Transcerebral exchange kinetics of large neutral amino acids during acute inspiratory

hypoxia in humans

Rasmus H. Dahl,1 MD; Ronan M. G. Berg,2,3,4 MD, PhD; Sarah Taudorf,5 MD, PhD; Damian M.

Bailey,4 PhD; Carsten Lundby,6 PhD; Mette Christensen,7 MSc; Fin S. Larsen,8 MD, DMSc; Kirsten

Møller,1 MD, PhD, DMSc

1Department of Neuroanaesthesiology, Rigshospitalet, Copenhagen, Denmark; 2Department of Clinical Physiology, Nuclear Medicine & PET,

Rigshospitalet, Copenhagen, Denmark; 3Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen,

Copenhagen, Denmark; 4Neurovascular Research Laboratory, Faculty of Life Sciences and Education, University of South Wales, UK; 5Department

of Neurology 2082, University Hospital Rigshospitalet, Copenhagen, Denmark; 6Centre for Physical Activity Research, Rigshospitalet, Copenhagen,

Denmark; 7Department of Clinical Genetics, Rigshospitalet, Copenhagen, Denmark; 8Department of Hepatology, Rigshospitalet, Copenhagen,

Denmark

Word count: ~2000; Figures: 0; Tables: 2; References: 30

Target journal: Scandinavian Journal of Clinical and Laboratory Investigation (original article)

Running title: BBB transport of LNAAs during hypoxia

Contact information:

Dr. Ronan M. G. Berg, MD, PhD

Department of Clinical Physiology, Nuclear Medicine & PET

Rigshospitalet

Blegdamsvej 9

DK-2100 Copenhagen

Denmark

Phone: (+45) 35 45 18 25

E-mails:

Rasmus H. Dahl: rhd@dadlnet.dk

Ronan M. G. Berg: ronan@dadlnet.dk

Sarah Taudorf: sarah.taudorf@regionh.dk

Damian M. Bailey: damian.bailey@southwales.ac.uk

Carsten Lundbycarsten.lundby@regionh.dk

Mette Christensen: mette.christensen.03@regionh.dk

Fin S. Larsen: fin.stolze.larsen@regionh.dk

Kirsten Møller: kirsten.moller@dadlnet.dk

Abstract

Hypoxaemia is present in many critically ill patients, and may contribute to encephalopathy.

Changes in the passage of large neutral amino acids (LNAAs) across the blood-brain barrier (BBB)

with an increased cerebral influx of aromatic amino acids into the brain may concurrently be

present and also contribute to encephalopathy, but it has not been established whether hypoxaemia

per se may trigger such changes.

We measured cerebral blood flow (CBF) in 11 healthy men using the Kety-Schmidt technique and

obtained paired arterial and jugular-venous blood samples for the determination of LNAA by high

performance liquid chromatography at baseline and after 9 hours of poikilocapnic normobaric

hypoxia (12% O2). Transcerebral net exchange was determined by the Fick principle, and transport

of LNAAs across the BBB was determined mathematically.

Hypoxia increased both the systemic and corresponding cerebral delivery of the aromatic amino

acid phenylalanine, and the branched-chain amino acids leucine and isoleucine. Despite this, the

transcerebral net exchange values and mathematically derived brain extracellular concentrations for

all LNAAs were unaffected.

In conclusion, the observed changes in circulating LNAAs triggered by hypoxaemia do not affect

the transcerebral exchange kinetics of LNAAs to such an extent that their brain extracellular

concentrations are affected.

Keywords (MeSH ID)

Aromatic amino acids (D024322), blood-brain barrier (D001812), branched-chain amino acids

(D000597), metabolism (D008660), hypoxia (D000860)

Introduction

Fulminant hepatic failure and sepsis are associated with encephalopathy ranging from impaired

attention to coma [1,2]. While the underlying mechanisms remain to be established, changes in the

passage of large neutral amino acids (LNAAs) across the blood-brain barrier (BBB) may be a

potential contributory factor [3–7]. The pathophysiology of encephalopathy is complex and may

involve disturbances in cerebral metabolism, neurotransmission and microcirculation possibly due

to altered plasma metabolites or damage to the BBB [1,2]. In both hepatic failure and sepsis, plasma

concentrations of aromatic amino acids (AAA) increase disproportionately relative to branched-

chain amino acids (BCAA), traditionally attributed to increased protein turnover in skeletal muscle

combined with increased utilisation of BCAAs for the synthesis of acute phase reactants in the liver

[8,9]. Given that AAAs and BCAAs are transported across the BBB through the same carrier in a

competitive manner [10], this may lead to AAA accumulation within the brain extracellular fluid to

such an extent that central noradrenergic circuits involved in wakefulness and arousal are attenuated

[11].

The primary goal of this study was to assess the BBB transport of LNAAs during acute inspiratory

hypoxia in healthy humans. Our secondary goal was to evaluate the effect of changes in the

transcerebral exchange kinetic on estimated brain extracellular fluid concentrations of LNAAs.

Hypoxaemia is a characteristic feature of most critically ill patients with either fulminant hepatic

failure or severe sepsis [12–15] and may contribute to the development of brain dysfunction.

However, before studying these effects in patients, it should be known, to what extent hypoxaemia

per se affects the passage of LNAAs into the brain in healthy humans, which has not previously

been examined. Thus, in the present study, we measured for the first time, changes in the systemic

concentrations of LNAAs and corresponding transcerebral net exchange and competitive transport

across the BBB during acute hypoxia. We hypothesised that hypoxaemia would increase the

cerebral delivery and influx of phenylalanine, similar to that observed during lipopolysaccharide-

induced systemic inflammation [16], in a group of healthy volunteers.

Materials and methods

Ethics

The study was approved by the Scientific-Ethics Committee of Copenhagen and Frederiksberg

Municipalities, Denmark (file number KF01-290/011). All participants were informed of the

purpose/risks of the experiment and signed an informed consent form, with all procedures adhering

to guidelines set forth in the Declaration of Helsinki.

Experimental design

The experimental design has been described previously [17] though the current study describes

entirely separate measurements to address an independent working hypothesis. We recruited 11

healthy males aged 27 (mean) ± (SD) 4 years. While plasma levels of LNAAs may vary

considerably with fitness level and dietary pattern [23,24], this was not taken into account in the

recruitment process. Global CBF was measured using the Kety-Schmidt technique [18] and paired

arterial and jugular-venous blood samples were obtained for arterial blood gas analysis (ABL 605

OSM, Radiometer, Brønshøj, Denmark) and LNAA quantitation.

Subjects attended the laboratory at 7 pm after a 12-hour overnight fast. Ultrasound-guided

catherization of the radial artery and right internal jugular vein was performed under local

anesthesia. Measurements of CBF were performed and paired arterial and jugular venous blood

samples were obtained after 30 min of supine rest in normoxia (21% O2) and again after 9-h hours

exposure to poikilocapnic normobaric hypoxia (12% O2, balanced nitrogen). The inspirates were

delivered through a tight-fitting mask connected to a non-rebreathing valve and a 500-liter

meteorological balloon that was supplied through compressed gas cylinders. Data on arterial blood

gases, global CBF, and cerebral oxidative metabolism have been published previously [17,19–22].

Amino acids

We measured all LNAAs with the exception of tryptophan (AAAs: phenylalanine and tyrosine;

BCAAs: valine, leucine, and isoleucine; others: histidine and methionine). Plasma was isolated

immediately after sampling of EDTA-blood, by centrifugation at 3000g for 10 min. at 4oC, and

stored at -20 °C until analysis. On the day of amino acid analysis, isolated EDTA-plasma was

thawed and deproteinised using sulfosalicylic acid (containing norleucine as internal standard).

Amino acid analysis was performed by high performance liquid chromatography (HPLC) with

fluorometric detection, utilizing post-column derivatization of free amino acids with o-

phthalaldehyde (OPA). Chromatographic separation was performed by ion-exchange

chromatography, using a lithium amino acid analysis HPLC-column (4.0 x 100 mm, 5 µm,

Pickering Laboratories). The HPLC system consisted of a Waters 510 dual piston pump system

controller, a Waters 420 fluorescence detector and a Waters 717 plus autosampler all controlled by

a Waters Empower software system (Milford, MA) for chromatographic analysis.

Calculations

The BCAA/AAA ratio was calculated as the ratio between the total arterial concentrations of the

BCAAs (valine, leucine and isoleucine) and the AAAs (phenylalanine and tyrosine):

BCAA / AAA=CVal+CLeu+C Ile

CPhe+CTyr

The transcerebral net exchange J of a given LNAA was calculated according to the Fick principle:

J=a jvD ⋅CBF

where a jvD is the arterial-to-jugular venous concentration difference. As in our previous study

[16], we took into account that only the plasma compartment of the cerebral capillaries is involved

in the transport of LNAAs [25], and that LNAAs equilibrate over the red blood cell membrane [26],

and adjusted the jugular-venous concentrations accordingly. Thereafter we calculated the cerebral

delivery of LNAAs by:

CD=Cp ⋅CPF

where C p is the plasma concentration, and CPF is the cerebral plasma flow (CBF ⋅[1−Hct ]¿.

BBB transport of LNAAs were based on a single membrane model of the BBB, where the transport

was considered entirely saturable in accordance with Michaelis-Menten kinetics, while assuming

steady-state conditions [27]. J depends on the balance of fluxes between the cerebral capillaries and

the brain extracellular fluid and can be determined as the difference between the unidirectional

influx and efflux (v1 and v2, respectively) across the BBB:

J=υ1−υ2

where υ1 and υ2 may be estimated from the permeability surface area for the given LNAA from

blood to brain and brain to blood (PS1and PS2, respectively), and the plasma and brain extracellular

concentration (Cb) of the LNAA:

υ1=P S1⋅C p υ2=P S2⋅Cb

P S1 for a given LNAA was calculated from its V max and its absolute Km-value using established

kinetic constants [10,28], which has previously yielded reliable estimates of the brain LNAA

transport.

P S1=V max

Km⋅ 1

Sp+1

The saturation of the LNAA transport system in plasma was evaluated by the substrate activity in

the plasma compartment, Sp, which is the sum of all LNAAs competing for the transporter weighted

by their individual absolute Km-values [29]:

Sp= ∑LNAAs

Cp

Km

In order to examine the unidirectional efflux and the brain extracellular fluid concentration we have

previously established a capillary model of LNAA transport based on the Renkin-Crone equation

[30,31]. Below C jv is the concentration of LNAAs in jugular-venous blood:

υ2=P S1⋅C jv−Cp ⋅α

1−α, α=exp(−P S1

CPF )In order to find the brain extracellular fluid concentration of LNAAs, we calculated the

permeability surface area product from brain extracellular fluid to plasma, P S2 defined as:

P S2=V max

Km⋅ 1

Sb+1

The saturation of the transport system measured by the brain substrate activity, Sb, is of great

importance for the permeability from brain to blood [16]. We calculated the saturable transport

parameter, ζ , as the sum of unidirectional effluxes of all LNAAs competing for the transport system

weighted by their individual V max-values [27].

ζ = ∑LNAAs

υ2

V max

Hereafter Sb was calculated using the expression:

Sb=1

ζ−1−1

Finally, Cb was calculated using the definition of the unidirectional efflux from the brain:

C b=υ2

P S2

This approach was specifically used to estimate the changes in Cb between normoxia and hypoxia

[27].

Statistics

A prospective power calculation with 80% power at the P < 0.05 level was performed based on a

pilot study measuring CBF and oxidative metabolites [17]. Measurement of LNAAs were not

included in the pilot study. We considered a hypoxia-mediated change of ±10% of the normoxic

mean arterial/venous values as “physiologically significant”. Our calculations indicated that 9

subjects were sufficient to detect a difference between conditions (normoxia vs. hypoxia) or sample

site (arterial vs. venous). A final sample size of 11 was chosen taking account for a potential “drop-

out” rate of 20%.

The majority of the data did not show normal distribution according to visual inspection of

normality plots and Shapiro–Wilk W test. Nonparametric analyses were performed with R statistical

software version 3.4.1 (R Foundation for Statistical Computing, Vienna, Austria). The Wilcoxon

matched pairs-signed-ranks test was used to compare differences between normoxia and hypoxia.

Data are presented as median (interquartile range) or median (95% Wilcoxon confidence interval),

and p < 0.05 was considered statistically significant.

Results

Arterial blood gas values and CBF

As reported elsewhere [17], inspiratory hypoxia induced a reduction in PaO2 with hyperventilation,

resulting in a mild respiratory alkalosis, while CBF was unaffected.

Systemic and cerebral metabolism of LNAAs

Hypoxia increased arterial concentrations of phenylalanine, leucine, and isoleucine, whereas

methionine decreased and all other LNAAs remained unchanged (Table 1). Consequently, the

cerebral delivery of phenylalanine and isoleucine increased from normoxia to hypoxia, whereas the

change in the cerebral delivery of leucine did not reach statistical significance (p = 0.06). Hypoxia

did not alter the transcerebral net exchange of LNAAs (Table 1) or the BCAA/AAA-ratio [4.4 (4.1-

5.0) vs 4.8 (4.4-5.0), p = 0.63].

BBB transport of LNAAs

Hypoxia increased the substrate activity in plasma (Sp) from 12.2 (11.4-13.0) to 14.0 (13.1-14.5) (p

< 0.01), and the P S1-values for all LNAAs were subsequently reduced (p < 0.05) (Table 2).

Hypoxia increased the unidirectional cerebral influx of phenylalanine, while the unidirectional

influxes of leucine and isoleucine were unaffected. The unidirectional influxes of tyrosine, valine,

histidine and methionine were reduced during hypoxia. No significant changes in Cb were observed

for any of the LNAAs.

Discussion

The present study has identified two novel observations. Firstly, hypoxaemia induced by 9 hours of

inspiratory hypoxia at an FIO2 of 12% increased the systemic concentration and corresponding

cerebral delivery of the AAA phenylalanine and the BCAAs leucine and isoleucine. Secondly, this

was associated with increased competition between the LNAAs for transport across the BBB

evident from the increase in substrate activity (Sp). Although hypoxia changed the cerebral delivery

and unidirectional efflux of some LNAAs, it did not alter the transcerebral net exchange or brain

extracellular fluid concentration of LNAAs.

The present study demonstrates that in contrast to fulminant hepatic failure and sepsis [4–6,32,33],

where plasma AAA levels, notably phenylalanine, increase while BCAAs decrease, hypoxaemia

per se does not change BCAA/AAA-ratio, given that both AAAs and BCAAs increase

proportionately in plasma. This implies that factors other than hypoxaemia, such as the acute phase

response, may be responsible for the disproportionate elevation in AAA levels observed in these

conditions. Indeed, we have previously identified that as little as an hour-lasting systemic

inflammatory response triggered by intravenous lipopolysaccharide infusion leads to a reduction in

the BCAA/AAA-ratio [34]. In this previous study, plasma phenylalanine increased, while most

other LNAAs decreased following the lipopolysaccharide infusion. The opposing changes in the

LNAA concentrations did not change the competition between the LNAAs as the saturation of the

BBB transport from blood to brain was unaltered (Spwas unaffected; unpublished data) after

lipopolysaccharide infusion. Hence, due to the increase in plasma phenylalanine, an increase in its

cerebral influx was evident, which subsequently appeared to be converted to tyrosine within the

brain [16]. In contrast, the BCAA/AAA-ratio was maintained in the present study, and the

competition between the LNAAs for transport into the brain increased as both phenylalanine,

leucine and isoleucine increased in plasma. We therefore speculate that the parallel increase in

plasma BCAAs and AAAs may prevent pathological accumulation of a single amino acid due to a

more even competition for the saturable transport system.

Our mathematical model shows that insignificant changes in the brain extracellular fluid

concentrations of LNAAs from normoxia to hypoxia were enough to obtain unidirectional effluxes

(under the assumption of constant absolute Km-values) explaining the difference between the

unidirectional influx and transcerebral net exchange. This may indicate that hypoxia only induces

minor changes to the LNAA transport system in healthy humans. The changes in unidirectional

influxes were matched by concomitant changes in the unidirectional effluxes without affecting the

transcerebral net exchange.

Studies of the abluminal membrane of the cerebral capillaries suggest that energy- and sodium-

dependent transport systems are responsible for LNAA transport [35–39], but these system are not

readily available for mathematical modelling. Abluminal transport may avoid AAA accumulation

when increased plasma levels of AAAs compete for the saturable transport system by increasing the

unidirectional efflux of LNAAs. Some studies suggests that dysfunction of the abluminal transport

system with decreased unidirectional efflux may account for the AAA accumulation in fulminant

hepatic failure [5,6]. In healthy humans, the hypoxia-induced plasma LNAA changes are within the

capacity of the abluminal transport system.

In critically ill patients LNAAs with sepsis or fulminant hepatic failure [4,5,32] changes in plasma

LNAAs are much greater than observed herein. We therefore believe that hypoxaemia does not

contribute to AAA accumulation in critically ill patients, unless hypoxaemia has an additive effect

on the plasma phenylalanine increase without having such effect on the BCAAs. The possible effect

of hypoxaemia on the energy-dependent abluminal transport systems should be assessed in future

ex vivo studies. Furthermore, recent advances in 1H-magnetic resonance spectroscopy of cerebral

phenylalanine content [39] may be combined with mathematical modelling of the LNAA transport

system in future studies to elucidate this, both in healthy humans and in critically ill patients.

Any mathematical model such as that employed in the present study for the assessment of BBB

transport of LNAAs is associated with unavoidable limitations. However, it provides an important

albeit derived estimation of the physiological changes that occur in compartments not immediately

accessible for sampling in humans. Most of these have been discussed in detail elsewhere [16,27]

and only the most critical limitations will be mentioned here. First, the estimated parameters

represent average whole-brain values, and due to the heterogeneity of both perfusion and metabolite

concentrations between various subcompartments within the brain (grey and white matter) [16], it

cannot be ruled out on the basis of our findings that hypoxaemia induces specific changes in the

LNAA transport and turnover in specific brain areas. Although acute inspiratory hypoxia notably

increases blood flow in the posterior cerebral circulation, we believe it is unlikely to affect the

global LNAA transport [40]. Second, our approach focuses exclusively on carrier-mediated

saturable LNAA transport across the BBB [27], and it must therefore be considered that the

cerebrovasculature contains areas without a BBB that are not accounted for. Third, we applied

established kinetic constants obtained from studies on anesthetised rats for our calculations in this

study, as these appear to provide the best estimates of LNAA transport in humans in vivo

[10,27,28]. However, it remains a possibility that hypoxaemia per se as well as other physiological

interventions could influence these kinetic constants. Finally, we did not include the LNAA

tryptophan in our calculations in the present study, as we were unable to measure it at the time

when the analyses were performed. As tryptophan competes for transport across the BBB through

the same carrier as the other LNAAs [10] this may alter the interpretation of our findings though

our calculations suggest that its contribution to the overall competition across the BBB amounts to

only ~10% (unpublished observations).

Only one other study has examined the effect of hypoxia on plasma LNAAs in human. In healthy

volunteers aged 66 (mean) ± (SD) 8 years a 2 hr exposure of 10% oxygen significantly decreased

plasma tyrosine levels, while other measured LNAAs remained unchanged [41]. Interestingly, the

baseline plasma concentrations of tyrosine, phenylalanine and methionine differ markedly from

those obtained in our study, possibly due to the age of the subjects. Future studies should assess the

reponse to longer exopsure of hypoxia in elderly healthy human.

In conclusion, we found that although hypoxaemia induced by acute inspiratory hypoxia causes

changes in several circulating LNAAs, these changes do not affect the transcerebral exchange

kinetics or BBB transport of LNAA to such an extent that the concentration of phenylaline

increases in brain extracellular fluid.

Author contributions

RHD defined the model, performed the calculations, interpreted the data, prepared tables and

figures, performed statistical analyses, and drafted the manuscript. RMGB conducted and conceived

of the study, handled supervision, interpreted the data, and drafted the manuscript. ST, DMB, and

CL conducted the study and acquired and interpreted the data. MC conducted the amino acid

analyses. FSL interpreted the data. KM conceived of and designed the research, conducted the

study, acquired, analyzed, and interpreted the data, drafted the manuscript, and handled funding and

supervision. All authors made critical revisions and read and approved the final manuscript.

Acknowledgements

None.

Conflict of interest

None.

Tables

Normoxia HypoxiaC p

μMCD

nmol /min/gJ

nmol /min/gC p

μMCD

nmol /min/gJ

nmol /min/gPheAAA 45.2 (41.2−48.4) 22.6 (18.5−25.5) 0.55 (-0.42−1.26) 52.4 (47.8−59.3) ** 29.2 (25.0−31.0) * 0.75 (-

0.03−1.71)TyrAAA 43.8 (17.6−20.9) 21.0 (18.1−26.0) -0.19 (-0.89−0.38) 42.4 (37.1−50.3) 22.7 (20.4−23.4) 0.56 (-

0.43−1.45)ValBCAA 229.3

(213.6−251.1)109.8 (95.3−139.7) 2.64 (-0.07−7.62) 240.6 (227.6−251.2) 130.8 (99.9−142.6) 3.15 (2.45−4.30)

LeuBCAA 114.6 (108.8−127.9)

56.2 (50.3−69.9) 3.20 (2.70−6.04) 137.9 (129.1−140.3) *

72.4 (58.8−80.0) 5.15 (3.14−6.34)

IleBCAA 57.9 (54.6−65.5) 30.3 (23.4−34.2) 1.12 (0.91−2.77) 67.6 (64.8−70.5) * 35.0 (32.4−39.1) * 1.85 (1.33−2.53)His 82.3 (80.6−85.3) 41.7 (34.2−44.5) 0.45 (-0.93−1.88) 84.1 (75.4−87.0) 44.5 (37.9−46.8) 0.71 (-

0.28−1.08)Met 18.5 (17.6−20.9) 9.4 (8.2−10.6) 0.28 (0.01−0.78) 18.3 (15.0−19.4) * 9.1 (8.1−10.3) 0.51 (0.12−0.73)

Table 1. Arterial concentration, cerebral delivery and transcerebral net exchange of large neutral amino acids

during normoxia and hypoxia. Results are presented as median (interquartile range). AAA: Aromatic amino acids;

BCAA: branched chain amino acids; CD: Cerebral delivery; C p: Plasma concentration; J : Transcerebral net exchange.

* p < 0.05 for normoxia vs. hypoxia. ** p < 0.01 for normoxia vs. hypoxia.

Δ P S1

mL /min/ gΔυ1

nmol /min/gΔCb

μMPheAAA -0.022 (-0.045−[-0.004]) * 1.06 (0.30−1.62) * 2.8 (-15.8−18.9)TyrAAA -0.010 (-0.018−[-0.001]) * -0.82 (-1.13−[-0.24]) * -2.1 (-18.0−23.4)ValBCAA -0.002 (-0.003−[-0.000]) * -0.36 (-0.53−[-0.08]) * 19.5 (-129.8−69.0)LeuBCAA -0.012 (-0.024−[-0.003]) * -0.20 (-0.76−1.42) 2.3 (-33.2−37.1)IleBCAA -0.006 (-0.13−[-0.001]) * 0.13 (-0.15−0.47) 1.2 (-20.5−16.7)His -0.004 (-0.008−[-0.001]) * -0.29 (-0.80−[-0.22]) ** 6.4 (-39.9−33.8)Met -0.004 (-0.008−[-0.002]) * -0.14 (-0.38−[-0.11]) ** 1.1 (-16.4−6.8)

Table 2. Differences in blood-brain barrier transport parameters between normoxia and hypoxia.

Results are presented as the median of differences (95% Wilcoxon confidence interval). All differences are

presented as normoxia minus hypoxia. AAA: Aromatic amino acids; BCAA: branched chain amino acids;

ΔCb: Difference in brain extracellular fluid concentration; Δ P S1: Difference in permeability surface area

product from blood to brain; Δυ1: Difference in unidirectional influx from blood to brain. * p < 0.05 for

normoxia vs. hypoxia. ** p < 0.01 for normoxia vs. hypoxia.

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