Large neutral amino acids block phenylalanine transport into brain tissue in patients with phenylketonuria

Abstract

Large neutral amino acids (LNAAs), including phenylalanine (Phe), compete for transport across the blood-brain barrier (BBB) via the L-type amino acid carrier. Accordingly, elevated plasma Phe impairs brain uptake of other LNAAs in patients with phenylketonuria (PKU). Direct effects of elevated brain Phe and depleted LNAAs are probably major causes for disturbed brain development and function in PKU. Competition for the carrier might conversely be put to use to lower Phe influx when the plasma concentrations of all other LNAAs are increased. This hypothesis was tested by measuring brain Phe in patients with PKU by quantitative 1H magnetic resonance spectroscopy during an oral Phe challenge with and without additional supplementation with all other LNAAs. Baseline plasma Phe was approximately 1,000 micromol/l and brain Phe was approximately 250 micromol/l in both series. Without LNAA supplementation, brain Phe increased to approximately 400 micromol/l after the oral Phe load. Electroencephalogram (EEG) spectral analysis revealed acutely disturbed brain activity. With concurrent LNAA supplementation, Phe influx was completely blocked and there was no slowing of EEG activity. These results are relevant for further characterization of the LNAA carrier and of the pathophysiology underlying brain dysfunction in PKU and for treatment of patients with PKU, as brain function might be improved by continued LNAA supplementation.

Figure 1

Study design. In the Phe_only series (left), plasma concentrations of Phe and the other LNAAs were determined during an oral Phe load. Brain tissue concentrations of Phe were measured with in vivo ¹H-MRS. Brain activity was monitored using spectral analysis of EEG activity. In the Phe_+LNAA series (right), this experiment was supplemented by LNAA treatment with 5 × 30 mg/kg body weight.

Figure 2

Downfield portion of the ¹H-MR spectrum. (a) Spectrum of a 26-year-old patient with PKU. (b) The averaged spectrum of 10 healthy age-matched subjects, and (c) the corresponding spectrum of an aqueous solution of Phe. The right side illustrates the model-fitting procedure used. Spectrum (d) contains the best-fitting model for spectrum a, which is composed of a parameterized spectrum of normal background and residual water signals (e) as well as the parameterized spectrum of Phe (f). Trace (g) contains the residuals of the best fit (i.e., a–d) for this case.

Figure 3

Time course of plasma and brain Phe and examples for LNAAs. (a) Steep increase of plasma Phe levels after an oral dose of L-Phe (time 0 h) in the Phe_only (dotted line) and Phe_+LNAA series (solid line). (b) Plasma concentrations of valine and (c) tryptophan are given as examples of LNAA increase during LNAA supplementation in the Phe_+LNAA series (solid line), compared with the Phe_only series (dotted line). (d) K_m-corrected Phe/LNAA ratio versus time after load. During Phe_only, this ratio increased because of the increase in plasma Phe; it decreased during LNAA treatment. (e) Brain Phe concentrations as determined by in vivo ¹H-MRS demonstrating a continuous increase during Phe_only and a block of Phe influx during LNAA treatment. (f) The ratio of plasma/brain Phe increased much more with LNAA treatment than without. All curves represent the average values from all six patients.

Figure 4

Averaged ¹H-MRS difference spectra (patients minus averaged normal spectra), acquired in vivo before as well as 6, 12, and 24 h after the oral Phe load. The increase of the Phe peak at 7.37 ppm during the Phe_only series (dotted line) contrasts with the unchanged Phe peak 6 and 12 h postload in the Phe_+LNAA series (solid line). When LNAA treatment was stopped, brain Phe also increased in this series (measurement at 24 h).

Figure 5

Averaged EEG power spectra from patients with PKU. During the Phe_only series (dotted line), a shift of the dominant peak of EEG background activity to the lower-frequency spectrum is demonstrated, which was prevented by LNAA treatment (solid line).