ORIGINAL ARTICLE

M.A. Stroud áP. Ritz á W.A. Coward áM.B. SawyerD. Constantin-Teodosiu áP.L. Greenha� á I.A. Macdonald

Energy expenditure using isotope-labelled water (2H218O),

exercise performance, skeletal muscle enzyme activitiesand plasma biochemical parameters in humans during 95 daysof endurance exercise with inadequate energy intake

Accepted: 15 February 1997

Abstract Two men, R.F. and M.S., pulled sledges eachwith starting masses of 222 kg, 2300 km across Ant-arctica. Exercise was performed for approximately 10 heach day for 95 days. Despite an average energy intakeof 21.3 MJ á day)1 both subjects lost more than 25% ofbody weight. Energy expenditure was measured usingenergy balance data (EB) and isotope-labelled water(2H2

18O). Isotope doses were taken on day 0 and day 50of the expedition. During the ®rst 50 days both methodsgave reasonable agreement, giving energy expendituresof 38.3 (EB) and 35.5 (2H2

18O) MJ á day)1 in R.F. and28.6 (EB) and 29.1 (2H2

18O) MJ á day)1 in M.S. Theisotope data for days 20±30 yielded exceptional values of44.6 MJ á day)1 in R.F. and 48.7 MJ á day)1 in M.S.Estimates of energy expenditure between day 51 and day96 were much lower and although the methods were inagreement for R.F. ± 24.1 (EB) and 23.1 (2H2

18O) MJ áday)1, there was poor agreement for MS ± 26.8 (EB) and18.8 (2H2

18O) MJ á day)1. However, some practical dif-®culties occurred during this second period and therewere also problems arising from marked increases inbody water that made estimates of body mass andcomposition change di�cult to interpret. The latterproblems were probably due to malnutrition, which mayhave also been responsible for surprising increases inurinary excretion of 2H and 18O observed in both men ataround day 81. These increases may re¯ect the release oflabel incorporated into molecules other than water

which do not normally freely exchange with the bodywater pool under the circumstances of marked mal-nourishment. Following the expedition, both menshowed declines in maximal O2 consumption ( _V O2max,53.6 to 41.2 ml O2 kg

)1 á min)1 in R.F., 58.1±46.0 mlO2 kg

)1 á min)1 in M.S.); maximal voluntary isometricforce production in di�erent muscle groups (up to19.9% in R.F. and 55.8% in M.S.) and both cytoplasmicand mitochondrial skeletal muscle enzyme activities (upto 56% in R.F. and 63% in M.S.). Plasma samples takenduring the expedition showed low glucose levels, inap-propriately high insulin levels, and declines in testoster-one and luteinizing hormone. Thyroxine, cholesterol,albumin and triglyceride levels remained normal.

Key words Energy expenditure á Isotope-labelledwater á Exercise performance á Oxygen-18 á Deuterium

Introduction

Over the Austral summer of 1992/93, two men hauledsledges 2300 km on foot across Antarctica, withoutthe aid of other men, animals or machines. Logisticsdictated that sledge weights and hence food supplieswere minimised and so the journey provided an oppor-tunity to examine the e�ects of prolonged low-intensityphysical exercise combined with negative energy bal-ance. The studies reported here describe measurementsof energy balance, body composition changes, exerciseperformance, skeletal muscle enzyme activities and bio-chemical parameters re¯ecting nutritional status andphysiological stress.

Since the diet was pre-packaged and repetitive, it waspossible to obtain accurate estimates of energy andmacronutrient intake which, linked with measurementsof body weight and composition changes, were likely toprovide accurate estimates of overall energy expendi-ture. These estimates could then be compared withmeasurements of energy expenditure made using iso-tope-labelled water (2H2

18O) ± a technique which pro-

Eur J Appl Physiol (1997) 76: 243±252 Ó Springer-Verlag 1997

M.A. Stroud (&)MA Stroud, Institute of Human Nutrition, Mailpoint 113, Level C,West Wing, Southampton General Hospital, Southampton,SO16 6YD, UK

P. RitzLaboratoire de Nutrition Humaine, Clermont Ferrand, France

W.A. Coward áM.B. SawyerMedical Research Council, Dunn Nutrition Centre, Milton Road,Cambridge CB4 1XJ, UK

D. Constantin-Teodosin áP.L. Greenha� á I.A. MacdonaldDepartments of Physiology and Pharmacology, Medical School,Queen's Medical Centre, Nottingham, UK

vides measures of energy expenditure in free-living in-dividuals over periods of several days to a few weeks(Schoeller 1983; Coward and Cole 1991). It was hopedthat this would provide an opportunity to validate theisotope technique at very high work outputs, since mostearlier validation studies had relied on comparing thetechnique with direct or indirect calorimetry and hencethere had been few which examined expenditure in highlyactive subjects. The few studies that have attempted thishave also been di�cult to interpret due to the problemsof obtaining accurate alternative measures of energyexpenditure in active individuals over prolonged periods(e.g. Westerterp et al. 1986; Forbes-Ewan et al. 1989).

In an earlier study attempting to make a comparisonof energy balance versus isotope assessments of energyexpenditure under similar circumstances of a 48-dayNorth Pole journey, problems occurred due to uncer-tainties in the estimates of the natural isotopic back-ground levels (Stroud et al. 1993). These problems weredue to the 10-day period prior to taking the isotope dosebeing inadequate for the subjects to equilibrate fromnormal UK background values of 2H and 18O to thevery low background values present in melted-snowwhich provides the water source on such Polar journeys.Predictions of ultimate background levels were thereforemade by extrapolation of post-dose isotope disappear-ance curves, although the approach was limited by thefact that the men took a repeat isotope dose after only20 days. Nevertheless, the novel approach to dataanalysis appeared to work in principal and it was con-cluded that accurate estimates of the low backgroundisotope levels could be obtained without prior equili-bration if repeat dosing was delayed to obtain moreextended predictive data. This modi®cation to thestandard isotope-labelled water method was thereforeadopted in this study.

Although it is well recognised that endurance trainingwill lead to an increase in maximal aerobic capacity� _V O2max� and increases in skeletal muscle mitochondrialenzymes involved in b-oxidation, the citric acid cycleand the electron transport chain (e.g. Holloszy 1981;Hoppeler et al. 1985; Wibom et al. 1992), these studieswere undertaken under conditions of normal energybalance. This expedition provided an opportunity toexamine whether the same changes occurred followingvery prolonged endurance exercise with high but rela-tively inadequate food intake. Measurements of _V O2max,isometric muscle strength and skeletal muscle enzymeactivities were therefore made before and after thejourney. A variety of biochemical markers of nutritionalstatus and stress were also measured before, during andafter the journey.

Materials and methods

The expedition

In November 1992, two men set o� from the Atlantic coast of theAntarctic pulling sledges weighing 222 kg each which contained

100 days of food and fuel and other essential survival equipment.Initially they travelled for 20 days and 350 km across the ¯atFilchner ice-shelf before meeting the Antarctic mainland coast anda 340-km ascent to the Polar plateau at 3000 m. There then fol-lowed 550 km across the plateau to arrive at the South Pole on day68. After the pole, the men travelled a further 480 km on the Polarplateau, before descending to the Paci®c coast of Antarctica andthe Ross ice-shelf which was reached on the 90th day. An attemptto cross this second ice-shelf, to reach the open Paci®c ocean, hadto be abandoned on day 95 when supplies were running low and itbecame evident that the men were su�ering from severe malnutri-tion. The expedition was the ®rst to successfully complete acrossing of Antarctica without the use of aircraft to ferry food andequipment, and was the longest unsupported walk ever made. Stillair temperatures ranged from )45°C to )10°C often accompaniedby high winds (Stroud 1993).

Subjects

The two men, R.F. and M.S., were 48 and 37 years old respectivelyand gave informed, written consent to participate in the experi-ments.

Energy expenditure from dietary intakeand body weight/composition changes

Body masses were recorded in the UK (Salter KR120 scales) priorto and following the expedition and were recorded at 10-day in-tervals during the journey using a miniature load-cell portable scale(Miniscale, Raviv-Aran, Israel) accurate to �0.1 kg. On each oc-casion, the measurements were made after overnight fast, defae-cation and the voiding of urine.

Body composition was measured 10 days before and 7 daysafter the expedition using underwater weighing (UWW), withmeasurements of residual lung volumes using a helium dilutionspirometer (Gould Pulmonet III). Estimates of body compositionfor the period of the walk were made from the dilution spaces ofisotopes administered as part of the isotope studies using the 18Ospace (litres) on day 0 and day 50 and the 2H space (litres) on day95. Total body water (TBW) was calculated from the equations:

TBW = 18O space/1.01

and

TBW = 2H space/1.04

with lean tissue mass taken as TBW/0.73 kg.During the expedition, the subjects ate a specially formulated

diet containing a high proportion of fat in order to provide a highenergy to weight ratio. This consisted of freeze-dried meals sup-plemented with butter, chocolate bars, biscuits, soups and hotchocolate drinks. Analysis was performed before departure withfood values taken from tables (Paul and Southgate 1978) in the caseof standard items, and from manufacturers' nutritional informa-tion where specialised items were used. All food was weighed andpackaged into daily ration bags at the beginning of the expeditionand each man ate identical rations. Under the circumstances ofthese expeditions, no food is ever wasted and hence an accurateaverage daily energy intake could be calculated for the period of theexpedition itself. However, accurate food intake data were notavailable for the periods between the UWWs and the beginning andend of the walk.

Mean daily energy expenditure was estimated for the ®rst 50days and the last 45 days of the expedition by combining the meandietary energy intake with the mean daily energy de®cits calculatedfrom estimates of lean tissue and fat losses over each period of theexpedition. Lean tissue losses were assumed to be 73% water and27% protein whereas fat losses were considered to be 100% fat.The calori®c value of protein was taken as 18.39 kJ/g and fat wastaken as 39.7 kJ/g (Brouwer 1965)

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Energy expenditure using isotope-labelled water

Estimates of energy expenditure were made using the 2H218O

method, following body isotope disappearance by daily collectionand later analysis of 2-ml urine samples. Each subject had twoseparate determinations of energy expenditure, one between days 1and 50 and the other between days 51 and 95. The doses given weredesigned to achieve a body water enrichment above pre-dose of300 ppm for 18O, and 130 ppm for 2H. Saliva samples were col-lected immediately prior to and 4, 5 and 6 h after each 2H2

18O dosein order to measure 18O dilution spaces. A dose of deuterated water(2H2O) was taken at the end of the expedition to measure the ®nal2H dilution space.

All samples were returned to the UK where 2H enrichmentswere measured with the standard uranium technique (Barrie andCoward 1985) and 18O enrichments were measured with the H2O±CO2 equilibration technique (Wong et al. 1987). Background levelsand disappearance rate constants for both 2H and 18O were thencalculated by using non-linear regression analysis (Genstat soft-ware, release 1.3; Lawes Agricultural Trust) of observed enrich-ments to calculate initial (E0) and in®nite time (E¥) asymptotes ofthe enrichment curves and their slopes (k) according to the equa-tion:

Et � �E0 ÿ E1�eÿkt � E1

The regression analysis software also produced standard errors forthese data. Isotope-dilution spaces were estimated from both thepost-dose saliva samples and the time-zero intercepts of the isotopedisappearance curves.

Carbon dioxide production was calculated for the entire ®rst setof 50 days' data, but for the second set only data up to day 80 couldbe used because of an unexpected rise in enrichments from days 81to 95 (see Results). R values were calculated from the compositionof the diet and the estimated contribution to metabolism of thechanging body energy stores. Energy expenditures were then cal-culated from CO2 production values using an R-related energyequivalent per litre CO2 (Elia 1991). Calculations were made al-lowing for assumed fractionation of either 20%, 30% or 40% ±values which were likely to cover the possible range under suchenvironmental conditions. Energy expenditure was also estimatedfor shorter 10-day intervals during the ®rst 50 days of completedata by performing the non-linear regression analysis on data fromdays 0±10, 0±20, 0±30, 0±40 and 0±50. Linear changes in dilutionspaces with time were assumed for these calculations.

_V O2max and isometric strength

_V O2max was measured from Douglas bag collections made duringtreadmill running using a continuous incremental exercise protocol(Woodway treadmill, UK; 1400 series paramagnetic O2 analyserand infra-red CO2 analyser, Servomex, UK; chain compensatedgasometer, Cranlea, UK). Dominant maximal voluntary isometricforce production (MVC) was measured for elbow ¯exion, elbowextension, abdominal ¯exion and leg extension using a Hermansenisometric rig (Hermansen et al. 1972); and for hand grip and up-right pull using specialised dynamometers (Digimeter, MIE, UK,and Takei, Japan respectively).

Skeletal muscle enzyme activities

Ten days prior to the expedition and 6 days following its comple-tion, muscle biopsy samples were taken from vastus lateralis usingthe percutaneous needle biopsy technique described by BergstroÈ m(1962). Samples were immediately frozen in liquid nitrogen andsubsequently freeze-dried. The muscle samples were then dissectedfree from visible blood and connective tissue, and powdered.Freeze-dried muscle (approx. 2 mg) was homogenised (1:400 drywt/vol) in a solution consisting of (mmol á l)1) either 50 KH2PO4,1 EDTA and 0.1% Triton X-100, or 100 KCl, 50 KH2PO4, 50 Tris,5 MgCl2 á 6H2O, 1 EDTA and 1.8 ATP. Enzyme activities were

assessed for four di�erent enzymes, chosen to be representative ofdi�erent components of muscle energy metabolism (Wibom et al.1992): glyceraldehyde-3-phosphate dehydrogenase (Gly3PDH, EC1.2.1.12) from glycolysis; b-hydroxyacyl-CoA dehydrogenase(HAD, EC 1.1.1.35) from b-oxidation of free fatty acids, citratesynthase (CS, EC 4.1.3.7) from the citric acid cycle; and cyto-chrome-c oxidase (COX, EC 1.9.3.1) from the electron transportchain. Gly3PDH and HAD, CS, and COX activities were deter-mined spectrophotometrically at 25°C, as described by Bass et al.(1969), Alp et al. (1976) and Gohil et al. (1981), respectively.

Plasma biochemical parameters

At 10-day intervals during the expedition, blood samples were ta-ken to assess hormonal and biochemical responses to the exercise,cold and relative undernutrition. Venepuncture was performedapproximately 30 min after the end of the daily 10±12 h of exerciseand at least 4 h after the last food intake. The samples were col-lected into tubes containing ¯uoride oxalate which were then hungin the roof of the tent for 2±3 h to allow partial red blood cellsedimentation. A small plasma sample of between 0.5 and 1.0 mlwas then pipetted o� and allowed to freeze. Following the experi-ment, the frozen samples were returned to the UK where standardenzymatic and radioimmuno assays were used to assess glucose,insulin, growth hormone (GH), cortisol, testosterone, luteinizinghormone (LH), thyroid function, cholesterol, triglycerides, totalprotein and albumin.

Results

Energy expenditure from dietary intakeand body weight/composition changes

The dietary intake provided an average over the wholejourney of 21.3 MJ per day of which 56.7% came fromfat, 35.5% from carbohydrate and 7.8% from protein.Over the ®rst 50 days, the average daily intake was19.9 MJ consisting of 95 g protein, 293 g fat and 425 gcarbohydrate. Over the second part of the expedition theaverage daily intake was 22.2 MJ consisting of 103 gprotein, 338 g fat and 557 g carbohydrate. Despite thehigh energy intake, both men lost considerable weightover the course of the expedition and became signi®-cantly debilitated by starvation.

Unfortunately, the saliva samples for R.F. followingthe dose of isotopes on day 50 were lost and, further-more, by day 50 it appears that M.S. had already de-veloped a degree of ¯uid retention. This was evidentfrom the fact that although there had been goodagreement between the estimates of TBW from salivaand the 18O regression intercept data for day 0, by day50 the estimates of TBW for M.S. from the 18O regres-sion intercept data were markedly lower than those fromhis saliva samples. The 18O regression intercept datawere therefore used to calculate TBW at day 0 and 50for both men.

By day 95, the problem of ¯uid retention had becomeeven more marked and peripheral oedema was clinicallyevident in both subjects. This led to the 2H saliva datagiving impossibly high estimates of TBW and hence evengreater di�culties in estimating likely body composition.However, since the UWW observations of 7 days later

245

suggested that the body fat of both men was still under3%, even after very vigorous refeeding, it seemed likelythat fat stores were essentially zero at the end of theexpedition. The appearance of oedema during the courseof the expedition was almost certainly due to malnutri-tion and would have also led to underestimates of weightloss in both men, particularly for the second period ofstudy.

Table 1 shows body weight measurements, the esti-mates of TBW from both 18O saliva data and the 18Oregression intercept data, and body composition esti-mates from both UWW and TBW. Using these ®gures,combined with the intake data, energy expendituresduring the ®rst 50 days were 38.3 MJ á day)1 for R.F.and 28.6 MJ á day)1 for M.S., whilst during the last 45days they were 24.1 MJ á day)1 for R.F. and26.8 MJ á day)1 for M.S.

Energy expenditure from isotope-labelled water

Figure 1 shows the decline in isotope concentrations andthe ®tted isotope disappearance curves for R.F. whileFig. 2 shows the same data for M.S. The non-linearregression analysis of the isotope disappearance curvesfor both R.F. and M.S. for the ®rst 50 days was verysuccessful and gave standard errors of around only0.8 ppm on the predicted background asymptotes, alevel of accuracy close to estimates of within-subjectvariation for 18O, although double that which would beexpected for 2H. However, following the second doses ofisotope-labelled water, the curves for both R.F. andM.S. showed an unexpected rise in both 2H and 18Oenrichments between days 80 and 95. Non-linear re-gression analysis was therefore limited to the periodbetween days 51 and 80, although this was successful inthat predictions of asymptotic values could still be madewhich were associated with very small estimates ofstandard error.

In line with the arguments presented above, the esti-mates of CO2 production were made allowing for iso-

tope dilution spaces calculated from the regressionintercepts. Changing the assumed levels of fractionationbetween 20% and 40% made virtually no di�erence tothe results, because of the unusually high rate constantfor the disappearance of 18O compared to 2H ± a con-sequence of the very high energy expenditures and henceloss of 18O through CO2 production. A value of 30%was therefore used to calculate the values of energy ex-penditure from the isotope studies which are shown inTable 2 along with the estimates based on energy bal-ance, and the calculated R values. Table 3 shows theresults of the further analysis of the isotope data for 10-day periods during the ®rst 50 days.

_V O2max and isometric strength

Following the expedition _V O2max had declined from4.77 l á min)1 to 3.14 l á min)1 in R.F. and from4.01 l á min)1 to 2.61 l á min)1 in M.S. These changescould not be attributed to changes in body weight alonewith declines from 53.6 to 41.2 ml O2 á kg)1 á min)1 inR.F. and from 58.1 to 46.0 ml O2, á kg)1 á min)1 inM.S. The MVC force production measured in di�erentmuscle groups showed greater declines in M.S than R.F.(Table 4).

Table 1 Body weight, total body water (TBW )18 from O salivadata (TBWs) and 18O regression intercept data (TBWr), and bodycomposition estimates from both underwater weighing (UWW )and TBWr

Subject Parameter Day

)10 0 50 95 +7

R.F:Mass(kg) 89.9 95.6 72.8 71.0 76.2TBWs ± 51.50 ± ± ±TBWr ± 51.45 51.54 ± ±%fat TBWr ± 26.2 3.0 ± ±% fat UWW 17.4 ± ± ± 1.9

M.S:Mass(kg) 69.0 74.8 56.6 53.0 58.8TBWs ± 43.60 42.05 ± ±TBWr ± 43.44 33.32 ± ±%fat TBWr ± 20.4 9.2 ± ±% fat UWW 16.3 ± ± ± 2.5

Fig. 1 Observed 2H and 18O enrichments in R.F. for the ®rst 50 days(top) and last 45 days (bottom).The ®tted lines from linear regressionare superimposed

246

Skeletal muscle enzyme changes

The skeletal muscle biopsy samples pre- and post-expe-dition showed marked decreases in cytoplasmic and mi-tochondrial enzyme activities in both subjects (Table 5).

Plasma biochemical parameters

A variety of changes occurred in the hormones andbiochemical markers measured in both men and theseare shown in Table 6 and Figs. 3±5.

Discussion

The undertakings of this expedition were most unusualand resulted in massive exercise-induced weight lossesdespite a very high energy intake. They also led to thedevelopment of ¯uid retention during the course of theexpedition which was quite marked by the end of thejourney. Such changes are known to occur to a smallextent with prolonged endurance exercise (Williams et al.1979) and to a much greater extent with the onset ofmalnourishment (McCance 1951).

Although the development of this ¯uid retentionmade evaluation of the body weight and compositionchanges di�cult, they were obviously extreme for eventhe Minnesota experiment of Keys et al. (1950) onlydescribed changes of 70% in fatness and 20% in musclemass after 52 weeks of chronic under-nutrition. Thedegree of change can probably be attributed to the veryhigh energy expenditures sustained during the period,which from both the energy balance and isotope resultsappeared to be most unusual. Certainly, the ®gures of44.6 MJ á day)1 for R.F. and 48.7 MJ á day)1 for M.S.estimated for the period between days 20 and 30 areextremely high compared to mean values reported forfour male cyclists in the Tour de France of only29.4 MJ á day)1 (Westerterp et al. 1986) and for fourmale elite cross-country skiers during a week of trainingof only 30.2 MJ á day)1 (Sjodin et al. 1994). They mustalso lie close to what is physiologically possible, al-though they do remain lower than the theoretical energyexpenditure ceiling of 58.5 MJ á day)1 that could be at-tained by ultra-long-distance runners (Davies andThompson 1979). They are nevertheless credible sincethey correspond to the period when the heavy sledgeswere dragged uphill from the ice-shelf to the plateau, atime that was matched by the most rapid weight losses.Later in the expedition, when the men were descendingfrom the Polar plateau with lighter sledges, energy ex-penditures were nearer normal and, overall, the energyexpenditure values for the whole journey do not lookunreasonable considering the circumstances.

When planning these experiments, it was hoped thatthe energy balance estimates of energy expenditurewould be accurate and that they could therefore be usedto validate the isotope method under these circum-

Fig. 2 Observed 2H and 18O enrichments in M.S. for the ®rst 50 days(top) and last 45 days (bottom). The ®tted lines from linear regressionare superimposed

Table 2 Energy expenditure form the energy balance techniqueand the isotope-labelled water technique, mean(SE) Isotopevalues assumed 30% fractionation and were calculated fromCO2 production using R values estimated form food intake/changes in body composition

Subject Energy expenditure MJ á day)1

Energybalance

Isotopemethod

R Energybalance

Isotopemethod

R

0±50 0±50 0±50 51±95 51±80 51±80R.F. 35.2 35.5 (2.1) 0.76 23.6 23.1 (1.9) 0.76M.S. 32.7 29.1 (2.3) 0.78 26.4 18.8 (2.4) 0.78

Table 3 Variation in energy expenditure measured using the iso-tope method [mean (SE)] during the ®rst 50 days

Days Energy expenditure MJ á day)1

R.F. M.S.

1±10 39.8 (7.00) 26.8 (6.54)1±20 26.9 (7.56) 22.0 (7.98)2±30 44.6 (3.44) 48.7 (5.42)3±40 36.4 (2.49) 26.7 (3.64)4±50 29.9 (2.66) 23.3 (3.14)

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Table 4 Changes in maximal voluntary contraction isometric force production (kg)

R.F. M.S.

Pre Post %Change Pre Post %Change

Elbow ¯exion 28.1 25.2 )10.4 27.4 16.3 )40.6Elbow extension 23.2 18.6 )19.9 20.2 14.7 )27.2Grip strength 46.1 45.4 )1.5 64.9 40.9 )27.0Leg extension 121.0 113.8 )6.0 192.1 85.0 )55.8Abdominal ¯exion 45.7 46.3 +1.3 52.5 42.7 )19.7Upright pull 144.7 131.3 )9.3 183.2 113.0 )38.3

Table 5 Enzymes activities in vastus lateralis muscle biopsy samples taken pre- and post-expedition (enzymes activites are expressed asmmol of substrate used min)1 á kg)1 wet muscle at 25°C), (Gly3PDH Glyceraldehyde-3-phosphate dehydrogenase, HAD b-hydroxyacyl-coA dehydrogenase, Cyt-c oxidase cytochrome-C oxidase)

Glyc3 PDH HAD Citrate synthase Cyt-c oxidase

Pre- Post- %D Pre- Post- %D Pre- Post- %D Pre Post %D

R.F. 195 104 47 6.04 3.10 49 20.14 13.10 35 8.42 3.74 56M.S. 216 96 56 5.86 4.91 16 23.33 20.22 13 7.48 2.80 63

Table 6 Plasma thyroxine (T4, T3), thyroid stimulating hormone, cholesterol, HDL cholesterol, triglycerides, albumin concentrations andtotal protein during and 6 days post-expedition. (N/A Insu�cient sample)

Day Free T4c Free T3

c TSHb Cholesterola HDL Chola Triglyceridesa Albumind Total proteind

R.F. M.S. R.F. M.S. R.F. M.S. R.F. M.S. R.F. M.S. R.F. M.S. R.F. M.S. R.F. M.S.

10 9.5 18.7 3.8 4.7 2.0 1.9 4.9 4.9 0.9 0.8 0.75 N/A 44 N/A 67 N/A20 8.0 19.6 3.2 4.4 2.0 2.3 5.5 4.9 1.1 0.8 0.85 0.75 44 45 66 7130 9.0 20.3 3.5 5.2 2.0 1.9 6.4 4.6 1.0 0.9 0.85 0.68 45 41 66 6440 8.7 19.2 3.3 4.6 1.5 2.4 6.0 5.6 1.3 1.2 0.93 0.81 45 44 66 6750 9.4 19.6 3.5 4.2 1.7 2.1 5.7 5.5 1.5 1.3 N/A 0.7 N/A 46 N/A 7160 8.8 17.5 3.3 4.3 1.5 1.7 5.4 5.5 1.7 1.3 0.85 0.75 43 42 65 6770 9.2 16.7 3.3 3.5 1.4 1.7 5.0 5.5 1.5 1.5 0.65 0.92 47 44 68 6895 10.5 14.6 2.9 3.0 1.7 1.8 5.2 4.1 1.0 1.1 0.6 0.69 41 40 63 61Post 13.2 19.7 4.4 6.3 1.8 1.4

Values are given in:ammol á l)1b IU á l)1c nmol á l)1d g á l)1

Fig. 3 Blood glucose and insu-lin concentrations during andafter the expedition

248

stances. However, the changes in body water that oc-curred with the onset of malnourishment meant thatvarious assumptions had to be made which obviouslyweaken the credibility of the results. Nevertheless, webelieve that the techniques and the ®ndings warrant re-porting along with a full discussion of the possiblesources of error particularly since the problems thatoccurred are not likely to have had a marked in¯uenceon the estimates of energy expenditure for the ®rst pe-riod of study.

The potential sources of error using the energy bal-ance technique resulted from the problems of estimatingbody weight and composition under circumstances inwhich there was evidence of increasing body water

during the course of the expedition. This would have ledto underestimates of body weight losses and consequentunderestimates of energy expenditure although thesemay have been o�set or even reversed by overestimatesof TBW with consequent overestimates of fat loss. Er-rors in either direction would probably have been smallfor the ®rst period of study when no oedema was clini-cally evident and when estimates of body compositionfrom the isotope regression intercepts seem reasonable.The higher estimates of TBW from the saliva data ofM.S. at day 50 and both men at day 95 may re¯ect ane�ect of the malnutrition on the distribution andquantity of body water that made post-dose equilibra-tion timing very abnormal.

Fig. 4 Cortisol and growthhormone concentrations duringand after the expedition

Fig. 5 Testosterone and lute-inising hormone (LH) concen-trations during and after theexpedition

249

For the second period of the expedition, the potentialscale of the errors was much greater. In particular, thepresence of clinical oedema suggests that the estimates ofbody weight losses were at least 2 kg too low (Julian1977) and the impossibly high estimates of TBW fromthe isotope dilution spaces also suggest profound bodywater changes. The estimates of body composition weretherefore made from the UWW performed 7 days later,but this itself was subject to potential problems since thetechnique assumes an unchanging density of lean tissues± a situation that may not have been true under theseextreme circumstances. Nevertheless, the men's fatcontent is likely to have been extremely low at the end ofthe expedition and, furthermore, even if 1 kg of weightloss was mistakenly assigned to fat rather than leantissues, the overestimate in daily energy expenditurewould only amount to 0.72 MJ á day)1.

Overall, we therefore believe that the estimates ofenergy expenditure from the energy balance results arelikely to be fairly accurate for the ®rst part of the ex-pedition, and probably low but reasonable for the sec-ond part.

The estimates of energy expenditure using the modi-®ed isotope-labelled water technique were fairly close tothose from the energy balance data in both men over the®rst 50-day study, and were in reasonable agreement forthe second period. The originality of the technique wasthat it neither relied on direct measurements of naturalabundance, nor on indirect prediction of that abundanceto estimate new background levels under circumstancesof a changing water source. Instead, it used the asymp-tote of exponential disappearance curves at in®nite timeand hence overcame the need to wait for a new isotopicequilibrium to be reached ± a situation that would havebeen impractical under these conditions of relativelyslow water turnover.

The isotope data themselves were internally consis-tent. Over the ®rst 50 days, the SEs estimated from theregression lines were very small, giving great con®dencein the estimates of CO2 production. The suggestion ofinternal accuracy was then further enhanced by the factthat the high CO2 production resulted in such largedi�erences in the rate constants for 2H and 18O, that the®ndings were remarkably insensitive to the e�ects ofeither fractionation or the probable errors in estimatesof the dilution spaces. Furthermore, the estimates of Rfrom the diet and overall body composition measure-ments would have been unusually accurate.

Unfortunately, there were sources of error in the useof the technique during the second period of the studywith the occurrence of the surprising rise in urinary 2Hand 18O levels. This occured at around day 80 whichmeant that the isotope expenditure estimates for theentire second period had to be based upon the data fordays 51±80. This limited period may not have beenrepresentative of the entire period up to day 95, al-though anecdotal accounts of the men's activities(Stroud 1993) suggest that no major change in expen-diture levels would have occurred at that time. Since the

geography of the walk makes it di�cult to envisage whya change in enrichments of the natural backgroundwater source (the snow and ice) should have occurredtowards the end of the expedition, it seems likely that theobserved increases in 2H and 18O were due to re-entry ofisotopes into the water pool from internal sequesteredsources. As such, the problem might also have a�ectedthe data prior to day 80, although there is no suggestionthat this phenomenon was occurring during the periodof the ®rst dose prior to day 50. The source of this in-ternal isotope release may have been structural proteinsor other molecules which do not usually exchange withbody water at a signi®cant rate and which had incor-porated higher isotope enrichments at the time of theirsynthesis either back in the UK, from the naturally higherbackground levels, or earlier in the expedition followingisotope dosing. The isotopes were then released as these``labelled'' materials were broken down, e.g. a cohort ofred blood cells produced at the beginning of the expedi-tion following the ®rst isotope dose may have beenreaching the end of their lifespan with the consequentbreakdown of ``labelled'' haemoglobin. However, it isdi�cult to explain the fact that the increases of 18O wereof a similar magnitude to those of 2H when sequesteredbody stores of 2H are likely to have been considerablygreater. Nevertheless, despite these di�culties, we believethat this study of energy expenditure in Polar travellerssupports the fact that the isotope-labelled water tech-nique can bemodi®ed to provide measures of total energyexpenditure in very active individuals, moving to remote®eld areas with unusual background isotope levels.

The changes in exercise performance, muscle enzymeactivities and some biochemical parameters followingthe prolonged period of endurance exercise and negativeenergy balance that the expedition entailed were alsovery striking. With regard to the studies on exerciseperformance and muscle function, the prolonged phys-ical work would have normally been expected to pro-duce an increase in mitochondrial protein content(Hoppeler et al. 1985), oxidative enzyme activities (Ho-lloszy et al. 1981; Holloszy and Booth 1976), _V O2max

(Davies et al. 1983) and probably maximal isometricstrength in the muscle groups directly involved in thesledge-hauling. The declines in exercise performance andmuscle enzyme activities observed in the present studywere therefore surprising. The enzyme decreases were inparticularly marked contrast to increases of 20±80%seen in the same enzymes after 6 weeks of endurancetraining in subjects with normal energy balance (Wibomet al. 1992), and it was notable that all of the enzymesmeasured declined, with no suggestion of preservation ofthe capacity for fat utilisation which would have beenthe primary muscle substrate under these circumstancesof sustained exercise on a high-fat diet. The observeddeclines in enzyme activities are uncorrected for theprotein content of the homogenates, and may thereforebe partly attributable to a relatively greater loss of thesenon-contractile protein elements compared to structural,connective tissue support, but it seems more likely that

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the changes can be attributed to the negative energybalance and the need for muscle protein oxidation too�set the energy de®cit.

The decrease in skeletal muscle mass and enzymeactivities would have directly a�ected peripheral oxygenutilisation and this must have contributed to the ob-served decline in _V O2max. However, under normal cir-cumstances, much of the increase in _V O2max that is seenwith training is attributable to increases in cardiac out-put and consequent oxygen delivery to the musculature.It is therefore possible that declines in cardiac outputalso contributed to the drop in _V O2max, since losses ofcardiac muscle and lowered cardiac outputs have beendescribed during weight loss induced by dietary restric-tion with a smaller degree of negative energy balance(Rahamadany et al. 1989). The decline in the activity ofthe skeletal muscle enzymes measured would not directlyaccount for the observed loss of isometric force pro-duction, but in view of the loss of muscle bulk and en-zyme activity it would seem likely that contractileproteins (actin, myosin, myosin ATPase, etc) were alsolost. It was particularly notable that M.S. showed muchgreater declines in isometric strength than R.F. and itseems likely that these re¯ect his greater lean tissuelosses of 6.5 kg compared to 4.3 kg in R.F. Thesegreater losses were accompanied by greater de®cits innitrogen balance which was recorded over ®ve 24-h pe-riods during the expedition and which are reportedelsewhere (Stroud et al. 1997).

The blood samples taken during the expeditionyielded some very unusual results. Most striking werethe low end of day blood glucose levels of around4.0 mmol á l)1 in both men for most of the journey, andthe grossly hypoglycaemic levels of around 0.3 mmo-l á l)1 in the latter stages on days 70 and 95. It is obvi-ously tempting to assume that these low values wereartefactual, and certainly they may have been exagger-ated by the cold causing low peripheral blood ¯ows andhence greater peripheral glucose extraction. However,they were accompanied on one occasion by raised cor-tisol and GH levels which would be appropriate re-sponses to marked hypoglycaemia and there was ageneral trend for increasing cortisol and GH levelsthroughout the expedition. There is also no evidencefrom the other assays that there were any problems withsample storage. It would therefore appear that the pro-longed exercise, combined with the relatively low car-bohydrate intake, did genuinely lead to severehypoglycaemia and that the men had adapted to the useof other substrates in the central nervous system such asketones and lactate. It is recognised that this is probablypossible, since it is only acute drops in blood glucosethat invariably lead to unconsciousness in diabetic pa-tients. However, we are not aware of reports describingpatients who are conscious at levels as low as thoseobserved on this study, although it would be very un-usual circumstances that would expose individuals tolow glucose values for adequate periods to permitmaximal adaptation.

The insulin levels of around 11 to 14 mlU á l)1 seenover the ®rst 70 days of the expedition were rather highconsidering the low glucose levels and this may re¯ect aloss of insulin sensitivity due to the very high fat diet.However, the insulin levels of 15 and 30 mlU á l)1 inR.F. and M.S. on day 95 would still seem totally inap-propriate in the face of glucose levels of only 0.3 and0.4 mmol á l)1 and, indeed, it would seem likely that thisinexplicable hyper-insulinaemia was causing the hypo-glycaemia. Certainly, the high insulin levels tend tosupport the fact that the men were genuinely hypo-glycaemic.

The striking decline in testosterone in both men wassimilar, although more marked, to that reported byAakvaag et al. (1978) in soldiers undergoing a combi-nation of physical stress and near-total sleep deprivationfor 5 days. However, LH also declined while Aakvaagreported little change or small increases in LH levels.

The preservation of plasma albumin levels despite theundernourishment is in considerable contrast to the fallsthat would be anticipated under circumstances of similarweight loss induced by dietary restriction. This probablyre¯ects the maintained levels of whole-body proteinsynthesis which were also measured during the ®ve ni-trogen balance studies and reported elsewhere (Stroudet al. 1997). The relatively stable cholesterol levels are ofnote in view of the enormously high intake of saturatedfat in the men's diet which corresponded to three timesthe UK average. It presumably re¯ects the e�ect of theexercise and the negative energy balance.

Overall, although there were only two subjects in thestudies, the ®ndings demonstrate that marked bio-chemical and physiological changes can occur when menwork hard in negative energy balance for a prolongedperiod. We therefore believe that they provide uniqueinformation regarding the body's capabilities to adaptunder circumstances which were probably close tophysiological limits.

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