and then what to do after you have achieved it … (by rene ... briefing notes.pdf · and then what...

Prepared as briefing notes only by: René Nevola (2003)

visit: http://www.unu.edu/unupress/food2/UID05E/UID05E00.HTM

And then what to do after you haveachieved it … (BY Rene Nevola)


Pre-dosing procedures and how to calculate the right dose:

1. Collect a urine sample and ensure that the subject has an empty bladder (ideally you should measure local WBGT at the

time of each urine sample) (D-2hrs).

2. Weigh the subject nude, to the nearest decimal place (this is important)

3. Use the The Doubly-labelled Water (DLW) Method for Measuring Energy Expenditure: A consensus Report by the IDECG working group - NAHRES-4, IAEA, Vienna (1990) recommendations and calculate:

Deuterium oxide (2H2O) 0.05g/kg Body Mass Oxygen-18 water (H2O18) 0.15g/kg Body Mass

4. Calculate the dose for your subjects:


Doubly labelled water dosing table - adults only

methodThe Doubly-labelled Water Method for Measuring Energy Expenditure: A consensus Report by the IDECG working

group - NAHRES-4, IAEA, Vienna (1990)Schoeller 1983

EquationsDeuterium oxide (2H2O) 0.05g/kg Body Mass 0.12g/kg Total Body Water

Oxygen-18 water (H2O18) 0.15g/kg Body Mass 0.3g/kg Total Body Water

Data to enterSubject nude weight (kg) 80

Subject Total Body Water (Litres) 36.7

Enrichment (% in solution)Deuterium oxide (2H2O) 99.9

Oxygen-18 water (H2O18) 11.01

Doses to prescribeDeuterium oxide (2H2O) 4 4.404

Oxygen-18 water (H2O18) 12 11.01

Weight of solution to measure in order to meet

prescribed dosage (g)Deuterium oxide (2H2O) 4.0 4.4

Oxygen-18 water (H2O18) 109.0 100.0

Rene’s Dose converter spreadsheet (MSExcel)(See René for a copy) YOU MUST make sure that the right enrichments have beeninput eg 11.01% of the water is O-18, otherwise you will miss calculate the dose)

Grammes


Use the sartorius scales (calibrated and levelled with a spirit level) toWEIGH the right dose (remember DLW is called ‘heavy water’ because 1ml >1g so do not use a measuring cylinder you MUST use scales).

1st label the 300ml plastic containers with the appropriate subject number anddose quantities (+ lids)

2nd Place the container on the scales, zero the weight, use a pipette to transferthe right dose of each isotope into the container - keeping an eye on the scales (avoidvibrations) - use a separate pipette for each isotope (ie use one pipette for deuteriumand 1 pipette for O-18). Place the lid on the container when the dose has beencorrectly made (AVOID spillage's). Speakman et al. believe that there is no reason toissue each isotope separately so for ease, weigh out and make up a combined doseof both isotopes as a single serving of DLW.

3rd Obtain a 2ml sample from each dose and label them appropriately (sub no,time, date, and dose), and freeze (avoid too much air in the container, but do not overfill as it will expand when frozen) - THIS IS ESSENTIAL to know what isotopes in partsper million (ppm) have actually been issued to the subjects and to ensure that this isgreater than the background enrichments.


4th Obtain a urine sample from the subjects prior to dosing them and record thelocal WBGT, time and date. (D-2hrs).

5th Sit all the subjects down in a line on the floor > arms length away from eachother and out of the risk of any wind, or traffic (ie people moving etc). Brief them, issuethe correct container to each subject - double check this, then one by one instruct thesubject to carefully take the lid off the container, move it to the mouth and drinkevery last drop of the water WITHOUT SPILLING it. Record the time, WBGT,date and any events. (D).

6th Collect the 300ml containers from each subject and weigh them once again onthe sartorius to see who much of the DLW was actually consumed.

7th Pour 200ml of potable water into each container, weigh them, issue them to theappropriate subjects and watch them all drink the water. Take a 10 ml sample of thewater (potable water) to store, freeze and analyse in the UK. Collect the containers onceagain and weigh them the the last time.

8th Instruct the subjects NOT to conduct hard physical work or to exposethemselves unduly to a heat stress that may illicit an unusually high rate of sweatingfrom this time until the next urine sample in 10 hours time (D+10hrs). This is theequilibrating period when the isotopes will be equilibrating with the subject’s endogenouswater stores. Remember to weigh the subjects whenever you collect a urine sample.


D-2hrsbaseline09 Oct 03

D (dose)ARCHERS

POSTsample of

DLW consumed for each subject09 Oct 03

D+10hrsurine sample

10hr post Dose09 Oct 03

D+ day110 Oct 03

D+ day211 Oct 03

D+ day312 Oct 03

D+ day413 Oct 03

D+ day515 Oct 03

D+ day616 Oct 03

D+ day717 Oct 03

D+ ENDEX25 Sep 02

Critical samples are marked in redIDEALLY: Same time of day + with body weight to the nearest decimal place... IF … it is possible to get an evening and and morning urine sample then please do it as thatsatisfies the best of the Maastricht Protocol of Westerterp et al. Otherwise a daily morning sampleis fine (as suggested by Coward et al and Speakman et al).

UKbaseline

urine and

source water

10 Sep 03

D+ day 818 Oct 03

D+ day 919 Oct 03

D+ day 1020 Oct 03

D+ day 1121 Oct 03

Doubly labelled water sampling programme for Ex Grand Prix IV (Kenya) 2003 (make sure that you get

the red samples)


5 polystyrene igloo containers packed with dry ice

The contents list for eachigloo is enclosed at the topof each container

20-ml urine samples (frozen)and arranged in order oftest day (date)

Box number 1 also has the 1-ml aliquot sample of thedose of DLW administered to each subject. Doses arenot the same as each was calculated on the basis of the subject’s body mass (ie per kg body mass)

The boxes are each markedwith a yellow label. United Courierswill package them in a 2 cardboard boxes for the purpose of delivery.(caution: handle with care : DRY ICE)

Packing the samples for analysis at Iso-Analytical Ltd (UK)


- WARNING: Urine samples, stored under DRY ICE -

- WARNING: Urine samples, stored under DRY ICE -

Please deliver to:


If undelivered please return asap to:

Mr V R NevolaQinetiQ LtdCody Technology ParkA50 building Room G012FarnboroughHAMPSHIRE GU14 0LX

tel 01252 39 6452mobile 07748246035

If undelivered please return asap to:

Mr V R NevolaQinetiQ LtdCody Technology ParkA50 building Room G012FarnboroughHAMPSHIRE GU14 0LX

tel 01252 39 6452mobile 07748246035


Box number: of in total

Contents:

Urine samples stored in DRY ICE - handle with care

Technical information:TEST reference :

Number of Urine samples enclosed :

Notes :


D-2hrsbaseline09 Oct 03

D (dose)ARCHERS

POSTsample of

DLW consumed for each subject09 Oct 03

D+10hrsurine sample

10hr post Dose09 Oct 03

D+ day110 Oct 03

D+ day211 Oct 03

D+ day312 Oct 03

D+ day413 Oct 03

D+ day515 Oct 03

D+ day616 Oct 03

D+ day717 Oct 03

D+ ENDEX25 Sep 02

Please analyse all samples BUT the CRITICAL samples are marked in red (send results to [email protected])

IDEALLY: Take samples at the same time of day + with body weight to the nearest decimal place

UKbaseline

urine and

source water

10 Sep 03

D+ day 818 Oct 03

D+ day 919 Oct 03

D+ day 1020 Oct 03

D+ day 1121 Oct 03

Doubly labelled water sampling that was achieved in the study(send this to Iso-Analytical Ltd with the samples)


Client: QinetiQ Ltd.

Contact: Rene Nevola

Analysis: Deuterium & Oxygen-18 of urine and doses

IA Ref. No.: 021008-1

Client Ref.: CU005-05924

From: Charles Belanger

October 30, 2002

We have completed the analysis of the urine and dose samples for Deuteriumand Oxygen-18 that arrived at our facility on the 8th of October 2002. Theresults of analysis can be found as an e-mail attachment in MS Excel 95format with the name 021008-1-Results.XLS. The analysis report can also befound as an attachment in MS Word 95 format with the name 021008-1-Report.DOC. The results are presented in both per mil and ppm notation andinclude results from duplicate analyses.

Eight samples were analysed per subject. These were; dilute dose, baseline,5 hours, day 2, day 4, day 6, day 8, and day 11. These sample points wherechosen as they provide sufficient range for energy expenditure calculation.Where no sample was present for a specific day, a sample from the followingday was used. The sample points used for each subject are indicated in theresults table. The analysis proceeded as follows:

Deuterium

A sample size of 0.3 ml sample was pipetted into septum sealed vials (induplicate) along with the catalyst Platinum (5%) on Alumina in insert vials.The vials were sealed and the headspace flushed with pure hydrogen.Reference waters (including a quality control standard) were prepared in thesame manner. Once all vials were flushed, they were left to equilibrate for aperiod of three days to ensure complete equilibration. The samples andreferences were then analysed by continuous flow using an Europa ScientificANCA-GSL and Geo 20-20 IRMS.

The deuterium enrichments of the samples were calibrated against our twolaboratory reference waters IA-R008 (3.7‰ δ2H vs. v-SMOW/v-SLAP) and IA-R012 (1017.2‰ δ2H vs. v-SMOW/v-SLAP). The accuracy of the analyses wascontrolled by measuring laboratory standard water IA-R010 (551.8 ‰ δ2H vs.v-SMOW/v-SLAP) as a check standard in each batch of samples. The datafor the check standard can be found at the bottom of the attached Excel file.

ISO-ANALYTICAL Laboratory Report Iso-Analytical Laboratory ReportClient Details

Name: QinetiQ Ltd.Contact: Rene Nevola

P.O. No.: CU005-05924Sample Details

Number: 214Material: Urine and Doses

Sample TrackingIA Reference No.: 021008-1

Date of Arrival: 08/10/2002Analysis Details

Isotope(s) : Deuterium and Oxygen-18Method: Equilibration IRMS

Subject Sample δδδδ -2H vs. 2H δδδδ -18O vs. 18O Wt. DoseIdentification Point V-SMOW (‰) (ppm) V-SMOW (‰) (ppm) (g/100mL)

1 Dilute Dose -40.88 149.37 -6.17 1988.87 0.2435-39.95 149.51 -6.24 1988.72

Baseline -23.16 152.13 -4.51 1992.18-29.89 151.08 -4.45 1992.31

5 hrs -25.22 151.81 -3.76 1993.68-23.81 152.03 -3.75 1993.70

Day 2 -25.91 151.70 -3.51 1994.17-25.74 151.73 -3.33 1994.54

Day 4 -22.23 152.27 -3.69 1993.82-23.38 152.10 -4.20 1992.79

Day 6 -17.25 153.05 -3.13 1994.94-23.22 152.12 -3.90 1993.39

Day 8 -24.93 151.85 -3.34 1994.52-23.71 152.04 -3.32 1994.56

Day 10 -24.56 151.91 -3.46 1994.27-24.76 151.88 -3.07 1995.06

Day 11 -20.79 152.50 -2.58 1996.03-20.04 152.62 -2.80 1995.60

D-ENDEX -22.79 152.19 -2.84 1995.52-25.22 151.81 -2.89 1995.42

2 Dilute Dose 483.99 231.09 118.44 2237.68 0.2481483.37 231.00 118.85 2238.49

Baseline -18.12 152.91 -3.76 1993.69-22.21 152.28 -3.74 1993.72

5 hrs 511.22 235.33 126.41 2253.59510.79 235.27 126.97 2254.71

Day 2 425.16 221.93 102.78 2206.41424.60 221.85 103.05 2206.95

Day 4 365.51 212.65 82.66 2166.25368.29 213.08 83.43 2167.79

Day 6 311.93 204.30 66.76 2134.51317.80 205.22 67.41 2135.81

Day 8 274.31 198.45 55.26 2111.55272.48 198.16 55.50 2112.03

Day 10 233.13 192.03 46.53 2094.10233.15 192.04 46.84 2094.73

Day 11 222.76 190.42 41.80 2084.65219.57 189.92 41.91 2084.88

D-ENDEX 204.75 187.62 38.26 2077.60204.01 187.50 37.86 2076.79

3 Dilute Dose 484.31 231.14 118.47 2237.75 0.2479484.95 231.24 118.65 2238.11

Baseline -26.77 151.57 -3.64 1993.92


The interpretation of the samples has been possible using the PCprogram provided free of charge by Speakman and Lemen1999.To get a copy of the program and the user guide see ReneNevola or visit: http://www.abdn.ac.uk/~nhi158/dlwprogram.htm

Interpreting the results

ie Getting MJ for Kcal from ppm


Two sample experiment:

If all you were able to achieve was thebaseline samples and the ENDEX samples,with body weights at the same time, thenuse the following procedures to calculate thetotal energy expenditure and to estimate themean daily expenditure … HOWEVER, ifyou achieved the daily sampling plan thenadvance to the procedures for analysingmultiple samples (= far more accurate)

Prepared as briefing notes only by: René Nevola (2003)Background datappm O18 in tap water

ppm H2 in tap water

ppm O18 in baseline urine

ppm H2 in baseline urine

ppm O18 in 5-hr urine

ppm H2 in 5-hr urine

Injectate (doubly labelled water):% concentrate O18 %

% concentrate H2 %

Mass of O18 in injectate g

Mass of H2 in injectate g

Mass of DLW g

Volume of DLW ml

Mass of vessel g

mass of vessel + DLW g

Date Time consumed:

Date Time sample takenMass of subject gppm O18 in initial urine sampleppm H2 in initial urine sample

Date Time sample taken:Mass of subject: g

ppm O18 in last urine sampleppm H2 in last urine sample

Subject ID:

Filename:

Deuterium / O-18 Enrichment (ppm) in injectate

ppm O18 in injectate

ppm H2 in injectate

Initial sample: (urine)

Final sample: (urine)


1

2


29787

92075


3


4


5


6


7


8 9

Prepared as briefing notes only by: René Nevola (2003)Analysis data:Species:

ID

Date:

Group file

Notes:

ko /hour (O18 turnover)

kh /hour (H2 turnover)

Oxygen dilution space (No) % of initial body mass (must be >50%)

Hydrogen dilution space (Nd) % of initial body mass (must be >50%)

Plateau dilution space ratio (must be between 1.0-1.0552)

Mass change 9

Energy equivalent if fat kJ/day

Method plateau-mass Mean (kJ/day) Coward et al 1985a

Food intake plateau-mass Mean (kJ/day)

Taken from Page 1

Taken from Page 2

Taken from Page 3


10


(a) Do all days for each subject

(b) open your group and put all data intothe same group

(c) run the analysis program

(d) store analyses in the group file

(e) re-run analysis using the group forcorrection factors

Prepared as briefing notes only by: René Nevola (2003)Background datappm O18 in tap water

ppm H2 in tap water

ppm O18 in baseline urine

ppm H2 in baseline urine

ppm O18 in 5-hr urine

ppm H2 in 5-hr urine

Injectate (doubly labelled water):% concentrate O18 %

% concentrate H2 %

Mass of O18 in injectate g

Mass of H2 in injectate g

Mass of DLW g

Volume of DLW ml

Mass of vessel g

mass of vessel + DLW g

Date Time consumed:

Date Time sample takenMass of subject gppm O18 in initial urine sampleppm H2 in initial urine sample

Subject ID:

Filename:

Deuterium / O-18 Enrichment (ppm) in injectate

ppm O18 in injectate

ppm H2 in injectate

Initial sample: (urine)

- add addition sample data overleaf -

Prepared as briefing notes only by: René Nevola (2003)Date Time sample takenMass of subject gppm O18 in urine sampleppm H2 in urine sample

Date Time sample takenMass of subject gppm O18 in urine sampleppm H2 in urine sample



Date Time sample taken:Mass of subject: g

ppm O18 in last urine sampleppm H2 in last urine sample

Subject ID:

Sample ID:

Sample ID:

Sample ID:

Sample ID:

Final sample: (urine)


1


2


3a3b


4


5


6a

6b 6c


87


2

and then what to do after you have achieved it … (by rene ... briefing notes.pdf · and then what...

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