Using this protocol, deuterium oxide, or D2O, a nonradioactive, stable isotope of water, can be used to estimate body composition and water consumption. This technique is noninvasive and therefore can be used to assess body composition in endangered species, as well as in humans and domestic species. A unique application of the deuterium oxide dilution technique is the ability to measure the water consumption of free-ranging wildlife with high recapture rates or of socially housed animals.
While anesthesia is not required, it may be easier for those unfamiliar with giving subcutaneous injections to sedate the animals before delivering the deuterium oxide. Demonstrating the procedure with Sarah Hooper and me will be Amanda Eshelman and Ashley Cowan, veterinary students, and Alicia Roistacher, a graduate student from my laboratory. To make a 50-milliliter stock solution of nine grams per liter salinated deuterium oxide, first weigh 450 milligrams of pharmaceutical-grade sodium chloride, and record the exact amount to four decimal places.
Transfer all of the salt into a sterilized 100-milliliter beaker, and measure out 50 grams of greater than or equal to 99.8%deuterium oxide in a sterile, graduated cylinder. Record the exact amount of deuterium oxide to four decimal places, and transfer the deuterium oxide to the sterile beaker of sodium chloride. Next, attach a 20-gauge needle to a nonpyrogenic, sterile disk filter with submicron pores fitted with a 10-milliliter syringe barrel, and insert the needle into the septum of a sterile, empty, 100-milliliter vial.
Attach a vacuum tube to a 22-gauge needle, and insert the needle into the septum of the 100-milliliter vial. Then load 10 milliliters of isosmotic strength sodium chloride into the syringe barrel, and slowly turn on the vacuum until the deuterium oxide solution begins to slowly filter into the vial. Continue pouring the deuterium oxide stock solution into the syringe barrel until the entire 50-milliliter volume has been filtered.
After confirming a lack of response to toe pinch in an anesthetized bat, use an alcohol prep pad to clean the uropatagium over the interfemoral vein, and allow the disinfected tail membrane to dry. Apply a thin layer of petroleum jelly over the interfemoral vein, and use a 29-gauge needle to puncture the dorsal portion of the interfemoral vein. Then use plastic sodium heparin capillary tubes to collect 100 microliters of blood.
To ensure an adequate mixing of the whole blood with the sodium heparin, gently roll each tube after the blood collection before labeling. After determining the mass of deuterium oxide in grams to inject, load an insulin syringe equipped with a 29-gauge needle with the appropriate volume of deuterium oxide. Weigh the deuterium-loaded insulin syringe and needle, recording the weight to four decimal places, and inject the entire volume of deuterium oxide subcutaneously over the dorsal hip region of the anesthetized bat.
We recommend using a precision scale with an antistatic draft shield and to make sure to pull back on the plunger to ensure negative pressure prior to injecting. Immediately after the injection, weigh the now-empty insulin syringe and needle, and record the weight to four decimal places. Within 30 minutes post-blood collection, use a hematocrit centrifuge to spin down each capillary tube at 10, 000 times g for five minutes.
Using sharp scissors, cut the plastic capillary tube between the whole blood and the plasma, and use a 200-microliter pipette to expel the plasma directly into a labeled, 500-microliter storage tube. After the equilibration period, collect and store another 100 microliters of blood from the interfemoral vein as just demonstrated. For FTIR spectrophotometry analysis, set the temperature of a sand bath to 60 degrees Celsius to facilitate distillation, and add 50 microliters of each plasma sample and standard into individual 1.5-milliliter conical microcentrifuge caps.
Keeping the microcentrifuge cap upside down, screw the 1.5-milliliter conical microcentrifuge tube onto the cap, and place the inverted tube in contact with the sand in the sand bath for a minimum of 12 hours. The next morning, place a new, clean cap onto each tube, and pulse the microcentrifuge tubes for 10 seconds in a microcentrifuge. Install a liquid transmission cell into the FTIR spectrometer, and fill the cell with methanol.
Connect the injection port, and slowly fill the cell with background water while carefully removing the methanol syringe to reduce the risk of air bubbles. Attach tubing to the output port to allow removal of the samples post-analysis, and open the FTIR spectrometer software. Set the parameters according to the table, and collect a background sample using the diluent, 0.22-micrometer-filtered, distilled water.
Inject 40 microliters of a zero parts per million deuterium oxide standard, and record and save the spectra as a comma-separated values file. After injecting and saving the spectra of all of the standards to create a standard curve, inject 40 microliters of each distilled sample into the liquid transmission cell, and save the spectra. With a known equilibration time, the total body water, lean body mass, and body fat mass for wild-caught big brown bats and captive big brown bats can be determined.
A negative body fat mass can be calculated due to one or more of the following reasons:not receiving the entire dose of deuterium oxide, becoming torpid during the equilibration phase, having abnormally large fat masses and minimal lean masses, or having under three to 5%body fat as determined by dual x-ray absorptiometry. Here, a representative percentage of body fat determined by the deuterium oxide dilution technique compared to dual x-ray absorptiometry is shown. The two techniques are well correlated, with the body fat mass showing strong correlations between the body fat and body weight and with the deuterium oxide dilution technique not consistently over or underestimating the body fat mass.
The deuterium oxide dilution method has been previously validated in cats, and the technique was adapted to allow measurement of the daily water consumption of socially housed cats during each dietary block of the experiment. Remember that this technique should be performed only on healthy animals and that you should accurately weigh, record, and deliver the full dose of deuterium oxide to each animal. A secondary method for determining body composition, such as complete carcass analysis or DEXA, can be completed to validate the deuterium oxide technique.
This technique allowed us to identify a socially housed cat that was consuming abnormally high quantities of water without separating each cat to measure their water consumptions.
