Effects of high-dose folic acid and pyridoxine on plasma and erythrocyte sulfur amino acids in hemodialysis patients.
In this investigation, sulfur amino acids (sAA) and sulfhydryls were determined in the plasma and erythrocytes (RBC) of 10 uremic patients on regular hemodialysis (HD) treatment and 10 healthy subjects, before and after supplementation with 15 mg/d of folic acid and 200 mg/d of pyridoxine for 4 wk. The basal total plasma concentrations of homocysteine (Hcy), cysteine (Cys), cysteinylglycine (Cys-Gly), gamma-glutamylcysteine (gamma-Glu-Cys), glutathione (GSH), and free cysteinesulfinic acid (CSA) were significantly higher in HD patients when compared to healthy subjects, whereas methionine (Met) and taurine (Tau) concentrations were the same in the two groups. HD patients showed significantly higher RBC levels of Hcy and Cys-Gly, whereas the RBC concentrations of Met, Cys, Tau, and GSH were not different from those in the healthy subjects. The plasma concentrations of sAA and sulfhydryls differed compared with RBC levels in the healthy subjects and HD patients. In both groups, supplementation with high doses of folic acid and pyridoxine reduced the plasma Hcy concentration. In addition, increased plasma concentrations of Cys-Gly and GSH were found in the HD patients and of CSA in the healthy subjects. After vitamin supplementation, the RBC concentrations of Hcy, Cys, and GSH increased and that of Tau decreased in healthy subjects. The only significant finding in RBC of HD patients was an increase in GSH levels after supplementation. This study shows several RBC and plasma sAA and sulfhydryl abnormalities in HD patients, which confirms earlier findings that RBC and plasma pools play independent roles in interorgan amino acid transport and metabolism. Moreover, high-dose supplementation with folic acid and pyridoxine significantly reduced Hcy levels, but did not restore the sAA and sulfhydryl abnormalities to normal levels. The increase that was observed in GSH after vitamin supplementation may have a beneficial effect in improving blood antioxidant status in uremic patients. Finally, the findings of elevated plasma Cys levels correlating to the elevated plasma Hcy levels in the presence of elevated plasma CSA levels, both before and after vitamin supplementation, led to the hypothesis that a block in decarboxylation of CSA is linked to hyperhomocysteinemia in end-stage renal failure. (+info)
Protein synthesis by native chemical ligation: expanded scope by using straightforward methodology.
The total chemical synthesis of proteins has great potential for increasing our understanding of the molecular basis of protein function. The introduction of native chemical ligation techniques to join unprotected peptides next to a cysteine residue has greatly facilitated the synthesis of proteins of moderate size. Here, we describe a straightforward methodology that has enabled us to rapidly analyze the compatibility of the native chemical ligation strategy for X-Cys ligation sites, where X is any of the 20 naturally occurring amino acids. The simplified methodology avoids the necessity of specific amino acid thioester linkers or alkylation of C-terminal thioacid peptides. Experiments using matrix-assisted laser-desorption ionization MS analysis of combinatorial ligations of LYRAX-C-terminal thioester peptides to the peptide CRANK show that all 20 amino acids are suitable for ligation, with Val, Ile, and Pro representing less favorable choices because of slow ligation rates. To illustrate the method's utility, two 124-aa proteins were manually synthesized by using a three-step, four-piece ligation to yield a fully active human secretory phospholipase A(2) and a catalytically inactive analog. The combination of flexibility in design with general access because of simplified methodology broadens the applicability and versatility of chemical protein synthesis. (+info)
Evaluation of feather meal as a source of sulfur amino acids for growing steers.
In situ and digestion studies were conducted to evaluate feather meal (FTH), blood meal (BM), and meat and bone meal (MBM) for escape protein content, amino acid composition of the escape protein, true protein digestibility, and digestibility of the individual amino acids. Following 12 h of ruminal incubation, escape protein values were 73.5, 92.4, and 60.8% of CP for FTH, BM, and MBM, respectively. Blood meal and MBM were poor sources of sulfur amino acids (SAA), whereas FTH was a good source. Most of the SAA of FTH, however, was Cys, with very little Met. True protein digestibilities were not different for the protein sources (P > .15), ranging from 86.7 to 94.0% of the CP. However, digestibilities of the individual amino acids were quite different. Two growth studies were conducted to evaluate FTH as a source of SAA for growing cattle. The first study used 120 steers (228 +/- 15 kg) supplemented with urea, MBM, MBM plus 1% FTH, or MBM plus 2% FTH. Additionally, incremental amounts of rumen-protected Met were added to treatments containing MBM. Supplementation of MBM increased (P < .05) ADG compared with the urea control. Addition of FTH to MBM resulted in a linear (P < .01) increase in ADG. However, addition of rumen-protected Met to MBM plus FTH treatments further improved gains. Although FTH is an effective source of SAA, Met probably was first-limiting. The second study used 90 steers (243 +/- 18 kg) supplemented with BM plus incremental amounts of SAA from either FTH or rumen-protected Met. Addition of SAA improved ADG compared with BM alone (P < .05). Rumen-protected Met as a source of SAA improved ADG compared with FTH (P < .05). The SAA from FTH promoted a gain response equal to 50% of the response obtained with rumen-protected Met. Formulation of ruminant diets for metabolizable amino acids must account for escape value and digestibility of each individual amino acid. Feather meal is an effective source of SAA; however, Cys supplies over five times the amount supplied by Met. (+info)
Dietary sulfur amino acid requirement of juvenile yellow perch fed the maximum cystine replacement value for methionine.
We conducted three separate experiments designed to determine the dietary methionine requirement, ability of cyst(e)ine to spare methionine, and the total sulfur amino acid requirement (TSAA) of juvenile yellow perch when fed the maximal amount of cyst(e)ine. The purified basal diet used in each experiment contained 33.6 g of crude protein/100 g diet and 12.0 g of lipid/100 g diet. In the first experiment,;>L-methionine was added to eight diets providing methionine concentrations ranging from 0.37 to 1.77 g/100 g diet in gradations of 0.2 g/100 g diet. Diets were fed for 12 wk to juvenile yellow perch initially weighing 4.7 g/fish. Broken-line analyses of weight gain and feed efficiency data indicated that the dietary methionine requirement was 1.0 g/100 g diet (3.1 g TSAA/100 g dietary protein) and 1.1 g/100 g diet (3.4 g TSAA/100 g dietary protein), respectively. In the second experiment, various ratios of L-cyst(e)ine and L-methionine were added to the basal diet and fed for 12 wk to determine the cyst(e)ine replacement value of yellow perch initially weighing 19.3 g/fish. Weight gain and feed efficiency (FE) data indicated that cyst(e)ine spared up to 51% of the methionine requirement. In the final experiment, graded levels of cyst(e)ine plus methionine in a ratio of 51:49 were added to the basal diet in gradations of 0.1 g/100 g diet (0.5 to 1.2 g TSAA/100 g diet) to determine the dietary total sulfur amino acid requirement. Diets were fed to satiation for 10 wk to fish initially weighing 8. 1 g. Broken-line analyses of weight gain, feed intake and FE data indicated that the dietary TSAA requirement was 0.85, 0.87 and 1.0 g of TSAA/100 g diet (2.5 to 3.0 g of TSAA/100 g of dietary protein), respectively. The majority of dietary TSAA requirements of fish are in the range of 2 to 4 g/100 g of dietary protein and are generally similar to those of both birds and swine, but lower than estimates for rodents. (+info)
Improved HPLC determination of acidic opines by phenylisothiocyanate derivatization and its application to marine animals.
We present here a reliable and sensitive method for the determination of acidic opines such as meso-alanopine, beta-alanopine, tauropine and strombine in biological samples. Interfering primary amino acids were eliminated by reaction with o-phthalaldehyde, and the derivatized compounds were passed through Sep-Pak Plus PS-1 cartridges. The acidic opines were recovered by flushing the cartridges with water, then determined by high performance liquid chromatography after a second derivatization with phenylisothiocyanate. All 4 acidic opines were detected within 30 min. This method ensured good separation and guaranteed almost full recovery of all acidic opines. This method was applied to analyze opines in marine animals and to test whether opines are metabolized in the livers of the rat and fish. (+info)
Oxoproline kinetics and oxoproline urinary excretion during glycine- or sulfur amino acid-free diets in humans.
L-5-oxoproline (L-5-OP) is an intermediate in glutathione synthesis, possibly limited by cysteine availability. Urinary 5-OP excretion has been proposed as a measure of glycine availability. We investigated whether 5 days of dietary sulfur amino acid (SAA-free) or glycine (Gly-free) restriction affects plasma kinetics of 5-OP and urinary excretion of L- and D-5-OP in 6 healthy men. On day 6, L-5-[1-(13)C]oxoproline and [3,3-(2)H(2)]cysteine were infused intravenously for 8 h (3 h fast/5 h fed). In a control study (adequate amino acid mixture), plasma oxoproline fluxes were 37.8 +/- 13.8 (SD) and 38.4 +/- 14.8 micromol x kg(-1) x h(-1); oxidation accounted for 85% of flux. Cysteine flux was 47.9 +/- 8.5 and 43.2 +/- 8.5 micromol x kg(-1) x h(-1) for fast and fed phases, respectively. Urinary excretion of L- and D-5-OP was 70 +/- 34 and 31.1 +/- 13.3 micromol/mmol creatinine, respectively, during days 3-5, and 46.4 +/- 13.9 and 22.4 +/- 8.3 micromol/mmol over the 8-h tracer study. The 5-OP flux for the Gly-free diet was higher (P = 0. 018) and tended to be higher for the SAA-free diet (P = 0.057) when compared with the control diet. Oxidation rates were higher on the Gly-free (P = 0.005) and SAA-free (P = 0.03) diets. Cysteine fluxes were lower on the the Gly-free (P = 0.01) and the SAA-free diets (P = 0.001) compared with the control diet. Rates of L-5-OP excretion were unchanged by withdrawal of SAA or Gly for 5 days but increased on day 6 (P = 0.005 and P = 0.019, respectively). Thus acute changes in the dietary availability of SAA and Gly alter oxoproline kinetics and urinary 5-OP excretion. (+info)
Blood glutathione synthesis rates in healthy adults receiving a sulfur amino acid-free diet.
The availability of cysteine is thought to be the rate limiting factor for synthesis of the tripeptide glutathione (GSH), based on studies in rodents. GSH status is compromised in various disease states and by certain medications leading to increased morbidity and poor survival. To determine the possible importance of dietary cyst(e)ine availability for whole blood glutathione synthesis in humans, we developed a convenient mass spectrometric method for measurement of the isotopic enrichment of intact GSH and then applied it in a controlled metabolic study. Seven healthy male subjects received during two separate 10-day periods an L-amino acid based diet supplying an adequate amino acid intake or a sulfur amino acid (SAA) (methionine and cysteine) free mixture (SAA-free). On day 10, L-[1-(13)C]cysteine was given as a primed, constant i.v. infusion (3 micromol x kg(-1) x h(-1)) for 6 h, and incorporation of label into whole blood GSH determined by GC/MS selected ion monitoring. The fractional synthesis rate (mean +/- SD; day(-1)) of whole blood GSH was 0.65 +/- 0.13 for the adequate diet and 0.49 +/- 0.13 for the SAA-free diet (P < 0.01). Whole blood GSH was 1,142 +/- 243 and 1,216 +/- 162 microM for the adequate and SAA-free periods (P > 0.05), and the absolute rate of GSH synthesis was 747 +/- 216 and 579 +/- 135 micromol x liter(-1) x day(-1), respectively (P < 0.05). Thus, a restricted dietary supply of SAA slows the rate of whole blood GSH synthesis and diminishes turnover, with maintenance of the GSH concentration in healthy subjects. (+info)
The effect of dietary sulfur-containing amino acids on the activity of drug-metabolizing enzymes in rat-liver microsomes.
Male Wistar rats were fed either a balanced diet whose protein source was a mixture of amino acids (diet 1), or a similar diet which differed only in having 48% less the quantity of the sulfur-containing amino acids methionine and cystine (diet 2). The diets were given either continuously for 1 month or for 15 days after a protein-free diet. Both diets 1 and 2 permitted good growth of rats and relatively stable microsomal protein content. Protein depletion decreased the total proteins, total phospholipids, and cytochrome P-450 content, and it strongly increased UDP-glucuronosyltransferase activity in rat-liver microsomes. Repletion with diet 1 restored those values, to the level found in control rats. However, diet 2, given continuously induced an increase in UDP-glucuronosyltransferase activity and in the cytochrome P-450 concentration. Since high UDP-glucuronosyltransferase activity was related to lessened amounts of sulfur-containing amino acids in the diet, we discuss the possible effect of methionine and cystine on the regulation of glucuronoconjugation in relation to sulfoconjugation. (+info)