Novel ion exchange chromatography method for nca arsenic separation.

A high-performance liquid chromatography (HPLC) device equipped with an anion exchange column was used to isolate nca 77As from reactor irradiated natGeO2 targets. The oxidation states of the isotope 77As during the process was verified by thin layer chromatography. The radionuclidic purity of the separated fractions was checked by gamma measurements and it was found to be 99.91% for the As fraction. The elaborated method was applied to separate the isotope 74As from cyclotron irradiated natGeO2 targets too.

Liquid chromatographic and mass spectrometric analysis of human serum acid alpha-1-glycoprotein.

Human serum acid alpha-1-glycoprotein (AGP, orosomucoid) content of healthy individuals and cancer patients was measured, isolated and purified using a protocol of fast and biocompatible sample preparation, ion exchange and dye-ligand affinity chromatographic methods. In comparison to the healthy individuals significantly higher serum AGP levels were found in a wide spectrum of cancer patients, indicating its diagnostic value in the malignant disease. Oligosaccharide content of AGP samples was separated following PNGase F enzyme digestion and analysed by RP-HPLC and MALDI-TOF mass spectrometry. RP-HPLC and MALDI-TOF mass spectrometric analysis of sugar constituents of AGP specimen originated from selected cancer patients with high serum AGP levels indicated the appearance of anomal distribution of bi-, tri- and tetra-antennary oligosaccharide structures compared to the healthy controls.

Use of serum 5-S-CD and S-100B protein levels to monitor the clinical course of malignant melanoma.

5-S-Cysteinyldopa (5-S-CD) is a precursor of pheomelanin. S-100B protein is a low molecular weight, acidic, calcium binding, cytoplasmic protein. In this study, the concentration changes of serum 5-S-CD and S-100B protein in melanomas of all stages were examined in parallel and patients were monitored during and after treatment. Serum samples were taken from 478 melanoma patients on 1924 occasions. Of these, 180 cases were regularly monitored. Concentrations of 5-S-CD were determined by high performance liquid chromatography (HPLC), S-100B protein by immunoluminometric assay. The mean/median concentrations of 5-S-CD and S-100B protein in Stage I, II and III patients and in the control group ranged around the normal level. In Stage IV patients, 58.4/50.6% sensitivity, 100% specificity and 100/86.6% positive predictive values were obtained concerning S-100B protein and 5-S-CD, respectively. Recurrence was observed in 57/180 of the regularly monitored patients in Stages I, II and III. In 10/57 (17.5%) of these patients suffering from any type of disease progression increases in both marker levels preceded the detection of metastasis by conventional methods. We can confirm that changes in both marker concentrations correlated with the stages of the patient. The markers are most sensitive in Stage IV patients and also have a high specificity in these patients. In Stage IV melanoma patients, 5-S-CD and S-100B protein levels are independent significant prognostic factors. In almost one fifth of patients both marker levels increased before the detection of metastatic disease with other appropriate, routinely scheduled investigations. This study suggests that serial serum marker measurements in the management and follow-up of melanoma patients should be examined further.

Clinical significance of 5-S-cysteinyldopa monitoring in patients with malignant melanoma.

5-S-cysteinyldopa is a precursor of melanin. Its serum and urinary level can reflect melanoma progression. In this study the concentration changes of 5-S-CD in melanomas of all stages were examined, and patients were monitored during and after treatment. Serum samples were taken from 479 melanoma patients with different Stages on 1924 occasions, from June 1996 to December 2000. Levels of 5-S-CD were determined by HPLC. The mean/median value of 5-S-CD in the Stage I-II-III patients and in the control group ranged around the normal level. Significant difference was found between Stage III and Stage IV as well as between patients with no evidence of disease and patients with tumor burden. In Stage IV 69.7% sensitivity, 61.5% specificity and 79.3% positive predictive value were evaluated. The survival of patients with normal 5-S-CD level (n=235) differed significantly from cases having elevated marker concentration (n=244). One hundred eighty cases were regularly monitored on 1210 occasions. Recurrence development was noticed in 57 patients. In 24.6% of these patients suffering from any type of disease progression the increasing marker level preceded the detection of metastasis by conventional methods. Serum 5-S-CD in Stage IV is sensitive enough to detect distant metastasis, and its predictive value has a great importance. It is a reliable marker for monitoring the clinical course in malignant melanoma.

Serum concentration of 5-S-cysteinyldopa in patients with melanoma.

BACKGROUND: 5-S-cysteinyldopa (5-S-DC) is a precursor of melanin. Its serum and urinary level can reflect melanoma progression. In this study we examined the concentration changes of 5-S-CD in melanomas of different clinical stages and in patients with different symptoms of melanoma, during and after treatment. METHOD: Serum samples were taken from 252 melanoma patients on 765 occasions, from June 1996 to July 1998. Levels of 5-S-CD were determined by HPLC. RESULTS: The value of 5-S-CD in patients with primary melanoma and in patients without symptoms ranged around the normal level. There was a significant difference between the values of patients with or without symptoms. There was also a significant difference between the 5-S-CD values at clinical Stage I and Stage III, as well as at clinical Stage II and Stage III, respectively. Analysing the values of patients with symptoms we found a significant difference between the mean values of primary tumour and stage III, between values in lymph node metastasis and stage III, between values in lung metastasis and stage III. The tumour burden was found to correlate with a rising marker level. In 7% of the symptomatic patients that had a marker level under the upper limit, amelanotic primary tumour was detected. CONCLUSION: According to the high marker level in lung and liver metastases, the marker might be useful in monitoring both patients with disease free ocular melanomas, to detect liver metastasis and high-risk patients after surgical removal of the primary tumour to reveal lung metastases.

Hydrophobic interaction chromatography of human serum alpha1-antitrypsin and alpha1-acid glycoprotein.

The relative hydrophobicity of human serum alpha1-antitrypsin (AAT) and alpha1-acid glycoprotein (AGP) in comparison to various reference proteins was determined by hydrophobic interaction chromatography (HIC). Apolar character of glycoproteins was generated using three different cosmotropic salts, ammonium sulfate, sodium sulfate, sodium citrate and isocratic, or reversed linear gradient elution techniques. Human serum AAT and AGP showed different apolar properties on Fractogel EMD phenyl and propyl columns modulated either by the type and concentration of cosmotropic salts, or by the pH of the mobile phase. According to its higher carbohydrate content AGP proved to be more polar than AAT. Human serum AAT and AGP were pre-separated by Cibacron Blue F3G-A dye ligand affinity chromatography and based on their different hydrophobicity were fractionated and purified by HIC.

Serum levels of S-100 protein and 5-S-cysteinyldopa as markers of melanoma progression.

Serum S-100 protein is widely used as a marker of melanoma and since 5-S-cysteinyldopa (5-S-CD) is a precursor of melanin its serum and urinary levels can reflect melanoma progression. In this study we examined the concentration changes of serum S-100 protein and 5-S-CD in 252 melanoma patients of different clinical stages. Serum samples were taken from 252 melanoma patients at 860 times, from June 1996 to July 1998. The serum S-100 protein was measured by the immunoluminometric assay, levels of 5-S-CD was determined by HPLC. The value of S-100 protein in patients with primary melanoma (0.11m mg/l) and in patients without symptoms (0.15 m mg/l) ranged around the normal level (0.01 0.12 m mg/l). There was a significant difference between the values of patients with or without symptoms. There was a similarly significant difference between the S-100 values of clinical Stage I (0.11 mg/l) and Stage III (2.91 mg/l) as well as between those of clinical Stage II (0.47 mg/l) and Stage III (2.91 mg/l), respectively. Analyzing the values of patients with symptoms we observed significant difference between the S-100 protein values of patients with primary tumor and those with solitary or multiple distant metastases. In case of 5-S-CD significant difference was found between clinical Stage I and III as well as clinical Stage II and III. Furthermore, there was a significant difference between the mean marker values of patients with primary tumor, lymph node, lung metastasis and clinical stage III.

[Determination of serum S-100 protein and 5-S-cysteinyl-DOPA levels in melanoma patients]. / S-100 protein és 5-S-ciszteinil-DOPA vizsgálata melanomás betegekben.

This was the first time that authors detected se-S-100 and 5-SCD values with patients with malignant melanoma in Hungary. They examined the change of serum S-100 and 5-SCD value parallel. Sera were obtained with 184 melanoma patients 326 times. Patients were ranked into groups on the basis of clinical symptoms: free of symptoms and suffering from it (primary tumour, regional lymph node metastasis, soliter or multiplex distant metastasis). On the basis of the initial results the following have been found: S-100 protein and 5-SCD serum levels had no prognostic value in patients with primary melanoma. Patients without symptoms showed values around the normal level. There was significant difference in both markers between patients with or without symptoms. Significant differences were found between clinical stage I and II, as well as in clinical stage II and III. In the case of S-100 protein there was significant difference between the values of patients with soliter and multiplex distant metastasis.

Effects of methylacetylenic putrescine, an ornithine decarboxylase inhibitor and potential novel anticancer agent, on human and mouse cancer cell lines.

Sensitivity of several human and mouse cancer cell lines to methylacetylenic putrescine (MAP) was evaluated using clonogenic, sulforhodamine B and cell counting assays. The effects of MAP on cell morphology, cell cycle phase distribution and changes in polyamine metabolism of xenografted MCF-7 and MDA-MB-231 human mammary tumor cells were also investigated. On the basis of IC50 values, BHT-101 human thyroid carcinoma cells were the most sensitive (9 micrograms/ml), followed by P388 mouse lymphoma (32 micrograms/ml), MCF-7 (48 micrograms/ml) and MDA-MB-231 (110 micrograms/ml) human breast carcinoma cell lines. MAP treatment led to accumulation of P388 cells in G1 phase. At higher doses, the cytoplasm of the cells became vacuolated followed by apoptosis. The foamy cytoplasm may suggest a rare type of cell death (Clarke III type) called non-apoptotic programmed cell death. MAP treatment resulted in a total inhibition of ornithine decarboxylase (ODC) activity with a concomitant decrease of intracellular polyamine (mostly putrescine and spermidine) content in the breast cancer cells, whilst the spermine concentration was shown to increase. MAP proved at least 10 times more potent than the formerly studied DL-alpha-difluoromethylornithine making it an attractive candidate for clinical testing.

[Serum acid alpha-1-glycoprotein (APG) levels in gastrointestinal tumors]. / A szérum savanyú alfa-1-glikoprotein (AGP) szintje gastrointestinalis tumorokban.

Using a new microanalytical method authors investigated the AGP level in the sera of 41 healthy individuals and 87 patients with and without malignant diseases of gastrointestinal tract verified clinically by other diagnostic (image forming) techniques. It could be stated that serum AGP levels measured in healthy persons were in good agreement with the data of others, and selecting 80 mg/dl concentration as upper limit of normal values (cut-off level, mean + 2SD) the specificity of the test for a healthy population was 95 per cent. AGP content in the sera of patients with various malignancies of the gastrointestinal tract, mainly of colon and stomach was significantly higher in comparison to the healthy controls. On the other hand, 16 patients from 22 gastrointestinal cases without malignant diseases represented serum AGP concentrations within the normal range, while 6 patients with acute phase of inflammation had specifically elevated AGP levels. According to these data specificity of 72.7 per cent and sensitivity of 78.5 per cent (for colorectal tumours 82.9 per cent) of the test were obtained in average for the gastrointestinal malignancies. The positive predictive value of the marker was 89.5 per cent. Our investigations demonstrated that a marked elevation of serum AGP level indicates malignant process in the diseases of gastrointestinal tract with high probability. Results presented here led to the conclusion hat determination of the serum AGP concentration performed and evaluated simultaneously with other diagnostic (image forming) procedures can be applied successfully in the recognition of gastrointestinal tumours.

A re-evaluation of the lipid-bound sialic acid determination.

The origin of serum sialic acid measured in the lipid-bound sialic acid determination reported by Katopodis et al. (1980) was investigated in detail. By varying the experimental conditions of sample preparation the protein, lipid and sialic acid contents of the methanol-water extract obtained from human sera were analyzed and compared in healthy controls and cancer patients. Using polyacrylamide gel electrophoretic and gel chromatographic methods it has been shown that most of the lipid-bound sialic acid was attributed to the acid alpha 1-glycoprotein (orosomucoid) fraction of human sera. Based on these observations a re-evaluation of the molecular background of the LBSA determination seems to be necessary.

Comparative studies on the polyamine metabolism and DFMO treatment of MCF-7 and MDA-MB-231 breast cancer cell lines and xenografts.

In order to characterize the estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cells and xenografts, their growth kinetic parameters and some biochemical characteristics concerning the receptor status and polyamine metabolism were determined and compared. The doubling times calculated from the growth curves showed higher proliferation rate of MDA-MB-231 cells, both in culture (21 hours) and in xenograft (9.7 days), in comparison to the MCF-7 cells which had values of 32 hours and 11.6 days, respectively. Growth-dependent changes observed in the intracellular putrescine, spermidine and spermine concentrations indicated a higher activity of polyamine metabolism in the MDA-MB-231 cells and xenograft as well. However, biosynthetic key-enzyme ornithine decarboxylase activity (ODC, EC 4.1.1.17) showed neither characteristic differences between the two types of breast cancer, nor consistent relationship with their proliferation rate. Metabolic alterations of the MCF-7 and MDA-MB-231 cell lines grown in vitro were also reflected in the polyamine composition of their culture medium. Independently of their receptor status, both types of breast cancer were responsive to difluoromethylornithine (DFMO) treatment. DFMO inhibited the ODC activity totally and depleted the cellular polyamine levels. MCF-7 cells in culture were more sensitive to the antitumoral effect of DFMO than the MDA-MB-231 line, while the rate of growth inhibition did not differ significantly in the xenografts. The present results provided further evidence on the different polyamine metabolism of ER-positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cells in vitro and in vivo, suggesting a correlation of hormonal modulation with polyamines as a determinant group of biological response modifiers.

Influence of luteinizing hormone-releasing hormone agonists on human mammary carcinoma cell lines and their xenografts.

The specific binding of luteinizing hormone-releasing hormone (LH-RH) agonist in estradiol-dependent MCF-7 and estradiol-independent MDA-MB-231 human breast cancer cells has been studied using [3H]Ovurelin [(D-3H-Phe6),des-Gly10-LH-RH- ethylamide]. The results of Scatchard analyses suggest the presence of a single class of receptor sites, both in cell suspensions and membrane fractions. Evaluation of these peptide receptors appears to reflect additional characteristics of biological behaviour of these human breast cancer cells. The synthetic LH-RH agonist Ovurelin [(D-Phe6),des-Gly10-LH-RH-ethylamide] can directly interfere (25-30%) with the proliferation of MDA-MB-231 human breast cancer cells in culture. The inhibitory effect of Ovurelin in vitro was negligible in the MCF-7 cell line. In the in vivo experiments the treated immunosuppressed mice bearing either MCF-7 or MDA-MB-231 xenografts responded to the high-dose LH-RH analogue Zoladex depot and Decapeptyl depot therapy. Since the MDA-MB-231 tumour was found to be ER-negative it seems possible that the regression of this xenograft results from the direct antitumor action of the LH-RH agonist.

Lipid-rich cell thyroid adenoma: histopathology with comparative lipid analysis.

A second case of the unique lipid-rich cell thyroid adenoma is described complemented by detailed lipid analysis. New observations were made. The cytoplasm of the tumour cells contained scattered, aggregated sudanophil crystals; under polarized light the frozen, unstained sections exhibited numerous birefringent lipid crystals; electron microscopy provided further evidence that the clear cell appearance was due to intracellular lipid droplets with scanty glycogen particles. Comparative lipid analysis by thin layer chromatography and high-pressure liquid chromatography (HPLC) revealed quantitative and qualitative differences in lipid composition of tumour cells when compared with goitre cells from normal thyroid gland and subcutane fat. Qualitative differences in triglyceride composition (by HPLC) between tumour cells and subcutaneous fat indicated that the fat accumulation in the follicle cells was not a result of simple storage, but an expression of altered intracellular lipid metabolism.

Application of the fast protein liquid chromatographic system and MonoQ HR 5/5 anion exchanger to the separation of nucleotides.

The applicability of a new type of anion exchanger, MonoQ HR 5/5, and the Pharmacia-LKB fast protein liquid chromatographic (FPLC) system to the separation of nucleotides is described. The elution characteristics of adenosine-5'-, cytidine-5'-, uridine-5'-, guanosine-5'-mono-, -di- and -triphosphates and inositol-5'-monophosphate reference compounds, and of nucleotides originating from various biological samples, are optimized by varying the concentration gradient programme with ammonium phosphate buffer. Some practical examples of biological interest for monitoring the metabolic changes of nucleotides are presented.

Induction of erythroid differentiation and modulation of gene expression by tiazofurin in K-562 leukemia cells.

Tiazofurin (2-beta-D-ribofuranosyl-4-thiazole-carboxamide; NSC 286193), an antitumor carbon-linked nucleoside that inhibits IMP dehydrogenase (IMP:NAD+ oxidoreductase; EC 1.1.1.205) and depletes guanylate levels, can activate the erythroid differentiation program of K-562 human leukemia cells. Tiazofurin-mediated cell differentiation is a multistep process. The inducer initiates early (less than 6 hr) metabolic changes that precede commitment to differentiation; among these early changes are decreases in IMP dehydrogenase activity and in GTP concentration, as well as alterations in the expression of certain protooncogenes (c-Ki-ras). K-562 cells do express commitment-i.e., cells exhibit differentiation without tiazofurin. Guanosine was effective in preventing the action of tiazofurin, thus providing evidence that the guanine nucleotides are critically involved in tiazofurin-initiated differentiation. Activation of transcription of the erythroid-specific gene that encodes A gamma-globin is a late (48 hr) but striking effect of tiazofurin. Down-regulation of the c-ras gene appears to be part of the complex process associated with tiazofurin-induced erythroid differentiation and relates to the perturbations of GTP metabolism.

Changes in the polyamine and nucleotide metabolism of P388 leukemia cells treated with DL-alpha-difluoromethylornithine in culture.

Influence of DL-alpha-difluoromethylornithine (DFMO) treatment on the growth kinetics, labelling index, extra- and intracellular polyamine and nucleotide concentrations was monitored in cultured P388 leukemia cells. A substantial decrease of cell proliferation was observed when the cells were continuously treated with 1-5 mM DFMO. Depletion of cellular polyamines, mostly of putrescine and spermidine, was seen with a concomitant but delayed increase of spermidine and spermine levels in the culture medium. Changes of DNA content and of labelling index of untreated and treated cells seem to indicate that DFMO arrested cells in G1/S transition. The results presented here provide additional in vitro evidence on the characteristic changes in the metabolic imbalance of ornithine in tumor cells induced by DFMO via inhibition of ornithine decarboxylase and ornithine carbamoyl transferase activities.

Permanent metachromatic effect of 1,9-dimethyl-methylene blue on acidic mucopolysaccharides of mastocytes.

The 1,9-dimethyl-methylene blue (SERVA) has a very strong metachromatic reaction with sulphated mucopolysaccharides of mastocytes at pH = 1 (Tris-HCl buffer). This metachromasia is retained also at increasing pH (2.5-5), and is not affected either by the quality or by the concentration of the ions present (1/240 M Tris-HCl, and 0.2 M phosphate buffer). The smears do not need any special mounting medium, only Canada balsam. The metachromasia is preserved for a considerable length of time.

Comparison of high-performance ion-exchange and ion-pair liquid chromatographic methods for the separation of tumour cell nucleotides.

Ion-exchange liquid chromatography (IELC) on a novel anion-exchanger, Polyanion SI HR 5/5, and the ion-pair technique (IPLC) using Hypersil ODS and/or MinoRPC reversed phases with tetrabutylammonium phosphate as pairing agent were compared for the separation of nucleotides. Modifications to the concentration gradient in IELC in the range 0.01-0.3 M ammonium phosphate resulted in the simultaneous separation of twelve to fourteen biologically important nucleotides. IPLC studies revealed that the capacity factors and resolution of nucleotides were more sensitive to the ionic strength than the methanol content. It was concluded that a well controlled ion concentration (0.08-0.09 M sodium chloride) should be maintained in the mobile phase and a linear methanol gradient ranging from 0 to 20% (v/v) was suitable for optimal resolution. Separations of four nucleotides and twelve nucleotides were further improved using a mixed-type reversed-phase column (C2/C18, MinoRPC). Using these complementary methods, it was possible to reveal the metabolic changes induced by different drug treatments (cyclophosphamide, DL-alpha-difluoromethylornithine) in the nucleotide pool of P388 leukaemia cells.

In vivo effects of DL-alpha-difluoromethylornithine on the polyamine and nucleotide phosphate metabolism in P388/S leukemia cells.

In vivo effects of DL-alpha-difluoromethylornithine (DFMO) on the metabolism of polyamines and nucleotide phosphates were monitored in P388/S leukemia cells grown intraperitoneally in BDF1 inbred male mice. Inhibiting the ornithine decarboxylase (ODC) activity DFMO depleted putrescine and spermidine to 30-50 and 50-60%, respectively, and increased spermine to 25-60% compared with the controls, when given as 2% solution in drinking water of the tumor-bearing animals. DFMO treatment caused a parallel 56% elevation of total nucleotide content in tumor cells with distinct and significant increase of some nucleotide phosphates. The most pronounced alterations were shown in the intracellular UTP (202%), CTP (103%), ADP (92%) and ATP (71%) concentrations. Changes in polyamine and nucleotide phosphate metabolisms were dependent on tumor progression. A possible explanation of the metabolic events induced by DFMO is discussed.