Inhibition of angiogenesis by the antineoplastic agents mitoxantrone and bisantrene

The effects of mitoxantrone and bisantrene on angiogenic responses induced by tumor cell-conditioned media in the avascular cornea of rat eye have been evaluated. Both mitoxantrone and bisantrene effectively inhibited, in a concentration-dependent manner, angiogenesis induced by conditioned media obtained from either a hamster buccal pouch carcinoma cell line or P388D1 murine macrophage-like cells. Whereas vessel ingrowth in corneas containing tumor cell-conditioned media was detected as early as day 2 or 3 and was maximal by day 7, inclusion of mitoxantrone or bisantrene in the conditioned media at a 1:1 ratio (160 microM mitoxantrone or 32 microM bisantrene) resulted in complete inhibition of angiogenesis throughout the 14-day evaluation period. When concentrations of 64 and 32 microM mitoxantrone or 13 and 6.4 microM bisantrene were employed there was a marked delay in the appearance of capillary blood vessels (day 5 to 7) and a reduction in the intensity of angiogenic responses. No untoward toxicity to the tissue was observed at the concentrations of mitoxantrone or bisantrene employed.     

Inhibition of adriamycin-stimulated microsomal lipid peroxidation by mitoxantrone and ametantrone,two new anthracenedione antineoplastic agents

Ametantrone and mitoxantrone, two new anthracenedione antineoplastic agents, produced a concentration-dependent inhibition of hepatic microsomal lipid peroxidation. Malondialdehyde production was diminished from 10.6 nmoles/mg/60 min to 3.3 and 5.4 nmoles/mg/60 min, in the presence of 100 μM mitoxantrone and ametantrone, respectively. Under similar conditions, Adriamycin stimulated lipid peroxidation over twofold. In addition, both mitoxantrone and ametantrone inhibited Adriamycin-stimulated lipid peroxidation, with 50% inhibition occurring at concentrations of 4 and 6 μM, respectively. Microsomal superoxide production was not significantly inhibited at anthracenedione concentrations which markedly decreased lipid peroxidation, suggesting that inhibition of lipid peroxidation was not the result of inhibition of superoxide generation. These results correlate with the lack of anthracenedione cardiotoxicity and also demonstrate anthracenedione inhibition of lipid peroxidation at micromolar concentrations; an observation with potential therapeutic significance.     

Improved procedure for the separation of phospholipids by high-performance liquid chromatography

We describe a rapid and efficient high-performance liquid chromatography procedure for the separation of phospholipids. The separation is accomplished on a microparticulate silica gel column using isocratic elution and UV detection at 203 nm. The solvent mixture is acetonitrile—methanol—85% phosphoric acid(130:5:1.5, v/v). Complete separation is achieved within 30 min of phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidyldimethylethanolamine, lysophosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine and sphingomyelin. The method is suitable for the analysis of phospholipids in tissue extracts.     

Improved method of analysis of biomass sugars using high-performance liquid chromatography

The precise quantitative analysis of biomass derived sugars is a very important step in the conversion of biomass feedstocks to fuels and chemicals. However, the most accurate method of biomass sugar analysis is based on the gas chromatography analysis of derivatized sugars either as alditol acetates or trimethylsilanes. The derivatization method is time-consuming but the alternative HPLC method cannot resolve most sugars found in biomass hydrolysates. We have demonstrated for the first time that by careful manipulation of the HPLC mobile phase, biomass monomeric sugars (arabinose, xylose, fructose, glucose, mannose, and galactose) can be analyzed quantitatively and there is excellent baseline resolution of all the sugars. This was demonstrated for both standard sugars and corn stover hydrolysates. Our method can also be used to analyze dimmeric sugars (cellobiose and sucrose).     

Direct determination of mitoxantrone and its mono- and dicarboxylic metabolites in plasma and urine by high-performance liquid chromatography

The simultaneous isolation and determination of mitoxantrone (Novantrone ®) and its two known metabolites (the mono- and dicarboxylic metabolites) were carried out using a high-performance liquid chromatographic (HPLC) system equipped with an automatic pre-column-switching system that permits drug analysis by direct injection of biological samples. Plasma or urine samples were injected directly on to an enrichment pre-column flushed with methanol-water (5:95, v/v) as the mobile phase. The maximum amount of endogenous water-soluble components was removed from biological samples within 9 min. Drugs specifically adsorbed on the pre-column were back-flushed on to an analytical column (Nucleosil C₁₈, 250x4.6 mm I.D.) with 1.6 M ammonium formate buffer (pH 4.0) (2.5% formic acid) containing 20% acetonitrile. Detection was effected at 655 nm. Chromatographic analysis was performed within 12 min. The detection limit of the method was about 4 ng/ml for urine and 10 ng/ml for plasma samples. The precision ranged from 3 to 11% depending on the amount of compound studied. This technique was applied to the monitoring of mitoxantrone in plasma and to the quantification of the unchanged compound and its two metabolites in urine from patients receiving 14 mg/m² of mitoxantrone by intravenous infusion for 10 min.     

Improved method for the analysis of furosemide in plasma by high-performance liquid chromatography

Modifications of existing rapid high-performance liquid chromatographic procedures for the determination of furosemide in plasma were made in order to achieve greater sensitivity. To a small volume of plasma was added an internal standard structurally related to furosemide. Then, following previously described procedures, acetonitrile was added to precipitate the proteins and the clear supernatant was separated. However prior to injection of the supernatant the pH and composition of the sample were adjusted. This modification of the sample enabled an injection volume of up to 300 μl of the supernatant to be injected onto the chromatographic column. The effluent was monitored spectrofluorimetrically. A standard linear calibration curve with a mean precision of ± 4.4% was obtained for plasma samples containing 20–900 ng/ml of furosemide. Two structurally related compounds were used as internal standards in the furosemide assay.     

Strontium-phosphate hydroxyapatite high-performance liquid chromatography

High-performance liquid chromatography using spherical aggregates of strontium-phosphate hydroxyapatite(SrHA) micro-crystals as adsorbent has been developed; preliminary performance tests were carried out by using several types of protein. It can be deduced that, in parallel with the case of usual calcium-phosphate hydroxyapatite(CaHA), with SrHA also, two types of effective surface, vector a (or vector b) and vector c surfaces, appear on the crystal: the same protein molecular generally shows slightly different chromatographic behaviors between the CaHA and the SrHA packed column. Combining the SrHA and the CaHA packed column would lead to an efficient fractionation of a particular molecule from an assembly of molecules with subtle structural differences from one another.     

Improved method for the analysis of estrogenic steroids in pregnancy urine by high-performance liquid chromatography

The use of microparticulate packing materials and large injection volumes gives significant improvements in the analysis of complex samples, such as urine extracts by high-performance liquid chromatography. Lower detection limits and improved accuracy can now be attained. In addition, the combined use of adsorption and reversed-phase chromatography leads to reduced uncertainty in peak identification and gives more reliable quantification.     

Histone fractionation by high-performance liquid chromatography

A method for the rapid chromatography of histones by high-performance liquid chromatography (HPLC) using a reverse-phase μBondapak C₁₈ column containing a packing of octadecylsilane chemically bonded to silica and a linear elution gradient running from water to acetonitrile is described. Two conditions were found to be necessary to achieve histone fractionation: (i) silylation of the active groups of the silica solid support, and (ii) trifluoroacetic acid (TFA) in the eluting solvents. Greater than 90% of the total [³H]lysine-labeled protein applied to the column was eluted from the column. The fractionation of the histones appears to be based on the hydrophobic properties of the proteins. The HPLC histone fractions (identified by their electrophoretic mobilities) were eluted from the column in the following order: H1, H2B, (LHP)H2A, (MHP)H2A + H4, (LHP)H3, and (MHP)H3 (where LHP and MHP refer to the less hydrophobic and more hydrophobic histone variants). Phosphorylated histone species were not resolved from their unmodified parental species. The volatile nature of the water/acetonitrile/TFA eluting solvent facilitated the recovery of salt-free histones from the eluted HPLC fractions by simple lyophilization. This system is very useful for the rapid isolation of the lysine-rich histones, H1 and H2B, and the variants of histone H3. With further development, this system is expected to extend the advantages of HPLC to the fractionation of histone H4 and the variants of histone H2A as well.     

Polarimetric detection in high-performance liquid chromatography

Chiroptical detection for HPLC is particularly useful as a selective detection method for chiral molecules, and in enantiomeric purity determination with partial chiral separation or without chiral separation. The recent development of laser-based polarimeters with microdegree sensitivity has increased the applicability of optical rotation detection in HPLC. The detection limit of these instruments is submicrogram on-column for many chiral compounds in analytical HPLC. A variety of applications of the selective detection of optically active molecules are reviewed. The use of polarimetric detection with partial chiral separation is considered, both as an aid to method development and for enantiomeric purity determination. Finally applications to enantiomeric purity determination without chiral separation are reviewed, with the dual use of nonchirally selective and chiroptical detectors to determine the total amount and optical purity of the analyte. Determinations of chiral purity for samples of high enantiomeric excess are described, which with laser-based instrumentation may give accuracies of better than +/- 1% with sample loadings of 50 micrograms on an achiral column. Applications to the study of enantioselective reactions are also considered, with determination of enantiomeric excess in near-racemates to better than +/- 0.1%.     

Chlorophyll analysis by high-performance liquid chromatography

The separation and determination of chlorophylls by high-performance liquid chromatography (HPLC) is described. Chlorophylls and their derivatives were separated by reversed-phase HPLC based on hydrophobic interaction between solute and support, using an octadecyl silica column and elution with 100% methanol. Separated pigments were detected fluorometrically with a sensitivity in the picomole range: the fluorescence response was linear over a wide pigment concentration range. Resolution of five chlorophylls a and four protochlorophyll species esterified with different alcohols was achieved within 22 min in a single experiment. This method can be used for the determination of chlorophyll b, bacteriochlorophyll a esters and products synthesized from chlorophyll, but not for nonesterified pigments, i.e., chlorophyllide, protochlorophyllide and chlorophyll c. The chromatographic mobility of chlorophyll a esterified with different alcohols increases with increasing number of carbon atoms in the esterifying alcohols. The plots obtained from the logarithm of the capacity factor (k′) of these pigments versus the numbers of carbon atoms of the alcohol molecule gave a straight line, thus permitting the estimation of the chain length of unknown pigment esterifying alcohols. This HPLC separation technique did not cause the formation of artifacts. The deviation of the individual retention time for each pigment is less than ±0.5%, thus making this method suitable for the rapid identification and quantification of unknown pigments.     

Reversed-phase high-performance liquid chromatography of chlorophylls and carotenoids

Reversed-phase high-performance liquid chromatography with octadecyl- or octylsilylated silica gel as the stationary phase provides a powerful tool in the analysis of chloroplast pigments from higher plants and green algae. Chromatographic columns packed with 10 μm chemically bonded silica gel particles allow the simultaneous separation of chlorophylls a and b, chlorophyll isomers, pheophytins a and b, α-carotene, β-carotene, lutein, violaxanthin, lutein-5,6-epoxide, antheraxanthin, neoxanthin and several minor carotenoids from a single sample within a short analysis time. The quantitative analysis requires a minimum of 1–5 pmol for carotenoids and 5–10 pmol for chlorophylls. Pigment degradation products, formed on polar stationary phases, are not found in reversed-phase high-performance liquid chromatography due to the weak hydrophobic forces on which the separation mechanism is based. The production of altered pigments however, either induced by various treatments or generated during the isolation, can be monitored as the reversed-phase system is selective enough to separate cis-isomers and oxidation products from their parent compounds. The reproducibility of the individual retention time for each pigment is better than ±1.5% which facilitates the identification of unknown pigments. The method is applied to the analysis of the pigment composition of Chlorella fusca, spinach (Spinacia oleracea) chloroplasts, and to the rapid determination of the ratio of chlorophyll a to chlorophyll b.     

Determination of cystamine by high-performance liquid chromatography

A highly sensitive and specific assay method for cystamine using high-performance liquid chromatography has been developed. The method is based on postcolumn derivatization of cystamine with o-phthaladehyde in the presence of 2-mercaptoethanol and sodium hypochlorite. The separation of cystamine was achieved using a cation exchange column (ISC-05/S0504). The assay was linear over the concentration range of 2 to 200 pmol. For the application of this assay method to biological materials, the pretreatment with a cation exchange column (Dowex 50W X 8) was necessary to remove interfering o-phthaladehyde-reactive substances. Since cysteamine in biological materials was quantitatively converted to cystamine during these sampling procedures, this method was found to be suitable for assaying the cysteamine plus cystamine content in various organs and tissues. The cysteamine-cystamine content in various tissues of rat determined by the present assay method has been presented.     

Determination of N-acetyl-L-glutamate using high-performance liquid chromatography

Acetylglutamate in HClO4 tissue extracts is first separated from glutamate by ion exchange. It is then deacylated with aminoacylase, and the resulting glutamate, after adsorption to and elution from an AG 50 column, is quantitated by a fast-HPLC method using o-phthaldialdehyde precolumn derivatization, separation in a C18 reverse-phase column, and fluorescence detection. A linear response is obtained up to 2 nmol, the detection limit is 5 pmol, and the method is suitable for assay in 1 mg liver tissue and thus for needle biopsies. When samples were analyzed by this procedure and by earlier procedures based upon detection of glutamate with glutamate dehydrogenase or upon activation of carbamoyl phosphate synthetase the results were similar. The method, which is highly specific, compares favorably in sensitivity, precision, and accuracy with all other published procedures. Using this assay, no acetylglutamate has been found in chicken liver and rat kidney.