Peter Stehle

Appraisal of four pre-column derivatization methods for the high-performance liquid chromatographic determination of free amino acids in biological materials

Reversed-phase high-performance liquid chromatography (RP-HPLC) is a powerful method for assaying physiological amino acid concentrations in biological fluids. Four pre-column derivatization methods, with o-phthaldialdehyde (OPA), 9-fluorenylmethyl chloroformate (FMOC-Cl), phenyl isothiocyanate (PITC) and 1-dimethylaminonaphthalene-5-sulphonyl chloride (dansyl-Cl), were assessed with respect to their applicability in biological research. The methods permit the measurement of 21-26 major amino acids in 13-40 min. The superior sensitivity favours the use of OPA, FMOC-Cl and dansyl-Cl techniques. Because of instability of the OPA adducts, automated on-line derivatization is required when using this method in general practice. Application of the PITC method, although less sensitive, is useful in clinical chemistry, where sample availability is rarely a problem. Cystine determination is not feasible when using OPA or FMOC-Cl and with PITC the reproducibility and linearity are poor, whereas the dansyl-Cl method allows reliable quantitation. The four methods are currently used to perform ca. 8000 OPA and 5000-6000 FMOC-Cl, PITC and dansyl-Cl analyses of biological samples per year. The results obtained with the RP-HPLC methods compare favourably with those derived from conventional ion-exchange amino acid analyses. When the guard column is regularly changed after 120 analyses, the separation remains satisfactory for at least 700 OPA, 800 FMOC-Cl, 150 PITC and 500 dansyl-Cl analyses. Careful control of factors and limitations inherent in the various methodologies is a prerequesite for proper identification and appropriate quantitation.

Effect of free glutamine and alanyl-glutamine dipeptide on mucosal proliferation of the human ileum and colon

BACKGROUND/AIMS Glutamine (Gln) is considered a trophic factor for small intestinal epithelia, which is important during severe illness. Its use in parenteral nutrition is precluded by its instability, a problem that may be overcome by use of the stable dipeptide L-alanyl-L-glutamine (Ala-Gln). The hypothesis was tested that Gln or Ala-Gln may stimulate cell proliferation not only in the ileum but also in the proximal and distal colon and, thus, may contribute to the gut barrier function. METHODS Biopsy samples from the normal human ileum, proximal colon, and rectosigmoid were incubated for 4 hours with Gln (2 mmol/L), Ala-Gln (2 mmol/L), and saline (control). Cells in S phase were labeled with bromodeoxyuridine. In longitudinal crypt sections labeled and quiescent cells were counted. RESULTS Gln as well as Ala-Gln stimulated crypt cell proliferation in the ileum, proximal colon, and rectosigmoid colon. In ileal specimens, labeling was greater in the entire crypt, whereas in both colonic regions, the trophic effect was confined to the basal crypt compartments. CONCLUSIONS Gln and Ala-Gln have trophic effects not only in the ileum, but also in the proximal and distal colon. This could be important during parenteral nutrition when mucosal atrophy may weaken the gut barrier.

Glutamine-containing dipeptides in parenteral nutrition.

Of the total pool of muscle free intracellular amino acids, glutamine represents about 60%. During catabolic stress, a marked reduction (50%) of this pool occurs; the depletion is not reversible by therapeutic efforts or conventional nutritional means. If maintenance of the intracellular glutamine pool promotes conservation of muscle protein, there is a theoretical case for use of glutamine supplements in the parenteral nutrition of patients with injury and infection. Glutamine is too unstable and poorly soluble for addition to existing preparations in its native form, but this drawback can be overcome by the use of synthetic stable and highly soluble glutamine-containing dipeptides. In vivo studies in humans and animals provide firm evidence that a synthetic glutamine-containing dipeptide, L-alanyl-L-glutamine (Ala-Gln), is readily hydrolyzed following its intravenous administration. The results also indicate a safe and efficient use of Ala-Gln as a source of free glutamine in parenteral nutrition. In clinical studies, nitrogen balance was more positive in catabolic patients receiving a peptide-supplemented solution than in control patients given isonitrogenous, isoenergetic total parenteral nutrition. Muscle glutamine concentrations were markedly decreased in the control groups. The intracellular concentrations were not influenced following severe injury, but were maintained in postoperative trauma. It is inferred that the increased intestinal requirement and cellular demand for metabolic fuel during catabolic stress is matched by an enhanced demand on muscle glutamine, resulting in intracellular glutamine depletion. Thus, the delivery of adequate amounts of glutamine is essential to maintain the integrity of intestinal mucosa and rapidly proliferating cells, to preserve the muscle glutamine pool, and to improve overall nitrogen economy during conditions of stress.

Papain-catalysed synthesis of dipeptides: a novel approach using free amino acids as nucleophiles.

For the first time, papain-catalysed synthesis of peptide bonds was successfully carried out using free amino acids as nucleophiles. In kinetically controlled experiments employing pH-Stat-mode, the ester substrates Z-Ala-OMe and Z-Gly-OMe were coupled with alanine, glutamine, and Cys(Acm)-OH, respectively. Under optimized reaction conditions (pH 9.2, high ratio nucleophile/carboxyl component, 10 mumol substrate mg-1 papain), the peptide yields ranged from 17% to 79%, depending on the structure of the amino and/or carboxyl component. The peptides formed were not hydrolysed under the chosen reaction conditions. With Z-Gly-OMe as the ester substrate, formation of the dipeptide was both rapid and high yielding. Papain-catalysed formation of peptide bonds applying free amino acids as nucleophiles might serve as an economic and easily manageable approach for the synthesis of short-chain peptides to be used in clinical nutrition.

Availability of glutamine supplied intravenously as alanylglutamine

In this review, new knowledge about the potential use of glutamine containing dipeptides as substrates in the frame of parenteral nutrition is presented. Using chemical and biotechnological methods, the stable and highly soluble peptide L-alanyl-L-glutamine (Ala-Gln) can be synthesized in high yields. Studies in experimental rats and dogs demonstrate the effective utilization of intravenously supplied Ala-Gln and the rapid provision of free glutamine for maintenance of the intracellular muscle-free glutamine pool in catabolic situations. Subsequent studies in healthy volunteers provide firm evidence that the infused Ala-Gln is rapidly eliminated from plasma (t1/2:3.8 minutes), associated by a prompt equimolar increase in the concentrations of free alanine and glutamine. Bolus injection and continuous infusion of the peptide was not accompanied by any side effects, and no complaints by the subjects were noted. These results may indicate a safe and efficient use of Ala-Gln as source of free glutamine in parenteral nutrition.

Whole-Body Autoradiography in Rats after Intravenous Administration of L-Alanyl-L-[U-14C]Glutamine

The uptake and distribution in rats of the radiolabelled dipeptide L-alanyl-L-[U-14C]glutamine were investigated by whole-body autoradiography. Rats were killed 5, 30 and 180 min following bolus injection of the peptide via the tail vein and, as a comparison, 180 min after injection of [U-14C]glutamine; 30-microns sections of the animals were autoradiographed against an X-ray film. Tissue specimens from the remainder of the carcass were completely combusted and the 14CO2 formed was analyzed by liquid scintillation counting. Visceral organs especially exocrine glands and the mucosa of the alimentary tract showed an initially high labelling (5 min), whereas skeletal muscle and brain exhibited maximum radioactivity 30 min after peptide bolus. Muscle and intestine (19 and 10% of the dose injected, respectively) showed the highest 14C incorporation of all tissues investigated. The broad conformity in 14C distribution observed after peptide and glutamine bolus strongly indicates similar utilization of free and peptide-bound glutamine.

Analytical control of enzyme-catalyzed peptide synthesis using capillary isotachophoresis

Abstract Analytical capillary isotachophoresis (ITP) was employed to control an enzyme-catalyzed peptide synthesis and the subsequent purification, as well as to evaluate the amino acid composition after acid and enzymatic hydrolysis. Compared with alternative chromatographic techniques. ITP offers certain advantages such as simultaneous detection of the synthetic peptide, amino acids, amino acid derivatives and contaminating inorganic ions in amounts of less than 200 ng. In addition, ITP provides quantitative information about the composition of the untreated samples directly analyzed without the mandatory use of suitable reference substances.

Isotachophoretic control of peptide synthesis and purification : A Novel approach using ultraviolet detection at 206 nm

Abstract The synthesis and purification of two glutamine-containing dipeptides were controlled by applying analytical capillary isotachophoresis. The use of a newly developed detector block allowed conductivity detection and, for the first time, UV measurement at 206 nm. This system facilitates qualitative and quantitative analysis of peptides not absorbing at 254 and 280 nm in amounts of less than 200 ng, thereby permitting a direct classification of the sample ions analysed. Optimization of peptide synthesis is significantly improved by this novel method, which enables simultaneous monitoring of the reaction products and contaminating inorganic ions during synthesis and purification.

Papain‐catalyzed synthesis of dipeptides

Abstract The cysteine proteinase papain (EC 3.4.22.2) was used to synthesize N‐protected dipeptides in a kinetically controlled reaction starting from N‐benzyloxycarbonyl‐L‐alanine methylester as the carboxyl component. Beside L‐amino acid amides, free L‐amino acids could be successfully applied to yield the corresponding Z‐dipeptides. The yields with free L‐amino acids ranged from 13–80%, obviously depending on the structure of the molecule. The evaluation of the reaction conditions for the synthesis of N‐benzyloxycarbony1‐L‐alanyl‐L‐arginine revealed that a pH of 9.5, a temperature of 25–40°C and 0.5 M L‐arginine were optimal.

The occurrence of neurotoxic pyroglutamic acid in patenteral amino acid solutions. Specific determination by means of capillary isotachophoresis

In certain commercially available amino acid solutions glutamic acid (Glu) represents an important source of nonessential nitrogen. Such preparations are generally heat-sterilized according to the regulation by health authorities and can be stored prior to use for up to five years. Since Glu in aqueous solutions is partly transformed into pyroglutamic acid (pGlu; 5-oxo-proline) at higher temperatures and storage [l-3], there is thus the potential risk that this intramolecular cyclization product may be formed in various amounts in parenteral amino acid solutions. Given the presumed neurotoxicity of pGlu [4-lo], it is pertinent to examine the occurrence of this solute in commercially available amino acid preparations. The lack of such control studies might be due to methodological difficulties with regard to a specific qualitative and quantitative determination of pGlu. In a previous communication, we applied capillary isotachophoresis (ITP) as an analytical tool Co simultaneously analyze Glu and pGlu in aqueous solutions [ll]. In the present work, ITP was employed to determine pGlu in commercial parenteral solutions in the presence of the major amino acids including Glu.