Analysis of lanthionine ketimine ethyl ester in mouse serum, whole blood and tissues using ultrahigh-pressure liquid chromatography/tandem mass spectrometry.

RATIONALE: Preclinical studies in the search for treatments for several neurodegenerative diseases have identified lanthionine ketimine (LK) and its monoethyl ester derivative (LKE) as potential candidates. An ultrahigh-pressure liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) assay was developed to evaluate bioavailability by measuring these compounds in mouse serum, whole blood and brain tissue. METHODS: Following administration of LKE to mice for 3 days in chow at 300 ppm, the animals were sacrificed, and LKE was extracted from serum, whole blood and brain tissues through protein precipitation using cold methanol. To enhance chromatographic separation and electrospray ionization, LK was methylated using diazomethane. Separations were carried out using C18 reversed-phase UHPLC, and quantitative measurements were obtained using on-line triple-quadruple mass spectrometry with positive ion electrospray ionization, collision-induced dissociation and selected reaction monitoring. Tolbutamide was used as internal standard. RESULTS: LKE showed good recovery ranging from 77-90% in serum and 82-88% in brain tissue. An eight-point standard curve ranging from 0.005 to 4.6 µM was linear (R2 0.998). The average LKE detected in mouse serum was 277.42 nM, while the concentration in whole blood was 38 nM. Neither LK nor LKE was detected in brain tissues. CONCLUSIONS: A rapid quantitative method to measure LKE in mouse serum, whole blood and brain tissues using UHPLC/MS/MS was developed and validated following FDA guidelines. This method is suitable for bioavailability and pharmacokinetic studies.

Effects of decreased availability of sulfur amino acids in severe childhood undernutrition.

In studies of glutathione (GSH) metabolism in children with severe childhood undernutrition (SCU), slower erythrocyte GSH synthesis in children with edema was associated with lower concentrations of cysteine, the rate-limiting precursor of GSH synthesis. This finding suggested a shortage of cysteine available for GSH synthesis in children with edematous SCU. The plasma concentration of methionine, the sulfur donor for cysteine synthesis, was also lower in children with edematous SCU, suggesting decreased availability of methionine for cysteine synthesis. It is also possible that reduced methionine availability will result in decreased synthesis of S-adenosylmethionine, which could lead to an overall defect in methylation reactions. This review focuses on the relationship between cysteine availability and GSH synthesis in children with SCU. It also examines whether there is an inadequate supply of cysteine in those with edematous SCU and, if so, whether this is due to a shortage of methionine due to a decreased release of methionine from protein breakdown. Finally, the review explores whether a shortage of methionine results in decreased synthesis of S-adenosylmethionine, the universal methyl donor.

Application of the indicator amino acid oxidation technique for the determination of metabolic availability of sulfur amino acids from casein versus soy protein isolate in adult men.

Our objective was to determine the metabolic availability (MA) of sulfur amino acids in dietary proteins using the indicator amino acid oxidation (IAAO) technique. Five to seven men received graded levels (20, 40, 60, and 70%) of the mean total sulfur amino acid (TSAA) requirement of 13 mg x kg(-1) x d(-1) as a crystalline AA mixture, casein, and soy protein isolate (SPI) (40, 50, 60, and 70%), respectively. Five of these subjects received 40% of TSAA requirement from SPI supplemented with methionine to the level of 40% of requirement. These 5 subjects also repeated the level of 60% TSAA requirements from both casein and SPI to assess repeatability. The mean MA of TSAA from SPI (71.8 +/- 3.6%) was lower than from casein (87.4 +/- 3.8%, P < 0.05). Supplementation of SPI with methionine decreased the IAAO (11.5 +/- 0.3% administered dose) compared with unsupplemented SPI (12.8 +/- 0.5% administered dose, P < 0.05). IAAO was similar for repeated measurements of casein and SPI, respectively, at the 60% TSAA intake level (10.8 +/- 1.0 vs. 10.7 +/- 1.2% for casein; 12.7 +/- 1.3 vs. 12.9 +/- 2.6% for SPI). In conclusion, the IAAO technique can be used to determine the MA of AA for protein synthesis in test proteins for humans.

Functions of sulfur-containing amino acids in lipid metabolism.

It is known that plasma lipid levels are controlled not only by dietary fat and carbohydrate but also by dietary protein and amino acids. Although it used to be thought that the source of protein was important, it is known that amino acid composition, amino acids themselves, and peptides from digested protein are more important than the protein source. Sulfur-containing amino acids (SAAs) are recognized to be some of the most potent modulators of lipid metabolism among amino acids. It has been demonstrated that SAAs have an increasing effect on HDL (high-density lipoprotein)-cholesterol and a decreasing effect on VLDL (very low-density lipoprotein)-cholesterol. These data lead us to propose that SAAs have some beneficial functions against atherosclerotic diseases and metabolic syndrome. Relative availability of SAAs (RASAA) as well as the amount of SAAs in dietary protein would determine lipid metabolism. Therefore, we propose RASAA as a feasible index for improvement of lipid metabolism by amino acids. Although it is not clear how SAAs influence gene expression and lipid metabolism at a molecular level, SAAs change the metabolic pathway through transcriptional stimulation and posttranslational modification of regulatory proteins.

Comparative species utilization and toxicity of sulfur amino acids.

Animal studies have shown that several methionine (Met) and cysteine (Cys) analogs or precursors have L-Met- and L-Cys-sparing activity. Relative oral bioavailability (RBV) values, with the L-isomer of Met and Cys set at 100% (isosulfurous basis), are near 100% for D-Met for animals but only about 30% for humans. Both the OH and keto analogs of Met have high RBV-sparing values, as does N-acetyl-L-Met (the D-isomer of acetylated Met has no bioactivity). L-Homocysteine has an RBV value of about 65% for Met sparing in rats and chicks, but D-homocysteine has little if any Met-sparing activity. S-Methyl-L-Met can partially spare Met, but only when fed under dietary conditions of choline/betaine deficiency. Relative to L-Cys, high RBV values exist for L-cystine, N-acetyl-L-Cys, L-homocysteine, L-Met, and glutathione, but D-cystine, the keto analog of Cys, L-cysteic acid, and taurine have no Cys-sparing activity. l-2-Oxothiazolidine-4-carboxylate has an RBV value of 75%, D-homocysteine 70%, and DL-lanthionine 35% as Cys precursors. Under dietary conditions of Cys deficiency and very low inorganic sulfate (SO4) ingestion, dietary SO4 supplementation has been shown to reduce the Cys requirement of several animal species as well as humans. Excessive ingestion of Met, Cys, or cystine has also been studied extensively in experimental animals, and these sulfur amino acids (SAA) are well established as being among the most toxic of all amino acids that have been studied. Even though Cys and its oxidized product (cystine) are equally efficacious at levels at or below their dietary requirements for maximal growth, Cys is far more toxic than cystine when administered orally in the pharmacologic dosing range. Isosulfurous (excess) levels of cystine, N-acetyl-L-Cys, or glutathione are far less growth depressing than L-Cys when 6 to 10 times the minimally required level of these SAA compounds are fed to chicks.

Urinary sulfur excretion and the nitrogen/sulfur balance ratio reveal nonprotein sulfur amino acid retention in piglets.

We evaluated the use of urinary sulfur (S) excretion as a measure of sulfur amino acid (SAA) catabolism and the nitrogen/sulfur (N/S) molar balance ratio as an indicator of nonprotein SAA storage in growing piglets. After confirming that an intravenous dose of sulfate is fully recovered in urinary sulfate, we measured urinary S recovery after an intravenous dose of methionine in 6 piglets fed an adequate protein (AP) diet and 6 piglets fed a low protein (LP) diet with normal energy provision. As measured over 48 h, recoveries of the methionine load as urinary total S was 106% in the AP group but only 69% in the LP group (P < 0.05). On the baseline diets the N/S balance ratio in the AP group was 36, whereas that in the LP group was 30 (P < 0.05); immediately after the methionine load, this ratio remained constant in the AP group but decreased further, to 26 (P < 0.05) in the LP group. These results indicate that protein-deficient piglets accumulate relatively more S than N from their diet, and under these conditions a significant portion of the S derived from a methionine load is retained in nonprotein compounds. Urinary S excretion, a simple nontracer measurement, can provide an accurate measure of SAA catabolism, and the N/S balance ratio is a potentially useful indicator of changes in nonprotein SAA stores of growing piglets.

Bioavailability of the digestible lysine and total sulfur amino acids in meat and bone meals varying in protein quality.

Experiments were conducted to determine whether the digestible Lys, Met, and TSAA in a high and low quality meat and bone meal (MBM) were totally bioavailable for protein synthesis in chicks. True digestibility of amino acids (AA) in the two MBM was determined by the precision-fed cecectomized rooster assay. Bioavailability of the digestible AA was then assessed in three slope-ratio chick growth assays using Lys-, Met-, or TSAA-deficient crystalline AA basal diets that were supplemented with varying levels of the test AA, high or low quality MBM, or AA mixtures simulating the mean digestible AA composition of the high and low quality MBM. Response parameters were weight gain, feed efficiency, body N gain, and body Lys gain in the Lys assay and weight gain and feed efficiency in the Met and TSAA assays. Bioavailability values for the digestible Lys, Met, and TSAA in the MBM and AA mixtures simulating MBM varied depending on response parameter, with values based on feed efficiency generally being highest. No consistent differences in bioavailability of the digestible AA were observed between the two MBM when all AA were considered; however, the bioavailability of the digestible Lys in the low quality MBM was lower than that in the high quality MBM for two of four performance criteria. When considering all response parameters and the AA mixture results, bioavailability of the digestible Lys and Met in the two MBM was generally 90% or greater, whereas bioavailability of the digestible TSAA was only 80% or less. The results of this study indicated that essentially all of the digestible Lys and Met in MBM were bioavailable for protein synthesis and metabolism but suggested that a significant amount of the TSAA, particularly Cys, was not bioavailable.

On the transformation of sulfur-containing amino acids and peptides to volatile sulfur compounds (VSC) in the human mouth.

Halitosis is most often caused by oral conditions. Volatile sulfur compounds (VSC), constituting the major components of oral malodor, are produced by anaerobic, gram-negative bacteria retained mainly in periodontal pockets or on the tongue dorsum. Sulfur-containing amino acids serve as substrate for these bacteria. VSC have also been found to have unfavorable effect on the tissue. The aim of this study was to examine whether normal, healthy individuals with no history of halitosis were able to produce VSC from cysteine, when applied as a mouthrinse. A further aim of the study was to investigate and compare the potential of other sulfur-containing amino acids and peptides as substrates for oral VSC production and to localize the odor-production sites. A portable sulfide monitor was used for VSC registration. Results showed that all test subjects produced high oral concentrations of VSC upon rinses with cysteine, which thus seems to be a major substrate for VSC production. The other sulfur-containing substrates had much less effect. It was found that the tongue was the major site for VSC production, and that saliva per se caused low VSC production.

Maximal portion of the young pig's sulfur amino acid requirement that can be furnished by cystine.

Three pig experiments were conducted using a chemically defined, amino acid diet under conditions in which all nutrients were 100% bioavailable to assess the maximal portion of the sulfur amino acid (SAA) requirement that could be furnished by cystine (Cys). In Exp. 1, a methionine (Met)-deficient diet containing .12% L-Met and .40% L-Cys was supplemented with graded levels of L-Met. Pigs weighing 10 kg initially responded quadratically (P less than .05) to Met supplementation. A two-slope, broken-line regression model (weight gain regressed on percentage of dietary Met) estimated an infection point at .23% dietary Met. A constant level of .46% dietary SAA with differing Met:Cys weight (wt:wt) ratios was used in Exp. 2. Pigs fed Met:Cys ratios of 60:40 and 50:50 had similar (P greater than .05) weight gains, but pigs fed a 40:60 Met:Cys ratio gained less (P less than .05) than those fed the other diets. Maintaining dietary sulfur at .111% in Exp. 3, pigs fed Met:Cys ratios (wt:wt) of 100:0, 55:45, 50:50, and 45:55 gained weight at similar (P greater than .05) rates, but pigs fed the 45:55 Met:Cys ratio had a tendency to produce lower weight gains. Regardless of whether a constant dietary SAA or sulfur level was maintained, no more than 50% of the young pig's total SAA requirement (wt:wt) could be furnished by Cys.

[The metabolism of sulfur containing amino acids in the rat lens].

To determine the metabolism of sulfur containing amino acids in cultured rat lenses, the author studied the uptake and incorporation of radioactive amino acids (3H-leucine, 14C-glycine, 14C-cystine and 35S-methionine) into whole and fractions of the lens, and the effects of diethyl maleate (DEM), the inhibitions of glutathione (GSH) synthesis on them. The uptake and incorporation of radioactive amino acids with the exception of cystine into whole and fractions of the lens were decreased, especially in DEM-additive group. In DEM group, the uptake of cystine into whole lens was suppressed but that in control group was not. The incorporation of cystine into protein fraction (WIS, WS) were increased in DEM group. It was supposed that the decrease in the incorporation of glycine and the increase in that of cystine into protein fractions were related to the formation of high molecular weight proteins. It was also assumed that the decrease of the uptake of cystine into TCA soluble fraction was due to the inhibition of the synthesis of GSH.

Development and biologic evaluation of a kit for preformed chelate technetium-99m radiolabeling of an antibody Fab fragment using a diamide dimercaptide chelating agent.

A kit has been developed for 99mTc antibody radiolabeling via defined chemistry using an N2S2 diamide dimercaptide bifunctional chelating agent and the performed chelate method. The process involved efficient transchelation of 99mTc from gluconate to 2,3,5,6-tetrafluorophenyl 4,5-bis-S-(1-ethoxyethyl) mercaptoacetamidopentanoate as an active ester ligand and subsequent conjugation to antibody lysine amine functional groups. The use of the ethoxyethyl group for sulfur protection allowed optimum yields of 99mTc N2S2 chelate formation with complete retention of the active ester. Subsequent addition of antibody Fab fragment gave 99mTc chelate conjugates indistinguishable from the stepwise in situ esterification and purification of the 99mTc N2S2 complex followed by conjugation as previously shown to give stable 99mTc antibody fragments with retained immunoreactivity and tumor-targeting properties.

Excretion of 3-mercaptolactate-cysteine mixed disulfide, sulfate and taurine in human urine before and after oral administration of sulfur-containing amino acids.

The excretion of 3-mercaptolactate-cysteine mixed disulfide [S-(2-hydroxy-2-carboxyethylthio)-L-cysteine, HCETC], sulfate and taurine in the urine of normal adults was investigated before and after oral administration of L-cysteine and related sulfur-containing amino acids. Before the loading of amino acids, the excretion (mean +/- SD) per kg of body weight per day of HCETC, free sulfate and taurine was 0.096 +/- 0.042, 305.7 +/- 66.1 and 31.9 +/- 8.7 mumols, respectively. After the loading of L-cysteine (800 mumols/kg of body weight), the average excretion in the 24-h urine of HCETC increased 2-fold and that of taurine increased 1.6-fold. The average excretion of free sulfate after the L-cysteine loading was 989.4 +/- 145.1 and 388.8 +/- 51.6 mumols/kg per day in the first and second 24-h urine, respectively, indicating that the sulfur corresponding to 85% of the L-cysteine loaded was excreted as free sulfate in 24 h. Administration of L-cystine (400 mumols/kg) resulted in similar results. The increase in HCETC after L-cysteine or L-cystine administration indicates that L-cysteine is metabolized in part through the transamination pathway (3-mercaptopyruvate pathway) and that an equilibrium exists between the intake and excretion of sulfur in humans.

L-[35S]cysteic acid selectively detects chloride-dependent L-glutamate transporters in synaptic membrane.

Na+-independent L-[35S]cysteic acid (CA) accumulation in rat cortical synaptic membrane was examined. In the absence of Cl-, the accumulation was not observed. Addition of Cl- revealed the accumulation in a dose-dependent manner. Br- and NO3- also did. Ca2+-enhanced the Cl- -dependent accumulation, whereas low concentrations of Na+ reduced it. L-[35S]CA accumulation was inhibited by quisqualate, L-glutamate (L-Glu), L-cysteine sulfinate, D,L-homocysteic acid and D,L-2-amino-4-phosphonobutyrate (D,L-APB) potently. L-CA inhibited L-[3H]Glu accumulation in synaptic membrane in the presence of Cl-. The maximal inhibition of L-CA was equal to that of D,L-APB, but L-CA did not inhibit L-[3H]Glu accumulation in the absence of Cl-. These results show that L-[35S]CA selectively detects the Cl- -dependent L-Glu transporters in synaptic membrane.