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Phenylalanineandtyrosinekineticsinrelationtoalteredproteinandphenylalanineandtyrosineintakesinhealthyyoungmen

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Am J C/in Nuir l992;56:517-25. Printed in USA. © 1992 American Society for Clinical Nutrition 517

Phenylalanine and tyrosine kinetics in relation to alteredprotein and phenylalanine and tyrosine intakesin healthy young men13

Joaquin Cortiella, J Sergio Marchini, Steven Branch, Thomas E Chapman, and Vernon R Young

ABSTRACT Plasma phenylalanine (Phe) and tyrosine (Tyr)

turnover and the rate ofconversion ofphenylalanine to tyrosine

(Phehyd) and of phenylalanine oxidation (Pheox) after reduced

intakes of Phe and Tyr were determined in a metabolic study

involving five healthy young adult men. In a pilot study, six

postabsorptive young men received either 1 2- or 4-h infusions

of[2H2]Phe and [l-’3C]Tyr or [1-’3CJPhe and [2H2]Tyro. From

these results a primed 8-h constant infusion of [l-’3CJPhe and

[2H2]Tyr and [2H3jleucine was used in the metabolic study (first

3 h fasted, the 5 h fed) at the end of l-wk periods during which

subjects received an adequate nitrogen L-amino acid based-diet

followed by a restricted intake of Phe and Tyr. This procedure

was again repeated after 1 and 3 wk when subjects were given

a diet low in both nitrogen and Phe and Tyr. Phe and Tyr fluxes

were not significantly affected by diet during the fasted metabolic

state but Tyr fluxes were lower when the restricted intakes were

given. Compared with the rate during the fasting state, Pheox

was significantly higher (P < 0.01) when the adequate diet was

consumed; Pheox and Phehyd for fed and fasted states were

similar when Phe and Tyr were restricted. Am J C/in Nutr

1992;56:5 17-25.

KEY WORDS Phenylalanine, tyrosine, hydroxylation, flux

oxidation, adult men, diet restriction

Introduction

The rate-limiting step in mammalian phenylalanine catabo-lism is hydroxylation, catalyzed by phenylalanine hydroxylase

(PAH, EC 1. 14. 16. 1) (1). Mutations that reduce the activity of

PAH, ofquinonoid dihydropteridine reductase (which maintains

the required pteridine cofactor tetrahydrobiopterin for PAH in

reduced form), and ofenzymes associated with tetrahydrobiop-

tern synthesis, cause hyperphenylalaninemia and/or phenyl-

ketonuria (PKU) because ofa diminished rate of phenylalanine

hydroxylation (2). Collective PKU frequencies are ‘� I in 10 000

births in Caucasians and 1 in 100 000 births in Japanese pop-

ulations (1).

Liver PAH is controlled through induction (3, 4), a phos-

phorylation-dephosphorylation cycle (5, 6), and activation by

phenylalanine (7). Additionally, tyrosine aminotransferase

(TAT) has been identified as rate limiting for the degradation

oftyrosine (8), and both PAH and TAT activities are modulated

by a range of nutritional and hormonal stimuli. However, these

two enzymes differ from each other in their sensitivity, time

course, and possibly direction ofchange, after a given stimulus,

and the human enzyme may be affected or regulated in a way

that differs from that for the rat enzyme (9). Further, Pogson et

al (9) point out that blood concentrations ofphenylalanine and

tyrosine do not change quantitatively in any simple predictable

way as a result of enzyme changes. Consistent with this view,

we reported earlier that free phenylalanine and tyrosine con-

centrations in plasma from postabsorptive subjects showed little

change when they had consumed an aromatic amino acid-free

diet for as long as 12 d, but the concentrations in plasma during

the prandial period were lower than during a control-diet period

( 10). Because the latter effect was observed soon after the first

aromatic amino acid-free meal, the composition of a meal can

have an immediate but short-term effect on aromatic amino

acid concentrations, as has been observed by others ( 1 1 , 12).

From an analysis of the control of hepatic aromatic amino acid

metabolism it has been concluded that transport across the

plasma membrane plays a crucial role in determining the oxi-

dation of these amino acids (9). Thus, phenylalanine oxidation

would probably be retarded during the prandial period when

phenylalanine intakes are reduced substantially but its oxidation

rate during the postabsorptive state might be little affected by

the immediate past daily phenylalanine intake. We wished to

explore this hypothesis and to determine how phenylalanine

and tyrosine fluxes respond to altered dietary protein and amino

acid intakes. The potential practical importance of this knowl-

edge is with respect to the improved therapeutic efficacy of long-

term dietary control in patients with phenylketonuria (13-15).

The validity of the current international recommendations for

the requirements for the aromatic amino acids (phenylalanine

and tyrosine) in healthy adults has been questioned (16-18),

making it more difficult to plan for safe and effective dietary

management of PKU patients.

I From the Laboratory of Human Nutrition and Clinical ResearchCenter, Massachusetts Institute ofTechnology, Cambridge, MA, and the

Shriners’ Burns Institute, Boston.2 Supported by NIH grants DK1 5856, DK42 101 , and RR88 and grants

from Shriners’ Hospitals for Crippled Children.3 Address reprint requests to VR Young, Massachusetts Institute of

Technology, Room E 18-6 13, Cambridge, MA 02139.Received August 16, 1991.Accepted for publication March 17, 1992.

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518 CORTIELLA ET AL

There have been few physiologic studies of plasma phenyl-

alanine and tyrosine kinetics, with particular reference to the

influence ofnutritional factors in healthy adult humans. Clarke

and Bier ( 19) developed a tracer model for measurement of phe-

nylalanine flux and estimation of the rate of phenylalanine hy-

droxylation, using stable isotopically labeled phenylalanine and

tyrosine. More recently, Thompson et al (20) described a re-

finement of the Clarke-Bier model. Hence, we have applied a

tracer approach, essentially according to Thompson et al (20),

to explore the dynamic status ofwhole-body phenylalanine me-

tabolism in healthy young men after 1) adjustment to an ade-

quate-protein diet, 2) a diet supplying adequate total nitrogen

but with reduced phenylalanine and tyrosine, and 3) a diet lowin nitrogen as well as phenylalanine and tyrosine.

Methods

Metabolic study

Subjects. Five healthy, young adult men participated in this

study (Table 1). They were alljudged to be in good health, based

on a medical history, a thorough physical examination, and rou-

tine blood biochemical screening and urinalysis. They gave their

written, informed consent and the study protocol was approved

by the Massachusetts Institute ofTechnology (MIT) Committee

on the Use of Humans as Experimental Subjects and the Ad-

visory Committee of the MIT Clinical Research Center (CRC).

Diets. Three experimental diets were used in this investigation,

each being based on an L-amino acid mixture (Table 2). The

composition of the amino acid mixtures is shown in Table 2

and it was given in amounts to supply 1 g - kg’ - d�. The ade-

quate diet (Adequate N) was formulated with an L-amino acid

mixture patterned as in egg protein and given for 1 wk before

conducting the first tracer study (see below). The second amino

acid diet (Adequate N: Low Phe/Tyr) immediately followed the

adequate-diet period. This was again given for 1 wk, supplying

the same nitrogen intake as during the first diet period but with

a reduced amount of phenylalanine and tyrosine so that the

daily intakes of these two aromatic amino acids would be es-

sentially as for the third amino acid diet, described below. The

nitrogen intake supplied by this second amino acid mixture was

maintained isonitrogenous to that of the Adequate N diet by

increasing the amount ofglutamate in the mixture. After about

a 5-7-d break period, during which subjects consumed a normal,

adequate diet, the third diet (Low N: Low Phe/Tyr) began for

3 wk, during which amino acids were supplied in the same pat-

TABLE 1Characteristics of young men studied for the effects of diet onphenylalanine and tyrosine kinetics

Subject Age

3,

Height

cm

Body

Initial

kg

weight

Final

kg

Estimated energy

intake

Mild (kcal/d)

1 22 173 71 71 11.72 (2800)

2 20 189 95 94 14.15 (3380)3 20 180 71 72 13.02 (3110)

4 20 174 71 71 12.32 (2943)

5 20 155 55 55 10.74 (2565)

TABLE 2

Daily intakes provided by the three L-amino acid-amino acid

mixtures

Adequate

Amino acidAdequate

NN:low Phe/

TyrLow

N:low Phe/Tyr

mg.kg’ . d’

L-Isoleucine 62.35 62.35 24.94

L-Leucine 82.74 82.74 33.10L-Lysine#{149}HC1 75.12 75.12 30.05L-Methionine 29.45 29.45 1 1.78L-Cystine 21.94 21.94 8.78L-Phenylalanine 54.24 2 1.70 21.70L-Tyrosine 40.42 16.17 16.17L-Threonine 46.73 46.73 18.69L-Vahne 69.72 69.72 27.89

L-Tryptophan 15.48 15.48 6.19

L-Histidine . 2H2O 30.43 30.43 12.17L-Arginine . HC1 74.54 74.54 29.82

L-Alanine 61.00 61.00 24.40

L-Aspartic acid 65.80 65.80 26.32L-Glutamatic acid 1 12.39 169.39 44.96

Glycine 33.00 33.00 13.20

L-Proline 41.62 41.62 16.65L-Serine 83.34 83.34 33.34

tern as for the Adequate N diet but total amino acid and nitrogen

intakes were reduced to an amount equivalent to �0.4 g egg

protein (N X 6.25). kg’ . d’.

In addition to the amino acid mixtures, which were preweighed

for each meal and mixed with water for consumption, the major

energy source was in the form of protein-free, wheat-starch

cookies. The daily energy intake, with �40% from fat sources

and 60% from carbohydrate, varied between subjects but was

kept constant for each individual. Vitamins were supplied as a

daily supplement, to meet or exceed recommended allowances

or safe and adequate intakes (2 1). Macromineral supplements

provided sodium, potassium, calcium, and phosphate in the

range of accepted allowances. A trace mineral mixture was for-

mulated and encapsulated to provide magnesium and those trace

minerals not provided by the vitamin and mineral supplements.

A choline supplement of 500 mg was given daily. The taste of

the amino acid mixture was improved by serving it with an

equal weight of sucrose and a flavoring agent (Vivonex flavor

packets; Norwich Eaton Pharmaceuticals, Norwich, NY). The

sources of the carbohydrate were beet sugar and wheat starch.

These were chosen for their relatively low ‘3C enrichment tominimize changes in the background enrichment ofbreath sam-

ples when the diet was given during the fed phase of the tracer-

infusion protocol (see below).

The total daily intake was consumed as four separate mealsat 0800, 1200, 1700, and between 2000 and 2200. At least two

of these meals were eaten in the CRC under supervision of the

dietary staff. Based on the good rapport developed between the

volunteer subjects and one of the investigators, as well as the

CRC dietary staffand through daily interviews, it is our judgmentthat all subjects complied with the requirements ofthe protocol.An objective measure of dietary compliance in studies of this

kind has not been easy to develop and this investigation was

initiated before we had evaluated use ofurinary p-aminobenzoic

acid excretion as one index of compliance (22).

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PHENYLALANINE KINETICS 519

Tracer-infusion studies

A primed continuous intravenous tracer-infusion approachwas used in these experiments. The infusion studies were per-

formed on the concluding morning ofthe first and second dietaryperiods, and after 1 and 3 wk for the third dietary period (LowN:low Phe/Tyr). The infusion-day protocol was the same for all

diets and subjects. In the metabolic study the isotope-infusion

period lasted a total of 8 h. For the first 3 h subjects remained

in the postabsorptive state, after an overnight fast, and this was

immediately followed by a 5-h fed state, tracer-infusion period.

The subjects entered the CRC infusion room at 0630; they were

then weighed, after voiding and asked to recline in bed. Intra-

venous lines were inserted after local anesthesia with 1% Xy-

locaine (Astra Pharmaceutical Products, Inc, Westborough, MA).

One line was used for tracer infusion via an antecubital vein,with a 20-gauge, 5-cm indwelling catheter, and for blood sam-pling a 20-gauge, 0.6-cm catheter was inserted retrograde into

an ipsilateral hand vein. The hand was maintained in a custom-

made warming box maintained at 60 ± 5 #{176}C,to obtain arter-

ialized blood. The patency of the sampling catheter was main-

tamed by slow infusion of normal saline.

Baseline blood and breath samples were then collected. At

0800 priming doses of [‘3Cjbicarbonate, [l-’3C]phenylalanine

(99 atoms percent), [l-13C]tyrosine (98 atoms percent), and [3,3-

2H2]tyrosine (98 atoms percent), all from Tracer Technologies,

Inc, Somerville MA, and [2H3]leucine (MSD Isotopes, Inc,

Montreal; 99 atoms percent) were given over 2 mm. This was

followed by the constant intravenous infusions of [ 1-

13C]phenylalanine, [2H2]tyrosine, and [2H3]leucine tracers, given

with the aid of a Harvard pump (Harvard, MA) for the total 8-h period. At 180 mm the subjects started eating small isoener-

getic, isonitrogenous meals every 30 mm. The hourly intake

represented one-twelfth ofthe total daily amino acid and energy

intake provided by the experimental diet during the preceding

5 d. The amino acids were provided in the form of a gel (23),

and subjects also received 30 mL beet sugar water, with a fla-

voring agent, per meal. This approach has been used in our

previous series of studies for achieving a fed-state condition and

is now also used by others (24).

Infusates of tracers were prepared from sterile powders of high

chemical purity and high isotopic enrichment. The bicarbonate

pool was primed with a sterile solution of sodium

[13C]bicarbonate (25 g sodium bicarbonate/L; 90 mole percent

excess); the dose was 0. 1 mg/kg. The priming doses (��moI/kg)

for [l-’3C]phenylalanine, [l-’3C]tyrosine, [2H2jtyrosine, and

[2H3jleucine were 3. 1 , 3. 1, 2.3, and 4.7, respectively. The known

constant infusions (Mmol - kg� . h�) were designed to supply

3. 1 for [‘3C]phenylalanine, 2.3 for [2H2]tyrosine, and 4.2 for

[2H3]leucine.

Blood and expired air samples

Blood samples were transferred from a syringe to heparinizedvacutainers (Venoject, T-218U; Terumo Medical, Elkton, MD),

being careful to minimize hemolysis. They were stored on ice

and centrifuged at 1000X g at 4 #{176}Cfor 15 mm. Aliquot samples

ofplasma were stored separately at -20 #{176}Cuntil used for analysis

for free amino acid concentrations and isotopic enrichment of

the tracers.

Samples of expired breath, for ‘ 3CO2 analysis, were collectedin a disposable rubber bag. Subsequent handling and analysis

ofsamples for ‘3C enrichment were as described previously (25).

Total carbon dioxide production (VCO2) and total oxygen

consumption (VO2) were determined with the aid ofa custom-

made, ventilated-hood, indirect-calorimeter system, as described

previously (26). For the postabsorptive phase the determinations

were made between 90 and 120 mm ofthe infusion period. For

the absorptive or fed phase, measurements were made between

360 and 440 mm of the tracer-infusion period.

Analysis

Plasma amino acid concentrations, and the amino acid content

of infusates, were analyzed by HPLC (model 334; Beckman,

Palo Alto, CA) by using an ion-exchange chromatographicmethod, involving postcolumn derivitization with o-phthalal-

dehyde (OPA), and quantitation with the aid of a fluorescence

detector.

The 3C enrichment of the expired carbon dioxide was mea-sured on a triple collector isotope ratio mass spectrometer (Fin-

nigan-MAT delta E; Finnigan, San Jose, CA). The ratio of m/z

44 to m/z 45 was determined relative to a working gas standard

of carbon dioxide. The baseline ‘3C02 values were subtracted

from each plateau sample to give a relative atom percent excess

(APE) of ‘3C.

For isotopic analysis ofphenylalanine and tyrosine, the plasmaamino acids were first isolated on a cation-exchange resin column

after acidifying 200 �z1 plasma with 1 mL 1 mol acetic acid/L

and then extracted with 3 mL NH4OH. The eluant was collected

in 3.7-mL vials and dried under a gentle flow of nitrogen gas at

80 #{176}Cin a heating block. The n-heptafluorobutyl, n-propyl ester

derivatives of phenylalanine and tyrosine were prepared by a

slight modification of the method of March (27). A l.0-mL al-

iquot sample of n-propanol:HCI (2: 1 molar ratio) was added to

the dry amino acids and heated at 1 10 #{176}Cfor 30 mm to form

the propyl esters. The cooled samples were dried at 60 #{176}Cunder

nitrogen flow. To the propyl esters 50 �tl heptafluorobutyric an-

hydride (HFBA) was added and these were heated at 60 #{176}Cfor

30 mm. Excess HFBA was removed by evaporation at room

temperature by using nitrogen and the derivative then dissolved

in 500 �l ethyl acetate. One microliter of the mixture was used

for injection into a Hewlett-Packard 5988 quadrupole gas chro-

matographic/mass spectrometric (GC/MS) system. This was

operated in the negative chemical ionization mode with the

source at 1 .0 torr; methane was the reagent gas and helium the

carrier at a flow rate of 1 .0 mL/min. The samples were chro-

matographed on a 30 m X 0.25 mm X 0.25 �m DB-210 capillary

column (I & W Scientific, Folson, CA). The initial oven tem-

perature was 1 30 #{176}Cand ramped at 10 #{176}C/minto 240 #{176}C;in-

jection temperature was 250 #{176}Cand interface temperature was

240 #{176}C.Selected ion monitoring was carried out at m/z 382,

383, and 384 for unlabeled and labeled phenylalanine, and m/

z 417, 418, and 419 for unlabeled and labeled tyrosine.The enrichment of 5,5,5-2H-ketoisocaproic acid (KIC) was

determined by preparation ofthe O-tert-butyldimethylsilyl qui-

noxalinol derivative (28, 29). A Hewlett-Packard 5970 quad-

rupole GC/MS was used for this purpose. Selected ion moni-

toring was carried out at m/z 259 and 262 for unlabeled and

labeled KIC, respectively. A 30 m X 0.25 mm X 0.25 �m, SE-

54 capillary column (Alltech, Deerfield, IL) was used in the gas

chromatograph. The initial GC oven temperature was 100 #{176}C;

the oven temperature ramped at 25 #{176}C/minto 300 #{176}Cand in-

jection temperature was 280 #{176}C,with a helium flow rate of 0.8

mL/min. Enrichment was calculated as molar fraction above

baseline from signal area ratios determined against a calibration

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520 CORTIELLA ET AL

curve made with standards of KIC and [2H3]KIC (Tracer Tech-

nologies, Inc, Somerville, MA).

Model: Pheny/alanine, tvrosine, and /eucine kinetics

The model of phenylalanine-tyrosine metabolism used herewas developed by Clarke and Bier (19) with modifications pro-

posed by Thompson et al (20). Briefly, the amino acid (phenyl-

alanine or tyrosine) flux (Q� or Qt :�tmol . kg � . h ‘) is calculatedby isotope dilution as previously described for leucine (30):

. I EiQ = ii - - 1

\ Ep

where i is the rate of tracer infusion (zmol - kg’ - h’) and Ei

and Ep are the enrichments of the infusate and plasma aminoacid, respectively.

The rate of phenylalanine conversion to tyrosine (Q�:

�tmol . kg’ . h1) was derived as follows:

- __� E�[i�+Q�]

where Qt and Q�, are the tyrosine and phenylalanine fluxes, re-

spectively, estimated from the primed constant infusions of

[2H2]tyrosine and [l-’3Cjphenylalanine; E� and E,�, are the plasmaenrichments of [l)3C]tyrosine and [l-’3C]phenylalanine, re-

spectively; i�, is the rate of infusion of [l-’3Cjphenylalanine. As

discussed by Thompson et al (20), the term Q�/(i� + Q�,) correctsfor the contribution of the phenylalanine tracer infusion to Q�.

The rate of ‘3CO2 released by the L-[l-’3C]phenylalanine tracer(or L-[l-’3C]tyrosine in the pilot studies, described below) andthe oxidation rate ofthe amino acid were estimated as for leucine

oxidation (30). The calculated rates were corrected for the re-

tention of ‘3CO2 in the body, based on our previous infusion

studies with NaH13CO3 (26). A factor of 0.71 was used for thefasted, or postabsorptive, state and 0.82 for the fed state.

Leucine flux was calculated as described previously (30) by

using the plasma enrichment of [2H3]KIC as an index of the

intracellular labeling ofthe mixed whole-body leucine pool (31).

There is no analogous plasma metabolite measure for estimatingintracellular phenylalanine and tyrosine enrichments. However,

from equation 2 it might be appreciated that the tyrosine flux

(Qt) calculated from the plasma [2H2]tyrosine enrichment will

underestimate the actual whole-body flux because the intracel-

lular tyrosine enrichment would be expected to be lower than

the value for tyrosine in plasma. Similarly the degree of enrich-

ment of phenylalanine within organs and particularly in liver

would be lower than the measured [l)3C]phenylalanine enrich-

ment in plasma, also giving rise to an underestimation of Q�,.

Thus, calculated conversion of phenylalanine to tyrosine (Q�;phenylalanine hydroxylation) as derived from equation 2 is likely

to be an underestimate of the true rate. The possible extent of

this underestimate is considered in the discussion below.

Pilot studies

Before the major phase ofthis investigation, pilot studies were

conducted to establish the suitability of relatively brief tracer-

infusion protocols for estimating phenylalanine and tyrosine ki-

netics. In these initial studies we used different combinations oftracers and periods of tracer infusion. Clarke and Bier (19) in-

fused tracer phenylalanine for 1 3-14 h coupled with a combined,

primed infusion oflabeled tyrosine during the final 4 h. There-

fore, six healthy young men ofcomparable age and body weightto those participating in the metabolic study were investigated

by using the following tracer protocols: 1) a continuous infusionof [2H2]phenylalanine for 12 h with a primed constant infusionof[l-’3C]tyrosine during the final 4 h, 2) [l-’3Cjphenylalanine

for 12 h with a primed constant infusion of[2H2]tyrosine duringthe final 4 h, and 3) primed continuous infusions of [1-‘3Cjphenylalanine and [2H2]tyrosine during a 4-h period. During

the conduct of this phase of our studies, Thompson et al (20)

described the results ofa series oftracer-infusion protocols rang-ing from 4- to 16-h periods for phenylalanine infusion, as well

as a shorter 4 h combined, primed infusion protocol with labeled(1) phenylalanine and tyrosine.

The three tracer protocols were applied in random order toeach subject. Before each study the subject received, for 2 d, ahouse diet supplying adequate amounts of all nutrients. The

subjects were given their last meal at 2000 on the evening when

the prolonged 12-h infusion of phenylalanine was given or at2200 on the evening before the 4-h infusions. They did not con-sume any food until termination ofthe tracer infusion but wereallowed to drink fluids. Measurements ofphenylalanine and ty-rosine kinetics were based on the isotopic data for the final 2 h

of the infusion protocol.With respect to the metabolic study, changes with metabolic

state (fast, fed) and diet effects were evaluated by using a repeated-measures analysis of variance blocking by subject followed by

post hoc, pair-wise comparisons among dietary means by usingNeuman-Keuls test. The differences in phenylalanine hydrox-

ylation and oxidation between the fed and fasted states for the

Adequate N diet were examined by using a paired t test.

Results

Pilot studies

As summarized in Table 3, estimates of phenylalanine and

tyrosine fluxes and of the measured rate of phenylalanine hy-droxylation were essentially the same irrespective of the tracercombinations or length of the phenylalanine-infusion period.

Furthermore, the estimate of phenylalanine hydroxylation based

on the measured rate of conversion of labeled phenylalanine totyrosine compared quite well with the estimation of[‘3Cjphenylalanine oxidation when data for expired ‘3CO2 for

this purpose were used. These results, therefore, supported the

use of a shortened tracer protocol, as proposed by Thompsonet al (20), in the follow-up, metabolic study ofthe effects of diet

on phenylalanine and tyrosine kinetics. Furthermore, from the

results obtained with these pilot studies, together with those ofour previous comparisons ofvarious phenylalanine tracers (32),when given by vein, and with those of Thompson et al (20), itwas evident that either [2H5]-, [2H2]-, or [‘3C]phenylalanine could

be used to estimate the phenylalanine flux and conversion of

phenylalanine to tyrosine. In our major metabolic study dis-

cussed below, we therefore used [‘3C]phenylalanine and[2H2]tyrosine to explore the impact of changes in diet on phe-

nylalanine homeostasis. Finally, as shown in Table 3 the estimate

oftyrosine oxidation, based on the infusion of[’3C]tyrosine, wasabout double that for phenylalanine hydroxylation or oxidation.

This would be expected because the tyrosine oxidation rate dur-

ing the postabsorptive state reflects the conversion of phenyl-

alanine to tyrosine and its subsequent oxidation together with

the oxidation of preformed tyrosine.

Metabolic study

The measured enrichments of plasma phenylalanine and ty-rosine, together with KIC, are summarized in Table 4 for each

(2)

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TABLE 4

Measured isotope enrichments (molar fraction) of plasma phenylalanine,

under different dietary conditions5

tyrosine, and a-ketoisocaproate (KIC) during the fasted and fed states

Diet

[2H3]KIC [‘3C]Phenylalanine [‘3C]Tyrosine [2H2]Tyrosine

Fasted Fed Fasted Fed Fasted Fed Fasted Fed

Adequate NAdequate N:Low

Phe/Tyr

Low N:Low Phd

Tyr

1 wk3wk

3.3 ± 0.1

3.6 ± 0.2

4.3 ± 0.44.3±0.3

2.7 ± 0.1

3.9 ± 0.2

4.0 ± 0.34.0±0.2

5.5 ± 0.3

5.1 ± 0.8

5.1 ± 0.2

5.5±0.1

4.9 ± 0.3

5.6 ± 0.9

4.2 ± 0.5

5.5±0.1

1.1 ± 0.2

0.85 ± 0.03

0.7 ± 0.1

1.1 ±0.2

1.2 ± 0.2

1.4 ± 0.4

0.6 ± 0.1

0.9±.1

4.8 ± 0.5

4.9 ± 0.4

5.2 ± 0.9

6.3±0.6

4.0 ± 0.6

6.3 ± 1.3

4.4 ± 0.4

5.9±0.9

PHENYLALANINE KINETICS 521

51±SE;n = 5.

TABLE 3Comparison of labels and tracer protocols with respect to estimates of phenylalanine and tyrosine kinetics: pilot studies

Tracer and protocol

Kinetic parameter

[‘3C]Phenylalanine or

(hours of infusion) Qp Qt Qpt [‘3C]Tyrosine oxidation

�mo/. kg’ . h’

[2H2]phenylalanine ( I 2 h):[1-’3-C]tyrosine (4 h) + prime 47 ± 4 40 ± 3 6 ± 0.8 13.7 ± 1

[‘3C]phenylalanine ( 12 h):[2H2jtyrosine (4 h) + prime 44 ± 3 31 ± 1 5 ± 0.6 8 ± 0.5

[‘3C]phenylalanine (4 h) + prime[2H2]tyrosine (4 h) + prime 48 ± 2 34 ± 3 5 ± .05 7 ± 0.7

5.�±SE;n = 6.

dietary phase ofthe investigation. The carbon dioxide production

rates and ‘3CO2-enrichment data are presented in Table 5. From

these isotopic data estimates of amino acid flux, the conversion

ofphenylalanine to tyrosine (phenylalanine hydroxylation) and

of phenylalanine oxidation, based on ‘3CO2-excretion data, weremade. The estimates of the fluxes are summarized in Table 6

and the phenylalanine hydroxylation and oxidation rates arepresent in Table 7.

Mean phenylalanine and tyrosine fluxes were 5 1 and 53�mol . kg� . h’, respectively, during the fasting state for subjectsreceiving the Adequate N diet and the fluxes were only slightly

but not significantly higher when measured for the fed state (Ta-

ble 6). The rate of phenylalanine oxidation (Table 7) differed

between the fed and fasted states when subjects consumed the

Adequate N diet, with the rate being significantly higher (P

< 0.01) for the fed condition. Phenylalanine hydroxylation was

more variable and although not statistically significant also

showed a similar pattern of change from fasted to fed states,

although these estimates were not numerically identical to those

for phenylalanine oxidation.

Reducing the amount of phenylalanine and tyrosine in the

diet for 1 wk but without altering the intake of all other indis-

pensable amino acids and of total nitrogen did not affect phe-

nylalanine or tyrosine flux in the fasted state but tyrosine flux

was lower (P � 0.05) during the fed condition. The conversion

of phenylalanine to tyrosine (hydroxylation) and the oxidation

of[’3C]phenylalanine for the fed period did not differ from that

for the fasting condition when dietary phenylalanine and tyrosine

intakes were restricted, in contrast to the differences observed

between these metabolic states for the Adequate N diet.

Phenylalanine fluxes remained essentially unchanged during

the 3-wk dietary period when the total nitrogen, indispensable

amino acid, and phenylalanine and tyrosine intakes all werereduced. There was a tendency for tyrosine flux to decline; in

the fasted state and in the fed state it was less (P � 0.05) than

when the diet supplied generous aromatic amino acid intakes.Also, leucine fluxes were lower when the restricted diets were

consumed. The conversion of phenylalanine to tyrosine (Q�;

hydroxylation) and ofphenylalanine oxidation was similar dur-ing the fed and fasted states for the restricted diets. Furthermore,the fasted and fed rates of phenylalanine hydroxylation and ox-idation did not differ (P > 0.05) between the Adequate N:Low

Phe/Tyr and the Low N:Low Phe/Tyr diets.

As summarized in Table 8, the alterations in the concentra-tions of free phenylalanine and tyrosine in circulating bloodplasma paralleled the changes described for phenylalanine hy-

droxylation and oxidation; the concentrations of phenylalanine

and tyrosine were higher in the fed than in the fasted state when

the supply of the amino acid was generous together with an

Adequate N intake. Fasting plasma phenylalanine and tyrosineconcentrations were similar throughout the various diet-meta-

bolic periods but they were lower during the fed state when

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522 CORTIELLA ET AL

51±SE.

t Significantly lower than Adequate N, P � 0.05.

TABLE 5Rate ofcarbon dioxide production and ‘3C enrichment ofexpired airin subjects receiving [‘3C]phenylalanine under different dietary

conditions5

Diet

Carbon dioxideproduction

13C Enrichmentof expired air

Fasted FedFasted Fed

mL/min atom % excess X 1O�

Adequate N 203 ± 5 286 ± 12 3.3 ± 0.4 5.0 ± 0.6

Adequate N:LowPhe/Tyr 193 ± 10 264 ± 8 3.1 ± 0.5 3.6 ± 0.7

Low N:Low Phe/Tyr

1 wk 197± 11 253± 13 2.4±0.3 2.5 ±0.1

3 wk 197 ± 13 254 ± 7 3.1 ± 0.5 3.1 ± 0.7

51±SE:n= 5.

phenylalanine and tyrosine intakes were reduced, compared with

those for the Adequate N diet period.

Further, in reference to leucine fluxes, based on measurement

of plasma [‘3C]KIC enrichment, for the fasted state, phenylal-anine flux was �47% of the leucine flux when subjects were

studied during the initial, Adequate N diet period. After 3 wk

at the Low N:low Phe/Tyr intake, phenylalanine fluxes during

the fasted and fed periods were 63% and 59% ofthe leucine flux,suggesting that the relationships between plasma and intracellular

labeling ofleucine and phenylalanine might differ according to

prior dietary history. Additional comments concerning intra-

cellular labeling of phenylalanine and tyrosine are given below.

Discussion

There have been few studies concerned with the changes of

whole-body phenylalanine and tyrosine kinetics in healthy adult

subjects given various intakes of protein (nitrogen) or of these

two aromatic amino acids. On the basis of an interpretation of

our previous studies on the kinetics of whole-body amino acidmetabolism, we have proposed substantial revisions, relative to

current recommendations (33-35), in the estimates of amino

acid requirements in adults (16-18, 36, 37). Hence, to begin to

explore further the metabolic basis of and quantitative require-

TABLE 6

ments for phenylalanine and tyrosine and their metabolic in-

terrelations, we considered it worthwhile to evaluate and apply

a phenylalanine-tyrosine stable-isotope tracer model as originallydescribed by Clarke and Bier (19) and recently modified by

Thompson et al (20). Our purpose was to investigate the impactof reduced nitrogen, phenylalanine, and tyrosine intakes onphenylalanine and tyrosine kinetics and phenylalanine hydrox-

ylation and oxidation in healthy young adult men.

In an initial series of pilot studies we assessed the suitability

ofconducting a shorter tracer protocol than that used originallyby Clarke and Bier ( 1 9), which involved infusing labeled phe-

nylalanine for a 12-h period. In support of the findings by

Thompson et al (20), we found that with a priming dose of theappropriate phenylalanine and tyrosine tracers estimates of phe-nylalanine and tyrosine fluxes, phenylalanine hydroxylation and

oxidation could be obtained within a 3-h period. The values for

the fluxes of phenylalanine and tyrosine and of phenylalanine

and hydroxylation obtained in these pilot studies, involving dif-ferent combinations of tracers and periods of infusion, ranging

from 12 to 4 h for phenylalanine, were generally similar to thosereported by Clarke and Bier (19) and Thompson et al (20).Furthermore, our estimates of phenylalanine hydroxylation,

and oxidation, for the postabsorptive condition, (�6-9jzmol - kg’ . h’) are also comparable to those given in these

published studies ( I 9, 20).As emphasized by Scriver et al (1), an estimate ofthe relative

distribution of phenylalanine disposal according to its incor-

poration into proteins, oxidation, and/or metabolic conversion

to tyrosine is of theoretical and practical interest. From Kauf-man’s (38) studies of rat liver, Scriver et al (1) and Scriver and

Clow (39) have suggested that, at physiological concentrations,the hydroxylation of phenylalanine accounts for about three-quarters of the disappearance of phenylalanine from the free

phenylalanine pool, whereas incorporation into proteins

amounts to about one-quarter. Although this appears to be rea-sonable for the liver, the relationships for whole-body phenyl-alanine metabolism in the human adult are different because

the rates ofdaily protein synthesis and degradation are of aboutthree or more times that ofa usual protein intake (40, 41). This

means that the rate of phenylalanine hydroxylation would bemuch lower, relative to the rate ofphenylalanine disappearancevia protein synthesis, at the whole-body level.

We find that phenylalanine oxidation and hydroxylation rates

were the components ofwhole-body phenylalanine kinetics most

affected by a reduced total protein or restricted phenylalanine

Phenylalanine, tyrosine, leucine fluxes in young men receiving different nitrogen and aromatic amino acid intakes5

Diet

Phenylalanine Tyrosine Leucine

Fasted Fed Fasted Fed Fasted Fed

�mol.kg’.h’

AdequateN 51±2 58±3 53±6 66±9 105±3 128±4

Adequate N:Low Phe/Tyr 57 ± 7 55 ± 1 1 47 ± 5 41 ± 8t 89 ± 7t 100 ± lotLow N:Low Phe/Tyr

lwk 51±1 65±8 47±7 53±4t 86±5t 91±4t3wk 49±1 50±2 36±2 39±2t 78±7t 85±8t

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PHENYLALANINE KINETICS 523

5 1 ± SE. Means in same column with different superscripts are sig-nificantly different from each other, P < 0.05.

TABLE 7

Phenylalanine hydroxylation and oxidation in young men receiving

different nitrogen and aromatic amino acid intakes5

Diet

Phenylalanine Phenylalanine

hydroxylation oxidation

Fasted Fed Fasted Fed

�zmol. kf’ .

Adequate N 9.3 ± 1.7 15.1 ± 3.3 5.9 ± 0.7 13.6 ± 2.3t

Adequate N:Low

Phe/Tyr 9.2 ± 1.5 8.8 ± 1.9 6.2 ± 1.6 7.3 ± 1.1

Low N:Low Phe/Tyr

1 wk 5.5 ± 0.6 7.4 ± 0.8 5.1 ± 0.3 6.9 ± 1.5

3 wk 6.5 ± 1.3 6.2 ± 1.1 6.0 ± 0.4 6.6 ± 2.0

*j;� SE.

t Significantly different from fasted, P < 0.01.

and tyrosine intake, especially during the fed state. Phenylalanine

oxidation was stimulated by the ingestion of the Adequate N

diet but with a restricted intake of phenylalanine and tyrosine,

either associated with maintenance of an adequate intake of

total nitrogen and of the other indispensable amino acids, or in

combination with lower nitrogen and other indispensable amino

acids, there was no meal-induced increase in phenylalanine hy-

droxylation and oxidation rates. Also there was a lack of any

further detectable changes in phenylalanine flux, hydroxylation,

and oxidation between 1 and 3 wk at low nitrogen and aromatic

amino acid intakes. These observations are consistent with the

findings of Zello et al (42), showing that oxidation of phenyl-

alanine during ingestion of test meals supplying various phe-

nylalanine intakes was not affected by first adapting subjects to

low phenylalanine intakes, either 4. 1 or 14 mg . kg � . d � in the

presence of excess tyrosine, for � 9 d. The present estimates of

fed-state phenylalanine fluxes, when subjects consumed the Ad-

equate N diet, also are in good agreement with those of �50-

60 �mol . kg’ . h1, reported by Zello et al (42).

The sensitivity ofphenylalanine hydroxylation and oxidation

to altered intakes ofthe amino acid has been demonstrated pre-

viously in humans and experimental animals (43), and our find-

ings provide further in vivo evidence for the regulation of phe-

nylalanine hydroxylation as a key step in the maintenance of

whole-body phenylalanine homeostasis under various dietary

conditions in healthy adults. We recognize that there are other

possible pathways of phenylalanine catabolism that might ac-

count for the lower oxidation ofphenylalanine during a restricted

amino acid intake. These include transamination ofthe amino

acid but this appears not to be a quantitatively important route,

at least in rats (43-45). Our comparative studies with various

isotopomers of phenylalanine support the view that a reversible

transamination ofphenylalanine does not contribute significantly

to the overall flux of phenylalanine in healthy adult men (32);

this alternative pathway ofphenylalanine removal may become

important, however, under conditions of an intrinsically low

PAH activity or in phenylalanine overload (43).The present tracer model, as pointed out by Thompson et al

(20), permits an estimation ofthe rate ofconversion of phenyl-

alanine to tyrosine and this requires only analysis ofblood sam-

ples. Thus, for purposes of estimating whole-body protein syn-

thesis, it offers, in theory, an advantage over the more widely

used [‘3C]leucine method (46). This latter tracer approach re-

quires collection ofexpired air (or plasma) for isotope-ratio mass

spectrometry analysis, and determination of carbon dioxide

production to calculate whole-body amino acid oxidation and

protein synthesis. These procedural and analytical requirements

add complexity to the tracer study, particularly where it might

be difficult to measure respiratory gas-exchange rates in certain

clinical settings, or if the analytical equipment required for gas

isotope measurements is not readily available. Any possible ad-

vantage of the phenylalanine-tyrosine model would depend,

however, on the accuracy of the estimate of phenylalanine hy-

droxylation, which, as Millward et al (47) have concluded, is

likely to be underestimated by the present model, thus overes-

timating the rate of uptake of phenylalanine into proteins. The

hydroxylation takes place principally in the liver and it is highly

probable that the plasma enrichment of[’3Cjphenylalanine and

of [2H]tyrosine is higher than that of the amino acids at the site

of phenylalanine hydroxylation and subsequent oxidation of

these amino acids. Thus, the measured value of hydroxylation,

based on equation 2 in Methods, will be less than the true ratebecause both the values used in the equation for phenylalanine

and tyrosine fluxes will be lower than actual whole-body fluxes.

For determination ofleucine flux and oxidation rates, the plasmaisotope enrichment of KIC has been used as an index of the

intracellular labeling ofleucine and this appears to be a generally

reliable approach (48-50). There is no analogous marker in

plasma for estimation of the intracellular enrichment of phe-

nylalanine or tyrosine and so the question arises as to the extent

of the underestimation of phenylalanine hydroxylation and of

its subsequent oxidation in the present study, as well as in the

published studies (19, 20, 42), referred to above.

Millward et al (47) have suggested an approach for developing

correction factors for adjustment oftyrosine and phenylalanine

fluxes for the postabsorptive state, based on the assumption that

KIC enrichment is a reliable estimate ofthe labeling ofthe whole-

body intracellular leucine pool and that the molar ratios of ty-

rosine to leucine and ofphenylalanine to leucine in body proteins

are 0.3 and 0.46, respectively. The correction factors proposed

by Millward et al (47) were �0.6 for the tyrosine flux and for

phenylalanine it was 0.8 (fed) and 1 (fast). From the results

of a recent study conducted in our laboratories using

[2H5]phenylalanine and [2H2]tyrosine (JS Marchini, I Cortiella,

TE Chapman, VR Young, unpublished observation, 1992), we

TABLE 8Plasma free phenylalanine and tyrosine concentrations under different

dietary conditions during fast and fed states5

Phenylalanine Tyrosine

Diet Fasted Fed Fasted Fed

Mmo//L

Adequate N 60 ± 4 78 ± 3� 60 ± 4 79 ± 7�Adequate:Low Phe/Tyr 56 ± 4 52 ± 3b 53 � 5 45 � 6�’Low N:Low Phe/Tyr

lwk 57±2 6l±3c 56±3 52±2k’

3wk 59±4 63±3c 58±6 54�4b

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524 CORTIELLA ET AL

have estimated, using a similar approach, that the correction

factor for the [2H2jtyrosine flux is �0.6 and the correction factor

for the plasma phenylalanine would be �0.78. Hence, if thesecorrection factors are appropriate, and they are similar to thoseproposed by Millward et al (47), the true rate of phenylalanine

hydroxylation would be

Q�t X 1/0.78 X 0.6 = Q�t x 1/0.47

or approximately twice the measured rate. It is not certain, how-

ever, whether these are satisfactory corrections but they suggestthe potential for a significant underestimate of the actual rates

of phenylalanine hydroxylation.

The uncertainty in calculating phenylalanine hydroxylationto tyrosine and the subsequent oxidation ofthe tyrosine arising

from that phenylalanine concerns, in part, the extent to which

the tyrosine derived from the phenylalanine equilibrates with

the free tissue tyrosine pool. Zello et al (42), based on their

[‘3C]phenylalanine infusions in healthy adults and from Mol-

dawer et al (5 1 ), whose studies were carried out in rats, haveproposed that the tyrosine formed via phenylalanine hydrox-ylation is preferentially oxidized without first equilibrating withthe tyrosine-free pool. If this is the case then the correction fac-

tors, especially for the tyrosine flux, proposed by Millward et al

(47) would overestimate the rate of phenylalanine hydroxylation

or oxidation. The similar, although not numerically identical,

estimates ofbody phenylalanine loss based on determination of

� in the recent study and on 13CO2 excretion from[13C]phenylalanine, suggests to us that the overall correction

factors (ie, 0.5-0.6) proposed by Millward et al (47) deserve fur-ther evaluation.

We might consider, therefore, an alternative approach, by

making the reasonable assumption that the Adequate N diet

supplied a more than just sufficient daily intake oftotal aromatic

amino acids (phenylalanine plus tyrosine) to meet minimum

physiological needs. This intake was 54 mg (327 �mol) phenyl-

alanine and 40 mg (220 �mol) tyrosine - kg� - d’ [a total of 94mg (�550 �tmol) aromatic amino acid - kg’ - d’], which com-

pares with the much lower international estimate ofthe aromatic

amino acid needs in healthy adults of 14 mg (�85

�zmol) . kg� - d�’ (33). A higher estimate for the physiologic needs

has been proposed by us, namely 39 mg (�236 �mol) - kg’ - d�,

(17), which is comparable to the 30-mg (� 182 j�mol) value sug-

gested by Zello et al (42). Thus, the Adequate N diet could be

reasonably assumed to supply aromatic amino acids in consid-

erable excess of the physiological requirement. Therefore, taking

the mean hydroxylation rates determined in subjects receiving

the Adequate N diet and assuming that the hourly rates measured

during the fasted and fed conditions can be extrapolated to each

1 2-h period (1 2 h fast and 12 h fed) of the day, our total daily

rate ofphenylalanine hydroxylation is estimated to be 293 Mmol/

kg. Hence, on the assumption that the subjects were in zero-

body phenylalanine balance, then it appears that our estimateof the daily rate of hydroxylation is lower than the intake by

� 10% [ie, (327-293/327) X 100]. The underestimation in this

case is calculated to be �28% when the ‘3C oxidation data,

which gave lower values for rates of phenylalanine oxidation,

were used. Also, it is likely that we may have overestimated

phenylalanine balance for the fed state because a portion of di-etary phenylalanine might undergo a first pass oxidation in thesplanchnic region before it equilibrates with the intravenous

tracer. Hence, this range of 10-28% for the underestimationmay be too high.

Finally, we found that the subjects were in apparent negative

phenylalanine balance (- 18 �mol . kg � . d ‘) at the end of the

3-wk-diet period. We did not expect this, given the amount of

dietary phenylalanine and tyrosine supplied, but current esti-mates ofthe average minimum requirements for aromatic amino

acids in healthy adults remain poorly established. In this context,

Zello et al (42), in agreement with our findings, have concludedthat the requirement for aromatic amino acids is significantly

higher than that proposed by FAO/WHO/UNU (33). However,

note that the phenylalanine oxidation rates reported by these

investigators are much lower than we find in our study, for corn-

parable phenylalanine intakes, and also they are lower than the

hydroxylation rates reported by Clarke and Bier (19) and

Thompson et al (20) for healthy subjects in the postabsorptive

state. Thus, the low rates of phenylalanine oxidation reported

by Zello et al (42) make interpretation of the nutritional irnpli-

cations of their findings rather difficult. Hence, further studies

directed toward a determination of the quantitative needs for

the aromatic amino acids would be highly desirable. Particularly,

it is important to define the extent to which tyrosine can “spare”

the requirement for phenylalanine, which, as emphasized by an

earlier expert group (52), has not been defined precisely. C]

We thank the volunteers for their willingness to participate in our

studies. The amino acids used were generously donated by Ajinomoto

USA, Inc, courtesy of E Gotto. The Vivonex flavor packets were kindly

donated by Norwich Eaton Pharmaceutical, Norwich, NY.

References

1 . Scriver CR, Kaufman 5, Woo SLC. 1989. The hyperphenylalanin-

emias. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The met-abolic basis ofinherited disease. 6th ed. Vol 1. New York: McGraw-Hill, Inc, 1989:495-546.

2. Wang T, Okano Y, Eisensmith RC, et al. 1991. Founder effect of aprevalent phenylketonuria mutation in the Oriental population. Proc

Natl Acad Sci USA 199l;88:2146-50.3. Freedland RA. Factors affectingthe activity in vitro ofthe tryptophan-

and phenylalanine-hydroxylating systems. Biochim Biophys Acta1963;73:7 1-5.

4. Greengard 0, Delvalle IA. The regulation of phenylalanine hy-droxylase in rat tissues in vivo. Substrate- and cortisol-induced dc-vations in phenylalanine hydroxylase activity. Biochem I l976;l54:

619-24.5. Abita J-P, Milstien 5, Chang N, Kaufman S. In vitro activation of

rat liver phenylalanine hydroxylase by phosphorylation. I Biol Cheml976;25 1:5310-4.

6. Pogson CL, Santana MA, Fisher MI. Phenylalanine hydroxylase:metabolic aspects. Biochem Soc Trans 1985;l3:439-41.

7. Tourian A. Activation of phenylalanine hydroxylase by phenylala-

nine. Biochim Biophys Acta 197 l;242:345-54.8. Goldsmith LA, Laberge C. Tyrosinemia and related disorders. In:

Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic basisof inherited disease. Vol 1. p. 547-562, New York: McGraw-Hill,

Inc, 1989:547-62.9. Pogson CI, Knowles RG, Salter M. The control of aromatic amino

acid catabolism and its relationship to neurotransmitter amine syn-thesis. Crit Rev Neurobiol 1989;5:29-64.

10. Ozalp I, Young VR, Nagchaudhuri I, Tontisirin K, Scrimshaw NS.Plasma amino acid response in young men given diets devoid ofsingle essential amino acids. I Nutr 197l;102:l 147-2258.

1 1. Fernstrom JD, Wurtman RI, Hammarstrom-Wiklund B, Rand WM,Munro HN, Davidson CS. Diurnal variations in plasma concentra-

tions of tryptophan, tyrosine, and other neutral amino acids: Effectofdietary protein intake. Am I Clin Nutr 1979;32:1912-22.

by guest on July 10, 2011w

ww

.ajcn.orgD

ownloaded from

PHENYLALANINE KINETICS

12. Stegink LD, Filer LT Jr. Brummel MC, et al. Plasma amino acidconcentrations and amino acid ratios in normal adults and adultsheterozygous for phenylketonuria ingesting a hamburger and milkshake meal. Am I Clin Nutr 1991;53:670-5.

13. Thompson AJ, Smith I, Brenton D, et al. Neurological deteriorationin young adults with phenylketonuria. Lancet l990;2:602-5.

14. Davidson DC. Maternal phenylketonuria. Postgrad Med I

1989;65(suppl 2):SlO-20.15. Clark BI, Cockburn F. Management ofinborn errors of metabolism

during pregnancy. Acta Pediatr Scand Suppl 199 1;373:43-52.16. Young VR. 1987 McCollum Award Lecture. Kinetics of human

amino acid metabolism: nutritional implications and some lessons.Am I Clin Nutr 1987;46:709-25.

17. Young VR, Bier DM, Pellett PL. A theoretical basis for increasingcurrent estimates ofthe amino acid requirements in adult man, with

experimental support. Am I Clin Nutr l989;50:80-92.18. Young VR, Marchini IS. Mechanisms and nutritional significance

ofmetabolic responses to altered intakes ofprotein and amino acids,with reference to nutritional adaptation in humans. Am J Clin Nutr

l990;5 1:270-89.

19. Clarke JTR, Bier DM. The conversion ofphenylalanine to tyrosine

in man. Direct measurement by continuous intravenous tracer in-

fusions ofL-[ring-2H5]phenylalanine and L-[l-�CJtyrosine in the post

absorptive state. Metabolism l982;3 1:999-1005.20. Thompson GN, Pacy P1, Merritt H, et al. Rapid measurements of

whole body and forearm protein turnover using a [2H5]phenylalanine

model. Am I Physiol l989;256:E63l-9.

21. National Research Council. Recommended dietary allowances. 9threvised ed. Washington, DC: National Academy ofSciences, 1980.

22. Roberts SB, Morrow FJ, Evans WI, et al. Use ofp-amino benzoic

acid to monitor compliance with prescribed dietary regimens duringmetabolic studies in man. Am J Clin Nutr l990;5 1:485-8.

23. Storch KJ, Wagner DA, Burke IF, Young yR. Quantitative studyin vivo of methionine cycle in human using [methyl-2H3]- and [1-

‘3C]methionine. Am I Physiol 1989;255:E322-31.24. Melville 5, McNurlan MA, McHardy KC, et al. The role of degra-

dation in the acute control ofprotein balance in adult man: Failure

of feeding to stimulate protein synthesis as assessed by L-[1-

3C]leucine infusion. Metabolism l989;38:248-55.

25. Meguid MM, Matthews DE, Bier DM, Meredith CN, Soeldner IS,Young VR. Leucine kinetics at graded leucine intakes in young

men. Am J Clin Nutr l986;43:770-80.

26. Hoerr RA, Yu Y-M, Wagner DA, Burke IF, Young VR. Recoveryof ‘3C in breath from NaH’3CO3 infused by gut and vein: effect offeeding. Am I Physiol 1989;257:E426-38.

27. March IF. A modified technique for quantitative analysis of amino

acid by gas chromatography using heptafluorobutyric n-propyl de-rivatives. Anal Biochem 1975;69:420-5.

28. Langenbeck U, Luthe H, Schaper G. Ketoacids in tissues and bio-

logicals fluids: O-i-butyldimethylsilyl quinoxalinols as derivatives

for sensitive gas chromatographic/mass spectrometric determination.Biomed Mass Spectrom l985;12:507-9.

29. Rocchiccioli F, Leroux IP, Carter P. Quantitation of 2-keto-acids

in biological fluids by gas chromatography chemical ionization massspectrometry of O-trimethylsilyl-quinoxalinol derivatives. Biomed

Mass Spectrom 1981:8:160-4.

30. Matthews DE, Motil KJ, Rohrbaugh D, Burke IF, Young VR, Bier

DM. Measurement of leucine metabolism in man from a primed,continuous infusion of L-[1-’3Cjleucine. Am I Physiol 1980;238:E473-9.

31. Matthews DE, Schwartz HP, Yang RD, Motil KJ, Young VR, Bier

DM. Relationship of plasma leucine and a-ketoisocaproate duringa L-[l-’3C]leucine infusion in man: a method for measuring humanintracellular leucine tracer enrichment. Metabolism 1982;31:l 105-

I 2.

525

32. Krempf M, Hoerr RA, Marks L, Young VR. Phenylalanine flux inadult men: estimates with different tracers and route of administra-tion. Metabolism 1990;39:560-2.

33. Food and Agricultural Organization/World Health Organization!United Nations University. Energy and protein requirements. Report

of a joint FAO/WHO/UNU expert consultation. WHO Tech Rep5cr l985;724: 1-204.

34. Rose WC. The amino acid requirements ofadult man. Nutr Abstr

Rev 1957;27:63l-47.35. Irwin MI, Hegsted DM. Amino acid requirements of man. I Nutr

197 1;lOl:539-66.

36. Young VR, Gucalp C, Rand WM, Matthews DE, Bier DM. Leucinekinetics during three weeks at submaintenance to maintenance in-

takes ofleucine in men. Hum Nutr Gin Nutr l987;4lC:l-l8.37. Cortiella I, Matthews DE, Hoerr RA, Bier DM, Young VR. Leucine

kinetics at graded intakes in young men: quantitative fate of dietaryleucine. Am I Gin Nutr 1988;48:998-1009.

38. Kaufman S. Phenylketonuria: biochemical mechanisms. Adv Neu-rochem 1976;2:1-13e.

39. Scriver CR, Clow CL. Phenylketonuria: epitome of human bio-chemical genetics. N Engl I Med l980;303: 1336-42, 1394-400.

40. Young VR, Meredith C, Hoerr R, Bier DM, Matthews DE. Aminoacid kinetics in relation to protein and amino acid requirements:

The primary importance of amino acid oxidation. In: Garrow IS,Halliday D, eds. Substrate and energy metabolism in man. London:John Libbey, 1985:1 19-34.

41. Waterlow IC. Protein turnover with special reference to man. Q I

Exp Physiol 1984;69:409-38.42. Zello GA, Pencharz PB, Ball RO. Phenylalanine flux, oxidation

and conversion to tyrosine in humans studied with L-[1-‘3C]phenylalanine. Am J Physiol 1990;259:E835-43.

43. Harper AE. Phenylalanine metabolism. In: Stagink LD, Filer U Jr.eds. Aspartame physiology and biochemistry. New York: MarcelDekker Inc, 1984:77-109.

44. Munro HN. Absorption and metabolism ofamino acids with special

emphasis on phenylalanine. I Toxicol Environ Health 1976;2:l89-206.

45. Haley CI, Harper AE. The importance of transamination and de-carboxylation in phenylalanine metabolism in vivo in the rat. Arch

Biochem Biophys 1978; 189:524-30.46. Young VR, Yu Y-M, Krempf M. Protein and amino acid turnover

using the stable isotopes ‘5N, ‘3C, and 2H as probes. In: WhiteheadRG, Prentice A, eds. New techniques in nutritional research. San

Diego: Academic Press, Inc, 1991:17-72.47. Millward DI, Price GM, Pacy PIH, Halhiday D. Whole body protein

and amino acid turnover; what can we measure with confidence?Proc Nutr Soc 199 l;50:197-2l6.

48. Schwenk WF, Beaufrere B, Haymond MW. Use of reciprocal poolspecific activities to model leucine metabolism in humans. Am IPhysiol 1985;249:E646-50.

49. Horber FF, Horber-Feyder CM, Kroyer 5, Schwenk WF, HaymondMW. Plasma reciprocal pool specific activity predicts that of intra-cellular free leucine for protein synthesis. Am I Physiol l989;257:E385-99.

50. Watt PW, Lindsay Y, Scrimgeour CM, et al. Isolation of aminoacyl-tRNA and its labeling with stable-isotope tracers: Use in studies

ofhuman tissue protein synthesis. Proc Natl Acad Sci USA 199 l;88:5892-6.

51. Moldawer LL, Kawamura I, Bistrian BR, Blackburn GL. The con-

tribution of phenylalanine to tyrosine metabolism in vivo. Studies

in the post-absorptive and phenylalanine-loaded rat. Biochem Il983;2l0:81 1-7.

52. Williams HH, Harper AE, Hegsted DM, Arroyave G, Holt LE Jr.Nitrogen and amino acid requirements. In: US Food and nutritionBoard, ed. Improvement ofprotein nutriture. Washington, DC: Na-

tional Academy of Sciences. 1974:23-63.

by guest on July 10, 2011w

ww

.ajcn.orgD

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