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D K Lloyd

Publications and source records attributed to D K Lloyd.

At least 19 recordsLinked to original sources
PubMed1998

The potentiation of the effect of radiation treatment by intratumoral delivery of cisplatin.

PURPOSE: To compare potentiation of the effects of acute or fractionated radiation by cisplatin when the drug was delivered intratumorally by implanted biodegradable polymer, by intraperitoneal injection, or by intraperitoneal osmotic pump. METHODS AND MATERIALS: Radiation was delivered to a mouse tumor (RIF-1) either in a single dose or in a fractionated regime in conjunction with cisplatin delivered either as a bolus injection, with an osmotic pump, or with a biodegradable polymer rod containing cisplatin. The osmotic pump was implanted in the intraperitoneal cavity of the mouse while the polymer implants were placed directly in the tumor. As the polymer degrades, the drug is released at the treatment site leading to high local concentrations. The osmotic pump, in contrast, leads to prolonged systemic exposure to the drug at low concentrations. Tumor growth delay (TGD) was used as an endpoint in these experiments. RESULTS: The most effective treatment protocol, in terms of potentiating the effects of radiation was cisplatin delivered by polymer implanted 2 days before an acute dose of radiation (growth modification factor [DMF] = 2.2). Comparison of single and multifraction regimes where polymer implant was on the same day as the commencement of treatment showed greater potentiation of the effect of fractionated than of acute radiation treatment with the DMF for fractionated treatment remaining relatively constant (1.5-1.9) for 5, 8, and 12 fraction treatments. Cisplatin delivered via the osmotic pump did not deliver a high enough dose of cisplatin to produce therapeutic effect in this mouse tumor model and had little impact on response to treatment. CONCLUSIONS: Our results indicated that cisplatin delivered intratumorally by biodegradable polymer implant was effective in potentiating the effect of both acute and fractionated radiation. For the fractionated treatments the effect was maintained with increasing fraction numbers and treatment time.

Animals↗
PubMed1998

Evaluation of beta-lactoglobulin as a stationary phase in high-performance liquid chromatography and as a buffer additive in capillary electrophoresis: observation of a surprising lack of stereoselectivity.

Previous studies have reported that alpha1-acid glycoprotein is quite similar in amino acid sequence and disulfide bond arrangements to members of a group of proteins which include beta-lactoglobulin (BLG). Since generally homologous proteins retain some similarity in function at the molecular level, we decided to evaluate the enantioselective properties of BLG as an high-performance liquid chromatographic chiral stationary phase (HPLC-CSP), and as an additive in capillary electrophoresis (CE). Two columns with differences in internal diameter and method of immobilisation on epoxide silica were prepared. Chiral acidic, basic and uncharged drugs were chromatographed and mobile phase parameters, namely pH and type of organic modifier, were varied in order to test the column performance. The CE approach has some advantages in that there is no need for immobilisation and only a small amount of protein is required. BLG was therefore tested as a CE buffer additive, using the same analytes as in the HPLC study. Although one would expect that a protein would display some enantioselectivity, BLG did not show any enantioselectivity whatsoever in either system; the protein has fairly weak interaction with the majority of the test solutes, as indicated by both techniques.

Animals↗
PubMed1998

Analysis of proteins in microsamples of rat airway surface fluid by capillary electrophoresis.

A thin layer of airway surface fluid (ASF) lining the pulmonary airways plays an important role in the primary defense mechanisms of the lung against bacterial infection. However, little is known about the composition of ASF due to the thinness (typically 5-30 microm in healthy animals) of the fluid layer and its relative inaccessibility, which causes considerable difficulties in sample collection and subsequent analysis. We have used a novel technique of capillary sampling coupled with capillary electrophoresis (CE) to analyze the protein composition of rat ASF. CE analyses were performed under two different conditions: a borate buffer, pH 9.1, or a phosphate buffer, pH 2.5, with 0.5 mM spermine. The different selectivities afforded by the two methods aid in peak identification, and quantitation of most of the major species was possible using both separation conditions. Albumin, transferrin and globulins are observed to be the major protein components in rat ASF, at concentrations of 28 mg ml(-1), 4.0 mg ml(-1) and 34 mg ml(-1) respectively, in comparison to 31 mg ml(-1), 3.1 mg ml(-1) and 40 mg ml(-1), respectively, in rat plasma.

Albumins↗
PubMed1997

Selectivity in capillary electrophoresis: the use of proteins.

Proteins, by their very diverse nature, provide a wide variety of options for generating selectivity in capillary electrophoresis (CE). Their use in different modes of CE will be considered in this review. Proteins added in solution to the background electrolyte allow separations to be made in a similar fashion to other electrokinetic chromatography methods, e.g., micellar separations. Alternatively, different immobilization schemes can be used to secure proteins within the capillary; these have included capillary electrochromatography with the protein grafted onto a silica support, or immobilization of the protein within a gel structure. Compounds varying in size from small inorganic ions to biopolymers may be bound by proteins. There is the potential for any sort of intermolecular interaction to play a role in the binding process (e.g., hydrophobic interactions, electrostatic interactions, etc.). Very specific high-affinity binding often occurs, but also there is often weaker, non-selective binding. Frequently the interactions of chiral compounds with proteins are stereoselective. Obtaining chiral selectivity has been one of the main applications of protein selectors in CE, and this use will be emphasized here in a discussion structured by type of protein. As well as utilizing the selectivity of proteins to develop separations, the role of CE in investigating ligand-protein interactions will be emphasized.

Animals↗
PubMed1997

Determination of p-aminosalicylic acid and its N-acetylated metabolite in human urine by capillary zone electrophoresis as a measure of in vivo N-acetyltransferase 1 activity.

A capillary zone electrophoresis method has been developed for the determination of p-aminosalicylic acid (PAS) and its metabolite, N-acetyl-p-aminosalicylic acid (N-acetyl-PAS), in urine. A linear relationship was observed between time-normalized peak area and the concentration of the parent and metabolite with correlation coefficients greater than 0.9990. The method could be applied to the determination of PAS and N-acetyl-PAS in human urine without any sample pretreatment. A good separation of the analytes is achieved in a run time of 12 min (15 min total, including capillary wash). Using PAS as a probe for N-acetyltransferase 1 activity, 20 healthy volunteers were phenotyped after oral administration of a 1 g dose. The preliminary results seem to indicate a bimodal distribution of N-acetyl-PAS/PAS molar ratios.

Acetamides↗
PubMed1997

Tumor treatment by sustained intratumoral release of cisplatin: effects of drug alone and combined with radiation.

PURPOSE: The effect of intratumoral delivery of cisplatin to a mouse tumor model (RIF-1) by means of a biodegradable polymer implant with and without radiation was studied. METHODS AND MATERIALS: The polymer bis (p-carboxyphenoxy) propane-sebacic acid (CPP:SA; 80:20) and its degradation products have been characterized. Polymer rods (8 x 0.5 mm) containing 17% cisplatin by weight were prepared by extrusion, and the in vitro degradation rate measured. The implants were placed into mouse tumors and their effect (with and without radiation) on tumor growth delay studied. The levels of Pt in the mouse kidney, tumor, and blood plasma at selected intervals after implant were also determined. These results were compared with those obtained when cisplatin was delivered systemically. RESULTS: When cisplatin was delivered by the polymer implants, higher levels were present in the tumor for longer time periods (cf. systemic delivery of the drug). For both nonirradiated and irradiated tumors, those treated with the polymer implants had significantly longer tumor growth delays compared to nonimplanted controls and to systematically treated tumors. CONCLUSIONS: The results show that intratumoral delivery of cisplatin is more efficient than systemic delivery. Using the biodegradable polymer implant, higher doses of cisplatin can be tolerated by the animal as the drug is localized within the tumor, and the high levels of the drug in the tumor can be maintained for an extended period of time. When radiation is given in conjunction with cisplatin, the tumor response is supraadditive for all modes of cisplatin administration but is potentiated to a greater extent when cisplatin is delivered through the polymer implant. The greatest effect is seen for treatment with cisplatin delivered by polymer implant combined with fractionated radiation.

Animals↗
PubMed1997

Microanalysis of lung airway surface fluid by capillary electrophoresis with conductivity detection.

The thin layer of fluid that covers the surface of the epithelia lining the conducting airways plays an important role in primary pulmonary defense, and its composition may be a critical factor in the pathogenesis of several lung diseases including cystic fibrosis. Despite its physiological importance, the composition of airway surface fluid (ASF) is poorly understood due to considerable difficulties in sample collection from the 5-30 microns thick layer and subsequent analysis. We have used a novel technique for sample collection and microanalysis of ASF (nanoliter sample required) by capillary electrophoresis with conductivity detection. Limitations on the diameters of capillary required for the sample injection process and for the conductivity detector require the use of coupled separation capillaries with different external diameters. Two different methods were used to construct a butt-joint coupling for capillaries of different outer diameters. Reasonable efficiency is observed with the coupled capillaries (N = 100000 plates m-1) compared to an unbroken single capillary (N = 180000 plates m-1). The use of conductivity detection allows greater flexibility in method development and the possibility of determining a greater variety of ions than with a previous indirect-UV method. In the present study, we describe the analysis of cations (Na+, K+, Ca2+, Mg2+) and anions (Cl-, NO2-, NO3-, SO4(2-), PO4(2-), HCO3-) in rat ASF. Particular attention was paid to developing washing procedures which limited fouling of the conductivity sensor. In healthy rats, ASF was found to be hypotonic compared to plasma levels, consistent with some observations made in human airways.

Animals↗
PubMed1997

Effect of organic modifiers on retention and enantiomeric separations by capillary electrophoresis with human serum albumin as a chiral selector in solution.

We have investigated the effect of methanol, ethanol, 1-propanol, 2-propanol and acetonitrile on the retention and enantiomeric separations of benzoin and propiomazine by capillary electrophoresis, using human serum albumin as a chiral selector. The effects of these modifiers on mobilities of analytes are rather difficult to interpret. Calculation of capacity factors reveals the underlying analyte-protein interactions; pitfalls in making such calculations are pointed out. In the case of benzoin and propiomazine binding to human serum albumin, capacity factors were observed to always decrease upon addition of organic modifiers, although the effects of 1- and 2-propanol suggest a possible specific interaction or modification of the protein conformation.

Acetonitriles↗
PubMed1997

A quantitative relationship between capacity factor and selector concentration in capillary electrophoresis and high performance liquid chromatography: evidence from the enantioselective resolution of benzoin using human serum albumin as a chiral selector.

Many selectors are used both in pressure-driven liquid chromatography (LC) and in electrokinetic chromatography (EKC), particularly chiral species such as cyclodextrins and proteins. It should be possible to readily apply information gleaned using one technique to the other, since in both techniques the underlying molecular interactions which lead to separations are expected to be the same. Superficially this may be the case, but an exact transfer of operating conditions, i.e., background electrolyte (BGE) composition/mobile phase composition, assuming that these meet certain minimum requirements for each technique, is not often possible. To investigate the reason for this we have measured retention (k') of a neutral solute (racemic benzoin) in HPLC and EKC using an identical range of BGE/mobile phase conditions in both techniques. The selector used was human serum albumin. The k' measurements obtained for each benzoin enantiomer were consistently higher in HPLC than in EKC. This can be explained very simply if one considers that retention in both systems is related to the selector concentration [S], by the expression k'=K[S], where K is the affinity constant. In EKC, [S] is simply the concentration of free selector in the BGE, while in LC, [S] = m(p)/Vo, where m(p) is the number of moles of accessible selector, and Vo is the column void volume. In LC, [S] is generally considerably higher than in EKC, leading to larger values of k'.

Benzoin↗
PubMed1997

Airway surface fluid composition in the rat determined by capillary electrophoresis.

The apical surface of respiratory epithelial cells is covered by a thin layer of low-viscosity fluid termed airway surface fluid (ASF), about which relatively little is known. We collected samples of ASF from anesthetized rats, which were then analyzed using capillary electrophoresis, a method that enables extremely small quantities of fluid to be analyzed. We found values for Na+ (40.57 +/- 3.08 mM), K+ (1.74 +/- 0.36 mM), and Cl- (45.16 +/- 1.81 mM), indicating that this fluid is hypotonic compared with rat plasma. In contrast, the concentrations of nitrite and nitrate within ASF were higher than reported plasma values. Additionally, intravenous administration of the cholinergic agonist methacholine (MCh) resulted in a dose-dependent increase in the concentration of Na+ and Cl- within the ASF. This increase is approximately 50% in these ions after a dose of 100 ng MCh/g body wt. This animal model, together with this microanalytical technique, may be useful for investigating the in vivo regulation of ASF composition.

Animals↗
PubMed1996

Capillary electrophoretic analyses of drugs in body fluids: sample pretreatment and methods for direct injection of biofluids.

A variety of strategies for the analysis of biological samples by capillary electrophoresis (CE) are described, with particular emphasis on the determination of drugs and metabolites. Analytical methods involving extensive sample pretreatment before CE analysis are considered, as well as strategies for directly injecting untreated biofluids. The application in CE of techniques common in liquid chromatography is first described, e.g. protein precipitation, liquid-liquid extraction and solid-phase extraction. On-capillary methods of sample concentration are considered. Approaches to performing CE assays of urine and plasma, without prior sample treatment, are described. The use of both capillary zone electrophoresis and micellar electrokinetic chromatography for direct-injection assays is compared for both urine and plasma analyses, and capillary washing strategies are discussed. Finally, direct-injection microanalyses are mentioned.

Body Fluids↗
PubMed1996

Separation of testosterone metabolites in microsomal incubates using a new capillary electrophoresis assay.

A capillary electrophoresis method has been developed to separate the products of liver microsomal testosterone metabolism. The microsomal mixture undergoes liquid-liquid extraction and pre-concentration, and then electrophoretic analysis takes less than 25 min including capillary conditioning steps. The development of the complex background electrolyte (Tris-HCl and borate buffers, sodium dodecyl sulfate, beta-cyclodextrin and ethanol) necessary for this separation is described. A z-type capillary flow cell is used to obtain adequate detection sensitivity. The proportion in which the metabolites are produced as determined by this method allows assignment of the relative activity of cytochrome P-450 enzymes in the microsomes. The technique is useful for comparison of activity in normal and abnormal hepatic microsomes.

Animals↗
PubMed1995

Quantitation of the diastereoisomers of L-buthionine-(R,S)-sulfoximine in human plasma: a validated assay by capillary electrophoresis.

An assay for the diastereoisomers of the biochemical modifier L-buthionine-(R,S)-sulfoximine (BSO) in human plasma has been developed using capillary electrophoresis (CE). Separation of the diastereoisomers is achieved by the micellar electrokinetic chromatography (MEKC) mode of CE. Plasma is injected directly onto the separation capillary without any extraction step, and BSO is detected directly by ultraviolet absorbance measurements at 190 nm without prior derivatization. The whole assay, including capillary conditioning, takes approximately 30 min. Intra- and inter-day R.S.D. values are approximately 7% at sample concentrations around 25 micrograms ml-1, and approximately 3% at sample concentrations around 500 micrograms ml-1. The limit of detection in plasma is 3.9 micrograms ml-1 (S/N = 2). The assay has been used to quantitate the diastereoisomers of BSO in patient samples in a pharmacokinetic study.

Electrophoresis, Capillary↗
PubMed1995

Determination of the inorganic ion composition of rat airway surface fluid by capillary electrophoresis: direct sample injection to allow multiple analyses from nanoliter volumes.

The pulmonary airways are covered by a layer of airway surface fluid (ASF) which is typically < 30 microm in thickness. ASF composition is an important factor in the pathogenesis of several lung diseases including cystic fibrosis. Because of the very small volume of ASF, it is difficult to determine ASF composition, Particularly for inorganic ions, since sampling by lavage is not suitable. With nanoliter injected volumes, capillary electrophoresis (CE) is ideally suited to ASF analysis. We have developed a novel technique using separate sampling and injection capillaries whereby submicroliter volumes of ASF (typically approximately 100 nL) can be collected from airways and then analyzed by CE. Cations (Na+, K+, Ca2+, Mg2+) and anions (including Cl-) are quantitated (RSD < 10%) using indirect UV detection. In healthy rat lungs, ASF was found to be hypotonic, consistent with observations made in human airways. This technique has been developed using rats, which have not previously been studied because their small size prohibits the use of other sampling techniques.

Animals↗
PubMed1995

Sodium dodecyl sulfate solution is an effective between-run rinse for capillary electrophoresis of samples in biological matrices.

It is common practice in capillary electrophoresis to perform some sort of capillary washing step(s) between separations. In many analyses little consideration is given to optimization of the wash, and typically a rather standard washing procedure is used involving a few minutes wash with 0.1 M NaOH followed by a few minutes reconditioning with the run buffer. As an alternative to this procedure, we have investigated the use of wash solutions containing sodium dodecyl sulfate (SDS). This type of wash has been used in the analyses of both small molecules and proteins, with encouraging results. After the SDS wash, the electroosmotic flow has been shown to be restored to values close to normal in a capillary which had previously been coated with plasma proteins. Separation efficiency for a test compound (dextromethorphan) is improved if an SDS rather than a HCl-NaOH wash is used after injection of plasma. In a direct-injection analysis of plasma proteins using a pH 10 borate buffer, an SDS-based washing procedure (total time, 1 min) gave better migration-time reproducibility than an NaOH-based wash, which took 5 min in total.

Blood Proteins↗
PubMed1995

Effect of pH and sodium dodecyl sulfate concentration on the analytical window in the direct-injection analysis of plasma samples by capillary electrophoresis.

Plasma samples can be analyzed with direct injection onto a capillary electrophoresis system if interactions between the plasma proteins and capillary walls are minimized. This can be achieved using micellar electrokinetic capillary chromatography with a surfactant such as sodium dodecyl sulfate. The surfactant complexes with the proteins, giving them a net negative charge, and thus causes them to be repelled from the negatively-charged fused-silica capillary walls. Migrating as anions, the complexed proteins appear late in the electropherogram. In such analyses, it is important to manipulate the mobility of the analyte(s) of interest such that they migrate in the useful analytical time window before the plasma proteins. In this article, a study of the effect of buffer pH and surfactant concentration on the width of the migration time window is reported. It is shown that at pH 6 the migration time window is wide, but reduced electroosmosis can result in inordinately long run times; pH 7 gives an acceptable analytical window with acceptable run times over a wide range of sodium dodecyl sulfate (SDS) concentrations; pH 8 and higher are most useful when higher concentrations of surfactant (> 50 mM SDS) are employed.

Acetaminophen↗