Marvin C. Brummel

Blood methanol concentrations in normal adult subjects administered abuse doses of aspartame

Blood methanol concentrations were measured in 30 normal adult subjects administered aspartame, a dipeptide methyl ester. The doses studied included the 99th percentile of projected daily ingestion (34 mg/kg body weight) and three doses considered to be in the abuse range (100, 150, and 200 mg/kg body weight). Methanol concentrations were below the level of detection (0.4 mg/dl) in the blood of the 12 normal subjects who ingested aspartame at 34 mg/kg. They were significantly elevated (p less than or equal to 0 .001) after ingestion of each abuse dose, with the mean peak blood methanol concentrations and the areas under the blood methanol concentration-time curve increasing in proportion to dose. Mean (+/- SD) peak blood methanol concentrations were 1.27 +/- 0.48 mg/dl at the 100 mg/kg dose, 2.14 +/- 0.35 mg/dl at the 150 mg/kg dose, and 2.58 +/- 0.78 mg/dl at the 200 mg/kg dose. Blood methanol concentrations returned to predosing levels by 8 h after administration of the 100 mg/kg dose. Methanol was still detected in the blood 8 h after the subjects had ingested aspartame at 150 or 200 mg/kg. Blood formate analyses were carried out in the 6 subjects who ingested aspartame at 200 mg/kg, since recent studies indicate that the toxic effects of methanol are due to formate accumulation. No significant increase in blood formate concentrations over predosing concentrations was noted. No changes were noted in any of the blood chemistry profile parameters measured 24 h after aspartame ingestion, compared to values noted before administration. Similarly, no differences were noted in ophthalmologic examinations carried out before and after aspartame loading.

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.

The temporal regulation of protein synthesis during synchronous bud or mycelium formation in the dimorphic yeast Candida albicans

Abstract When stationary phase cells of the dimorphic yeast Candida albicans are diluted into fresh medium at 37°C at either pH 4.5 or pH 6.5, they evaginate at exactly the same time and with the same synchrony. However, they then grow in the budding yeast form at the former pH and in the elongate mycelium form at the latter pH. Three phases of protein synthesis are distinguished for cells forming either buds or mycelia: an initial 50-min period (phase I) during which total cell protein remains constant and the rate of incorporation of labeled amino acid into protein is virtually zero; a second period (phase II) during which there is a slow but constant increase in both total cell protein and the rate of incorporation; and a third period (phase III) during which there is a dramatic increase in both total cell protein and the rate of incorporation. The transition from phase I to phase II occurs at the same time for cells forming either buds or mycelia, but the transition from phase II to phase III occurs 20 to 30 min later in the mycelium than in the bud forming population, the same temporal difference observed for phenotypic commitment. The polypeptides synthesized during phases II and III were first analyzed by one-dimensional polyacrylamide gel electrophoresis. The patterns are similar for the two phenotypes. The major polypeptides synthesized during phase II are also synthesized during phase III, but in addition, a group of at least four new major polypeptides appear during phase III for both phenotypes. The minor polypeptides synthesized during phase III were also compared between the two phenotypes by two-dimensional polyacrylamide gel electrophoresis. The patterns, including roughly 200 distinguishable polypeptides, were similar. The similarities in the patterns of protein synthesis and the delay in the onset of phase III in mycelium forming cells are discussed in terms of phenotypic commitment. From these considerations, alternate hypotheses for the regulation of fungal dimorphism, in particular, and cell divergence, in general, are proposed.

Toxicity of protein hydrolysate solutions: correlation of glutamate dose and neuronal necrosis to plasma amino acid levels in young mice.

Abstract Casein and fibrin hydrolysate preparations currently used for human total parenteral alimentation therapy contain varying quantities of glutamate and aspartate. The administration of large quantities of these amino acids to the neonatal mouse either orally or by injection, produces a variety of neurotoxic effects, the most marked of which is an acute hypothalamic neuronal necrosis. Olney et al. have recently reported a dose-related incidence of such neuronal necrosis in infant mice injected with these protein hydrolysates, suggesting a possible hazard to the young human infant infused with such preparations. Since the neonatal mouse is acutely sensitive to glutamate-induced neuronal damage, we have measured plasma amino acid levels following injection of these protein hydrolysates in an attempt to determine the threshold plasma glutamate level which first results in neuronal damage. Casein and fibrin hydrolysates were administered subcutaneously to 9- to 11-day-old mice at 3 dose levels (20, 50, and 100 μl/g body weight), and plasma amino acid levels were determined at appropriate time intervals thereafter. Control animals were injected with isotonic saline at 50 μl/g body weight. Comparison of the maximal glutamate levels obtained in this study with the extent of neuronal necrosis reported by Olney et al. , indicated that no neuronal damage would be expected at plasma glutamate levels ranging from 24 to just under 50 μmoles/dl. This comparison indicates that plasma glutamate levels reached 50 to 52 μmoles/dl (normal, 6–12 μmoles/dl) during the hydrolysate injection reported to produce the smallest lesion, and thus approximate a minimal threshold value for the neonatal mouse. The data of Olney et al. indicate that most animals (11 of 12) were able to sustain this level without neuronal damage. Older mice (25 days) showed a marked improvement in ability to metabolize glutamate following injection when compared to 9- to 11-day-old animals. The latter phenomenon likely accounts for the decreased glutamate susceptibility noted in the adult mouse. Since plasma glutamate and aspartate levels remain within normal limits in the human infant during parenteral alimentation, and since the acutely sensitive neonatal mouse tolerates blood glutamate levels of at least 24 μmoles/dl, there appears to be little danger to the human infant during parenteral alimentation therapy.

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.

The dependency of nuclear division on volume in the dimorphic yeast Candida albicans

Abstract When stationary phase cells of the dimorphic yeast Candida albicans are induced to synchronously form mycelia, over 90% of the cells undergo nuclear division. However, when stationary phase cells are induced to synchronously form buds, less than half undergo nuclear division even though all form buds. The majority of cells which do not undergo nuclear division form buds with volumes below a threshold value and the majority of cells which do undergo nuclear division form buds with volumes above this threshold value. In this report, we have investigated the possibilities that cells which form small buds do not attain a particular mass threshold. Cell cultures were examined for DNA replication, dry weight, and protein content during synchronous bud and during synchronous mycelium formation. Evidence is presented which indicates that the lack of nuclear division in over half of a budding population is due to low daughter cell volumes or to low surface areas, and not to their failure to attain a mass threshold or to replicate their DNA. The dependency of nuclear division on daughter cell volume is discussed.

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.

Placental transfer of aspartate and its metabolites in the primate

Abstract The placental transfer of aspartate was tested in pregnant monkeys infused maternally with sodium aspartate. In five animals infused at 100 mg/kg/hr, maternal plasma aspartate levels increased from 0.36 ± 0.19 to 80.2 ± 11.5 μmole/dl (mean ± SD). However, fetal plasma aspartate levels increased only slightly from 0.42 ± 0.31 to 0.98 ± 0.24 μmole/dl ( p = 0.02). Erythrocyte aspartate levels were unchanged in both fetal and maternal circulation. In two animals infused at 200 mg/kg/hr, maternal plasma aspartate levels increased from 0.28 and 0.31 μmole/dl to values of 141 and 237 μmole/dl, respectively. This increase produced a significant ( p = 0.001) increase in fetal plasma aspartate levels from 0.53 and 0.67 to 3.3 and 4.5 μmole/dl, respectively. Maternal plasma aspartate levels in two animals infused at 400 mg/kg/hr increased from 0.5 and 0.7 μmole/dl to 400 and 750 μmole/dl, respectively, at the end of the infusion. Fetal plasma aspartate levels increased from 0.21 and 0.25 μmole/dl to 60 and 92 μmole/dl, respectively. Maternal aspartate infusion at each level increased maternal, but not fetal, plasma taurine levels. The increase in maternal taurine levels was not in proportion to the dose of aspartate infused. Aspartate metabolites, glucose, and lactate were readily transferred across the placenta. The data indicate that aspartate, like glutamate but unlike most amino acids, is not concentrated toward the fetal circulation in the pregnant primate, and suggest that a barrier to aspartate transfer exists unless maternal plasma levels are grossly elevated.

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.

Chicken heart H4 lactate dehydrogenase: N-terminal and C-terminal residues

Abstract N-Terminal analysis of chicken heart H 4 lactate dehydrogenase demonstrated the absence of a free amino acid or pyrrolidone carboxylic acid residue at the amino terminus of the protein. Quantitative determination of bound acetyl groups gave a value of 4.9 mole/mole of protein (mol wt of 135,000). The N-terminal peptide, characterized as N -acetyl-alanylthreonine, was isolated from pronase digests of the protein, and quantitative recovery experiments indicate that nearly all acetyl residues are found at the amino termini of the protein. The presence of leucinc as the C-terminal amino acid was confirmed using a tritium exchange method.