Saliva indices track hypohydration during 48 h of fluid restriction or combined fluid and energy restriction

To investigate whether unstimulated whole saliva flow rate (UFR) and osmolality (Sosm) track changes in hydration status during 48 h of restricted fluid intake (RF) or combined fluid and energy restriction (RF + RE). Following the 48 h periods, UFR
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  This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:  Author's personal copy Saliva indices track hypohydration during 48 h of fluidrestriction or combined fluid and energy restriction Samuel J. Oliver  a, * , Stewart J. Laing a , Sally Wilson a , James L.J. Bilzon b , Neil P. Walsh a a School of Sport, Health and Exercise Sciences, Bangor University, Bangor LL57 2PZ, UK b Headquarters Army Recruiting and Training Agency, Upavon SN9 6BE, UK 1. Introduction The development of a hydration assessment techniquerequires the marker to be valid to track changes in hydrationevoked by common causes of water deficits including restricted access to fluid and food, heat exposure and exerciseinduced sweat loss with inadequate fluid replacement. Themarker should also be able to identify water deficits of only 2–3% body mass loss (BML) as water deficits of this magnitudeareassociated withdisease, 1 decreasedexercisecapacity 2 andcognitive-motor performance. 3 To be of practical use, hydra-tion markers should also reliably identify hydration status inindividuals with a single non-invasive measurement thatprovides immediate results and requires little technicalexpertise.Unstimulated whole saliva parameters have been identi-fied as potential markers of hydration status. 4,5 Decreases insalivaflowrate and increases in salivaosmolality wereshowntotrackdehydrationequalto1,2,and3%ofbodymassevokedby exercise and heat stress lasting lessthan 3 h. Unstimulatedwhole saliva flow rate and osmolality were also shown tocorrelate strongly with plasma and urine osmolality 4 : two archives of oral biology 53 (2008) 975–980 a r t i c l e i n f o  Article history: Accepted 3 May 2008 Keywords: DehydrationExerciseFlow rateOsmolality a b s t r a c t Objective:  To investigate whether unstimulated whole saliva flow rate (UFR) and osmolality(Sosm) track changes in hydration status during 48 h of restricted fluid intake (RF) orcombined fluid and energy restriction (RF + RE). Following the 48 h periods, UFR and Sosmwere assessed after acute exercise dehydration and rehydration. Design:  Thirteen healthy males completed three trials in a randomised order: control (CON)where participants received their estimated energy (12,154  230 kJ/d: mean  S.E.M) andfluid (3912  140 ml/d) requirements, RF trial where participants received their energyrequirements and 193  19 ml/d water to drink (total fluid 960  15 ml/d) and RF + RE whereparticipants received 1214  25 kJ/d and 962  16 ml/d. After 48 h, participants completed30 min of maximal exercise followed by rehydration (0–2 h) and refeeding (2–6 h). Results:  At48 hbodymasslossexceeded3%onRFandRF + RE.UFRdecreasedduring48 honRF (510  122 to 169  37 m l/min) and RF + RE (452  92 to 265  53 m l/min) and was lowerthan CON at 48 h (441  90 m l/min:  P < 0.05). Sosm increased during 48 h on RF (54  3 to73  5 mOsmol/kg) and RF + RE (52  3 to 68  5 mOsmol/kg) and was greater than CON at48 h(52  2 mOsmol/kg: P < 0.05).UnlikeUFR,Sosmidentifiedtheadditionalhypohydrationassociated with exercise ( P < 0.05) and returned to within 0 h values with rehydration. Conclusions:  Sosm, and to a lesser extent UFR, track hydration status during a 48 h period of RF or RF + RE and after subsequent exercise and rehydration. # 2008 Elsevier Ltd. All rights reserved.*  Corresponding author . Tel.: +44 1248 383965; fax: +44 1248 371053.E-mail address: (S.J. Oliver). available at www.sciencedirect.comjournal homepage: 0003–9969/$ – see front matter # 2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.archoralbio.2008.05.002  Author's personal copy widelyacceptedhydrationindices. 6 Forsalivamarkerstobeof use to track changes in hydration status in an applied setting they must also be shown to be valid and sensitive to chronicdehydration ( > 3 h). Parotid saliva flow rate has been shown todecrease in response to a 24 h period of fasting  7,8 ; however, itis unclear as to whether these findings are representative of changesin wholesalivaas parotidsalivacontributesonly 20%oftotalunstimulatedsalivaproduction. 9 Also,itisunfortunatethat saliva osmolality was not assessed 7,8 because salivaosmolality exhibits smaller individual variation comparedwith unstimulated whole saliva flow rate 4,5 and thus showsmore promise as an indicator of hydration status. Moreover,the effect of fluid loss alone on unstimulated whole salivahydration indices remains unclear as the previous studiesincorporated both fluid and energy restriction. 7,8 The primary aim of this investigation was to assesswhether unstimulated whole saliva flow rate and osmolalitywould track changes in hydration status during a 48 h periodof either fluid restriction alone or combined fluid and energyrestriction. It was hypothesised that saliva flow rate andosmolalitydeterminedfromunstimulatedwholesalivawouldtrackandidentifymodesthypohydrationevokedbyprolongedfluid restriction alone and combined fluid and energyrestriction. It is also unknown whether saliva flow rate andosmolality are able to identify further changes in hydrationstatus evoked by acute exercise after periods of fluidrestriction and fluid and energy restriction. Therefore, asecondary aim of this investigation was to assess the validityof saliva hydration markers to identify further dehydrationevoked by exercise following the 48 h periods of fluidrestriction and combined fluid and energy restriction. It washypothesised that saliva flow rate and osmolality wouldidentify further dehydration associated with exercise follow-ing the 48 h periods of restricted fluid alone or combined fluidand energy restriction. 2. Methods 2.1. Participants Thirteen recreationally active healthy males (mean    S.E.M:age, 21    1 years; height, 179    2 cm; body mass, 74.7    2.2 kg;body fat, 16.8    1.4%;  ˙V  O 2max , 50.9    1.2 ml/kg/min) volun-teeredtoparticipateinthestudy.Allparticipantsgavewritteninformedconsentbeforethestudy,whichreceivedlocalEthicsCommittee approval. 2.2. Preliminary measurements Prior to the experimental trials, maximal oxygen uptake ð  ˙V  O 2max Þ wasestimatedbymeansofacontinuousincrementalexercisetestonatreadmillasdescribedpreviously. 10 Fromthe ˙V  O 2  – work rate relationship, the work rate equivalent to 50% ˙V  O 2max  was estimated and used for submaximal exerciseduring the experimental trials. On a separate day, 7–10 daysprior to beginning the experimental trials, participantsreturned to the laboratory for individual energy expenditureestimation and familiarisation. Participants arrived euhy-drated at 08:00 h after an overnight fast, having consumedwater equal to 40 ml/kg of body mass the previous day. Onarrival and after voiding, anthropometric measurements of height and nude body mass were collected. Following thesemeasures, body composition was estimated by dual energy X-rayabsorptiometry(Hologic,QDR1500,softwareversionV5.72,Bedford,USA)andrestingmetabolicrate(RMR) wasestimatedfor 10 min as previously recommended 11 using a portablebreath-by-breath system (Cortex Metamax 3B, Biophysik,Leipzig, Germany). After breakfast, participants performed a1.5 h treadmill walk at 50%  ˙V  O 2max  during which energyexpenditure was estimated (Cortex Metalyser 3B, Biophysik,Leipzig, Germany). For short periods during the day partici-pants wore the portable breath-by-breath system (CortexMetamax 3B, Biophysik, Leipzig, Germany) to estimate theenergy expenditure incurred during habitual living in thelaboratory environment (e.g. light office work and watching T.V.). These additional energy expenditure data were used,along with the RMR data, to estimate the total energy intakerequired for the experimental trials. In addition, during this24 h period fluid requirements were estimated by assessing changesinbodymassathourlyintervals.Physicalactivitywasstandardised throughout the familiarisation and all experi-mental trials by recording 24 h step counts with pedometers(Digi-walker SW-200, Yamax, Tokyo, Japan). 2.3. Experimental trials and procedures Separated by 7–23 days, participants were required tocomplete three experimental trials in a random order(Table 1). The three dietary interventions included a controltrial (CON), a restricted fluid intake trial (RF) and a restrictedfluidandenergyintaketrial(RF + RE).Dietarycompositionwasestimated using software (Dietmaster, Version 4.0, SwiftComputer Systems, Surrey, UK). On the day prior to eachexperimental trial, participants were instructed to consumeonlythefoodandwaterprovidedandto refrainfromexercise.Participants were provided with their estimated energyrequirements (11346    197 kJ per day where 49, 36, 15% of energy was derived from carbohydrate, fat and protein, Table 1 – The nutrient intake for a 24 h period duringeach experimental trial CON RF RF + RE FluidFluid consumed (ml) 3912    140 960    15 962    16Water to drink (ml) 3145    134 193    19 885    15Water in food (ml) 767    11 767    11 77    1EnergyEnergy consumed (kJ) 12154    230 12154    230 1214    25Carbohydrates (g) 387    8 387    8 39    1Fat (g) 119    3 119    2 12    1Protein (g) 104    2 104    2 10    1Values are mean and standard errors of the mean (S.E.M.),  n  = 13.Abbreviations: CON, control trial; RF, restricted fluid intake trial;RF + RE, restricted fluid and energy intake trial. The percentage of energy derived from carbohydrate, fat and protein was the sameacross all trials and equal to 50, 36 and 14%, respectively. Adaptedfrom Oliver et al. 17 archives of oral biology 53 (2008) 975–980 976  Author's personal copy respectively) and water equal to 40 ml/kg of body mass. Totalenergy intake was provided as three prepared meals con-sumed between 09:00–09:30, 13:00–13:30 and 17:00–17:30 h,respectively. To ensure water was consumed at regularintervals throughout the day, water was divided equallybetween six bottles with instructions to consume one bottlewith each meal and a further bottle following each meal.Participants arrived at the laboratory at 22:00 h the evening prior to each experimental trial. On the evening prior to eachexperimental trial participants slept for 8 h in a temperatelaboratory (19.7  0.3  8 C, 59  2% RH).The intervention began at 08:30 h the following morning after participants had voided and after a nude body mass wasobtained.Thereafter,duringwakinghoursnudebodymasswasrecorded at 2-hourly intervals. Euhydration was verified byensuring all participants’ urine osmolality was less than 700mOsmol/kg. 6 Participants were seated for 10 min after whichbaseline (0 h) blood and saliva samples were obtained. Furthercollectionsoffirst-morningbloodandsalivaweremadeafter24h(08:30h,day2)and48h(08:30h,day3)(Fig.1).Ondayoneandtwo of each experimental trial participants were instructed toconsume only the food and water provided (Table 1).To simulate active populations (e.g. military recruits onfield exercise) participants performeda 1.5 h treadmillwalk atasetworkloadequivalentto50%  ˙V  O 2max  afterbreakfastondayoneandtwo.Waterwasconsumedtooffsetfluidlosseswhilstwalking on CON whereas no fluids were provided whilstwalking on RF and RF + RE. Following lunch and evening meals, participants also completed a 20 min walk. Afterproviding 48 h samples of blood and saliva participantsperformed a 30 min maximal exercise bout the data fromwhich have been presented elsewhere. 10 Participants wereinstructed to ‘‘run as far as possible in 30 min’’ and to controlthe speed of the treadmill (gradient set at 1%) as and whenthey felt appropriate. No fluids were consumed during thisexercisebout.Furtherbloodandsalivasampleswereobtainedimmediatelypost (post-EXER), 2 h post (2 h post-EXER) and6 hpost (6 h post-EXER) the 30 min maximal exercise bout. All1.5 h walks and 30 min maximal exercise bouts were per-formedin an air-conditionedlaboratory (19.7  0.3  8 C,59  2%RH).During the first 2 h of recovery, fluid was provided as acitrus-flavouredelectrolyte–onlysolution(50 mmol/lsodium,ScienceinSport,Blackburn,UK).Therehydrationsolutionwasdivided evenly across the 2 h and consumed equal to 100%BML or up to 29 ml/kg of body mass; which reflects theapproximate maximal gastric emptying rate for this solu-tion. 12 During 2–3 and 4–5 h of recovery, participants con-sumedatotalof8164  155 kJ,where49,36,15%ofenergywasderived from carbohydrate, fat and protein, respectively,dividedequally intotwo meals.Waterwas available ad-libitum during these two meals. 2.4. Analytical methods 2.4.1. Saliva Unstimulated whole saliva samples were collected using pre-weighed salivette tubes (Sarstedt, Leics, UK). All salivasamples were collected while the subject sat quietly in thelaboratory. After thoroughly rinsing the mouth with watereach subject was asked to swallow in order to standardise theamountofresidualsalivapriortocollection.Thesalivasamplewas collected by the participants placing the cotton swab(diameter 1 cm, length 2.5 cm) under the tongue for exactly2 min with minimal orofacial movements during the collec-tion. Saliva volume was estimated by weighing the salivettetube immediately after collection to the nearest milligram,and saliva density was assumed to be 1.00 g/ml. 13 From this,the saliva flow rate was determined by dividing the volume of saliva by the collection time. The salivette centrifuge tubecomprising the inner snap seal container and the cotton swabwasspunat3000  g for5 minat5  8 Callowingthecollectionof salivainthebottomofthetube.Salivawasthenaspiratedintoeppendorfs and stored at   80  8 C for further analysis. Salivaosmolality measurements were made using a freezing pointdepression Osmometer with an intra-assay CV of 1.5%. 2.4.2. Blood Blood samples were collected without venestasis by vene-puncture from an antecubital vein into vacutainer tubescontaining lithium heparin (Becton Dickinson, Oxford, UK).The blood in the lithium heparin tube was immediatelycentrifuged (1500  g  for 10 min at 5  8 C), plasma aspirated andstoredat  80  8 Cforfurtheranalysis.Osmolalitymeasurementon heparinised plasma was made using a freezing pointdepression Osmometer (Model 3 MO, Advanced Instruments,Massachusetts, USA) with an intra-assay coefficient of variation (CV)  < 1.0%. 2.4.3. Urine Urinesampleswerecollectedmid-flowintouniversalcontain-ers, aspirated into eppendorfs and frozen at  80  8 C for furtheranalysis. Urine osmolality measurement was made using afreezing point depression Osmometer with an intra-assay CVof 2.0%. 2.4.4. Statistical analysis One-way repeated-measures analysis of variance (ANOVA)was performed on pre-experimental body mass and trial Fig.1 –Orderofexperimentaltrialevents.Abbreviations:B-fast, breakfast; WALK, 20 min walks following lunch anddinner; EXER, 30 min maximal exercise bout. Adaptedfrom Oliver et al. 17 archives of oral biology 53 (2008) 975–980  977  Author's personal copy pedometer counts. Two-way repeated-measures ANOVA (3trials  6 sample collections) was performed on BML, plasmaosmolality, saliva flow rate and saliva osmolality. Appropriateadjustments to the degrees of freedom were made in caseswhere the assumptions of sphericity and normality wereviolated.PosthocTukey’sHSDandBonferroniadjusted t  -testswere used where appropriate. Data in tables and figures arepresented as mean and standard errors of mean (  S.E.M.).Significance was accepted at  P < 0.05. 3. Results 3.1. Body mass loss, fluid replacement and physicalactivity At baseline (0 h) body mass was not significantly differentamong trials (CON: 73.4  2.0; RF: 73.9  2.0; RF + RE:73.7  2.1 kg,  P > 0.05). Body mass was relatively well main-tained over the 48 h period during CON (  0.6  0.4%; Fig. 2).The 48 h period on RF and RF + RE evoked linear BML that wassignificantly different from CON by 24 h ( P < 0.05). Post-EXER,BMLwassignificantlyincreasedcomparedwithbaselineonalltrials ( P < 0.05).Rehydration following the maximal exercise bout was1363  89 ml on CON, 2056  56 ml on RF and 2037  53 ml onRF + RE; replacing 100, 69, 63% total BML on CON, RF andRF + RE, respectively.  Ad-libitum  water intake between 2 and6 hpost-EXERwas830  72 mlonCON,1031  95 mlonRFand914  123 ml on F + ER. Following this  ad-libitum  water intakeat 6 h post-EXER total fluid replacement was equal to 104 and91% of total BML on RF and RF + RE, respectively.Mean experimental trial physical activity ranged between18,459 and 19,230 steps per day and was not significantlydifferent among the trials ( P > 0.05). 3.2. Saliva flow rate Due to insufficient saliva volume for osmolality analysis at alltime points in one participant, the data presented are from 12participants where we have a complete data set. Saliva flowrate decreased on RF and RF + RE by 48 h ( P < 0.05) remaining unaltered on CON (Fig. 3). Post-EXER saliva flow rate wassignificantly decreased compared with 48 h on CON ( P < 0.05)but was not significantly decreased on RF and RF + RE,respectively ( P > 0.05). Saliva flow rate returned to withinbaseline values on all trials by 6 h post-EXER. 3.3. Saliva osmolality SalivaosmolalityincreasedonRFandRF + REby48 h( P < 0.05)whilst remaining unaltered on CON (Fig. 4). Post-EXER salivaosmolality was significantly increased compared with 48 h onCON ( P < 0.05), RF and RF + RE ( P < 0.05), returning to withinbaseline values on all trials by 2 h post-EXER. 3.4. Plasma osmolality Plasma osmolality increased on RF by 24 h compared with allother trials ( P < 0.05) but remained unaltered during the 48 hperiod on CON and RF + RE (Table 2). Post-EXER plasmaosmolality increased significantly compared with 48 h on alltrials ( P < 0.05). Plasma osmolality returned to within baseline(0 h) values by 6 h post-EXER. 4. Discussion The primary aim of the present study was to investigatewhether unstimulated whole saliva flow rate and osmolalitywere able to track changes in whole body hydration statusevoked by 48 h period of fluid or combined fluid and energy Fig. 2 – The effects of a 48 h period of fluid restriction (  ~  ),fluid and energy restriction (  ^  ) compared with control (  &  )and subsequent 30 min exercise bout, rehydration andrefeeding on body mass loss (%). Values are means for 13subjects, with standard errors represented by vertical bars. Mean value was significantly different from control:* P  < 0.05. Mean value was significantly different from fluidrestriction:  § P  < 0.05. Mean value was significantlydifferent from 0 h:  y P  < 0.05. Mean value was significantlydifferent from 48 h:  z P  < 0.05.Fig. 3 – The effects of a 48 h period of fluid restriction (  ~  ),fluid and energy restriction (  ^  ) compared with control (  &  )and subsequent 30 min exercise bout, rehydration andrefeeding on saliva flow rate. Values are means for 12subjects, with standard errors represented by vertical bars. Mean value was significantly different from control:* P  < 0.05. Mean value was significantly different from 0 h: y P  < 0.05. Mean value was significantly different from 48 h: z P  < 0.05. archives of oral biology 53 (2008) 975–980 978
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