Z. Deyl

Profiling of amino acids in body fluids and tissues by means of liquid chromatography

The needs of urgent diagnoses and the needs emerging from acute forms of diseases have directed progress in amino acid profiling to modern, rapid, automated analyses that can be done at reasonable cost. The first step in this direction was the short programmes of classical ion-exchange chromatography. At the beginning of this review we attempted to survey methods of sample preparation and sample treatment, as these are frequently neglected stages where artefacts or erroneous results may arise. There are basically the following approaches in amino acid profiling by liquid chromatographic techniques. For preliminary screening of a large number of samples in clinical routine planar procedures are the methods of choice, as they allow large numbers of samples to be handled with minimum effort and at very reasonable cost. For more precise profiling, particularly where quantitative data are essential, one can choose between some of the modern procedures for separating underivatized amino acids using modern equipment for cation-exchange chromatography, by making use of a stepped series of lithium citrate buffers with ninhydrin, o-phthalaldehyde or 4-fluoro-7-nitrobenzo-2,1,3-oxadiazole detection. Ninhydrin detection is preferred in those situations where the demands on sensitivity are not high. Where, however, only small amounts of samples are available or high sensitivity is required, one of the latter two methods is preferred. The o-phthalaldehyde procedure is not suitable for the detection of secondary amines and, if these are of interest, then diazole derivatization is to be preferred. At present, however, the ninhydrin and o-phthalaldehyde detection procedures are the most popular. The other choice is to use one of the sophisticated HPLC systems equipped with fluorescence detection and to separate amino acids as derivatives. Here o-phthalaldehyde and 4-fluoro-7-nitrobenzo-2,1,3-oxadiazole derivatives offer the most versatile possibilities. Automation and computerization have penetrated both categories of liquid column separation and are applied to automated sample delivery, automated and computerized gradient formation and quantitation of the data obtained. The tables of metabolic disorders of amino acids and the roles of different amino acids in these disorders should provide preliminary information for clinical chemists.

Studies on the chemical nature of elastin fluorescence

Two fluorescent fractions were found in total acid hydrolysate of elastin. The fraction with higher chromatography mobility in isopropyl alcohol/conc. ammonia/water (9 : 1 : 2) was purified by multiple preparative paper chromatography in the same solvent system, and by gel chromatography on Sephadex G-25, ion-exchange chromatography on phosphocellulose and another gel chromatography on Sephadex G-10. The purified material was chromatographically homogeneous, had an ultraviolet absorption maximum at 315 nm and exhibited a strong 320/405 nm fluorescence. 1H- and 13C-NMR spectra were in good agreement with those published previously [6] for pyridinoline, a lysine derived fluorescent compound in collagen. The major part of the fluorescent material present in acid hydrolysate of elastin was always contaminated, even after complex purification procedures. It is concluded that elastin contains several fluorophores, one of which is a cross-linking tricarboxylic amino acid with a pyridinium ring having very probably the structure of 3-(2-amino-2-carboxyethyl)-1-(5-amino-5-carboxy-2-hydroxy-pentyl)-4-(3-amino-3- carboxypropyl)-5-hydroxypyridinium. The position of 2-amino-2-carboxyethyl and 3-amino-3-carboxypropyl residues has not been definitely established and can be interchanged.

Separation of collagens by capillary zone electrophoresis

Collagen (types I, II, V, IX and XI) constituting polypeptide chains and their polymers and cyanogen bromide-cleaved peptides of collagen type I and type III were investigated by means of capillary zone electrophoresis. Separations were effected in 2.5 mM sodium tetraborate buffer in less than 15 min. A 50 cm x 0.1 mm I.D. fused-silica capillary was used. The separations were run at 18 kV per capillary. The results of the separation were monitored at 220 nm with an on-tube detection system. Using the Offord equation, relative retention times of cyanogen bromide cleavage fragments were plotted against M(2-3)/Z, where M is the molecular mass of a polypeptide and Z its valency. A linear relationship was observed. Collagen alpha-chains and their polymers were also satisfactorily resolved.

Capillary zone electrophoresis: its applicability and potential in biochemical analysis

Recent developments in capillary zone electrophoresis (CZE) are reviewed, starting with available instrumentation, a description of different operational modes and the most commonly used detection systems. Appropriate attention is paid to CZE-mass spectrometry coupling and coupling of electrophoretic and chromatographic procedures. The possibility of separating chiral molecules is also discussed. Examples of applications concern mainly amino acids, peptides, proteins, nucleic acids and their constituents.

Simple apparatus for capillary zone electrophoresis and its application to protein analysis

The construction of a simple apparatus for capillary zone electrophoresis is described, consisting of an optical system allowing direct absorbance measurement in the capillary in UV light, an evaluating electronic module and a high-potential source. An attempt was made to achieve maximum sensitivity with a simple construction. In the electronics, care was taken to obtain a quiet baseline and to optimize the signal-to-noise ratio. That part of the noise which is of the frequency band of the signal is filtered off. Both suction and electrophoretic sample introduction are possible. According to operators choice, the apparatus can be run under constant voltage or current and is protected against overloading. The high-potential electrode chamber contains separate buffer and sample compartments and its construction offers an easy interchange between the running and sampling positions. The applicability of the system to the separation of amino acids as phenylthiocarbamyl derivatives, peptides and both artificial and naturally occurring protein mixtures is demonstrated.

The effect of low protein-high dextrin diet and subsequent food restriction upon life prolongation in Fischer 344 male rats

Fischer 344 rats fed low protein-high dextrin diet exhibit a higher median (but not 10th percentile) survival as compared to controls. The effect of this diet appears already if the diet is administered between 6 weeks and 6 months of age; after this treatment median survival of experimental animals is increased by 96 days while the 10th percentile is not different from standard diet-fed controls. Further treatment of animals with the same diet has minimum effect as animals that lived on this regimen throughout the whole life exhibited a median lifespan increase by 120 days and increase in the 10th percentile by 41 days. However, if such animals at the age of 6 months are transferred to a restricted (60%) food intake regimen (control diet, not carbohydrate enriched) a further increase in median and 10th percentile lifespan prolongation can be observed reaching +328 and +396 days respectively as compared to controls. The effects of this early feeding (6 weeks to 6 months) with a low protein-high carbohydrate diet available ad libitum and the food restricted regimen (standard diet 60% controls) fed from the age 6 months onwards are additive, the final results being identical to those obtained if the animals were kept on the 60% food restricted intake throughout the whole life. The fact that animals fed the low protein-high carbohydrate diet and those kept on 60% standard diet food restriction had different survival though they were equal in daily (identical) protein intake is emphasized.

Two-dimensional thin-layer chromatography of Dns-amino acids on reversed-phase silica gel

Ready-for-use reversed-phase high-performance thin-layer chromatographic plates were used for the separation of Dns-amino acids. The development was performed by using methanol-2% acetic acid (75.25) or methanol-0.01 M Na2HPO4 (75:25). Reversed-phase plates were used also for adsorption chromatography using a non-aqueous solvent system such as benzene-chloroform-acetic acid (50:48:2) or n-heptane-ethyl acetate-acetic acid (65:33:2). Good separations were achieved by using both principles in the two-dimensional arrangement.

Change with age of UV absorbance and fluorescence of collagen and accumulation of ϵ-hexosyllysine in collagen from wistar rats living on different food restriction regimes

Accumulation of glycation products (as revealed by the thiobarbituric test and hexosyllysine assay) and the pigmented products (350 nm UV absorbance and 370ex/440em nm fluorescence) in aortal and skin collagen was investigated under the conditions of different nutritional regimes. Four groups of animals were tested: (1) ad libitum fed controls, (2) animals which were food restricted throughout their whole life (50% food intake), (3) animals fed ad libitum during their first year of life and then food restricted and (4) animals food restricted when young and fed ad libitum from the age of 1 year onwards. It was shown that all food-restricted animals showed lower levels of glycation and pigmentation products in collagen preparations from skin and aorta. The lowest accumulation was observed in group 4 which exhibited the longest 50% survival (29.4 months, as compared with 18.3 months in normally-fed controls). Of particular interest is the fact that in this group the decreased rate of accumulation of the glycated and pigmented products was preserved even after 1 year of life, i.e., when the animals had a free access to food. Though not directly supporting the glycation theory of aging (Cerami, 1985), our data are indicative of the involvement of glucose metabolism in the ageing process. Correlation between the levels of glycated and pigmented products in aortal and skin collagen as well as the correlation between the rate of accumulation of these products and 50% survival was impossible to establish. Nevertheless, each time that food restriction was imposed on the animals it always resulted in decreased accumulation of glycated and pigmented products and increased 50% survival. Possible mechanisms for this process are discussed.

Application of the sorption properties of spheron gels in high-resolution liquid column chromatography of naturally occurring macromolecular species

Abstract Sorption effects, in addition to molecular sieving properties, of Spheron P 1000 gel (grain size less than 25 μm) were exploited for the separation of complex naturally occurring mixtures of high-molecular-weight compounds. The applicability of this approach was demonstrated on the separation of glycosaminoglycans and proteoglycan. In both examples, separations exceeding those obtained with conventional sorbents and ion exchangers were achieved.

Increased glycation and pigmentation of collagen in aged and young parabiotic rats and mice

Changes of non-enzymatic collagen glycosylation were followed in 2- and 24-month-old rats and mice and in parabiotic animals of the same age. With advancing age increased glycation of collagen was observed in both old male Wistar rats and white mice. Further it was demonstrated that both aortal and skin collagen of young animals is rapidly non-enzymatically glycosylated in the common milieu created by parabiotic animals and the proportion of non-enzymatically incorporated glucose approaches in the young counterparts the level found in old individuals. Similar trends as with non-enzymatic glycosylation were found with a fluorescent (370/440 nm) product present in both categories of collagen preparations. This fluorescence was higher in old animals and was considerably increased in the young counterpart of the parabiotic couple 6 weeks after operation. The nature of the fluorescent product appears different from pyridinoline and remains to be elucidated.