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 have
achieved it … (BY Rene Nevola)
Prepared as briefing notes only by: René Nevola (2003)
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:
Prepared as briefing notes only by: René Nevola (2003)
Rene’s Dose converter spreadsheet (MSExcel)
(See René for a copy) YOU MUST make sure that the right enrichments have been
input eg 11.01% of the water is O-18, otherwise you will miss calculate the dose)
Doubly labelled water dosing table - adults only
method
Equations
Deuterium oxide (2H2O)
18
Oxygen-18 water (H2O )
The Doubly-labelled Water Method for Measuring Energy
Expenditure: A consensus Report by the IDECG working
group - NAHRES-4, IAEA, Vienna (1990)
Schoeller 1983
0.05g/kg Body Mass
0.12g/kg Total Body Water
0.15g/kg Body Mass
0.3g/kg Total Body Water
Data to enter
Subject nude weight (kg)
Subject Total Body Water (Litres)
Enrichment (% in solution)
Deuterium oxide (2H2O)
18
Oxygen-18 water (H2O )
80
36.7
99.9
11.01
Doses to prescribe
Deuterium oxide (2H2O)
18
Oxygen-18 water (H2O )
4
4.404
12
11.01
4.0
4.4
109.0
100.0
Weight of solution to
measure in order to meet
prescribed dosage (g)
2
Deuterium oxide ( H2O)
18
Oxygen-18 water (H2O )
Grammes
Prepared as briefing notes only by: René Nevola (2003)
Use the sartorius scales (calibrated and levelled with a spirit level) to
WEIGH 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 and
dose quantities (+ lids)
2nd
Place the container on the scales, zero the weight, use a pipette to transfer
the right dose of each isotope into the container - keeping an eye on the scales (avoid
vibrations) - use a separate pipette for each isotope (ie use one pipette for deuterium
and 1 pipette for O-18). Place the lid on the container when the dose has been
correctly made (AVOID spillage's). Speakman et al. believe that there is no reason to
issue each isotope separately so for ease, weigh out and make up a combined dose
of 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 over
fill as it will expand when frozen) - THIS IS ESSENTIAL to know what isotopes in parts
per million (ppm) have actually been issued to the subjects and to ensure that this is
greater than the background enrichments.
Prepared as briefing notes only by: René Nevola (2003)
4th
Obtain a urine sample from the subjects prior to dosing them and record the
local WBGT, time and date. (D-2hrs).
5th
Sit all the subjects down in a line on the floor > arms length away from each
other and out of the risk of any wind, or traffic (ie people moving etc). Brief them, issue
the correct container to each subject - double check this, then one by one instruct the
subject to carefully take the lid off the container, move it to the mouth and drink
every 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 on
the 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 the
appropriate subjects and watch them all drink the water. Take a 10 ml sample of the
water (potable water) to store, freeze and analyse in the UK. Collect the containers once
again and weigh them the the last time.
8th
Instruct the subjects NOT to conduct hard physical work or to expose
themselves unduly to a heat stress that may illicit an unusually high rate of sweating
from this time until the next urine sample in 10 hours time (D+10hrs). This is the
equilibrating period when the isotopes will be equilibrating with the subject’s endogenous
water stores. Remember to weigh the subjects whenever you collect a urine sample.
Prepared as briefing notes only by: René Nevola (2003)
Doubly labelled water sampling programme for
Ex Grand Prix IV (Kenya) 2003 (make sure that you get
the red samples)
UK
baseline
urine
and
source
water
10 Sep 03
D-2hrs
baseline
09 Oct 03
D (dose)
ARCHERS
POST
sample of
DLW
consumed for
each subject
D+10hrs
urine sample
10hr post Dose
09 Oct 03
D+ day1
D+ day2
D+ day3
D+ day4
D+ day5
D+ day6
10 Oct 03
11 Oct 03
12 Oct 03
13 Oct 03
15 Oct 03
16 Oct 03
09 Oct 03
D+ day7
D+ ENDEX
D+ day 8
D+ day 9
D+ day 10
D+ day 11
17 Oct 03
25 Sep 02
18 Oct 03
19 Oct 03
20 Oct 03
21 Oct 03
Critical samples are marked in red
IDEALLY: 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 that
satisfies the best of the Maastricht Protocol of Westerterp et al. Otherwise a daily morning sample
is fine (as suggested by Coward et al and Speakman et al).
Prepared as briefing notes only by: René Nevola (2003)
Packing the samples for analysis at Iso-Analytical Ltd (UK)
5 polystyrene igloo
containers packed
with dry ice
The contents list for each
igloo is enclosed at the top
of each container
Box number 1 also has the 1-ml aliquot sample of the
dose of DLW administered to each subject. Doses are
not the same as each was calculated on the basis of
the subject’s body mass (ie per kg body mass)
20-ml urine samples (frozen)
and arranged in order of
test day (date)
The boxes are each marked
with a yellow label. United Couriers
will package them in a 2 cardboard
boxes for the purpose of delivery.
(caution: handle with care : DRY ICE)
Prepared as briefing notes only by: René Nevola (2003)
- WARNING: Urine samples, stored under DRY ICE Please deliver to:
- WARNING: Urine samples, stored under DRY ICE -
Prepared as briefing notes only by: René Nevola (2003)
If undelivered please return asap to:
If undelivered please return asap to:
Mr V R Nevola
QinetiQ Ltd
Cody Technology Park
A50 building Room G012
Farnborough
HAMPSHIRE GU14 0LX
Mr V R Nevola
QinetiQ Ltd
Cody Technology Park
A50 building Room G012
Farnborough
HAMPSHIRE GU14 0LX
tel
01252 39 6452
mobile 07748246035
tel
01252 39 6452
mobile 07748246035
Prepared as briefing notes only by: René Nevola (2003)
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
:
Prepared as briefing notes only by: René Nevola (2003)
Doubly labelled water sampling that was achieved in the study
(send this to Iso-Analytical Ltd with the samples)
UK
baseline
urine
and
source
water
10 Sep 03
D-2hrs
baseline
09 Oct 03
D (dose)
ARCHERS
POST
sample of
DLW
consumed for
each subject
D+10hrs
urine sample
10hr post Dose
09 Oct 03
D+ day1
D+ day2
D+ day3
D+ day4
D+ day5
D+ day6
10 Oct 03
11 Oct 03
12 Oct 03
13 Oct 03
15 Oct 03
16 Oct 03
09 Oct 03
D+ day7
D+ ENDEX
D+ day 8
D+ day 9
D+ day 10
D+ day 11
17 Oct 03
25 Sep 02
18 Oct 03
19 Oct 03
20 Oct 03
21 Oct 03
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
Prepared as briefing notes only by: René Nevola (2003)
Prepared as briefing notes only by: René Nevola (2003)
ISO-ANALYTICAL
Laboratory Report
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
Iso-Analytical Laboratory Report
Client Details
Name:
Contact:
P.O. No.:
Sample Details
Number:
Material:
Sample Tracking
IA Reference No.:
Date of Arrival:
Analysis Details
Isotope(s) :
Method:
Subject
Identification
1
October 30, 2002
QinetiQ Ltd.
Rene Nevola
CU005-05924
214
Urine and Doses
021008-1
08/10/2002
Deuterium and Oxygen-18
Equilibration IRMS
Sample
Point
Dilute Dose
Baseline
We have completed the analysis of the urine and dose samples for Deuterium
and Oxygen-18 that arrived at our facility on the 8th of October 2002. The
results of analysis can be found as an e-mail attachment in MS Excel 95
format with the name 021008-1-Results.XLS. The analysis report can also be
found as an attachment in MS Word 95 format with the name 021008-1Report.DOC. The results are presented in both per mil and ppm notation and
include results from duplicate analyses.
5 hrs
Day 2
Day 4
Day 6
Day 8
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 where
chosen as they provide sufficient range for energy expenditure calculation.
Where no sample was present for a specific day, a sample from the following
day was used. The sample points used for each subject are indicated in the
results table. The analysis proceeded as follows:
Day 10
Day 11
D-ENDEX
2
Deuterium
Baseline
A sample size of 0.3 ml sample was pipetted into septum sealed vials (in
duplicate) 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 the
same manner. Once all vials were flushed, they were left to equilibrate for a
period of three days to ensure complete equilibration. The samples and
references were then analysed by continuous flow using an Europa Scientific
ANCA-GSL and Geo 20-20 IRMS.
The deuterium enrichments of the samples were calibrated against our two
laboratory reference waters IA-R008 (3.7‰ δ2H vs. v-SMOW/v-SLAP) and IAR012 (1017.2‰ δ2H vs. v-SMOW/v-SLAP). The accuracy of the analyses was
controlled 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 data
for the check standard can be found at the bottom of the attached Excel file.
Dilute Dose
5 hrs
Day 2
Day 4
Day 6
Day 8
Day 10
Day 11
D-ENDEX
3
Dilute Dose
Baseline
2
δ - H vs.
V-SMOW (‰)
-40.88
-39.95
-23.16
-29.89
-25.22
-23.81
-25.91
-25.74
-22.23
-23.38
-17.25
-23.22
-24.93
-23.71
-24.56
-24.76
-20.79
-20.04
-22.79
-25.22
483.99
483.37
-18.12
-22.21
511.22
510.79
425.16
424.60
365.51
368.29
311.93
317.80
274.31
272.48
233.13
233.15
222.76
219.57
204.75
204.01
484.31
484.95
-26.77
2
H
(ppm)
149.37
149.51
152.13
151.08
151.81
152.03
151.70
151.73
152.27
152.10
153.05
152.12
151.85
152.04
151.91
151.88
152.50
152.62
152.19
151.81
231.09
231.00
152.91
152.28
235.33
235.27
221.93
221.85
212.65
213.08
204.30
205.22
198.45
198.16
192.03
192.04
190.42
189.92
187.62
187.50
231.14
231.24
151.57
18
δ - O vs.
V-SMOW (‰)
-6.17
-6.24
-4.51
-4.45
-3.76
-3.75
-3.51
-3.33
-3.69
-4.20
-3.13
-3.90
-3.34
-3.32
-3.46
-3.07
-2.58
-2.80
-2.84
-2.89
118.44
118.85
-3.76
-3.74
126.41
126.97
102.78
103.05
82.66
83.43
66.76
67.41
55.26
55.50
46.53
46.84
41.80
41.91
38.26
37.86
118.47
118.65
-3.64
18
O
Wt. Dose
(ppm) (g/100mL)
1988.87
0.2435
1988.72
1992.18
1992.31
1993.68
1993.70
1994.17
1994.54
1993.82
1992.79
1994.94
1993.39
1994.52
1994.56
1994.27
1995.06
1996.03
1995.60
1995.52
1995.42
2237.68
0.2481
2238.49
1993.69
1993.72
2253.59
2254.71
2206.41
2206.95
2166.25
2167.79
2134.51
2135.81
2111.55
2112.03
2094.10
2094.73
2084.65
2084.88
2077.60
2076.79
2237.75
0.2479
2238.11
1993.92
Prepared as briefing notes only by: René Nevola (2003)
Interpreting the results
ie
Getting MJ for Kcal from ppm
The interpretation of the samples has been possible using the PC
program provided free of charge by Speakman and Lemen
1999.To get a copy of the program and the user guide see Rene
Nevola or visit: http://www.abdn.ac.uk/~nhi158/dlwprogram.htm
Prepared as briefing notes only by: René Nevola (2003)
Two sample experiment:
If all you were able to achieve was the
baseline samples and the ENDEX samples,
with body weights at the same time, then
use the following procedures to calculate the
total energy expenditure and to estimate the
mean daily expenditure … HOWEVER, if
you achieved the daily sampling plan then
advance to the procedures for analysing
multiple samples (= far more accurate)
Background data
Prepared as briefing notes only by: René Nevola (2003)
Subject ID:
ppm O18 in tap water
ppm H2 in tap water
ppm O18 in baseline urine
Filename:
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 taken
Mass of subject
g
ppm O18 in initial urine sample
Deuterium / O-18 Enrichment (ppm) in injectate
ppm O18 in injectate
ppm H2 in injectate
Initial sample: (urine)
ppm H2 in initial urine sample
Date
Mass of subject:
ppm O18 in last urine sample
ppm H2 in last urine sample
Time sample taken:
g
Final sample: (urine)
Prepared as briefing notes only by: René Nevola (2003)
1
2
Prepared as briefing notes only by: René Nevola (2003)
29787
92075
Prepared as briefing notes only by: René Nevola (2003)
3
Prepared as briefing notes only by: René Nevola (2003)
4
Prepared as briefing notes only by: René Nevola (2003)
5
Prepared as briefing notes only by: René Nevola (2003)
6
Prepared as briefing notes only by: René Nevola (2003)
7
Prepared as briefing notes only by: René Nevola (2003)
8
9
Analysis data:
Prepared as briefing notes only by: René Nevola (2003)
Taken from Page 1
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)
Food intake plateau-mass
Mean (kJ/day)
Taken from Page 2
Coward et al 1985a
Taken from Page 3
Prepared as briefing notes only by: René Nevola (2003)
10
Prepared as briefing notes only by: René Nevola (2003)
(a)
Do all days for each subject
(b)
open your group and put all data into
the same group
(c)
run the analysis program
(d)
store analyses in the group file
(e)
re-run analysis using the group for
correction factors
Prepared as briefing notes only by: René Nevola (2003)
Background data
Prepared as briefing notes only by: René Nevola (2003)
Subject ID:
ppm O18 in tap water
ppm H2 in tap water
ppm O18 in baseline urine
Filename:
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 taken
Mass of subject
ppm O18 in initial urine sample
g
Deuterium / O-18 Enrichment (ppm) in injectate
ppm O18 in injectate
ppm H2 in injectate
Initial sample: (urine)
ppm H2 in initial urine sample
- add addition sample data overleaf -
Prepared as briefing notes only by: René Nevola (2003)
Date
Subject ID:
Time sample taken
Mass of subject
g
ppm O18 in urine sample
Sample ID:
ppm H2 in urine sample
Date
Time sample taken
Mass of subject
g
Sample ID:
ppm O18 in urine sample
ppm H2 in urine sample
Date
Time sample taken
Mass of subject
g
Sample ID:
ppm O18 in urine sample
ppm H2 in urine sample
Date
Time sample taken
Mass of subject
g
ppm O18 in urine sample
Sample ID:
ppm H2 in urine sample
Date
Mass of subject:
ppm O18 in last urine sample
ppm H2 in last urine sample
Time sample taken:
g
Final sample: (urine)
Prepared as briefing notes only by: René Nevola (2003)
1
Prepared as briefing notes only by: René Nevola (2003)
Prepared as briefing notes only by: René Nevola (2003)
2
Prepared as briefing notes only by: René Nevola (2003)
Prepared as briefing notes only by: René Nevola (2003)
3a
3b
Prepared as briefing notes only by: René Nevola (2003)
4
Prepared as briefing notes only by: René Nevola (2003)
5
Prepared as briefing notes only by: René Nevola (2003)
6b
6a
6c
Prepared as briefing notes only by: René Nevola (2003)
7
8
Prepared as briefing notes only by: René Nevola (2003)
2
Prepared as briefing notes only by: René Nevola (2003)
Prepared as briefing notes only by: René Nevola (2003)
Prepared as briefing notes only by: René Nevola (2003)