Polyamine biomarkers as indicators of human disease.

The significant increase of periodontitis, chronic kidney disease (CKD), Alzheimer's disease and cancer can be attributed to an ageing population. Each disease produces a range of biomarkers that can be indicative of disease onset and progression. Biomarkers are defined as cellular (intra/extracellular components and whole cells), biochemical (metabolites, ions and toxins) or molecular (nucleic acids, proteins and lipids) alterations which are measurable in biological media such as human tissues, cells or fluids. An interesting group of biomarkers that merit further investigation are the polyamines. Polyamines are a group of molecules consisting of cadaverine, putrescine, spermine and spermidine and have been implicated in the development of a range of systemic diseases, in part due to their production in periodontitis. Cadaverine and putrescine within the periodontal environment have demonstrated cell signalling interfering abilities, by way of leukocyte migration disruption. The polyamines spermine and spermidine in tumour cells have been shown to inhibit cellular apoptosis, effectively prolonging tumorigenesis and continuation of cancer within the host. Polyamine degradation products such as acrolein have been shown to exacerbate renal damage in CKD patients. Thus, the use of such molecules has merit to be utilized in the early indication of such diseases in patients.

Gender-Related Differences on Polyamine Metabolome in Liquid Biopsies by a Simple and Sensitive Two-Step Liquid-Liquid Extraction and LC-MS/MS.

Polyamines are involved in the regulation of many cellular functions and are promising biomarkers of numerous physiological conditions. Since the concentrations of these compounds in biological fluids are low, sample extraction is one of the most critical steps of their analysis. Here, we developed a comprehensive, sensitive, robust, and high-throughput LC-MS/MS stable-isotope dilution method for the simultaneous determination of 19 metabolites related to polyamine metabolism, including polyamines, acetylated and diacetylated polyamines, precursors, and catabolites from liquid biopsies. The sample extraction was optimized to remove interfering compounds and to reduce matrix effects, thus being useful for large clinical studies. The method consists of two-step liquid-liquid extraction with a Folch extraction and ethyl acetate partitioning combined with dansyl chloride derivatization. The developed method was applied to a small gender-related trial concerning human serum and urine samples from 40 obese subjects. Sex differences were found for cadaverine, putrescine, 1,3-diaminopropane, γ-aminobutyric acid, N8-acetylspermidine, and N-acetylcadaverine in urine; N1-acetylspermine in serum; and spermine in both serum and urine. The results demonstrate that the developed method can be used to analyze biological samples for the study of polyamine metabolism and its association with human diseases.

Reductive Desulfuration as an Important Tool in Detection of Small Molecule Modifications to Payload of Antibody Drug Conjugates.

A high resolution accurate mass LC-MS method was developed to facilitate the characterization of a subset of antibody drug conjugate (ADC) biotherapeutics, where the payload is linked to the antibody by a thioether bond. Desulfuration of the thioether linker was optimized for release of the payload to take advantage of the high resolution and high mass accuracy of the Orbitrap to characterize metabolism of the payload. Two model ADCs, trastuzumab emtansine (T-DM1) and SigmaMAb dansyl-cadavarine-SMCC (SigmaMAb ADC mimic) were selected for optimization of the desulfuration reaction as a function of reaction time, pH, organic solvent, and chaotropic reagents (urea, guanidine HCl) by monitoring the yield of released desulfurated DM1 from T-DM1 and desulfurated dansyl-cadaverine-SMCC from SigmaMAb ADC mimic, respectively. The optimized desulfuration technique was successfully applied to enable characterization of the ADC following its incubation in hepatocytes, liver microsomes, and buffers, as illustrated by the identification of a hydrolyzed thiosuccinimide ring of SigmaMAb ADC mimic following incubation in buffer for 48 h. The results from this study demonstrate that the chemical cleavage of thioether bond by desulfuration is simple, efficient, and specific. This technique is useful in characterization of metabolism on the payload of ADC to provide guidance for improvement of its biopharmaceutical profile. This is the first report on characterization of modification to payload of ADC following desulfuration.

Serum nuclear magnetic resonance-based metabolomics and outcome in diffuse large B-cell lymphoma patients - a pilot study.

The prognosis for diffuse large B-cell lymphoma (DLBCL) patients with early relapse or refractory disease is dismal. To determine if clinical outcome correlated to diverse serum metabolomic profiles, we used (1)H nuclear magnetic resonance (NMR) spectroscopy and compared two groups of DLBCL patients treated with immunochemotherapy: i) refractory/early relapse (REF/REL; n=27) and ii) long-term progression-free (CURED; n = 60). A supervised multivariate analysis showed a separation between the groups. Among discriminating metabolites higher in the REF/REL group were the amino acids lysine and arginine, the degradation product cadaverine and a compound in oxidative stress (2-hydroxybutyrate). In contrast, the amino acids aspartate, valine and ornithine, and a metabolite in the glutathione cycle, pyroglutamate, were higher in CURED patients. Together, our data indicate that NMR-based serum metabolomics can identify a signature for DLBCL patients with high-risk of failing immunochemotherapy, prompting for larger validating studies which could lead to more individualized treatment of this disease.

[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.

Arginase expression in peritoneal macrophages and increase in circulating polyamine levels in mice infected with Schistosoma mansoni.

We investigated the nitric oxide (NO) synthase and arginase pathways in resident peritoneal macrophages of mice infected with the tropical parasite Schistosoma mansoni. The two enzymes may have opposite effects, insofar as NO may be involved in the killing of the parasite whereas arginase may stimulate parasite growth via polyamine synthesis. We determined the effects of the infection on the expression and activity of the two enzymes in macrophages, before and after cytokine activation. Cells from infected mice expressed the hepatic type I arginase, whereas in control cells, the enzyme was expressed only after cytokine activation, as were NO synthase II and type II arginase in both groups of cells. Moreover, we found that in infected mice, arginase expression in macrophages was associated with a ten fold increase in the concentration of circulating ornithine-derived polyamines. This may be of pathological importance, since parasitic helminths are though to be dependent on their hosts for the uptake and interconversion of polyamines.

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.

Capillary gas chromatographic determination of putrescine and cadaverine in serum of cancer patients using trifluoroacetylacetone as derivatizing reagent.

Trifluoroacetylacetone (FAA) derivatives of 1,4-diaminobutane (putrescine) (Pu) and 1,5-diaminopentane (cadaverine) (CA) were prepared and characterized by elemental microanalysis, IR, and mass spectrometry. Diamine derivatives were eluted from capillary gas chromatographic (CGC) column BP1 (12 m x 0.22 mm I.D.) or BP5 (50 m x 0.22 mm) with layer thickness 0.25 microm, using nitrogen as a carrier gas and flame ionization detection (FID). A solvent extraction procedure was developed for the extraction of Pu and CA from aqueous solution with a linear calibration range 0-20 microg/0.2 ml of extract with a detection limit of 0.5-0.6 ng/injection. The method was applied for the determination of Pu and CA in the serum of five cancer patients before and after radiotherapy. The serum of two healthy persons was also analyzed for Pu and CA contents. Pu and CA concentrations were found within the range 1.16-3.96 microg/ml and 0.88-1.46 microg/ml in cancer patients as compared to 0.11-0.16 microg/ml and 0.06-0.075 microg/ml respectively in healthy persons with a coefficient of variation (CV) within 0.62-5.47%. Pu and CA concentrations decreased on radiotherapy in cancer patients, but were much higher than in healthy persons.

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.

The elimination of spermidine from plasma in rats.

Infused spermidine in rats with bypassed livers was eliminated very slowly, whereas endogenous spermidine in saline-infused controls with bypassed livers increased. In the same animals endogenous putrescine plasma levels did not differ significantly between spermidine- or saline-infused animals, whereas endogenous cadaverine levels increased during spermidine infusion and decreased afterwards. It is concluded that the liver is the most important site for plasma spermidine elimination.

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.

Serum levels of polyamines in patients with chronic renal failure.

Serum levels of total putrescine, cadaverine, spermidine and spermine were measured in 10 normal subjects, 11 nondialyzed patients with chronic renal failure, and 25 patients undergoing maintenance hemodialysis. The measurement of the serum levels of four polyamines was done with high-performance cation-exchange chromatographic separation and fluorometric detection using o-phtalaldehyde. In normal subjects, the serum levels of putrescine and spermine were 0.24 +/- 0.09 and 0.20 +/- 0.05 nmoles/ml. In patients with chronic renal failure, the levels of polyamines were obtained in the order of putrescine, cadaverine, spermidine, and spermine: 0.51 +/- 0.15; 0.05 +/- 0.01; 0.34 +/- 0.08; and 0.05 +/- 0.04 nmoles/ml. In dialyzed patients, predialysis values of polyamines in the same order as above were: 0.88 +/- 0.31; 0.12 +/- 0.10; 0.67 +/- 0.31; and 0.09 +/- 0.08 nmoles/ml. The results show that, compared to normal subjects, the serum levels of all four polyamines are significantly elevated either in nondialyzed patients with chronic renal failure or in dialyzed patients. In dialyzed patients, the postdialysis putrescine and spermidine levels are significantly low. Serum levels of putrescine and spermidine are both significantly correlated with serum urea nitrogen and serum creatinine in a combined group of normal subjects and patients with chronic renal failure. In dialyzed patients, none of the four polyamine serum levels show correlations either with serum urea nitrogen or serum creatinine; with hematocrit, only spermine exhibits a correlationship, whereas other polyamines do not.

Determination of polyamines in plasma by capillary gas chromatography and some applications.

An analytical method is presented making it possible to measure spermidine concentration in 0.2 ml plasma. After derivatization and extraction following an existing procedure, spermidine was measured by gas chromatography using nitrogen-phosphorus detection. Mounting capillary columns decreased the limit of determination to 0.05 nanomole/ml for routine determination. Some applications of the method in animal model systems are shown. The method is also applicable to other polyamines.

[Polyamine levels in blood and plasma of patients with malignant melanoma]. / Polyaminspiegel im Blut und Plasma von Patienten mit malignem Melanom.

The polyamines putrescine, cadaverine, spermidine, spermine, and the amine histamine in whole blood and plasma of 42 patients with malignant melanoma were analysed after cold acid extraction and ion-exchange chromatography by fluorescence detection. Depending on the degree of tumor spreading the patients were grouped into 5 subpopulations; results were statistically compared with the values obtained from a normal population. The mean values of polyamine concentrations in the patient groups were generally not distinguishable from the mean values of the controls. Solely the mean of the spermidine values of postoperative patients with or without medical therapy and the spermine mean value for postoperative patients in tumor stage I and III were elevated. Cadaverine was never detected. The individual frequency of elevated polyamine levels in the plasma of single patients and variable, depending on patient subpopulation and polyamine under consideration. Spermidine was most frequently elevated (44%) in patients of tumor stage I under immunetherapy. We conclude that due to the low frequency of polyamine elevations to be found in plasma of melanoma patients by our method no diagnostic or prognostic significance and only limited importance for monitoring patients under therapy can be claimed for.

Alterations of red blood cells' polyamines during pregnancy and neonatal period.

Red blood cell polyamines were measured using high-performance liquid chromatography. Spermidine and spermine continued to increase with pregnancy and retained a high level during 24-35 weeks of gestation, then decreased at term; putrescine, however, kept the low nonpregnant level during pregnancy and puerperium. At delivery, umbilical blood showed a significantly higher polyamine level than maternal blood (p less than 0.005 or p less than 0.001). In the neonatal period, each polyamine showed a slight increase on the 1st day after birth, then spermidine and spermine decreased day by day, but putrescine kept the same level. Alterations of polyamines seemed to have some relation to the erythropoiesis in bone marrow and rapid cell proliferations.

Rapid detection of polyamines in the sera of patients with colorectal carcinoma by liquid ion-exchange chromatography.

The polyamines spermidine and spermine and the diamines putrescine and cadaverine have been determined in their free and unbound state in the sera of patients with colorectal carcinoma. The obtained values were compared with normal control levels. Elevated levels of putrescine and spermidine were found in untreated patients and declined to the control range after surgical and chemotherapeutic treatment. Spermine levels remained unchanged throughout the observation at normal control levels.

High-performance liquid chromatographic determination of free and total polyamines in human serum as fluorescamine derivatives.

A highly sensitive and simple fluorimetric method for the determination of free and total polyamines, spermidine, spermine, putrescine and cadaverine, in human serum by high-performance liquid chromatography is described. The polyamines, obtained after clean-up of deproteinized serum by Cellex P column chromatography, are converted to their fluorescamine derivatives in the presence of nickel ion which inhibits the reaction of interfering amines with fluorescamine, and the derivatives are separated simultaneously by reversed-phase chromatography (LiChrosorb RP-18) with a linear gradient elution. The lower limits of detection are 10 and 15 pmole for spermine and the others in 0.5 ml of serum, respectively.

Monoacylcadaverines in schizophrenia.

Monoacetylcadaverine and monopropionylcadaverine were found in the blood of schizophrenic patients in higher concentrations than in the blood of nonschizophrenic subjects. The blood levels of both monoacylcadaverines in schizophrenics were relatively higher during exacerbation of the illness and were relatively lower during improvement.

[Aliphatic diamines and histamine in the serum of patients with chronic polyarthritis]. / Die Untersuchung der aliphatischen Diamine und des Histamins im Serum von Patienten mit chronischer Polyarthritis.

The concentration of histamine, cadaverine, putrescine as well as spermidine and spermine was measured in blood serum of patients with rheumatoid arthritis (RA). In comparison to normals there is no statistically significant difference in the concentration of putrescine and spermidine in blood serum of RA patients. Spermine which could be detected in all samples too did not show distinct abnormalities from healthy subjects. Histamine and cadaverine present in measurable amounts were only in particular cases. A relation between increased histamine and basophile count was not demonstrable. There is apparently no correlation between histidinemia which is reported in the literature for RA patients in blood serum and plasma, and histamine production in the blood.

Monoacylcadaverines in the blood of schizophrenic patients.

Concentrations of cadaverine, monoacetylcadaverine and monopropionylcadaverine in the blood of schizophrenic and nonschizophrenic subjects were measured. Two groups, one from the U.S.A. the other from Japan, were tested. Monoacetylcadaverine and monopropionylcadaverine were found elevated in the blood of some schizophrenic patients in comparison with those in controls in each group. Their increase could be caused by a reduced monoamine oxidase activity or by an increased acylation in schizophrenic patients.