Edited by: Guido Santos-Rosales, University Hospital Erlangen, Germany
Reviewed by: Stefan Broer, Australian National University, Australia; Natalia Jarzebska, Dresden University of Technology, Germany
^{†}These authors have contributed equally to this work
This article was submitted to Membrane Physiology and Membrane Biophysics, a section of the journal Frontiers in Physiology
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Phenylketonuria (PKU) is an inherited metabolic disease characterized by abnormally high concentrations of the essential amino acid L-phenylalanine (Phe) in blood plasma caused by reduced activity of phenylalanine hydroxylase (PAH). While numerous studies have shown association between high plasma Phe concentration and intellectual impairment, it is not clear whether increased Phe fluctuations also observed in PKU affect the brain as well. To investigate this, time-resolved
Phenylketonuria (PKU) is the most common disorder of amino acid (AA) metabolism, resulting from severely reduced activity of the liver enzyme phenylalanine hydroxylase (PAH), which leads to abnormal accumulation of the essential amino acid L-phenylalanine (Phe) in the blood plasma (
The dysfunction of the PAH enzyme is also the cause of higher than normal fluctuations in Phe plasma concentration, where patterns greatly vary among patients in relation to age, diet, genotype and level of PAH defect (
The propagation of Phe fluctuations from plasma into the brain is critically influenced by the competition between this AA and its competing LNAAs (CL, i.e., L-leucine, L-isoleucine, L-tyrosine, L-tryptophan, L-valine, L-histidine, and L-methionine) for transporters at NVU cell membranes (
Schematic representation of the neurovascular unit and the therein expressed dominant Phe and CL transporters. The dominant Phe and competing large neutral amino acid (CL) transporters involved in the pathophysiology of PKU disorder include the Na^{+}-independent antiporter LAT1 (SLC7A5) in microvascular brain endothelial cells (MBECs), the Na^{+}-independent antiporter LAT2 (SLC7A8) in astrocytes and the Na^{+}-dependent symporter B^{0}AT2 (SLC6A15) in neurons. The arrows specify Phe and CL transmembrane pathways. The plasma concentration profiles of Phe and CL are taken as the input to the neurovascular unit (NVU), based on which concentrations in the NVU compartments are calculated.
To go around these hurdles, we have employed a previously developed computational model of NVU-LNAAs homeostasis (
We employed a previously developed compartmental model of NVU-LNAA homeostasis in adult rats (
Model parameters.
Value | Unit | Reference | |
---|---|---|---|
11 | μM | ||
V_{max,LAT1,lum,Phe} | 0.075 | μmol/min | |
52.9^{∗} | μM | ||
V_{max,LAT1,lum,CL} | 0.129^{∗} | μmol/min | |
RK_{LAT1} | 80 | – | |
110.2^{∗} | μM | ||
V_{max,LAT2,Phe} | 0.1128 | μmol/min | |
185.9^{∗} | μM | ||
V_{max,LAT2,CL} | 0.1494^{∗} | μmol/min | |
1050 | μM | ||
V_{max,B0AT2,Phe} | 0.0086^{∗} | μmol/min | |
126.2^{∗} | μM | ||
V_{max,B0AT2,CL} | 0.0186^{∗} | μmol/min | |
1050 | μM | ||
ΔΨ | −70 | mV | |
β | 0.6^{∗} | mV | |
[Na]^{ISF} | 141 | mM | |
[Na]^{Neu} | 40 | mM | |
V_{MBEC} | 3.5 | μl | |
V_{ISF} | 352.6 | μl | |
V_{Ast} | 742 | μl | |
V_{Neu} | 441.7 | μl |
Model calculations were performed as follows: We first determined steady state (ss) concentrations of Phe and CL in the individual NVU compartments
We then prescribed fluctuating plasma Phe concentrations as
where c_{f} is a coefficient that can take on values between 0 and 1, and thereby scales the fluctuation amplitude (relative to mean), and
where n is an integer, f_{o} is the fundamental frequency of Phe fluctuation, and a_{n} and b_{n} are the Fourier coefficients.
For presentation purposes, we initially considered three distinct plasma Phe fluctuation profiles referred to as cases c1–c3, with values of steady state concentration, frequency and relative amplitude as given in
Fluctuation indices of exemplary Phe plasma concentration profiles.
Case number | f_{0} (cycles/day) | c_{f} (−) | |
---|---|---|---|
c1 | 300 | 1 | 0.99 |
c2 | 800 | 3 | 0.3 |
c3 | 1600 | 0.14 | 0.6 |
Steady state normal physiologic (baseline) concentration values of Phe and CL in the NVU.
Compartment | Parameter | Concentration | Unit |
---|---|---|---|
Microvascular brain endothelial cell | 24.6 ± 146.8 | μM | |
235.7 ± 1408.8 | μM | ||
Brain interstitial fluid | 0.03 ± 0.4 | μM | |
0.3 ± 3.8 | μM | ||
Astrocyte | 4.8 ± 40.6 | μM | |
46.0 ± 389.7 | μM | ||
Neuron | 7.0 ± 53.4 | μM | |
46.9 ± 368.7 | μM |
To further investigate the effect of fluctuations, we considered a PKU case for which we fixed the non-fluctuating plasma Phe and CL concentrations and varied the fluctuation indices (fundamental frequency and amplitude-to-mean ratio) of a purely sinusoidal signal oscillation,
Finally, we employed the model to investigate the impact of therapeutic supplementation of non-Phe LNAAs on the concentrations of Phe and CL in NVU cells. To this end, we considered the supplemented LNAAs (SL) as CL with the same kinetics, and prescribed it as constant input (representing the effective plasma concentration of SL) to the model. We then determined steady state and fluctuating responses in the NVU, considering, as model input, different values for the effective steady state concentrations of SL in the plasma (0.5, 2, and 5 mM) and of both steady state (
We evaluated the sensitivity of the reported results on the choice of literature-reported model parameter values. To this end, we calculated model output for 100 cases in which the nominal model input parameters [maximum transport rates, Michaelis–Menten binding constants and steady state physiologic concentration values of LNAAs in individual compartments (
Model calculation of the steady state concentration of Phe and CL in neurons in relation to Phe concentration in the plasma. The bold solid lines correspond to results obtained with nominal model parameter values. The lower and upper bounds indicate standard deviation determined by sensitivity analysis (see section Materials and Methods). Similar relations between Phe and CL steady state concentrations are also observed in the other NVU compartments (see
After the assessment of the NVU steady state response to steady state plasma Phe concentrations, we calculated the dynamic changes of Phe and CL in the NVU in response to plasma Phe fluctuations.
Dynamic changes in the concentrations of Phe and CL in MBECs, ISF, astrocytes and neurons in response to fluctuations of Phe concentration in the plasma. Panel
To study how perturbations in Phe and CL concentrations in the NVU relate to plasma Phe fluctuation indices, we simultaneously varied f_{0} and c_{f} of sinusoidally fluctuating plasma Phe concentration, and then calculated the corresponding changes in concentration of Phe and CL in the NVU compartments. We thereby quantified the associations between Phe and CL excursion and the plasma Phe frequency and amplitude-to-mean ratio.
To elucidate the impact of non-Phe LNAA supplementation, we determined Phe and CL concentrations in neurons (
Impact of LNAA supplementation on Phe and CL concentrations in the NVU. Panels
Failure of AA homeostasis in general negatively affects cell function (
We first showed that with increasing Phe concentration in plasma, the steady state concentration of Phe in the NVU and in whole brain increases, while CL concentration decreases. The growth trend we determined for Phe in our
The computational model has been built with a number of simplifying assumptions. In particular, we focused on the pathways mediated by dominant transporters and thus disregarded pathways related to metabolism, diffusion and NVU transporters with low levels of expression, which have been shown to be of lesser importance (
Using a computational model of adult rat NVU-LNAA homeostasis, we investigated the effects of plasma Phe fluctuations on the dynamics of LNAAs in MBECs, brain ISF, astrocytes and neurons in PKU. Comparable
MT implemented the computational model and performed the calculations with SB. FV and VK directed the research. All authors conceived and designed the study, analyzed the data, wrote the manuscript, and approved the final version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We would like to thank Nenad Blau for helpful discussions on the fluctuation of Phe concentration in blood.
The Supplementary Material for this article can be found online at: