Plasma Phe Concentrations Research Articles

L-Phenylalanine concentration in blood of phenylketonuria patients: a modified enzyme colorimetric assay compared with amino acid analysis, tandem mass spectrometry, and HPLC methods

Phenylketonuria (PKU) is an autosomal recessive disorder caused by an impaired conversion of L-phenylalanine (Phe) to L-tyrosine, typically resulting from a deficiency in activity of a hepatic and renal enzyme L-phenylalanine hydroxylase. The disease is characterized by an increased concentration of Phe and its metabolites in body fluids. A modified assay based on an enzymatic-colorimetric methodology was developed for measuring blood Phe levels in PKU patients; this method is designed for use with undeproteinized samples and avoids the use of solvents or amphiphilic agents. Thus, the method could be suitable for incorporation into a simple home-monitoring device. We report here on a comparison of blood Phe concentrations in PKU patients measured in undeproteinized plasma using this enzyme colorimetric assay (ECA), with values determined by amino acid analysis (AAA) of deproteinized samples, and HPLC and tandem mass spectrometry (MS/MS) analyses of dried blood spot (DBS) eluates. Pearson correlation coefficients of 0.951, 0.976 and 0.988 were obtained when AAA-measured Phe concentrations were compared with the ECA-, HPLC- or MS/MS-measured values, respectively. A Bland-Altman analysis revealed that mean Phe concentrations determined using AAA were on average 65 μmol/L lower than values measured by our ECA. These results may be the result of minimizing the manipulations performed on the patient sample compared with AAA, HPLC, and MS/MS methods, which involve plasma deproteinization or DBS elution and derivatization. The results reported here confirm that Phe concentrations determined by our ECA method are comparable to those determined by other widely used methods for a broad range of plasma Phe concentrations.

Unresponsiveness to tetrahydrobiopterin of phenylalanine hydroxylase deficiency

Conflicting results have been reported concerning the efficacy of tetrahydrobiopterin (BH4), the cofactor of phenylalanine hydroxylase, for reducing phenylalanine (Phe) concentration in phenylketonuria (PKU). We aimed to test quantitatively the effects of BH4 in PKU patients. Seven fully characterized patients were selected among a population of 130 PKU subjects as harboring PKU mutations predicted as BH4 responsive and previously considered responsive to a cofactor challenge. They received a simple Phe (100 mg/kg) and 2 combined Phe (100 mg/kg) and BH4 (20 mg/kg) oral loading tests. Cofactor was administered either before or after the amino acid. The concentrations of Phe, tyrosine (Tyr), and biopterin were measured over 24 hours after loading. The comparative analysis of the loading tests showed that in all patients plasma Phe concentrations peaked within 3 hours, and fell within 24 hours by about 50% in benign, 20% in mild, and 15% in severe phenylalanine hydroxylase deficiency regardless of BH4 administration. A consistent or moderate increase of plasma Tyr, again independent of the cofactor challenge, was observed only in the less severe forms of PAH deficiency. Mean blood biopterin concentration increased 6 times after simple Phe and 34 to 39 times after combined loading tests. The administration of BH4 does not alter Phe and Tyr metabolism in PKU patients. The clearance of plasma Phe after oral loading and, as well as Tyr production, is not related to cofactor challenge but to patient's phenotype. The assessment of BH4 responsiveness by the methods so far used is not reliable, and the occurrence of BH4-responsive forms of PKU still has to be definitely proven.

Phenylalanine hydroxylase activity and expression in chicks subjected to phenylalanine imbalance or phenylalanine toxicity

Experiments were performed to investigate the activity of hepatic Phe hydroxylase (PAH) and plasma amino acid concentrations under conditions of Phe imbalance or toxicity in chicks fed on experimental diets from 7 to 14 or 16 d of age. In experiment 1, Phe imbalance was created by adding 10% of a mixture of indispensable amino acids lacking Phe (IAA - Phe) to a basal diet containing 0.46% Phe. The activity of PAH was not significantly affected by the imbalance. Correcting the imbalance by adding 1.12% Phe to the diet prevented the growth impairment and increased the activity of PAH. In experiment 2, growth was reduced by the addition of excess (2%) Phe to the basal diet. Correcting the excess by adding the IAA - Phe to the diet prevented the growth reduction. The activity of PAH was not significantly affected by 2% Phe, but it increased in chicks fed the corrected diet. The levels of PAH mRNA were not affected by the dietary treatments. A factorial arrangement of treatments with 3 dietary levels of Phe (0.46, 1.58, and 2.46%) with or without the IAA - Phe was used in experiment 3. The effects on growth were similar to those of the same treatments in experiments 1 and 2. The addition of Phe significantly increased hepatic PAH activity, but there was no detectable main effect of the IAA - Phe and no interaction. Plasma Phe concentration was increased by dietary Phe and decreased by the IAA - Phe mixture. We conclude that hepatic PAH activity in chicks variably increases in response to Phe or a 10% dietary supplement of indispensable amino acids including Phe but does not increase in response to IAA - Phe when the amino acids are added to a diet that is marginally adequate in Phe. The increased activity does not involve changes in PAH mRNA. The effects of IAA - Phe on plasma Phe concentrations appear to be independent of hepatic PAH activity as measured in vitro.

Safety and efficacy of 22 weeks of treatment with sapropterin dihydrochloride in patients with phenylketonuria

Phenylketonuria (PKU) is an inherited metabolic disease characterized by phenylalanine (Phe) accumulation, which can lead to neurocognitive and neuromotor impairment. Sapropterin dihydrochloride, an FDA-approved synthetic formulation of tetrahydrobiopterin (6R-BH4, herein referred to as sapropterin) is effective in reducing plasma Phe concentrations in patients with hyperphenylalaninemia due to tetrahydrobiopterin (BH4)-responsive PKU, offering potential for improved metabolic control. Eighty patients, > or =8 years old, who had participated in a 6-week, randomized, placebo-controlled study of sapropterin, were enrolled in this 22-week, multicenter, open-label extension study comprising a 6-week forced dose-titration phase (5, 20, and 10 mg/kg/day of study drug consecutively for 2 weeks each), a 4-week dose-analysis phase (10 mg/kg/day), and a 12-week fixed-dose phase (patients received doses of 5, 10, or 20 mg/kg/day based on their plasma Phe concentrations during the dose titration). Dose-dependent reductions in plasma Phe concentrations were observed in the forced dose-titration phase. Mean (SD) plasma Phe concentration decreased from 844.0 (398.0) micromol/L (week 0) to 645.2 (393.4) micromol/L (week 10); the mean was maintained at this level during the study's final 12 weeks (652.2 [382.5] micromol/L at week 22). Sixty-eight (85%) patients had at least one adverse event (AE). All AEs, except one, were mild or moderate in severity. Neither the severe AE nor any of the three serious AEs was considered related to sapropterin. No AE led to treatment discontinuation. Sapropterin is effective in reducing plasma Phe concentrations in a dose-dependent manner and is well tolerated at doses of 5-20 mg/kg/day over 22 weeks in BH4-responsive patients with PKU.

Increased spontaneous osteoclastogenesis from peripheral blood mononuclear cells in phenylketonuria

Phenylketonuria (PKU) is commonly complicated by a progressive bone impairment of uncertain aetiology. The therapeutic phenylalanine (Phe)-restricted diet and the possible noxious effects of high plasma Phe concentrations on bone have previously been suggested as possible determinant factors. Since osteoclasts are involved in bone reabsorption, they could play a role in determining bone damage in PKU. The reported increased excretion of bone resorption markers in PKU patients is consistent with this hypothesis. Although different diseases characterized by bone loss have been related to increased spontaneous osteoclastogenesis from peripheral blood mononuclear cells (PBMCs), to date there is no evidence of increased osteoclast formation in PKU. In this study, we compared the spontaneous osteoclastogenesis from PBMCs in 20 patients affected by PKU with that observed in age- and sex-matched healthy subjects. Phenylketonuric patients showed the number of osteoclasts to be almost double that observed in controls (159.9 ± 79.5 and 87.8 ± 44.7, respectively; p = 0.001). Moreover, a strict direct correlation between the spontaneous osteoclastogenesis in PKU patients and the mean blood Phe concentrations in the preceding year was observed (r = 0.576; p = 0.010). An imbalance between bone formation and bone resorption might explain, at least in part, the pathogenesis of bone loss in this disease. These findings could provide new insights into the biological mechanisms underlying bone damage in PKU.

Performance and nitrogen metabolism of calves fed conventionally or following an enhanced-growth feeding program during the preweaning period

Thirty-seven Holstein and seven crossbred female calves (16.1 ± 4.60 days, and an initial BW of 36.5 ± 3.19) were used to study the effects of conventional (CF) vs enhanced-growth feeding programs (EF) on performance, plasma amino acid (AA) concentrations, and rumen microbial development. After 1 week of adaptation to milk replacer (MR), the CF calves received 4 l/day of MR at 12.5% DM throughout the preweaning period, and the EF calves were offered MR at 18% DM: 6 l/day from 1 to 6 days, 8 l/day from 7 to 26 days, and 4 l/day from 27 days to weaning day (38 days). Calf starter and water were offered ad libitum throughout the study (87 days). Calves fed EF were heavier ( P < 0.05) than CF calves at the end of the study (111.7 vs 102.6 ± 1.72 kg, respectively). Until the 27 days, average daily gain (ADG) was greater ( P < 0.001) for EF than for CF calves (1.00 vs 0.49 ± 0.061 kg/day, respectively), but it was lower ( P < 0.001) from days 27 to 45 of the study (0.32 vs 0.71 ± 0.061 kg/day, respectively), coinciding with the days around weaning. Starter intake was greater ( P < 0.001) for CF than for EF calves during the first 45 days of the study (0.60 vs 0.27 ± 0.061 kg/day, respectively) but similar afterwards. As a consequence, EF treatment may have delayed rumen function as suggested by total daily purine derivatives urinary excretion (49.52 vs 33.27 ± 3.095 mmol/day, in CF and EF calves, respectively). Linear regression analyses showed a positive relationship between plasma Trp and Phe concentrations and ADG, and a negative relationship between these two AA and plasma urea concentrations, suggesting that Trp and Phe could be limiting growth in calves fed conventional feeding programs.

207. Long-Term Therapy for PKU in Mice by rAAV Pseudotype 8 Gene Transfer to Liver

Phenylketonuria (PKU) is caused by an autosomal recessive deficiency of the hepatic phenylalanine hydroxylase (PAH) leading to high serum phenylalanine and irreversible damage of the brain. We recently reported on the use of recombinant AAV2 to deliver the PAH gene to the liver of PKU mice (C57Bl/6-Pah-enu2), which is an exceptionally faithful model for the human disease (Ref: Ding, Georgiev and Thony, Gene Ther, advanced online publication Dec 2005). In this study we used an AAV2/8 hybrid vector expressing the murine PAH-cDNA under the control of the composite CMV enhancer-chicken beta-actin promoter and which was pseudotyped with capsids from serotype 8 and delivered to the liver of PKU mice via single injections of portal vein with 5x10e12 vg per mouse or tail vein with 2x10e13 vg per mouse. Untreated PKU mice exhibit normal expression of mutant Pah-mRNA and PAH protein, but no detectable enzyme activity. They consequently had Phe levels between 1500 to 2500 micromol/l (25 to 42 mg/dl), and we (and others) have noticed that Phe concentrations in PKU mice were slightly higher for females (1866 micromol/l) than males (1695 micromol/l) rendering females putatively more resistive to PKU treatment. Two weeks post hepatic or systemic delivery of AAV2/8 a complete phenotypic correction was observed in PKU mice with blood Phe levels decreasing from high to normal values (<120 micromol/l or 2mg/dl). The therapeutic levels of plasma Phe concentrations (360 micromol/l or 6 mg/dl), as determined approximately every four weeks, were observed for a period of at least 7-10 months. Here we report on the follow-up of this gene therapy approach during a period of over a year where animals were successively euthanized for analyzing their hepatic viral load and PAH expression, and only two females were kept in each study until the end of the experiment. For tail vein injection, one female started to rise Phe concentrations after 35 weeks, and at 56 weeks the two females had Phe blood levels of 1203 and 1453 micromol/l. For portal vein infusion, mice remained at therapeutic levels at least until week 61, and three weeks later the remaining two females had Phe values of 284 and 517 micromol/l. Thus far we have collected data on viral titers after 8 and 46 weeks for portal vein injection, and found a decrease from 3400 to below 1000 viral genome copies per diploid hepatocytes genome. PAH expression 8 weeks after portal vein injection was found to be moderate with a 32% increase of Pah-mRNA and a 2.2-fold increase of PAH protein, and PAH enzyme activity comparable to wild-type mice. Expression analysis of PAH and parameter important for the enzymatic activity at different treatment intervals are under way. In conclusion, a 4-fold lower concentration of the AAV2/8 vector directly injected into the portal vein as compared to tail vein resulted in a more efficient long-term correction of serum Phe levels. Furthermore, understanding the mechanism for the re-rise of blood Phe levels might help to improve a gene therapy protocol to eventually perform complete and persistent correction of PKU by hepatic gene transfer with AAV vectors.

Platelet serotonin concentrations in PKU patients under dietary control and tetrahydrobiopterin treatment

Tetrahydrobiopterin (BH4) supplementation has been applied in PKU treatment, resulting in successful control of blood phenylalanine (Phe) concentrations. We evaluated serotonin status in PKU patients under classical dietary treatment (n = 40) and in a group of 11 PKU patients under BH4 treatment, both during a 6-month period. Platelet serotonin values were significantly lower in PKU patients under dietary treatment when compared with controls. A negative correlation was observed between plasma Phe and platelet serotonin concentrations (r = -0.367, p = 0.017) in PKU patients. Platelet serotonin concentration increased significantly after both 1 and 6 months of BH4 therapy when compared with baseline conditions (Wilcoxon test: p = 0.013 and p = 0.021, respectively), while no differences were observed when comparing plasma Phe concentrations at the different points. Our results indicate that PKU patients under classical treatment have decreased platelet serotonin concentrations, probably owing to continued high Phe values, while BH4 supplementation restored platelet serotonin values.

Clinical and nutritional evaluation of phenylketonuric patients on tetrahydrobiopterin monotherapy

The clinical, nutritional, and neuropsychological data of 11 mild/moderate PKU patients after one year of treatment with BH4 are evaluated. BH4 monotherapy was introduced at 5 mg/kg/day in 14 PKU patients. In 11/14 patients, Phe tolerance increased significantly from 356 ± 172 to 1546 ± 192 mg/day ( p = 0.004), and special PKU formula was gradually reduced until complete removal. In them, mean plasma Phe concentrations remained below 360 μmol/L at 5 mg BH4/kg/day (7 mg/kg/day in one patient). BH4 therapy was stopped in three patients (V388M/P362T and R243Q/IVS10-11G > A genotypes) because it was not possible to improve Phe tolerance and to remove formula intake. Serum micronutrients were not significantly different at the start of treatment and at one year follow-up, except for selenium, which increased significantly after one year of therapy ( p = 0.017). Anthropometric, and nutritional measurements were within the age- and sex-specific percentiles for a healthy population after one year therapy. Neuropsychological follow-up indicated that intelligence scores persisted within normal limits. In terms of patients’ genotype, we confirmed that the P275S mutation combined with R408W was associated with long-term BH4 responsiveness, while the combination of P362T/V388M, and R243Q/IVS10-11G > A resulted in poor metabolic control in long-term BH4 therapy. In summary, our data confirm that BH4 is a safe, and effective therapy in a selected group of mild, and moderate PKU patients who respond to the BH4 loading test. Low doses of BH4 in monotherapy permit withdrawal of the special formula and guarantee a good clinical and nutritional outcome with no adverse side effects in PKU patients.

Fractional Protein Synthesis Rates Measured by an Intraperitoneal Injection of a Flooding Dose of L-[ring-2H5]Phenylalanine in Pigs

Our objectives were to examine the effect of an i.p. injection of a flooding dose of l-phenylalanine (Phe) containing l-[ring-(2)H(5)]Phe on time courses of physiologic responses, the tracer Phe enrichments, and fractional protein synthesis rates (FSR) in plasma, visceral organs, and muscles. In a randomized complete block design, 5 blocks of 5 littermate piglets were weaned at 16 d of age and injected i.p. with a flooding dose of l-Phe (1.5 mmol/kg body weight) on d 8 postweaning under fed conditions. Tissues were collected at 15, 30, 45, 60, and 75 min postinjection. Plasma glucose concentration increased (cubic effect, P < 0.05) from 4.8 preinjection to 5.8 mmol/L 15 min postinjection and returned to preinjection levels thereafter. Plasma insulin concentration did not change (P > 0.05) over time. Plasma Phe concentration increased logarithmically (P < 0.05) from 85 to 711 micromol/L and reached 95% of the maximum concentration 48 min postinjection, but no changes (P > 0.05) in tissue contents of other free amino acids were observed. The Phe free pools in plasma, visceral organs, and muscles were evenly enriched (32.3 +/- 1.4 mol%) with l-[(2)H(5)]Phe 15 min after the i.p. injection. The FSR in visceral organs did not change (P > 0.05), whereas plasma and muscle protein FSR decreased (P < 0.05) over time. We conclude that the i.p. injected tracer Phe rapidly distributed into plasma and intra- and extracellular spaces, and was effective for measuring FSR in visceral organs, but not in plasma and muscles of pigs.

Influence of knowledge of the disease on metabolic control in phenylketonuria.

In patients with phenylketonuria (PKU) knowledge of the disease and its treatment is not a major independent predictor for dietary compliance. PKU is an inborn error of amino-acid metabolism caused by a deficiency of phenylalanine hydroxylase (PAH), resulting in high plasma phenylalanine (Phe) concentrations and consequently severe mental retardation. Treatment comprises a life-long diet and PKU patients maintaining good metabolic control develop normally [3]. Poor dietary compliance is a regularly encountered problem. Increasing knowledge of the disease and its treatment may be a possibility to improve dietary compliance. Until now few studies have investigated this issue in PKU and the results are not conclusive [2, 4, 5, 6]. We therefore conducted a study to assess knowledge of the disease and its treatment in a large population of PKU patients and their parents and to determine the association between knowledge and the plasma Phe concentration as an indicator of dietary compliance in PKU. Parents of 161 PKU patients (aged 1 to 22 years) and 62 PKU patients (aged 12 to 22 years) completed a questionnaire to assess knowledge. The mean plasma Phe concentration during a 3-year period (1994 to 1996) was taken as an indicator of metabolic control. Table 1 shows the questions and the number of parents and patients correctly answering each separate question. We had expected that the greater part of the patients and their parents would correctly answer the questions. However, only 52% of the parents and 29% of the patients answered more than 11 of the 14 questions adequately. Linear regression showed that higher knowledge was associated with lower Phe concentrations, but this association was statistically significant only in the parent group (parents: b=)28, 95% CI)39/)17; patients: b=)20, 95% CI)42/2). After adjustment for confounders (pre-treatment Phe concentrations and dietary Phe tolerance, both as indicators for the severity of the enzyme deficiency, patient’s age, parental educational level and ethnicity) this association disappeared (parents: b=)0.4, 95% CI)8/ 7; patients: b=)1.5, 95% CI)10/7) and pre-treatment Phe concentrations, Phe tolerance and ethnicity were the strongest predictors for Phe concentration.

Elevated plasma phenylalanine concentrations may adversely affect bone status of phenylketonuric mice.

Children with phenylketonuric (PKU) are at risk for fractures. This study used a PKU murine model (PAH(enu-2)) to evaluate effects of moderate dietary protein restriction and elevated plasma phenylalanine concentration impact upon bone status. Fifty-four male weanling PKU and control mice were assigned to either an elemental phenylalanine (Phe)-restricted diet (treated) or Phe-unrestricted diet (untreated) with low or normal protein levels for 56 days. Untreated mice and control mice received equal amounts of dietary Phe; treated mice consumed prescribed dietary Phe to maintain plasma Phe concentrations between 120 and 480micromol/L. Plasma Phe, osteocalcin, and urine deoxypyridinoline (DPD)/creatinine were analysed at baseline and at days 28 and 56. Femur strength, bone mineral density (BMD) and bone mineral content (BMC) were analysed at day 56. Moderate protein restriction did not significantly affect bone status. Mean plasma Phe concentrations were significantly greater in untreated vs treated and control mice (p < 0.0001). Total body weight was significantly less in untreated vs control mice (p < 0.01). Mean femur weight was reduced in untreated mice vs both treated and control mice (p < 0.03). Untreated mice had smaller mean femur length than control mice (p < 0.002). Femur strength was greater in treated mice compared to control mice (p < 0.01) but not compared to untreated mice. No significant difference among groups was found in BMD and BMC. At day 56 there was a statistical trend (p < 0.056) towards higher urine DPD/creatinine excretion in untreated mice than in treated mice. Plasma Phe concentration was positively correlated with urine DPD/creatinine. These data suggested that hyperphenylalaninaemia may adversely affect bone status in PKU mice.

Effect of Storage on Phenylalanine and Tyrosine Measurements in Whole-Blood Samples

With an incidence of 1 in 6600 newborns, phenylketonuria (PKU) is among the most common inborn errors of metabolism. PKU is caused by a deficiency of hepatic phenylalanine hydroxylase (1). The increase in the blood Phe concentration leads to permanent structural damage of the central nervous system as a result of disturbed myelination and neurotransmitter deficiency (2). If plasma Phe concentrations are normalized by a low-protein diet with supplementation of essential amino acids before 3 weeks of age, irreversible mental retardation is prevented (2). Still, strict metabolic control is mandatory throughout childhood (2) and perhaps into adult life (3). This is achieved by regular measurement of blood Phe and Tyr concentrations. Tyr is monitored because Phe hydroxylase deficiency renders it an essential amino acid in PKU. Recommendations for the duration and intensity of dietary control are not uniform (2); likewise, there is some variation in the local practices of PKU monitoring. The current guidelines of the German “Arbeitsgemeinschaft fur Padiatrische Stoffwechselerkrankungen” (The German Working Group for Metabolic Diseases) set a target range for Phe concentrations of 40–250 μmol/L, at least until age 10 (≤900 μmol/L is acceptable during adolescence) (4). These recommendations were based on data from samples that were analyzed immediately after sampling (Udo Wendel, University Children’s Hospital, Dusseldorf, Germany, personal communication). Pregnant women with even mild hyperphenylalaninemia also require strict dietary control of Phe concentrations to prevent PKU-induced fetopathy (1). To ensure optimal metabolic control, patients are monitored in specialized clinics where dietary …

Cerebral protein synthesis in a genetic mouse model of phenylketonuria.

Local rates of cerebral protein synthesis (lCPS(leu)) were measured with the quantitative autoradiographic [1-(14)C]leucine method in a genetic mouse model (Pah(enu2)) of phenylketonuria. As in the human disease, Pah(enu2) mice have a mutation in the gene for phenylalanine hydroxylase. We compared adult homozygous (HMZ) and heterozygous (HTZ) Pah(enu2) mice with the background strain (BTBR). Arterial plasma concentrations of phenylalanine (Phe) were elevated in both HMZ and HTZ mutants by 21 times and 38%, respectively. In the total acid-soluble pool in brain concentrations of Phe were higher and other neutral amino acids lower in HMZ mice compared with either HTZ or BTBR mice indicating a partial saturation of the l-amino acid carrier at the blood brain barrier by the elevated plasma Phe concentrations. In a series of steady-state experiments, the contribution of leucine from the arterial plasma to the tRNA-bound pool in brain was found to be statistically significantly reduced in HMZ mice compared with the other groups, indicating that a greater fraction of leucine in the precursor pool for protein synthesis is derived from protein degradation. We found reductions in lCPS(leu) of about 20% throughout the brain in the HMZ mice compared with the other two groups, but no reductions in brain concentrations of tRNA-bound neutral amino acids. Our results in the mouse model suggest that in untreated phenylketonuria in adults, the partial saturation of the l-amino acid transporter at the blood-brain barrier may not underlie a reduction in cerebral protein synthesis.

Branched chain amino acids decrease tardive dyskinesia symptoms.

Prior studies had suggested (a) that a lessened ability to clear ingested forms of the large neutral amino acid (LNAA), phenylalanine (Phe), was associated with having tardive dyskinesia (TD), and (b) that greater availability of a group of LNAA, the branched chain amino acids (BCAA), concomitant with the lower availability of Phe to the brain are associated with a decrease in TD symptoms. The present study was then conducted to test whether increasing the daily intake of the BCAA would decrease the symptoms of TD. A 2-week trial of a BCAA medical food administered three times a day was conducted in nine men with long neuroleptic treatment histories. Frequency counts of TD movements were collected by videotape throughout the trial and these tapes were analyzed in blind random sequence for both patient and time for TD symptom level changes subsequent to completion of the trial. Plasma levels of the LNAA were also collected throughout the trial. A statistically significant decrease in the level of TD symptoms was observed for the sample. The symptom changes were also clinically significant in that six of the nine subjects had symptom decreases of at least 58%, with all subjects having a decrease of at least 38%. BCAA administration increased plasma BCAA concentrations and BCAA/LNAA ratios and decreased plasma Phe concentrations and the Phe/LNAA ratio. Analyses indicated a strong significant correlation between the percent increase in the plasma BCAA values at the first administration and the percent improvement in TD over the trial in eight of the nine subjects. The BCAA show promise as a treatment for TD. The decrease in TD symptoms seen in the trial may have been modulated by the BCAA treatment-induced increased availability of the BCAA and decreased availability of Phe to the brain.

Subclinical visual impairment in phenylketonuria. A neurophysiological study (VEP-P) with clinical, biochemical, and neuroradiological (MRI) correlations.

During detailed visual function testing, pattern-reversal visual evoked potentials (VEP), generated by different spatial frequencies (3 c/d, 1 c/d and 0.6 c/d) and visual contrasts (100% and 10%) were recorded in 21 adolescent and young adult phenylketonuric (PKU) patients (11 females and 10 males; mean age 14.8 years, range 9-22.8) on and off diet. In 14 of the 21 patients, disease had been detected at neonatal screening and in 7 later. Ten age-matched healthy subjects acted as controls. Recordings in more than 40% of eyes in the whole group and 30% of eyes in the screening subgroup showed a prolonged P100 latency. All visual pattern stimuli elicited a significantly longer P100 latency in PKU patients than in controls. VEP latencies to 3 c/d, 1 c/d and 1 c/d with 10% contrast--but not to 0.6 c/d--were longer in patients off diet than in patients on diet. No differences were found between VEP latencies in early- and later-detected subjects. To study the link between biochemical variables and VEP latencies, we envisaged either a linear relationship between recent exposure to phenylalanine (Phe) and VEP abnormalities or a threshold model considering phenylalanine (Phe) concentrations among the factors influencing VEP latencies. The correlation analysis detected an association between plasma Phe concentrations and abnormal VEP latencies, predicting that plasma Phe concentrations > 901 mumol/L would prolong VEP latencies to 1 c/d; concentrations > 879 mumol/L would prolong latencies to 3 c/d; and concentrations > 898 mumol/L would prolong latencies to 1 c/d with 10% contrast.

Does impaired growth of PKU patients correlate with the strictness of dietary treatment? National Dutch PKU Steering Committee.

To assess whether growth retardation in patients with phenylketonuria (PKU) is related to the strictness of their dietary treatment, the relationship between Z scores for height up to 3 y of age and different indices of dietary control in 103 early treated Dutch PKU patients was studied. As indices of dietary control, the mean phenylalanine (Phe) concentration, the frequency of plasma Phe concentrations < 200 and < 120 micromol/l, and the standard deviation of the individual plasma Phe concentrations were studied. These measures of the dietary control were divided into quartiles. The mean Z score of the studied patients showed a decrease of 0.18/y (SD 0.36). No statistically significant difference between any of the quartiles of the studied indices with growth retardation was found. None of the used indices of dietary control based on plasma Phe concentrations showed a relationship between different degrees of the strictness of dietary treatment with growth retardation in Dutch PKU patients.

Hyperglucagonemia stimulates phenylalanine oxidation in humans.

Glucagon stimulates in vitro liver phenylalanine (Phe) degradation, thus inducing net protein catabolism. Whether these effects occur also in vivo in humans is not known. Therefore, we studied the effects of physiological hyperglucagonemia on Phe rate of appearance (Ra), hydroxylation, and oxidation in seven normal volunteers during infusions of somatostatin with replacement doses of insulin and growth hormone. Steady-state Phe kinetics were evaluated using the L-[1-14C]Phe tracer both at the end of a 3-h basal glucagon replacement period (glucagon concentration: 212 +/- 115 ng/l) and after a 3-h hormone infusion at the rate of approximately 3 ng x kg-1 x min-1 (--> 654 +/- 280 ng/l). Hyperglucagonemia did not change plasma Phe concentration and Ra but increased Phe oxidation by approximately 30% (P < 0.01). Oxidation was also increased by approximately 24% (P < 0.01) using plasma [14C]tyrosine (Tyr) specific activity as a precursor pool. Phe hydroxylation to Tyr estimated by assuming a fixed ratio of Tyr to Phe Ra (0.73) did not change. Nonhydroxylated Phe disposal decreased by approximately 6% (P = 0.08). These data show that in humans in the postabsorptive state, hyperglucagonemia, with near maintenance of basal insulin and growth hormone concentrations, stimulates Phe oxidation but not Phe hydroxylation, suggesting a different regulation of these two Phe catabolic steps. Glucagon may also reduce Phe availability for protein synthesis.

Nutrition and reproductive outcome in maternal phenylketonuria.

Maternal weight gain and intake of selected nutrients were correlated with plasma phenylalanine (Phe) concentrations and reproductive outcomes (in 150 and 142 subjects respectively) in the Maternal PKU Collaborative Study. Daily protein intake was negatively correlated with plasma Phe concentration. Birth length, weight and head circumference of offspring were negatively influenced by the length of time required for the maternal plasma Phe to decline below 600 mumol/l (10 mg/dl) and positively influenced by weight gain of mother as a percentage of recommended weight gain. Birth weight and length were positively correlated with maternal protein and energy intakes. During the first trimester, intakes of fat, calcium, phosphorus, vitamin A and folate were significantly greater in women who had a good reproductive outcome than by women who had a poor outcome. In addition to plasma Phe control in maternal phenylketonuria (PKU), maternal weight gain and dietary intake of protein, energy and fat were correlated with outcome. Therefore, nutrient intake and maternal weight gain should be considered along with plasma Phe concentration when managing the therapy of a pregnant woman with PKU.

Neurotransmitter positron emission tomographic-studies in adults with phenylketonuria, a pilot study

Patients with phenylketonuria (PKU) may suffer from cognitive and neurological deficits which are related to reduced intracerebral concentrations of catecholamines. The function of phenylalanine (Phe) as an inhibitor of the uptake of the precursor amino acid tyrosine (Tyr) through the blood-brain barrier as well as an inhibitor of the expression of dopamine receptors in the brain is under investigation. Positron emission tomography (PET) is a method for quantitatively determining biochemical and physiological processes in vivo. In the current pilot study, L-[1-11C]-Tyr and 18F-fluoro-ethyl-spiperone (FESP) have been used. The metabolic pathway of carboxylic labelled Tyr is mainly incorporation into protein. From the measured tissue and plasma activity as a function of time in combination with a compartimental model the Protein Synthesis Rate (PSR) for Tyr can be calculated. FESP is a ligand which binds irreversibly to the dopamine D2-receptor and has also a low non specific binding, although affinity to the serotonin receptor has been described. The ratio of FESP concentration in striatum and in cerebellum is a measure of the receptor status in vivo. In patients with plasma Phe levels above the maximum therapeutic concentration (> 700 mumol/l) the PSR for Tyr was decreased as compared to controls and patients with plasma Phe concentrations within the therapeutic range, indicating a decreased availability of Tyr for neurotransmitter synthesis, and hence explaining the reduced cerebral concentration of catecholamines.