Doubly Labeled Water
Protocol
Pontzer Lab, Duke University
July 2025
Planning & Preparation: You will need enough dose material (isotope-enriched water) to dose each of the participants in your study (see Dosing Table). It may be easiest to bottle individual doses for all of the participants ahead of time, while you have access to a high-precision scale. You need to know how much dose was given to the nearest 0.1 gram. You can pre-measure the doses to match the range of bodyweights you are likely to encounter. If you are within ~10% of the recommended dose size, it will work fine. It’s always better to give an over-sized dose than an under-sized dose.
Other field supplies you will need
Urine collection cups: You can buy urine sample cups from a medical or lab supply source, but any dry, clean, plastic cups, including disposable drinking cups, will work. Never reuse cups! One clean, new cup per sample.
Cryovials: Any lab-grade vial that doesn’t leak and can withstand -80C freezing should work. We use internally threaded 5ml vials with a white field on the side that you can write on for notes (be sure to have sharpies to write on them.) You could use 2ml vials if space or bulk is a concern (we prefer ~4ml samples; ~1 ml is the minimum.) It’s often wise to collect duplicate vials of each sample.
Transfer pipettes: One clean, dry, new transfer pipette per sample to transfer the urine from the collection cup to the cryovial. Never reuse transfer pipettes between participants or samples.
In the Field: If you pre-measured the dose bottles, try to check that they haven’t leaked. If the bottles are marked with a “Gross Weight”, and you have access to a scale, you can check that the gross weight hasn’t changed. If the bottle is marked with a fill line, you can check if the level of dose in the bottle still reaches the fill line. If the bottle has leaked, don’t use the dose until you can re-weigh the bottle to determine the exact amount of dose to the 0.1g.
Once you’ve checked the dose and consented and enrolled the participant, you’re ready to start:
Step 1: Anthropometrics
Collect the following data for each subject:
- Body Mass (to 0.1kg if possible), 2. Height, 3. Age, 4. Sex.
Optional but recommended: 1. Medications, 2. Illness, 3. Health metrics (blood pressure, etc.)
If possible, collect Body Mass again at the end of the DLW period (e.g. at the final urine collection). This lets us know if the subject is weight stable during the DLW period, and can be used to calculate food intake (kcal/d) from energy expenditure. Not mandatory but strongly encouraged.
Step 2: Collect a Baseline Urine Sample
We aim for 4 ml, but you can do 2 ml. (Minimum for analysis is 1 ml.) Time of day / fasting / resting and other considerations do not matter. Any urine sample will do. And you can collect it any time (minutes, hours, days) before Step 3. Write Subject ID, date, and “Baseline” on these sample vials.
Step 3: Drink the DLW Dose
The participant needs to drink every last drop of the DLW dose – no spilling whatsoever. Have the participant drink the DLW dose directly from the bottle, then pour some bottled (or clean tap) water in the dose bottle, swish it around, and have them drink that, then pour more water in the dose bottle, swish it around, and have them drink that. This way you know every last DLW drop has been flushed out of the bottle and consumed. Once that’s done, record:
- Date and time (to the minute) that the dose was consumed
- Participant ID
- Dose amount given, to the 0.1 gram.
- If possible, note other info from the dose bottle: Bottle ID, Net dose, Material, etc.
- Note any spills and estimate amount spilled – hopefully this never happens
Step 4: Post-dose urine samples (collected after the DLW dose was consumed).
Plan to collect 3 to 5 post-dose urine samples, evenly spaced over the next 7 to 14 days. The first and last post-dose samples are the most important.
- Collect the first post-dose sample ~6 hours after the dose is given (no earlier than 4 hours). Ideally, the participant should urinate between the time they drink the dose and the time they provide the first post-dose sample. If you can’t collect the first post-dose sample at ~6 hours after dosing, you have until ~24 hours after dosing to do it, but ~6 hours post-dose is best.
- Collect every 2 to 3 days after the dose is given. Ideally, these would be collected at around the same time of day that the dose was given. If they are collected in the morning, ideally these samples would be the second void after waking. If samples need to be collected at other times due to logistical constraints, that’s ok.
- Collect the final sample between 7 and 14 days after the dose is given. This will mark the end of the energy expenditure measurement period. Ideally, this last sample should be collected near the same time of the day that the dose was given. If it is collected in the morning, ideally these samples would be the second (or third) void after waking. If you can measure and record body weight when you collect this final sample, that is strongly
For each urine sample, record the following – both in your notes AND on the vials:
- Subject ID
- Date and Time (to the minute) the sample was collected
- Sample # (1, 2, 3…) for this subject
Other Considerations
Avoid Contamination: ANY foreign fluid in the urine samples (rain water, cleaning water, urine from a different sample, etc.) will ruin a urine sample for analysis. A bit of dry dirt is OK (probably) but should still be avoided. Use new, clean, dry collection cups and pipettes for each urine sample.
Don’t overfill the vials! Leave 0.5 to 1.0 cm of air in the tube (“headspace”). Otherwise, when they freeze and the urine expands, the vials may leak.
Sample Storage and Shipping: Samples should be sealed tightly in cryovials and kept cold, ideally frozen (if possible) until they come back to the U.S. If they thaw that’s ok (but try to avoid it), and food-grade freezers are fine. The goal is to prevent bacteria activity.
Often, the samples can be brought back from the field in checked luggage. They will thaw on the flight, but if you put them right back into a freezer when you get home they will be ok. You need to check the regulations in the country in which you’re working regarding permissions to export samples. There may also be regulations (e.g., CITES) regarding non-human samples, depending on the species. Importing human samples into the U.S. is generally simple: As of July 2025, no permits are needed to bring non-infectious human urine samples into the U.S.
Another option is to ship samples (e.g., DHL, FedEx, etc.) to the analysis lab. If you go that route, pack the samples into an insulated (e.g., Styrofoam) biological samples box. If possible, include a frozen gel cold pack, the kind you use for first-aid or to ice a sprained ankle. Do not use water ice, which will melt and can contaminate the samples. Dry ice and liquid nitrogen are not necessary. Ship the samples express / overnight (the fastest option possible), and be sure to alert the analysis lab they are coming and provide a tracking number.
Activity Data: DLW measurements are more useful when you have data on daily activity. Accelerometry is preferred. If you’re unsure what accelerometry device or protocol to use, let us know.
Diet: DLW measures the amount of carbon dioxide (CO2) the body produces. To convert that rate of CO2 to a rate of energy expenditure (kcal/day) we need to know the macronutrient (carbs, fats, protein) make-up of the diet. We can estimate this, but it’s best if you can collect or access data on dietary intake for your population. You don’t need individualized data (unless individuals vary greatly in their diet) but a population average for the % of calories from carbs / fats / proteins is very helpful. If you want help with this, or are unsure what data to collect, let us know.
