Subject dosing
Making up the dose
We make up doses for studies here in Aberdeen and supply these pre-weighed to collaborators, based on supplied subject characteristics. If you are making up your own doses then it is important to follow strict procedures to ensure the dose is not contaminated and can be delivered quantitatively to the subjects.
Normally human subjects are dosed using 10APE oxygen18 which has 99.9% deuterium added to it. It is best if a batch of dose solution is made up for an entire experiment. To make up the batch of doses calculate the required quantity of oxygen18 water. For example if a study involves 30 children who will each receive 70 mls of 10APE oxygen18 water you will need 30 x 70 mls = 2100 mls. In fact you also require an extra dose to charcaterise the mix of isotopes so in this case you need 2170 mls of 10 APE oxygen18. Place this amount of oxygen-18 water into a clean dry vessel. The actual dose of oxygen18 is 7mls per child. The required dose of deuterium is half the dose of oxygen18 – in this case 3.5 mls per subject. So the total required amount of deuterated water (99.9%) is 3.5 x 31 subjects = 108.5 mls. Add this quantity of deuterium to the oxygen-18 water and mix them using a clean glass rod. This is the dose solution. The exact quantities that are added together are not crucial because exactly how much oxygen18 and deuterium is in the dose is assessed later using mass spectrometry – that is why an extra dose is made up.
If you have concerns about sterility then this is the stage to autoclave or filter the dose solution. Opinion is divided about the necessity to do this. In line with many other laboratories we do not routinely autoclave or filter the doses.
The individual doses then need to be aliquotted into dose bottles. At this stage it is important to know exactly how much isotope has been put into each dose bottle. The doses are prepared roughly in line with the body weights of the subjects. If exact subject weights are known then a personalised dose can be prepared for a given subject. If individual weights are not known then use an average expected weight.
Suggested dose rates are as follows.
Neonates and very young children : 4 mls 10APE oxygen18 per kg body weight
Children (up to age of 15) and very active athletes : 2.5 mls 10APE oxygen18 per kg body weight
Adults : 1.8 mls 10APE oxygen18 per kg bodyweight
Elderly : 1.6 mls 10APE oxygen18 per kg bodyweight
These doses are sufficient for study durations of 7-14 days. Dose bottles should be made of shatterproof glass with a rubber sealed top. They should be dry and at room temperature. We normally dry ours at 60 oC overnight and then allow them to cool in a dessicator before use.
Weigh the dose bottle (2 figure balance). Write it down.
Add the required quantity of the dose water approximately using a calibrated measuring cylinder.
Weigh the dose bottle again containing the dose to obtain the exact weight of the dose. Write it down. Do the subtraction to make sure it is in the correct range required. Adjust if necessary by adding more dose to the bottle or pouring some out. Make a final weight and a final estimate of the dose weight.
Write down the final dose weight and the identification number of the dose bottle.
Seal the bottle. We use crimp seals which are completely water tight. Rubber sealed glass bottles are also suitable.
Write on the bottle the ID number for the dose and also if appropriate the subject details who will receive it.
Keep one bottle of the dose for characterisation
Doses do not need to be frozen for storage prior to use. It is advisable however to minimise the time between dose preparation and dose administration. This minimises any risk of dose contamination or leakage before administration.
The dose rate is calculated according to the body weight of the subject and also an evaluation of their likely activity levels.
When to dose
Opinion is divided over the ideal timing of the dose. Two protocols are commonly used.
a) The subjects are dosed in the morning on an empty stomach. Pre-dose urines are taken as the first or second void of the day. Initial post-dose samples are then collected at 3-4 hours post dosing. Subjects are allowed light beverage and food consumption during the period between dosing and initial sample collection. Post dose urines are collected slightly later in heavily obese, pregnant or the elderly (ie at 4-5 hours post dose) where equilibration may take slightly longer.
b) The subjects are dosed last thing at night before retiring. Pre-dose urines are collected before dose consumption. In the morning the first void is discarded and then the second void is used for the post dose sample.
Record the time when the dose was administered using the 24h clock. This avoids any confusion over am and pm. A 12h error in timing due to mixing up am/pm can introduce a 6% error in the CO2 production estimate.
The dosing procedure itself
When you receive the doses from our lab they will be supplied in crimp sealed bottles, or screw top bottles with an internal rubber seal. Exceptionally you may be sent two bottles for a given subject if the dose is very high. You will be informed of this in advance if it is applicable in your study. These bottles and their contents have been pre-weighed. The aim when dosing is to get the entire contents of this bottle safely into the subjects body without any spillage. You may wish to give adult subjects written instructions about what your aims are in the dosing and the importance of them consuming the dose completely. You may wish to also remind the subjects that there are no expected adverse effects from consuming the doubly-labelled water. The bottles are individually labelled. The exact weights of the bottles vary. It is important to know which subject received which dose. You must make sure that the bottle ID is correctly associated with the relevant subject in the case notes.
To dose the subject proceed as follows
1) Have a weighed paper towel available in case there is any spillage.
2) Check the dose bottle to make sure there is no sign of leakage. If you suspect the dose bottle has leaked then do not use it. Write down the dose bottle ID against the subject ID and get a second person to confirm these are both correct. Ideally both parties should initial the notes to confirm this has been done.
3) Tap the bottle to dislodge any of the dose water that may be attached to the lid or rubber seal.
4) Remove the crimp seal or screw top. Be careful. Check the cap for any signs of dose water and if there is any make sure that it is not spilled.
5) Pour the entire contents of the bottle into a drinking vessel and add to it any water from the cap. The doses vary between about 50 mls for small children up to 180 mls for obese adults. For children it is advised to use a cup with a sealed lid to prevent spillage during the dosing process.
6) If there is any spillage of the dose at this stage mop it up with the weighed towel and then reweigh the towel to estimate the weight of dose that was lost. Make a note that this happened and write down the calculation of how much dose was spilled.
7) Take about 50 mls of local tap or bottled water if you have concerns about sterility and add this to the empty dose bottle. Replace the cap or lid and shake this up. This will catch any residual water that was in the dose bottle and not transferred to the drinking vessel.
8) Transfer this added water from the dose bottle also to the drinking vessel.
9) You may add flavouring to the dose if you wish at this stage. This is particularly useful for dosing children but adults aregenerally content to drink the water without flavouring.
10) OBSERVE THE SUBJECT drink the dose. It is important that dose consumption is supervised.
11) Make sure the entire dose has been consumed. This can be facilitated by providing the subjects with a drinking straw to consume the last drops. If any of the dose remains in the drinking vessel add some tap water to it (about 50 mls) and get the subject to consume this additional water.
12) Record in the case notes who supervised the dosing (ideally the identity of the dose bottle number and the subject should be cross checked at the time of dosing by a second observer).
13) Record anything abnormal that happened. Particularly record if there was any spillage of the dose and the weight of dose that was recovered by the paper towel.
The most important thing about dosing is to ensure you record exactly which subject got which dose and also that you supervise the dose consumption to ensure the entire dose was consumed. We strongly advocate two people doing the dosing with everything being cross checked and signed off in the laboratory notes or case notes by both parties. This ensures no mistakes are made in the dosing.
It is important to remember that if the wrong dose is administered the error in the estimated metabolic rate will probably exceed every other error in the measurement. If you give the subject 10% more or less isotope than you think you have given this will translate directly to a 10% error in the estimated CO2 production and metabolic rate. Dosing mistakes are not easy to spot when they are being made. You would not for example easily spot if you had accidentally administered 120 rather than 130 mls of dose. Moreover the standard QC procedures at the mass spectrometry stage will not always pick up dosing errors.