RESUMEN

OBJECTIVES: Elevated concentrations of polyamines have been found in urine of patients with malignant tumors, including ovarian cancer. Previous research has suffered from poorly standardized detection methods. Our liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is capable of simultaneous standardized analysis of most known polyamines. Liquid chromatography-tandem mass spectrometry has not previously been used in the differential diagnostics of ovarian tumors in postmenopausal women. MATERIALS AND METHODS: In this prospective study, postmenopausal women (n = 71) presenting with an adnexal mass and, as controls, women with genital prolapse or urinary incontinence scheduled for surgery (n = 22) were recruited in the study. For analysis of the polyamines, a morning urine sample was obtained before surgery. Preoperative serum CA125 concentrations were determined in the study group. RESULTS: Twenty-three women with benign and 37 with malignant ovarian tumors were eligible. Of all analyzed polyamines, only urinary N,N-diacetylspermine showed statistically significant differences between all groups except controls versus benign tumors. N,N-diacetylspermine was elevated in malignant versus benign tumors (P < 0.001), in high-grade versus low malignant potential tumors (P < 0.001), in stage III to IV versus stage I to II cancers (P < 0.001), and even in early-stage cancer (stage I-II) versus benign tumors (P = 0.017). N,N-diacetylspermine had better sensitivity (86.5%) but lower specificity (65.2%) for distinguishing benign and malignant ovarian tumors than CA125 with a cut-off value of 35 kU/L (sensitivity, 75.7%; specificity, 69.6%). CONCLUSIONS: Urinary N,N-diacetylspermine seems to be able to distinguish benign and malignant ovarian tumors as well as early and advanced stage, and low malignant potential and high-grade ovarian cancers from each other, respectively.

Asunto(s)

RESUMEN

For the first time, CEC was coupled with tris(2,2-bipyridyl) ruthenium(II) (Ru(bpy)(3)(2+) electrochemiluminescence detection. Efficient CEC separations of proline, putrescine, spermidine and spermine were achieved when the pH of the mobile phase is in the range of 3.5-7.0. The optimum mobile phase for CEC separation is much less acidic than that for CZE separation, which matches better with the optimum pH for Ru(bpy)(3)(2+) electrochemiluminescence detection and dramatically shortens the analysis time because of larger EOF at higher pH. The time for CEC separation of the polyamines is less than 12.5 min, which is about half as much as the time needed for CZE. The detection limits were 1.7, 0.2, and 0.2 microM for putrescine, spermidine, and spermine, respectively. The RSD of retention time and peak height of these polyamines were less than 0.85 and 6.1%, respectively. The column showed good long-term stability, and the RSD of retention time is below 5% for 150 runs over one-month use. The method was successfully used for the determination of polyamines in urine samples.

Asunto(s)

RESUMEN

The physiological response of the human body to several diseases can be reflected by the metabolite pattern in biological fluids. Cancer, like other diseases accompanied by metabolic disorders, causes characteristic effects on cell turnover rate, activity of modifying enzymes, and RNA/DNA modifications. This results in an altered excretion of modified nucleosides and biochemically related compounds. In the course of our metabolic profiling project, we screened 24-h urine of patients suffering from lung, rectal, or head and neck cancer for previously unknown ribosylated metabolites. Therefore, we developed a sample preparation procedure based on boronate affinity chromatography followed by additional prepurification with preparative TLC. The isolated metabolites were analyzed by ion trap mass spectrometry (IT MS) and Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). IT MS was applied for LC-auto MS(3) screening runs and MS(n(n=4-6)) syringe pump infusion experiments, yielding characteristic fragmentation patterns. FTICR MS measurements enabled the calculation of corresponding molecular formulae based on accurate mass determination (mass accuracy: 1-5 ppm for external and sub-ppm values for internal calibration). We were able to identify 22 metabolites deriving from cellular RNA metabolism and related metabolic pathways like histidine metabolism, purine biosynthesis, methionine/polyamine cycle, and nicotinate/nicotinamide metabolism. The compounds 1-ribosyl-3-hydroxypyridinium, 1-ribosyl-pyridinium, and 3-ribosyl-1-methyl-l-histidinium as well as a series of ribosylated histamines, conjugated to carboxylic acids at the N(omega)-position were found as novel urinary constituents. The occurrence of the modified nucleosides 2-methylthio-N(6)-(cis-hydroxyisopentenyl)-adenosine, 5-methoxycarbonylmethyl-2-thiouridine, N(6)-methyl-N(6)-threonylcarbamoyladenosine, and 2-methylthio-N(6)-threonylcarbamoyladenosine in human urine is verified for the first time.

Asunto(s)

Asunto(s)

Asunto(s)

RESUMEN

The derivatization of biogenic amines such as putrescine, cadaverine, spermidine and spermine with dansyl chloride in solid phase extraction cartridges is described. Different types of filling materials were tested in order to have the highest retention of the different analytes. The best results were obtained by using C18 cartridges. The optimal conditions were: amine solution buffered at pH 12, 2 mM dansyl chloride (acetone-bicarbonate solution 20 mM (pH 9-9.5), 2 + 3 v/v) as reagent concentration, room temperature and 30 min reaction time. The developed procedure was applied to the determination of these polyamines in urine samples from healthy controls and cancer patients using HPLC with 1,7-diaminoheptane as internal standard. The concentrations ranged from 0.5 to 5 micrograms mL-1 and the detection limits were 10 ng mL-1 for all polyamines. By concentrating the urine extracts, the detection limits were improved down to 2 ng mL-1. The accuracy and the precision of the method were tested. The proposed dansylation method is advantageous with respect to solution dansylation. It improves the total analysis time, avoids high temperatures that can affect the thermal stability of the derivatives and could make possible the automation of the procedure.

Asunto(s)

RESUMEN

We assessed the effects of dietary arginine supplementation on protein turnover and organ protein synthesis in burned rats. Male Wistar rats weighing about 200 g underwent catheter jejunostomy and received scald burns covering 30% of the whole-body surface area. Animals were divided into a control group (n = 9) and an arginine group (n = 9) and continuously received total enteral nutrition for 7 d (250 kcal.kg-1.d-1, 1.72 gN.kg-1.d-1). Changes in body weight, plasma total protein, plasma albumin, urinary excretion of polyamines, nitrogen balance, whole-body protein kinetics, and tissue protein synthesis rates were determined. Whole-body protein kinetics and tissue fractional protein synthetic rates (Ks, percent/d) were estimated using a 24-h constant enteral infusion of 15N glycine on the last day. The changes in body weight were not different between the control and arginine groups. The urinary excretion of polyamines was higher in the arginine group than in the control group (P < 0.01). Burned rats enterally fed arginine-supplemented diet yielded significantly greater cumulative and daily nitrogen balance on days 3 and 5 than those fed a control diet (cumulative, P < 0.05; day 3, P < 0.01; day 5, P < 0.01). Whole-body protein turnover rate was significantly elevated in the arginine group as compared to that in the control group (P < 0.05). The Ks of rectus abdominis muscles were significantly increased in the arginine group in comparison to the control group (P < 0.01). We have shown that dietary arginine supplementation improved protein anabolism and attenuated muscle protein catabolism after thermal injury.

Asunto(s)

RESUMEN

Eleven urinary polyamine levels were determined in controls (32 cases) and 43 patients with varying stages of leukemia including initial, relapse and complete remission, using gas chromatography nitrogen-phosphorus detection. Also, to indirectly evaluate the possible involvement of enzymes, precursor to product concentration ratios were compared between controls and patients with each stage of leukemia. As a result, it is confirmed that the ratio of N1-acSpd/N8-acSpd could be used as a diagnostic marker and the level of N1,N12-diacetylspermine could be used for determining disease stage and as a malignancy marker for leukemia. An altered metabolic pathway related to leukemia is also proposed in which N1,N12-diacetylspermine can be produced directly from spermine and N1,N12-diacetylspermine is a major source of N1-acetylspermidine.

Asunto(s)

RESUMEN

The urinary concentrations of 16 estrogens and 11 polyamines were quantitatively determined by gas chromatography-mass spectrometry and gas chromatography with nitrogen-phosphorus detection. Samples from patients with stages I-IV of breast cancer (35 cases, aged 27-65 years) as well as from age-matched normal female subjects (25 cases, aged 22-61 years) were tested. Also, the ratios of precursor to product metabolite including 16alpha-OH E1 to 2-OH E1, which are linked to estrogen and polyamine biosynthetic pathways, were determined to explore enzyme involvement in breast cancer and to evaluate the potential usefulness of these ratios and concentrations as disease staging markers. It was confirmed that major estrogens and 16a-OH E1 were positively associated with breast cancer and catechol estrogens including 2-OH E1 were inversely associated with breast cancer. The ratios of N1-acSp/Spd and 16alpha-OH E1/2-OH E1 might be a useful dual marker for staging of breast cancer. From the variation of the relative ratios of polyamines, it is suggested that alteration in polyamine oxidase (PAO) activity may play an important role in the development of breast cancer.

Asunto(s)

RESUMEN

An analytical system developed for fractionating free and monoacetylated polyamines [Hiramatsu, K. et al. (1994) J. Biochem. 115, 584-589] was proved useful also in detecting diacetylpolyamines, namely N1,N8-diacetylspermidine (diAcSpd) and N1,N12-diacetylspermine (diAcSpm). Detection limits were 0.9 and 0.6 pmol (S/N = 5) for diAcSpd and diAcSpm, respectively. Analytical recovery and within-run variation were also satisfactory. Human urine samples were found to contain diAcSpd and diAcSpm. These polyamines were identified on the basis of the following observations: (i) their retention times were coincident with those of authentic samples; (ii) they were deacetylated to N8-acetylspermidine and monoacetyl- and free spermine, respectively, by acetylpolyamine amidohydrolase; and (iii) they were practically inert to direct oxidation by bacterial polyamine oxidase as were authentic samples. The amounts of eleven polyamine species including diAcSpd and diAcSpm in urine samples from 52 healthy persons were determined. Mean values for the major polyamine components were consistent with those reported by others. Although the amounts of diAcSpd and diAcSpm were very small, comprising only 1.4 and 0.46% of total polyamines, respectively, these two compounds were found to be always present in healthy human urine as regular constituents. Moreover, variation in their content among individuals was small, suggesting that excretion of these components in urine is strictly regulated.

Asunto(s)

RESUMEN

Urinary polyamine excretion has been suggested to reflect hypermetabolism or catabolism in different illnesses. In the present study, the excretion of urinary polyamines was examined in 12 obese subjects (3 men, 9 women aged 32-55 y, body mass index 33.3-64.7 kg m-2) before and during a very low calorie diet (the total calorie intake 2100-3350 kJ). In addition, nitrogen balance, basal energy expenditure (BEE) and serum thyroid hormone levels were examined. During the first week on a very low calorie diet (VLCD) the mean body weight declined from 121.8 +/- 27.3 to 117.4 +/- 26.2 kg (mean +/- SD, p < 0.001), and after 12 weeks of treatment body weight was 106.6 +/- 24.6 kg. Immediate reduction of BEE from 1.44 +/- 0.24 to 1.34 +/- 0.24 kcal min-1 (p < 0.001) was found within the first week of therapy and BEE measured on weight-maintaining diet remained lower at 12 weeks (1.25 +/- 0.27 kcal min-1, p < 0.01). Serum free T3 decreased and reverse T3 increased significantly after starting VLCD. Nitrogen balance remained negative during the first 2 weeks on VLCD. A significant increase in total (38%), and in N1-acetyl- and N8-acetylspermidine excretions in the urine (40% and 27%, respectively, p < 0.05) was found during the first week, but later on the levels were not significantly different from the baseline levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

RESUMEN

Polyamines have been known to play an important role in hepatic regeneration. In the present study, we measured the amount of urinary polyamine excretion in various liver diseases using a simple enzymatic method. Urinary polyamine excretion was elevated above the normal range in 21 out of 47 cases with fulminant hepatic failure, acute hepatitis, chronic active hepatitis, and liver cirrhosis. No change, however, was observed in 11 patients with chronic inactive hepatitis. In fulminant hepatic failure, two patients with urinary polyamine concentrations above 100 mumoles/g.cr. recovered, while two patients with concentrations of 56.2 and 26.7 mumoles/g.cr., died. In acute hepatitis, urinary polyamine excretion was significantly less in the recovery stage compared with the acute stage. When insulin and glucagon infusion therapy was performed in patients with liver cirrhosis without ascites, urinary polyamine excretion was significantly elevated after three days. These results suggest that measuring the amount of polyamine in urine is clinically useful for monitoring hepatic regeneration.

Asunto(s)

RESUMEN

Urinary polyamine (UPA) excretion patterns were measured in 39 patients with clinically evaluable epithelial ovarian cancer immediately before they were treated with a cycle of chemotherapy and 24-48 h after chemotherapy to ascertain if changes in UPA excretion patterns correlated with eventual response to treatment. Almost all of the 19 patients who responded to chemotherapy had a rise in the excretion of all UPA fractions after treatment while most patients with chemoresistant cancer showed only an increase in the excretion of the putrescine and spermine fractions. However, a two-fold increase in excretion of the spermidine fractions occurred exclusively in patients who would eventually respond to chemotherapy. This phenomenon was not seen in patients with chemoresistant cancer. If, 48 h after chemotherapy, a patient with epithelial ovarian cancer does not show at least a doubling of the urinary levels of spermidine, acetylspermidine or total polyamine excretion that chemotherapy should be stopped since it is unlikely to be effective.

Asunto(s)

RESUMEN

Polyamines are recognized as cell growth factors in relation to cell proliferation, differentiation, regeneration and malignant transformation. Polyamines play an important role in the growth of normal cells like vascular endothelial cells and also exert various effects on the proliferation and metastasis of malignant cells. The recent studies on the biosynthesis have clearly elucidated its mechanism at the gane levels, which reflects to the development of the inhibitors of the polyamine biosynthesis. One of the main purposes of the studies on the various polyamine synthesis inhibitors is for the development of new anti-cancer agents, based on the characteristics of the polyamine functions. The clinical effects of several inhibitors, however, have not been shown to be satisfactory and the reason is now the most important research subject in this field. The measurement of the polyamine contents in biological fluids including urine and blood has been shown to be useful as the tumor marker. The recent studies have indicated that the mechanism of increased secretion of urinary polyamines is due to the release from the degraded cancer cells. The results now stimulated the research which aims to elucidate the usefulness of the measurement of urinary polyamines as the parameters of the sensitivity to the anticancer drugs in patients with cancer.

Asunto(s)

RESUMEN

The HHH syndrome (hyperornithinemia associated with hyperammonemia and homocitrullinuria) is characterized by a very rare genetic defect of ornithine transport in mitochondrial membrane. We first demonstrated that a patient with HHH syndrome excreted about 6 times higher amount of polyamines in urine than the control when supplemented with high protein diets and ornithine loading. Each urinary polyamine fraction measured by HPLC method in HHH syndrome appears to be increased, as compared with those of the control. These data suggest that increased urinary excretion of polyamines in this syndrome is closely related to overflowing of plasma polyamine due to an ornithine transport defect in the mitochondrial membrane.

Asunto(s)

RESUMEN

1,2-Dimethylhydrazine (DMH) is a potent procarcinogen with selectivity for the colon. Recently, it has been demonstrated that levels of N1-acetylspermidine were elevated 2-3-fold in colonic tumors induced by this agent compared to control tissues. To determine whether alterations in the urinary levels of this acetylated polyamine or other polyamines were useful biochemical markers for colon cancer in this experimental model, rats were given s.c. injections of DMH (20 mg/kg body weight/week) or diluent for 26 weeks. One week after the last injection, control and DMH-treated animals were placed in separate metabolic cages and their urine was collected for 24 h. The urinary levels (expressed as nmol/mg creatinine) of putrescine, spermidine, spermine, N1-acetylspermidine, and N8-acetylspermidine were then analyzed by high-performance liquid chromatography. Animals from each group were then sacrificed and their colons were examined for tumors. The results of these studies demonstrated that the urinary level of N1-acetylspermidine was an excellent biochemical marker for colonic tumors induced by DMH. At 18.3 nmol/mg creatinine, N1-acetylspermidine was 100% sensitive and specific for colon cancer. Moreover, urinary levels of N1-acetylspermidine were better for this purpose than the N1-acetylspermidine/N8-acetylspermidine molar ratio, a marker previously suggested to be more specific for certain cancers than free polyamines.

Asunto(s)

RESUMEN

This simple, rapid liquid-chromatographic assay of urinary polyamines (putrescine, spermidine, spermine, and cadaverine) involves electrochemical detection with a post-column immobilized enzyme, polyamine oxidase (EC 1.4.3.6) from soybean seedlings. Polyamines are separated by isocratic ion-pairing reversed-phase chromatography, then enzymatically converted, with release of hydrogen peroxide, via the post-column reactor with immobilized polyamine oxidase; the hydrogen peroxide is detected by electrochemical oxidation on a platinum electrode. The detection limits for injected putrescine, spermidine, and spermine were 0.3, 0.5, 0.6, and 4 pmol, respectively, with linear ranges of two to three orders of magnitude. Reproducibility was also good, with CV values less than 7%. The efficiency of the immobilized enzyme column was not decreased after analysis of 300 urine samples. Putrescine and spermidine excretion in urine from patients with blood cancers and solid cancers was significantly increased.

Asunto(s)

RESUMEN

Proliferation end products are of considerable value as tumour markers. Hexosaminidase and polyamines were significantly elevated in urine of patients with leukemia and lymphoma (p less than 0.001). Interfering low molecular weight compounds were eliminated by a microcolumn centrifuge method. Urinary polyamines were estimated by high voltage electrophoresis technique. Marked increase in these markers in untreated patients and subsequent decrease on effective chemotherapy support the use of these two tumour markers to monitor therapeutic response.

Asunto(s)

Asunto(s)