Assessment of magnesium influence on fatty acid content in isolated rat hepatocytes subjected to incubation.

Magnesium salts are components of many dietary supplements used in treatment or prevention of magnesium deficiency. Hypomagnesemia usually results from an improper lifestyle, including unbalanced diet. Isolated hepatocytes of animals or humans are the preferred model used to study the in vitro effects of exogenous factors on cellular metabolic changes. The aim of this study was to evaluate the content of saturated, monounsaturated and polyunsaturated fatty acids and their esters in isolated rat hepatocytes influenced by different magnesium concentrations. The isolated rat hepatocytes were used as the test material. Hepatocytes were prepared in culture medium (Hepatocyte Medium) + MgCl(2) solution to concentrations of 2 mM/dm(3) MgCl(2), 4 mM/dm(3) MgCl(2). After incubation with different concentrations of magnesium ions, changes in the content of fatty acids and their esters were found for the whole hepatocytes and hepatocyte membranes. Despite changes in the fatty acid content in the whole hepatocytes and their membranes, there were no changes in the coefficient of degree of saturation of fatty acids when different concentrations of MgCl2 were used.

The profile of melatonin receptors gene expression and genes associated with their activity in colorectal cancer: a preliminary report.

The antiproliferative and immunomodulatory effects of melatonin (MLT) have been demonstrated in a variety of neoplasms including colorectal cancer (CRC). In humans and other mammals, MLT acts on target tissues through membrane and retinoid nuclear receptors. The aim of this study was to evaluate transcription activity of melatonin receptors and genes associated with regulation of their activity in colorectal adenocarcinoma tissues in relation to clinical stage of cancer. A total of 24 pairs of surgically removed tumoral and healthy (marginal) tissue samples from colorectal cancer patients at clinical stages I-II and III-IV were collected. As an additional control, twenty normal samples were tak¬en from people whose large intestine tissues were reported as non-tumoral after colonoscopy. Expression of mRNA genes was studied by microarray HG-U133A analysis. The analysis of gene expression profile was performed using commercially available oligonucleotide microarrays of HG-U133A. High increase of MT1 mRNA expression levels in all cancerous samples vs non-cancerous tissues was observed. The MT2 mRNA expression levels increased slightly in marginal and malignant samples. Among the genes participating in the cascade of signal transfer in cells activated by MLT via melatonin receptors, we found encoding genes (GNA11, OXTR, TPH1) only for differentiating stage III - IV of CRC. Monitoring the expression levels of genes that are related to melatonin receptors may offer a strategy to anticipate tumour development and estimate the molecular changes that occur during carcinogenesis. The mechanism behind this association needs further elucidation.

Expression of ADAM28 and IGFBP-3 genes in patients with colorectal cancer - a preliminary report.

Adamalisynes (ADAMs) play an important role in inter-membrane interactions, cell adhesion and fusion processes and protein shedding from the cell surface. Many reports indicate that members of the ADAMs family are overexpressed in human cancer. The aim of the present study was to evaluate ADAM28 and Insulin Like Growth Factor Binding Protein-3 (IGFBP-3)) gene expression in colorectal carcinoma tissues with regard to the overweight or obese status of the patients using an oligonucleotide microarray technique. Fresh tissue specimens were obtained from colorectal cancer patients during surgical treatment. Eighteen specimens from tumour and 18 normal tissue specimens from colorectal cancer patients at clinical stages III and IV were analysed. The examined patients were divided into two groups; those with BMI greater than or equal to 25 and those with normal BMI. The control group consisted of 18 specimens of non-neoplastic colon tissues, which were divided between overweight/obese and normal body weight patients. The gene transcriptional activity from the specimens was analysed using an oligonucleotide microarray technique. Microarrays and rinsing and marking solutions were prepared according to the procedure in the Gene Expression Analysis Technical Manual. The following conclusions were made: i) change of ADAM28 and IGFBP-3 genes expression are present in the normal tissue in overweight/obese patients with colorectal cancer only; ii) the observed molecular variability of ADAM28 and IGFBP-3 expression may be an initial process of cancer proliferation; iii) the histopathologically normal surgical margin in this group of patients was not equal to the molecular margin.

Changes in the content of short, medium and long-chain fatty acids in isolated hepatocytes incubated in the presence of magnesium ions and/or ethanol.

Magnesium is one of the commonly used dietary supplements. Therefore, this study was to evaluate the content of short, medium and long-chain fatty acids and their esters in isolated rat hepatocytes induced by magnesium and/or ethanol. Isolation of hepatocytes was carried out by the Seglen's enzymatic method using collagenase. To thus prepared samples ethanol and/or MgCl2 solution were added, respectively, so that their concentrations were as follows: 150 mM/dm3 ethanol and/or 2 mM/dm3 MgCl2, 4 mM/dm3 MgCl2. The contents of short, medium and long-chain fatty acids and those of ester-bound acids were determined. The statistical evaluation of the experiment was made by comparing the area normalized for the analysed fatty acids in hepatocytes incubated for 5 h in the presence of the test substances. The effect of magnesium ions on the content of fatty acids and their esters in isolated hepatocytes incubated for 5 h depended on their concentration in the medium. A normalizing effect of magnesium ions on ethanol-induced changes in the content of C14-C17, C18-C20 and C21-C24 fatty acids was demonstrated. A normalizing effect of magnesium on ethanol-induced changes in the content of ester-bound fatty acids in hepatocytes was not confirmed.

Effect of magnesium ions on ethanol-induced changes in the pool of saturated, mono- and polyunsaturated fatty acids in isolated rat hepatocytes.

The aim of the paper was the assessment of the effect of magnesium ions on ethanol-induced changes in the content of ester-bound fatty acids in isolated rat hepatocytes and their cell membranes. Hepatocytes were isolated by means of the enzymatic method of Selgen using collagenase. The number and viability of isolated rat hepatocytes, incubated for 5 hours in culture media (Hepatocyte Medium) with ethanol and MgCl2 solutions with concentration amounting respectively to: 150 mM/dm3 of ethanol and/or 2 mM/dm3 of MgCl2, 4 mM/dm3 of MgCl2, were determined. Biochemical tests of hepatocytes were performed, consisting in the determination of the total content of ester-bound fatty acids in whole hepatocytes and their cell membranes after incubation. Confirmed normalising action of magnesium ions with respect to the effects induced in hepatocytes and their membranes by the presence of ethanol should be attributed to the important role of magnesium which it performs during reactions taking place with participation of ATP.

Expression of genes associated with H factor in fibroblasts infected with Borrelia spirochaetes.

Infection with Borrelia spirochaetes leads to the launch of specific and non-specific immunological response in humans. Activation of the complement system is one of the first defence mechanisms against penetrating pathogens. The aim of this study was to select genes related to the alternative pathway of the complement system [including complement factor H (CFH)], differentiating the type of infection in the system model, that is, a culture of normal human diploid fibroblasts infected with three different spirochaete genospecies: Borrelia afzelii, Borrelia garinii and Borrelia burgdorferii sensu stricto, by comparing the infected fibroblast culture with the control fibroblast one. With the use of oligonucleotide microarrays HGU-133A, the differences in the expression of genes selected on the basis of a scientific database Affymetrix were studied by comparing transcriptomes from the four cultures of fibroblasts. In the result of infection of fibroblast cultivation with a specific Borrelia genospecies, a variable expression of certain CFH and complement system-associated genes, specific for one genospecies only, B. afzelii- C1QBP, CD59, C2, CD46 and FHL1; B. garinii - C1S and CLU; Borrelia burgeforii- CFB, A2M and VSIG4, was observed. CFH differentiates infections induced by B. afzelii and B. garinii from infections induced by B. burgdorferii sensu stricto.

Analysis of expression profile of gene encoding proteins of signal cascades activated by insulin-like growth factors in colorectal cancer.

The aim of the study is to analyse gene typing with the use of the microarray technique (HG-U133A, Affymetrix), differentiating colorectal cancer tissues from tissues assessed histopathologically as healthy ones among a panel of 93 mRNA of gene encoding proteins involved in the activation of cellular signal transduction pathways by insulin-like growth factors. The study was conducted on a group of 8 colorectal cancer patients. Frozen tumor and healthy specimens from the patients were used in molecular tests. Transcript IGF2 differentiated cancer from healthy tissue. Among the genes participating in the cascade of signal transfer in cells activated by IGF, GRB10, PIK3R3, PIK3R1, and IRS1 were qualified as differentiating transcripts. IRS1 indicated over-expression in tumour. Transcript SMAD2 showed a significant changed in tumour samples (increased expression).

Statistical analysis of differential gene expression in colorectal cancer using CLEAR-test.

CLEAR test provides a novel method of analysis by combining inference for differential expression and variability. Frozen tumor specimens from 14 (3 coded Stage I, 5 Stage II, 2 Stage III and 4 Stage IV) colon cancer patients were obtained. Archived primary tumor samples were collected at the time of surgery and normal colon mucosae (controls specimens) were also collected. The studied transcriptomes were clustered using hierarchical agglomeration with Ward's method and Tchebychev distance. The separable groups of transcriptomes were classified as high clinical stage of adenocarcinoma (HCS; stages II-IV), low clinical stage of adenocarcinoma (LCS; stages I and 3 controls), and two normal colon mucosae (controls N1 and N2). The results of the CLEAR-test algorithm in normal colon specimens and adenocarcinoma specimens with low and high clinical stage showed 50 most and 50 least significant genes. The list of differential genes (p<0.01) in normal colon specimens and adenocarcinoma specimens with low and high clinical stage presented 58 genes.

Magnesium content in daily food portions and the influence of supplementation.

Magnesium is one of the most important cations for an organism. The aim of our study is to evaluate whether the use of a magnesium formulation as a diet supplement or medical treatment is necessary. The 24-hour recall method was used to obtain information regarding the daily magnesium consumption of 949 people. The results were compared with the Estimated Average Requirement (EAR) and Recommended Daily Allowance (RDA) values. The average daily requirement for magnesium was exceeded by 292 (183 women and 109 men) of the 949 respondents. This research confirmed excessive magnesium intake by both men and women that exceeded both the EAR and the RDA. Uncontrolled, excessive dietary supplementation or medical treatment with magnesium by this group may constitute a health threat.

Leptin and its receptors in obese patients with colorectal cancer.

The purpose of this study is to examine serum concentration of leptin and that of the soluble form, the Ob-Re receptor, in patients with colorectal cancer, as well as to examine the level of leptin mRNA and that of its receptors, Ob-Ra and Ob-Rb, in large intestine specimens collected from patients with colorectal cancer, depending on cancer clinical and pathological progression and BMI. A total of 146 patients with colorectal cancer in a I-IV stage scale according to the TNM Classification were enrolled. The patients were divided into two groups according to BMI calculations based on body weight and height: a Study group (BMI greater than or equal to 25 kg/m2) of 75 patients aged 57 plus or minus 4.5 years and a Control group (20 less than BMI less than 25 kg/m2) of 71 patients aged 60 plus or minus 5 years. The experimental part of the work was performed in two stages: Stage I regarding the assay of leptin concentration and that of its soluble receptor, Ob-Re, in the serum of patients with the use of the ELISA method; and Stage II to determine the number of leptin mRNA copies and two isoforms of leptin receptors, Ob-Ra and Ob-Rb, using the QRT-PCR method in tissue specimens collected from 146 patients. In our results the concentration of serum leptin and Ob-Re was not dependent on the stage of clinical and pathological progression of the cancer. There was a statistically significant higher serum leptin level in colon cancer patients who were overweight or obese compared to patients with normal weight. No presence of mRNA of the gene encoding leptin was found in tissues collected from colorectal cancer patients. The number of mRNA copies of Ob-Rb was statistically significantly higher in all the study groups compared to the reference tissues.

Influence of magnesium on fatty acids and their esters in isolated rat hepatocytes.

The aim of the study is to analyse the changes in the profile of fatty acids and their esters in rat hepatocytes that were incubated for 5 hours with different concentrations of MgCl2 (2 and 4mM) in hepatocyte culture medium. The methyl esters of fatty acids were identified with a GC-MS system included in the Hewlett?Packard quadrupolar mass spectrometer, coupled with a Hewlett-Pacard 5890 gas chromatograph with an ionisation potential of 70 eV and recorded on a Vectra 386 computer. We observed differences in the amount of saturated, monounsaturated and polyunsaturated fatty acids among the examined samples. In the control sample, the largest component consisted of the pool of saturated and monounsaturated fatty acids. Analysing the changes in the profile of ester-bound fatty acids, we found statistically significant differences when 4 mM MgCl2 was presented. The amount of C18:2, C18:1b and C20:4a decreased in comparison with the control sample.

Ethanol-induced changes in profile of fatty acids in isolated rat hepatocytes and the influence of magnesium ions.

The influence of magnesium and ethanol on the fatty acid content in isolated rat hepatocytes was examined in this study. The isolated liver cells were obtained according to the Selgen method and then subjected to ethanol alone or both ethanol and magnesium activity. MgCl2 was used in two concentrations: 2 and 4 mM. The profile of fatty acids in the hepatocytes was evaluated after 5 hours of incubation. Our results revealed that magnesium ions presented together with ethanol in hepatocyte medium changed the hepatocyte fatty acid profile. The total amounts depended on the concentration of magnesium ions.

The availability "in vitro" of magnesium formulations using various leaching media.

The aim of this study was to determine the availability "in vitro" of magnesium ions from five tablet formulations. In our experiment the flow-through method according to Grzesiczak was used. The leaching media in above method were: an aqueous solution of hydrochloric acid at pH 1.0 used as artificial stomach fluid, an aqueous solution of hydrochloric acid at pH 4.0 and physiological salt solution.

Bioavailability of magnesium ions from solid dosage forms.

The bioavailability of magnesium ions from three formulations: I - the granulate, II - the frothy mixture and III - the tablets was determined. To calculate the cmax, tmax and AUC the computer programs AUC-RPP and KINPAK were used.

[The dynamics of the drug release from tablets. Part 6: Liberation of glaucine from Tussiglauzin Dragées (author's transl)]. / Dynamik der Arzneimittelabgabe aus Tabletten. 6. Mitteilung: Liberation von Glaucin aus Tussiglauzin-Dragees.

The liberation of glaucine hydrobromide from Tussiglauzin dragées has been verified. The release to artificial gastric juice was determined by three different procedures: method using the apparatus according to Grzesiczak, stationary-basket method and rotating-basket method. All three methods showed that the largest proportion of the drug (greater than 90%) is released within 2.5 h, the maximum amount determined by means of the rotating-basket method being 99.2%.

[The dynamics of the drug release from tablets. Part 7: Comparison of three liberation models on Cholestil tablets (author's transl)]. / Dynamik der Arzneimittelabgabe aus Tabletten. 7. Mitteilung: Vergleich von drei Liberationsmodellen am Beispiel der Cholestil-Tabletten.

The liberation of hymecromone from Cholestil tablets (0.2 g) has been determined by three procedures; half-change method, stationary-basket method, and rotating-basket method. The release rate was highest when determined by the rotating-basket method. The other two methods yielded comparable, but considerably lower values. Artificial gastric and intestinal juices according to the Polish Pharmacopoeia IV have been used in this study.