Study of normal and modified nucleosides in serum by RP-HPLC

Modified nucleosides derived predominantly from transfer ribonucleic acid (tRNA) have been studied as possible tumor markers. In this paper a reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been applied to study 15 normal and modified nucleosides in serum. The nucleoside levels in normal human serum were established, and the concentrations of 15 nucleosides in serum from 19 cancer patients were determined, it was found that the concentrations of modified nucleosides were significantly higher in patient sera. Based on 15 nucleoside concentrations in serum, factor analysis could classify correctly 90% of cancer patients from the normal humans Further work showed that urine was slightly better than serum when determining nucleosides as biological marker candidates. More work is ongoing to determine the clinical usefulness of modified nucleosides as tumor markers.

Synthesis and characteristics of the human serum albumin-triazine chiral stationary phase

Human serum albumin (HSA) was successfully bonded to silica with s-triazine as activator. The coupling reaction by this method was rapid and effective. The triazine-activated silica is relatively stable and can be installed for at least 1 month without obvious loss of reactivity when stored below 30 degreesC, pH below 7. It was observed that the amount of bound HSA reached 120 mg/g silica calculated from the UV absorbance difference of the HSA solution. d,l-tryptophan was selected as the probe solute to characterize the properties of HSA bonded s-triazine chiral stationary phase, and separation factor of 9.4 was obtained for d,l-tryptophan. Furthermore, the amount of effective HSA on silica was measured by high-performance frontal analysis, and only 16.8 mg/g silica was responsible for the resolution of d,l-tryptophan. These results indicate that the amount of both the bound and effective HSA on silica with triazine as activator was much higher than those by the Schiff base coupling method. Different kinds of enantiomers were resolved successfully on the aminopropylsilica-bonded HSA s-triazine chiral stationary phase. (C) 2000 Wiley-Liss, Inc.

Artificial neural network classification based on high-performance liquid chromatography of urinary and serum nucleosides for the clinical diagnosis of cancer

Nucleosides in human urine and serum have frequently been studied as a possible biomedical marker for cancer, acquired immune deficiency syndrome (AIDS) and the whole-body turnover of RNAs. Fifteen normal and modified nucleosides were determined in 69 urine and 42 serum samples using high-performance liquid chromatography (HPLC). Artificial neural networks have been used as a powerful pattern recognition tool to distinguish cancer patients from healthy persons. The recognition rate for the training set reached 100%. In the validating set, 95.8 and 92.9% of people were correctly classified into cancer patients and healthy persons when urine and serum were used as the sample for measuring the nucleosides. The results show that the artificial neural network technique is better than principal component analysis for the classification of healthy persons and cancer patients based on nucleoside data. (C) 2002 Elsevier Science B.V. All rights reserved.

Effects of pH on the Interactions and Conformation of Bovine Serum Albumin: Comparison between Chemical Force Microscopy and Small-Angle Neutron Scattering

The conformation of bovine serum albumin (BSA), as well as its interactions with negatively charged mica surfaces in saline solutions of different pH values, have been studied by small-angle neutron scattering (SANS) and chemical force microscopy (CFM), respectively. A new approach to extract the contribution of elementary interactions from the statistically averaged force-extension curves through self-consistent fitting was proposed and used to understand the effects of pH on the interactions and conformation of BSA in saline solutions. When pH increases, the SANS results reveal that the sizes of BSA molecules increase slightly, while the statistical analysis of the CFM results shows that the averaged pull-off force for the elongation monotonously decreases. The decrease of pull-off force with the increase of pH results from the decrease in the strength of hydrogen bonding and the number of interaction pairs, as well as the slight increase of the strength of van der Waals interaction. When pH approaches the isoelectric point (pI) of BSA, results from both SANS and CFM suggest a loss of long-range interactions in BSA molecules. Our results also suggest that the force-extension curve is mainly contributed by the van der Waals interaction. The combination of SANS and CFM provides new insight to understand the interactions and conformation of BSA molecules

Studies on interaction of bovine serum albumin with indo-1 by fluorescence spectroscopic method

The binding-site number was calculated by using fluorescence spectroscopic method with bovine serum albumin(BSA) and Indo-1 as protein and ligand models, respectively. The method for calculating binding-site number in BSA for Indo-1 was developed based on the relationships between the changes of Indo-1 fluorescence intensity and the analytical concentration of BSA. And the interaction of BSA with Indo-1 was investigated comprehensively by using fluorescence techniques as well as fluorescence resonance energy transfer, and the thermodynamic parameters were calculated according to the changes of enthalpy on temperature.,

Toxicological effects of cinnabar in rats by NMR-based metabolic profiling of urine and serum

Cinnabar, an important traditional Chinese mineral medicine, has been widely used as a Chinese patent medicine ingredient for sedative therapy. However, the pharmaceutical and toxicological effects of cinnabar, especially in the whole organism, were subjected to few investigations. In this study, an NMR-based metabolomics approach has been applied to investigate the toxicological effects of cinnabar after intragastrical administration (dosed at 0.5, 2 and 5 g/kg body weight) on male Wistar rats.

Affinity and Specificity of Levamlodipine-Human Serum Albumin Interactions: Insights into Its Carrier Function

The affinity and specificity of drugs with human serum albumin (HSA) are crucial factors influencing the bioactivity of drugs. To gain insight into the carrier function of HSA, the binding of levamlodipine with HSA has been investigated as a model system by a combined experimental and theoretical/computational approach. The fluorescence properties of HSA and the binding parameters of levamlodipine indicate that the binding is characterized by one binding site with static quenching mechanism, which is related to the energy transfer. As indicated by the thermodynamic analysis, hydrophobic interaction is the predominant force in levamiodipine-HSA complex, which is in agreement with the computational results. And the hydrogen bonds can be confirmed by computational approach between levamlodipine and HSA. Compared to predicted binding energies and binding energy spectra at seven sites on HSA, levamlodipine binding HSA at site I has a high affinity regime and the highest specificity characterized by the largest intrinsic specificity ratio (ISR). The binding characteristics at site I guarantee that drugs can be carried and released from HSA to carry out their specific bioactivity.

Label-free electrochemical detection of DNA in blood serum via target-induced resolution of an electrode-bound DNA pseudoknot

We have demonstrated a fully covalent, signal-on E-DNA architecture based on the target-induced resolution of a DNA pseudokont. In the absence of target, the electrode-bound DNA probe adopts a pseudoknot conformation that segregates an attached methylene blue (MB) from the electrode. Upon target binding, the pseudoknot is resolved, leading to the formation of a single-stranded DNA element that supports electron transfer from the methylene blue to the electrode.

Metabolic profiling of serum from gadolinium chloride-treated rats by H-1 NMR spectroscopy

Metabolic profiling of serum from gadolinium chloride (GdCl3, 10 and 50 mg/kg body weight, intraperitoneal [i.p.])-treated rats was investigated by the NMR spectroscopic-based metabonomic strategy. Serum samples were collected at 48, 96, and 168 h postdose (p.d.) after exposure to GdCl3. H-1 NMR spectra of serum were analyzed by pattern recognition using principal components analysis. The studies showed that there was a dose-related biochemical effect of GdCl3 treatment on the levels of a range of low-molecular weight compounds in serum. The liver damage induced by GdCl3 was characterized by the elevation of lactate, pyruvate, and creatine as well as the decrease of branched-chain amino acids (valine and isoleucine), alanine, glucose, and trimethylamine-N-oxide concentration in serum samples. The biochemical effects of GdCl3 in rats could be consulted when evaluating the biochemical profile of gadolinium-containing compounds that are being developed for nuclear magnetic resonance imaging.

Drug-human serum albumin binding studied by capillary electrophoresis with electrochemiluminescence detection

A new technique for investigating drug-protein binding was developed employing capillary electrophoresis (CE) coupled with tris(2,2'-bipyridyl) ruthenium(II) [Ru(bPY)(3)(2+)] electrochemiluminescence (ECL) (CE-ECL) detection after equilibrium dialysis. Three basic drugs, namely pridinol, procyclidine and its analogue trihexyphenidyl, were successfully separated by capillary zone electrophoresis with end-column Ru(bPY)(3)(2+) ECL detection. The relative drug binding to human serum albumin (HSA) for each single drug as well'as for the three drugs binding simultaneously was calculated. It was found that the three antiparkinsonian drugs compete for the same binding site on HSA. This work demonstrated that Ru(bPY)(3)(2+) CE-ECL can be a suitable technique for studying drug-protein binding.

In situ circular dichroic electrochemical study of bilirubin and bovine serum albumin complex

The electrooxidation of bilirubin (BR) and bovine serum albumin (BSA) complexes was studied by in situ circular dichroism (CD) spectroelectrochemistry. The result showed that the mechanism of the whole electrooxidation process of this complex corresponded to electrochemical processes (EE mechanism) in aqueous solution. Some parameters of the process were obtained by double logarithm method, differential method and nonlinear regression method. In visible region, CD spectra of the two enantiomeric components of the complex and their fraction distribution against applied potentials were obtained by singular value decomposition least-square (SVDLS) method. Meanwhile, the distribution of the five components of secondary structure was also obtained by the same method in far-UV region. The peak potential gotten from EE mechanism corresponds to a turning point for the component transition, beyond which the whole reaction reaches a new equilibrium. Under applied positive potentials, the enantiomeric equilibrium between M and P form is broken and M form transfers to its enantiomer of P, while the fraction of alpha-helix increases and that improves the transition to P form.

In vitro study on the binding of neutral red to bovine serum albumin by molecular spectroscopy

In this paper, the binding of neutral red (NR) to bovine serum albumin (BSA) under physiological conditions has been studied by spectroscopy method including fluorescence, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. The Stern-Volmer fluorescence quenching constant (K-SV), binding constant (K-b) and the number of binding sites (It) were measured by fluorescence quenching method. Fluorescence experiments were also performed at different ionic strengths. It was found K-SV was ionic strength dependent, which indicated the electrostatic interactions were part of the binding forces. The distance r between donor (BSA) and acceptor (NR) was obtained according to Foster's non-radiative energy transfer theory. CD spectroscopy and FT-IR spectroscopy were used to investigate the structural information of BSA molecules on the binding of NR, and the results showed no change of BSA conformation in our experimental conditions.

H-1 NMR analysis for metabolites in serum and urine from rats administrated chronically with La(NO3)(3)

H-1 NMR spectroscopy has been used to assess long-term toxicological effects of a rare earth. Male Wistar rats were administrated orally with La(NO3)(3) at doses of 0.1, 0.2, 2.0, 10, and 20 mg/kg body wt, resp., for 3-6 months. Urine was collected at 1, 2, and 3 months and serum samples were taken after 6 months. Numerous low-M-r metabolites in rats serum and rats urine, including creatinine, citrate, glucose, ketone bodies, trimethylamine N-oxide (TMAO), and various amino acids, were identified on 400- and 500-MHz H-1 NMR spectra. La3+-induced renal and liver damage is characterized by an increase in the amounts of the excreted ketone bodies, amino acids, lactate, ethanol, succinate, TMAO, dimethylamine, and taurine and a decrease in citrate, glucose, urea, and allantoin. Information on the molecular basis of the long-term toxicity of La(NO3)(3) was derived from the abnormal patterns of metabolite excretions. An assay of some biochemical indexes and analysis of some enzymes in plasma supported NMR results.