples using Blood and Eosin Methylene Blue agars for checking the presence of bacterial infections. We measured thiobarbituric acid reactive substances (TBARs) and activities of xanthine oxidase (XO), superoxide dismutase (SOD), glutathione peroxidase (GSH‐PX), and catalase (CAT) in cancerous tissues of 25 bladder cancer patients, in noncancerous adjacent bladder tissues of 13 out of these 25 patients, and in control bladder tissues of 15 patients with a non‐neoplastic genitourinary disease. TBARs levels increased and XO, SOD, GSH‐PX, and CAT activities decreased significantly in cancerous bladder tissues. TBARS, XO, and SOD levels were not significantly different between noncancerous adjacent tissue and control bladder tissue. Statistically significantly lower GSH‐PX and higher CAT activities were observed in noncancerous adjacent bladder tissue compared with cancerous tissue. GSH‐PX level of tumor tissue was correlated significantly with tumor grade (r=−0.425, P=0.034). Results suggested that pathway activity of free radicals were accelerated in the cancerous human bladder tissues via increased TBARs levels and decreased enzyme activities of XO, SOD, GSH‐PX, and CAT, which implicated a severe exposure of cancerous tissues to oxidative stress. J. Clin. Lab. Anal. 24:25–30, 2010. © 2010 Wiley‐Liss, Inc.     

Analytical and clinical evaluation of a new urinary tumor marker: bladder tumor fibronectin in diagnosis and follow-up of bladder cancer.

Bladder cancer is the fourth most common malignant neoplasm in men and the tenth most common in women. Cystoscopy presents the gold standard for detection and monitoring of bladder cancer. However, it is an invasive and expensive procedure. Therefore, development of biomarkers for the purposes of screening, diagnosis and prediction of the prognosis in bladder cancer is required. Bladder tumor fibronectin is one of the new urinary tumor markers. The aim of this study is to evaluate the diagnostic performance of urinary bladder tumor fibronectin in detecting and monitoring bladder cancer. A total of 75 patients with the diagnosis of bladder cancer, 20 patients with the diagnosis of benign prostatic hyperplasia, 7 patients with the diagnosis of prostate cancer between the years 1996-2000, and 28 age-matched healthy individuals, were enrolled in the study. The patients were diagnosed by cystoscopy, with histopathological evaluation of the tumor, as having superficial or invasive bladder cancer. Patients were followed-up clinically with data pertinent to disease recurrence and progression. Bladder tumor fibronectin (BTF; ng/ml) was determined by solid phase, two-site chemiluminescent immunometric commercial diagnostic assay developed for the Immulite automated immunoassay system (Diagnostic Products Corporation, Los Angeles, CA, USA). All measured values were normalized by urinary creatinine, and the obtained data were evaluated by receiver-operating characteristics (ROC) curve analysis. Optimal cut-off was established at 43.4 ng/mg. This cut-off rendered overall sensitivities of 72% and specificity of 82.1%. The analytical evaluation of the BTF test displayed promising results in terms of a non-invasive in vitro test in the diagnosis of bladder cancer. Although it was not satisfactory in prediction of recurrence or progression of the disease, it correlated well with the stage, one of the most reliable prognostic factors. In conclusion, the urinary bladder tumor fibronectin test warrants further clinical evaluation.     

Urinary arylsulfatase in normal children and in patients with pediatric malignant disease

Urinary arylsulfatase (AS) activities were measured in 20 normal children and 10 patients with malignant disease, consisting of leukemia (6), Hodgkin's disease (1), neuroblastoma (2), and malignant teratoma (1), Seven of the patients showed a significantly high activity, and a serial measurement carried out in 4 patients showed a well correlated relationship between AS activity and the activity of disease. Thus the measurement of urinary AS activity could be a laboratory test for monitoring the activity of malignant diseases because of its simple and rapid procedure.