Plasma Phenylalanine Concentrations Research Articles

Amino acid supply to individual cerebral structures in awake and anesthetized rats.

The movement of phenylalanine from plasma into various cerebral structures was examined in detail in normal alert rats as well as in rats anesthetized with nitrous oxide, halothane, and sodium pentobarbital. Radioactive phenylalanine was infused intravenously in such a way as to rapidly establish and maintain a trace concentration in arterial blood. The rat was decapitated after 1.5 min, the brain removed, frozen, and thin sections cut for quantitative autoradiography. The plasma concentrations of phenylalanine as well as other neutral amino acids that compete with phenylalanine for entry into brain were measured. Phenylalanine influx varied considerably between structures, ranging from 5 nmol.min-1.g-1 in the globus pallidus to 12 nmol.min-1.g-1 in the inferior colliculus. Anesthetics reduced phenylalanine influx, but this effect was primarily due to alterations in the circulating neutral amino acid pattern. The anesthetics had little, if any, direct effect on the neutral amino acid transport process itself.

Renal transport of aromatic acids in patients with phenylketonuria.

Renal clearance of phenylpyruvic acid is maximal at a plasma concentration of 40-60 mumol/l. This concentration is obtained with plasma phenylalanine concentrations of 1.0-1.2 mmol/l, the threshold for separating classical phenylketonuria from phenylketonuria variants.

Plasma and cerebrospinal fluid amino acid concentrations in phenylketonuria during the newborn period

Plasma and cerebrospinal fluid amino acid values were determined in 29 infants 9 to 30 days of age with a confirmed diagnosis of phenylketonuria. Phenylalanine concentrations in plasma and cerebrospinal fluid were markedly elevated; the degree of elevation in the cerebrospinal fluid had a significant relationship to that of the plasma. The only other significant deviations in the plasma were reductions in the threonine and tyrosine values. Cerebrospinal fluid threonine, alanine, and arginine concentrations were reduced, whereas those of serine, isoleucine, and histidine were elevated. This combined deficiency and excess of amino acids in the central nervous system may have a significant effect on protein synthesis at a time in life when this synthesis and turnover is most active.

Unsuccessful treatment of phenylketonuria with tyrosine

ON E of the earliest questions in the treatment of phenylketonuria was whether the severe mental retardation characteristic of this disorder was caused by phenylalanine accumulation or by tyrosine deficiency. Although early therapy with a diet low in phenylalanine and supplemented with tyrosine is effective in preventing mental retardation in PKU, 1, ~ the role of a tyrosinesupplemented diet alone has not been evaluated. We present the case of a child born in 1967 whose parents elected 1o have him treated with tyrosine supplementation rather than with phenylalanine restriction. CASE REPORT The patient was the product of an uncomplicated term pregnancy, labor, and delivery in a gravida 3, para 2, 25-year-old mother. Birth weight was 4.3 kg an d the Apgar score was 10 at 5 minutes. The neonatal course was uncomplicated. However, plasma phenylatanine concentrations at 3 and 10 days of age were 1,400 and 3,030 ~M, respectively (normal range 26 to 61 /~M). Plasma tyros!no concentration was 70/xM at 10 days of age (normal range 31 to 71 /~M). The child was placed on the standard phenylalanine-restricted diet for two weeks. However, the parents noted the child to be irritable and lethargic and requested that the diet be terminated. They questioned whether another form of therapy might be available, and consented to a trial of tyrosine supplementation alone, c-Tyrosine, as a free base, was given with meals at a dose of 200 mg/day. Plasma tyrosine concentration rose to 214 ~M. Tyrosine supplementation was continued until 4 years of age at doses ranging from 200 to 300 mg/day. During this time plasma phenylalanine concentrations ranged from 1,370 to 3,818 tsM and plasma tyrosine values from 62 to 219/~M. Therapy was discontinued at four years when it became apparent that the child was severely retarded. Developmental milestones were delayed from infancy. Motor skills were always less affected than were language skills. The

Plasma and erythrocyte concentrations of free amino acids in adult humans administered abuse doses of aspartame.

Plasma and erythrocyte concentrations of amino acids were measured in 18 fasting adult subjects (9 male, 9 female) administered abuse doses of aspartame (100, 150, and 200 mg/kg body weight) dissolved in 500 ml orange juice. Six subjects were studied at each dose. Plasma aspartate concentrations increased significantly (p less than or equal to 0.05) over baseline values after ingestion of each dose. However, the increase was small in each case, and maximal levels observed were below those noted postprandially in formula-fed infants. No significant changes (p greater than 0.05) were noted in erythrocyte glutamate, or erythrocyte aspartate concentrations after any dose. Plasma phenylalanine concentrations increased significantly over fasting concentrations (p less than 0.01) from 15 min to 6 h after each dose, and the increase was proportional to dose. Mean (+/- SD) peak plasma phenylalanine concentrations were 20.3 +/- 2.03, 35.1 +/- 11.3, and 48.7 +/- 15.5 mumol/dl, respectively, after aspartame doses of 100, 150, and 200 mg/kg. Erythrocyte phenylalanine concentrations showed similar changes. Although these phenylalanine concentrations are considerably above the normal postprandial range (12 +/- 3 mumol/dl), they are below values associated with toxic findings. These data indicate little risk to normal subjects from excessive aspartate or phenylalanine levels after ingestion of single abuse loads of aspartame.

Plasma phenylalanine levels in phenylketonuric heterozygous and normal adults administered aspartame at 34 mg/kg body weight

Following administration of aspartame (34 mg/kg body wt) in orange juice, plasma concentrations of free amino acids were measured in 12 female subjects known to be heterozygous for phenylketonuria and 22 normal subjects (12 male, 10 female). No change in fasting plasma aspartate concentrations were noted after aspartame loading in either group. In normal male subjects, the mean (±S.D.) plasma phenylalanine concentration increased from a fasting value of 5.86 ± 1.25 μmol/dl to a mean peak value of 12.0 ± 3.79 μmol/dl. Plasma phenylalanine levels in normal female subjects increased from a mean fasting concentration of 4.83 ± 0.84 μmol/dl to a mean peak value of 8.95 ± 1.49 μmol/dl suggesting a more rapid absorption, metabolism, and/or clearance of phenylalanine by females. In female heterozygous subjects, the mean peak plasma phenylalanine concentration was significantly higher than in normal females. Plasma phenylalanine values increased from a mean fasting value of 5.92 ± 1.51 μmol/dl to a mean peak value of 15.1 ± 4.67 μmol/dl. Similarly, the area under the plasma phenylalanine concentration-time curve was significantly greater in heterozygous female subjects (21.36 ± 5.10 IU) than in normal female subjects (10.84 ± 2.32 IU). However, peak plasma phenylalanine levels were well below those associated with toxic effects in all cases.

Behavioral alteration of plasma phenylalanine concentration

The concentration of 13 neutral and acidic plasma amino acids was measured before, during and either 40 min of control relaxation or 40 min of the process known as transcendental meditation (TM). An electro-oculogram, electroencephalogram, and electromyogram were simultaneously monitored in these subjects. Increased phenylalanine concentration was noted during TM practice with no change during control relaxation: no difference between the groups of total time slept or sleep stage percent was observed. The stability of phenylalanine concentration in controls and lack of correlation of increased phenylalanine with sleep in the long-term practitioners seem to suggest a relationship of the phenylalanine increase to TM practice.

Detection of heterozygotes for phenylketonuria by constant intravenous infusion of L-phenylalanine.

We measured the rate of elimination of phenylalanine by constant intravenous infusion of L-phenylalanine in 14 parents of children with phenylketonuria and in 21 subjects with a negative family history for this disease. When reciprocals of the observed elimination rates were plotted against the reciprocals of the increase in the plasma phenylalanine concentrations, approximately straight lines resulted. The theoretical maximum elimination rate, the mean value for which was 32 mmol/h in the reference subjects, was reduced by 41% in the phenylketonuric heterozygotes. The elimination rate at an increase in plasma phenylalanine concentration of 0.5 mmol/liter discriminated the phenylketonuric heterozygotes from normal homozygotes, with no overlap between the groups. A lower plasma tyrosine concentration in the phenylketonuric heterozygotes than in the reference subjects at the same rate of elimination of phenylalanine indicated an increased rate of elimination of tyrosine at a fixed concentration of this amino acid in these subjects.

Detection of heterozygotes for phenylketonuria. Total body phenylalanine clearance and concentrations of phenylalanine and tyrosine in the plasms of fasting subjects compared.

Two tests have been compared for detection of heterozygotes for phenylketonuria, one based on determination of plasma phenylalanine and tyrosine concentrations in fasting individuals and the other on kinetic evaluation of the plasma elimination curve after intravenous loading with L-phenylalanine. The plasma elimination curve was biexponential and the kinetics were evaluated according to the two-compartment model. The constant, beta, expressing the rate of elimination from plasma at pseudo-equilibrium, the rate constant for the elimination from the central compartment, and the total body clearance were determined. Of these three, total body clearance, which on the average was reduced by 32% in the phenylketonuric heterozygotes, showed the best discriminatory ability, but was not better than the information on concentrations of phenylalanine and tyrosine in detecting heterozygotes for phenylketonuria.

Kinetics of intravenous administered l-phenylalanine in patients with cirrhosis of the liver

The metabolism of intravenously administered l-phenylalanine has been studied in 13 patients with cirrhosis of the liver and in 17 control subjects. The decline in the plasma phenylalanine concentration followed a biexponential curve and the kinetics could be evaluated assuming a two-compartment model. The volume of the central compartment was reduced in the cirrhotic patients, reflecting a diminished liver pool size. The total phenylalanine clearance was lower in the cirrhotic patients, due mainly to a reduction of the liver cell mass or to changes in the intrahepatic membrane permeability and only to a minor extent to a reduced hepatic blood flow. The elimination rate constant from the central compartment was similar in both groups, suggesting that the remaining liver cells, had a normal capacity to hydroxylate phenylalanine to tyrosine. There was considerable overlapping in the kinetic parameters between the two groups, and even in patients with well established cirrhosis an apparently normal phenylalanine metabolism was observed.

Milk Production, Nitrogen Utilization and Glucose Synthesis in Lactating Cows Infused Postruminally with Sodium Caseinate and Glucose

Four lactating Holstein cows were used in a 4 X 4 Latin Square design to determine the effects of postruminally administering sodium caseinate and/or glucose on milk production, milk composition, nitrogen utilization, amino acid utilization by the lactating mammary gland and glucose turnover rate. An 8.5% increase in milk yield and a 13.3% increase in milk protein production were obtained during infusion of sodium caseinate. No significant production responses were attributed to abomasal infusion of glucose. Arterial concentrations of most essential amino acids were increased during infusion of sodium caseinate. Uptake of phenylalanine, methionine and lysine by the mammary gland most closely paralleled their output in milk. The relative concentrations of methionine, lysine and phenylalanine in arterial plasma were considerably less than their concentrations in milk which resulted in a large percentage extraction of these amino acids by the mammary gland. If the availability of essential amino acids to the mammary gland, per se, was limiting the synthesis of milk protein, methionine, lysine and phenylalanine may have been the three amino acids most limiting. Measurements of glucose entry rate showed a trend toward increased glucose flux when either glucose, sodium caseinate or glucose plus sodium caseinate were infused abomasally. The similarity in glucose entry rates obtained during infusion of glucose and sodium caseinate suggest that the increase in milk production was not due totally to increased glucose flux resulting from sodium caseinate infusion.

Effect of cotrimoxazole on the response to phenylalanine loading in man

Cotrimoxazole impairs the L-phenylalanine tolerance of control subjects as judged by the time course of the plasma phenylalanine concentration and the plasma ratios [phenylalanine] / [tyrosine] after oral and intravenous loading with phenylalanine. These subjects are presumed not to be heterozygous for any of the abnormal recessive genes which cause hyperphenylalaninaemia when they are present in the homozygous state. Patients with classical phenylketonuria did not show this effect. Cotrimoxazole did not affect the renal clearance of phenylalanine. The effect appears to be virtually entirely due to the trimethoprim component as opposed to the sulphamethoxazole component of Cotrimoxazole. The evidence presented is compatible with the view that the drug inhibits phenylalanine hydroxylation in the patients' tissues, presumably by inhibiting dihydrobiopterin reductase. We suggest that it may be possible to utilise the observed effect of cotrimoxazole on phenylalanine hydroxylation to identify in vivo those hyperphenylalaninaemic patients who have residual phenylalanine hydroxylase activity.

The regulation of phenylalanine hydroxylase in rat tissues in vivo. The maintenance of high plasma phenylalanine concentrations in suckling rats: a model for phenylketonuria.

Maximum inhibition of phenylalanine hydroxylase activity in the liver (85%) and in the kidney (50%) of suckling rats required the administration of over 9 mumol of p-chlorophenylalanine/10g body weight. Despite the decrease in the total activity from 184 to 34 units per 10g body weight, the injection of as much as 26 mumol of phenylalanine was required for its concentration in plasma to be still considerably elevated 12h later. In rats injected with p-chlorophenylalanine every 48h and with phenylalanine every 24h from 3 to 18 days of age, the hepatic and renal phenylalanine hydroxylase remained inhibited, whereas the activities of three other hepatic enzymes were unchanged. There was about 20% inhibition of brain and body growth, but no interference with the developmental formation of several cerebral enzymes (four dehydrogenases, hexokinase and glutaminase) was detected. In the course of this prolonged treatment, the phenylalanine concentrations in plasma increased gradually; on day 2 and day 8 (measured 12h after the last injection) they were 800 and 1395 nmol/ml respectively; on day 15, 12 and 18h after the usual injection, the values were 2030 and 1030 respectively as opposed to the 96 nmol in untreated rats. This degree of hyperphenylalaninaemia, persisting for 18h per day throughout a critical period of development, fulfils the primary criterion of a suitable animal model for phenylketonuria.

Concentrations of phenylalanine and tyrosine in plasma and brain of rats treated with phenylalanine loads.

Concentrations of phenylalanine and tyrosine in plasma and brain of rats treated with phenylalanine loads.

The phenylalanine and tyrosine content of maternal and fetal body fluids from rabbits fed aspartame

The methyl ester of aspartylphenylalanine (aspartame; SC-18862; 3-amino- N-(α-carboxyphenethyl)-succinamic acid, methyl ester) is a sweetening agent which organoleptically has about 180 times the sweetness of sugar. Since this compounds is a food additive its metabolism in pregnancy has been evaluated. Pregnant female rabbits were fed aspartame at a level of 6% in the diet beginning on day 6 of pregnancy. Maternal plasma samples taken at days 6, 9, 16, and 20 were analyzed for phenylalanine and tyrosine. Fetal amniotic fluids were similarly analyzed on days 16 and 20 as were fetal plasma samples at day 20. Maternal plasma phenylalanine and tyrosine increased as a result of the treatment, reaching a peak on day 9. These values returned toward normal levels by day 20. The ratios of fetal/maternal plasma phenylalanine and tyrosine concentrations were unaffected by the treatment. No evidence of substrate inhibition of phenylalanine hydroxylase was seen in maternal or fetal plasma analyses or in in vitro studies of maternal liver homogenates. Tyrosine concentrations in maternal and fetal plasma rose higher with treatment than phenylalanine concentrations. Both amino acids accumulated in the amniotic fluid in amounts greater than, but proportional to, plasma concentrations.

Impairment of hepatic pyruvate metabolism in phenylketonuria

In patients with untreated classical phenylketonuria, elevated plasma levels of pyruvate, lactate, phenylalanine and phenylpyruvate were observed. After about 10 days on a low-phenylalanine diet, the levels of pyruvate, lactate and phenylpyruvate in plasma of treated patients returned to normal; the concentrations of phenylalanine in plasma were markedly lowered. In plasma from hyperphenylalaninemic subjects, phenylpyruvate was not detectable; pyruvate and lactate were within normal limits. Phenylpyruvate at a concentration of about 1 mM inhibited pyruvate carboxylation by human and rat liver homogenates by about 50%; phenylalanine had no effect on this process. The values of apparent Km for pyruvate and Ki for phenylpyruvate of human liver pyruvate carboxylase were approximately 0.27 mM and 1.4 mM, respectively. These studies suggest an impairment in hepatic pyruvate metabolism in untreated phenylketonuric patients.

Simulation of Phenylketonuria in Rats by Extended p-Chlorophenylalanine Treatment

The brain damage which occurs in untreated phenylketonuria probably results from the high plasma phenylalanine concentration prevailing in this condition, as treatment with a low-phenylalanine diet mitigates the situation. The actual cause of the brain damage is not known, but investigations have been carried out with high phenylalanine loads to simulate phenylketonuria and so elucidate the nature of the ensuing abnormalities. Hypotheses involving phenylalanine or its toxic metabolites in the impairment of vital metabolic processes have been tested (Agrawal et al., 1970; Weber et al., 1970). The results obtained, however, under short-term acute loads probably do not reflect the situation prevailing in phenylketonuria; although the plasma phenylalanine reaches phenylketonuria values for short periods, tyrosine also rises appreciably. A number of workers have used p-chlorophenylalanine as an inhibitor of phenylalanine hydroxylase in vivo (Lipton et al., 1967), hoping to produce a more sustained hyperphenylalaninaemia with a near normal tyrosine concentration, i.e. a more valid model phenylketonuria. However, we found that a single pretreatment of young rats with p-chlorophenylalanine does not significantly alter the response to a phenylalanine load. We have extended this by carrying out phenylalanine tolerance tests on weanling rats after treatment with p-chlorophenylalanine for up to 20 days. Four groups of weanling rats were treated daily, by intraperitoneal injection, with (a) 300mg of p-chlorophenylalanine/kg body wt., or (6) 300mg ofp-chlorophenylalanine/kg plus 200mg of L-phenylalanine/kg, or (c) 200mg of L-phenylalanine/kg, or ( d ) an equal volume of saline. Animals from each group were killed on different days and their liver phenylalanine hydroxylase activity was determined by using ~-[U-'~C]phenylalanine as substrate, separating the product chromatographically and counting the radioactivity in both substrate and product (Bender & Coulson, 1972). Liver phenylalanine hydroxylase activity reached a minimum between 5 and 7 days in groups (a) and (6) (about 15% of control values), whereas group (c) did not differ from control value. After 7 days of p-chlorophenylalanine treatment the activity gradually increased towards the control values and cessation of treatment resulted in a rapid return to control values (2 days). Phenylalanine tolerance tests were performed by injecting rats with 500mg of L-phenylalanine/kg, and removing blood at 30min intervals. Plasma phenylalanine and pchlorophenylalanine were determined by the method of McCaman & Robins (1962), after separation on t.1.c. (this eliminates the mutual interference between phenylalanine and p-chlorophenylalanine). Tyrosine was determined by the method of Wong et al. (1964). Figs. l(a) and l(6) show phenylalanine and phenylalanine/tyrosine ratios after phenylalanine loads in control animals and in rats after 1 and 7 days ofp-chlorophenylalanine treatment. Both are of the same order as those observed in untreated phenylketonuria children. The basal phenylalanine concentration (before injection of the phenylalanine load) was subtracted from each value, after the peak, and the net elevated values plotted against time on semi-logarithmic paper. The slope of the line gives the first-order rate constant for the disappearance of phenylalanine load ( k ) (Woolf et al., 1967). Fig. 2 shows phenylalanine tolerance curves for animals treated with p-chlorophenylalanine for different periods and for controls. Phenylalanine loads produce a sustained hyperphenylalaninaemia over several hours in the 5-7-day-treated animals. Further, there is a good correlation (r = 0.89; P<O.OOl) between k and the liver phenylalanine hydroxylase activity. This supports the view that the main metabolic pathway of phenylalanine in these animals is by liver hydroxylation to tyrosine. From these studies we concluded that extended treatment withp-chlorophenylalanine over 5-7 days causes a decrease in liver phenylalanine hydroxylase activity which consequently lowers phenylalanine tolerance so that loading doses of phenylalanine produce a simulated phenylketonuria, over a period of several hours.

A Relationship between the Granulocyte Phenylalanine Content and the Degree of Disability in Phenylketonuria

A method has been developed to assess clinically the degree of neuropsychiatric damage in phenylketonuria, which takes account of factors other than the intelligence quotient. It can be applied both to severely retarded patients in whom the intelligence quotient is a poor discriminant, and to those whose intelligence can be measured accurately. The numerical value obtained is termed the ‘ability score’. Plasma and intracellular (granulocyte) levels of phenylalanine and the ability scores were measured in 42 cases of phenylketonuria. The ability score correlated significantly with the granulocyte phenylalanine content, but not with the plasma phenylalanine concentration. We suggest that our findings support the concept that high intracellular phenylalanine levels may be a factor in causing brain damage in phenylketonuria. The granulocyte phenylalanine content may be helpful prognostically, and in the control of dietary therapy; and studying the granulocyte as well as the plasma levels may help to clarify the relationship between variant forms of classical phenylketonuria and persistent hyperphenylalaninaemia.

Maternal hyperphenylalaninaemia in the normal and phenylketonuric mother and its influence on maternal plasma and fetal fluid amino acid concentrations.

SummaryAn oral load of L‐phenylalanine (150 mg. per kg. body weight) was given to five women three hours before delivery by hysterotomy of their 16–18 week conceptuses. A sixth woman was delivered of a 16‐week conceptus after two hours continuous intravenous infusion of L‐phenylalanine (75 mg. per kg. body weight per hour).Concentrations of free amino acids were measured in maternal venous plasma, umbilical arterial plasma, fetal urine, amniotic fluid and cerebrospinal fluid. Free amino acid concentrations were also measured in maternal and umbilical vein plasma and amniotic fluid of a treated phenylketonuric mother delivered spontaneously at 36 weeks gestation of a non‐phenylketonuric infant.Increased concentrations of phenylalanine were measured in all maternal plasma samples obtained one, two and three hours after ingestion of L‐phenylalanine and during intravenous infusion. There were correspondingly high concentrations of phenylalanine in fetal plasma, fetal urine, amniotic fluid and fetal cerebrospinal fluid at delivery.Tyrosine concentrations were also increased in maternal and fetal plasma, fetal urine, amniotic fluid and fetal cerebrospinal fluid of mothers given phenylalanine. Although maternal plasma, fetal plasma and amniotic fluid from the phenylketonuric mother contained increased concentrations of phenylalanine, there was no increase in the tyrosine concentrations in these fluids.The mean umbilical artery plasma‐maternal vein plasma ratios for phenylalanine was 2·35 in normal 15‐ to 20‐week pregnancies, 2·26 three hours after oral L‐phenylalanine, 1·29 after intravenous infusion of L‐phenylalanine and 1·45 in the phenylketonuric pregnancy. Corresponding ratios for tyrosine were 2·52, 2·30, 2·15 and 2·90. In the acute tolerance studies the mean fetal plasma phenylalanine‐tyrosine ratio was 2·47 and in the phenylketonuric fetus 8·16.There were reductions in maternal plasma concentrations of most amino acids three hours after the oral phenylalanine and these reductions were significant for methionine, lysine, histidine and arginine. These changes were not reflected in the amniotic fluid but there were significant reductions in fetal plasma threonine and arginine and in fetal urine threonine and taurine. Methionine concentrations were increased in the maternal plasma, fetal plasma and amniotic fluid of the treated phenylketonuric mother.The human placenta does not “protect” the fetus from increased maternal plasma concentrations of phenylalanine in the normal or phenylketonuric mother.

Hereditary tyrosinemia: Metabolic studies in a patient with partial p-hydroxyphenylpyruvate hydroxylase activity

A child with hereditary tyrosinemia who was also heterozygous for hyperphenylalaninemiais described. The patient had an extensive renal tubular dysfunction and an increased urinary excretion of σ-aminolevulinic acid and catecholamines. A moderate restriction of the tyrosine and phenylalanine intake caused a rapid improvement of the renal tubular function and reduced the excretion of σ-aminolevulinic acid and catecholamines. The disappearance rate of phenylalanine from plasma was normal; during dietary treatment the plasma phenylalanine concentration was much below the normal range. The enzyme activity of p-hydroxyphenylpyruvate hydroxylase (E.C. 1.14.2.2) in liver was decreased.