Analysis of hepatotoxicity in maternal and fetal rats after glucocorticoid administration by lipid histochemistry and thin layer chromatography

Changes in lipid metabolism of fetal and maternal rat livers were investigated on day 20 of pregnancy after administration of either 3 mg/kg or 24 mg/kg triamcinolone-acetonide or 124 mg/kg hydrocortisone in crystalline suspension to the mothers on day 15 of pregnancy. Sudan black B and Nile red as well as the UV-Schiff reaction and thin layer chromatography were used to study qualitatively the response of lipids to these glucocorticoids. Generally, after application of triamcinolone-acetonide fetal livers accumulated more lipids as toxic response to this glucocorticoid than the maternal organ; the degree of lipid accumulation was clearly dose-dependent in the fetuses. After hydrocortisone treatment, lipids in maternal livers were slightly, those in the fetuses were not affected. Histochemistry and thin layer chromatography revealed an accumulation of neutral lipids, especially of triglycerides and fatty acids which both contained increased amounts of ethylene bonds after treatment with triamcinolone-acetonide. The results also show that using combined histochemistry and thin layer chromatography, the analysis of hepatic lipids is a promising tool for the assessment of toxic effects of glucocorticoids on fetal and maternal hepatocytes in rats.     

大枣中氨基酸类成分的薄层色谱分析

中药大枣中含有多种氨基酸类成分,建立大枣中主要氨基酸类成分的薄层色谱鉴定方法,为大枣的相关研究提供依据。采用薄层色谱法对大枣中氨基酸类成分进行定性鉴别,本法操作简单,重复性好,特征明显,可以作为大枣中四种氨基酸的薄层色谱鉴别依据。     

The morphology and histochemistry of adult rats neurocytes after BCNU administration

Studies were performed on adult rats of Wistar strain given four 7-days-spaced intraperitoneal doses of BCNU with single dose resembling to used in clinical practice. The animals were sacrificed at the seventh day after the last dose of the drug. Morphological alterations were evaluated in H + E or cresyl violet stained sections. In frozen microtome sections histoenzymatic reactions were performed to detect enzymatic activity of some phosphatases and esterases. Karyo- and cytophotometric measurements of pyramidal cell nuclei in frontal and parietal cortex and of motor neurons in trigeminal nerve nucleus were performed in sections subjected to Feulgen reaction, using automatic microscopic image analyzer "Morphoquant" (VEB Carl Zeiss, Jena). The performed studies showed that administration of multiple therapeutic doses of BCNU lead to primary injury of vascular wall as oedema and proliferation of endothelium and small perivascular haemorrhages. The cytostatic drug induced a decrease in NsE and AlkP enzymatic activities, increased activity of AChE, ChE, AcP and ATPase and topographically variable changes in intensity of TPPase enzymatic reaction. Several karyo- and cytophotometric alterations were observed also in neurocyte cell nuclei which became elongated and acquired a more round shape. This was associated with a decrease in relative DNA content, loosening of nuclear chromatin structure and with shifting chromatin lumps toward periphery of cell nuclei.     

Timing of glucocorticoid administration determines severity of lipid metabolism and behavioral effects in rats

Glucocorticoids (GCs) are a group of steroid hormones secreted by the adrenal glands in circadian cycles, and the dysregulation of GC signaling has been suggested to cause metabolic syndrome. Even though prolonged GC exposure is associated with serious side effects such as metabolic syndrome and central nervous system disorders, the use of GCs in anti-inflammatory and immunosuppressive therapies has been continuously rising. Meanwhile, the exact mechanisms by which GCs can influence the lipid metabolism as well as behavior and how they are affected by time remain unknown. In this study, the effects of two different long-term GC dosing regimens on lipid metabolism and behavior were investigated. Male Wistar rats received daily administrations of the GC dexamethasone sodium phosphate (DEX, 0.5 mg/kg body weight) at either ZT0 (Dex0) or ZT12 (Dex12). After 6 weeks of treatment, DEX-treated rats, especially those treated at ZT0, had higher hepatic lipid accumulation and serum triglyceride levels and less locomotor activity than did control rats. In addition, serum levels of corticosterone, 5-hydroxy tryptamine and norepinephrine were decreased in the Dex0 group but not in the Dex12 group compared to the control group. Furthermore, quantitative real-time polymerase chain reaction analysis indicated that the chronic administration of GCs at ZT0 upregulated genes related to glycolysis and lipid synthesis and downregulated genes related to fatty acid β-oxidation in the liver more remarkably than administration at ZT12. Both DEX-treated groups displayed severely altered expression patterns of the core clock genes Bmal1 and Per2 in the liver and in fat. In addition, the expression of glutamate aspartate transporter, glial fibrillary acidic protein and glutamate transporter-1, astrocyte-related genes important for maintaining nervous system functions, was drastically decreased in the hippocampus of DEX-treated rats, especially when DEX was given at ZT0. In conclusion, our findings confirm that the severity of side effects, indicated by altered lipid metabolism and behavioral activity, depends on the timing of GC administration and is associated with the degree of glucocorticoid receptor dysfunction after dosing at disparate time points.     

Analysis of brain and myelin lipids by high-performance thin layer chromatography and densitometry

We have developed a simple method involving high-performance thin layer chromatographic separation of total brain and myelin lipids. Only two solvent systems consisting of chloroform: methanol: acetic acid and water at different concentrations were needed. The plate was then stained with three sequential procedures to visualize phospholipids, cholesterol and galactolipids. Densitometric procedure at each step of staining was utilized to obtain quantitative analysis of brain and myelin samples.     

Micronucleus formation in fetal maternal rat erythroblasts after norfloxacin transplacental administration

Single oral doses of norfloxacin (4, 2, 1, 0.5 mmole/kg) administered to pregnant rats significantly increase the frequency of micronucleated polychromatic erythrocytes both in fetal liver and in maternal bone marrow. Therefore norfloxacin in this work has been found to be a genotoxic agent.     

Analysis of Fusarium toxins in maize and wheat using thin layer chromatography

Thin layer chromatography (TLC) methods for identifying and quantifying deoxynivalenol (DON), fumonisin B1 (FB1) and zearalenone in grain samples were compared to immunoassay (ELISA) and high performance liquid chromatography (HPLC) methods to determine the reliability of the less expensive TLC. There was a very good agreement between levels of DON measured by TLC and competitive-direct ELISA, and between levels of fumonisin B1 measured by TLC and HPLC, over a wide range of concentrations. Correlation coefficients (Pearson's) were 0.978, 0.914 and 0.953 for DON in maize, DON in wheat and FB1 in maize respectively. A lower correlation coefficient (r = 0.672) was obtained when zearalenone was quantified by TLC and HPLC. Possible reasons for this are discussed. A cost comparison of the various methods revealed that TLC was the least expensive for sample analysis. It is recommended that researchers choose which analytical method to use based upon individual considerations of cost and precision. This revised version was published online in June 2006 with corrections to the Cover Date.     

Detection of toxigenic Fusarium isolates by thin layer chromatography

A simple screening method was used to investigate mycotoxin production among 114 Fusarium isolates. Zearalenone, T-2 toxin, HT-2 toxin, diacetoxyscirpenol, neosolaniol, deoxynivalenol, nivalenol, fusarenon-X, butenolide, moniliformin and equisetin were detected. The importance of the use of a range of different substrates for mycotoxin production was proved.     

Analysis of the lipid composition of human and boar spermatozoa by MALDI-TOF mass spectrometry, thin layer chromatography and 31P NMR spectroscopy

Alterations in the phospholipid (PL) composition of spermatozoal membranes occur during the fertilization process. Furthermore, membrane lipid composition is of high interest with respect to cryopreservation. The PL and fatty acid compositions of human and boar spermatozoa are compared by using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) in combination with thin-layer chromatography and 31P NMR spectroscopy. The extreme sensitivity of alkenyl-linked PL against acid treatment was used to estimate the plasmalogen content of spermatozoa. Compared with humans, boar spermatozoa are characterized by a lower variability of their PL and fatty acid composition. Additionally, boar spermatozoa contain much higher moieties of alkyl-linked compounds, e.g. 1-palmityl-2-docosapentaenoyl-sn-glycero-3-phosphocholine and 1-palmityl-2-docosahexaenoyl-sn-glycero-3-phosphocholine as well as the corresponding phosphatidylethanolamine (PE), while human spermatozoa are characterized by high contents of diacyl-PL, e.g. 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine. A considerable plasmalogen moiety, for instance 1-palmitenyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine is a typical feature of both, human and boar spermatozoa. It will be shown that these differences in PL composition can be very rapidly and conveniently assessed by MALDI-TOF MS in combination with TLC and also by 31P NMR.     

In utero glucocorticoid exposure reduces fetal skeletal muscle mass in rats independent of effects on maternal nutrition

Maternal stress and undernutrition can occur together and expose the fetus to high glucocorticoid (GLC) levels during this vulnerable period. To determine the consequences of GLC exposure on fetal skeletal muscle independently of maternal food intake, groups of timed-pregnant Sprague-Dawley rats (n = 7/group) were studied: ad libitum food intake (control, CON); ad libitum food intake with 1 mg dexamethasone/l drinking water from embryonic day (ED)13 to ED21 (DEX); pair-fed (PF) to DEX from ED13 to ED21. On ED22, dams were injected with [(3)H]phenylalanine for measurements of fetal leg muscle and diaphragm fractional protein synthesis rates (FSR). Fetal muscles were analyzed for protein and RNA contents, [(3)H]phenylalanine incorporation, and MuRF1 and atrogin-1 (MAFbx) mRNA expression. Fetal liver tyrosine aminotransferase (TAT) expression was quantified to assess fetal exposure to GLCs. DEX treatment reduced maternal food intake by 13% (P < 0.001) and significantly reduced placental mass relative to CON and PF dams. Liver TAT expression was elevated only in DEX fetuses (P < 0.01). DEX muscle protein masses were 56% and 70% than those of CON (P < 0.01) and PF (P < 0.05) fetuses, respectively; PF muscles were 80% of CON (P < 0.01). Muscle FSR decreased by 35% in DEX fetuses (P < 0.001) but were not different between PF and CON. Only atrogin-1 expression was increased in DEX fetus muscles. We conclude that high maternal GLC levels and inadequate maternal food intake impair fetal skeletal muscle growth, most likely through different mechanisms. When combined, the effects of decreased maternal intake and maternal GLC intake on fetal muscle growth are additive.     

Complete analysis of cellular nucleotides by two-dimensional thin layer chromatography

We describe methods for the complete analysis of cellular nucleotides from as few as 10(6) 32Pi-labeled cells in a simple 2-day experiment. Nucleotides are extracted with acid, neutralized, and resolved by two-dimensional thin layer chromatography on polyethyleneimine cellulose. In the first dimension the nucleotides are separated based on the negative charge of their phosphate groups (i.e. cyclic, mono-, di, and triphosphates) and in the second dimension on their content of nucleobases (i.e. Ura, Cyt, Thy, Gua, and Ade). Because the separation is logical, one can predict the chromatographic migration of most nucleotides. By running standards we have determined the chromatographic location of over 90 biologically important nucleotides, nucleotide derivatives, and modified nucleotides from tRNA. We also developed a set of enzymatic and chemical methods to be used in conjunction with the chromatographic separations for verifying the identity of nucleotides and characterizing novel nucleotides. In this paper we use these methods to analyze and inventory the nucleotide content of Salmonella typhimurium in balanced log phase growth. Other potential uses of the method are also described.     

Densitometric quantification of brain gangliosides separated by two-dimensional thin layer chromatography

A procedure for accurate densitometric quantification of gangliosides separated by two-dimensional thin layer chromatography is reported. The procedure was set up employing 9 different pure gangliosides and was applied to the analysis of calf and pig brain gangliosides. Silica gel high performance thin layer plates, 10 × 10 cm. were two-dimensionally developed at 18–20 C with the following solvents: chloroform methanol 0.2% aqueous CaCl₂, 50/40/10 by volume, for the first run; n-propanol 17 M NH₄OH/water, 6/2/1 by volume for the second run. Ganglioside spots were visualized by spraying with an Ehrlich reagent, which is specific for sialic acid, and heating at 120 C for 15 min. The spots were quantified by sequential scanning densitometry, linear responses being obtained for ganglioside amounts on the plate ranging from 0.1 to 6 nmol as bound sialic acid. The reproducibility of densitometric responses resulted to be acceptable since the standard deviation values were lower than ± 15% of the mean values also for those ganglioside species contained in minor proportions. The ganglioside mixtures of calf and pig brain were resolved in about 20 spots. Of these 9 corresponded to gangliosides GM3, GM2, GM1, Fuc-GM1, GD1a, GD1b, Fuc-GD1b, GT1b and GQ1b, which were identified with certainty and quantified. The identification of GM3 (carrying N-glycolylneuraminic acid), GD3, GD1a (carrying N-acetyl- and N-glycolyl-neuraminic acid) and GT1a was only tentative. All the other spots corresponded to unidentified gangliosides, some of them possibly new species.