Mary A. Hilton

A simple method for detection of heterozygous carriers of the gene for classic phenylketonuria

Plasma phenylalanine (Phe) and tyrosine (Tyr) concentrations were measured by high-performance liquid chromatography in arteriolar blood obtained by finger prick from 78 normal volunteers and 36 heterozygote carriers of phenylketonuria (PKU). Subjects were tested at midday, 3 to 4 hours after their breakfast. Values for the micromolar ratio of Phe/Tyr and Phe2/Tyr for control subjects fell below 1.2 and 80, respectively; values for all carriers of PKU were higher for one or both ratios. This method should be applicable to widespread screening of the general population for carriers of the gene for PKU.

Taurine concentrations in hearts of big brown bats (Eptesicus fuscus) during hibernation, arousal and normothermic behavior

Abstract 1. 1. Hearts from bats (a) hibernating in a cave at 0°C, (b) after additional weeks in a hibernaculum at 2°C, (c) removed from the hibernaculum and exposed to room temperature for periods ranging from 8 to 120min, and (d) emerging from a summer roost, were rapidly removed, dissected and frozen. 2. 2. Mean concentrations of taurine in ventricles of hearts of bats from these five groups did not differ significantly. 3. 3. Plasma from a small subset of animals from the hibernaculum, sampled 0–120 min after transfer to room temperature, contained 324–1269 μM taurine. There was no apparent relationship between plasma taurine concentration and time at 23°C.

Use of the soluble peptide γ-L-glutamyl-L-tyrosine to provide tyrosine in total parenteral nutrition in rats

Limited solubility restricts amounts of tyrosine (Tyr) in amino acid solutions used in total parenteral nutrition (TPN). Excess phenylalanine (Phe) is included in TPN for conversion to Tyr by liver Phe hydroxylase. However, this conversion is limited, especially in infants. We have confirmed that infants receiving TPN have low Tyr concentrations and high Phe/Tyr ratios in plasma compared with published values for enterally fed neonates. Tyr is important in the synthesis of proteins and other biomolecules, including catecholamines in the brain. We tested the soluble peptide gamma-glutamyl-tyrosine (Glu(Tyr)) as a possible precursor of Tyr in TPN. Groups of five rats were given infusions of TPN containing an amino acid mixture simulating a commercial formulation (group A), TPN in which Glu(Tyr) was substituted for half the Phe in the group A solution) (group B), or saline (group C). Control animals (group C) were fed rodent chow. Blood was sampled at 0 time and daily for 4 days. Brains were collected at 96 hours, and aromatic amino acids in plasma and brains were measured by high-performance liquid chromatography. Throughout the experiment, plasma of animals in group A had significantly elevated Phe and reduced Tyr concentrations compared with control values; plasma concentrations in groups B and C were similar. In groups A and B, brain Tyr levels were 31% and 63% of control values, respectively. In group B, Glu(Tyr) was not detected in brains. These data suggest that supplementing current TPN mixtures with Glu(Tyr), which is stable in solution, can produce normal plasma Tyr concentrations and Phe/Tyr ratios and improve the supply of Tyr to the brain.

Use of the stable peptide, γ-l-glutamyl-l-tyrosine, as an intravenous source of tyrosine in mice

The relative insolubility of tyrosine (Tyr) at neutral pH limits amounts of this amino acid in solutions used for total parenteral nutrition (TPN). We have tested the potential of the natural peptide, gamma-L-glutamyl-L-tyrosine (Glu(Tyr], to release Tyr in vivo by making 20-microL injections, containing 2.9 mumol Glu(Tyr) (approximately 80 mumol/kg body weight), into the external jugular veins of mice. Mean concentrations of Glu(Tyr) in plasma were 138.5 and 11.4 mumol/L after 10 and 60 minutes, respectively; plasma Tyr was significantly elevated at 10 minutes, but returned to control levels at 60 minutes. When 5.8 mumol of Glu(Tyr) was injected, levels of Glu(Tyr) and of Tyr were significantly higher at both 10 and minutes than when 2.9 mumol of peptide was injected. Animals showed no evidence of toxicity. Two percent or less of the peptide could be detected in the urine, even in mice injected with 5.8 mumol Glu(Tyr). Pretreatment of mice with acivicin, a potent inhibitor of gamma-glutamyl transpeptidase (GGTase), prevented the increase in plasma Tyr seen after injection of 2.9 mumol Glu(Tyr) and led to higher levels of Glu(Tyr) in the plasma both at 10 and at 60 minutes than seen in mice given the same amount of Glu(Tyr) but no acivicin. The presence of the inhibitor also led to loss of as much as 48% of the administered peptide in the urine in 60 minutes. These data suggest that GGTase catalyzes hydrolysis of intravenous (IV) Glu(Tyr) to release Tyr in vivo. Glu(Tyr) in the blood is not partitioned into red blood cells; it remains in the plasma, available to GGTase, which functions at the external surface of cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Intravenous γ-glutamyl-tyrosine elevates brain tyrosine but not catecholamine concentrations in normal rats☆

A number of clinical situations may benefit from intravenous supplements of tyrosine (Tyr). In total parenteral nutrition (TPN), the supply of Tyr is limited by its poor solubility. In both rats and infants maintained on pediatric TPN, plasma Tyr levels are approximately 30% of normal, and in rat brains Tyr concentrations are similarly reduced. We reported previously that supplementing a TPN solution with the soluble peptide, gamma-glutamyl-Tyr [Glu(Tyr)], normalizes plasma Tyr and doubles brain Tyr in rats. To assess more fully the behavior of intravenous Glu(Tyr) in vivo, 20 mmol/L Glu(Tyr) was infused into the inferior vena cava of rats at rates increased every 2 hours over an 8-hour period (300 to 450 mumol Glu(Tyr)/kg body weight/h). The surgical procedure for catheterization is described. At the maximum rate of infusion, plasma Tyr and Glu(Tyr) concentrations reached mean plateau values of 326 and 252 mumol/L, respectively. Brain Tyr concentrations were 71 and 264 nmol/g wet weight in control rats infused with heparinized saline (SAL group) and rats infused with Glu(Tyr) (PEP group) respectively. No differences were found in concentrations of norepinephrine (NE), dopamine (DA), or homovanillic acid (HVA) in prefrontal cortex (PFC), striatum (STR), or remaining brain (RB) tissue in PEP and SAL rats. We did not detect undergraded Glu(Tyr) in the brain, and less than 0.5% of infused Glu(Tyr) appeared in the urine.

The effect of tyrosine-deficient total parenteral nutrition on the synthesis of dihydroxyphenylalanine in neural tissue and the activities of tyrosine and branched-chain aminotransferases

The poor solubility of tyrosine (Tyr) limits the amount of this amino acid in total parenteral nutrition (TPN). In rats maintained on a standard pediatric TPN mixture, plasma and brain concentrations of Tyr are reduced to about 25% of the levels in chow-fed controls. To determine whether these low concentrations of Tyr affect the synthesis of catecholamines in neural tissue, the rate-limiting step (conversion of Tyr to dihydroxyphenylalanine [DOPA]) is studied by administering NSD-1015 to block the pyridoxal phosphate (PLP)-dependent decarboxylation of DOPA. However, in TPN rats, plasma concentrations of Tyr are increased by drug treatment. Because brain Tyr is also increased, these and other experiments using NSD-1015 clearly overestimate the rate of DOPA synthesis for drug-free rats on TPN. Nevertheless, in TPN rats, there is less DOPA in the brain in one experiment and less DOPA in the olfactory bulbs in another, versus control rats. Further examination of the metabolic effects of NSD-1015 reveals that the drug also elevates the concentration of branched-chain amino acids (BCAAs) in the plasma of TPN rats. These findings result from inhibition by NSD-1015 of the PLP-dependent aminotransferases that initiate catabolism of Tyr in the liver and BCAAs in the muscle. Despite the pronounced reduction in plasma Tyr, TPN rats showed a marked increase in the activity of hepatic Tyr aminotransferase compared with chow-fed controls. Conversely, although TPN elevates BCAA concentrations in plasma, the activity of branched-chain aminotransferase (BCAT) in the heart muscle of TPN rats is not different from control values. Different values but the same relationships are seen in drug-free rats.

Taurine in Hearts and Bodies of Embryonic through Early Postpartum CF1 Mice

Abstract The hearts and remaining bodies of embryonic and fetal mice of known gestational age and of neonatal mice up to the age of 8.5 days were freeze-dried, weighed, and analyzed for the amino acid, taurine, by high performance liquid chromatography. Although cardiac taurine is only a small fraction of the taurine in the rest of the body in all animals studied, the concentration of taurine in the heart is similar to that in the rest of the body (40–45 nmole/mg freeze-dried wt) in embryos through Day 14.5 of gestation. Cardiac taurine concentration then begins to exceed that of the remainder of the body which gradually declines throughout the period studied. A doubling of cardiac taurine concentration is seen at birth (Day 19.5) when the cardiac to body taurine ratio rises markedly and is maintained at 2–4 throughout the period of observation. A maximum concentration of cardiac taurine (110 nmole/mg freeze-dried wt) is recorded 2.5 days after birth. The dramatic increase in cardiac taurine concentration at the time of birth follows the reported appearance in neonatal mouse hearts of adult levels of β-adrenergic receptors and the increased work load of the heart.

Plasma amino acids during high-dose methotrexate-citrovorum “rescue”

Abstract Plasma amino acids were monitored over a 4-day period in a patient with osteogenic sarcoma just before and during infusion of a high dose of methotrexate and during subsequent rescue with citrovorum factor. The concentrations in the plasma of phenylalanine rose seven-fold to a maximum observed at 22 hr and of cysteine rose almost threefold in 98 hr. The phenylalanine concentration maintained a threefold elevation during the 24-hr period following completion of the citrovorum rescue. These data indicate the inhibition by high dose methotrexate of liver phenylalanine hydroxylase, an enzyme system which is apparently not rescued by citrovorum factor. Plasma levels of tyrosine throughout the study did not fall below the pretreatment value.

Permeability of rat liver mitochondria to α-ketoglutarate, antipyrine, and carnitine☆

Abstract The study of the permeability of rat liver mitochondria under conditions supporting oxidative phosphorylation compares the uptake of 14 C-labeled α-ketoglutarate, antipyrine and carnitine by mitochondria incubated briefly at 0 ° and those incubated up to 90 min at 30 ° Uptake is measured in pellets produced by centrifugation in the cold. The α-ketoglutarate was incubated in the presence of malonate with mitochondria in which oxygen consumption was followed with an oxygen electrode. Less than 4% of the isotope equivalent to the amount of substrate oxidized remains with the unwashed pellets. Antipyrine uptake appears to be the same at 0 ° and at 30 °, and is greater than would be predicted if simple diffusion were responsible; adsorption to the mitochondrial membranes is indicated. The uptake of carnitine is enhanced at the higher temperature and is greater in mitochondria carrying out oxidative phosphorylation than in those suspended in 0.25 m sucrose. The extent to which these compounds can be removed from the pellets by washing with isotonic sucrose varies.