The photochemical attachment of the O-glucoside of 3-hydroxykynurenine to alpha-crystallin: a model for lenticular aging

The young human lens contains a small metabolite from tryptophan called the O-glucoside of 3-hydroxykynurenine (3-HKG). Its function is to absorb most radiation between 295 and 400 nm, preventing it from reaching the retina. With age the concentration of this component decreases while the lens crystallins acquire covalently attached chromophores. This study investigates the photochemical attachment of 3-HKG to lens alpha-crystallin. Initial studies showed that alpha-crystallin photolyzed in the presence of 3-HKG developed a fluorescence (emission, 440 nm) and UV-visible spectrum similar to that found in aged human lens proteins. Extensive studies were then performed on the tryptic HPLC maps as monitored by photodiode array and fluorescent detection. Numerous photoproducts with either blue (emission, > 400 nm) or green (emission, > 500 nm) fluorescence were formed in addition to nonfluorescent compounds with absorption maxima above 300 nm. Comparisons were made between these model photoproducts and peptide maps from alpha-crystallin isolated from old human lenses. In terms of retention time and UV-visible spectra at least two of the peptides that appear in the model system are also present in the human samples. It is concluded that one of the aging processes in the human lens is the photochemically induced attachment of 3-HKG to lens proteins.     

MODEL STUDIES ON THE PHOTOCHEMICAL PRODUCTION OF LENTICULAR FLUOROPHORES

Abstract With aging, human lens proteins accumulate fluorophores having blue and green emissions. Model studies were undertaken to determine the role of 3-hydroxykynurenine (3-HK) and its glucoside (3-HKG) in the photochemical production of those fluorophores. Experiments were carried out using 10⁻³ M 3-HK solutions in the presence or absence of glycine (1 M ), which was used to mimic the environment of the lens. The solutions were photolyzed (transmission above 295 nm) for various periods of time while the loss of starting material and the formation of fluorescent photoproducts (blue emission at 470 nm, and green emission at 520 nm) were monitored using fluorescence and UV-visible spectroscopy and thin-layer and high-pressure liquid chromatography analysis. Several parameters were varied such as oxygen tension and the addition of the free radical scavenger, penicillamine. The photolytic loss of 3-HK in the absence of glycine occurred approximately 5-10 times faster than in its presence. Conversely, blue and green fluorophores formed in irradiated solutions containing glycine but not with the photolysis of 3-HK alone. The blue fluorophore was formed first and appeared then to be photochemically converted to the green one, with the rate of formation of the latter increasing with an increase in UV dosage or oxidizing conditions. The addition of penicillamine drastically reduced the photochemical formation of both fluorophores.
Both the blue and green fluorophores appear to result from the photochemically induced covalent attachment of 3-HK to glycine. In the human lens, these reactions can explain the age-related loss of 3-HKG with the concomitant formation of fluorophores covalently attached to lens proteins, probably via the amino group of lysine.     

STUDIES ON HUMAN LENS: I. ORIGIN AND DEVELOPMENT OF FLUORESCENT PIGMENTS

Abstract
Fluorescence spectra of normal mature human lenses have been measured and at least eight species with distinct emission characteristics identified. To determine the specific photochemical and photophysical processes responsible for the origin and development of these fluorophores, emission behavior of the products generated by successive irradiation of young human lenses (3–6 y old) as well as of L-tryptophan solution have been systematically monitored. Fluorescent products that resulted from this irradiation were comparable to many of the fluorophores detected in aged lenses, indicating that light plays a major role in the development of these pigments. In addition to photogenerated species, there are other compounds in human lenses, presumably advanced glycosylated end products, with marked fluorescence properties.
Several oxidation products of tryptophan including N -formylkynurenine or its derivatives, β-carboline or its derivatives, and anthranilic acid have been identified in the mature human lens. The development of several photoproducts also was attributed to endogenous ascorbate-mediated Maillard reaction products, which undergo photoconversion by the visible light. Although some of these chromophores could act as photosensitaizers, the sensitizing efficiency of many are low. Conversely, the near-UV filtering capability of these colored compounds conceivably could protect the vitreous and retina from development of any photochemical lesion.     

STUDIES ON HUMAN LENS: I. ORIGIN AND DEVELOPMENT OF FLUORESCENT PIGMENTS

Fluorescence spectra of normal mature human lenses have been measured and at least eight species with distinct emission characteristics identified. To determine the specific photochemical and photophysical processes responsible for the origin and development of these fluorophores, emission behavior of the products generated by successive irradiation of young human lenses (3-6 y old) as well as of L-tryptophan solution have been systematically monitored. Fluorescent products that resulted from this irradiation were comparable to many of the fluorophores detected in aged lenses, indicating that light plays a major role in the development of these pigments. In addition to photogenerated species, there are other compounds in human lenses, presumably advanced glycosylated end products, with marked fluorescence properties. Several oxidation products of tryptophan including N-formylkynurenine or its derivatives, beta-carboline or its derivatives, and anthranilic acid have been identified in the mature human lens. The development of several photoproducts also was attributed to endogenous ascorbate-mediated Maillard reaction products, which undergo photoconversion by the visible light. Although some of these chromophores could act as photosensitizers, the sensitizing efficiency of many are low. Conversely, the near-UV filtering capability of these colored compounds conceivably could protect the vitreous and retina from development of any photochemical lesion.     

Isolation of experimental anti-AIDS glycerophospholipids by micro-preparative reversed-phase high-performance liquid chromatography.

The experimental anti-AIDS glycerophosphatidic acid: nucleoside (sn-1/sn-2 diacylglycerol:dideoxynucleotide) drugs 3'-azido-3'-deoxythymidine monophosphate diglyceride (AZT-MP-DG) and 2',3'-dideoxycytidine monophosphate diglyceride (ddC-MP-DG) were isolated and purified by reversed-phase high-performance liquid chromatography (HPLC). The chromatographic separation was based on the glycerophospholipid moiety of the drugs and detection of the nucleoside component. The separations were optimized on method development columns packed with the stationary phase to be used in the micro-preparative column and monitored by a UV detector. Fractions were collected and analyzed for purity by analytical-scale HPLC and by thin-layer chromatography (TLC). The purity of the recovered drugs based on UV and light-scattering detection and on TLC was greater than 99%. The purified compounds were isolated for studies on structure confirmation, physical, biophysical and formulation properties and anti-HIV efficacy in culture.     

PHOTOCHEMICAL AND PHOTOPHYSICAL STUDIES ON HUMAN LENS CONSTITUENTS

The young human lens contains species (3-hydroxy kynurenine; 3-HK and its glucoside; 3-HKG) which absorb most light between 300 and 400 nm. Photochemical studies have indicated that these compounds are relatively inefficient sensitizers of lens proteins. An investigation of the fluorescent properties of 3-HKG indicate that it contains a fast deactivation pathway (ps) which would be expected to have minimal photochemical effect on the integrity of the lens. Further phot physical studies on 3-HK indicates that it has an even faster fluorescent lifetime (less than 10 ps) with a much lower quantum yield of fluorescence (0.001 vs 0.03 for 3-HKG). With aging, the human lens proteins undergo numerous changes including a generalized yellowing. These chromophores exhibit a higher quantum yield of fluorescence, an increase in the fluorescent lifetime by 2 orders of magnitude and the formation of two long lived transient species (microsecond). These species might be expected to drastically increase the susceptibility of the human lens to ambient radiation. Based upon quantitative experimental comparisons with 3-HK this does not seem to be the case. Further time resolved studies on old lens proteins indicate that the two transient species are interconnected in that the first transient species is the precursor to the second. The implications of this mechanism on the integrity of the lens and origin of those chromophores is discussed.     

DSC study of radiostability of 1,4-dihydropyridine derivatives

The effect of sterilisation by irradiation has been studied for the seven most often used in medicine derivatives of 1,4-dihydropyridine (nifedipine, nisoldipine, nicardipine, nitrendipine, nimodipine, felodipine and amlodipine). The sterilisation was performed for the compounds in the solid phase with an electron beam of the energy 10 MeV, at room temperature, using the irradiation doses from 20 to 400 kGy. The effects of the irradiation were studied by the methods SEM, DSC, XRD and TLC. The sterilisation with doses 20-100 kGy was found to cause no changes in the physico-chemical properties of the compounds, while the irradiation with higher doses (200-400 kGy) was found to induce changes in the colour, DSC spectrum and TLC picture. As follows from the TLC results, the main product of radiolysis of the compounds studied was a pyridine nitrozoderivative, which indicates the same mechanism of decomposition as in the process of photodegradation. The results prove that the 1,4-dihydropyridine derivatives being highly sensitive to visible and UV radiation are generally resistant to ionising radiation and thus can be subjected to sterilisation by irradiation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of the UV Modification of α-Crystallin on Its Ability to Suppress Nonspecific Aggregation

Recent studies have shown that structural modifications of α-crystallin during lens aging decrease it's effectiveness as a molecular chaperone. Some of these posttranslational modifications have been linked to UV radiation, and this study was undertaken to investigate the effect of UV irradiation on the ability of α-crystallin to suppress nonspecific aggregation. The effect of 3-hydroxykynurenine (3-HK) was also investigated as a model for its glucoside (3-HKG), a main lens chromophore that has been linked to photochemical changes in the human lens. Alpha- and γ-crystallin solutions (1 mg/mL, 1:0.125 wt/wt) were photolyzed (transmission above 295 nm) for various time intervals. Thermal denaturation of γ-crystallin with or without α-crystallin was carried out at 70°C and increases in light scattering were measured at 360 nm. We found that (1) irradiation of γ-crystallin increased its susceptibility to heat-induced scattering. The addition of α-crystallin protects it against thermal denaturation, although its ability to do so decreases the longer γ-crystallin is irradiated and (2) irradiation of α-crystallin decreases its ability to suppress nonspecific aggregation and the presence of 3-HK during irradiation decreases it further. Our results indicate that posttranslational modifications of α-crystallin due to UV irradiation affect the sites and mechanisms by which it interacts with γ-crystallin. The kinetics of γ-crystallin unfolding during thermal denaturation were also analyzed. We found that a simple two state model applies for nonirradiated γ-crystallin. This model does not hold when γ-crystallin is irradiated in the presence or absence of α-crystallin. In these cases, two step or multistep mechanisms are more likely.     

Rapid,simple, and highly sensitive analysis of drugs in biological samples using thin-layer chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry

Rapid and precise identification of toxic substances is necessary for urgent diagnosis and treatment of poisoning cases and for establishing the cause of death in postmortem examinations. However, identification of compounds in biological samples using gas chromatography and liquid chromatography coupled with mass spectrometry entails time-consuming and labor-intensive sample preparations. In this study, we examined a simple preparation and highly sensitive analysis of drugs in biological samples such as urine, plasma, and organs using thin-layer chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry (TLC/MALDI/MS). When the urine containing 3,4-methylenedioxymethamphetamine (MDMA) without sample dilution was spotted on a thin-layer chromatography (TLC) plate and was analyzed by TLC/MALDI/MS, the detection limit of the MDMA spot was 0.05 ng/spot. The value was the same as that in aqueous solution spotted on a stainless steel plate. All the 11 psychotropic compounds tested (MDMA, 4-hydroxy-3-methoxymethamphetamine, 3,4-methylenedioxyamphetamine, methamphetamine, p-hydroxymethamphetamine, amphetamine, ketamine, caffeine, chlorpromazine, triazolam, and morphine) on a TLC plate were detected at levels of 0.05 − 5 ng, and the type (layer thickness and fluorescence) of TLC plate did not affect detection sensitivity. In addition, when rat liver homogenate obtained after MDMA administration (10 mg/kg) was spotted on a TLC plate, MDMA and its main metabolites were identified using TLC/MALDI/MS, and the spots on a TLC plate were visualized by MALDI/imaging MS. The total analytical time from spotting of intact biological samples to the output of analytical results was within 30 min. TLC/MALDI/MS enabled rapid, simple, and highly sensitive analysis of drugs from intact biological samples and crude extracts. Accordingly, this method could be applied to rapid drug screening and precise identification of toxic substances in poisoning cases and postmortem examinations.     

Screening search for organic fluorophores: syntheses and fluorescence properties of 3-azolyl-7-diethylaminocoumarin derivatives

In order to find a highly sensitive fluorophore, 3-azolyl-7-diethylaminocoumarin derivatives were synthesized. Both the absorption and fluorescence maxima of the coumarin-thiazole compounds showed red shifts with increases of the molar absorptivities and fluorescence intensities, in comparison with those of the corresponding coumarin-oxazole compounds. Among them, 3-(5-ethoxycarbonyl-1,3-thiazol-2-yl)-7-diethylamino-2H-chromen -2-one (3e) was one of the most promising candidates as a fluorophore accessible for analytical purposes in the fields of analytical and biological chemistry.     

Lipophilicity analysis of newly synthetized quinobenzothiazines by use of TLC

Lipophilicity is a very important property of chemical compound taking into consideration in drugs design. Relationships between biological activity, among others lipophilicity, and chemical structure (QSAR) of the compound are very often used by researches. Especially important is the kind of substituents connected to the basic structural fragment and how it changes the lipophilicity of the compound. The aim of this study was to determine the parameters of lipophilicity of quinobenzothiazine derivatives using reversed phase - thin-layer chromatography (RP-TLC), which would enable one to determine the structure–activity relationship. The objective of our work is a series of 15 newly synthetized quinobenzothiazines. They were analyzed by thin-layer chromatography (TLC) with the use of two different mobile phases consisting of methanol or acetone as organic modifiers. For all compounds investigated, the values of lipophilicity obtained from computational method were also determined. Cluster analysis was carried out too for all data of lipophilicity obtained. Low correlation was found between values of experimental lipophilicity and lipophilicity from computational methods for newly synthetized compounds.     

Preparative-scale chromatography of ecdysteroids of Serratula wolffli andrae

Numerous ecdysteroids are isolated from the herb of Serratula wolffii Andrae, a cultivated plant. The isolation procedure includes a variety of low-pressure liquid chromatography, thin-layer chromatography (TLC), gel chromatography, and high-performance liquid chromatography (HPLC) methods. The progress of separation is monitored by TLC, and the final proof of purity is carried out by HPLC. The isolation process involves the removal of proteins, flavonoids, chlorophylls, other sterines, etc. The purification also includes the separation of the target ecdysteroids from each other. Isolation of the pure compounds requires 2-8 chromatographic steps. The consecutive steps are based on the different physicochemical properties of the ecdysteroids. In some cases, a special peak-cut method employing a flush of dichloromethane into the dichloromethane-isopropanol-water mobile phase is used. This flush of dichloromethane leads to an almost perfect separation of otherwise unresolved peaks. Two ecdysteroids, 25-hydroxydacryhainansterone and 14-epi-20-hydroxyecdysone, are identified as natural products for the first time. The structure-chiroptical relationships for some ecdysteroids are also discussed.     

Evaluation of lipid removal from animal fats for the determination of organochlorine, pesticides, and polychlorinated biphenyl indicators by gas chromatography with electron capture detector

For cleanup of animal fat before GC analysis of organochlorine pesticides and polychlorinated biphenyls, freezing-lipid filtration with solid-phase extraction and matrix solid-phase dispersion were evaluated to replace the official Brazilian methodology that uses preparative alumina column chromatography. General drawbacks associated with this last technique, such as the use of large amounts of solvent, laborious and time-consuming procedure could be avoided by using these alternative approaches. Experiments were carried out to study the performance by using different combinations of sorbents and elution solvents. Efficiency of alternative extraction methods in terms of fat removal and recovery capability was monitored by gravimetry, TLC, and GC with electron capture detection. Freezing-lipid filtration with solid-phase extraction afforded better clean up efficiency with recoveries in a range of 54.5 to 103.6% with the relative standard deviation of less than 10% for all compounds under investigation.     

Identification of some polycyclic nitrogen-containing compounds in coal-derived oil

A systematic method for the identification of aza-arenes in coal-derived oil was developed. The basic nitrogen-containing substances were extracted with 6 M hydrochloric acid and fractionated sequentially by using gel chromatography and thin-layer chromatography (TLC) on an alumina plate. The aza-arenes in these fractions were separated by using glass capillary gas chromatography. Individual compounds in the column effluent were trapped in a system consisting of a valve for flow switching and a trapping tube made from a glass capillary. The fluorescence spectra of nanogram to subnanogram amounts of trapped compounds were measured. Some attempts were made to identify components based on their TLC RF values and their fluorescence spectra, in addition to their mass spectra.     

A new procedure for separation of complex mixtures of pesticides by multidimensional planar chromatography

The objective of this communication is to report a new procedure for the separation of complex mixtures of pesticides by a combination of different modes of multidimensional planar chromatography. Complete separation of a mixture of pesticides was carried out by multidimensional planar chromatography on silica gel plates. The plates were videoscanned to show a real picture of the experimental results. Complete separation of all compounds was not possible by a single development on silica gel. The separation can be characterized as [PC x (nPC + PC + PC + PC)]. With the aid of this new procedure a complex mixture of fourteen compounds was separated on a 10 x 10 cm TLC plate.     

分离与鉴定柴油基体中的微量芥子气类化合物

本文叙述了制备和分析柴油和芳香烃混合溶剂基体中痕量芥子气及芥子气二硫醚样品的方法。样品用硅胶色谱柱预分离及正已烷洗脱除去大量的基体；并用制备薄层色谱分离得到纯样。样品经ＧＣ－ＭＳ，质子ＮＭＲ法等作结构鉴定。分析结果与标准样品相一致。     

Effect of γ-irradiation on volatile compounds of dried Welsh onion (Allium fistulosum L.)

The volatile compounds of γ-irradiated dried Welsh onion were isolated by simultaneous distillation–extraction (SDE) technique and then analyzed by gas chromatography–mass spectrometry (GC–MS) along with their non-irradiated counterparts. A total of 35 volatile compounds were identified in non-irradiated and 1 kGy irradiated samples and 36 volatile compounds were identified in 3, 5, 10 and 20 kGy irradiated samples so far belong to chemical classes of acid, alcohol, aldehyde, ester, furan, ketone and S-containing compound. S-containing compounds were detected as major volatile compounds of all experimental samples. Though the content of several compounds was increased after irradiation, content of major S-containing compounds was found to decreased in the process. Application of high-dose irradiation if required for microbial decontamination of dried Welsh onion is feasible as it enhanced the total concentration of volatile compounds by 31.60% and 24.85% at 10 and 20 kGy, respectively.     

Paper electrophoretic separation of some diastereomers

Summary Gas chromatography (GC) and thin-layer chromatography (TLC) have received most attention as techniques for separation of stereoisomers. Indirect separations by GC of diastereomers have been extensively reviewed by Gil-Av and Nurok [1] while Lochmüller and Souter recently reviewed direct GC resolution methods [2]. TLC work involving diastereomeric compounds of various classes: -methyltryptophans [3], di- and tripeptides [4], some aliphatic compounds [5], and thymidine hydrates [6] have been reported. Although much attention is given to diastereomer separations by GC and TLC, no reports exist of such applications by electrophoresis. This report describes some high voltage electrophoretic separations of diastereomers of several pharmaceutically and biochemically useful compounds.     

Rapid and sensitive determination of tryptophan, serotonin and psychoactive tryptamines by thin-layer chromatography/fluorescence detection

A rapid, sensitive and selective method for the determination of tryptophan (Trp), serotonin (5-HT) and psychoactive tryptamines (PATs) by thin-layer chromatography (TLC) with fluorescence detection is proposed. These compounds form fluorophores on the developing plate by heating after spraying with sodium hypochlorite, hydrogen peroxide or potassium hexacyanoferrate(III)-sodium hydroxide reagent. Fluorescent spots (vivid blue) were observed by irradiation with ultraviolet light (365 nm). The detection limits of Trp, 5-HT and PATs were in the range from 0.01 microg to 0.06 microg. This method was effectively applied to the detection of confiscated PAT powder and PAT in abusers' urine samples.