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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.12, #3-4)

Effect of amino acid imbalances on the stimulatory effect of L-tryptophan on hepatic protein synthesis by H. Sidransky M. D.; E. Verney (pp. 205-212).

Earlier it was reported that mice or rats tube-fed a single feeding of L-tryptophan (TRP) demonstrated a stimulation of hepatic protein syn thesis. The present study was concerned with whether dietary imbalances induced by tube-feeding different ratios of L-alanine (ALA) or L-leucine (LEU) in relation to TRP would affect TRP's stimulatory effect on hepatic protein synthesis. Male Swiss mice, food-deprived overnight, were tube-fed one feeding of solution keeping TRP constant and varing ratios of ALA/TRP of 0.4, 2.1, or 4 or ratios of LEU/TRP of 4.8, 7.2, or 9.6. After 1 h, mice were killed and protein synthesis (¹⁴C-leucine incorporation into proteins in vitro using microsomes of livers) was measured. TRP alone stimulated hepatic protein synthesis by 83 % while ALA/TRP ratios of 2.1 or 4 but not of 0.4 and LEU/TRP ratios of 9.6 but not of 4.8 or 7.2 caused significant decreases in the stimulation of hepatic protein synthesis. Measurements of serum and hepatic free TRP concentrations in the experimental groups were similar in all groups tube-fed TRP alone or in combinations.

Keywords: Amino acids; L-Tryptophan; L-Alanine; L-Leucine; Protein synthesis; Liver; Mice

Origin ofd-serine present in urine of mutant mice lackingd-amino-acid oxidase activity by S. Asakura; Dr. R. Konno (pp. 213-223).

Urine of ddY/DAO mice lackingd-amino-acid oxidase contained 5.7 times more serine than that of normal ddY/DAO⁺ mice. Most of the serine wasd-isomer. The origin of this^d-serine was examined. Oral administration of 0.02% amoxicillin and 0.004% minocycline to the ddY/ DAO^- mice for 7 days did not reduce the urinaryd-serine, indicating that thed-serine was not of intestinal bacterial origin. When the mouse diet was changed to one with different compositions, the urinaryd-serine was considerably reduced. Furthermore, starvation of the ddY/DAO^- mice for 24 hours reduced the urinaryd-serine to 33% of the original level. These results indicate that most of the urinaryd-serine comes from the diet. However, the urine of the starved ddY/DAO^- mice still contained 4.6 times mored-serine than that of the ddY/DAO⁺ mice, suggesting a part of the D-serine have an endogenous origin.

Keywords: d-Serine; Urine; Mouse; d-Amino-acid oxidase; d-Amino acids

Structural elucidation of a novel lysine-lysine crosslink generated in a glycation reaction with L-threose by M. Prabhakaram Ph.D.; Qi Cheng; M. S. Feather; B. J. Ortwerth (pp. 225-236).

A novel lysine-lysine crosslink was isolated from a reaction containing L-threose (a degradation product of ascorbic acid) and N-α acetyllysine. The compound, after chromatographic purification, had a relative molecular mass of 628 as determined by fast atom bombardment spectrometry. Structural analysis by NMR spectroscopy (¹H,¹³C, COSY, HETCOR and DEPT) suggests that the compound is composed of 3 threose residues and 2 N-α-acetyllysines with an empirical formula of C₂₈H₄₅N₄O₁₂. The compound, referred to as threosidine (2,5-dihydroxy-5,6,7,8-tetrahydro 1,7-naphthyridinium as the core molecule) has an absorption maximum at 328 nm with an excitation and emission maxima at 328 nm and 402 nm respectively. Threosidine was stable upon acid hydrolysis, and it was not found in the reactions containing threose and amino acids other than N-α-acetyllysine. Threosidine was also not found in the reactions containing N-α-acetyllysine and sugars other than threose.

Keywords: Amino acids; Lysine; L-threose; Glycation; Crosslinking; AGE

Proline metabolism in sepsis by Dr. C. Chiarla; I. Giovannini; J. H. Siegel; G. Boldrini; M. Castagneto (pp. 237-247).

Sepsis is characterized by an abnormal increase in plasma proline (PRO) level, which tends to be related to the severity of disease. This study has been performed to assess the relationship between changes in plasma PRO and levels and doses of other amino acids (AA) in critically ill septic patients undergoing total parenteral nutrition (TPN).Sixteen septic patients receiving TPN were randomly divided into two groups: 8 patients (Group A) received TPN with a standard AA solution, and 8 patients (Group B) with a modified AA solution (isonitrogenous, branchedchain AA enriched, with unchanged PRO concentration). Serial determinations of plasma AA profiles and of other variables were performed in each patient for a total of 396 measurements. In Group A mean plasma PRO level was 372μM/L; changes in PRO were tightly correlated with changes in the levels of most of the other AA, and the highest PRO levels characterized the more severely unbalanced septic metabolic profiles. In Group B, plasma levels of PRO and of the other AA (except glutamate, aspartate, taurine and the three branched-chain AA) decreased. The decrease in PRO level was well correlated with the increased branched-chain AA dose and with simultaneous decreases in plasma lactate and respiratory quotient. These changes could be related to a specific effect of branched-chain AA on septic metabolic derangement and on PRO metabolism, and to an improved balance between protein synthesis and catabolism.

Keywords: Amino acids; Proline; Sepsis; Lactate; Parenteral nutrition; Amino acid metabolism; Branched-chain amino acids; Respiratory quotient

Effect of Simazine on the production of lysine and methionine byAzotobacter chroococcum andAzotobacter vinelandii by R. Murcia; B. Rodelas; V. Salmerón; C. Pozo; Prof. J. González-López (pp. 249-255).

Production of lysine and methionine byAzotobacter chroococcum strain H23 andA. vinelandii strain ATCC 12837 was studied in chemically-defined medium and dialysed-soil medium, amended with different concentrations of Simazine. Responses on production due to Simazine were different for each strain and were fairly conditioned by culture media composition. Quantitative production of amino acids was significantly affected by the xenobiotic only at higher doses (50–100,μg/ml). The effect of Simazine on methionine production by strain H23 was very pronounced when bacteria were grown in dialysed-soil medium, which was specially formulated to reproduce the natural habitat of the organisms.

Keywords: Amino acids; Lysine; Methionine; Azotobacter ; Simazine

Synthesis ofα-triazolylα-amino acid derivatives by S. Achamlal; A. Elachgar; Pr. A. El Hallaoui; S. El Hajji; M. L. Roumestant; Ph. Viallefont (pp. 257-263).

We report the synthesis ofα-triazolylα-amino esters by 1,3 dipolar cycloaddition of acetylenic compounds andα-azidoα-amino esters.

Keywords: Amino acids; α-Azidoα-amino esters; α-Triazolylα-amino esters; Cycloaddition

Stimulation of renal amino acid reabsorption after treatment with triiodothyronine or dexamethasone in amino acid loaded rats by Prof. Dr. Ch. Fleck; M. Aurich; M. Schwertfeger (pp. 265-279).

In adult female anaestetized rats, the influence of triiodothyronine or dexamethasone on renal amino acid handling was investigated in leucine (20mg/100g b.wt.) or glutamine (45mg/100g b.wt.) loaded animals. Bolus injections of both amino acids were followed by temporary increase in fractional excretion of the administered amino acids as well of the endogenous amino acids which were not administered.Under load conditions (leucine and glutamine), dexamethasone treatment (60 μg/100 g b.wt. for 3 days, i.p. once daily) was followed by a stimulation of renal amino acid reabsorption. The increase in fractional amino acid excretion after amino acid load was significantly lower than in untreated rats. The effect of triiodothyronine (20,μg/1008 b.wt. for 3 days, i.p. once daily) was different in leucine and glutamine loaded animals: after leucine bolus injection a comparable stimulatory effect as shown for dexamethasone could be demonstrated, but after glutamine administration the stimulatory action of T3 was masked. T3 even increases fractional amino acid excretion in glutamine loaded rats as a sign of enhanced “house-keeping” in the renal tubular cells. These results confirm previous findings and indicate different effects of both hormones on the renal handling of amino acids.

Keywords: Amino acid transport; Kidney; Triiodothyronine; Dexamethasone; Amino acid load; Rats

Role of taurine in osmoregulation in brain cells: Mechanisms and functional implications by H. Pasantes-Morales; Dr. A. Schousboe D.Sc. (pp. 281-292).

All cells including neurons and glial cells are able to keep their volume within a very limited range. The volume regulatory mechanism involves changes in the concentration of osmolytes of which taurine appears to be of particular importance in brain cells. Swelling in brain cells may occur as a result of depolarization or small fluctuations in osmolarity. In isolated brain cells these conditions will always lead to a release of taurine, the time course of which is superimposable on that of the volume regulatory decrease which follows the initial cell swelling. The mechanism responsible for taurine release associated with swelling has not been fully elucidated but a large body of evidence seems to exclude participation of the taurme high affinity carrier. Using a number of inhibitors of anion exchangers it has been demonstrated that both volume regulation and taurine release in brain cells are inhibited by these drugs, implicating an anion channel in the process. It has be controversial issue as to whether or not taurine release is Ca++ dependent. Recent evidence appears to suggest that the release process is not associated with Ca⁺⁺ or Ca⁺⁺ channels. It is, however, quite possible that the swelling process may involve the Ca⁺⁺ calmodulin system or other second messengers. Taurine also contributes to volume regulation after shrinkage of brain cells, in this case by increasing its intracellular concentration. This change is accomplished byan upregulation of the Na+/taurine cotransporter, together with reduced passive fluxes and increased endogenous synthesis.

Keywords: Amino acids; Taurine; Swelling; Astrocytes; Neurons

Effect of extra- and intra-mitochondrial calcium on citrulline synthesis by A. Saavedra-Molina; T. M. Devlin (pp. 293-298).

A quantitative relationship between rat liver mitochondrial matrix free Ca²⁺ ([Ca²⁺]_m) and citrullinogenesis has been observed. Maximum citrulline synthesis occurred at 100 to 200nM [Ca²⁺]_m; higher [Ca²⁺]_m caused inhibition. When [Ca²⁺]_m was decreased to below 50nM, by addition of A23187 and EGTA, inhibition also occurred. By incubating mitochondria with ruthenium red ([Ca²⁺]_m = 200nM) prior to addition of extramitochondrial free Ca²⁺ ([Ca²⁺]_o) it was found that high external Ca²⁺ (800nM) did not inhibit citrulline synthesis thus demonstrating that [Ca2+]m, not [Ca²⁺]_o was controlling citrullinogenesis.

Keywords: Amino acids; Liver; Mitochondria; Calcium; Citrulline; Ruthenium red

Studies on propylamine transfer activity in anti-AdoDATO antibodies by Dr. G. Pontoni; L. Lopalco; S. De Maria; U. Zambardino; M. Carteni-Farina; A. G. Siccardi; V. Zappia (pp. 299-308).

Based on a structural similarity to the transition state of a propylamine transfer reaction involved in polyamine biosynthesis, Sadenosyl-(5′)-1,8-diamino-3-thiooctane (AdoDATO), the most potent inhibitor of spermidine synthases, was used as a hapten for mice immunization. From immunized mice sera, the IgG fractions were purified by means of affinity (protein A/G) chromatography. Sera and purified polyclonal antibodies from several mice were found to exert spermidine synthase-like activity. Moreover, by means means of hybridoma technology, 19 anti-AdoDATO hybridoma clones have been screened for propylamine transfer activity and at least 6 were found to produce catalytic antibodies. These findings indicate the presence in the sera of active spermidine synthase-like catalytic antibodies. The reported results for the first time evidence the feasibility of preparation of N-alkylating antibodies, widening the biotechnological perspectives of antibodies as biocatalysts.

Keywords: Amino acids; Catalytic antibodies; Transition state analogs; Aminopropyl transferase; Polyamines

Effects of thiazolidine-4(R)-carboxylates and other low-molecular-weight sulfur compounds on the activity of mercaptopyruvate sulfurtransferase, rhodanese and cystathionase in Ehrlich ascites tumor cells and tumor-bearing mouse liver by Dr. M. Wróbel; L. Włodek (pp. 309-314).

Changes of the specific activity of 3-mercaptopyruvate sulfurtransferase (MPST), rhodanese and cystathionase in Ehrlich ascites tumor cells (EATC) and tumor-bearing mouse liver after intraperitoneal administration of thiazolidine derivatives, L-cysteine, D,L-methionine, thiocystine or thiosulfate were estimated. Thiazolidine derivatives used were: thiazolidine-4-carboxylic acid (CF), 2-methyl-thiazolidine-2,4-dicarboxylic acid (CP) and 2-methyl-thiazolidine-4-carboxylic acid (CA). In the liver, the activity of MPST was significantly increased by all the studied compounds, whereas the activity of rhodanese was by CF and thiocystine and that of cystathionase was by the administration of cysteine and CP. Un the other hand, cysteine lowered the rhodanese activity and the activity of cystathionase was decreased by the administration of methionine and thiocystine. Activities of MPST and rhodanese were even lower in EATC than those in the liver of tumor-bearing mouse and the activity of cystathionase in EATC was not be detected. The thiazolidine derivatives significantly increased the level of MPST activity in EATC, but decreased the rhodanese activity. Thiosulfate also increased the activity of MPST to a lesser degree, but cysteine, methionine and thiocystine gave little change in the activity. The rhodanese activity in EATC was slightly increased only by thiocystine. These findings suggest that the sulfur metabolism in the tumor-bearing mouse liver is different from that in the normal mouse liver, and that sulfur compounds are minimally metabolized to sulfane sulfur, a labile sulfur, in EATC.

Keywords: Amino acids; Thiazolidine-4(R)-carboxylates; Mercaptopyruvate sulfurtransferase; Rhodanese; Cystathionase Ehrlich-ascites tumor cell; Mouse liver

Increased postprandial homocysteinemia in a group of depressed patients by Dr. M. Candito; V. Aubin-Brunet; F. Beaulieu; T. Chevalier; D. Pringuey; G. Darcourt (pp. 315-321).

Elevated tissue and serum concentrations of homocysteine (HCY) are associated with neuropsychiatric disorders as well as with premature occlusive vascular disease, as seen in homocystinuria. In order to study dietary-related modifications in plasma HCY, total HCY was assayed in the fasted state and 2 hr after meals in 12 depressed female patients aged 54 to 81 yr and in 12 female controls aged 50 to 85 yr. Fasting HCY was also studied in 4 patients with dementia. Postprandial HCY varied only slightly in the controls compared with their fasting values, whereas a significant increase was noted in the depressives. To study the influence of normal and low protein diets on this abnormality, fasting and postprandial HCY were investigated in 4 of the depressives after one week of a normal diet, after a week on a diet without meat, fish or eggs, and then again after return to a normal diet for one week. Persistence of the abnormal increase in postprandial HCY in 2 of these 4 patients while on the low-protein diet may have been due to an inherited defect in HCY metabolism. Folate deficiency can also cause hyperhomocysteinemia, and as folate supplements constantly lower HCY concentrations, nutritional counseling and folate therapy might prove helpful in the treatment of depression.

Keywords: Amino acids; Homocysteine; Depression; Dementia

Adaptation of siblings of female rats given ethanol effect of N-acetyl-L-cysteine by V. Rajakrishnan; P. Viswanathan; Dr. V. P. Menon (pp. 323-341).

Ethanol administration to female rats before and during pregnancy resulted in decreased number of litters and increased activities of serum GOT, GPT and ALP. The hepatotoxicity of ethanol was indicated by the histological alterations both in the mother and siblings. There was increased levels of tissue lipids in mother and litters born to alcoholic rats. The concentration of TBARS in the liver and kidney were significantly increased in alcohol treated rats and their litters. The activities of the anti-peroxidative enzymes SOD and CAT were decreased on alcohol treatment in female rats. The glutathione content in liver and kidney decreased significantly in litters born to alcoholic rats.We have observed that the treatment with N-acetylcysteine offers protection against the toxic effect of alcohol in female rats during pregnancy and litters born to them. In N-acetylcysteine treated rats the number of litters as well as the average birth weight were close to that of control animals. Nacetylcysteine decreases the activities of serum GOT, GPT and ALP in female rats. We have also observed decreased levels of tissue lipids in mother and litters born to alcoholic rats given N-acetylcysteine when compared to alcoholic rats. The levels of TBARS in liver, kidney were also decreased both in mother and litter born to alcohol + N-acetylcysteine, while the activities of SOD and CAT were increased in liver of alcoholic rats given N-acetylcysteine when compared to alcoholic rats. Histopathological studies also showed the protective effect of N-acetylcysteine in both mother and litter in liver and kidney against alcoholic induced toxicity.

Keywords: Amino acids; Catalase; Ethanol; Glutathione; N-Acetyl-Lcysteine; Superoxide dismutase; Thiobarbituric acid reactive substances

4-Thiaproline reduces heart lipid peroxidation and collagen accumulation in the diabetic db/db mouse by B. Lubec MD; M. Hjelm; H. Hoeger; J. Gialamas; G. Lubec (pp. 343-351).

Collagen accumulation is a main pathological feature of diabetic cardiomyopathy. The underlying mechanisms seem to be increased cross linking by reactive carbonyles. The purpose of the study was to decrease the collagen content of total ventricular tissue by the oral administration of thiaproline, which could reduce collagen due to its functions as a proline analogue, blocking collagen production and as a free oxygen radical scavenger, blocking reactive carbonyles and oxygen species and subsequently collagen cross linking.Thiaproline was administered to genetically diabetic db/db mice and compared to untreated animals. Total ventricular collagen as expressed by hydroxyproline was significantly lower in the treated group (means 0.23 micromoles/10 tissue in the treated vs 0.35 micromoles/100 mg tissue in the untreated group, p < 0.001). Significantly more collagen could be eluted in the treated group (p < 0.001) and carboxymethyllysine was significantly reduced in the treated group (p < 0.001). Di-tyrosine and glycemic control did not differ between the groups. Glutathione was significantly increased in the TP treated experimental group (p < 0.001) and lipid peroxidation products were significantly decreased (means 0.221 absorbance in the treated group versus 0.321 absorbance in the untreated diabetic group) correlating with total ventricular collagen content (r = 0.87, p < 0.01).We conclude that thiaproline reduced total ventricular collagen content by inhibiting collagen cross linking as reflected by increased solubility of collagen and expressed by higher elution quantity of collagen. Thiaproline, and/or its metabolites induced increase of heart glutathione which may well have been scavenging reactive carbonyles derived from lipid peroxidation and advanced stage nonenzymatic glycosylation as shown by decreased total ventricular carboxy-methyllysine and lipid peroxidation products paralleling reduced heart collagen content.It remains to be shown that the successful reduction of heart collagen by thiaproline is paralleled by improved functional properties.

Change of serum L-tryptophan levels following the development and recovery of acute puromycin aminonucleoside nephrosis in rats by E. Sasaki; Y. Ohta Ph.D.; R. Shinohara; I. Ishiguro (pp. 353-361).

It is known that total L-tryptophan (Trp) levels decrease with a decrease in albumin-bound Trp levels and an increase in free Trp levels in the plasma or serum of nephrotic children. We, therefore, examined the change of serum Trp levels following the development and recovery of acute nephrosis in 6-week-old male Wistar rats injected once with puromycin aminonucleoside (100mg/kg body weight) and checked the levels of 16 amino acids including Trp in the serum and the levels of Trp in the liver, kidney, and urine under nephrotic conditions. In this study, the development and recovery of nephrosis were checked by the changes of levels of urinary protein and serum protein and albumin. Total serum Trp and albumin-bound serum Trp levels decreased with the development of nephrosis and these decreased levels returned to the normal level with its recovery. In contrast, free serum Trp levels increased with the development of nephrosis and this increased level returned to the normal level with its recovery. In the serum of nephrotic rats, the decrease of albumin-bound Trp levels and the increase of free Trp levels were well consistent with a decrease in albumin levels and an increase in the level of non-esterified fatty acids which are known to weaken the binding of Trp to albumin and among 16 amino acids studied, only Trp showed a significant change in its levels. Trp levels increased in the liver and kidney but not in the urine under nephrotic conditions. These results indicate that the change of serum Trp levels should be closely related to the condition of nephrosis and that although serum Trp is lost under nephrotic conditions, the lost serum Trp is accumulated in the liver and kidney.

Keywords: Amino acids; L-Tryptophan; Albumin; Non-esterified fatty acids; Puromycin aminonucleoside; Experimental nephrosis (rat)

Synthesis of some racemicγ-fluoro-α-amino acids by Prof. Dr. S. Kröger; G. Haufe (pp. 363-372).

Versatile three-step procedures for syntheses of seven racemiγ-fluoro-a-amino acids are described. Alkylation oftert-butyl N-(diphenylmethylene) glycinate with 1-bromo-2-fluoroalkanes gave N-protected aminoacid esters both in anhydrous medium using lithium-diisopropylamide as base at low temperature or in a two phase system of 50% aqueous sodium hydroxide and methylene chloride with triethylbenzylammonium chloride as the phase transfer catalyst at room temperature. Subsequent two-step deprotection with citric acid and hydrochloric acid gave the title compounds in 13–33% overall yields.

Keywords: Fluorinated amino acids; Alkylation; tert-Butyl N-(diphenylmethylene)glycinate; 1-Bromo-2-fluoroalkanes; Phase transfer catalysis

Amino acids and plasma antioxidant capacity by Dr. E. Meucci; M. C. Mele (pp. 373-377).

Amino acids antioxidant capacity has been investigated and compared with the chain-breaking antioxidant activity of known compounds as ascorbic acid and Trolox. Basic and acidic amino acids and most of neutral ones showed no antioxidant capacity. On the contrary, tryptophan, tyrosine, cysteine and homocysteine showed antioxidant ability at concentrations which are within the usually reported physiological ranges.These findings are discussed in connection with the antioxidant capacity ascribed to plasma proteins, as human serum albumin.

Keywords: Amino acids; Antioxidant capacity; Oxidative stress; Plasma proteins; Human serum albumin

Reaction pathway of tryptophanase degrading D-tryptophan by A. Shimada; H. Kogure; H. Shishido; I. Nakamura (pp. 379-383).

Tryptophanase, which has the very strict stereospecificity to L-tryptophan under ordinary condition, becomes active to D-tryptophan in highly concentrated diammoniumhydrogen phosphate solution. The reaction process of D-tryptophan degradation is studied in terms of kinetics. Diammoniumhydrogen phosphate acts on tryptophanase as activator below 3.1 M, and as noncompetitive inhibitor over it. Additionally, the pathway of the reaction is provided on the basis of kinetic parameters.

Keywords: Amino acids; Tryptophanase; D-tryptophan; Diammonium-hydrogen phosphate; Kinetic analysis; Stereospecificity

In Memoriam (pp. 385-386).

Abstracts 30th Meeting of the European Metabolic Group (pp. 387-392).

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.12, #3-4)