Method for isotopic analysis of water in bodily fluids
Biological fluid samples containing proteins (e.g., blood plasma or saliva) are prepared for isotopic analysis by precipitating the proteins while leaving the isotopic ratio unaffected. This precipitation can involve adding metal ions, salts, organic solvents, or organic polymers. The sample is then centrifuged to allow transfer of the supernatant for isotopic analysis, e.g. by tunable diode laser absorption spectrometry to obtain a quantitative measure of the 2 H and 18 O isotope levels in the water relative to reference standards.
1. A method of performing isotopic analysis of water from biological fluid samples, comprising:
obtaining a biological fluid sample, wherein the sample contains water and proteins;
preparing the sample prior to analysis by precipitating the proteins in the sample, wherein a protein-precipitating agent added to the sample leaves isotope ratios unaffected for all isotopes that have been selected to be analyzed, and centrifuging the sample sufficient to separate precipitated protein from supernatant liquid;
without using any distillation of the sample or of the supernatant liquid prior to quantitative isotopic analysis, transferring, once the centrifuging has stopped, an amount of supernatant liquid from the centrifuged sample to a vial; and
conducting a quantitative isotopic analysis of at least one of δ 18 O, δ 17 O and δ 2 H in the undistilled supernatant liquid using laser absorption spectroscopy of water.
2. The method as in claim 1 , wherein the volume of sample in a range from 30 μL to 250 μL is inserted into a microcentrifuge tube.
3. The method as in claim 1 , wherein precipitating the proteins in the sample involves metal-induced affinity protein precipitation by adding a metal-cation salt to the sample.
4. The method as in claim 3 , wherein the metal-cation salt is any of a zinc, copper, calcium or aluminum salt.
5. The method as in claim 4 , wherein zinc sulfate monohydrate is added to the sample at a rate of 5 mg per 200 μL of sample, followed by vortexing of the microcentrifuge tube.
6. The method as in claim 1 , wherein precipitating the proteins in the sample involves salting the solution with any of a citrate, phosphate or sulfate salt.
7. The method as in claim 1 , wherein precipitating the proteins in the sample involves adding an organic solvent incapable of hydrogen exchange to the sample.
8. The method as in claim 7 , wherein the organic solvent is selected from any of acetone, ketones, dioxanes and ethers.
9. The method as in claim 1 , wherein precipitating the proteins in the sample involves adding an organic polymer agent incapable of hydrogen exchange to the sample.
10. The method as in claim 9 , wherein the organic polymer agent is polyethylene glycol.
11. The method as in claim 1 , wherein the sample is centrifuged at 5000-10000 rpm for 8-12 minutes.
12. The method as in claim 1 , wherein 10 μL to 75 μL of supernatant liquid is transferred to a vial for measurement.
13. The method as in claim 1 , wherein the quantitative analysis is calibrated using one or more reference standards.
14. The method as in claim 1 , wherein the quantitative analysis is conducted using cavity-enhanced absorption spectroscopy.
15. The method as in claim 14 , wherein the cavity-enhanced absorption spectroscopy is selected from any of cavity ringdown spectroscopy, integrated cavity output spectroscopy, off-axis integrated cavity output spectroscopy, and cavity-attenuated phase-shift spectroscopy.
16. The method as in claim 1 , wherein the laser absorption spectroscopy for conducting quantitative analysis of the sample is any of photoacoustic spectroscopy, and photothermal spectroscopy.