Amino Acids L-leucine Research Articles

Biochemical and Immunological Study on the Effects of Barley and its Components as Hypoglycemic Agents in Diabetic Rats

The present study was carried out to investigate the hypoglycemic effect of barley (Hordeum vulgare L) and some of its components such as amino acids (L-leucine and L-glutamine) and chromium picolinate on some biochemical and immunological parameters of alloxan induced diabetic rats. Alloxan-diabetic rats were treated with barley water (10% w/v) at a dose 10 ml Kg -1 b.wt., chromium picolinate at 15 μg Kg -1 b.wt., L-leucine plus L-glutamine at 4.5 mg & 15 mg Kg -1 b.wt., and/or the combination of barley plus chromium plus L-leucine and L-glutamine at the same previous doses in the same water volume, respectively. Rats received the treatments in their drinking water for four weeks. The levels of glucose, immunoglobulin G (IgG), total lipids (TL), cholesterol, triglycerides (TG), low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) were significantly (P<0.05) increased, while high-density lipoprotein (HDL) decreased in plasma of alloxan-diabetic rats compared to control group. The activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), acid phosphatase (AcP) and alkaline phosphatase (AlP) were significantly (P<0.05) decreased in both plasma and liver of alloxan-diabetic rats. On the other hand, the activity of lactate dehydrogenase (LDH) decreased in plasma and increased in liver of alloxan- diabetic rats. Treatment of the diabetic rats with repeated doses of any one of the three treatments alone could restore the changes of the above parameters to their normal levels after four weeks of treatment. However, treatment with the combination of all together did not show complete restoration. Furthermore, the electron microscope results were supported biochemical and immunological findings. The present results showed that barley, amino acids and chromium picolinate exerted antihyperglycemic effects and consequently may alleviate liver damage caused by alloxan-induced diabetes.

A combined [formula omitted] RAPT and MQ-MAS NMR study of l-leucine

We report the application of rotor-assisted population transfer (RAPT) to measure the quadrupolar coupling constant ( C q ) for spin 5 2 nuclei. Results from numerical simulations are presented on the magnitude of enhancement factor as a function of frequency offsets, i.e. the RAPT profile. Experimental O 17 RAPT profile is traced for the amino acid l-leucine. In addition, results from MQ-MAS experiments are incorporated to determine the quadrupolar asymmetry parameter ( η q ) . Unlike previous reports, the O 17 NMR parameters for an amino acid, l-leucine, is reported at a relatively low field of 9.4 T.

Nucleating characteristics of Pseudomonas aeruginosa above 0 °C and the role of l-leucine

It has been reported earlier that the bacteria Pseudomonas aeruginosa have the ability to nucleate ice over a long temperature range below 0 °C. In the present work, the same bacteria are shown to have nucleating ability even above 0 °C. It is found to reach a peak at 4 °C and then gradually falls off as the temperature is raised, vanishing altogether at 12 °C. When the bacteria are irradiated by UV-rays, the nucleating ability above 0 °C has the same variational pattern with change of temperature, though the ability is significantly less. When the bacterial medium is boiled for 20 min, the bacteria loose their nucleating ability completely. Since ice cannot exist above 0 °C, some supporting experiments are performed to establish the identity of the nucleated crystals: (a) Crystals grown around a small sample of cultured P. aeruginosa in an incubator were shown to be identical with the nucleated crystals in the Cold Room experiments. (b) X-ray diffraction (XRD) spectrum of the grown crystals is found to be consistent with the presence of amino acids like l-leucine, isoleucine and l-isoleucine, amongst which l-leucine is the most abundant. When droplets of an aqueous solution of l-leucine are seeded, their nucleation characteristics are similar to those with P. aeruginosa. From XRD, nuclear magnetic resonance (NMR) and thermal analysis, one can conclude that the crystals formed by P. aeruginosa above 0 °C are hydrate crystals with water phase caging around the seeded agent; the amino acid l-leucine present in the protein of the bacteria plays the dominant role in the nucleation.

Prolonged oral treatment with an essential amino acid l-leucine does not affect female reproductive function and embryo-fetal development in rats

l-leucine, an essential amino acid, is one of the most popular ingredients in dietary supplements. To investigate a possibility of its embryo-fetal toxicity in rats, 11- to 12-week old dams were orally administered an aqueous solution of l-leucine at doses of 300 or 1000 mg/kg body weight on gestational days 7–17. Body weight and feed intake was evaluated throughout the whole course of pregnancy (days 0–20). l-Leucine did not influence body weight, but at a dose of 1000 mg/kg, slightly enhanced feed intake on days 14 and 18 of pregnancy. Caesarean section (day 20) revealed no influences on the litter size and weight of live-born fetuses, the number of corpora lutea, implantation index or the quality of placenta, and the minor increase in feed intake was considered irrelevant to the pregnancy outcomes. Fetuses were evaluated in a battery of external, visceral and skeletal examinations. No effects of l-leucine on gender ratio and external abnormalities, and no significant treatment-related variations in visceral and skeletal pathologies were observed. These results suggested that l-leucine, administered orally during organogenesis at doses up to 1000 mg/kg body weight, did not affect the outcome of pregnancy and did not cause fetotoxicity in rats.

Effect of heavy metals on the uptake of [ 3H]- l-histidine by the polychaete Nereis succinea

Integumentary uptake of [ 3H]- l-histidine by Nereis succinea was measured in the presence and absence of selected heavy metals and the amino acid l-leucine in 60% artificial seawater (ASW). The time course of 10 μM [ 3H]- l-histidine uptake into worms over a 60 min incubation was approximately doubled in the presence of 0.5 μM zinc and when calcium in the incubation medium was reduced from 6 mM to 5 μM the stimulatory effect of zinc on amino acid accumulation was reduced and uptake under the latter conditions was approximately half that of the control. Zinc stimulation of [ 3H]- l-histidine influx was a hyperbolic function of zinc concentration over the range 0 to 50 μM metal and displayed an apparent activation or affinity constant of 385±127 nM Zn 2+. The hyperbolic stimulatory effect of 1 μM Zn 2+ on the time course of 10 μM [ 3H]- l-histidine uptake was abolished in the presence of 25 μM l-leucine, suggesting that this amino acid shared the same transport system as [ 3H]- l-histidine and acted as a potential competitive inhibitor. Influx of [ 3H]- l-histidine was a hyperbolic function of external amino acid concentration and displayed an apparent affinity constant ( K m ) of 23.71±5.02 μM and an apparent maximal velocity ( J max) of 4701±449 pmol/g dry wt.×15 min. Addition of 0.5 μM zinc resulted in a four-fold increase in J max and a doubling of K m , suggesting the effect of the metal was mostly on the rate of amino acid transport. [ 3H]- l-histidine influx was mildly stimulated by Fe 2+ (0.5 μM), but was unaffected by either Ag + or Al 3+ (both at 0.5 μM). These results suggest that [ 3H]- l-histidine uptake into worm integument may take place by the classical Na +-independent l-transport system shared by l-leucine and regulated by exogenous calcium and other divalent metal concentrations.

Inhibition of l‐arginine transport by reactive oxygen species in rat anococcygeus muscle

The effect of L-arginine on nitrergic transmission and its alteration with reactive oxygen species (ROS) were investigated. L-arginine potentiated the relaxation response induced by electrical field stimulation in rat anococygeus muscle. This effect was inhibited by L-lysine, a cationic amino acid using y+ L and y+ transport systems in a similar way with L-arginine. The neutral amino acid L-leucine, which uses only y+ L system as a transport mechanism, inhibited this potentiation at only low frequency stimulation. Electrolysis of the physiological solution did not change the responses to electrical field stimulation, but inhibited the potentiation elicited by L-arginine that was prevented in the presence of mannitol and N-acetyl-L-cysteine. In conclusion, L-arginine is transported via y+ system predominantly to potentiate the relaxation response to nitrergic nerve stimulation in rat anococcygeus muscle. ROS, primarily hydroxyl radicals inhibited L-arginine-induced potentiation probably by interacting with the y+ amino acid transport system.

Complexation of Some Amino Acids and Peptides by p -Sulfonatocalix[4]arene and Hexasodium p -Sulfonatocalix[6]arene in Aqueous Solution

The stability constants (log K), the reaction enthalpy(Δ H) and entropy (Δ S) of the complexesformed between some amino acids (glycine, L-alanine,L-valine, L-leucine, L-phenylalanine, L-tryptophan,L-threonine, and L-lysine) and peptides (glycyl-glycine,glycyl-L-alanine, glycyl-L-leucine, glycyl-L-phenylalanine,L-leucyl-glycine, L-leucyl-L-alanine, glycyl-L-valine,L-leucyl-glycyl-glycine, and glycyl-glycyl-glycine) withp-sulfonatocalix[4]arene and hexasodiump-sulfonatocalix[6]arene in aqueous solutions by meansof calorimetric titration have been investigated. The reportedresults demonstrate that the amino acids and peptides under studyform complexes with both p-sulfonatocalix[4]areneand hexasodium p-sulfonatocalix[6]arene. In the case of theamino acids and peptides the complexation with water-solublecalixarenes in aqueous solution is favored by enthalpiccontributions and disfavored by entropic contributions. However,no influence of the ring size of the calixarenes upon thecomplexation is observed. By comparison with the reaction ofthe sodium salt of phenol-4-sulfonic acid with amino acids amacrocyclic effect in case of the calixarenes is possible.

Electron Ionization Mass Spectra of N(O,S)-Isobutoxycarbonyl Isobutyl Esters of Amino Acids

Mass spectra of N(O,S)-isobutoxycarbonyl isobutyl esters of 17 amino acids (L-alanine, glycine, L-valine, L-norvaline, L-leucine, L-isoleucine, L-norleucine, L-proline, L-asparagine, L-methionine, L-threonine, L-serine, L-phenylalanine, L-lysine, L-tryptophan, L-tyrosine and L-cystine) were obtained in the electron ionization mode. These derivatives were found suitable for the analysis of amino acids in aqueous media allowing proper identification and quantitation from the mass spectral characteristics.

Leucine-derived cyano glucosides in barley.

Barley (Hordeum vulgare) seedlings contain five cyano glucosides derived from the amino acid L-leucine (Leu). The chemical structure and the relative abundance of the cyano glucosides were investigated by liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses using spring barley cultivars with high, medium, and low cyanide potential. The barley cultivars showed a 10-fold difference in their cyano glucoside content, but the relative content of the individual cyano glucosides remained constant. Epiheterodendrin, the only cyanogenic glucoside present, comprised 12% to 18% of the total content of cyano glucosides. It is proposed that the aglycones of all five cyano glucosides are formed by the initial action of a cytochrome P450 enzyme of the CYP79 family converting L-Leu into Z-3-methylbutanal oxime and subsequent action of a less specific CYP71E enzyme converting the oxime into 3-methylbutyro nitrile and mediating subsequent hydroxylations at the alpha-, as well as beta- and gamma-, carbon atoms. Presence of cyano glucosides in the barley seedlings was restricted to leaf tissue, with 99% confined to the epidermis cell layers of the leaf blade. Microsomal preparations from epidermal cells were not able to convert L-[(14)C]Leu into the biosynthetic intermediate, Z-3-methylbutanal-oxime. This was only achieved using microsomal preparations from other cell types in the basal leaf segment, demonstrating translocation of the cyano glucosides to the epidermal cell layers after biosynthesis. A beta-glucosidase able to degrade epiheterodendrin was detected exclusively in yet a third compartment, the endosperm of the germinating seed. Therefore, in barley, a putative function of cyano glucosides in plant defense is not linked to cyanide release.

Distribution of Isomorphic Amino Acids between a Crystal Phase and an Aqueous Solution

We present distribution coefficients and crystal purity data for systems containing two of the three near-isomorphic amino acids l-leucine (LEU), l-isoleucine (ILE), and l-valine (VAL) in aqueous s...

The Complexation of Some Amino Acids by Isomers of Dicyclohexano-18-Crown-6 and 18-Crown-6 in Methanol

The complexation of some amino acids (glycine, L-alanine,L-leucine, L-valine, and L-serine) by the cis-syn-cisand cis-anti-cis isomers of dicyclohexano-18-crown-6 (DC18C6) in methanol was studied by calorimetric titrationmeasurements. Both isomers exhibited a significant ability to bind the amino acidsmentioned above. The results obtained demonstrate that the structural isomers ofDC18C6 are significantly different in terms of thermodynamics concerning their complexationwith the amino acids. The stability constants and the thermodynamic data forthe reaction of the amino acids under study in protonated form and 18-crown-6 (18C6) are reported.

Stimulation of lipid peroxidation in vitro in rat brain by the metabolites accumulating in maple syrup urine disease.

In this study we investigated the in vitro effects of the metabolites accumulating in maple syrup urine disease on lipid peroxidation in brain of young rats. Chemiluminescence and thiobarbituric acid-reactive substances were measured in brain homogenates from 7- and 30-day-old rats in the presence of 10 mM of the branched-chain amino acids L-leucine, L-isoleucine, or L-valine; their keto acids L-2-ketoisocaproic acid, L-2-keto-3-methylvaleric acid, or L-2-ketoisovaleric acid; or the hydroxy derivatives L-2-hydroxyisocaproic acid, L-2-hydroxy-3-methylvaleric acid, or L-2-hydroxyisovaleric acid separately added to the incubation medium. We observed that all amino acids, keto acids, and hydroxy acids accumulating in this disease stimulate to a variable degree the in vitro parameters of lipid peroxidation tested in homogenates of rat brain. The results indicate a possible participation of oxidative stress in the neuropathology of maple syrup urine disease patients, especially during a crisis, when the metabolites are highly increased, and point to the use of antioxidant drugs as a possible adjuvant therapy in such situations to improve the neurological status of the patients and to prevent sequelae.

Zur N-reduzierten Fütterung von Mastschweinen bei Einsatz freier Aminosäuren

Abstract. Title of the Paper: Investigation on N-reduced feeding and use of free amino acids in fattening pigs The aim of the present study was to investigate the N-reduced feeding by fattening pigs feed by cereals rieh feedmixture supplemented with the free amino-acids L-lysine, DL-methionine, L-threonine, L-tryptophane, Lleucine, L-isoleucine, L-histidine and L-valine. It was to test extreme low crude protein content by requirement sufficient supply on amino-aeid. The reaction of the animals in growth, carcass quality and the reduction of Nexeretion was to investigate. The pigs were divided in two groups (control and experimental group) with 24 animals per group (12 females and 12 castrates). The experiment was divided into three periods (25–60, 60–85 and 85–110 kg body weight). The protein content of the feedmixture was by the control group 18.3, 15.6 and 13.6% and by the experimental group 14.0, 11.8 and 10.7%, respectively. The lysine-energy ratio of the feedmixture was by all three feeding periods by 0.72 or 0.74, 0.61 or 0.62 and 0.55 or 0.57 g lysine per MJ ME in control feedmixture or experimental feedmixture, respectively. By a high Performance are the average daily life weight gain (control group 869 and experimental group 863 g) and the lean meat rate (control group 55.2 and experimental group 55:0%) not influenced by the high of crude protein supply. The calculated N-excretion were by the control group 4.16 kg (as 100%) and by experimental group 2.60 kg (62.5%) per 100 kg body weight gain.

The Role of Cationic Amino Acid Transporters in the Regulation of Blood Flow and Nitric Oxide in Rat Kidney

P94 Previous studies from our laboratory have indicated that nitric oxide (NO) production by isolated inner medullary collecting duct cells is dependent on cellular uptake of L-arginine via cationic amino acid (CAT) transporters ( FASEB J 13:A723,1999). In the present study we performed experiments to determine if CAT transport regulates NO production and renal hemodynamics in the renal medulla in vivo . The effects of renal medullary interstitial infusion of cationic amino acids (100 μg/kg/min) on renal medullary blood flow and NO levels were examined in anesthetized Sprague-Dawley rats by laser-Doppler flowmetry and a microdialysis-oxyhemoglobin trapping technique. Furthermore, we studied the effect of chronic intramedullary infusion of L-ornithine (50 μg/kg/min) on arterial pressure in uninephrectomized SD rats. Renal medullary blood flow was significantly reduced during the acute intramedullary infusion (30 min) of the cationic amino acids L-ornithine (-24±3%, N=4), L-lysine (-20±1%, N=4), and L-homoarginine (-21±2%, N=4), and during infusion of the NOS inhibitor N ω -nitro-L-arginine methyl ester (L-NAME; -38±4%, N=6). In contrast, infusion of L-arginine significantly increased medullary blood flow (+17±4%, N=4) and the neutral amino acids L-leucine, L-serine, and L-glycine (N=4 each) had no effect on renal medullary blood flow. NO concentration in the renal medulla was significantly decreased by the intramedullary infusion of L-ornithine (for 60 min; 336 ± 25 vs 232 ± 34 nmol/L, N=6, p&lt;0.05). In contrast, NO concentration in renal cortex was not significantly affected by the intracortical infusion of L-ornithine (227 ± 31 vs 240 ± 60 nmol/L, p=NS). Similar changes in NO were observed with medullary and cortical infusion of L-lysine. Finally, chronic infusion of L-ornithine into the renal medulla of uninephrectomized SD rats for 7 days led to a sustained increase in mean arterial pressure (168±5 mmHg, N=5) when compared to vehicle treated rats (125±3 mmHg, N=6). These results suggest that CAT transport in the renal inner medulla may play an important role in the production of NO, the maintainence of blood flow, and the level of mean arterial pressure.

Characterization of L-arginine transporters in rat renal inner medullary collecting duct.

Previous work from our laboratory has demonstrated that the inner medullary collecting duct (IMCD) expresses a large amount of nitric oxide synthase (NOS) activity. The present study was designed to characterize the transport of NOS substrate, L-arginine, in a suspension of bulk-isolated IMCD cells from the Sprague-Dawley rat kidney. Biochemical transport studies demonstrated an L-arginine transport system in IMCD cells that was saturable and Na(+) independent (n = 6). L-Arginine uptake by IMCD cells was inhibited by the cationic amino acids L-lysine, L-homoarginine, and L-ornithine (10 mmol/l each) and unaffected by the neutral amino acids L-leucine, L-serine, and L-glutamine. Both L-ornithine (n = 6) and L-lysine (n = 6) inhibited NOS enzymatic activity in a dose-dependent manner in IMCD cells, supporting the important role of L-arginine transport for NO production by this tubular segment. Furthermore, RT-PCR of microdissected IMCD confirmed the presence of cationic amino acid transporter CAT1 mRNA, whereas CAT2A, CAT2B, and CAT3 were not detected. These results indicate that L-arginine uptake by IMCD cells occurs via system y(+), is encoded by CAT1, and may participate in the regulation of NO production in this renal segment.

A Simple Chemical Method for Rendering Wild-Type Yeast Permeable to Brefeldin A That Does Not Require the Presence of an erg6 Mutation.

The present work aims to develop a growth medium to render a wild-type strain of Saccharomyces cerevisiae permeable to the antifungal drug Brefeldin A. In the current study, a synthetic medium containing 0.1% L-proline and supplemented with 3.0 × 10-3% SDS is employed. When Brefeldin A is added to this medium, a wild-type strain shows increased growth sensitivity and a diminished transport of the amino acid L-leucine. Since Brefeldin A exerts its effect on the endoplasmic reticulum and the Golgi apparatus, the medium permits the study of the drug effect on the intracellular traffic of L-leucine permeases.

Molecular aggregation in selected crystalline 1:1 complexes of hydrophobicD- andL-amino acids. III. TheL-leucine andL-valine series

The amino acids L-leucine (L-Leu) and L-valine (L-Val) have been cocrystallized with D-aminobutanoic acid (D-Abu), D-2-aminopentanoic acid (D-Nva) and D-methionine (D-Met) to form six complexes, L-Leu:D-Abu, C 6 H 13 NO 2 .C 4 H 9 NO 2 , L-Leu:D-Nva, C 6 H 13 NO 2 .C 5 H 11 -NO 2 , L-Leu:D-Met, C 6 H 13 NO 2 .C 5 H 11 NO 2 S, L-Val:-D-Abu, C 5 H 11 NO 2 .C 4 H 9 NO 2 , L-Val:D-Nva, C 5 H 11 NO 2 .-C 5 H 11 NO 2 and L-Val:D-Met, C 5 H 11 NO 2 .C 5 H 11 NO 2 S. A fourth L-Leu complex, with D-Val, C 6 H 13 NO 2 .C 5 H 11 -NO 2 has also been studied. The crystals of these amino acid complexes are all divided into distinct hydrophilic and hydrophobic layers. The D- and L-amino acids are all related by pseudo-inversion in the L-Leu complexes and by pseudo-glide planes in the L-Val series. Similarities and differences in the crystal packing and molecular conformations of L-Leu/L-Val, as well as of the partner molecules, are discussed.

Synthetic and Mechanistic Studies of (2R,3S)-3-Vinylmalic Acid as a Mechanism-Based Inhibitor of 3-Isopropylmalate Dehydrogenase

(2R,3S)-3-Vinylmalic acid (VM) was designed as a mechanism-based inhibitor of threo-3-isopropylmalate dehydrogenase (IPMDH), the rate-determining enzyme responsible for the penultimate step in the biosynthetic pathway of the essential amino acid l-leucine. The synthesis of VM was achieved in six steps from diethyl (R)-malate. Besides its weak activity as a substrate, VM was shown to be a mechanism-based inhibitor (KI = 1.20 mM) for IPMDH as deduced from the time-dependent and kinetic analyses. 2-Oxo-3-pentenoate was identified as the enzyme reaction product of VM by GC−MS analysis and 1H NMR spectroscopy, but the supplemented 2-oxo-3-pentenoate was not, in contrast, inhibitive to the enzyme reaction. It seems likely from these results that an activated or nucleophilic amino acid of IPMDH by deprotonation during the ordinary enzyme reaction process participates in a strong interaction with the 2-oxo-3-pentenoate product, probably by the formation of a transient covalent bond, which in turn gives rise to inhibition of the enzyme reaction.

Synthesis of cyclopropane substrate analog for 3-isopropylmalate dehydrogenase and its mechanism-based inhibition

A cyclopropane substrate analog 1 was designed as a mechanism-based inhibitor for 3-isopropylmalate dehydrogenase (IPMDH), the rate-determining enzyme responsible for the penultimate step in the biosynthetic pathway of the essential amino acid l-leucine. The synthesis of 1 was pursued in 5 steps from diethyl ( R)-malate. As was deduced from the time-dependent and kinetic analyses, 1 appeared to be a competitive and mechanism-based inhibitor for IPMDH ( Ki = 1.9 mM). Thus, 1 was recognized as a substrate and oxidized by IPMDH. Inhibitory mechanism is suggested to be slowering the decarboxylation step.

Maintenance of Digestive Performance in the Turtles Chelydra serpentina, Sternotherus odoratus, and Trachemys scripta

In our continued investigation of the adaptive interplay between feeding ecology and digestive physiology, we measured postfeeding responses of juvenile Chelydra serpentina, adult Sternotherus odoratus, and subadult Trachemys scripta, three aquatic turtle species that feed at frequent intervals and consume a catholic diet of plants and animals. In this study, we measured 02 consumption rates from fasting and digesting individuals and compared intestinal nutrient uptake rates and organ masses of turtles fasted for one month with turtles sacrificed one day after the ingestion of a meal equivalent to 5-11% of body mass. 02 consumption during digestion peaked at rates 3.4, 2.1, and 2.7 times fasting values, respectively, for C. serpentina, S. odoratus, and T. scripta-factors much smaller than those documented previously for reptile species that normally consume large meals at long intervals. None of the turtle species experienced significant postfeeding changes in intestinal uptake of amino acids or D-glucose. Ratios of amino acid uptake rates to D-glucose uptake rates were much greater than 1.0 for each species, either fasted or fed, a finding characteristic of other carnivores as well. Total intestinal capacity to transport the amino acids L-leucine and L-proline and the sugar D-glucose did not change with feeding for any of the turtle species. None of the species experienced significant differences in intestinal mass or enterocyte morphology between fasted and fed individuals. Chelydra serpentina and T. scripta experienced no significant postfeeding changes in organ masses, and the only changes for S. odoratus were 59% and 42% increases in stomach wet and dry masses, respectively, upon feeding. Thus, juvenile C. serpentina, adult S. odoratus, and subadult T. scripta maintain the functional and morphological integrity of their guts during fasting and exhibit only modest metabolic responses to feeding. We hypothesize that these are adaptive traits characteristic of species that frequently consume and digest small meals.