Heterozygotes For Phenylketonuria Research Articles

Phenylalanine and Tyrosine Metabolism Analysis in Heterozygotes for Phenylketonuria and in Healthy Individuals

Phenylketonuria (PKU) is an inherited metabolic disorder derived from a deficiency in the enzyme phenylalanine hydroxylase, which converts the amino acid phenylalanine (Phe) into tyrosine (Tyr). Here we aimed to examine the metabolism of Phe and Tyr in heterozygotes for PKU during fasting and after oral overload of Phe (25 mg/kg). Plasma concentration of Phe and Tyr and Phe2–Tyr ratio were determined under fasting condition or 30, 45, 60, and 90 minutes after Phe overload. The sample consisted of 50 participants: 23 heterozygotes for PKU (10 men and 13 women) and a control group of 27 healthy individuals (13 men and 14 women). The dosage of Phe at 45 and 90 minutes and the micromolar fraction of Phe2/Tyr after 90 minutes of overload efficiently differentiated PKU heterozygotes. The discriminant function revealed 86% of accuracy. In fact, 94.4% of heterozygotes for PKU were correctly classified.

Phenylalanine and tyrosine metabolism in phenylketonuria heterozygotes: influence of different phenylalanine hydroxylase mutations.

Phenylalanine and tyrosine metabolism in phenylketonuria heterozygotes: influence of different phenylalanine hydroxylase mutations.

Aberrant phenylalanine metabolism in phenylketonuria heterozygotes.

The wide variation in phenylalanine hydroxylating capacity observed among patients with phenylketonuria (PKU) is primarily due to allelic heterogeneity at the phenylalanine hydroxylase (PAH) locus. In this study, we examined phenylalanine metabolism after an oral phenylalanine load in 148 carriers of known PAH gene mutations. As a group, heterozygotes formed less tyrosine than normozygotes (p < 0.001), and there was a tendency that carriers of a severe PAH mutation formed less tyrosine than carriers of a mild mutation. Nevertheless, the interindividual variation was extensive, and we identified a group of individuals who formed no or very little tyrosine after the phenylalanine load. This tyrosine response was accompanied by a decreased ability to eliminate the phenylalanine test dose but did not correlate with the intrinsic severity of the mutant PAH allele. Examination of the entire coding region of the PAH gene revealed no additional sequence alterations in these subjects. Our data suggest that a subset of PKU heterozygotes have reduced phenylalanine hydroxylating capacity approaching or equalling the levels observed in genetic compounds with non-PKU mild hyperphenylalaninaemia (MHP). Awareness of this phenotypic overlap between PKU carriers and genetic compounds with two mutant alleles may be useful for clinicians and paediatricians involved in diagnosis and genetic counselling.

Evaluation of an aspartame loading test for the detection of heterozygotes for classical phenylketonuria.

Classical phenylketonuria (PKU) is an inborn error of metabolism of autosomal recessive inheritance characterized by the accumulation of phenylalanine (Phe) in tissues due to Phe-4-hydroxylase deficiency. Several methods have been developed for the detection of PKU heterozygotes based on the determination of plasma Phe and tyrosine (Tyr) levels, on the analysis of the Phe/Tyr and Phe2/Tyr ratios and on the use of discriminant functions. The objective of the present study was to test the value of loading with aspartame (a sweetener consisting of Phe, aspartate and methanol) for the identification of PKU carriers. The study was conducted on 22 obligate heterozygotes and 27 controls. Two blood samples were collected (under fasting conditions and 30 min after the loading) for fluorometric determination of Phe and Tyr. Phe, Phe/Tyr and Phe2/Tyr values were higher in heterozygotes, whereas Tyr was higher in controls in both situations investigated. Linear discriminant function was considered to be the best parameter for differentiation of the individuals in the two groups. Under the conditions employed in the present study, aspartame loading did not show any advantages in discriminating between PKU carriers and normal individuals when compared to the same analysis performed under fasting conditions.

Inherited metabolic diseases affecting the carrier.

The objective of this review is to draw attention to those inherited metabolic traits which are potentially harmful also for the carrier, and to outline preventive measures, at least for obligate heterozygotes, i.e. parents of homozygous children. Concerning carriers of food-dependent abnormalities, early vascular disease in homocystinuria, hyperammonaemic episodes in ornithine transcarbamylase deficiency, presenile cataracts in galactosaemia as well as galactokinase deficiency, spastic paraparesis in X-linked adrenoleukodystrophy, and HELLP syndrome in mothers of babies with long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency have to be mentioned. In the group of food-independent disorders, clinical features in carriers may be paraesthesias and corneal dystrophy in Fabry disease, lens clouding in Lowe syndrome, lung and/or liver diseases in alpha 1-antitrypsin deficiency, and renal stones in cystinuria type II and III. Finally, two monogenic carrier states are known which in pregnant individuals could possibly afflict the developing fetus, i.e. heterozygosity for galactosaemia and for phenylketonuria. Elevated levels of galactose-1-phosphate have been found in red blood cells of infants heterozygous for galactosaemia born to heterozygous mothers. Aspartame in very high doses is reported to increase blood phenylalanine levels in heterozygotes for phenylketonuria, thus being a risk for the fetus of a heterozygous mother. For some of these carrier states preventive measures can be recommended, e.g. restriction of lactose in parents and heterozygous grandparents of children with galactosaemia and galactokinase deficiency as well as transiently in infants heterozygous for galactosaemia, dietary supplementation with monounsaturated fatty acids in symptomatic carriers for X-linked adrenoleukodystrophy, avoidance of smoking and alcohol in heterozygotes for alpha 1-antitrypsin deficiency, avoidance of episodes of dehydration in heterozygotes for cystinuria, and restriction of aspartame in pregnant women.

Effect of high-protein meal plus aspartame ingestion on plasma phenylalanine concentrations in obligate heterozygotes for phenylketonuria

The effect of a protein-rich meal alone or in combination with 85 μmol/kg body weight aspartame (APM) on plasma phenylalanine and large neutral amino acids (LNAA) was evaluated in obligate heterozygotes for phenylketonuria (PKU) and normal subjects (controls). Thirteen PKU heterozygotes (seven women, six men) and 13 controls (five women, eight men) ingested a 12-noon meal providing approximately 303 μmol/kg Phe. In addition, 10 PKU heterozygotes (five women, five men) and 10 controls (five women, five men) ingested the same meal with 85 μmol/kg APM (providing 75 μmol/kg Phe). Plasma amino acids were analyzed at baseline (−4 and 0 hours) and at 1, 3, and 20 hours after the meal or meal plus APM. Compared with the meal alone, ingestion of the meal plus APM significantly increased plasma Phe concentrations in both controls and PKU heterozygotes. Mean plasma Phe values were higher for controls at 1 hour (95 ± 7 μmol/L) and for PKU heterozygotes at 3 hours (153 ± 21 μmol/L). After the addition of APM to the meal, the highest mean plasma Phe concentration was only slightly greater than the usual postprandial range for both controls and PKU heterozygotes. Ingestion of the meal did not increase the plasma Phe/LNAA ratio in either controls or PKU heterozygotes. Compared with baseline, the plasma Phe/LNAA ratio increased significantly 1 hour after combined ingestion of the meal plus APM in both groups ( P = .020 and P = .008, respectively); however, the ratios were well below the range of Phe/LNAA values in individuals with mild hyperphenylalaninemia, who are clinically normal and do not require a Phe-restricted diet. In conclusion, AMP in doses up to 10 times current consumption (90th percentile) resulted in plasma Phe concentrations well below values associated with known adverse effects.

Severity of mutation in the phenylalanine hydroxylase gene influences phenylalanine metabolism in phenylketonuria and hyperphenylalaninaemia heterozygotes.

We examined whether the degree of residual activity from the mutant phenylalanine hydroxylase (PAH) allele affected phenylalanine metabolism in heterozygotes for phenylketonuria (PKU) or non-PKU hyperphenylalaninaemia (HPA). Discriminant analysis was carried out to find the function of fasting plasma concentrations of phenylalanine (PHE) and tyrosine (TYR) that best separated carriers from non-carriers. This function (0.103TYR -0.214-PHECORR -4.499) was subsequently used as the dependent variable, with the in vitro activity of the expressed mutant PAH as the independent variable, in a regression analysis performed on heterozygotes for mutations that had been studied in a eukaryotic cell expression system. This analysis showed a significant correlation (r = 0.40, n = 140, p < 0.001), although there was a wide spread of values within each of the two major groups of carriers and a considerable overlap between the groups. We conclude that the severity of the mutation, as determined by in vitro expression analysis, in the mutant PAH gene is reflected in the biochemical phenotype of heterozygotes. This result emphasizes the relevance of the cell expression system used for establishing the relative severities of most mutations at the PAH locus. Differences in the activities from the carried mutant PAH allele on phenylalanine metabolism in heterozygotes are, however, small compared to the activity from the normal PAH allele and are easily obscured by other factors leading to inter- or intra-individual variation in phenylalanine metabolism. Fasting plasma concentrations of phenylalanine and tyrosine thus can not be used to predict the severity of the carried PAH mutation in individual PKU or HPA heterozygotes.

Improved identification of heterozygotes for phenylketonuria using blood neopterin and biopterin.

A novel approach that combines information provided by the metabolism of pteridines and that of phenylalanine has been applied to the detection of heterozygotes for phenylketonuria. Phenylalanine, tyrosine, biopterin and neopterin have been measured in serum from normal controls and heterozygotes for classical phenylketonuria, before and after a phenylalanine oral load. Significant differences in neopterin and biopterin mean values in fasting serum and in the mean increase of biopterin induced by the phenylalanine load were found between groups. Inclusion of pteridine data in the discriminant analysis significantly improved the resolution of the classical phenylalanine loading test for the detection of heterozygotes for phenylketonuria.

Measurement of Neopterin and Biopterin in Urine from Phenylketonuria Heterozygotes and Normal Controls

The effect of an oral load of phenylalanine (100 mg/kg body weight) on the levels of neopterin and biopterin in urine has been determined in 8 heterozygotes for classical phenylketonuria and 25 supposed normal controls. In basal conditions, neopterin and biopterin levels were significantly different between males and females. A significant increase in urinary biopterin was found two hours after the phenylalanine load, both in heterozygotes and in normal homozygotes. This increase was maintained at least until the fourth hour. Neopterin levels did not suffer any change during that period. Comparison of urinary pteridines from normal controls and phenylketonuria carriers showed that there were no significant differences between both groups neither before nor after the phenylalanine load. From these data we concluded that measurement of biopterin and neopterin in urine cannot help in the identification of heterozygotes for phenylketonuria.

MODERATE CHANGES IN PLASMA PHENYLALANINE CONCENTRATIONS AFFECT BRAIN ELECTRICAL DISCHARGE

We previously demonstrated that large changes in blood phenylalanine (phe) concentrations (500 - 2000 pM) decreased plasma L-DOPA, slowed brain electrical discharge and prolonged performance (Fed. Res. 20:112, 1986). In this study we ask whether moderately elevated plasma phe affects brain function. Six heterozygotes for phenylketonuria (PKU) and 2 normal controls were studied in a crossover, blinded protocol of 4, 2-week intervals. Volunteers ingested a constant diet of 40-50 mg/kg/day phe supplemented either with 100 mg/kg/day phe or placebo. On the last three days of each period the mean power frequency of three electroencephalograms (MPF) were measured. Plasma phe rose in both heterozygotes and controls on ingesting supplemental phe from a mean placebo phe concentration of 99 ± 20 to 140 ± 166 μM. The range of changes was from −200 to +400 μM. There was an inverse relationship between changes in plasma phe above 50 μM and changes in the MPF. By regression analysis 100 μM change in plasma phe produced an inverse change of 0.12 CPS in MPF. We conclude that plasma phe concentrations are raised by phe ingestion in both PKU heterozygotes and homozygous normal, and that alterations in brain electrical discharge are induced by intermediate changes in blood phe concentrations supporting a linear rather than threshold relationship to altered brain function.

Impaired phenylalanine-tyrosine conversion in patients with iron-deficiency anemia studied by a L-(2H5)phenylalanine-loading test

Ten patients with manifest iron deficiency and without documented relationship to phenylketonuria patients were orally loaded with 25 mg/kg of L-(2H5)phenylalanine. Before loading, the fasting phenylalanine-tyrosine plasma ratio was determined and after loading, the concentrations of labeled and nonlabeled phenylalanine and tyrosine were determined in five consecutive plasma samples. With respect to the fasting phenylalanine-tyrosine ratio and to the post-load ratios of labeled phenylalanine over labeled tyrosine, the iron-deficient patients showed data intermediate between those of normals and heterozygotes for phenylketonuria. Compared to a 100% in vivo activity of phenylalanine hydroxylase in normals and a circa 37% activity in heterozygotes for classic phenylketuria, iron-deficient patients with an average hemoglobin of 8.6 ± 1 g/dl showed an activity of circa 56%. After normalization of their iron status, four patients were subjected again to the L-(2H5)phenylalanine-loading test. For three of these individuals, test results shifted into the range of normal.

Metabolic conversion of l-[U- 14C]phenylalanine to respiratory 14CO 2 in healthy subjects, phenylketonuria heterozygotes and classic phenylketonurics

Ten healthy volunteers, 12 classic phenylketonuria (PKU) heterozygotes, and 5 classic phenylketonurics have been loaded orally with a mixture of 5 μCi of l-[U- 14C]phenylalanine plus 25 mg/kg of l-[ 2H 5]phenylalanine. For 3 h thereafter, carbon-14 activity in expired air and total carbon dioxide were measured continuously and the levels of l-phenylalanine and l-tyrosine in plasma were determined in six blood samples. After 3 h, 15.1 ± 2.1% of the applied dose of radioactivity was recovered in the expired air of the healthy subjects, compared to 10.1 ± 2.2% for PKU heterozygotes and 0.32 ± 0.18% for classic phenylketonurics. The integrated activity expired provides a discrimination between normals and PKU heterozygotes with a classification error of about 13% compared to an error of about 9% based on the fasting l-phenylalanine over l-tyrosine ratio. A combination of these two parameters in a two-dimensional discriminatory analysis reduces the classification error to < 1%. An intraindividual correlation between the absolute activity expired and the formation of l-[ 2H 4]tyrosine formed is shown, confirming that ring hydroxylation of l-phenylalanine to l-tyrosine is mandatory in the catabolism of l-phenylalanine to carbon dioxide.

Plasma phenylalanine, tyrosine, and tryptophan in schizophrenia.

Plasma phenylalanine, tyrosine, and tryptophan concentrations were measured in chronic schizophrenic patients, normal controls, and heterozygotes for phenylketonuria. Schizophrenic patients' plasma concentrations of these amino acids could not be distinguished from those of normal controls, either when fasting or following oral or intravenous (IV) phenylalanine challenge. No neuroleptic effect was observed. Plasma phenylalanine-tyrosine ratios following IV phenylalanine challenge could easily distinguish heterozygotes from schizophrenic and normal control subjects but could not distinguish schizophrenic subjects from normal control subjects. No overlap between heterozygotes' values and those of the schizophrenic and normal subjects was observed. These studies find no evidence of abnormal phenylalanine metabolism in schizophrenic persons. Phenylalanine challenge did not affect the abstraction or judgment capacities of the subjects.

Studies on the molecular defect in phenylketonuria and hyperphenylalaninaemia using antibodies against phenylalanine hydroxylase.

Monospecific antibodies against human phenylalanine hydroxylase were prepared. Liver extract of 10 patients with phenylketonuria (PKU) and of two patients with hyperphenylalaninaemia were examined for reactivity toward the antibodies in crossed immunoelectrophoresis. In all patients phenylalanine hydroxylase could be detected. In nine out of 10 patients with PKU the mutant enzyme did not differ from the wild type enzyme with respect to the electrophoretic mobility. In one case the mutant enzyme had a more negative charge. In the patients with hyperphenylalaninaemia the enzyme of one patient had normal electrophoretic mobility and the enzyme of the other differed from the normal enzyme. No difference could be shown in heterozygotes for PKU.

An enzymatic assay of plasma phenylalanine and tyrosine for the detection and management of phenylketonuria

An enzymatic method for the determination of plasma phenylalanine (Phe) and tyrosine (Tyr) in newborns and phenylketonurics is described. The method uses yeast phenylalanine ammonia-lyase to catalyze the conversion of l-Phe to transcinnamic acid and l-Tyr to p-coumaric acid, which are then determined by differential spectrophotometry at 290 and 315 nm. This method, requiring only one drop of blood, is simple, quantitative, and time efficient, and it has been adopted for the management of phenylketonuria (PKU). It can be used for monitoring plasma Phe and Tyr levels of PKU patients during dietary treatment, for diagnosis of classical PKU through the Phe-loading test, and for detection of PKU heterozygotes. A total of 350 plasma samples collected from 31 PKU patients over a 4 year period were analyzed and yielded results similar to those obtained by automated amino acid analysis. In an exploratory study, 1084 newborn infants were screened for PKU by our enzymatic method and the Guthrie test. In this group we detected 5 infants with Phe levels greater than 8 mg/dl plasma and 2 infants with Tyr levels greater than 8 mg/dl plasma, while the Guthrie test on the same subjects gave no positives.

Intracellular phenylalanine and tyrosine concentrations in 19 heterozygotes for phenylketonuria (PKU) and 26 normals. Do the higher values in heterozygotes explain their lowered intellectual level?

Intracellular phenylalanine and tyrosine was determined in lymphocytes of 10 heterozygotes (parents) for PKU and in 26 randomly collected apparently normal persons. In cells from the heterozygotes the concentrations of both phenylalanine and tyrosine were higher than in those from the normals, the difference being statistically highly significant. It is argued that this could be responsible for the slight, though statistically significant, intellectual inferiority of heterozygotes for PKU.

The high frequency of phenylketonuria in Ireland and Western Scotland.

Fewer than 5% of individuals with untreated typical ('classical') phenylketonuria (PKU) reproduce. Hence nearly two PKU genes are removed from the population pool per affected individual, which would be expected to lead to a decline in the incidence of the disease towards an equilibrium level maintained by mutation at between one in 60000 and one in 250000 live births (Woolf 1976). In Ireland and Western Scotland the incidence of PKU is about one in 6000 live births (Lindsay, 1970; Cahalane, 1976). The calculated rate of decline of the gene frequency makes it highly improbable (/9 < 0.00003 ~, where n = number of generations) that this high incidence results from chance (i.e. genetic drift) or the introduction of a fbw PKU genes into a population by, say, invaders or immigrants (Woolf, 1976). The known history of Ireland excludes the founder or bottleneck effect. Since mutation cannot account fbr the high PKU gene frequency, the sole remaining explanation is that the heterozygote must possess some advantage over the normal individual (Woolfet al., 1975); a 1.28% advantage (i.e. 1.28% more offspring) would maintain the present high gene frequency (Woolfand Goodwin, 1967). In a search for the nature of the heterozygote advantage, the obstetric-histories of 112 families with PKU children in Ireland and Western Scotland were compared with those of 863 matched control families. The average numbers of pregnancies were very nearly equal after correcting for incomplete ascertainment, an average of 3.09 pregnancies per PKU family and 3.1 pregnancies per control family (the uncorrected figure for PKU families would be 3.76). However in the PKU families 88.2% of pregnancies led to liveborn infants; in the control families there were 82.1% livebirths (both values corrected, Table 1). This difference is significant (p < 0.01). There appears to be some local hazard to fetal life against which maternal heterozygosity for PKU is protective. The only known biochemical or physiological difl);rence between PKU heterozygotes and normal individuals is a somewhat higher concentration ofphenylalanine in the blood and tissues in the former (Knox and Messinger, 1958), the fasting blood concentrations being 1.333_+0.23 and 0.818 _+ 0.19 mg/100 ml respectively (Woolf et al., 1967); this difference is a consequence of the smaller amount of phenylalanine hydroxylase in the liver of heterozygotes. We may hypothesize that the higher concentration of phenylalanine in the maternal circulation protects the fetus against some environmental hazard. It may be significant that Tocci and Beber (1973) found an association between congenital malformations in infants and relatively low blood phenylalanine concentrations in their mothers after a loading dose. The reduction of fetal mortality in PKU families constitutes a heterozygote advantage of magnitude 5.04°~, significantly greater than the 1.28% required for genetic equilibrium (p < 0.01). Hence the PKU gene frequency and the incidence of the disease may be rising in Ireland and Western Scotland; the rates of rise can be calculated from the equation in Table 2. I f the heterozygote advantage remains at 5.04%, the incidence will double to one in 3000 livebirths in from nine generations, say 240 years, i fPKU homozygotes are untreated, to seven generations, if all PKU homozygotes are treated and have the average number of offspring (Figure t). Halving the heterozygote advantage would extend the doubling period to about 20 generations; if the heterozygote advantage were reduced to 1.2% it would take about 90 generations (say 2500 years) to increase the

Influence of the phenylalanine dose on the aromatic acid excretion in phenylketonuric heterozygotes and controls after oral loading.

Heterozygotes for phenylketonuria and controls were given oral loads of 100 mg and 200 mg L-phenylalanine per kilogram body weight. The concentrations of urinary aromatic acids were determined by gas-chromatography after isolation by ion-exchange chromatography and ethylacetate extraction. On an intake of 100 mg L-phenylalanine per kilogram, controls and carriers of classical phenylketonuria excreted nearly the same amounts of aromatic acids (P greater than 0.05). However on an intake of 200 mg per kilogram L-phenylalanine they could be distinguished from one another (P less than 0.001).

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.

Heterozygote advantage for the phenylketonuria allele.

Mean weight at birth of unaffected (normal homozygous and PKU heterozygous) offspring of parents heterozygous for the phenylketonuria (PKU) allele averages significantly above that of Norwegian neonates, with no significant difference in mean age of mothers or in mean parity. It approaches the optimal birthweight--that which confers the minimum overall mortality in the pre-, peri-, and postnatal periods. This near-optimal birthweight together with the possibly higher effective fertility observed in PKU heterozygous couples (at least in those who married before 1940), has apparently more than outweighed the disadvantages of the allele in PKU homozygous offspring as shown, for example, in an excessive number of pre- and perinatal deaths among the total offspring of PKU heterozygotes, to say nothing of the PKU survivors who, often, used to die young. The two effects--fertility and viability--apparently both contribute in the same direction, to give a biological fitness in excess of 1 for the heterozygote. This heterozygote advantage presumably explains the presence of the allele at frequencies above those to be expected from the simple replacement of a homozygously-lethal allele by mutation alone.