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Biomedical subjects

T M Jefferies

Publications and source records attributed to T M Jefferies.

At least 19 recordsLinked to original sources
PubMed2000

Seminal plasma reduces exogenous oxidative damage to human sperm, determined by the measurement of DNA strand breaks and lipid peroxidation.

Exposure of spermatozoa to reactive oxygen species (ROS) has been associated with cellular injury, that includes DNA damage and lipid peroxidation. In addition, sperm preparation techniques such as centrifugation, commonly used prior to in vitro fertilization and scientific studies, are associated with the generation of ROS and an increase in the level of DNA damage. The preservation, therefore, of sperm in vitro that might decrease the potential for oxidative DNA damage to arise and allow for an improvement in semen quality used for artificial insemination, is of importance. Seminal plasma is a rich source of antioxidants, which, potentially, safeguards sperm from oxidative attack during storage and once ejaculated. We have investigated the protection of human spermatozoa from ROS afforded by seminal plasma. Sperm were exposed to exogenous ROS by incubating the cells with hydrogen peroxide in the presence of ferrous sulfate and ADP. Aliquots of seminal plasma were added to the incubation mixture in differing amounts, and the generation of DNA strand breaks and thiobarbituric acid reactive species (TBARS), indicative of lipid peroxidation, determined. Incubation of sperm with exogenous ROS resulted in a significant generation of DNA strand breaks and lipid peroxidation compared to basal levels of damage (P<0.05). Addition of seminal plasma to the incubation media produced a significant decrease in DNA strand breaks and TBARS (P<0. 05), when the amount of plasma added exceeded 60% v/v. The results indicate that spermatozoal oxidative damage induced by exogenous ROS, specifically DNA damage and lipid peroxidation, is reduced by the presence of seminal plasma.

DNA↗
PubMed1999

Sperm chromatin damage associated with male smoking.

Cigarette smoke is a rich source of mutagens and carcinogens; thus, we have investigated the effects of male smoking on the DNA of human sperm. This was performed using the sperm chromatin structure assay (SCSA), which measures the sensitivity of sperm DNA to acid induced denaturation, and the terminal deoxynucleotidyl transferase assay (TdTA), which measures DNA strand breaks by addition of the biotinylated nucleotide dUTP to 3'-OH ends of DNA, sites of DNA breakage, using the enzyme terminal deoxynucleotidyl transferase. Sperm from subjects who smoked were significantly more sensitive to acid induced denaturation than non-smokers (P<0.02) and possessed higher levels of DNA strand breaks (P<0.05). We hypothesise that smoking damages the chromatin structure and produces endogenous DNA strand breaks in human sperm. These changes may result in sperm DNA mutations, that predispose offspring to greater risk of malformations, cancer and genetic diseases.

Chromatin↗
PubMed1999

Extra-epididymal spermatozoa express nuclear abnormalities.

Extra-epididymal spermatozoa account for approximately a third of all spermatozoa found in the normal human ejaculate. Whilst remaining outside of the testes at core body temperature, the functional competence of spermatozoa, including cell motility and fertilizing capacity, diminishes. By examining spermatozoa found in the seminal fluid of recently vasectomized men, this study has investigated the nuclear changes that occur in spermatozoa whilst persisting in sites distal to the epididymis. Spectral recordings of spermatozoa stained with the nucleic acid dye, toluidine blue and the sperm chromatin structure assay (SCSA) were performed. Toluidine blue staining of human sperm DNA is an effective predictor of abnormal protamine disulphide crosslinking and chromatin condensation. Using flow cytometry, the SCSA determines the sensitivity of sperm DNA to acid-induced denaturation, providing a measure of chromatin and DNA damage. Abnormal protamine disulphide crosslinking and chromatin condensation was significantly higher in spermatozoa from patients after vasectomy when compared to normozoospermic controls (p < 0.01). Additionally, spermatozoa from vasectomized donors were significantly more sensitive to acid-induced denaturation than were normozoospermic donors (p < 0.05). The results indicate that spermatozoa surviving in extra-epididymal sites are more likely to possess DNA and chromatin abnormalities than those present in the testes and epididymis. These changes may partly explain the depletion of cell viability and fertilizing capacity of extra-epididymal spermatozoa which has been reported previously.

Cell Nucleus↗
PubMed1999

Antioxidant capacity of the epididymis.

The human epididymis provides an optimal environment for the storage and maturation of spermatozoa. However, the ability of the epididymis to protect spermatozoa from oxidative attack whilst stored at this site, through the local actions of antioxidants, has not thus far been well studied. This study assessed the contribution of the epididymis to seminal plasma antioxidant activity, by comparing the semen of normozoospermic and vasectomized men. Total seminal plasma antioxidant activity was measured, as were concentrations of urate, ascorbate and thiols, antioxidants that are abundant in human semen. Thiobarbituric acid reactive species (TBARS) were measured to indicate lipid peroxidation. Total antioxidant activity and thiol content were significantly lower (P < 0.05) in the plasma from vasectomized men compared with that of normozoospermic donors. Ascorbate and urate were found at similar concentrations in the plasma of both groups. The concentration of TBARS was significantly higher (P < 0.001) in the semen from vasectomized individuals compared with the normozoospermic group. The results indicate that the epididymis contributes to the antioxidant capacity of seminal plasma and possesses region-specific antioxidant activity, which may potentially protect spermatozoa from oxidative attack during storage at this site.

Antioxidants↗
PubMed1997

Degradation pathways of ampicillin in alkaline solutions.

Ampicillin trihydrate, sodium salt, in aqueous solution has a pH of about 8. No complete degradation pathway has been proposed to explain the degradation of ampicillin under alkaline conditions and the information available explains the formation of only certain products. The present work was carried out with the aim of providing this information. The formation of degradation products of ampicillin trihydrate, sodium salt, produced in aqueous solutions (pH 12 and 37 degrees C) have been studied as an accelerated form of the possible degradation that may occur in aqueous solutions at pH 8. Some of the degradation products formed under these conditions were then obtained either by synthesis or by degradation of ampicillin sodium followed by isolation using semi-preparative HPLC. These compounds were characterized by 1H NMR spectroscopy. The information obtained from the experiments by HPLC and NMR spectroscopy made it possible to propose a degradation pathway for ampicillin under the conditions described above. 5R-penicilloic acid is the first degradation product of ampicillin and subsequently undergoes epimerization at C-5 to form the 5S isomer via the imine tautomer. Mechanisms for the formation of compounds previously believed to form only under acidic conditions are proposed, i.e. ampicillin penilloic acid and 2-hydroxy-3-phenylpyrazine. The formation of ampicillin polymers was detected in dilute solutions (1% w/v) within a few hours of dissolution. The presence of ampicillin penicillenic acid and ampicillin penamaldic acid was detected by 1H NMR and their main spectroscopic features determined.

Ampicillin↗
PubMed1994

Chiral discrimination of phenethylamines with beta-cyclodextrin and heptakis(2,3-di-O-acetyl)beta-cyclodextrin by capillary electrophoresis and NMR spectroscopy.

The resolution of nine sympathomimetic phenethylamine racemates by beta-cyclodextrin and heptakis(2,3-di-O-acetyl)beta-cyclodextrin has been investigated by capillary electrophoresis and 1H NMR spectroscopy. The NMR and capillary electrophoresis results showed that beta-cyclodextrin probably formed stronger complexes with the amines than did heptakis(2,3-di-O-acetyl)beta-cyclodextrin but was a poorer chiral discrimination agent in both techniques. The addition of heptakis(2,3-di-O-acetyl)beta-cyclodextrin to the capillary electrophoresis buffer gave baseline resolution of enantiomer peaks for seven of the compounds studied while beta-cyclodextrin resolved only three of the racemates.

Cyclodextrins↗
PubMed1993

Rapid isolation of polychlorinated biphenyls from milk by a combination of supercritical-fluid extraction and supercritical-fluid chromatography.

The extraction and isolation of polychlorinated biphenyls (PCBs) from sample matrices such as freeze-dried milk is a lengthy and expensive process that conventionally requires the isolation of microgram amounts of PCBs from large volumes of flammable organic solvents used in the extraction and chromatographic stages. Supercritical-fluid extraction (SFE) and chromatography (SFC) using carbon dioxide has been investigated as a replacement procedure using a Simplex optimisation approach to optimise the working conditions. The SFE conditions required to extract PCBs and milk fat from freeze-dried milk were investigated, and it was found that although extraction was rapid (10 min), the conditions required were identical so that there was no separation of PCBs and fat. However, SFC conditions have been developed that permit the resolution of PCBs from milk fat in 15 min. Thus, a combination of SFE and SFC have the potential to replace the existing methods of extraction and isolation with procedures that are quicker, cheaper, and avoid the use of flammable organic solvents.

Animals↗
PubMed1991

HPLC and 1H-NMR study of chiral recognition in some thromboxane antagonists induced by beta-cyclodextrin.

The interaction of beta-cyclodextrin with a series of structurally related chiral thromboxane antagonists was investigated using NMR and RP-HPLC. HPLC studies used both a cyclodextrin bonded phase (Cyclobond I), and beta-cyclodextrin as a mobile phase additive with an achiral C8 column. Many of the compounds exhibited chiral recognition with beta-cyclodextrin in each technique, but only partial correlations between the three data sets were observed. HPLC and ROESY NMR data suggested the possibility of bimodal inclusion.

Chromatography, High Pressure Liquid↗
PubMed1990

Performance evaluation of an aqueous-organic phase separator for post-column reactions in high-performance liquid chromatography, and its application to the enhanced detection of some basic drugs of abuse.

A phase separator is described that is suitable for post-column HPLC applications. It operates with commonly used HPLC eluents and immiscible organic solvents as long as the two phases remain immiscible. It is compatible with gradient elution systems. Separation efficiency is routinely better than 0.8, which ensures that analyte peak heights are about 95% of the maximum height under these conditions. An application for the detection of pethidine, cocaine, methadone, piritramide and dipipanone at 0.8-1.8 ng on-column loadings is described.

Chromatography, High Pressure Liquid↗
PubMed1990

On-line recovery of trimeprazine enantiomers following chiral separation by reversed-phase high-performance liquid chromatography using a beta-cyclodextrin-containing mobile phase.

A procedure is described which allows the on-line recovery of enantiomers following semi-preparative chiral separation by RP-HPLC using beta-cyclodextrin in the mobile phase. By this method, the phenothiazine antihistamine trimeprazine (I) was resolved into its antipodes at greater than 95% optical purity at a throughput of more than 1 mg of each enantiomer per hour, using 4 mm i.d. columns. The recovered trimeprazine was found to be free from cyclodextrin.

Chromatography, High Pressure Liquid↗
PubMed1988

Sensitive HPLC analysis of basic drugs of abuse having weak UV absorptivity using columns with low carbon loadings monitored at 205 nm.

The selection of columns containing cyanopropyl-bonded silica (3.5% carbon loading) and their use under reversed-phase conditions has permitted short retention times with good resolution to be obtained for 16 basic drugs of abuse and three metabolites. Predominantly aqueous mobile phases enable the determination of these weakly UV-absorbing compounds over the concentration range 0.7-18 microg ml(-1) with detection at 205 nm. Trimethylsilyl-bonded silica (2.6% carbon loading) also provided good resolution but required longer analysis times.

Journal Article↗
PubMed1988

Analysis of tetracycline antibiotics and their common impurities by high-performance liquid chromatography using a polymeric column.

The chromatographic separation of tetracycline, oxytetracycline, 6-demethyl-chlortetracycline, methacycline and minocycline, and the common impurities of tetracycline, oxytetracycline and chlortetracycline has been optimised on a Hamilton PRP-1 column using citrate-phosphate buffer with propan-2-ol and tetrahydrofuran as organic modifiers. Column efficiency was approximately doubled by the inclusion of 1% v/v dichloromethane in the mobile phase.

Journal Article↗