[Determination and correlation analysis of contents of putrescine, cadaverine, and histamine in necrotic tissue, blood, and urine of patients with diabetic foot].

OBJECTIVE: To determine and perform a correlation analysis of the contents of putrescine, cadaverine, and histamine in necrotic tissue, blood, and urine of patients with diabetic foot (DF). METHODS: Ten patients with severe wet necrotizing DF hospitalized from January 2011 to January 2012 were assigned as group DF, and 10 orthopedic patients with scar but without diabetes or skin ulcer hospitalized in the same period were assigned as control group. Samples of necrotic tissue from feet of patients in group DF and normal tissue from extremities of patients in control group, and samples of blood and 24-hour urine of patients in both groups were collected, and the amount of each sample was 10 mL. Contents of putrescine, cadaverine, and histamine were determined with high performance liquid chromatography-mass spectrometry. The data got from the determination of blood and urine were processed with t test, and those from necrotic or normal tissue with Wilcoxon rank sum test. The correlation of contents of polyamines between necrotic tissue and blood, blood and urine were processed with simple linear regression analysis. RESULTS: (1) Contents of putrescine, cadaverine, and histamine in the necrotic tissue of group DF were (186.1 ± 26.8), (78.553 ± 12.441), (33 ± 10) mg/kg, which were significantly higher than those in normal tissue of control group [(2.2 ± 1.2), (1.168 ± 0.014), 0 mg/kg, with Z values respectively -3.780, -3.781, -4.038, P values all below 0.01]. The content of putrescine in necrotic tissue of group DF was significantly higher than those of cadaverine and histamine (with Z values respectively -3.780, -3.630, P values all below 0.01). (2) Contents of putrescine, cadaverine, and histamine in the blood of group DF were (0.075 ± 0.013), (0.022 ± 0.003), (0.052 ± 0.014) mg/L, and they were significantly higher than those in the blood of control group [(0.014 ± 0.009), (0.013 ± 0.003), (0.016 ± 0.008) mg/L, with t values respectively 6.591, 2.207, 3.568, P < 0.05 or P<0.01]. The content of putrescine in the blood of group DF was significantly higher than those of cadaverine and histamine (with t values respectively 13.204, 3.096, P values all below 0.01). (3) Contents of putrescine, cadaverine, and histamine in the urine of group DF were (0.735 ± 0.088), (0.450 ± 0.012), (0.1623 ± 0.0091) mg/L, and only the contents of putrescine and cadaverine were significantly higher than those in the urine of control group [(0.050 ± 0.014), (0.035 ± 0.007) mg/L, with t values respectively 3.270, 4.705, P<0.05 or P<0.01]. The content of putrescine in the urine of group DF was significantly higher than that of cadaverine (t = 6.686, P < 0.01). (4) There were significant and positive correlations in contents of putrescine, cadaverine, and histamine between necrotic tissue and blood in patients of group DF (with r values respectively 0.981, 0.994, 0.821, P values all below 0.01). There were no significant correlations in contents of putrescine, cadaverine, and histamine between blood and urine in patients of group DF (with r values respectively 0.150, 0.239, 0.177, P values all above 0.05). CONCLUSIONS: Putrescine, cadaverine, and histamine exist in the necrotic tissue of patients with DF in high concentrations, among which putrescine predominates. These polyamines can be absorbed into the blood through wound and excreted through the urine.

Possible role of polyamines in gyrate atrophy.

PURPOSE: Gyrate atrophy (GA) is marked by hyperornithinemia and lowered ornithine amino transferase (OAT). However there are patients of GA without hyperornithinemia and those with hyperornithinemia without GA. Some cases of GA have been reported to have low lysine. The purpose of the study was to determine if polyamines, the metabolites of ornithine, and lysine have any diagnostic role in GA. METHODS: Ornithine in plasma was estimated by two-dimensional paper chromatography, with elution of the coloured spot, and the absorbance measured using a spectrophotometer at 560 nm. OAT assay in lymphocytes was done spectrophotometrically using ornithine as substrate. Blood and urinary polyamines were extracted with n-butanol, benzoylated and analysed with HPLC; putrescine, spermine, spermidine, and cadaverine were assayed individually at 254 nm with the UV detector using ODS, G18 column with 63% methanol as solvent. RESULTS: Of the 7 patients investigated, 6 had features typical of GA. One was diagnosed to have atypical retinitis pigmentosa (case 3). The first five cases had elevated ornithine and diminished OAT, but cases 6 and 7 had near-normal ornithine and case 7 had near-normal OAT. However, all 7 patients had increased levels of total polyamines in urine compared to normals. Five had increased putrescine and three had increased spermine. All the 7 had decreased cadaverine in urine. Thus, though there were inconsistencies with ornithine and OAT, all the 7 patients had elevated polyamines from ornithine and decreased cadaverine. CONCLUSION: In addition to estimating ornithine and OAT in GA, it is suggested that urinary polyamines may be analysed as the latter appears to correlate better with the clinical condition and help in the diagnosis to a greater extent. Moreover, while ornithine is an innocuous amino acid, polyamines are known to damage DNA and proteins.

Polyamines in colorectal cancer. Evaluation of polyamine concentrations in the colon tissue, serum, and urine of 50 patients with colorectal cancer.

Total, free, and acetylated polyamine concentrations were measured simultaneously in colon tissue, serum, and urine of 50 patients with histologically proven colorectal cancer, 40 patients with nonmalignant gastrointestinal diseases, and 30 healthy volunteers. Compared with histologically unaffected colon tissue, concentrations were significantly (P less than 0.001) higher for putrescine, elevated for cadaverine, and nearly identical for spermidine and spermine in colon carcinoma, whereas N1-acetylated and N8-acetylated spermidine were detectable in cancer tissue only. Serum and urine concentrations of all polyamines except total cadaverine and spermine in serum and free spermine in urine were significantly elevated compared with healthy controls and highest sensitivity for colon cancer was found for total spermidine (89.15%) in serum and acetylputrescine (84.5%), total putrescine (84.0%), N1-acetylspermidine (79.3%), and total spermidine (92.1%) in urine. However, nonmalignant gastrointestinal diseases partly showed similar elevations which resulted in a low specificity for polyamines in colorectal cancer. Therefore, polyamines are of little value only as diagnostic markers in colorectal carcinoma. Since polyamine concentrations in serum and urine normalized in patients after curative operation while they were further elevated in patients with proven tumor relapse or metastases, these substances might play a clinical role in predicting therapeutic success or indicating relapse of the tumor. Although a significant dependency of polyamine concentrations in serum or urine to Dukes' classification, tumor localization, CEA, CA 19-9, or CA 125 did not exist, a significant linear correlation was found for tumor size.

Polymeric benzotriazole reagent for the off-line high-performance liquid chromatographic derivatization of polyamines and related nucleophiles in biological fluids.

A polymeric benzotriazole reagent containing a 9-fluorenylmethyleneoxycarbonyl (FMOC) group has been synthesized, characterized, and its derivatizations, off-line, for three polyamines, have been optimized with regard to solvent, time, and temperature. An authentic FMOC derivative of cadaverine has been prepared, characterized, and used as the external standard for quantitation of off-line derivatizations and identification of final derivatives. Actual percent derivatizations have been determined, rather than just percent disappearance of starting material. The polyamines in urine or other biological fluids can be derivatized without organic solvent or solid phase extraction, but rather in situ by the simple addition of the polymeric reagent to the fluid, incubation for a few minutes at room or elevated temperature, filtration and direct injection. Derivatizations could also be performed by transferring a small volume of the hydrolyzed and filtered biological fluid to a disposable pipette containing the polymeric reagent. Derivatization was then followed by elution, filtration, and direct injection onto the high-performance liquid chromatographic (HPLC) system. Automation of the overall polymeric derivatization, filtration, HPLC injection, separation, detection, quantitation, and data acquisition-interpretation is suggested. The polymeric reagent has been utilized for the qualitative and quantitative determination of cadaverine and putrescine, normally occurring polyamines, in human urine. These levels were compared with the corresponding literature values for healthy human subjects, and the values were found to be in excellent agreement. This novel derivatization approach, though off-line, provides for a much simpler, more rapid, and more efficient conversion of these and related polyamines or nucleophiles to derivatives having vastly improved chromatographic detection properties in HPLC. The final derivatives contain the FMOC group, making them extremely chromophoric and fluorophoric, and providing trace detection at ppb (microgram/l) and sub-ppb levels. The overall approach is recommended for these and other biologically occurring polyamines, in fluids and tissues, as well as related bioorganic and biologically active nucleophiles, including drugs and their metabolites.

Clinical usefulness of an enzymatic determination of total urinary polyamines, excluding cadaverine.

In one widely used enzymatic method for urinary polyamines, the total concentrations of four polyamines--putrescine, spermidine, spermine, and cadaverine--are determined. We report here a simple enzymatic method for measuring the total concentrations of urinary polyamines except cadaverine. The coefficients of variation (CV) for within-run measurements by this method were 4.3% (means = 17.2 mumol/L) and 1.5% (means = 66.5 mumol/L), between-run CVs were 4.8% (means = 16.8 mumol/L) and 1.8% (means = 67.5 mumol/L). The central 95% normal reference interval was 12.3-29.1 mumol/g creatinine for men and 14.1-36.8 mumol/g creatinine for women. In some cases, physiological variations in urinary polyamine excretion were large, mainly because of variations in cadaverine excretion, even in health. Pathological changes in polyamine production in the body may therefore be more easily shown by the excretion of total polyamines excluding cadaverine than by that including cadaverine.

[Urinary total polyamines levels in estrous cycle and during pregnancy in female rats].

Urinary total polyamines; putrescine, spermidine and cadaverine, levels were determined in the estrous cycle and during the pregnancy in female Wistar rats. Urinary total polyamines level in 12 estrous cycles in 4 female rats revealed the definite cyclic changes, showing high levels at estrous and low levels at diestrous stage. After the ovariectomy those cyclic changes disappeared. Urinary total polyamines levels were constantly low during the first half of the pregnancy of 8 female rats, whereas the levels increase abruptly from 10 days before parturition. After fetusectomy at 15th and 17th days of the pregnancy, those levels decreased gradually. These data suggested that the urinary total polyamines levels were closely related with the pituitary-ovarian function and the growth of fetus of rats.

Polyamine levels in gynecologic malignancies.

Polyamines are closely related to many aspects of cell growth. Since increased amounts of polyamines in the urine of human cancer patients were reported in 1971, polyamines have been studied from the standpoint of tumor markers. In this study, polyamines in erythrocytes, plasma and urine were determined in 42 controls and 105 patients with gynecologic malignant tumors. The changes in polyamine levels were investigated before and after treatment. With advances in the stage of uterine cervical cancer, the frequency of abnormal levels of polyamines (concentrations greater than two standard deviations above the mean control level) became greater, and reached nearly 80% in recurrent and ovarian cancer. In the early stage of cancer, the diagnostic value was low. Comparison with carcinoembryonic antigen (CEA) was also performed. The polyamines lack specificity for malignant diseases, but they can be used to some extent as a tumor marker in the gynecologic field.

Influence of decontamination of the digestive tract on the urinary excretion of histamine and some of its metabolites.

Urinary excretions of histamine, N tau-methylhistamine and N tau-methylimidazoleacetic acid have been determined for 8 healthy volunteers during 14 consecutive days. Selective decontamination of the digestive tract was performed from day 3 to day 6, followed by total decontamination from day 7 to day 10. Urinary excretions of N tau-methylhistamine and N tau-methylimidazoleacetic acid decreased to a small though significant degree (about 15-20%) after total decontamination, suggesting a histamine production by anaerobic bacteria. Cadaverine decreased for about 70% under both selective and total decontamination, suggesting that this amine in human urine mainly originates from aerobic bacteria in the intestinal tract.

Total polyamines and their non-alpha-amino acid metabolites simultaneously determined in urine by capillary gas chromatography, with nitrogen-phosphorus detector; and some clinical applications.

A capillary gas-chromatographic method with nitrogen-phosphorus detection is used here to simultaneously determine 1,3-diaminopropane, putrescine, cadaverine, spermidine, spermine, isoputreanine , putreanine , and N-(3-aminopropyl)-N'-(2-carboxyethyl)-1,4- diaminobutane in urine. After acid hydrolysis the compounds are isolated by adsorption onto silica gel and converted into their methyl-heptafluorobutyryl derivatives. We give quality-control data and age-dependent values for urinary excretion of these analytes by 76 apparently healthy controls. Circadian rhythmicity in the excretion of spermidine and (especially) isoputreanine was established and is discussed in the light of its implications for monitoring therapy of cancer. Investigation of menstrual-cycle-dependent diurnal variation in one normal woman showed no distinct, consistent fluctuations. We applied the method to monitor (by use of 24-h urine specimens) an uncomplicated, normally progressing pregnancy, a patient with metastatic melanoma being treated with cytostatic drugs, and (in more detail) the treatment of a patient with high-grade non-Hodgkin lymphoma.

Amine metabolite profile of normal and uremic urine using gas chromatography--mass spectrometry.

A method for the simultaneous analysis of phenolic amines and aliphatic amines in human urine is described. The amine metabolites in urine were extracted using Dowex 50W-X8 cationic resin, derivatized and analyzed by a gas chromatographic--mass spectrometric--computer system. The amine metabolites profile of 5 ml of urine was obtained with good gas chromatographic separation. The gas chromatographic method described here separates urinary phenolic amines, di- and polyamines and methylguanidine in a single chromatographic separation. The urinary levels of methylguanidine, putrescine, cadaverine, spermidine, p-tyramine, dopamine, and 3-methoxytyramine were quantitated by using a mass spectrometric technique. In uremic patients, only the urinary excretion of methylguanidine was increased in comparison with normal subjects, although the urinary excretion of other amines was decreased in uremic patients.

Gas chromatographic method for the determination of urinary acetylpolyamines.

A gas chromatographic method was developed for the determination of monoacetylputrescine, monoacetylcadaverine, N1-acetylspermidine and N8-acetylspermidine in human urine. The amines were isolated from urine by silica gel column chromatography. 1,10-Diaminodecane was used as internal standard. The amines were reacted with ethyl chloroformate in aqueous medium to four ethyloxycarbonyl derivatives prior to application to gas chromatography using a flame ionization detector. Separation and determination of the derivatives were carried out on a Uniport HP column (1.0 m) impregnated with 0.5% SP-1000 under temperature-programmed conditions. The monoacetylpolyamines could be measured accurately at the nanomole level. The method was used for the determination of the monoacetylpolyamines in urine of healthy volunteers. The values obtained were in the range of the published data.

On the biogenesis of diamines and polyamines in the pregnant rat.

The urinary excretion of the diamines histamine, methylhistamine, putrescine and cadaverine and the polyamines spermidine and spermine was studied in rats which on the 19th day of pregnancy were subjected to various ectomizing operations. In sham-operated rats the urinary excretion of all the amines studied except spermine was highly elevated on the day preceding the sham-operation and on the 3 days studied post-operatively, i.e. sham-operation did not affect the elevated urinary amine excretion during pregnancy. Foetectomy resulted in an abolished increase in the urinary excretion of histamine and methylhistamine while the excretion of putrescine, cadaverine and spermidine was still significantly increased. Removal of both the foetuses and placentae reduced the excretion of putrescine, cadaverine and spermidine towards the level of non-pregnant rats. Combined hysterectomy and ovariectomy did not cause any additional effects to those after removal of the foetuses and the placentae except for cadaverine, the excretion of which was further reduced.

A fluorometric assay for total diamines in human urine using human placental diamine oxidase.

The detailed procedure for a new fluorometric assay for total diamines in human urine is described. The diamines were purified from the urine by cation-exchange chromatography and incubated with human placental diamine oxidase. Hydrogen peroxide formed in the diamine oxidase reaction was measured fluorometrically by converting homovanillic acid to a highly fluorescent compound in the presence of peroxidase. Because of its simplicity and high sensitivity, our present method seems useful for routine clinical investigation. The data obtained from normal subjects and patients suffering from various forms of cancer are also presented.

Putrescine turnover in kidneys of testosterone-treated mice.

Putrescine turnover was studied in testosterone stimulated mouse kidney after labeling the endogenous pool of putrescine with the polyamine precursor [3H]ornithine. Renal putrescine was rapidly labeled after the administration of radioactive ornithine, reaching maximal activities at the time of the earliest measurement (5 min after injection). The content of labeled putrescine then declined extremely rapid with a half-life of about 15 min. Treatment with the diamine oxidase inhibitor, aminoguanidine, did not affect this rapid turnover. The decline in radioactive putrescine was not accompanied by an increase in labeled spermine. However, testosterone treatment resulted in a marked increase in urinary content of both labeled and unlabeled putrescine indicating that most of the newly synthesized renal putrescine is excreted in the urine.

Biologic markers in breast carcinoma: clinical correlations with urinary polyamines.

Urinary polyamine levels were evaluated in patients with breast carcinoma. The individual levels of putrescine, spermidine, spermine, and cadaverine, and the product/precursor levels of putrescine, spermidine, and spermine were analyzed. Elevations of one or more individual polyamines or of the ratios were found in 50% of patients with metastatic disease, 38.5% of preoperative patients, and 35.7% of 5--24 week postoperative N + patients. Sequential sampling of patients with metastatic disease suggested that changes in elevated polyamine levels tend to reflect the clinical course of the disease, especially for the association of treatment failure with rising elevated values. The presence of one or more elevated parameters prior to treatment of metastatic disease tended to be associated with a higher response rate (85.7 vs. 68.4%) than all normal levels. Five of nine patients who recurred postoperatively had preceding postoperative polyamine elevations. In addition, there was a trend for a shorter disease-free time among patients with one of more elevated polyamine parameters between 5--24 weeks postoperatively than among patients with normal parameters. These data suggest that measurement of urinary polyamine levels, including calculation of the product/precursor levels, may be a useful clinical adjunct in the management of patients with breast carcinoma.

Polyamine-pyridoxal Schiff bases in urine.

Schiff bases of the diamines 1,3-diaminopropane, putrescine, and cadaverine and the polyamines spermidine and spermine with pyridoxal or pyridoxal phosphate occur in human urine, as shown by gas chromatographic/mass spectrometric selected ion-monitoring techniques. By use of synthetic standards, procedures were devised for conversion of the Schiff bases to stable derivatives amenable to gas chromatographic/mass spectrometric analysis. These procedures involve borohydride reduction of the C = N double bond, hydrolytic removal of the phosphate group, chromatographic separation from the bulk of urinary constituents, and trifluoroacetylation of polar functional groups. The levels of the polyamine-pyridoxal Schiff bases were estimated to be in the range of pmol/ml or urine.

Increased formation of diamines and polyamines in the pregnant rat.

1. The urinary excretion of histamine, methylhistamine, putrescine, cadaverine, spermidine and spermine was examined before, during and after pregnancy in rats. 2. During the last third of undisturbed pregnancy a distinct and steep rise occurred in the excretion of all amines studied except spermine. The peak values were found a few days before the birth of the young. In spermidine excretion a second peak was observed one or two days after delivery. 3. Before and during the first 2 weeks of gestation on a molar basis putrescine excretion was the greatest one. During the last trimester histamine was excreted in the largest amount. 4. Under the influence of the diamine oxidase inhibitor aminoguanidine the general pattern of excretion of diamines and polyamines in pregnant rats remained essentially unchanged but the total amount excreted increased. Most conspicuous was the great elevation of urinary contents of putrescine and cadaverine.