dehydrating

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O R I G I N A L A R T I C L E

Comparison between blood and urinary fluid balance indicesduring dehydrating exercise and the subsequent hypohydration

when fluid is not restored

Nassim Hamouti Juan Del Coso

Ricardo Mora-Rodriguez

Received: 17 March 2012/ Accepted: 23 July 2012 / Published online: 11 August 2012

Springer-Verlag 2012

Abstract Blood serum osmolality (SOSM) is the gold stan-

dard to assessbody fluid balance. Urine specific gravity (USG)is also a body fluid balance index but it is not invasive.

However, USG capability to detect the minimal level of

dehydration that affects athletic performance (i.e., 2 %)

remains untested. We collected urine and blood samples in

eighteen euhydrated trained athletes in the morning and that

evening while dehydrating by 1, 2, and 3 % of body mass by

cycling (60 % _VO2peak) in the heat (32 C, 46 % rh,

2.5 m s-1 air flow). At 9:00 pm, subjects left the laboratory

and went to bed after ingesting 0.7 0.2 L of a sports drink.

The next morning, subjects awoke 3 % hypohydrated, and

blood and urine samples were collected and test terminated.

We found that 2 % dehydration increased SOSM and USGabove exercise-baseline values (P\0.05). The next morn-

ing,SOSM and USG remained elevated compared to the first

morning while euhydrated (287 5 vs. 282 3 mO-

smol kg-1 H2O and 1.028 0.003 vs. 1.017 0.005,

respectively, P\0.05). However, when comparing 3 %

dehydration (end of exercise) to 3 % hypohydration (next

morning),USGincreased (1.025 0.003 to 1.028 0.003;

P\ 0.05) whileSOSMdecreased (295 5 to 287 5 mO-

smol kg-1 H2O; P\0.05). In summary, during exercise-

induced dehydration,USG isassensitiveas SOSM to detect low

levels of dehydration (i.e., 2 %). Both indices maintain the

ability to detect a 3 % overnight hypohydration although

SOSM approaches euhydration values, while USG remains a

superior index to detect hypohydration.

Keywords Urine specific gravity Blood serum

osmolality Body fluid shift Hydration status Renal water reabsorption

Introduction

Despite recent criticisms (Maughan et al. 2007), percent

reduction in body weight after exercise is a simple and non-

invasive measure of dehydration (Baker et al. 2009). In

addition, fasting morning body weight is a reliable index of

euhydration (i.e., *0.6 % CV) when assessed during 39

consecutive days (Cheuvront et al. 2004; Hamouti et al.

2010a). However, the assessment of hydration status based

only on body weight has several limitations. First, a log of

body weights during 39 days is not always feasible.

Often, we need to assess hypohydration in people from

which we do not know their habitual body weight. Second,

in people participating in a exercise program, body weight

can change over the course of a season as a consequence of

body fat loss (Koutedakis 1995) or muscle hypertrophy

independently of changes in body water. In those cases,

biochemical indices of hydration status based on concen-

tration and composition of body fluids (i.e., blood and

urine) become a necessary alternative to monitor body fluid

balance.

Urine specific gravity (USG) is frequently monitored in

laboratory (Armstrong et al. 1998; Bartok et al. 2004;

Hamouti et al.2010a) and field studies (Godek et al.2005;

Stover et al. 2006) to detect athletes body fluid deficit.

Unlike blood indices of fluid deficit (e.g., serum osmolal-

ity, SOSM), USG does not need invasive sample collection.

In addition, the USG apparatus is easy to operate and rel-

atively inexpensive which may explain its increased pop-

ularity among the athletic community (Kutlu and Guler

Communicated by Narihiko Kondo.

N. Hamouti J. Del Coso R. Mora-Rodriguez (&)Exercise Physiology Laboratory, University of Castilla-La

Mancha, Avda. Carlos III s/n., 45071 Toledo, Spain

e-mail: [email protected]

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Eur J Appl Physiol (2013) 113:611620

DOI 10.1007/s00421-012-2467-9


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2006; Osterberg et al. 2009). However, USG sensitivity to

detect low levels of dehydration during exercise in the heat

has been questioned (Popowski et al. 2001; Oppliger et al.

2005). In these studies, USG did not significantly increase

above euhydration levels until participants reached 3 %

dehydration. However, in these studies 2 % dehydration

was not tested. Thus, it is unclear ifUSGis sensitive enough

to detect a 2 % levels of dehydration which athletes oftenincur (Hamouti et al. 2010b) and that could reduce their

athletic performance (Walsh et al. 1994; Dougherty et al.

2006; Baker et al. 2007).

Blood serum osmolality (SOSM) is the gold standard

measurement for the detection of body fluid deficit caused

by prolonged exercise (Oppliger and Bartok2002; Sawka

et al. 2007). However, SOSM seems to lose its sensitivity

when used to assess long-term body fluid deficit (i.e.,

hypohydration). Francesconi et al. (1987) reported morning

values ofUSGand SOSMfrom army personnel during several

days of military field training at moderate altitude. They

found that, despite participants reduced body mass by 3 %,their SOSM did not increase while USG was markedly

increased. However, their reductions in body mass might be

affected by the negative caloric balance due to exercise

rather than dehydration since soldiers did not increase

caloric intake to match their daily caloric needs over the

experimental interval. Likewise, Armstrong et al. (1994)

could not find a correlation between USGand SOSMin tennis

players when urine and blood samples were collected in the

morning during three consecutive days of a tournament in a

hot environment. It is well established that soon after

dehydrating exercise, half of the reduced plasma volume is

recovered even if no fluid is ingested possibly due to the

bodys ability to shift fluid from the intracellular to the

vasculature fluid space (Nose et al.1988). It is possible that

this defense of plasma volume in the face of sustained

dehydration could explain the data from Francesconi et al.

(1987) and Armstrong et al. (1994). It is important to

determine if SOSM is able to correctly detect overnight

hypohydration since it is not uncommon for athletes

(Hamouti et al. 2010b) and other occupational personnel

(i.e., soldiers, Lieberman et al. 2005; and firefighters Horn

et al.2012) to neglect rehydration after an evening workout

and remain hypohydrated overnight. Detection of hypohy-

dration permits correcting the fluid deficit avoiding poten-

tial negative effects on athletic performance.

The purpose of this study was to determine the ability of

USG to detect the low levels of dehydration (2 % of body

mass loss) induced during the early stages of prolonged

exercise in the heat. Additionally, we sought to determine

ifUSG reflects long-term (i.e., 11 h) body fluid deficit (i.e.,

hypohydration) better than SOSM. Indices of renal function

(i.e., water retention and sodium reabsorption) and fluid

shift between body compartments (intracellular and

extracellular) were calculated to help explain the USG and

SOSM responses.

Methods

Participants

Eighteen aerobically trained male athletes volunteered to

participate in this study. Participants mean SD for age,

height, body weight, percent body fatand peak oxygenuptake

were 20.3 1.9 years, 1.76 0.06 m, 74.5 17.0 kg,

9.2 3.1 % and 58.4 10.3 mL kg-1 min-1, respec-

tively. They were non-smokershad no history of renal disease

and were not taking medication during the study. All partic-

ipants were fully informed of any risks and discomforts

associated with the experiment before giving their informed

written consent to participate. The study was approved by the

local Hospital Research Ethics Committee and conducted inaccordance with the guidelines of the revised Declaration of

Helsinki.

Preliminary testing

Body mass, height and six skinfolds (i.e., subscapular,

suprailiac, abdominal, triceps, front thigh and medial calf)

were measured to estimate percent body fat (Carter1982).

Following, oxygen uptake _VO2 was measured during acontinuous incremental exercise test to volitional exhaus-

tion on an electronically braked cycle ergometer (Ergose-

lect 200, Ergoline, Germany). After a 5-min warm-up at75 W, participants began cycling at 100 W with incre-

ments of 25 W every 1 min. Gas exchange data were

collected using an automated breath by breath system

(Quark b2, Cosmed, Italy) and averaged every 15 s. At rest

and at the end of each exercise stage, an ECG recording

(Quark T12, Cosmed, Italy) and blood pressure (Gamma

GST, Heine, Germany) were measured to discard abnormal

cardiovascular responses. The peak O2 uptake _VO2peak

was defined as the highest value reached during the test.

The preliminary test was carried out at 10:0012:00 am and

at least 2 days before the experimental protocol and par-

ticipants refrained from hard physical activity the day prior

to testing.

Experimental protocol

Participants withdrew from all dietary sources of caffeine

and alcohol for 48 h before the experimental. The night

before the beginning of the experiment, participants

ingested a packed dinner provided by the experimenters

consisting of 300 g of meat lasagne (i.e., 414 kcal,

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carbohydrate 30 g, protein 20 g, fat 26 g, sodium 1 g,

chloride 0.7 g, potassium 0.4 g), with one glass of tap

water (*150 mL) and a piece of fresh fruit. After dinner

and before participants went to bed, they voided and

drank 500 mL of bottled water (also provided by the

experimenters) to ensure that the next morning urine

specimen was representative of steady state hydration

(i.e., after *8 h of resting). Participants reported to thelaboratory on three occasions. On the first visit (i.e.,

morning of day 1), participants arrived to the laboratory

fasted at 8:00 am while nude body mass, urine and blood

samples were collected. Urine specimens were immedi-

ately measured in duplicate for USG to ensure euhydration

(i.e., USG\ 1.020; Sawka et al. 2007). Then, participants

were offered a breakfast with fluids. Thereafter, partici-

pants attended their university courses in an air condi-

tioning environment and ingested lunch at the university

canteen.

On the second visit (i.e., evening of day 1), participants

exercised in a hot environment (32 1 C, 46 % rh,2.5 m s-1 air flow) to induce dehydration. Three hours

before arriving to the laboratory (3:00 pm), participants

ingested a bowl of pasta (i.e., 250 g; 177 g of carbohy-

drate) to maximize blood glucose and muscle glycogen

levels before prolonged cycling in the heat. In addition,

2 h before arriving to the laboratory, participants drank

500 mL of water to increase the likelihood that they

would begin the trial in a euhydrated state. Two hours

elapsed between water ingestion and the start of exercise

to allow excretion of the excess fluid by urination. Upon

arrival to the laboratory (6:00 pm), participants voided

into a sterilized container and USG was immediately

measured in duplicate to confirm euhydration (i.e.,

USG\ 1.020; Sawka et al. 2007). Then, they were cath-

eterized (BD Insyte, BectonDickinson, Spain) in an

antecubital vein of the right arm and their nude body mass

was measured using a 0.05 kg sensitive scale (Seca 704,

SECA, Germany). The participants then put on cycling

shorts and a sweat patch was attached to their lower back

(TegadermTM ? Pad, 3M, USA). Following instrumenta-

tion, they entered the climatic chamber, and sat quietly on

the cycle ergometer for 20 min. Then, a 5-mL blood

sample was withdrawn and participants started the dehy-

drating exercise protocol.

During the dehydrating protocol, participants cycled

for bouts of 20 min at a constant work rate (i.e.,

161 29 W, *60 % _VO2peak) interspersed with 10 min

of rest. This pattern of activity was repeated until they

lost 3 % of their initial body mass. At the end of each

bout of exercise, a venous blood sample (5 mL) was

drawn and the catheter was flushed (sterile 0.9 %, saline;

Grifols, Spain) to maintain patency and plasma volume.

During the 10-min rest periods, participants toweled-dry

and nude body mass and a urine sample were collected.

During these periods, participants remained in the cli-

matic chamber to avoid body cooling. Since sweat rate

differed among participants (i.e., 0.61.0 L h-1), the time

to achieve a 3 % body weight loss varied from 120 to

160 min. At the beginning (i.e., 10 min) and at the end

of exercise, oxygen uptake _VO2 and carbon dioxide

production _VCO2 were measured using the computer-

ized open-circuit indirect calorimetry system described

previously.

After attainment of at least 3 % body mass loss, par-

ticipants exited the climatic chamber and were allowed to

rehydrate only the equivalent to 1 % of their individual

body mass loss (0.7 0.2 L) by ingesting a commercially

available sports drink (i.e., Aquarius; 221 63 kcal,

carbohydrates 59 16 g, sodium 0.16 0.05 g, chloride

0.24 0.07 g, potassium 0.02 0.005 g). Subject left the

laboratory at *9:00 pm with the request of avoiding fluid

and food ingestion to remain hypohydrated overnight. The

next morning (day 2), participants arrived fasted to the

laboratory at 8:00 am and their nude body mass and urine

samples were collected. In addition, a 5-mL blood sample

was withdrawn after 20 min of seated rest. At this time, the

experiment finalized and participants were offered a

breakfast with abundant fluids.

Blood analysis

A portion (0.5 mL) of each venous blood sample (5 mL)

was immediately analyzed in duplicate for hemoglobin

concentration (i.e., [Hb]; ABL-520, Radiometer, Den-

mark). Hematocrit was measured in triplicate by micro-

centrifugation (Biocen, Alresa, Spain). Relative changes in

plasma volume (PV) were calculated with the equations

outlined by Dill and Costill (1974). The remaining blood

sample was allowed to clot into serum tubes (Z Serum Sep

Clot Activator Vacuette, Greiner Bio-One GmbH, Aus-

tria) and then spun at 2,0009g for 10 min in a refrigerated

(4 C) centrifuge (MPW-350R, Med. Instruments, Poland)

to separate the serum portion. Blood serum samples were

analyzed in duplicate for serum osmolality (SOSM) by

freezing point depression osmometry (Model 3300,

Advanced Instruments, USA), electrolyte concentration

([Na?]serum, [K?]serum and [Cl

-]serum) using an ion-selec-

tive analyzer (Easylyte Plus, Medica Corporation, USA),

serum total protein concentration ([Total protein]serum) by

refractometry (Master-Sur/Na, Atago, Japan; Armstrong

et al. 1998), and protein metabolite concentration ([Creat-

inine]serum, [Urea]serum and [Uric acid]serum) by spectro-

photometry (WPA-S2000, Biochrom, UK) using enzymatic

assays (BioSystems, Spain).

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Urinary analysis

Urine volume (Urinev) collected before, during, and

immediately after exercise was measured using a graduated

cylinder (Symax; Proton). Urine specimens were analyzed

in duplicate for specific gravity (USG) using the hand-held

refractometer (Master-Sur/Na, Atago, Japan). The same

measurement apparatus and assays cited for blood analysiswere used to measure osmolality (UOSM), electrolytes

([Na?]urine, [K?]urine and [Cl

-]urine) and protein metabolite

concentration ([Creatinine]urine, [Urea]urine and [Uric

acid]urine) in urine.

Renal water and sodium reabsorption

Urine-to-serum creatinine ratio and the fractional excretion

of sodium FENa% were calculated to determine renal

water and sodium reabsorption, respectively (Moitra et al.

2006), as follow:

1. Urine-to-serum creatinine ratio= [Creatinine]urine/

[Creatinine]serum2. FENa(%) = [([Na

?]urine/[Na?]serum)/([Creatinine]urine/

[Creatinine]serum)] 9 100

Sweat collection and analysis

Sweat rate was calculated by subtracting pre- from post-

exercise nude body mass correcting for metabolic and

respiratory water losses (Mitchell et al. 1972). Before

exercise, participants lower back was cleaned with alco-

hol, rinsed with distilled water and dried with a sterile

gauze (Montain et al. 2007). Then, a sweat patch (Tega-

dermTM ? Pad, 3M, USA) was attached to the skin. After

90 min of exercise, sweat patch was removed using clean

tweezers and immediately placed in sealed tubes. The tubes

were centrifuged (2,0009g for 10 min at 4 C) and sweat

transferred into clean tubes (Eppendorf). Sweat samples

were analyzed for sweat sodium ([Na?]sweat) and chloride

concentration ([Cl-]sweat) using an ion-selective analyzer

(Easylyte Plus, Medica Corporation, USA). Lower back

[Cl-]sweat was extrapolated to whole-body [Cl-]sweat using

a specific site equation proposed by Patterson et al. (2000).

Fluid shift

Total body water loss (DTW) was estimated from body

mass loss, assuming that 1 kg of body mass loss is

equivalent to 1 L of water loss. At each dehydration stage,

urine Cl- losses were calculated by multiplying the urine

excreted in each stage by the [Cl-]urine. Likewise, sweat

Cl- losses were calculated by multiplying the sweat loss in

each stage by the [Cl-]sweat after 90 min of exercise

assuming unchanged [Cl-]sweat. Recently, we found that

sweat electrolyte concentration does not differ between 60

and 90 min of dehydrating exercise in a similar hot envi-

ronment (Mora-Rodriguez et al.2008). Relative changes in

the interstitial (DISF), extracellular (DECF) and intracel-

lular (DICF) fluids spaces were determined by the Cl-

method (Costill et al.1976), as follow:

1. DClECF-

= DClurine-

? DClsweat-

2. DClISF-

= DClECF-

- D Clserum-

3. DISF = (Donnan factor 9 DClISF- /DClserum

- ) 9 DPV

4. DECF = DPV ? DISF

5. DICF = DTW - DECF

These equations require the following assumptions: (1)

Cl- loss in sweat and urine came from only the ECF space,

(2) the Donnan factor for Cl- between plasma and inter-

stitial fluid (ISF) is 0.95 during exercise (Sanders et al.

2001) and 1.05 at rest (Nose et al.1988), (3) Cl- loss from

the plasma and ISF space is proportional to the water loss

from each space. The calculations of fluid shift resultingfrom these equations should be regarded as an approxi-

mation of what happens to the different compartment

volumes when fluid is lost through sweating during exer-

cise (Sanders et al. 2001) and when dehydration is main-

tained several hours after exercise.

Statistical analysis

Differences in all variables collected before, during and

after exercise were analyzed using one-way repeated-

measures ANOVA. After a significant Ftest (Greenhouse-

Geisser adjustment for sphericity) pairwise differences

were identified using Tukeys post hoc procedure (Vincent

1999). To determine the degree of association between

variables, we used Pearson correlation coefficient (r). Data

are presented as mean standard deviation (SD). The

significance level was set at P\ 0.05.

Results

Fluid balance and sweat electrolytes

Participants body mass in the morning and in the evening

prior to exercise (day 1) was similar (i.e., 74.5 17 vs.

74.9 17 kg, respectively). After 60 min of exercise in

the heat, participants lost 1.2 0.1 % of their initial body

mass, 2.1 0.1 % after 95 15 min and 2.9 0.1 %

after 140 15 min to finish with 72.7 17 kg. Average

sweat rate during exercise-induced dehydration was

0.8 0.1 L h-1. The next morning (day 2), body mass

was similar than at the end of the dehydrating exercise on

day 1 (i.e., 72.6 17 vs. 72.7 17 kg, respectively).


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After 90 min of exercise, [Na?]sweat and [Cl-]sweat were

65 21 and 34 11 mmol L-1, respectively.

Hematological responses

For all subjects,SOSM in the morning and in the evening prior

to exercise was similar (i.e., 282 3 vs. 281 3 mO-

smol kg-1 H2O, respectively; Fig. 1a). During exercise, PVdeclined progressively to -12.9 4.7 % from exercise-

baseline values after 3 % dehydration (Fig. 1a; P\ 0.05).

This progressive reduction in PV was paralleled by a pro-

gressive and significant increase in SOSM from exercise-

baseline values (288 5 mOsmol kg-1 H2O) to 292

5 mOsmol kg-1 H2O after 2 % dehydration (P\0.05)

reaching 295 5 mOsmol kg-1 H2O after 3 % dehydration

(Fig.1a;P\0.05). Similarly, serum electrolyte concentra-

tion ([Na?]serum, [K?]serumand [Cl

-]serum), protein metabo-

lites ([Urea]serum and [Uric acid]serum) and [Total

protein]serum increased progressively with dehydration

respect to exercise-baseline values (Table 1; P\0.05). Thenext morning, despite maintaining the body mass deficit (i.e.,

3 % hypohydration), most hematological parameters were

recovered (Table1; P\0.05). However, [Urea]serum and

[Uric acid]serum remained significantly higher than when

euhydrated (Table1;P\ 0.05). Likewise,SOSMwhen hyp-

ohydrated was slightly recovered although it remained still

elevated with respect to morning euhydrated values (287 5

vs. 282 3 mOsmol kg-1 H2O, respectively; Fig.1a;

P\ 0.05).

Renal responses

During exercise-induced dehydration, Urinev declined pro-

gressively from exercise-baseline values (i.e., 70 23 vs.34 27, 14 7and17 15 mL atbaseline vs. 1,2 and 3 %

dehydration, respectively;P\0.05). In accordance with the

reductions in Urinev, USG increased (P\0.05) and UOSMtended to increase through each stage of dehydration (Fig.1b).

In addition, the exercise-induced progressive dehydration

increased our renal indices of water and sodium reabsorption

([Creatinine]urine/[Creatinine]serum and FENa , respectively;

Fig.2;P\0.05). Progressive dehydration was accompanied

by a progressive reduction in [Na?]urineas expected from the

FENa data (Table2). In contrast, [K?]urine and [Creati-

nine]urine increased through each stage of dehydration from

exercise-baseline values (Table2; P\0.05). [Urea]urine didnot increase from exercise-baseline through each stage of

dehydration (Table2). Finally, [Uric acid]urinedid not change

significantly with progressive dehydration (Table2).

The next morning (i.e., 3 % hypohydration), renal water

reabsorption index, USG and UOSM were still elevated and

significantly higher than the morning euhydrated values

Fig. 1 a Blood serum

osmolality and plasma volume

changes, b Urine specific

gravity and osmolality response

at morning euhydration, during

progressive dehydration (i.e.,

1.0, 2.0 and 3.0 % of body mass

loss) by exercise in the heat and

11 h after exercise while

remaining hypohydrated (i.e.,

3.0 % body mass loss). Data are

presented as mean SD.

Different from morning

euhydration day 1 (*P\ 0.05);

different from exercise-baseline

value (P\ 0.05); different

from 1.0 % dehydration

(P\ 0.05); different from

3.0 % dehydration ($P\0.05)


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(Figs. 1b, 2; P\

0.05). The components of urine thataccounted for the rise in UOSM at 3 % hypohydration in

comparison to the morning euhydration state were elec-

trolytes and protein metabolites (Table 2; P\ 0.05) with

[Urea]urine playing a significant role.

Fluid shift

During the exercise-induced dehydration calculated ECF

volume progressively declined from exercise-baseline values

to peak at 3 % dehydration (-

1.2

0.3 L; Fig. 3). In thesame fashion, ICF volume decreased significantly below

exercise-baseline values after 2 % dehydration (P\0.05), a

reduction that peaked after 3 % dehydration (-0.97 0.2 L;

Fig.3; P\0.05). At the next morning (i.e., 3 % hypohy-

dration), despite that body mass remained similar to the level

at the end of exercise, ECF volume returned towards euhy-

dration values (-0.2 0.2 L; Fig. 3). In contrast, ICF vol-

ume markedly fell overnight to values twice lower than at the

end of exercise (-2.0 0.2 L; Fig. 3; P\0.05).

Table 1 Blood serum total protein, electrolytes and protein metabolite concentration at morning euhydration, during progressive dehydration (i.e.,

1.0, 2.0 and 3.0 % of body mass loss) by exercise in the heat and 11 h after exercise while remaining hypohydrated (i.e., 3.0 % body mass loss)

Variable Euhydration

(morning

day 1)

Dehydration (% body mass loss)

Before exercise

(evening day 1)

Baseline

after

beginning

(20 min)

1.0 % 2.0 % 3.0 % 3.0 %

hypohydration

(morning day 2)

[Total protein]serum(g dL-

1)

7.1 0.3 7.1 0.5 7.5 0.5 7.6 0.5 7.7 0.4 7.8 0.4

7.3 0.5$

[Na?

]serum (mmol L-1

) 140.4 2.3 140.4 2.0 141.5 2.2 142.7 1.8 143.1 1.4 143.5 1.2 141.8 2.6$

[Cl-]serum (mmol L-1) 100.1 3.0 100.5 2.3 103.5 2.3 104.3 2.4 105.5 2.6 105.7 2.4 101.5 3.6$

[K?]serum (mmol L-1) 6.0 1.5 5.3 0.5* 6.2 0.6 6.5 0.5 7.1 0.4 7.3 0.5 4.9 1.0*

$

[Creatinine]serum(mmol L-1)

0.12 0.02 0.13 0.03 0.11 0.03 0.12 0.03 0.12 0.03 0.14 0.03 0.12 0.03

[Urea]serum (mmol L-1

) 6.0 1.1 6.1 1.2 6.2 1.2 6.6 1.1 6.9 1.2 7.4 1.3 7.1 1.3*

[Uric acid]serum(mmol L

-1)

0.25 0.05 0.25 0.04 0.27 0.05 0.28 0.04 0.29 0.05 0.31 0.05 0.29 0.05*

Data are presented as mean SD

[Total protein]serum, blood serum total protein concentration; [Na?]serum, [K

?]serum and [Cl-]serum, blood serum electrolyte concentration;

[Creatinine]serum, [Urea]serum and [Uric acid]serum, blood serum protein metabolite concentration

* Different from morning euhydration day 1 (P\ 0.05)

Different from exercise-baseline value (P\0.05) Different from 1.0 % dehydration (P\0.05)$

Different from 3.0 % dehydration (P\0.05)

Fig. 2 Renal water retention

([Creatinine]urine/

[Creatinine]serum) and sodium

reabsorption (FENa ) indices at

morning euhydration, during


1.0, 2.0 and 3.0 % of body massloss) by exercise in the heat and





Different from morning

euhydration day 1 (*P\ 0.05);

different from exercise-baseline



(P\ 0.05)


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Correlations

During dehydrating exercise, urinary (USG and UOSM) and

blood (SOSM) markers of dehydration were significantly

associated (USG and SOSM r= 0.99, P = 0.001 and UOSMand SOSM r= 0.97, P = 0.006). However, those correla-

tions lost all significance if the data of the morning (i.e.,

3 % hypohydrated) were included since SOSM declined

(Fig.1a) while USG and UOSM kept increasing (Fig.1b).

Still, the association between USG and UOSM was not

altered by hypohydration (r= 0.97, P = 0.001).

Discussion

Urine specific gravity (USG) is a non-invasive, fast and

inexpensive index of whole-body fluid balance. However,the sensitivity of USG to detect low levels of exercise-

induced dehydration has been questioned (Popowski et al.

2001; Oppliger et al.2005). The first purpose of this study

was to determine the capacity ofUSG to detect not only

large but also the small levels of dehydration (*2 % of

body mass) that habitually occur before *90 min of

exercise has been completed. We found that USG was as

sensitive as SOSM, considered the gold standard index for

dehydration, on detecting exercise-induced dehydration.

Both SOSM and USG statistically increased above their

exercise-baseline value (i.e., 20 min of exercise) when

subjects dehydrated by 2 % (Fig. 1a, b; P\0.05). In

addition, we found that USG remains a responsive index to

detect a 3 % overnight hypohydration and that, in thatsituation, it is even more trustworthy than SOSM.

Popowski et al. (2001) and Oppliger et al. (2005)

reported a significant increase from resting values in SOSMafter 1 % dehydration and an increase in USG after 3 %

dehydration. They conclude that USG is not as sensitive as

SOSM to detect exercise-induced dehydration arguably

because of the inability of the renal system to respond

rapidly to reductions in body water. We confirm that

during exercise-induced dehydration SOSM seems to

increase more readily than USG. However, we argue that

USG is sensitive enough to detect 2 % dehydration

(Fig.1b). Popowski et al. (2001) and Oppliger et al.(2005) reported USG and SOSM after 1, 3, and 5 %

dehydration but did not test 2 % dehydration. Currently,

we focused our measurements in the low percent dehy-

dration (i.e., 1, 2, and 3 % dehydration) and the new

finding is that 2 % dehydration can be detected by USG.

This range of dehydration is commonly reported to

already affect performance (Walsh et al. 1994; Dougherty

et al.2006; Baker et al. 2007) and thus its detection using

the non-invasiveUSG seems relevant.

Table 2 Urine electrolytes and protein metabolite concentration at morning euhydration, during progressive dehydration (i.e., 1.0, 2.0 and 3.0 %

of body mass loss) by exercise in the heat and 11 h after exercise while remaining hypohydrated (i.e., 3.0 % body mass loss)

Variable Euhydration

(morning

day 1)

Dehydration (% of body mass loss)

Before

exercise

(evening

day 1)

Baseline

after

beginning

(20 min)

1.0 % 2.0 % 3.0 % 3.0 %

hypohydration

(next morning)

[Na?

]urine(mmol L-

1)

79.0 41.4 104.6 70.4 137.3 58.2 95.3 49.5

73.9 40.9

66.3 34.3

123.2 50.6*$

[Cl-

]urine(mmol L-1)

81.6 33.3 105.6 65.8 150.0 51.3 147.3 42.4 155.1 34.7 159.5 32.9 141.9 34.6*

[K?

]urine (mmol L-1

) 26.4 14.5 39.8 21.5 69.0 33.0 84.8 38.4 118.2 27.6 40.7 27.0 91.3 48.1*$

[Creatinine]urine(mmol L

-1)

10.4 4.0 6.6 3.8 12.0 3.1 15.7 6.7 23.4 11.3 32.5 18.4

# 22.3 3.5*$

[Urea]urine(mmol L-

1)

327.5 141.3 180.0 84.5* 305.8 84.3 326.5 106.0 316.2 80.0 286.7 101.0 522.4 81.0*$

[Uric acid]urine(mmol L-1)

2.1 1.2 2.1 1.0 2.6 1.1 2.1 0.8 1.9 0.5 2.0 0.4 3.0 0.9*$

Data are presented as mean SD

[Na?]urine, [K?]urine and [Cl-]urine, urine electrolyte concentration; [Creatinine]urine, [Urea]urine and [Uric acid]urine, urine protein metaboliteconcentration

* Different from morning euhydration day 1 (P\ 0.05) Different from exercise-baseline value (P\0.05) Different from 1.0 % dehydration (P\0.05)# Different from 2.0 % dehydration (P\0.05)$

Different from 3.0 % dehydration (P\0.05)


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We could not detect an increase in SOSM after 1 %

dehydration in contrast to previous studies (Popowski et al.

2001; Oppliger et al.2005) which may lead to question the

sensitivity of our SOSMmeasurement. We chose to compare

our blood and urinary indices of dehydration to samplescollected after 20 min of exercise instead of the pre-exer-

cise sample as previously reported (Popowski et al.2001;

Oppliger et al. 2005). Due to the increase in arterial pres-

sure in the transition from rest to exercise, plasma volume

is forced out of the vasculature (58 %) which raises SOSMindependently of dehydration (Montain and Coyle 1992).

In addition, exercise onset reduces glomerular filtration rate

and urine output (Guyton and Hall 2006) raising urine

density. Therefore, using this criterion, our data suggest

that the threshold for detecting dehydration with both SOSMand USG index coincided in a 2 %.

Our results argue against the concept that during exer-cise the renal system is not able to respond rapidly to

reductions in body water. At 2 % dehydration, SOSMincreased 4 3 mOsmol kg-1 H2O above exercise-base-

line values which was accompanied by a marked reduction

in urine volume (i.e., 43 %; P\ 0.05) and a concomitant

increase in USG and UOSM (Fig.1b; P\ 0.05). Robertson

et al. (1982) reported that an increase in SOSM of 3 mO-

smol kg-1 H2O above basal is sufficient to evoke a

detectable increase in blood arginine-vasopressin concen-

tration. We did not measure arginine-vasopressin or aldo-

sterone concentration but we measured renal water

retention ([Creatinine]urine/[Creatinine]serum ratio) andsodium reabsorption (FENa) which are surrogate indices of

the actions of those hormones (Moitra et al. 2006). Both

indices increased suggesting that the corresponding hor-

mone activity was increased upon 2 % dehydration (Fig. 2;

P\ 0.05). Other investigators have reported increase in

renal water retention and sodium reabsorption at the end of

dehydrating exercise in the heat (Stachenfeld et al. 1996;

Melin et al. 2001), however, at larger than 2 %

dehydration.

The second purpose of this study was to determine if

USG reflects long-term body fluid deficit (i.e., hypohydra-

tion). For that purpose, we restricted fluid and food after

dehydrating exercise so that participants remained hypo-

hydrated overnight (i.e., 3 % hypohydrated). The nextmorning, USG was higher than at the end of dehydrating

exercise (3 % dehydration vs. 3 % hypohydration) and

higher than the morning of the first day when subjects were

euhydrated (Fig.1b; P\ 0.05). In contrast, the next

morning,SOSMdecreased in comparison to the levels at the

end of the dehydrating exercise (Fig. 1a; P\ 0.05).

Nevertheless, SOSM remained elevated with respect

to morning euhydrated values (287 5 vs. 282 3

mOsmol kg-1 H2O; Fig.1a; P\ 0.05). Our data suggest

that urinary (USG) as well as blood markers (SOSM) keep

detecting dehydration 11 h after it was produced; however,

SOSM tended to return to euhydration values losing part ofits sensitivity as an index of hypohydration.

Based on the Cl- method, we estimated the shift

between the ECF and ICF spaces during and 11 h after

dehydration. During exercise-induced dehydration our

calculated ECF volume declined progressively from exer-

cise-baseline value (Fig.3) likely due to water losses

through sweating while SOSM increased (Fig. 1a;

P\ 0.05). Interestingly, 11 h after dehydrating exercise, in

the morning of the second day, ECF volume was almost

recovered coinciding with a pronounced reduction of ICF

volume (Fig.3). This suggests that, 11 h after dehydration,

the osmotic gradient between fluid spaces draws waterfrom the ICF space to the intravascular fluid space as it has

been previously reported 1 h after exercise (Nose et al.

1988). Additionally, the recovery of SOSM towards euhy-

dration values could in part be explained by the increased

renal water retention (Fig. 2). Our data suggest that, 11 h

after dehydration, both mechanisms (i.e., shift in body fluid

compartments and renal water retention) are involved in

the defense of intravascular fluid volume at the expense of

reductions in ICF fluid space and by concentrating urine.

Fig. 3 Extracellular fluid

(ECF) and intracellular fluid

(ICF) volume during


1.0, 2.0 and 3.0 % of body mass

loss) by exercise in the heat and





Different from exercise-baseline



(P\ 0.05); different from

3.0 % dehydration ($P\0.05)


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Our findings are in agreement with observations from

Francesconi et al. (1987) after 1, 20 and 44 days of a

military field study conducted at 2,000 m altitude. They

found that plasma osmolality did not increase in associa-

tion with the large increases in urine concentration due to

the chronic dehydration. However, it should be noted that

long-term exposures at altitude could produce isotonic

diuresis which could maintain SOSM unchanged despitechronic dehydration. Likewise, our findings are similar to

data from Armstrong et al. (1994) that found no correlation

between USG and SOSM in tennis players when urine and

blood samples were collected in the morning during three

consecutive days of a tournament in a hot environment.

However, Armstrong et al. (1994) induced only modest

dehydration (*2 %) which may have not been enough to

increase SOSM. Based on our data and the cited researches,

we cannot rule SOSM out as a valid indicator of hypohy-

dration; however, our data support that USG is a superior

index at least after 11 h of food and water restriction after

dehydrating exercise.Overnight after exercise-dehydration, the urine kept

concentrating and at the next morning UOSM was higher

(morning hypohydration vs. morning euhydration; Fig1b).

The urine component responsible for this increase inUOSMwas mainly [Urea]urine that increased to 195 mmol L

-1 in

comparison to the values found at morning euhydration

first day (Table 2; P\ 0.05). Hypohydration reduced ICF

volume which likely participated in the recovery of the

volume of the ECF space (Fig. 3) and thus the lowering of

SOSM(Fig. 1a). It is becoming evident that the reduction of

intracellular water content induces an increase in the rate

of protein catabolism (i.e., proteolysis) in human liver and

myocytes although the underlying mechanisms are unclear

(Haussinger et al. 1993, 1994). With hypohydration, not

only [Urea]urineand [Uric acid]urine increased (Table 2) but

also [Urea]serum and [Uric acid]serum (Table1) despite the

slight recovery of intravascular fluid volume (Figs.1a, 3).

To maintain concentrations in an expanding pool of plasma

(i.e., 6.1 % of plasma volume recovered between the 3 %

dehydration and 3 % hypohydration conditions; Fig. 1a),

the rate of appearance of urea in plasma should increase.

All these argue in favor of the existence of proteolysis

induced by the 11 h of maintained 3 % hypohydration.

In conclusion, USG is as sensitive as SOSM for the

detection of low levels of exercise-induce dehydration (i.e.,

2 %) reported to affect athletic performance. The rapid

increases in urine concentration at low levels of dehydra-

tion (i.e., 2 %) seemed to be mediated by an early activa-

tion of renal water and sodium reabsorption. In addition,

11 h after dehydrating exercise, both urinary (USG and

UOSM) and blood (SOSM) indices maintained the ability to

detect long-term body fluid deficit. However,SOSM tended

to return to euhydration values despite participants

remained 3 % hypohydrated. The recovery ofSOSM seems

to be mediated by transfer of fluid from ICF to the ECF

space in conjunction with renal water retention. This

defense of PV lowers the sensitivity ofSOSMas an index of

hypohydration remaining, in this specific situation,USG as

a better index of fluid deficit.

Acknowledgments The authors wish to thank the participants fortheir invaluable contribution to the study. Nassim Hamouti and Juan

Del Coso were supported by a predoctoral fellowship from the

Castilla-La Mancha government in Spain. The assistance of Andrea

Avila and Emma Estevez is greatly appreciated.

Conflict of interest The authors of this study declare that they have

no financial, professional or other personal interest of any nature in

any product, service and/or company that could be construed as

influencing the position presented in this manuscript.

Ethical standards The authors of this study declare that the

experiments comply with the current laws of the country in which

they were performed. The study was approved by the local Hospital

Research Ethics Committee and conducted in accordance with the

guidelines of the revised Declaration of Helsinki.

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