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.     

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.     

Therapy of prostate carcinoma with polyamine synthesis inhibitors. I. Physiological and pathophysiological principles

The therapeutic concept of irreversible inhibition of both ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) by alpha-difluoromethylornithine (DFMO) and methylglyoxal bis-guanylhydrazone (MGBG) is based on pathologic activities of these enzymes in tumor tissue. The polyamines putrescine, spermidine and spermine are measured in highest concentration in the prostate of both men and animals, with a significant increase of spermine in benign hyperplasia of the prostate. Patients with metastatic cancer of the prostate have elevated putrescine levels in the 24-hour urine. Treatment with 3 or 1% DFMO or 11 mg/kg MGBG in transplantable human and experimental cancer of the prostate demonstrated a significant anti-growth effect. A combination of DFMO and MGBG is tumor-destructive. The combination of 1% DFMO and 11 mg/kg MGBG distinctly reduces the activity of ODC and SAMDC and significantly lowers the levels of putrescine, spermidine and spermine in the tumor.     

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.     

The clinical significance of tissue, blood and urine polyamine in renal cell carcinoma]

With the use of a newly developed and convenient enzymatic method, tissue, urine and blood polyamine (diamine, spermidine and spermine) levels were evaluated as a tumor marker of renal cell carcinoma (RCC) in 50 cases with the disease. Furthermore, blood and urine polyamines were periodically determined and evaluated as a follow-up marker. The pretreatment three polyamine levels in tissue, blood and urine of the patients were all significantly higher than those of the controls. However, because of their low sensitivities, they were not always decisive for biochemical diagnosis of RCC. Tissue spermidine levels were increased with the advance of the stages. Tissue diamine level also showed a good correlation with the pathological grade. Tissue diamine was found to predict distant metastasis. Blood spermidine and urine diamine were useful as follow-up markers. In conclusion, combined determination of tissue, blood and urine polyamine levels was thought to be useful as tumor markers of RCC.     

Changes in plasma, erythrocytes and tissue polyamine levels in patients with colorectal cancer

Polyamines (putrescine, spermidine, spermine) are closely linked to cellular synthesis of DNA, RNA and protein, and are thought to be an indicator of cell proliferation. Plasma, erythrocytes and tissue polyamine levels in 58 patients with colorectal cancer were measured to survey the relationship between polyamines and stage classification. The polyamine levels of plasma, erythrocytes and tissue in patients were increased significantly compared with those of controls. Plasma spermine and erythrocytes spermidine and spermine levels were increased with the advance of stage. In plasma and erythrocytes, spermidine/spermine ratios were decreased in accordance with the stages. On the contrary, the polyamine levels and the ratio in cancer tissue were not varied in all stages. These results present the following conclusions. Although cancer tissue has higher proliferative activity than normal mucosa, these activities of main tumors in each stage are not fluctuated. The polyamine levels in plasma and erythrocytes are possibly influenced by tumor burden and therefore those could be an useful marker for indicating the stage of colorectal cancer.     

Effect of difluoromethylornithine on host and tumor polyamine metabolism during total parenteral nutrition

Clinical and experimental data suggest that erythrocyte (RBC) polyamine (PA) levels are markers of tumor proliferation during total parenteral nutrition (TPN). The purpose of this experiment was to determine whether the inhibition of PA synthesis during TPN was greater in tumors than in normal host tissue. Rats bearing a subcutaneous fibrosarcoma were randomized to receive a chow diet (n = 5), TPN (n = 5), or TPN + difluoromethylornithine (DFMO) (an irreversible inhibitor of ornithine decarboxylase (ODC), at 1000 mg/kg body wt/day n = 4) for 6 days by continuous central venous infusion. TPN + DFMO resulted in a higher plasma albumin level and lower tumor ODC activity compared with chow feeding or TPN. Liver ODC activity was similar for the chow fed, TPN, and TPN + DFMO groups. RBC putrescine, tumor putrescine, and tumor spermidine levels were significantly lower in the TPN + DFMO group compared with the chow fed and TPN groups. RBC spermidine, RBC spermine, and tumor spermine levels were significantly increased with TPN + DFMO compared with TPN alone. DFMO did not produce diarrhea or weight loss. Increased RBC spermidine may indicate a toxic effect of DFMO on the tumor, resulting in leakage of tumor spermidine into the extracellular space. The data suggest that DFMO during TPN can selectively inhibit tumor PA synthesis and may improve host utilization of nutrients.     

Detection of polyamines by a new enzymatic differential assay. (8) Studies on tissue polyamine concentrations in patients with genitourinary malignant diseases

Polyamine concentrations of human cancerous and non-cancerous tissues from the kidney, ureter, bladder were measured by a new enzymatic method for isolation and determination of polyamines. In cancerous and non-cancerous tissue of the organs studied, the spermine level was highest followed by the spermidine and diamine levels. The concentrations of diamine, spermidine and spermine in cancerous tissues were significantly higher than those in non-cancerous tissues, but there was no significant difference in the spermidine/spermine ratio between the cancerous and non-cancerous tissues. These data suggest that polyamines are produced above the normal levels in pathological conditions such as renal cell carcinoma, ureteral cancer and bladder cancer.     

Detection of urinary polyamine by a new enzymatic differential assay. (III). Studies on urinary polyamines in patients with malignant genitourinary diseases

A new enzymatic method for isolation and determination of urinary polyamines was presented and basically studied in previous report 1 and 2 in comparison with existing techniques. Using the new method, urinary polyamines were isolated and determined in 56 patients with genitourinary cancer. Urinary polyamines were also determined in 63 controls consisting of 20 normal subjects, 25 patients with benign urological disease and 18 patients with BPH. The mean concentrations of Diamine, Spermidine, Spermine in 20 normal subjects were 16.6 +/- 5.8 mumoles/g Cr, 4.7 +/- 2.0 mumoles/g Cr and 0.99 +/- 0.51 mumoles/g Cr respectively. To emphasize the specificity to cancer, the level of positiveness was modified to a higher value than M+3SD. The positive values thus calculated were 40 mumoles/g Cr for Diamine, 15 mumoles/g Cr for Spermidine and 3 mumoles/g Cr for Spermine. The positive ratios of Diamine in patients with early cancer were 43% in renal cell cancer, 20% in pelvic and ureter cancer, 0% in bladder cancer and 20% in prostatic cancer. Those of Spermidine were 29% in renal cell cancer, 0% in pelvic and ureter cancer, 20% in bladder cancer and 40% in prostatic cancer. Those of Spermine were 29% in renal cell cancer, 20% in pelvic and ureter cancer, 20% in bladder cancer and 0% in prostatic cancer. In early diagnoses, Diamine indicated high positive ratios to renal cell cancer and Spermidine to prostatic cancer. Relatively high positive ratios were demonstrated, when any one of the isolated polyamines was found positive: namely, 57% in renal cell cancer, 20% in pelvic and ureter cancer, 30% in bladder cancer and 40% in prostatic cancer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyamine metabolism in carcinoma of the oral cavity compared with adjacent and normal oral mucosa

In this study, polyamine biosynthesis required for cellular proliferation showed elevated levels in neoplastic cells. Putrescine, spermidine, and spermine, as well as the rate-limiting enzyme ornithine decarboxylase, were measured to evaluate differences in tissue concentration in squamous cell carcinoma of the oral cavity, and in the normal adjacent, buccal, and retromolar trigone tissues. Mean polyamine levels (nanomoles per gram of tissue +/- standard error of the mean) were significantly elevated in tumor tissue at 136 +/- 42 nmol/g for putrescine compared with 41 +/- 9 nmol/g in adjacent, 25 +/- 5 nmol/g in buccal, and 41 +/- 14 nmol/g in retromolar trigone tissues. Tumor spermidine was 415 +/- 41 nmol/g compared with 192 +/- 34 nmol/g in adjacent, 184 +/- 34 nmol/g in buccal, and 214 +/- 63 nmol/g in retromolar trigone tissues. Tumor spermine was 461 +/- 41 nmol/g compared with 236 +/- 30 nmol/g in adjacent, 233 +/- 35 nmol/g in buccal, and 269 +/- 59 nmol/g in retromolar trigone samples. Ornithine decarboxylase activity was highly variable in tumor tissues. High levels of polyamines appear to be specific for this malignancy, whereas ornithine decarboxylase activity is not. Measurement of polyamine content may be useful in evaluating epithelial changes in the oral cavity.     

Differential release of polyamines by cultured rat Sertoli cells

Cellular and media concentrations of polyamines in Sertoli cell cultures were determined by fluorescent spectroscopy of dansylated compounds after separation by high-performance liquid chromatography. In spite of low cellular levels of putrescine, the Sertoli cells released relatively large amounts of putrescine and spermidine even after several media changes. The inclusion in the culture media of cortisol, insulin, and thyroxine significantly elevated cellular polyamine levels, altered the spermidine to spermine ratio, and enhanced putrescine release by 3- to 4-fold. No spermine, however, was detected in the media under any of the conditions studied. The polyamine concentrations in cultured Sertoli cells from 13-day-old rats and the pattern of polyamine release by these cells differed significantly from those in the Sertoli cells from 46-day-old rats. These data demonstrate the differential release of polyamines by cultured rat Sertoli cells. The profiles of polyamine secretion appear to be age-dependent, and the significance of this phenomenon is discussed.     

Polyamines, polyamine antimetabolites and urogenital tumors. State of research and clinical results

The polyamine metabolism is pathologically changed in tumor tissues, and especially putrescine and spermidine demonstrate abnormally high values in kidney, bladder, and prostate cancer. The inductive processes which activate the biosynthetic polyamine enzymes in cancer are completely unknown. Of therapeutic interest is the fact that increased enzyme activities through irreversible inhibitors become significantly reduced, which consequently slows the tumor growth. Experimental therapy, especially in transplantable bladder and prostate cancer, displayed a 50% tumor destruction. In clinical studies using inhibitors of the polyamine biosynthesis, the dose had to be significantly reduced because of expressed toxicity. Additional investigations which tried a combination of reversible and irreversible inhibitors proved a similar antitumor activity, but less severe side effects.     

Polyamine levels and gastrin receptors in colon cancers.

Polyamines and gastrin receptors (GR) were studied in samples of colon cancer and mucosa from 40 patients and in control mucosa from 11 patients without cancer. Polyamines (i.e., putrescine, spermidine, spermine) are essential for growth and differentiation. The concentration of polyamines is elevated in rapidly proliferating normal tissues and in some cancers. The presence of GR in human colon cancers has been previously reported. The purpose of the present study was twofold: (1) to determine whether polyamine levels are elevated in colon cancers and in adjacent normal colon mucosa compared to colon mucosa from patients without cancer; and (2) to examine the relationship between polyamine levels and GR in colon cancers. Polyamine levels in colon cancers were significantly higher than in the normal colon mucosa from the same patients. The polyamines, spermidine and spermine, were significantly higher in colon mucosa from patients with cancer compared to patients without cancer. Spermidine and the spermidine:spermine ratio, an index of cell proliferation, were increased in colon cancers with GR compared to cancers without GR. There were no significant correlations between polyamine levels and the following: patient age, CEA level, site of cancer, stage, or differentiation. Because polyamine levels are increased in colon mucosa from patients with cancer, measurement of polyamines may detect patients at risk for subsequent development of colon cancer. Increased levels of polyamines in colon cancers with GR is evidence that gastrin may play a trophic role in human colon cancers.     

Ornithine decarboxylase activity and polyamine levels in human thyroid tumor tissue

Production of polyamines such as putrescine (PUT), spermidine (SPD) and spermine (SPM) primarily from ornithine by ornithine decarboxylase (ODC) is correlated with cell proliferation. Polyamine levels and ODC activities were measured to determine the degree of biological malignancy in 186 thyroid tumor tissues. Carcinoma showed significantly higher ODC activity and higher levels of PUT, SPD and SPM than benign tumors. PUT levels showed 2.28 nmol/mg protein in anaplastic carcinoma, 0.66 in papillary carcinoma, 0.11 in follicular adenoma, 0.06 in adenomatous goiter and 0.04 in normal thyroid tissue. Anaplastic and papillary carcinomas showed higher PUT/SPD and SPD/SPM ratios than benign tumors. Poorly differentiated carcinoma showed significantly higher PUT level and PUT/SPD and SPD/SPM ratios than well differentiated carcinoma. No correlation was found among polyamine levels, ages and sex in papillary carcinoma. In female patients with papillary carcinoma, no significant difference in polyamine levels was observed between patients above and below 50 years old. These results suggest that ODC activity and polyamine levels may provide useful information to determine the degree of biological malignancy of thyroid tumors.     

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).     

Effect of diethylstilbestrol on polyamine metabolism in hamster epididymis

Aim: To investigate the effect of diethylstilbestrol (DES), one of the most potent endocrine disruptors, on the metabolism of polyamines in hamster epididymis. Methods: Male golden hamsters of 7-week-old were kept under a light and dark cycle of 14 h and 10 h for 1 week to stimulate maximally the gonadal function. DES was injected subcutaneously at doses of 0.01 mg·kg-1·day-1, 0.1 mg·kg-1·day-1 and 1 mg·kg-1·day-1 for one week. Results: DES treatment caused a significant decrease in the weight of epididymis. The activity of epididymal ornithine decar boxylase (ODC) increased 1 day after DES treatment, kept at a high level for 4 days and then decreased to nearly normal level at day 7. The activity of spermidine/spermine N1-acetyltransferase (SSAT) also increased transiently after DES treatment. The contents of putrescine, spermidine, spermine and N1-acetylspermidine were increased 1 day -4 days after DES treatment and restored to normal at day 7. All these changes showed a marked difference between     

Overexpression of SSAT in kidney cells recapitulates various phenotypic aspects of kidney ischemia-reperfusion injury

To ascertain the role of spermidine/spermine N-1-acetyl-transferase (SSAT; the rate-limiting enzyme in polyamine catabolism) in cell injury, cultured kidney (HEK 293) cells conditionally overexpressing SSAT were generated. The SSAT expression was induced and its enzymatic activity increased 24 h after addition of tetracycline and remained elevated over the length of the experiments. Induction of SSAT upregulated the expression of polyamine oxidase and resulted in the reduction of cellular concentration of spermidine and spermine, increased concentration of putrescine, and inhibited cell growth. SSAT overexpression increased the expression of heme oxygenase-1 (HO-1) by 350% 24 h after addition of tetracycline, indicating the induction of oxidative stress. The presence of catalase significantly prevented the upregulation of HO-1 in SSAT overexpressing cells, indicating that generation of H2O2 is partially responsible for the induction of oxidative stress. Overexpression of SSAT caused rounding and loss of cell anchorage and significantly altered the morphology of actin-containing filopodia, suggesting an adhesion defect. SSAT upregulation may mediate majority of the oxidative stress in kidney ischemia-reperfusion injury (IRI) as manifested by decreased cell growth, generation of toxic metabolites (H2O2 and putrescine), upregulation of HO-1, disruption of cell anchorage, and defect in cell adhesion. These data point to the inhibition of polyamine catabolism as a therapeutic approach for the prevention of tissue injury in kidney IRI.     

Ornithine decarboxylase activity and its gene expression are increased in benign hyperplastic prostate

BACKGROUND: Ornithine decarboxylase (ODC) is the first key enzyme in the polyamine biosynthesis pathway. Polyamine is believed to participate in cellular proliferation and differentiation. To study the relationship between ODC and the pathogenesis of benign prostatic hyperplasia (BPH), the polyamine levels, ODC activities, and expression of ODC mRNA in benign hyperplastic and normal human prostates were assayed. METHODS: Polyamine contents and ODC activities in tissue extracts were determined by reverse-phase high-performance liquid chromatography and spectrophotometric procedures, respectively. The ODC mRNA levels were assayed by Northern blot analysis. RESULTS: The contents of putrescine, spermidine, and spermine in BPH tissues were 2.2, 3.4, and 6.0 times higher than those in normal tissues, respectively; the ODC activity of BPH tissue was about 3.2 times higher than in normal tissue; the expression level of ODC mRNA in the BPH tissues was greater than that of normal tissues. CONCLUSIONS: The findings imply that 1) the increased ODC activity and polyamine content in prostatic tissue may correlate with the pathogenesis of BPH, and 2) the high level of ODC activity is induced by the overexpression of ODC mRNA.     

Urinary vascular endothelial growth factor and its correlation with bladder cancer recurrence rates

PURPOSE: Vascular endothelial growth factor (VEGF) is a principal growth factor mediating tumor angiogenesis. The high expression of VEGF within bladder tumors is associated with a poor prognosis. We quantified urinary VEGF and determined its potential as a prognostic marker in bladder cancer. MATERIALS AND METHODS: VEGF was measured by enzyme-linked immunosorbent assay in the urine of 261 patients, including 153 undergoing cystoscopic surveillance for bladder cancer and 108 with another advanced malignancy or a benign urological condition. The source of urinary VEGF was studied through its quantification in bladder tumors and normal bladders. RESULTS: Urinary VEGF was higher in patients undergoing cystoscopic surveillance for bladder cancer than in those with an advanced nonbladder malignancy (p <0.0001) or a benign urological condition (p = 0.004). The highest levels were noted in patients with bladder cancer compared to those with clear cystoscopy (p <0.0001). In 26 cases the correlation between VEGF protein levels in bladder cancer and urine (r = 0.67, p = 0.003) suggested that the tumor is a source of urinary VEGF. Increased VEGF protein in normal urothelium in 22 patients with bladder cancer compared to that in 7 cadaveric organ donors (p = 0.002) indicates that urinary VEGF may also be derived from nonmalignant urothelium. In 61 cases we established a correlation between urinary VEGF and stage T1 or less superficial bladder tumor recurrence rates (r = 0.45, p <0.0001). CONCLUSIONS: Our study demonstrates that VEGF is high in the urine of patients with bladder cancer and it correlates with tumor recurrence rates. VEGF is implicated in the pathogenesis of bladder cancer recurrence. Its quantification may provide a valuable noninvasive marker for the early detection of bladder tumor recurrence as well as a therapy target.