Blood—Brain Barrier Phenylalanine Transport and Individual Vulnerability in Phenylketonuria

Abstract

In vivo nuclear magnetic resonance spectroscopy can be used to measure intracerebral phenylalanine (Phe) concentrations in patients with phenylketonuria (PKU). Stationary levels, obtained under free nutrition, as well as time courses after an oral Phe load (100 mg/kg) were investigated in 11 PKU patients and were correlated with the individual clinical outcome. At blood levels around 1.2 mmol/L, brain Phe was 0.41 to 0.73 mmol/L in clinically “typical” patients, but less than 0.15 mmol/L in three untreated, normally intelligent, adult women. Kinetic investigations revealed higher transport Michaelis constants and lower ratios of the brain influx and consumption rates in these women than in the “typical” control patients (K_t,app = 0.45 to 1.10 mmol/L versus 0.10 mmol/L; T_max/ν_met = 2.55 to 3.19 versus 7.8 to 14.0). Such variations seem to be major causative factors for the individual vulnerability to PKU.

Phenylketonuria (PKU), caused by a deficiency of the liver enzyme phenylalanine hydroxylase, which converts phenylalanine (Phe) to tyrosine (Tyr), is the most frequent inborn error of amino acid metabolism with an incidence of 1:8,000 in whites. In untreated classic PKU, this block in the hepatic catabolism of Phe leads to elevated serum levels greater than 1.2 mmol/L (normal value: less than 0.1 mmol/L) with catastrophic consequences for the developing brain, usually resulting in severe mental and psychomotoric retardation. If patients are subjected to a diet strictly reduced in Phe soon after birth their neurologic and intellectual development is, however, close to normal. Although introduced more than three decades ago, duration and strictness of the diet are still under discussion. Until recently, the policy was to relax the diet after 10 years of age, but current recommendations suggest maintaining the strict diet as long as possible (Medical Research Council Working Party on Phenylketonuria, 1993).

An individual vulnerability of patients to elevated blood Phe is well known. Several studies demonstrated different degrees of intellectual and neuropsychological deficits, cerebral white matter changes, and EEG abnormalities in patients with PKU who had similar metabolic control (Bick et al., 1993; Pietz et al., 1993; Ris et al., 1994). Single case reports described untreated patients with classic PKU and normal intelligence (Primrose, 1983). The pathogenesis of different clinical outcome in spite of comparable blood Phe levels, [Phe]_blood, is still unclear. One possible explanation could be interindividual variations in brain concentrations of Phe, which crosses the blood—brain barrier (BBB) slowly, mediated by a saturable transport system.

Since the introduction of newborn screening programs in the mid 1960s, an enlarging number of patients has assembled for whom there is a need to optimize therapy. Dietary compliance is monitored measuring [Phe]_blood by amino acid analysis. Up to now, it had been impossible to give individual diet recommendations. However, this would be most desirable because the burdensome diet is associated with an elevated risk of psychosocial maladjustment. Using an animal model of hyperphenylalaninemia, Avison et al. (1990) were able to show that Phe is detectable by ¹H nuclear magnetic resonance (NMR) spectroscopy in the brain in vivo, providing intracerebral Phe concentrations, [Phe]_brain, which closely correlated with postmortem data. In the meantime, several human studies demonstrated that this technique offers a new approach for quantifying [Phe]_brain noninvasively in PKU patients (Kreis et al., 1995; Möller et al., 1995, 1997; Novotny et al., 1995; Pietz et al., 1995, Ullrich et al., 1994).

During the course of a maternal PKU study, we found three untreated women with classic PKU but normal or nearly normal intelligence. Such highly uncommon cases, which seem to be “protected” without adhering to any diet, are ideally suited for an investigation of the relation between brain Phe and individual vulnerability in PKU. We therefore performed a series of stationary and dynamic NMR experiments to examine the blood—brain correlation of Phe concentrations and Phe transport kinetics at the BBB. For comparison, data from two groups of “typical” PKU patients were additionally included in our study.

METHODS

Patient characteristics

Eleven patients were investigated. Genotypes as well as extensive blood Phe testing during the years before the examination indicated that all patients had classic PKU. None of them had any history of other factors disturbing brain development. Clinical data for all individuals are summarized in Table 1. Intelligence quotient (IQ) was tested by the German version of the Wechsler Intelligence Scale (Tewes, 1983; Wechsler, 1991). Patients of group 1 had never received any dietary treatment and were still untreated at the time of the study. However, they were almost unaffected clinically and reached normal or nearly normal intelligence scores. Patients of group 2a were untreated in early infancy and were retarded. All subjects of these groups were either born before a screening program had been launched (patients 1–4) or came from a country without systematic newborn screening. Group 2b consisted of early treated adults who had stopped the diet 7 to 20 years ago. Only patient 11 reached a good dietary control during the first 10 years of life. Intelligence quotients were within or close to the normal range in this group. Changes with T₂-weighted magnetic resonance imaging (MRI) were mildest in the untreated, “atypical” group 1. Patients of groups 2a and 2b had abnormalities of cerebral white matter to a variable extent depending on their current diet status. In all individuals, brain NMR spectroscopy yielded normal signal intensities of all routinely observed metabolites, including N-acetyl-l-aspartate, total creatine, total choline, and myo-inositol. In blood, all amino acids other than Phe were within the normal range. Informed written consent was obtained in all cases after the nature and possible consequences of the study were fully explained.

TABLE 1.

Clinical data of patients with phenylketonuria and stationary phenylalanine levels

					Long-term mean [Phe]blood (mmol/L)
Patient	Gender	Age	Genotype	Start of diet*	<10 y	≥10y	IQ	MRI†	Actual [Phe]blood (mmol/L)	Actual [Phe]brain (mmol/L)
				Group I (untreated, “atypical” patients)-
1	F	33	R408W/R261Q	No diet	—	—	100	Ø/+	1.21	<0.15‡
2	F	31	R261Q/F39L	No diet	—	—	105	+	1.20	<0.15‡
3	F	37	R408W/A104D	No diet	—	—	75	+	1.04	<0.15‡
				Group 2a (untreated in early infancy)
4	F	34	IVS12nt1g > a/R261Q	No diet	—	1.25	58	+++	1.21	0.64
5	F	15	R408W/P281L	11 y	—	>1.20§0.75∥	60	+	1.34	0.59
6	M	11	R408W/P281L	7y	>1.20§0.36∥	0.84	50	+	1.32	0.62
7	F	6	R408W/R408W	485 d	>1.20§0.33§	—	retarded¶	+++	1.26	0.54
				Group 2b (diet until an age of 10 years)
8	M	30	R408W/R408W	12 d	0.88	1.42	77	+++	1.25	0.739
9	F	17	R408W/R408W	14 d	0.81	1.16	90	++	1.38	0.52
10	M	25	IVS12nt1g > a/R261Q	32 d	0.68	1.17	85	++	1.16	0.41
11	M	23	R408W/I12	32 d	0.38	1.32	109	++	1.29	0.71

IQ, intelligence quotient; MRI, magnetic resonance imaging; Phe, phenylalanine.

*

d, day of life; y, year of life.

†

Ø, no changes in cerebral white matter; +, mild changes, confined to parieto-occipital region; ++, moderate changes, extending frontally; +++, severe changes, subcortical white matter affected (Bick et al., 1993).

‡

[Phe]_brain at or below limit of detection.

§

Minimal four blood Phe checks before the beginning of the diet.

∥

On diet.

¶

IQ not obtainable because of severe psychomotoric retardation.

Nuclear magnetic resonance spectroscopy

A 1.5 T whole-body NMR imager (MAGNETOM 63 SP, Siemens, Erlangen, Germany) and a standard head coil were used to record localized proton spectra applying the stimulated echo technique (Frahm et al., 1990). All spectra were recorded under identical conditions (echo time 20 ms, repetition time 1.6 seconds, 512 acquisitions) and from similar volumes-of-interest (36 cm³) in parieto-occipital periventricular brain with predominantly white matter. Intracerebral Phe concentrations were determined from difference spectroscopy using spectra from healthy volunteers as a baseline reference as described in detail elsewhere (Möller et al., 1995, 1997). For quantification, the signal from total creatine at 3.01 ppm was used as an internal reference, and a correction for the normal [Phe]_brain in healthy volunteers, which was assumed to be 0.05 mmol/L from previous biopsy data (McKean, 1972), was applied. Parallel to NMR spectroscopy, complete blood amino acid profiles, including [Phe]_blood, were measured quantitatively by HPLC.

In addition to the measurements of stationary [Phe]_brain, time courses of [Phe]_blood and [Phe]_brain were determined after an oral loading test with l-phenylalanine (100 mg/kg body weight). Five patients could be investigated in this second part of the study, including all clinically unaffected, “atypical” patients as well as patients 8 and 9, serving as clinically “typical” control subjects. Loading was performed after baseline measurements, and [Phe]_blood and [Phe]_brain levels were recorded repeatedly starting 1 hour after loading and extending up to 75 hours. The patients had adhered to a fasting period of 12 hours before loading and returned to their normal nutrition (40 to 45 mg Phe per kg body weight per day) after the first postload scan.

Kinetic analysis of time courses

Numerous experimental studies have revealed that the large neutral amino acids are transported across the BBB by means of a common saturable carrier. Because of the competition between amino acids, an apparent Michaelis transport constant, K_t,app, is measured under in vivo conditions, which is determined by:

$$K_{t,app}=K_t^{Phe}(1+\sum \frac{[AA]}{K_t^{AA}})$$

(1)

where K_t,app is the plasma Phe concentration for half-maximal transport measured in the presence of other competing amino acids sharing the same carrier, K^Phe_t is the absolute Michaelis transport constant for Phe in the absence of competing amino acids, [AA] is the concentration of each competing amino acid, and K_t^AA is the absolute Michaelis constant of each amino acid (Pardridge, 1983). Recently, we showed that the relationship between [Phe]_blood and [Phe]_brain can be analyzed quantitatively using a symmetric Michaelis-Menten model and a constant Phe consumption velocity, ν_met, in the brain cells (Möller et al., 1997):

$$\frac{d[Phe{]}_{brain}}{dt}=\frac{T_{max}[Phe{]}_{blood}}{K_{t,app}+[Phe{]}_{blood}}-\frac{T_{max}[Phe{]}_{brain}}{K_{t,app}+[Phe{]}_{brain}}-v_{met}$$

(2)

where T_max is the maximal transport velocity. Phenylalanine transport into and out of the brain is assumed to be characterized by identical kinetic parameters, K_t,app and T_max. Equation 2 may be regarded as the simplest quantitative description of the consequences of amino acid brain uptake and metabolism. Agreement with experimental results was already obtained under steady-state conditions (Möller et al., 1997). The mathematical treatment is equivalent to the standard model of glucose transport kinetics at the BBB (Lund-Andersen, 1979), which was proven to be useful for the analysis of in vivo NMR data on brain glucose influx and consumption (Gruetter et al., 1993, 1996; Mason et al., 1993). A symmetric Michaelis-Menten model has also been used previously in computer simulations of amino acid uptake and metabolic pathways in the brain (Hommes and Lee, 1990a, 1990b).

Fitting of the time course predicted by equation 2 to the experimental data to obtain kinetic parameters was performed by minimizing the sum of squared residuals, χ², over a grid search (Bevington, 1969) with K_t,app = 0.05 to 5.0 mmol/L, T_max = 0.02 to 2.0 mmol·L⁻¹·min⁻¹, and ν_met = 0.002 to 0.2 mmol·L⁻¹·min⁻¹. Estimates of standard deviations were based on the range over which the parameters could be varied without significantly increasing χ². Brain Phe concentrations were determined with an accuracy of approximately ±0.15 mmol/L, which was taken into consideration for the error estimation of the kinetic parameters.

RESULTS AND DISCUSSION

Stationary brain Phe concentrations

In the first part of the study, [Phe]_brain was investigated in all patients while having comparable stationary [Phe]_blood around 1.2 mmol/L (mean values ± SD, 1.15 ± 0.10 mmol/L in group 1, 1.28 ± 0.06 mmol/L in group 2a, and 1.27 ± 0.09 mmol/L in group 2b). Representative spectra are shown in Fig. 1. Quantitative results are included in Table 1. As expected, no differences in mean [Phe]_brain were observed between groups 2a (0.60 ± 0.04 mmol/L) and 2b (0.59 ± 0.15 mmol/L) with typical outcomes depending on the individual diet status. Concentration ratios [Phe]_blood/[Phe]_brain varied between 1.9 and 2.3 for group 2a and between 1.7 and 2.8 for group 2b, in excellent agreement with results from previous studies (Kreis et al., 1995; Möller et al., 1995; Novotny et al., 1995; Pietz et al., 1995; Ullrich et al., 1994). In contrast, brain Phe was hardly detectable in any of the “atypical” patients. Therefore, [Phe]_brain must have been less than the estimated detection limit of our method of approximately 0.15 mmol/L, which is less than 25% of the mean values observed in the other two groups (P < 0.001). This excellent correlation with the clinical parameters underlines the paramount importance of stationary [Phe]_brain as one key explanatory factor for the outcome in classic PKU.

FIG. 1.

Representative difference spectra obtained at stationary blood Phe levels around 1.2 mmol/L in (A) patient 1 ([Phe]_blood = 1.21 mmol/L; [Phe]_brain < 0.15 mmol/L) of group 1, (B) patient 5 ([Phe]_blood = 1.34 mmol/L; [Phe]_brain = 0.59 mmol/L) of group 2a, and (C) patient 11 ([Phe]_blood = 1.29 mmol/L; [Phe]_brain = 0.71 mmol/L) of group 2b. An identical vertical scale is used for all spectra. The position of the Phe resonance at 7.36 ppm is indicated by an arrow. In patient 1, Phe is barely detectable.

Phenylalanine can interfere with the development and function of the CNS by different mechanisms. However, no single process by itself seems sufficient to explain the brain phenotype in PKU (Scriver et al., 1995). High Phe levels in the brain lead to dysmyelination and may also result in decreased neurotransmitter receptor density and cell connectivity (Hommes, 1994). Besides such morphologic changes, elevated Phe may cause an imbalance in neurotransmitter metabolism. Phenylalanine inhibits the uptake of the precursor amino acids Tyr and tryptophan into the brain with a resultant loss of dopamine and serotonin and possibly their neuronal release (Paans et al., 1996).

Time courses of brain Phe after oral loading

The CNS supply of Phe is a function of the plasma concentration and BBB transport processes. Because all patients presented with almost identical blood levels, we may conclude that the depression in [Phe]_brain seen in group 1 is related to transport characteristics. To test this hypothesis, we derived brain Phe transport kinetics from the time courses of [Phe]_blood and [Phe]_brain measured after the oral loading tests.

A general finding on fitting the experimental time courses to equation 2 was that T_max and ν_met could be varied over a wide range without significantly affecting the accuracy of the fit as long as the ratio of both velocities remained unchanged. This observation indicates an insufficient time resolution during the initial rise in Phe concentrations after the oral load, which results from prolonged signal averaging required to obtain reliable brain data of the low-concentration metabolite Phe. Consequently, most measurements were performed under conditions approaching a steady-state. In this situation, [Phe]_brain would only depend on K_t,app and the ratio T_max/ν_met (Möller et al., 1997):

$$[Phe{]}_{brain}^{steady-state}=K_{t,app}\frac{\{(T_{max}/v_{met})-1\}[Phe{]}_{blood}-K_{t,app}}{\{(T_{max}/v_{met})+1\}{K}_{t,app}+[Phe{]}_{blood}}$$

(3)

Our data analysis therefore included additional least-squares fits to the steady-state solution, equation 3. Results are summarized in Fig. 2 and Table 2. A comparison of the predicted time courses during the initial postload period indicated a delayed response of brain Phe after the rapid change in [Phe]_blood, which confirms previous observations (Pietz et al., 1995). Although only the more general equation 2 is appropriate to analyze this non—steady-state situation after the load, similar numerical results were found with both methods.

FIG. 2.

Dynamic changes of [Phe]_blood and [Phe]_brain on oral Phe loading. Exemplary results from patients 1 (A) and 3 (B) belonging to group 1 and from patient 8 (C) of group 2b are shown. Patients reached maximum [Phe]_blood (2.08 to 2.91 mmol/L) within 1 to 4 hours after loading. Similar to the stationary results, postload [Phe]_brain in group 1 remained always clearly below values observed in the “typical” control patients. Both blood and brain Phe relaxed quicker toward the baseline level in group 1 than in group 2. Besides the experimental [Phe]_blood (open circles) and [Phe]_brain data (filled circles), the graphs further show fitted time courses of brain Phe obtained with equation 2 (solid lines) and, under the steady-state approximation, equation 3 (dotted lines).

TABLE 2.

Results of the numerical analyses of the time courses

Patient	Kt,app (mmol/L)		Tmax/Vmet
	Group 1 (untreated, “atypical” patients)
1	0.87 ± 0.50	[1.25]	2.55 ± 0.41	[2.56]
2	0.45 ± 0.29	[0.65]	2.86 ± 0.85	[3.09]
3	1.10 ± 0.73	[1.50]	3.19 ± 0.54	[3.19]
	Group 2b (diet until an age of 10 years)
8	0.10±0.03	[0.09]	14.0±3.4	[14.2]
9	0.10± 0.04	[0.09]	7.8±2.2	[9.3]

Besides values and their standard deviations determined with the general Equation 2, additional results obtained with the steady-state approximation, Equation 3, are given in parentheses.

The range of velocities for Phe brain transport and consumption obtained with equation 2 from the grid search was T_max ≈ 0.040 to 0.25 mmol·L⁻¹·min⁻¹ (mean, 0.12 mmol·L⁻¹·min⁻¹) and ν_met ≈ 0.014 to 0.098 mmol·L⁻¹·min⁻¹ (mean, 0.046 mmol·L⁻¹·min⁻¹) for group 1, and t_max ≈ 0.070 to 0.086 mmol·L⁻¹·min⁻¹ (mean, 0.078 mmol·L⁻¹) and ν_met ≈ 0.005 to 0.011 mmol·L⁻¹min⁻¹ (mean, 0.008 mmol·L⁻¹·min⁻¹) for group 2. These values should be considered only as rough estimates because large errors have to be considered owing to the previously mentioned insufficient time resolution. Hence, the small variation obtained for T_max in group 2b may be accidental. Note that large interindividual variabilities in kinetic parameters were also reported for healthy volunteers examined with the double-indicator technique (Knudsen et al., 1995).

Other limitations of determining kinetic parameters from the observed relationship between plasma and brain Phe concentrations may result from several simplifying assumptions inherent in the model function, equation 2, and require further discussion.

First, in the brain, incorporation of Phe into peptides (e.g., γ-glutamylphenylalanine) or proteins and metabolite conversions (e.g., via hydroxylation or decarboxylation) provide a drain of the Phe level within the cell. In the normal state, the kinetics of the different components of this runout may be described by a Michaelis-Menten model (Kaufman, 1977; Salter et al., 1986). In contrast, the overall intracerebral metabolic rate for Phe was assumed to be constant in equation 2. This seems to be justified, knowing that brain Phe levels are increased by an order of magnitude in classic PKU as compared with healthy controls. Under such conditions we may assume that ν_met approaches its maximal velocity, V_max· This is consistent with theoretical simulations from rat data for [Phe]_blood greater than 1 mmol/L (Hommes and Lee, 1990a). Patients of group 1, however, showed substantially reduced [Phe]_brain even at high blood levels. Although the quality of the fits did not indicate systematic divergences and the introduction of further variables did not lead to significant reductions in χ², we cannot completely exclude the possibility of deviations from a constant intracerebral metabolic rate in these cases.

Second, another simplification results from the use of a symmetric Michaelis-Menten model for Phe transport at the BBB, which assumes that Phe influx and efflux are characterized by identical kinetic parameters, K_t,app and T_max. This model has been used successfully to describe steady-state data of Phe transport at the human BBB (Möller et al., 1997). Systematic deviations from the predicted saturation curve as recently reported for steady-state glucose BBB transport (Gruetter et al., 1997) did not occur in the Phe study, with [Phe]_blood ranging from 0.47 to 2.24 mmol/L. A symmetric model also agrees perfectly with l-(2-¹⁸F)-fluorophenylalanine positron emission tomography investigations of amino acid BBB transport in human volunteers (Ito et al., 1995), in which identical rate constants were obtained for influx and efflux. However, we cannot completely exclude the possibility that gradients in the amino acid concentrations across the BBB lead to differences in K_t,app for both transport directions.

The values for K_t,app and T_max/ν_met differ substantially among individuals of the same group. Pooling the data within the two groups to obtain mean values yielded standard deviations similar to those given in Table 2, which were obtained individually from the experimental accuracy (group 1, K_t,app = 0.81 ± 0.33 mmol/L, T_max/ν_met = 2.87 ± 0.33; group 2b, K_t,app = 0.10 ± 0.04 mmol/L, T_max/ν_met = 11.3 ± 4.4; the largest individual error was chosen as “standard deviation” for K_t,app in group 2b). Biologic variations may contribute to the observed range of K_t,app and T_max/ν_met within the groups. Further clarification of this point, however, requires larger data sets. Within the limitations of the very small database, statistical comparisons (Student's t test) of the mean values indicated that the obtained differences between both groups were significant (K_t,app, P < 0.04; T_max/ν_met' P < 0.03).

The kinetic parameters recorded from group 2 agree well with recent NMR results (K_t,app = 0.16 ± 0.11 mmol/L; T_max/ν_met = 9.0 ± 4.1) measured in nine clinically “typical” PKU patients under steady-state conditions (Möller et al., 1997). They are also consistent with the double-indicator measurements after Phe loading in three healthy volunteers, yielding K_t,app in a range of 0.03 to 0.58 mmol/L and T_max of 0.0144 to 0.0943 mmol·kg⁻¹·min⁻¹ (Knudsen et al., 1995). In rat parietal cortex, similar values of K_t,app of 0.218 ± 0.009 mmol/L and T_max of 0.041 ± 0.003 mmol·kg⁻¹·min⁻¹ were determined with the in situ brain perfusion technique (Momma et al., 1987).

Compared with these data, K_t,app appeared to be significantly larger in the “atypical” PKU patients. High BBB transport Michaelis constants make the brain uptake of amino acids in these individuals less sensitive to the effects of competition (Choi and Pardridge, 1986), which correlates well with their almost normal clinical outcome. Transport-induced brain amino acid imbalances may lead to changes in neurotransmitter metabolism and may thus influence brain function. The importance of competitive interactions, however, is still unclear. From theoretical calculations it was concluded that transport effects cannot explain Phe neurotoxicity (Hommes, 1989; Hommes and Lee, 1990a, 1990b), although others observed a significant lowering of rat brain Phe after injections of competing large neutral amino acids (Andersen and Avins, 1976).

The theoretical estimates of K_t,app at various concentrations of competing amino acids made by Hommes (1989) using equation 1 and rat data yielded values between 0.44 and 1.33 mmol/L. This matches the range observed in our study for group 1. Computer simulations demonstrated that variations of K_t,app over this range had very little effect on the Phe concentration in the brain. Hence, differences in K_t,app alone seem to be insufficient to explain the low [Phe]_brain values found in group 1.

Our data provide further evidence that the excess of the influx rate over the metabolic rate is substantially reduced in group 1. From the symmetric Michaelis-Menten model (equation 2) and our crude estimates of T_max and ν_met, values, this reduction in T_max/ν_met seems more likely to be caused by increased brain Phe consumption rates than depressed Phe influx in the “atypical” patients. However, an asymmetric transport at the BBB, with T_out more than T_in in group 1, might also explain the observed differences. Our ¹H NMR data do not distinguish between individual processes contributing to the drain of the intracerebral Phe level; hence, other methods are needed for further investigation of both alternatives.

In the normal state, dominant contributions to the Phe runout are hydroxylation in the liver and protein incorporation (Kaufman, 1977). It is still an unsolved question whether alternative pathways, involving transamination or decarboxylation, reflect metabolism in the body or whether it is mainly a metabolic product of bacteria in the gut. However, it has been concluded that such pathways are quantitatively only very minor ones (Kaufman, 1989). High brain Phe metabolic rates might indicate the intracerebral existence of a genetic enzyme system, restoring a more or less normal amino acid balance via hydroxylation (Kutter, 1978). Phenylalanine hydroxylation in human brain tissue has been reported from immunochemical studies leading to the assumption of an intracerebral isoenzyme of Phe hydroxylase (Bessman et al., 1977; Chestkov and Laptev, 1988; Petruschka et al., 1990), a possibility that was ruled out by others (Abita et al., 1974; Scriver et al., 1995). Alternatively, various areas of brain were shown to contain Tyr hydroxylase, which is capable of catalyzing the conversion of Phe to Tyr at a rate comparable to Tyr hydroxylation (Bagchi and Zarycki, 1973; Ikeda et al., 1965; Katz et al., 1976; McGeer et al., 1971). Although our NMR results do not provide information about the specific reactions involved in brain Phe metabolism, they are consistent with the assumption that full activity of any of those enzymes would lead to an optimal protection of the brain against neurotoxic consequences of permanently elevated [Phe]_blood.

CONCLUSION

In summary, our results suggest that interindividual variations in the kinetics of Phe uptake and metabolism do exist, leading to different brain concentrations of the neurotoxin Phe at comparable blood levels. Such variations seem to be a major causative factor in the pathogenesis of PKU, providing novel insights into the enigmatic phenotypic heterogeneity of the disease. An important advantage of in vivo NMR spectroscopy, which is widely available, is that the method is essentially noninvasive. Future use of spectroscopy may include an investigation of competition interactions directly in PKU patients by performing Phe loading studies with and without additional application of competing amino acids. Dynamic NMR measurements of [Phe]_brain therefore have a potential for optimizing individual treatment strategies.