Amino- and carboxyl-terminal analyses of hepatoma lactate dehydrogenase isozymes.

The M4 isozyme of lactate dehydrogenase was purified to homogeneity from normal rat liver and from two Morris hepatomas (7777 and 7793). Amino-terminal analyses with fluorodinitrobenzene failed to detect the presence of free amino-terminal residues in each enzyme studied. Each enzyme contained between 3.7 and 4.1 moles of protein-bound acetyl groups per mole of enzyme. The amino-terminal peptide, characterized as N-acetylalanylalanine, was isolated from Pronase digests of each isozyme preparation, and quantitative recovery experiments indicated that all acetyl residues were bound at the amino termini. Carboxylterminal analyses demonstrated phenylalanine to be the carboxyl-terminal residue in each enzyme studied. These data indicate no differences in either amino- or carboxyl-terminal regions of the hepatoma M4 isozymes compared to normal liver M4 isozyme.

Rabbit muscle pyruvate kinase. Amino- and carboxyl-terminal studies.

Amino-terminal analysis of rabbit muscle pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) failed to detect the presence of any free amino-terminal residues. Acetyl group analysis demonstrated the presence of between 3.7 and 4.0 mol of acetyl groups per mol of enzyme. The acetylated amino-terminal residue was isolated from pronase digests of the enzyme and identified as N-acetylserine. Quantitative recovery experiments indicated that all acetyl residues are found at the amino termini. Carboxyl-terminal analyses using the tritium exchange method suggested the presence of a blocked carboxyl-terminal residue, supporting previous hydrazinolysis and carboxypeptidase studies.

Erythrocyte L-aspartyl-L-phenylalanine hydrolase activity and plasma phenylalanine and aspartate concentrations in children consuming diets high in aspartame.

A deficit of alpha-aspartyl-phenylalanine (alpha-Asp-Phe) hydrolase activity has been suggested as a cause of possible adverse effects of aspartame ingestion. Twenty-five normal preschool children and 23 school-age children described by their parents as sensitive to sugar were fed diets high in sucrose, aspartame, or saccharin for three successive 3-wk periods. Blood samples were obtained at baseline (fasting) and within the last 3 d of each dietary period (postprandial). alpha-Asp-Phe concentrations were below detection limits (0.5 mumol/L) in all plasma samples and Phe and Asp concentrations remained within normal limits, alpha-Asp-Phe hydrolase activities in baseline hemolysate samples did not differ between groups. One subject had a plasma alpha-Asp-Phe hydrolase activity > 2 SD below the mean. Despite this low activity, this subject did not show consistent cognitive or behavioral anomalies that could be linked to low hydrolase activity.

Utilization of D-methionine during total parenteral nutrition in postsurgical patients.

Utilization of intravenously administered D-methionine was measured by morbidly obese subjects fed parenterally after elective gastric bypass surgery. Five patients were infused with a 25% glucose--4.25% amino acid solution containing DL-methionine, and four were treated with a 25% glucose--3.5% amino acid solution containing only L-methionine. Mean (+/- SD) total daily methionine excretion was 0.06 +/- 0.04 mmoles (of 28 +/- 4 mmoles infused) in patients treated with the L-methionine containing solution, and was 15.2 +/- 4.2 mmoles/day (of 45.2 +/- 5 mmoles DL-isomer infused) in patients treated with the DL-methionine containing solution. In these latter patients, 90 to 98% of the excreted methionine was the D-isomer. The data indicate 64 +/- 23% of infused D-methionine is excreted in the urine. Four patients excreted between 70 to 85% of infused D-methionine in the urine, but one patient excreted only 35 to 55%, suggesting better utilization. Plasma methionine levels were higher (9.9 +/- 1.9 mumoles/100 ml) in patients infused with solutions containing DL-methionine than those infused with the L-methionine solution (4.5 +/- 1.0 mumoles/100 ml). In the former case, 49% of plasma methionine was the D-isomer. The data indicate poor D-methionine utilization by postsurgical patients during total parenteral nutrition when given as DL-methionine in the presence of other amino acids and glucose.

Effect of sucrose on the metabolic disposition of aspartame.

Twelve normal adult subjects ingested a beverage providing 0.136 mmol aspartame/kg body wt on 2 different days. On 1 study day the beverage provided only aspartame, on the other the beverage provided both aspartame and 3.51 mmol sucrose/kg body wt. The high mean plasma phenylalanine concentrations were similar after administration of aspartame alone (158 +/- 28.9 mumol/L, mean +/- SD) and administration of aspartame plus sucrose (134 +/- 44.1 mumol/L). Evaluation of the area under the plasma concentration-time curve (AUC) for phenylalanine also showed no significant difference between groups (197 +/- 49.1 vs 182 +/- 28.3 mumol.L-1.h for aspartame alone and aspartame plus sucrose, respectively). Similarly, the high mean ratio of phenylalanine to large neutral amino acids (Phe:LNAA) in plasma did not differ significantly (0.265 +/- 0.046 for aspartame alone, 0.275 +/- 0.107 for aspartame plus sucrose). However, there was a small but significant difference between groups for the 4-h AUC values for plasma Phe:LNAA. The simultaneous ingestion of sucrose with aspartame had only minor effects on aspartame's metabolic disposition.

Plasma amino acid concentrations and amino acid ratios in normal adults and adults heterozygous for phenylketonuria ingesting a hamburger and milk shake meal.

Plasma amino acid concentrations were measured and selected amino acid ratios were calculated in 12 normal adults and 12 adults heterozygous for phenylketonuria (PKU) ingesting a hamburger and milk shake meal providing 1 g protein/kg body wt. Plasma concentrations of all amino acids increased significantly over baseline after meal ingestion in both groups, reaching the highest mean values 3-5 h after meal ingestion. Plasma phenylalanine concentrations were significantly higher in heterozygous than in normal subjects both before and at all times after meal ingestion. The absolute increase in plasma phenylalanine concentration over baseline and the area under the plasma phenylalanine concentration-time curve were approximately twice as large in heterozygous as in normal subjects. However, the molar ratio of the plasma phenylalanine concentration to the sum of the plasma concentrations of the other large neutral amino acids did not increase significantly over baseline, but rather decreased.

Aspartame ingestion with and without carbohydrate in phenylketonuric and normal subjects: effect on plasma concentrations of amino acids, glucose, and insulin.

Seven subjects homozygous for phenylketonuria (PKU) and seven normal subjects were administered four beverage regimens after an overnight fast: unsweetened beverage, beverage providing carbohydrate (CHO), beverage providing aspartame (APM), and beverage providing APM plus CHO. The APM dose (200 mg) was the amount provided in 12 oz of diet beverage; the CHO was partially hydrolyzed starch (60 g). Plasma amino acid concentrations were determined after dosing and the molar plasma phenylalanine (Phe) to large neutral amino acid (LNAA) ratio calculated. APM administration without CHO did not increase plasma Phe concentrations over baseline values in either normal or PKU subjects (5.48 +/- 0.85 and 150 +/- 23.0 mumols/dL, respectively). Similarly, the Phe/LNAA did not increase significantly. Ingestion of beverage providing APM and CHO did not significantly increase plasma Phe concentrations over baseline values in either normal or PKU subjects. However, ingestion of beverage providing CHO (with or without APM) significantly decreased plasma levels of valine, isoleucine, and leucine 1.5 to 4 hours after dosing in both normal and PKU subjects, thereby increasing the Phe/LNAA ratio significantly. These data indicate that changes noted in Phe/LNAA values after ingestion of beverage providing APM plus CHO were due to CHO. The plasma insulin response to beverage providing CHO (with or without APM) was significantly higher in PKU subjects than in normals.

Repeated ingestion of aspartame-sweetened beverage: effect on plasma amino acid concentrations in individuals heterozygous for phenylketonuria.

It has been suggested that excessive use of aspartame (APM) (N-L-alpha-aspartyl-L-phenylalanine methyl ester) might grossly elevate plasma aspartate and phenylalanine concentrations in individuals heterozygous for phenylketonuria (PKUH). In study 1 six adult PKUH (three males; three females) ingested three successive 12-oz servings of beverage at 2-h intervals. The study was carried out in two parts in a randomized crossover design. In one arm the beverage was not sweetened. In the other the beverage provided 10 mg APM/kg body weight per serving. The addition of APM to the beverage did not significantly increase plasma aspartate concentration but did increase plasma phenylalanine levels 2.3 to 4.1 mumol/dL above baseline values 30 to 45 min after each dose. The high mean plasma phenylalanine level after repeated APM dosing (13.9 +/- 2.15 mumol/dL) was slightly, but not significantly, above the normal postprandial range for PKUH (12.6 +/- 2.11 mumol/dL). In study 2 six different adult PKUH ingested beverage providing 30 mg APM/kg body weight as a single bolus. The high mean plasma phenylalanine concentration and the phenylalanine to large neutral amino acid ratio were significantly higher when APM was ingested as a single bolus than when ingested as a divided dose.

In search of a physiological function for L-ergothioneine.

Since its discovery at the turn of the century, attempts to define a physiological function for L-ergothioneine have been unsuccessful. This paper suggests several possible functions for this enigmatic compound or its metabolites. These include: transport of cations or carbon dioxide, catalysis of carboxylation or decarboxylation reactions, mediation of thyroid or antithyroid function, histaminic or antihistaminic action, and cholinergic or anticholinergic action.

In search of a physiological function for L-ergothioneine--II.

Recently a number of possible functions for ergothioneine (ERT) have been suggested (1). This paper elaborates on some of these in light of overlooked or recent publications and presents additional hypotheses including: 1. Reduced ERT (ergothionol) may be an acyl carrier. 2. ERT, in conjunction with thyroid hormone and iodine, may be a cofactor in peroxidative and oxidative reactions. 3. ERT and thyroid hormone may be required for the oxidation of reduced pyridine nucleotides and the coupling of this to oxygen consumption (respiration) and ATP generation/ATPase action (heat production). 4. ERT may be required for both gene expression and repair. 5. 2-Thioimidazoles (ERT and 2-thiourocanic acid in particular) may be immunoregulatory. 6. ERT may be involved in the protection from oxidation (inactivation) of methionine and methionine containing chemoattractants, hormones, tRNA, etc. Some future research activities are suggested.

Ergothioneine distribution in bovine and porcine ocular tissues.

Ergothioneine (ERT), is a low molecular weight, sulfur-containing antioxidant occurring in up to millimolar amounts in mammalian tissues. Using an improved HPLC assay, ERT levels have been measured and compared in bovine and porcine eyes and erythrocytes. The rank order of ERT levels in bovine ocular tissue was lens > retina = cornea > pigmented retinal epithelium (RPE) > aqueous humor (AQ) > vitreous humor (VIT) > sclera. In porcine ocular tissue, the rank order was retina > AQ > VIT > RPE > cornea > lens > sclera. ERT levels in bovine lens were about 250 x > that in porcine lens. Porcine erythrocyte levels were 5.5 x > bovine levels. Species differences were also observed in the retina, VIT and AQ where porcine levels were 2 to 10-fold greater than bovine levels. ERT in bovine lens and cornea was 35 and 14 times greater than the corresponding level of reduced glutathione (GSH). Porcine lens had 45 times more GSH than ERT. Values for ERT and GSH in other tissues from both species were of the same order of magnitude. These results are consistent with a role for ERT in prevention of oxidative damage to the eye.

Blood methanol concentrations in one-year-old infants administered graded doses of aspartame.

Blood methanol concentrations were measured in 24 1-year-old infants administered aspartame, a dipeptide methyl ester sweetener. The doses studied included a dose projected to be the 99th percentile of daily ingestion for adults (34 mg/kg body weight), a very high use dose (50 mg/kg body weight) and a dose considered to be in the abuse range (100 mg/kg body weight). Blood methanol values in infants were compared to values observed previously in adults administered equivalent doses of aspartame. Methanol concentrations were below the level of detection (0.35 mg/dl) in the blood of 10 infants administered aspartame at 34 mg/kg body weight, but were significantly elevated (P less than or equal to 0.05) after ingestion of aspartame at 50 and 100 mg/kg body weight. At the latter doses, mean peak blood methanol concentrations and the area under the blood methanol concentration-time curve increased in proportion to dose. Mean (+/- SEM) peak blood methanol concentration was 0.30 +/- 0.10 mg/100 ml at a 50 mg/kg body weight aspartame dose (n = 6) and 1.02 +/- 0.28 mg/ml at the 100 mg/kg body weight dose (n = 8). Blood methanol values in infants were similar to those observed in normal adults.

Monosodium glutamate metabolism in the neonatal monkey.

Monosodium glutamate (MSG) administered by gastric tube to 10 infant monkeys at doses of 1-4 g/kg produced rapid increases in plasma glutamate (17- to 33-fold) and aspartate (50- to 90-fold) levels. The degree of elevation was proportional to the dose administered. Levels of other amino acids were unaffected. Two of the monkeys exhibited high fasting glutamate levels and abnormal glutamate tolerance curves. Despite this apparent decreased ability to metabolize glutamate, neither these animals nor any of the others for whom morphologic studies have been previously reported demonstrated neurotoxicity. Studies using 14-C-labeled glutamated indicated conversion of administered glutamate to two ninhydrin-negative compounds identified as glucose and lactate, as well as to asparate.

Placental transfer of glutamate and its metabolites in the primate.

When radioactive glutamate was infused into pregnant rhesus monkeys, 69 to 88 per cent of radioactivity in the maternal plasma remained in association with glutamate while 10 to 22 per cent was converted to glucose. In the fetal plasma, glucose and lactate accounted for more than 80 per cent of radioactivity, with less than 2 per cent of the label found in glutamate. Maternal glutamate infusions resulting in a ten- to twenty-fold increase in maternal plasma glutamate levels (60 to 100 mumoles per 100 ml.) had no effect upon fetal glutamate levels. Infusions producing maternal glutamate levels 70 times normal (280 mumoles per 100 ml.) did result in some transfer of glutamate to the fetal circulation. Labeled glutamate administered to the fetus at 1.5 to 2.4 Gm. per kilogram of fetal weight did not result in glutamate transfer to the maternal circulation. Infusion of glutamate to the fetus at 5 Gm. per kilogram of fetal weight increased fetal plasma glutamate levels to 2, 000 mumoles per 100 ml. and resulted in some transfer of glutamate to maternal circulation. Glutamate metabolites (lactate and glucose) were readily transferred across the placenta in either direction. These studies indicate that the primate placenta is virtually impermeable to glutamate unless extreme elevations of plasma glutamate are induced.

Aspartame-sweetened beverage: effect on plasma amino acid concentrations in normal adults and adults heterozygous for phenylketonuria.

Twelve normal subjects ingested either unsweetened beverage (n = 6) or beverage providing 4 mg/kg body weight as aspartame (APM) (n = 6). Neither beverage had any significant effect on plasma aspartate or phenylalanine concentrations. After this study, eight normal and six obligate phenylketonuric (PKU) heterozygous adults each ingested a 354-mL (12-oz) beverage serving on two occasions in a randomized cross-over design. On one occasion the beverage was not sweetened; on the other occasion, the beverage provided 10 mg APM/kg body weight. Plasma amino acid concentrations were measured throughout the 2-h study period. The addition of 10 mg APM/kg body weight to the beverage had no significant effect on plasma aspartate concentration. APM ingestion increased plasma phenylalanine levels of normal subjects from a mean +/- SD baseline value of 5.09 +/- 0.82 mumol/dL to a high mean value of 6.73 +/- 0.75 mumol/dL. In PKU heterozygous subjects the plasma phenylalanine level increased from a mean +/- SD of 9.04 +/- 1.71 to a high mean value of 12.1 +/- 2.08 mumol/dL. The data indicate ready metabolism of the aspartate and phenylalanine portion of APM when administered at levels likely to be ingested by individuals who drink diet beverages.