Applications of the doubly labelled water method in studies of human energy metabolism

Applications of the doubly=labelled water method

in

studies

of human energy metabolism

Medical Researcl) Council, Dunn Clinical Nutrition Centre Hills Road - Cambndge CB2 2DH - UK

RESUMEN: El método del agua doblemente marca- da (%H, j80) para medir el gasto energético total, es fiable, seguro, no invasivo. Por su versatilidad se ha utilizado en estudios muy variados, en todas las épo- cas de la vida desde niños prematuros hasta la edad más avanzada; en mujeres embarazadas y lactantes, en pacientes obesos y otros con anorexia nerviosa; en todos los niveles de actividad física, desde pacien- tes en cama hasta ciclistas participantes en el -Tour de France,,; en astronautas y en campesinos de paí- ses en desarrollo. Además de ser un método avanza- do para la medición del gasto energético, el agua do- blemente marcada también tieneotras aplicaciones: validación de otros métodos como la monitorización del ritmo cardíaco, monitorización de la actividad dia- ria; medida de la composición corporal; medida de la secreción láctea, y, como un biomarcador indepen- diente, permite valorar la validez de la aportación ener- gética que se ha indicado. Esta revisión resume el fundamento de este método y lo ilustra con algunas de sus aplicaciones.

SUMMARY:The double-labelled water (DLW, 2H,1BO) method of measuring total energy expenditure is accurate, safe, non-invasive and non-intrusive. Because of its versatility it has been used in a huge range of studies worldwide- across the lifespan from premature infants to the very elderly; in pregnant and lactating women; in obese patients and those with anorexia nervosa; throughout the spectrum of activity levels from bedbound hospital patients to Tour de France cyclists; in astronauts and in subsistence farmers in developing countries. In addition to being the elite technique for measuring free-living energy expenditure, DLW also has a number of other applications. These include: the validation of other field methods such as heart rate monitoring, activity monitoring and activity diaries; measurement of body

44 REV MED UNlV NAVARRAIVOL.43, NP4, 1999,4451

composition; measurement of breast milk output; and as an independent biomarker to assess the validity of reported energy intake. This review summarises the background to the method and illustrates Come of its applications.

Palabras clave

Biomarcaje, agua can dol71e marcale, metabolismo.

K e y words

Biomarlcers, double-labelled water, metabolism.

Correspondencia Gail Goldberg Addtes.; as above Te1 ++1223 415695

Fax: ++1223 413763

Introducción

The

DLW

mbthod

Ln prlneiple the DLW tedinique appess to be verg simple @@te 1) It SnvoIves enriohing &e body w t e r of ii ~ u b j e ~ t with &bk i s d q ~ e s of hydrogsn @&m, zl$ and aay-n ('%>. F&wing &e h d i h ~ 3 dose C u m l l y d J of2H,0 and H, PBO dlt) eoneentrattons af

aH

and "@ in

the

body

&en

decrease e~panentially towarda natural ba-clng~ound leveis, fypietlty aver 10-

14 daye. Deqterkum labels the úody's water pea1 PNJ

Fig. 2

w its tiamenti-arian decretases at; a Zesult ~f'riilutton

by

new unlabdltd water wnsumed a8 Fapd and dnnk Water labeíied wid1% is alria last as watei vapour by ewapmtion from Eungs and skin, prnd by exautions and6eoietkms luiine, m t , aind bmastmikb Qxtgen-

18 labeis both the

mm

a d bhrúoiiatft goolx @TJ.

M ~ s t l80 15 lmt as waw, h t because Coa in bodp fluids is Tn iwmpfe equilibririni wfth bo& water,

C.&

the e w m e caibonlc anhydmse~ sonw is alm k t a@ c~rl>on ~lioxklc. l'hc <lis:ippc~i.;iii<e uf ilic two isi)toq>es honi tlie l>ociy is asscscrd I)y n x t . ~ s[wctii)iiu~tnc :<milysis ofthe mrchrnent&sersl mine oi saliva smples (F* re 23. The rate nf %H dimppearance (1Q cqn be crrleulatetl by 1% tninsformation of emichment against tíme a& provides

a

rnwure QE

the

iw

O af water

prdrnion CrF&O). The

rate @f IlaQ diappearanee .siroitai.ly bg transformed, measuies the sum of water

and carbon &mide p d u c t i a n (TH~O

+

rCO,). Thus &e d q i e in Fi$ure 2 is meper for % RBmppearance. -he dffetence bctween thae two ratw (slapesl tiuikre representli producrion and tilis in&sc~t mm~re

of fneP~bQlic

me

can ben emv-si-iad

m

UN.& of ewrgy apendture.

Thwe are

inra m i n wap

of

calculating data

-

the wo-pomt and the muIti-pofnt methods. It is importan

m note tkmt

&e

~xlnilatiw are díffe~ent and that tbe

h

HPO o CO"0

k, E rCO, +rH,O

4

'

1

1

where k = experimentally determined rate constant and r= product~on rate

Principie of the doubly-lobelled water method.

two-point method is not simply an abbreviated multtpoint one, In essence, the tm-point method measures the lotal flux between two points severa1 days apart. Samples are collected anci analysed only at the beginning and end of the measurement period. The resultant race constants

CK,

and

%

in Figure

11

are multiplied by a single mealred ebtimate of total budy water Grom the dilution spaces N, and N,). Whilst the two-point method is genaally considered to be more accurate, large changes fiom the average could produ- re biased results. The multi-point method calculates the rate constants as the best statistical fit ta enrichment values measured in daily samples and applies these to separate estimates of the *H ami 1 8 0 dilution spaces. Since water intake, excretion and CO, production change with time these should be detectzfble in the isotopic dia-ppearancce m e s . If the devíations from average are srnall they can be consideied as random phy4io-logical changes Since the multipoint method gives a mezan mlue per day, random mrom become less hpomnt. Whilst the multlpoint inethod iU pmbably more precise and has a number of practica1 and theorr?tical advantages, these may be outweighed by tlie additional analytical time and coat implications.

Although the DLV method is superficially simple, in practice, it is notas straighforward as first appears. The analysls and interpretaticm dDLW data 1s complex and requires the combined expertise of mass spectrometrists, stausticians and mathematiaans and of course those who have an unders-tanding QT the physiology and envkonment of the organism being studied. Lifson's model on which tkmethod is based makes a number af assumptrons. Wme of these are quite cairect in biological systems and like aJi niles, they can be, and are, frequently broken duimg m 1 lí e and during ex- perimental ptotocols. For example, assumptions 1 and

2 state that the volume of tlie budy water pool and outflow mes of w a w and CO, pioductlon ai-e constant. This is nor w e for any living organism Waier intake, excretion and CO, production change with time, d e p e n d ' f on its rate of energy expenditure and when and how much it eats and drinks.

In the early days o£ the appíication of DLW in human studies a workshop was convened by the Internatioml Diera17 Energy Consultancy Group QDECGI and thc Internaanona1 Atomic Eneigy Agency (IAEA). The paiticipantc were reserirchers from al1 the major laboratories conducting recearch usmg DLW. The outcame was a consensus ducunlent: a ~ u i d e to the DLW merhod, published in 1990

(0.

Since then

investigators have continued to refine calculations and analytical protocols, debate the various merits of &e multipoidt vs two-point method of anaíym, discuss how ta evaluare the quality of data and cal~ulate the limits of lilrely errors. These issuec are too complex to desui- be here, but the IDECG document, Speaknian's book and two papei-s in p;rrticular by Coward

CJ, 81

are al1 useful m d practica1 sources of infarmation, even for a mvice. These

referentes

alsoall contain comprehensive bibliographies for further reading, mcluding the many studies that have been carried out in wild and domesticated animals, reptiles, amphibians, birdi and hects.

Although the DLW m e t h d is complex, an in-depth undersanding and interest in mathematics is not a prerequisite for a11 those using the technique. However it is important that physiologists, clinícians and nutritionists intending to use DLW apprecrate the implications and biohgical rdevance of the assumptions and othw methodologcal issues before d-ning study protoeals Ci", 8). Some examples of the relevanee ta human ctudies are discussed bnefly below.

Advantages and disadvantages

oT

DLW

DLW is not an appropl-iale t e c w u e to use in some elmical settuigs paiticularly in acure conditions and where the need ta know a patienls energy requirement is required quickly. In such cases bedside calorírnetric techniqucs (9) or labelled bicartxonatc that gives estimates of energy expenditure over one day oor multiples of one &y periods (10) should be used. It should also be rememhered that RLW and indirect caloiimetiy meth& are mt mutually exclusive, but complementary, DLW @ves a measure of total energy expenditure but CanAOt partiuoned Chis into its components CBMR, DIT energl e~pended on physical activityj. In order to nieasure basa], resting or sleeping metabolic rate, or the energy expended ciuring exercise or a f t a a meal, some fomi of indirect calorimetiy Cwhole-body, portable apparatus, ventilated hoods etc) is required (91. Even though TEE-BMR gives a measure of the e n e m expended on physical activity, and TEE/

RMR an idea of physical activity level, because DLW

Study duration

Cimrly when designing s~udy pmocols ít is impottant fur both analytical purposes and

b

r

the investigators and subj~cts in the Eeld to consider how 10np DLW measurements W dbe mavle for Optin~al prectsion is obtained when the measurement period is between 2.5 and 3.0 btological haf-¡¡ves for ea& icotope. In practlce

thii

means typical vaIues are 6-10 dadys for Wants; 12-16 days for adults; 6-10 days far exmmely actiw aduitb, 16-20 days fui íxxüLtive elderly s~hfects Furthermore m tropical mvironmenw a n d h undm conditions

of

extreme physiml exetrtion where watn tunover is very k h , thcse pertads may be cansiderably s h o ~ t a .

Respiratory quotient (RQJ

Tha energy equivalents of CO, vaty markedly according to the substrate mkture being oxidised. Therefore conversíon of CO, production raie data ta units of energy expenditure rquires a knowledge or assumption

of

tlre subjectslRQ. Altha~gh thiiscan vary substantidly in &e short tai-m, in indiuiduals in enetgy balance, when averaged over l m g periods, it resembles the RQ of their dier (bod quutient, F a . The iapottant practica1 points

to

comider are the nnmber of days of o b s e r ~ t i i n ~ q u i r e d to deremine FQ, wliich dtetafy intake method to use, and the &ect of possible macronuuient-spe-&fc misieprthg ol dietaty inBke. Because the sources of maeronutdmts are both dietaiy and endopehous, a furthei consideration 1s the

eonsequence of body fa1 gniins or bsses in the expe- rimental pesiod T'he impact o£ using an inappqmate RQlFQ can be calculated and it is only in excepüonal arcumstances that this 1s ltkely tn cantribure subtanü;llly to significani mom C?, 8, 111.

Losses of Esotope

not

lnvolving water or CO, loss

Assi~mptions 3 and 4 sute thar losses of 'H and lHO are oniy as water and rarbun dioxide However, some tsotope may be dostt by being exrhanged, sequestered with, or crxported as, pwtetn, fat dr CHO. Exchangr 1s

more likely to occur with ZH thqn 180 becaiise the

hydrogen in water can exchangz: with many intermediates in biosynthetie pathways and hacome bound toa t b o n . Regarding wquesrration, in some animal cpecies dd

nouo

li~sogenesis poses

a

signioficant pmblem. However, this is much les* lil<ely to be the case in humans where wrualS all body Fat deposited

i lkely to come from the Anotlier souice of mror

has to be considered in iactating women. As well as boiog squesrrsrad prirh, T I can aiso exchange with, müs< p t e i n a m i lactose, m d he exported from the body. Isotopic exchange, sequestration and expar are al1 interpretesi as a l o s of water, thereby overestmatiqg water lms and underestimating 60, pmduction (ami therefare mmgy expenditure). The errws are small, but sime &ey are aU 1n rhe same direction in some ~ircumstances significanx bias could resirir

Fractionation

Fractionation 1s the reiati~e gbllity a€ is~topicaily labelled compJred with uníabelled moleniles to take part in physical exchange proms$es.

Assumptlon S is b t lassea of 4i and INO occur gt the same enriclimait as those existing at rhe same time in body water. However, in reaiity because rrf the differences bctween tram atoms &e. 'H v ?S; v 'Bol some water and CO, losses are fmctiomted, So correctiuns have to be made for the fractionation o£ %H leaving the body as water vapoui; 180 leaving as wam vapuur, l80 l&ng as watervapoiirand oT1W e x c h z e between wdtw and carbon dioxide. The mrrection6 are usually based o n die relationship between lung water losses and CO, production, and upon fradionateá skin water losses W n g depmdent on body size.

In

pi'actice,

iF

cbe tnie proporlion of water fractionated is less b t asqumed, then @O, prductian Cand thexefore energy expendihire3 will be unde~stimatad:

Background enrichment

The enrichment of water with aH and '80 depends upan dimatic mnditlons, pmipitation, mposation, and thetemperatu~e of seas and oceans. At the polar regiom heavicr isotopes are depleted, rn the tropics they are rckatively enrkhed Brcduse stable isotopes occur naturaily thcy are m e v e ~ w n g we eat, drink and breathe. Therefom the baekgfound e&ihmen?s have to be considered and correcred for in DLW studies Ideally, the mcentration of 2H and 'PO in the dsw given u> the subje~t should be Par in excess of back ground levels. However, because "O is so expenxíve, anly limted amounts can be given, and so towards

the

end of a measuremcnt peilod observaíions are close ro natural abun-dance.

geograpliicaI location duiing a ahidy andlor the soui-cc. of their water s~ipply changes. Wtlier study situntionv that niay be relevant are 13 in dinical seuings where paticntc a i ~ @ven inu-avenou~i fluids of an isotopie

composition difEerent f i ~ m noln7al

a21

and 23 m infanfs malring the tramition f r ~ n bi~ast feeding ts weaning. Brea~t fed babies are nldre eniiched than buttle fed on& (13). Ritz et al liave recently disnissed tlie efFec.ts of the magnitucie and covariance of aH and IsO natural abundances and the impact &ese liave un the precision of DLW measlueii~ents

(14,

151.

Applications of the

DLW

method in humans

The DLW nlethod is complex Sor the investigatois who analyse and interpret the data, but foi those applying it in the "fielde and partinilarly for the subjects, ir is an ideal teclrnique for measufing

comt

energy expendituie CEE). Tlie tnethnd is c;afe, non-íntiusive and non-im~asive, does not require subjects to wear appai-atus, keep diaiies orbe inonitorcd by otliers. TEE can I>e measured undei genuinely free-liwng conditions withthe rninimum of inconveniente er hindrance. Afrer dosing wirh DLW, al1 that i-, required is the colleaon

of a dngle daily uríne or saliva safnple for the mpired n u d e r of days. The sublects can Eoiget4 and indeed la the case of childreii and babies, be totally lmaware o£ being nxeasuied in any way. The mvst difficult cmponent of TEE to quanrifv has always been the eneigy expended onphycical activity. The conbinatiun of m e a ~ r e d oi p~edicted hdsai metabolic rete @M@

and TEE aliows the eneigy expended on physical aüivity and thermogenesis to be caíenlated rrs TEE-EMR. Since thermogenesis is a mal1 and i-eiisonably constant componmt of TEE, vaiiatiuns in TEE-BMR can

be

ascfibmi

m

chawes in physical aaivit)~. Phys~eil activiry levels (PALsl, ciBn aiso he e?lulaied ftoni these data as

TEEBIMR.

Wver tlie past 15 years DLW has heen used thruughnul the wmld by many investigators in a d e variety of wbjects and ciirumstances. The studies are fas too aurnenous to lisr here, but a wmprehensive review and meta-analysis by fllack e/ al C16) also contams an extensive list of orighal cítations. The ULW rnethod has undoubtedly revolutionised ~tndies of hunian energy nie~almitsm í\nd our under-ndin~

of

lmw enern hlance is regulated. It 11% not really led

to the genelation

of

tfw liyprl~eses, ratl3er it has allowed us ta answer k o ~ - l ~ e l d questions in a diEment

and more accepiable way. DLW fs also u.wd I ~ J validate o t h a Geld nlethods of e n e w expenclitm sucfias liart-

a

R E V M ~ U N I V NAVARRAIVM.

a,

N P B ~ tew, u41

ratc and activity monitorlng 07-131. In rnany instnnres DIAV has provided -new answers to old questi~nss., m some cases by exploding myths, nr disnussing long held assumptlons. Some emtnples of oui uwn shidies are gíven belww.

ObesPty and metabolSc defacts

Researc11ers in energy phyaiology spent many years weking deFecís in ihe replation of energy e@p1%6Itturs, O e. hypo-and hyper-netabolisd to explan h u m n obesity and wasting Cdue to disease or naiima) respectixwly. In 1986 DLW studies deiiionstrated conclusively for the fúst time the answer to a paradmr, naniely that o b n c mbjects appaiently cxistqd on energy inmkes that were the same as, or less than, their lean peers Despite deades of researcli no defmfs in energy expendicure wefe idenufied which wet'e o£ stifficient inagnitude to explain tliese differences. Prentide ot a1 C20) fonnd tlrat reponed energy inxakes in weight-stahle obese sublec$ were a mean of 3.5 MJ/day leas ilian ineasuiements of TEE. In contras a groiip of lean women demonstrated excellent aeyeement between energy intalce and TEE. Since then othet DLW studies in which al1 the componen@ of energy balance CuiBlíe, expenditure and body energy stores) have been messured have shown that there are no mafnr defectc in the iegulation of huinan eneigy e@endi.?uw that can adequately explain abesity or wasting (21-23). Disregulauon of en- balanrre 1s more likely to he

caused by defects in the regulation or provisiun of energy

Znfak.

Energy

costs

of

pregnancy

DLW meamrements made longi~dinaiiy h a n a pre- pitgnant !.?asehe have demstmted p i m m n d intm- individual differen~e rn fat changes and in TEE, mnd theiefuse m tlie tatal energy costs bf pregnancy 5242.

inralces tliat cotild not passibly be i'eprcsentíltive of uue habitual intalce [w belrrni) 1ncie;tres in self-iepited eneigy inmk accr>unted fog kss than 5CI% of tlie total wsts of pregnancy.

Average leuels of free-living energy erpenditure in people from aífluent sacieties

A recent paper by Blnck #

d

presentcd an analysis of al1 DLW measurements of TEE avaílable up to the i~iiddle oF 1994. It incinded ali avíiilablepublislied dala together wxh some pir~riously uiipiibliol1ed resulrs from investigaton fmib a m n d tlle world. The &ta comprised 1614 individual measi1-renlenu in total and the authors cklculatcd the average levels of fme-living energy expendituir in p w l e frmn affluent souaics, fiw-li-

ving PALs can i q e f f o m 1 2 to greater thiin 2.2

x

BNIR.

Upper and lower Iimits of free-living energy expenditure

DLW, in addition to pioviding data un avemge lavels of eneigy expenditure, las fm the fim tune embled the uppermd lower limíts of h l y eneigy expendituir to

be

esrablished -ta from non-amhulant subjects,

fiom elite endurance athletcs and subjects measwcd under othei cucunlsiances that elieited nitremdy higli levels of en= wrpndituse, iadicated tkiat mean PAts ranged f r m 1.22 to 4 . 6 9 ~ BMR and individual valuec weie as high a5 5.0. Of coiiwe the distinction 1x38 to be made between the maximum FAL wlrich c m be achíeved over a defined pellod and the maximum susíarnable habitual PAL awming physical fitness and adeqitate foodintake. Black's meta-analysis showedtliat a PAL o€ 2.5 indicated an extremeiy physically aaive lifescyle and probably the maaimiun sustainable in the long ~erm (e.g extended periozls of traimng m atliletes 2nd soldiers and during pealt harvesf. activities in subsismce farmers) A PAT. of 2.0 is likly ammg ma- nual workers in doveloped co~muies whereactivitynray vary widely Frrm dayto-&y or week-CO-wetlk.

Other applicatlons of DLW in studies

of human energy metabolism

Body composiffon measui'ements

There are many techniqiles Eor aasesaing body co~nposition, one of which is isotope d+liitíbn W8). This imrol\tes giving a dob'e of 'Be> or 'H labelled water. Tlie isotopic eimchment of iirine or saliva sainples is a fnncpion of themount oF body water. Body cornpo8i~iitiun

- -

be caleiiiated hom total body water CTHW) ancl tlie Iiydracini~ ooei'cient leither rneasurecl oi assuinedj of ciie fatfiee mass. Fat is caleulated as the difference hetween body weight tind fidt-fiee mass in inany studies in whith body compositim is ti1e piime meafiu~etiient, 2H or 180 is given only ta asV:ss TBW. A s E'iure 1 sliows, this is xlso incasui'ed as p r t o£ a DLw measuirmenl. Tlins an ebTh~ate of body coniposifion

is autaiatic%Uy obtained as a consequence of udng DLW ro ineasuie TEE

Breast milk output

When DLW is given to lactating women al1 tlie ccmpomts of tlieir enei;$y biidget can be a s $ d (29,30) Not only their TEE and body conipositi~ri, but also their breast n~iikoutpur and theTEE of theirinfaiirs can be meayurul. After tlie mother is d o s ~ d wícb DLW

hoth ieotopes n t e i tlie baby vía hreastdk and mix with the baby's tmdy water. The rate of appearance of isoiope m the baby, mi'asiii.ed in spot urbe or saliva san~ples, is proportioud to its inillr intalae dwe-&e- mother methad- ofmeasuring bi~iist fed infants' intake and expcnditnse was Erst dcsc~+bed usingsingly lahelled watm Q1S and has been cross-validated and used UI the field (32,331. Bisme.tlioci has huge sdvíintages over more conventionsl ohes for estinmting breast milk intake. These involve weighing the baby before and aftn each Ceed wliicli is time-cmsuming, inaccitrate, and interferes with the inociicr's normal activides. Test- weighing psocdui-es are also inappcopriate when tí= baby is constantly c h e to its mother anJ 1s Eed on demand. The dose-to-tile-mother method is much inore acceptable and practical, no appitranis other tlian containers to collect aamples in is required. The measiirement is alw tnom aceunte, since l&e tlie me~lsui'enient of TEE, the hahy's milk intake Onmher's oulpu~l 1s integrared over 10-14 days.

Evaluation

of

energy íntakes

Not only I1a8 DLW led to

a

inucli greata undcrs- randiiig of encrgy expenditure under vaiious plrysiological and experhentaíly irnpased condiüoils, it 1x33 & e n insiglrts into tlre valihcy and inteqxetation

of e n w y intake CEI) ineasuirmenrs (34). Betrause tlie nieaaurerneilu of TEE is integrated m~er relati\rely long prriods, ir 18 lilceiy, Yi the majority of subjects, to be repiesentative of thcir Imbitual Qong term average] energy erpenditure. DLW therdore also giws an indqendent meamise of energy i*quiremenís and can I

AppnEatlonsd IKadoubly~labslled wakrmethod In Mudb d human ener#metaboliam 5.R Qoldbsm

Prmtice and others

in

relatively small groups of vnlunteer subfeas confirined what energy physiologht and sume clie~rüans had long suspeaed; that obese persons tend to under-report their food intakc 0 0 , 35,

381

However, in

1490

underreporting waa demonsrrated

in a randomly seleded s-ample of men and women taking part in a laige snidy of dret and health. mis was cleatly an issue of p~tentially far grriater conrern with serious impliiiatioas for preuíous and ongoing studia,

1. Lífson N, Gordon FrU, McWinto~k

R. Measurement of total caibon dioxide produetion by mans u+ Da *O. Journal of Applred Physiolo@! 1955; 7. M47lO.

2 Mson N, Littlr WS, Lwiü UG, Henderm RM D, '9 method For C02 output in small animal6 and wnomic

fembility inn man. Journal of ApplKod Physiology 1975; 38: 657-663.

3. Schoeiler DA, van Santen E Mea- surement of engzgy expendinire rn humans by the daublylabekd water methd. Journal of App1,lied Physiolqg

1982; 53 955-959

4. Piennce AM, Coward VA, Davies

HL, et al Empectedly low lwels of

energy 'expenditure in healthy women, 1985; i: 1419-1422.

5. Speakamh JR. Unuhly labelled watel: theory and practica Lodon. Chapman aud Hall, 1997

6. IDECG. Internacional Uietaiy Energy Gomultancy Group The Doubly-Labelled Water Meihod for Measuring Energy Expenditllre: TL&-

nical recommendatmns for use in humanx. Carnbr~dgr IDECGIIAEA, Vienna, 19% (Prenuce AM, ed).

7 Cowaid WA, Cde

TJ.

The doubly- labelled water method

for

the m-- rement of energy expenditiire in

humsns: n.& and henefits. In: Whi- tehead BG, Prenhce A, ed. N- temí-

l

boa1 large and sma11 (37) Sincc DLW is

m«

expensive a n d technically demanding to use rouunely U> validate measure-mems of f dintake, w e piopnsed evaluating El by compaiing it with p~esumed enei-ayrequirements,

bmh expressed as multiples of BMñ (383. This method

of evaluation has recently bem enhanced because Rlack's meia-analysis of DLW data has enabled a more infonned dwice of PAL, to be used as comparrson (39).

ques in numtional r e s e a d . BrisLol- Myers SquibWMead Johnson Nutrition Symposia. Cambci&:e: Academic Prw Inc, 1991: 135176.

8 Ciipraid WA. Meauuremenr of energy expend$ture the doubly labelled water method in clinicnl pi,actice. Pirreeedings of the Nutrition Socieiy,

l791, 50. 227-237.

9 Murgaüoyd PR, Shetty PS, P m i c e

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