25
DOUBLY-LABELLED WATER
CALCULATION PROGRAM
USER GUIDE
(version 1.0)
J.R. SPEAKMAN
And
C. LEMEN
Funded by UK NERC Scientific Services
& The University of Aberdeen, Scotland UK
January 1999
CONTENTS
1. Background 3
2. Getting started 4-5
3. Estimating injectate enrichments 24-25
4. Two sample experiments 6
4.1 Entering new data
4.2 Dilution 7
4.3 Group 7
4.4 Background worksheet 8-9
4.5 Set Options 10
4.6 Initial worksheet 10-12
4.7 Final worksheet 13
4.8 Save data 14
4.9 Run analysis 14-22
5. Multiple sample experiments
6. Tritium
7. Acknowledgements
1. Background
This program calculates the CO2 production and inferred daily energy expenditure and food intake from isotope enrichment data derived from doubly-labelled water studies. The program uses all the alternative equations that had been published by 1997. Details of the derivation of the equations along with a discussion of which equations are most appropriate under differing circumstances can be found in Speakman (1997)
The program was written by C. Lemen of Natureware Inc, USA
Funding for this project was provided by the United Kingdom Natural Environmental Research Council (NERC) Scientific Services and The University of Aberdeen, Scotland, UK.
The program is provided free of charge to bona fide users of the DLW method without guarantees or liability. The only condition of using the program is that its use is acknowledged in any publication. This User Guide may serve as an appropriate citation.
The program works in the windows environment of any PC. Minimum system requirements are a 486 machine with 8 Mb RAM running windows 3.1
2. Getting started
To load the program transfer the DLW.exe file from the disc, or directly from the web site on to the hard drive of your PC. Do not locate it in a special folder. Create an icon, from which the program can be accessed directly, or use the Start and run options (command line dlw.exe) to start the program. When running correctly the program should open up a window with a standard task bar at the top of it and four pairs of icons below.
Check the version number under Help and About
The version should be 1.0 or above.
These pairs of icons allow direct access into the different operations that are available. Alternative access is via the File command on the task bar.
2.1 Two sample studies
The first pair of icons. These are protocols where the subject has been sampled for background, injected with isotope, sampled for an initial isotope enrichment and then released to be recaptured and sampled again at some later time.
The first icon concerns new data.
The second recovers saved data from a file. This second option only recovers data that has been saved previously using this program. Filenames of recovered files must have the form ********.dl2
Alternatively the same two options can be accessed by clicking on File on the task bar and selecting “New 2 sample” or “Open 2 sample”
2.2 Multiple sample studies.
The second pair of icons. These are protocols where the subject has been sampled repeatedly across several days with the intention of determining the elimination rates of the isotopes using regression. This does not refer to essentially two sample studies where it happened that the animal was recaptured twice. For such analyses the “Two sample protocol” should be used twice (but beware of lack independence of the derived estimates). Again there are two icons – the first is for entering new data and the second is for recovering saved data from a file. This second option only recovers files that have been saved by the program and have the form ********.dlm
Alternatively these same two options can be accessed under File on the task bar and selecting “New multi-sample“ or “Open multi-sample“.
2.3 Isotope enrichments in injectate solutions
The third pair of icons. These are intended to be used with data derived from dilution experiments where known amounts of the injectate have been injected into known amounts of water at background enrichment. Again there are two icons – the first is for entering new data and the second is for recovering saved data from a file. This second option only recovers files that have been saved by the program and have the form ********.enr
Alternatively these same two options can be accessed under File on the task bar and selecting “New estimate enrich“ or “Open enrichment“.
2.4 Group properties
The last two icons. Some calculations of CO2 production using DLW depend on parameters derived from the local group of animals in a particular study. This means calculations must be performed using the two sample or multiple sample options, and the data appended to a group file for each individual. Once a group has been completed the local parameters can be derived and the calculations which depend on these local group parameters estimated by re-running the two or multiple sample options and specifying the group file name at the appropriate point (see below). These two icons allow manual creation of a group file, or inspection of a file that has already been created. Alternatively the same functions can be accessed under File on the task bar and selecting “New Group” or “Open group”
3. ESTIMATING INJECTATE DILUTIONS
The module for estimating injectate enrichments is accessed either via the 3rd set of paired icons or from the task bar under file and choosing either new estimate enrich or open enrichment
NEW ESTIMATES OF ENRICHMENT
This module allows estimation of the enrichment of an injectate solution using a convergence iterative algorithm. The algorithm starts with estimates of the enrichments of both isotopes to set the molecular weights and then iterates the calculation using the derived enrichments to generate new molecular weights. The program performs 30 iterations. Convergence on a reasonable estimate normally occurs before 4 iterations are completed.
The window consists of 11 boxes for entry of data derived from an injectate dilution experiment. The program can operate with enrichment data either in ppm or in delta. In the case of delta it is necessary to include the known isotope ratios for the reference compounds to which delta refers. The default data included in the windows when it is first opened are the reference ratios known for SMOW.
On the line following these ratios are the estimated enrichments of deuterium and oxygen-18 in the injectate. These can be the manufacturers stated enrichment estimates or any other guesses of the likely enrichments in the solutions. Closer estimates minimise time to convergence but as the time is generally not an issue any values can be entered here. If a completely unknown solution is analysed the recommended values to use are 50 and 50.
The next three boxes require weights for the vessel into which the background water and injectate were mixed. The first box requires a mass for the vessel alone. The next box for the vessel plus the background water, and finally the vessel plus the background water plus the injectate. The boxes are designed like this because this is typically the procedure followed in a dilution experiment and it obviates the need for the user to manually calculate how much background water and injectate are involved in the mix. Masses should be entered to four decimal places.
The buttons above the following 4 boxes specify if the following data are in delta or ppm. Clicking on the buttons switches between the two types of unit. If ppm is selected the ratio at the top of the sheet becomes irrelevant.
It is not possible to mix the data units. All enrichments must either be in delta or ppm.
The four boxes require enrichment estimates for both isotopes in first the background water that is used as a diluent. These boxes are labelled
Water 2H and water 18O
and second the mix of diluent and injectate which are labelled
Water plus injectate 2H and Water plus injectate 18O
Once the data entry is completed there are 3 options.
SAVE will save the data in the boxes into a file appended ********.enr
These files can be reloaded into the worksheet using the open enrichment command under file or the second enrichment icon on the task bar.
Cancel leaves the worksheet unaltered
Analyse performs an estimation of the enrichments of deuterium and oxygen-18 in the injectate solution. Analysis time is generally instantaneous.
The output from analyse consists of six lines of data output. A typical example follows
Enrichment of injectate
2H ppm in water = 155.4
18O ppm in water = 2005.5
2H ppm in water plus injectate = 1999.0
18O ppm in water plus injectate = 6003.1
ppm 2H enrichment in injectate = 130543
ppm 18O enrichment in injectate = 284734
The first four lines represent the mean values derived from the four input boxes.
The final two lines represent the iterated calculation of enrichment of the injectate to the nearest ppm.
If the input data are delta units then the output data are also in delta units.
NOTE : the output program has a bug here and lists the output enrichments as ppm irrespective of whether the input is delta or ppm.
The file can be printed to the default device printer on the PC by selecting PRINT
Alternatively the data can be saved to a file using SAVE.
(Note in version 1.0 this capability is disabled)
Alternatively data can be entered into the window from an existing file by selecting the OPEN ENRICHMENT option under file or the second icon of the pairing on the task bar. (Note : there is a bug in the module which is used to re-open these files. If activated the module opens a window which is headed “Save as” and says the file type is “dilution” rather than enrichment. However, files listed in the window will be previously saved ********.enr files and they should open correctly).
4. TWO SAMPLE EXPERIMENTS
4.1 Entering new data
Opening this option reveals a window labelled IDD-t2main. This window contains 5 boxes in which information can be entered plus 8 buttons which access further data entry screens.
The 5 boxes are intended to contain information on the subject and the experiment it was involved in and are labelled :
Box Max input
Species 33 characters
ID # 14 characters
Location 76 characters
Date 10 characters
Notes 3 lines x 46 characters on each
Any information typed into these boxes will appear on the header of the output from the main calculation (section 4.9) under the appropriate heading. None of these boxes are compulsory fields. It is possible to make calculations with all the boxes empty. None of the information entered in these fields (e.g. date) is used in the calculations.
4.2 Dilution
The dilution data entry button opens a small window where it is necessary to enter the enrichments of the two isotopes in the injectate solution. There are two boxes labelled hydrogen2 ppm and Oxygen18 ppm. These fields must contain the ppm enrichments of the injectate for the program calculations to continue.
Data can be entered in 3 ways/
1) Manually into the boxes.
2) The output from the injectate enrichment module can be entered directly into these fields by accessing the appropriate ********.enr file. Clicking on the button “Open dilution file” allows access to the file searcher which will seek any files appended ********.enr. (Note : not possible in version 1.0).
3) The output from a ********.dln file that was saved from the dilution worksheet of a previous animal can be loaded directly into the boxes following the same procedures as detailed in 2).
The second button “Save dilution file” allows manually entered data in these fields to be saved to a ********.dln file for future reference.
Cancel closes the window without saving the current settings in the fields.
OK accepts the most recent values entered into the fields.
Both fields are mandatory for the calculation to proceed. The enrichment must be entered as ppm. Other units of enrichment (e.g. delta SMOW-SLAP) are not acceptable.
4.3 GROUP
The group box allows specification of a local group from which parameters can be derived for some of the DLW calculations. Clicking on GROUP opens a window in which an analysis is performed of the characteristics of any particular group of data.
Within this window there is a button called Select group search window for files with the append ********.grp. Selecting the group performs an analysis of the data held in the group file of the relationship between the dilution space and body mass of animals in the group. Four analyses are performed using the hydrogen and oxygen data for both plateau and intercept dilution spaces.
A plot shows the relationships derived. Plateau data are in green, intercept data in red, oxygen symbols o and hydrogen symbols +.
The regression parameters for these four plots are located in a table to the left of the plot. These show the intercept, gradient and r2 for each relationship.
By inspecting these plots one can decide if using these group relationships might enhance the predictions of the final body water space from mass. Generally this will be useful if the r2 of the relationships are good (> 0.8). Clicking on the button above the table allows the selection or not of using these group data to derive the final dilution spaces.