source and amount of dietary nonspecific nitrogen in relation to whole-body leucine, phenylalanine,...

Am J Clin Nuir 1994;59:l347-55. Printed in USA. © 1994 American Society for Clinical Nutrition I 347

Source and amount of dietary nonspecific nitrogen inrelation to whole-body leucine, phenylalanine, and tyrosinekinetics in young men13

Takeyuki Hiramatsu, Joaquin Cortiella, J S#{233}rgioMarchini, Thomas E Chapman, and Vernon R Young

ABSTRACT We studied the effects of amount and source

of nonspecific nitrogen (NSN) on the oxidation of leucine and

hydroxylation of phenylalanine. In phase 1, seven adult males

received for 6 d diets providing indispensable amino acid intakes

to meet the 1985 FAOIWHO/UNU (FAO) requirements or our

proposed requirement values (MIT). During one diet period with

each diet, the NSN of the basal diets (total nitrogen intake: 107

mg N ‘ kg ‘ . d ‘) was increased to a total of 160 mg N . kg ‘ . d ‘.

On the morning of day 7, an 8-h constant intravenous tracer-

infusion protocol (3-h fast; 5-h fed state) was conducted with L-

F1 -‘3C]leucine, L-[ring-2H5]phenylalanine, and

sine as tracers. In phase 2, six subjects were given three diets for

6 d, supplying 107 mg Nkg� d’; NSN was a mixture of dis-

pensable amino acids in which glutamine accounted for 0%,

12.5%, and 100% of total NSN. Leucine oxidation and phenyl-

alanine hydroxylation rates and whole-body leucine and phen-

ylalanine balances were unaffected by addition of supplemental

NSN to the diets in phase 1 or by amino acid source of NSN in

phase 2. Leucine and phenylalanine balances were lower (P

< 0.05) for FAO compared with MIT diets. Am J Clin Nutr

l994;59: 1347-55.

KEY WORDS Leucine, phenylalanine, amino acids, kinet-

ics, requirements

Introduction

For a particular pathophysiological state the minimum intakes

of protein and of the nutritionally indispensable amino acids re-

quired to maintain nutritional status can be affected by diet.

These include the amount and source of the major dietary, en-

ergy-yielding substrates (1 -3) and, under some circumstance, the

balance, or pattern, of the indispensable amino acid intake (4, 5).

Further, the amount and source of the so-called, nonspecific (or

nonessential) nitrogen (NSN) component (6) of the total nitrogen

intake can determine the status of body nitrogen balance (7, 8).

High intakes of NSN have been thought to spare the human re-

quirement for indispensable amino acids (9), and growth studies

in animals have demonstrated the importance of providing a suf-

ficient dietary intake of NSN, or mixture of dispensable amino

acids (10-12). In human infants, Snyderman et al (13) found that

a relatively low-protein diet promoted nitrogen balance when it

was supplemented with NSN given as urea or glycine.

We conducted previously a series of isotope tracer studies to

reassess the minimum physiological requirements for various in-

dispensable amino acids (14, 15) and concluded that the most

recent estimates by national ( 1 6) and international expert groups

( 1 7) for requirements in adult humans are far too low, except for

the sulfur amino acids. However, the validity of our conclusion

has been questioned (9), particularly because the experimental

diets used in our studies may have contained insufficient NSN to

promote maximum efficiency of retention of indispensable amino

acids, in comparison with the far lower requirement estimates

made by Rose (18). In that study a relatively high amount of

NSN was often present in the experimental diets. We consider

this suggestion to be an unlikely reason for the substantial dif-

ferences between the requirements proposed by Rose ( 18) and

our new, tentative requirement values (14, 15). However, there

have not been any direct studies of measurement of the effects

of raising the dietary intake of alternative sources of NSN on the

oxidation of indispensable amino acids in healthy adult humans.

Here we describe the results of an investigation designed to

examine whether our isotopically derived amino acid require-

ment values, using leucine and phenylalanine as the test tracer

amino acids, are affected by the amount of dietary NSN. For this

purpose the test leucine and phenylalanine intakes, as well those

for the other indispensable amino acids, were either set to meet

the FAOIWHOIUNU (17) adult requirement values or the higher

values proposed by us (14, 15). This latter intake may even be

marginal for some amino acids, because we have found some

subjects, for example, that are in negative leucine balance after

receiving 40 mg leucine . kg ‘ . d ‘ for a period of � 1 wk or less

(19, 20). However, we considered it desirable to study a margin-

1 From the Laboratory of Human Nutrition and Clinical Research Cen-ter, Massachusetts Institute of Technology, Cambridge, MA, and Shrin-

ers’ Burns Institute, Boston.

2 Supported by NIH grants DK15856, DK42lOl, and RR88. andgrants from the Shriners’ Hospitals for Crippled Children (15897 and15843). The L-amino acids were donated by Ajinomoto Inc. USA. Tea-neck, NJ. TH was supported by a fellowship from Otsuka PharmaceuticalFactory and JSM was supported by NIH grants I 5F05TW04236 and

CNPqBR 200535-88.9.

3 Reprints not available. Address correspondence to VR Young, RoomE18-6l3, Massachusetts Institute of Technology, Cambridge, MA02139.

Received January 14, 1993.Accepted for publication November 22, 1993.

by guest on July 14, 2011w

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1348 HIRAMATSU ET AL

ally limiting intake of leucine rather than one well in excess of

the requirement in order to assess whether NSN spares, to a nu-

tritionally significant extent, the oxidation of indispensable

amino acids.

Additionally. we examined the kinetics of leucine, phenylala-

nine, and tyrosine metabolism in relation to the dietary source of

NSN and especially when glutamine served as a major source of

dietary NSN. The glutamine pool plays an important homeostatic

role in the amino acid and nitrogen economy oforganisms, acting

as a significant source of nitrogen within the free amino acid pool

in muscle and as a vehicle for nitrogen transport in times of stress

(21 -23). Furthermore, there is now increased evidence suggest-

ing that glutamine is best classified as a ‘ ‘conditionally indispen-

sable’ ‘ amino acid (24) and that exogenous glutamine can have

a beneficial effect in catabolic states (25).

Subjects and methods

Subjects

Thirteen adult male volunteers (aged 20-27 y, 64.7-80.5 kg

body wt) participated in one of the two phases of this study. They

were studied as outpatients at the Clinical Research Center

(CRC) of the Massachusetts Institute of Technology (MIT). All

were in good health as determined by a medical history, physical

examination, and screening laboratory tests (complete blood

count, and a complete chemistry profile including liver enzymes,

negative hepatitis-B surface antigen) performed before entry into

the study. None of the subjects had a history of recent weight

loss, unusual dietary practices, endocrine disorders, pharmaco-

logical therapy, or hormonal treatment. Their daily energy intake

was designed to maintain body weight, based on a dietary history

and an estimate of the level of physical activity. The subjects

were encouraged to maintain their usual levels of physical activ-

ity, but were not permitted to participate in competitive sports.

The potential risks involved were explained fully to each subject.

Signed informed consent was obtained for participation in the

study in full accordance with, and approval of, the MIT Com-

mittee on the Use of Humans as Experimental Subjects and the

Advisory Committee of the MIT Clinical Research Center. The

subjects received financial compensation for their participation

in the experiment. They remained healthy throughout the inves-

tigation.

Diets

The study was conducted in two phases, phase 1 examined

whether a change in the intake of NSN affected the rates of leu-

cine oxidation and phenylalanine hydroxylation at one of two

combined, intakes of leucine (14 and 40 mg . kg ‘ . d’), respec-

tively. The supply of the other indispensable amino acids was

either to meet the 1985 FAO/WHOIUNU (17) values (at the

lower leucine and phenylalanine intakes) or to reach the higher,

tentative requirement values that we have proposed (14, 15); 40

mg leucine and 26 mg phenylalanine and 13 mg tyrosine. Hence,

four diets were studied; each was given to seven subjects for 6

d in random order. On the morning of the seventh day a tracer-

infusion study was conducted as described below. Between each

7-d period the subjects were given a break period of a few days,

during which they consumed a fully adequate diet based on usual

foods.

The total ‘ ‘protein’ ‘ (nitrogen) intake varied with the experi-

mental diets. Each subject received a series of isoenergetic diets

based on a crystalline L-amino acid mixture (amino acids were

donated by Ajinomoto, Inc, Teaneck, NJ), (Table 1), and given

with an equal weight of beet sucrose and a flavoring agent (Vi-

vonex flavor packets; donated by Norwich Eaton Pharmaceutical,

Norwich, NY). Diet I was formulated with an L-amino acid mix-

ture to supply 107 mg Nkg� d� [equivalent (N x 6.25) to

0.65 g protein�kg� d’J and contained indispensable amino ac-

ids to meet the FAOIWHOIUNU (17) requirements, together

with dispensable or ‘ ‘conditionally’ ‘ indispensable amino acids

(arginine, alanine, aspartic acid, cystine, glutamic acid, glycine,

proline, serine, and tyrosine). The diet 2 amino acid mixture was

essentially the same as diet 1 , except that the content of seven

dispensable amino acids, while maintaining constant cystine and

tyrosine, was increased so that the diet supplied 160 mg

N ‘ kg ‘ . d ‘, (or I .0 g protein . kg ‘ ‘ d ‘). Diet 3 contained the

same amount of nitrogen as diet I , but the amount of indispen-

sable amino acids was adjusted to meet the new, tentative re-

quirements that we proposed for healthy adults (15). Diet 4 was

essentially the same as diet 3, except that the dispensable amino

acid component was increased, as in diet 2, to supply 160 mg

Nkg’ d�.

The daily energy intake varied among subjects but was kept

constant within each individual. The energy intake was designed

to approximate their habitual intakes, which ranged from 170 to

209 kJ kg’ d�, as estimated by the dietitian according to in-

dividual needs after careful dietary history. The energy source

was derived from beet sucrose, protein-free cookies (made from

wheat starch), and essentially protein-free beverages, as previ-

ously described (1 7). These sources were chosen for their rela-

tively low ‘3C enrichment, to minimize change in the ‘3CO2 en-

TABLE I

Amino acid mixtures used to study effects of varying intakes ofdispensable amino acids in young men (phase 1)

Amino acid Diet 1 Diet 2 Diet 3 Diet 4

mgkg’ b ody wtd�’Indispensable amino acids

L-Histidine 12 12 12 12

L-Isoleucine 10 10 23 23L-Leucine 14 14 40 40L-Vahne 10 10 20 20

L-Lysine 12 12 30 30L-Methionine 13 13 13 13L-Phenylalanme 8 8 26 26L-Threonine 7 7 15 15L-Tryptophan 3.5 3.5 6 6

Conditionally indispensable ordispensable amino acids

L-Alanine 83 134 72 123L-Arginine 49 79 43 73L-Asparagine 13 199 108 184L-Cysteine 13 13 13 13L-Glutamate 136 220 119 203

Glycine 70 113 61 104L-Proline 107 173 93 159L-Serine 97 157 85 145L-Tyrosine 5 5 13 13

Total amino acids 772 1182 792 1201

Total nitrogen 107 160 107 160


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NITROGEN AND AMINO ACID KINETICS 1349

richment of breath samples when the diet was given during the

fed phase of the isotope tracer protocol. The subjects were given

three isoenergetic, isonitrogenous meals per day, at 0800, 1200,

and 1700, except on the day of the tracer studies, as described

below. Subjects consumed all meals in the MIT CRC.

The diets supplied all essential vitamins and minerals, to meet

or exceed current recommended dietary allowances (26). These

were supplied as supplements of multivitamins with minerals.

Water intake was ad libitum. Decaffeinated coffee, without milk

or sugar, was allowed.

On the night before the tracer infusion, the subjects did not eat

or drink fluids other than water, after 2200 until the tracer-infu-

sion study began on the following morning. After infusion the

subject returned to the experimental diet dictated by the protocol

until the next infusion.

In phase 2 we wished to determine whether the composition

of the amino acid mixture supplying the NSN had an impact on

the dynamic status of whole-body leucine and phenylalanine me-

tabolism; three experimental periods were included, each pro-

vided during a 6-d diet period that was followed by an isotope

tracer study on the seventh day. Each subject received, in a ran-

domized order, the three different isoenergetic, isonitrogenous

diets differing in glutamine content (Table 2). Between each ex-

perimental diet period these subjects were also given a break

period of a few days, during which they consumed an adequate

diet based on normal foods. For all three diets, total nitrogen

intake was 107 mg N . kg ‘ . d F, with the indispensable amino

acid content formulated to meet our proposed requirements (14,

15). In the first diet the NSN component was formulated without

glutamine. In the second, glutamine accounted for 12.5% of NSN

and in the third all of the NSN was supplied as glutamine. The

lower value of total dietary nitrogen was chosen for this study

because it has been demonstrated in nitrogen balance studies (7)

that as total nitrogen intake increases, any differences in the ef-

fectiveness of varying sources of supplemental NSN become less

apparent or are eliminated.

Tracer studies

The primed continuous tracer-infusion approach, using L-[ 1 -

‘3C]leucine, L-[ring-2H5jphenylalanine, and L-[2H2]tyrosine, was

used as previously described ( 1 ). Isotopes were purchased from

Cambridge Isotopes Laboratories, Woburn, MA. The chemical

and isotopic purity of the labeled leucine was confirmed by gas

chromatography - mass spectrometry. Infusates were demon-

strated to be sterile and free of pyrogens before use in the ex-

periments. Isomeric purity was assessed by gas chromatography

using a chiral column. Further details concerning preparation of

the isotopes for infusion and their administration are described

elsewhere (19).

Each subject fasted during a 10- 12-h period overnight before

the isotope infusion study. On this morning subjects arrived at

the CRC infusion room at 0600. Before isotope infusion, baseline

blood and expired air samples were obtained. They were then

stored on ice until the plasma samples were separated and kept

frozen (-80 #{176}C)until they were analyzed. Several measurements

of respiratory exchange were also obtained by open-circuit, in-

direct calorimetry (30 mm) by using a ventilated hood during the

infusion.

At 0800, priming doses of NaH’3CO3 (1.2 j�molIkg), L-

[3,3,2H2]tyrosine (2.4 ,umol/kg), L-[-’3C]leucine (4.0 j.�molIkg),

and L-[ring-2H5]phenylalanine (2.6 �zmolIkg) were given. fol-

TABLE 2Amino acid mixtures used to study effects of varying sources of

nonspecific nitrogen in young men (phase 2)

Amino acid Diet 1 Diet 2 Diet 3

m gkg bod�’ w’�’

Indispensable amino acids

L-Histidine I 2 12 12

L-Isoleucine 23 23 23

L-Leucine 40 40 40

L-Valine 20 20 20

L-Lysine 30 30 30

L-Cysteine I 3 13 13

L-Methionine 13 13 13L-Phenylalanine 26 26 26

L-Tyrosine I 3 13 13

L-Threonine 15 15 15

L-Tryptophan 6 6 6

Sources of nonspecific nitrogenL-Alanine 72 63 0

L-Arginine 43 37 0

L-Asparagine 108 94 0

L-Glutamate I 19 104 0

Glycine 61 53 0

L-Prohfle 93 82 0

L-Serine 85 74 0

L-Glutamine 0 52 414

Total amino acids 791 771 625

Nitrogen 107 107 107

lowed immediately by constant infusions of 4.0, 2.6, and 2.4

�mol . kg F . h F of the amino acid tracers, respectively. Also, 0.42

4umol L-[ring-2H4ltyrosine/kg was used to prime the tyrosine pool

formed via hydroxylation of L-[ring-2H5Jphenylalanine. The con-

tinuous intravenous infusions of tracers lasted for 8 h. After 3 h

the subjects received their respective experimental diets. The diet

was consumed by each subject at a rate to supply the equivalent

of one-twelfth the total daily amino acid and energy intake per

hour for 5 h. The diets were given in the form of cookies and the

appropriate amino acid mixture that were consumed at hourly

intervals. Using this protocol we could explore whether leucine

and/or phenylalanine metabolism differed among the various diet

groups during the postabsorptive (fasted) or absorptive (fed)

phases of amino acid metabolism.

Blood collections

Before tracers were administered, three baseline blood samples

(each 4 mL) were collected in heparinized evacuated tubes (T-

218U; Venoject, Terumo Medical Corp. Elkton, MD) and then

again at 60, 1 20, 140, 160, 1 80, 360, 420, 440, 460, and 480 mm

into the tracer-infusion period. They were stored on ice and cen-

trifuged at 4 #{176}Cfor 15 mm at 1000 x g. Aliquots of plasma were

stored separately at - 80 #{176}Cuntil analyzed for free amino acid

concentrations, and isotopic enrichment of phenylalanine, tyro-

sine, and a-ketoisocaproate (MC).

Breath samples

Samples of expired breath for ‘3C02 analysis were collected in

a disposable rubber bag, with subjects occluding their nostrils

during collection. In addition to the two baseline breath samples,

expired air was collected at each blood collection time point and


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introduced into 20-mL red-top evacuated tubes. Subsequent han-

dling and analysis of samples for ‘3C enrichment was as de-

scribed previously (27).

Total carbon dioxide production (VCO2) and oxygen con-

sumption (V02) were determined with the aid of a custom-made,

ventilated-hood, indirect calorimeter system described previ-

ously (27). For the postabsorptive phase this determination was

made between 90 and 120 mm of the infusion period; for the

absorptive phase it was between 360 and 420 mm of the tracer

period.

Amino acid isolation, derivatization, and analysis

For isotopic analysis of MC, phenylalanine, and tyrosine the

procedures were as follows: All measurements of plasma amino

acids and MC enrichment were carried out on a Hewlett-Packard

gas chromatograph (HP 5890 Series II) coupled to an HP 5988A

quadrupole mass spectrometer and an HP RTE-A data system

(Palo Alto, CA). Electron impact ionization was carried out at

120 eV.

KIC. Deproteinization of 300 �L plasma was carried out with

methanol. After centrifugation, the supernate was evaporated to

dryness under nitrogen and redissolved in 200 j.tL distilled water.

A quinoxalinol derivative was prepared (28) by using a 1% so-

lution of 1,2-phenylenediamine in hydrochloric acid and heating.

The solution was extracted twice with ethyl acetate and evapo-

rated to dryness. The residue was taken up in 50 �.tL N-methyl-

N-tert-(butyldimethylsilyl) trifluroacetamide (MTBSTFA,

Pierce, Rockford, IL) and 50 �iL pyridine and the quinoxalinol-

t-butyldimethylsilyl derivative was formed by leaving it to stand

overnight at room temperature. One microliter was sufficient for

gas chromatograph-mass spectrometer analyses by selective ion

monitoring (SIM). Analysis of MC was carried out on a 30 m

x 0.25 mm DB 1701 fused silica capillary column (J and W

Scientific, Folson, CA)temperature programmed from 140 to 300

#{176}Cat 10 #{176}C/min.MC eluted at 7.2 mm and was monitored at its

base peak m/z 259[M-571� and the ‘3C-labeled species at m/z

260, corresponding to loss of the t-butyl group. Plasma MC en-

richments were determined against standard mixtures containing

from 0% to 10% labeled [1-’3C]KIC. The calibration graphs had

slopes close to unity, y intercepts of 22%, and correlation coef-

ficients > 0.99. Each sample was measured in duplicate and four

different samples were measured during the isotopic plateau.

Phenylalanine and tyrosine. To 200 j�L plasma, 1 mL 1 mol

acetic acid/L was added and the mixture passed over an ion-

exchange column (AG5OW-X8 resin; BioRad, Richmond, CA)

(29). The fraction containing the amino acids was eluted from

the column with ammonium hydroxide and was then evaporated

to dryness. A tertiary butyldimethylsilyl derivative of the amino

acids was prepared in a one-step procedure by heating the residue

for 1 h at 60 #{176}Cwith 50 �iL MTSBSTFA and 50 �tL acetonitrile.

Two to three microliters of the mixture was sufficient for gas

chromatograph-mass spectrometer analysis, which was carried

out in one run by using a 30 m x 0.25 mm DB 1301 fused silica

capillary column (J and W Scientific), temperature programmed

from 200 to 300 #{176}Cat 15 #{176}C/min.Phenylalanine and tyrosine

eluted at 5.6 and 7.8 mm, respectively. SIM of both amino acids

was carried out on the M-57 fragment ion. SIM was carried out

at m/z 336 and m/z 341 for natural and [2H5]phenylalanine, re-

spectively. Tyrosine was monitored at m/z 466, m/z 468, and

m/z 470 for natural, [2H2]-, and [2H4]tyrosine, respectively. Stan-

dards containing 0% to 9% [2H5J-phenylalanine were used to con-

struct calibration graphs; measured slopes and y intercepts were

1.1% and 0.04%, respectively. The correlation coefficients were

> 0.99. Tyrosine and [2H2]tyrosine were measured at m/z 466

and m/z 468, respectively, against standards prepared containing

0% to 9% [2H2]tyrosine. The slopes and y intercepts were 1.0%

and 18%, respectively. The correlation coefficients were > 0.99.

Tyrosine and [2H4]tyrosine were measured at m/z 466 and m/z

470, respectively. Again, standards containing 0% to 2.5%

[2H4tyrosine were used to construct calibration graphs. The

slopes, y intercepts, and correlation coefficients were 0.8, 1.1%,

and > 0.99, respectively.

Because the ion clusters of natural, [2H2j, and [2H4]tyrosine,

which occur around the nominal masses m/z 466, m/z 468, and

m/z 470, respectively, all overlap at m/z 470, use of the

[2H4]tyrosine calibration graph alone will result in an overesti-

mate in the [2H4jtyrosine enrichment. The problem is due to the

natural isotopes in the derivatized [2H2]tyrosine tracer contrib-

uting to the signal at m/z 470, where the [2H4tyrosine tracer is

principally measured. Hence, [2H4]tyrosine enrichment was cal-

culated as follows: first, the [2H2]tyrosine enrichment ([2H2]tyr)

was determined from the [2H2jtyrosine calibration graph. The in-

creased signal at m/z 470 due to the natural isotopes of the de-

rivatized [2H2�tyrosine was subtracted from the measured m/z

470:m/z 466 to give a corrected ratio, by using the following

equation:

Corrected m/z 470:m/z 466

= measured m/z 470:m/z 466 - ([2H2]tyr F)

where the correction factor F was derived from the y intercept of

the [2H2jtyrosine graph (a two mass shift), or from the slope of

the [2H2]tyrosine graph determined by plotting m/z 470:m/z 466

against percent enrichment. The [2l-l.�]tyrosine enrichment was

then calculated by using the [2H�]tyrosine calibration graph, tak-

ing into account the corrected ratio. This approach was validated

by using known mixtures of natural, [2H2], and [2H.�]tyrosine.

Calculated enrichments of [2H2] and [2H4tyrosine were within

3% and 5% of expected values, respectively.

Plasma amino acids

Plasma concentrations of selected free amino acids and the

leucine, phenylalanine, and tyrosine contents of infusates were

determined by HPLC (model #334; Beckman, Palo Alto, CA)

using an ion-exchange chromatographic method, with postcol-

umn derivatization with o-phthalaldehyde and quantitation with

a fluorescence detector.

Calculations ofkinetic values

The whole-body kinetic values for leucine flux and oxidation

were calculated by using the equations reported earlier (30, 31).

In brief, the model used in this study is based on the steady-state

whole-body model of amino acid metabolism (32), which as-

sumes a common metabolic acid pool through which all amino

acids move, either to enter from the diet (intake, I), or organ and

tissue protein breakdown (B), or to exit for protein synthesis (S)

or oxidative catabolism (C). Movement through the metabolic

pool is flux (Q) and thus 0 = S + C = B + I. Here we are

principally interested in C or the rate of leucine oxidation. Fi-

nally, estimates of the status of whole-body leucine balance dur-

ing the fasted and fed phases of the tracer study were made as

previously described (30). The dietary intake plus the amount of


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TABLE 3

Leucine (Leu) kinetics and balance in young men receiving different amounts and sources of nonspecific nitrogen’

Phase,

condition,and diet #{176}C022

atompercentexcess

[#{176}C]KIC

molfracrion

Leu flux

jmzolkg’h’

Leu intake

�nolkg’h’

Leu ox

p.inolkg’h’

Leu balance

p.inolkg’h’

Phase 1, fasted

(n = 7)

1 4.3 ± 1.1 3.4 ± 0.2 114.5 ± 72A 4�#{216}� 0.1 12.8 ± 34A -8.8 ± 34A

2 4.5 ± 0.7 3.3 ± 0.3 116.4 ± 102A 4.0 ± 0.1 14.1 ± 2.2� -10.1 ± 2.2�3 5.5 ± 1.0 3.2 ± 0.4 120.6 ± 15.5” 3.9 ± 0.2 18.1 ± 4.0k -14.2 ± 41B

4 5.3 ± 1.0 3.2 ± 0.3 119.4 ± 96A 4.0 ± 0.2 16.5 ± 2.8� -12.5 ± 2.8�

Phase 1, fed(n = 7)

I 3.4 ± 1.3 3.8 ± 0.6 102.4 ± 137A 12.9 ± 0.1 9.2 ± 44B +3.6 ± 44A

2 3.1 ± 0.9 3.9 ± 0.5 100.5 ± 12.7A 12.9 ± 0.2 8.8 ± 35B +4.0 ± 3.4”

3 4.6 ± 1.4 3.3 ± 0.3 116.2 ± 116A 29.2 ± 0.5 15.6 ± 5.2”‘ +13.6 ± 49B

4 4.4 ± 1.1 3.4 ± 0.3 112.9 ± 11.6A 29.2 ± 0.6 13.3 ± 3.1� +16.0 ± 2.8k

Phase 2, fasted

(n = 6)

1 5.2 ± 0.9 3.2 ± 0.4 120.2 ± 16.0k 4.0 ± 0.2 17.7 ± 4.1’ -13.7 ± 4.1

2 5.2 ± 0.5 3.3 ± 0.3 114.6 ± 13.0’ 3.8 ± 0.2 17.1 ± 3.2’ -13.2 ± 3.2

3 5.3 ± 0.9 3.1 ± 0.4 123.2 ± 16.3’ 4.0 ± 0.2 18.5 ± 5.2’ -14.5 ± 5.2

Phase 2, fed(n = 6)

1 4.7 ± 0.7 3.4 ± 0.4 114.4 ± 14.4a 29.1 ± 0.3 15.7 ± 3.4’ +13.4 ± 3.4

2 4.6 ± 0.8 3.3 ± 0.4 112.2 ± 15.P 29.0 ± 0.4 14.9 ± 3.5’ +14.0 ± 3.63 5.1 ± 1.7 3.2 ± 0.6 124.6 ± 28.4’ 29.4 ± 0.5 18.1 ± 5.5’ +11.3 ± 5.9

I j� � SD. Means with different letter superscripts are significantly different, P < 0.05; capital letters compare diets in phase 1 for fasted or fed

conditions; lower-case letters compare diet in phase 2 for fasted or fed conditions.2 xi000.

[‘3C]leucine given during the continuous infusion were included

as total leucine intake.

The model of phenylalanine-tyrosine metabolism used here

was developed by Clarke and Bier (33), with modifications in

the tracer protocol as proposed by Thompson et al (34). As de-

scribed recently in detail (35) we followed the approach of

Thompson et al (34), with phenylalanine balance being derived

in a way analogous to that for leucine, except that phenylalanine

catabolism is determined from the rate of conversion (hydroxy-

lation) of [2H5]phenylalanine to [2H4tyrosine (35). According to

recent studies in our laboratories (36), it is necessary to correct

the measured fasted and fed state hydroxylation rates by factors

of 2.2 and 1.8, respectively, to obtain an estimate of the rate in

vivo. This is required because of the secondary deuterium isotope

kinetic effects introduced by use of the multideuterated phenyl-

alanine tracer. With these corrections the phenylalanine hydrox-

ylation rate is similar to the measured rate of phenylalanine ox-

idation, using in this latter case [1-’3C]phenylalanine as tracer

(35, 36). Without these corrections it is possible, of course, to

evaluate patterns of phenylalanine balance among the diets

groups but not the absolute balance. A further discussion of these

correction factors will be given later.

Statistical methods

The data were analyzed by two-factor (diet and fasted or fed

state) analysis of variance with repeated measures on both factors

to examine the differences in amino acid kinetic parameters, with

post hoc pairwise comparisons among diet means by using Tu-key’s test. A value of P < 0.05 was accepted as significant.

Results

The results for leucine flux and oxidation are summarized in

Table 3, for both phases 1 and 2. In phase 1 the rates of leucine

oxidation were lower (P < 0.05) in the fed state, for diets 1 and

2 supplying the FAO/WHOIUNU (17) requirement amount of

leucine (14 mgkg� d�) as compared with diet 3, which pro-

vided leucine to meet our tentative, higher requirement value of

40 mg . kg’ . d� (14, 15). These differences were expected be-

cause we observed previously (30) that leucine oxidation rates

are higher in subjects during the fed state while receiving the

MIT tentative requirement intakes as compared with the rate

measured during an intake with the lower FAO/WHO/UNU (17)

amount. Leucine oxidation was unaffected by addition of NSN

to either diet 1 or diet 3 in phase 1. Thus, addition of NSN did

not spare the dietary requirement for leucine because its oxida-

tion was unaffected by the dietary supplement. Similarly, for

phase 2, leucine flux and oxidation did not differ between the

three diets containing different sources of NSN. Here total nitro-

gen and indispensable amino acid intakes were maintained at

constant values. The lack of differences in leucine fluxes and in

oxidation rates between diets supplying different intakes of NSN

(jthase 1) or source of NSN (phase 2) indicate that these dietary

factors did not affect the status of whole-body protein turnover.

The kinetics of phenylalanine and tyrosine metabolism were

evaluated simultaneously with those for leucine. These results

are summarized in Table 4. In neither phase 1 nor phase 2 were

phenylalanine or tyrosine fluxes, for fasted and fed states, aS-fected significantly by amount or source of NSN. Similarly, the


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TABLE 4

Phenylananine (Phe) and tyrosine (Tyr) kinetic and phenylalanine balance in young men receiving different amounts and sourcesof nonspecific nitrogen’

Phase,condition

and diet 2H5PHEPheflux

Pheintake Hydroxy 2}LTyr 2H2Tyr

Tyr

flux

Tyr

intake

Phe

balance

mol pjnol p.mol pinol mol mol �imol p.�’nol 1wnol

fraction kg’h’ kg’h’ kg’h’ fraction fraction kg’h’ kg’h’ kg�’h�’Phase 1, fasted

(n = 7)

1 8.5 ± 1.0 28.7 ± 37A 2.6 ± 0.2 7.7 ± 1.9 1.0 ± 0.2 7.3 ± 1.1 30.3 ± 37A 2.3 ± 0.1 -5.1 ± 1.92 8.6 ± 1.4 26.9 ± 49A 2.5 ± 0.2 8.7 ± 1.6 1.0 ± 0.2 6.7 ± 0.8 33.1 ± 49B 2.3 ± 0.1 -6.2 ± 1.8

3 8.0 ± 1.0 30.5 ± 6.1A 2.6 ± 0.2 9.3 ± 1.7 1.0 ± 0.1 6.4 ± 0.6 35.3 ± 37B 2.4 ± 0.1 -6.7 ± 1.8

4 8.3 ± 1.0 28.8 ± 49A 2.6 ± 0.1 7.9 ± 1.3 0.9 ± 0.1 6.9 ± 0.5 31.3 ± 2.6� 2.3 ± 0.1 -5.3 ± 1.3

Phase 1, fed

(n = 7)1 9.1 ± 1.4 26.8 ± 47A 6.7 ± 0.2 5.7 ± 1.4A 1.0 ± 0.2 7.9 ± 1.0 27.6 ± 3#{149}7A 4.6 ± 0.1 +1.0 ± 1.4A2 9.0 ± 1.0 25.6 ± 43A 6.5 ± 0.3 6.4 ± 0.9A ± 0.2 7.0 ± 0.4 30.9 ± 3.2A 4.6 ± 0.2 +0.1 ± 1.1A3 7.4 ± 0.7 32.5 ± 5.1” 15.7 ± 0.4 8.2 ± i.i� 0.9 ± 0.1 6.2 ± 0.4 36.1 ± 14B 8.3 ± 0.1 +7.5 ± 1.1”4 7.8 ± 0.7 30.4 ± 34A 15.6 ± 0.3 7.0 ± 1.0� 0.9 ± 0.1 6.7 ± 0.4 32.1 ± 1.6� 8.3 ± 0.1 +8.6 ± 0.9’s

Phase 2, fasted

(n = 6)1 7.3 ± 1.1 32.8 ± 7.9’ 2.5 ± 0.2 10.2 ± 2.1’ 1.0 ± 0.1 6.6 ± 0.9 33.6 ± 4.6a 2.3 ± 0.1 -7.7 ± 2.0

2 7.4 ± 0.9 32.5 ± 3.4 2.6 ± 0.2 10.3 ± 1.9� 1.0 ± 0.1 6.4 ± 1.0 34.0 ± 4.7’ 2.3 ± 0.0 -7.8 ± 2.13 7.3 ± 0.7 32.6 ± 1.5’ 2.6 ± 0.3 11.1 ± 2.7’ 1.0 ± 0.1 6.0 ± 1.3 38.3 ± 12.0 2.3 ± 0.1 -8.5 ± 2.9

Phase 2, fed

(n = 6)1 6.7 ± 0.8 35.8 ± 6.7’ 15.5 ± 0.3 8.7 ± 1.9’ 1.0 ± 0.2 6.9 ± 0.7 31.7 ± 3.7’ 8.3 ± 0.1 +6.8 ± 1.8

2 7.2 ± 0.9 33.7 ± 4.3’ 15.6 ± 0.2 8.1 ± 1.4’ 1.0 ± 0.2 6.9 ± 0.6 31.1 ± 2.7’ 8.2 ± 0.1 +7.5 ± 1.43 6.9 ± 0.7 34.7 ± 3.0’ 15.7 ± 0.2 8.4 ± 2.2’ 1.0 ± 0.2 6.7 ± 0.8 32.4 ± 3.7’ 8.3 ± 0.1 +7.3 ± 2.2

‘ 1� ± SD. Values with different letter superscripts are significantly different, P < 0.05; capital letters compare diets in phase 1; lower-case letterscompare diets in phase 2.

phenylalanine hydroxylation rates were not affected by addition

of NSN, but in the fed state the rate was lower with diet 1 as

compared with diet 3 (jthase 1). The sources of NSN investi-

gated in phase 2 did not affect the rate of phenylalanine hydroxyl-

ation.

It follows from the above results for leucine and phenylalanine

catabolism that the estimates of leucine and phenylalanine bal-

ances during the fasted and fed periods and over the entire 24-h

period were unaffected by addition of NSN to either of the diets

(FAO and MIT) in phase 1 (Tables 3 and 4). However, leucine

balances for the fed state and the balances for the 24-h day (data

not shown) were higher (P < 0.05) for the MIT (diet 4) vs the

FAO diet (diet 2), when total nitrogen intake was adequate (160

mg N . kg� . d’). Phenylalanine balances during the fed state and

over the 24-h day (data not shown) were significantly lower (P

< 0.05) for the FAO vs MIT diets (Table 4). Again, NSN ad-

dition to either of these basal diets was without a detectable effect

on phenylalanine balance.

In phase 2 we did not observe an effect of the chemical form

of the NSN component of the diet on the kinetics or body balance

of either leucine (Table 3) or phenylalanine (Table 5). These

findings indicate that glutamine was neither more nor less effec-

tive as a source of NSN, compared with a mixture of dispensable

amino acids, under these experimental conditions in healthy

young adults.

Plasma amino acid data for phase 1 are given in Table 6. There

were no apparent effects on the free amino acids measured of

addition of NSN. However, the lower intakes of isoleucine, leu-

cine, phenylalanine, and tyrosine provided by diets 1 and 2 as

compared with diets 3 and 4 were reflected by their lower con-

centrations during the fed state. Despite different valine intakes,

the fed state concentrations did not differ between the FAO (diets

1 and 2) and MIT (diets 3 and 4) intakes. For phase 2, no sig-

nificant differences in the plasma amino acid concentrations were

observed among the diet groups, either for the fasted or fed states.

Hence, these data are not presented here.

The relationship between the whole-body phenylalanine and

leucine balance, expressed as a molar ratio was examined (Table

6). During the fasted state in which balance is essentially a mea-

sure of leucine oxidation and phenylalanine hydroxylation, the

mean ratios of the phenylalanine to leucine balance were �0.4

to 0.6. Because the molar ratio of phenylalanine to leucine in

mixed body proteins is �0.4-0.5 (37), it is to be expected that

values for the rate of phenylalanine hydroxylation, or phenylala-

nine balance, in the fasted state would approximate 0.4-0.5 of

the value for leucine oxidation, or balance. Hence, the general

agreement between the measured ratio (Table 6) and that pre-

dicted from the content of leucine and phenylalanine in mixed

body proteins suggests that we have corrected appropriately the

measured rate of phenylalanine hydroxylation from the multi-

deuterated tracer in this study.

Discussion

Studies in growing rats (1 1, 12) chicks (10), human infants

(13) and adults (7, 8) have shown the importance of an adequate


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‘ 1� ± SD. Molar ratio of phenylalanine to leucine balance.

TABLESSelected plasma free amino acids in young men receiving different

amounts of nonspecific nitrogen (phase 1 )‘

Amino acid

and condition

Diet

1 2 3 4

�tmol/L

Valine

Fasted 159 ± 29 164 ± 31 157 ± 33 150 ± 52

Fed 115 ± 20 122 ± 19 130 ± 17 129 ± 27

Isoleucine

Fasted 48 ± 6 51 ± 9 47 ± 13 52 ± 8

Fed 29±7”‘ 28±8A 49�6B41�9B

Leucine

Fasted 111 ± 25 118 ± 23 124 ± 21 125 ± 18

Fed 67 ± 15A 68 ± 12A 113 ± 13B 105 ± 20B

MethionineFasted 28±9A 19�3H 22±3� 21�2AB

Fed 27±3 25±4 25±4 22±3

PhenylalanineFasted 54±10 52±7 58±9 60±9

Fed 42±6A 37#{247}5A f,4#{247}5B57±88

Tyrosine

Fasted 46 ± 11 47 ± 9 55 ± 10 52 ± 7

Fed 35#{247}7A35#{247}7A 48�8B

46±88

C � � SD. n = 7. Values in same row with different letter superscriptsare significantly different, P < 0.05. Fasted, before tracer infusion: fed.end of 5-h fed period.

amount and appropriate dietary source of NSN for maintenance

of an adequate state of body protein balance and nutriture. Ap-

parently, an array of dispensable amino acids is required to pro-

mote maximum growth in rats, mice, and cats, whereas glutamate

alone is sufficient for pigs and chicks (38-42).

In reference to human studies, the nutritional value of diets

that supply low total nitrogen intakes, or those containing pre-

dominantly indispensable amino acids, are improved when NSN

is added. This has led to a view that NSN lowers the quantitative

dietary need for specific indispensable (essential) amino acids (7,

9). Indeed, the relatively low requirements for indispensable

amino acids in human adults as proposed by national (16) and

international (17) expert groups, has been justified and explained

by the ability of NSN to spare indispensable amino acids, allow-

ing whole-body amino acid balance to be achieved apparently at

low dietary intakes (9). Clearly, if total NSN is limiting then an

improvement in nitrogen balance after dietary supplementation

with NSN would be expected. In this case, the efficiency of util-

ization of indispensable amino acids would also be increased,

associated with lowered rates of oxidation of these amino acids.

Also, Jackson (43, 44) hypothesized that the endogenous synthe-

sis of glycine in preterm infants may be inadequate to meet the

needs for growth and that the glycine content of breast milk may

be insufficient to meet their requirements. If this is the case, and

NSN is limiting, supplementation with NSN should improve ni-

trogen balance and growth; there is evidence in support of this

hypothesis (13).

It was shown using isotopic methods that dietary urea nitrogen

can be used for amino synthesis, albeit to a limited extent, in

breast-fed (45) and formula-fed (46) infants. Additionally, when

rats were fed with an L-amino acid diet that was devoid of glycine

and serine their capacity to synthesize hippurate was limited (47)

and we reported reduced rates of glycine synthesis at marginal

intakes of total and dispensable amino acid nitrogen in healthy

adult men (48). Clearly, dietary NSN is important for the nitrogen

economy of the organism, when its supply is limiting there are

profound effects on the metabolism of indispensable amino acids

and on body nitrogen balance.

However, it is a distinctly separate issue whether NSN can

actually spare, or reduce, the minimum physiological require-

ment for one, or all, of the indispensable amino acids, providing

that the initial requirement estimates were established at a nitro-

gen intake sufficient to meet, but not greatly exceed. the indivi-

dual’s minimum total nitrogen needs. Thus, careful thought must

be given to the design of previous nitrogen balance studies when

attempting to interpret the nutritional significance of NSN. For

example, Kies et al (49) reported increased nitrogen retention in

adults when nitrogen intake was increased from 6 to 8 g nitrogen!

d, while the indispensable amino acid intake remained at a con-

stant level equivalent to that in 20 g egg protein. Rather than the

improved nitrogen retention being due to a sparing effect of NSN

indispensable amino acids, we interpret their results to mean that

the intake of total nitrogen was initially both low (�8l mg

N . kg ‘ . d ‘) and inadequate and that addition of NSN (supplied

as diammonium citrate and glycine) increased total dietary nitro-

gen to a more satisfactory level of � 107 mg N . kg F . d ‘. This

latter value compares with the 1985 FAO/WHOIUNU (17) safe

protein intake of 130- 137 mg N . kg ‘ . d ‘ for young adults.

Hence, in our view these careful studies by Kies et al (49) cannot,

in contrast to what appears to have been done by others (9), be

used to support the contention that the minimum physiological

requirements for indispensable amino acids are actually lowered

by the addition of high amounts of NSN to the diet.

It has also been claimed (9) that the requirement estimates

derived from nitrogen balance studies by Rose (18), which are

substantially lower than our new tentative figures (14, 15), are a

consequence of a major sparing effect of NSN on indispensable

amino acid oxidation. However, this appears to be unlikely; Rose

and Wixom (50) concluded that, when the indispensable amino

acid intake was at a safe level, or double their minimum require-

ment values, the need for NSN was between �2.28 and 2.55 g

nitrogen/d. These investigators remarked in their paper that this

requirement was ‘ ‘surprisingly small.’ ‘ Furthermore, it should be

noted that the total dietary nitrogen (from indispensable and dis-

TABLE 6Relation between balances of leucine and phenylalanine during fasted

and fed states in young men receiving different amounts and sources

of nonspecific nitrogen’

Phase and diet Fasted Fed

Phase 1 (n = 7)

1 0.6 ± 0.3’ 0.3 ± 0.12 0.6±0.1 0.0±0.23 0.5 ± 0.2 0.7 ± 0.5

4 0.4±0.1 0.6±0.1Phase 2 (n = 6)

I 0.6±0.1 0.6±0.22 0.6±0.2 0.5±0.13 0.7 ± 0.3 1.1 ± 1.2


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pensable amino acids) that they estimated to be necessary for

apparent nitrogen balance was 3.5 g daily or �40-50 mg

N . kg ‘ . d ‘ . Because obligatory nitrogen losses in adults have

been estimated to exceed 50 mg N . kg’ . d� (51) and it is now

well established that nitrogen intakes above these losses are re-

quired to achieve body nitrogen balance (17, 52), the nutritional

and metabolic significance of the study by Rose and Wixom (46)

remains unclear. Given the limitations of these earlier nitrogen

balance studies we are unable to identify any published, conclu-

sive evidence to support the view that a ‘ ‘high’ ‘ intake of NSN

can actually reduce the minimum physiological requirements for

indispensable amino acids, when these have been established in

the first instance under conditions of adequate, but not excessive,

intakes of total dietary nitrogen.

In summary, the present investigation was carried out to eval-

uate the effects of amount and source of NSN on the oxidation

of two indispensable amino acids, leucine and phenylalanine,

when consumed at requirement and limiting, but not markedly

deficient, intakes. From the findings in phase 1, we conclude that

a so called sparing effect of NSN (7, 9) does not account for the

relatively low requirement values for the indispensable amino

acids that were derived by Rose (18), as contrasted with our

higher values (14, 15). Finally, in the planning of phase 2, we

thought that addition of glutamine might improve body amino

acid homeostasis, in view of the reported beneficial effects of

glutamine on nitrogen balance and tissue function in various

pathophysiological states (23-25). We were not able to detect a

differential between sources of NSN tested. However, it should

be appreciated that we examined nonstressed, healthy subjects,

in which case endogenous glutamine synthesis and its tissue

availability evidently were adequate for these experimental con-

ditions. U

We thank the subjects for their commitment to these studies and the

staff of the CRC for their considerable assistance in the conduct of thisinvestigation.

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