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Without a doubt, the most difficult problem facing new users of the doubly-labelled water method has been obtaining isotopic analyses that are sufficiently accurate and precise. Small errors in the isotopic determinations lead to large errors in energy expenditure because the method depends on calculating the difference between the kinetics of 18O and 2H.
Accurate isotopic analyses are desirable if baseline isotopic abundances are to be interpretable against the extensive literature on isotopic hydrology (Chapter 8) and because day-to-day intra-laboratory and inter-laboratory comparisons are improved. Accuracy, however, is not an absolute requirement if all samples and the standard dilution of the dose are measured within the same day and if it can be assumed that the mass spectrometer calibration does not change. Changes in calibration occur due to linear offsets in the calibration which move the scale up or down a fixed amount regardless of the abundance, or proportional errors which introduce a constant percentage error in the abundance relative to the working standard. Linear offset errors are cancelled when the enrichment of any sample is calculated relative to the pre-dose background abundance. Proportional errors are removed from elimination rates because this calculation involves a natural logarithmic transformation. However, proportional errors will produce errors in the apparent isotope dilution space. This is overcome if an aliquot of the loading dose given to the subject is gravimetrically diluted in a similar proportion to its in vivo dilution, and if this is analysed on the same day to determine the dose administered. Under these circumstances any proportional errors in the dilution space will cancel.
Despite the decreased importance of absolute accuracy, investigators should strive to achieve accuracy for the reasons stated above. This can be done by obtaining international isotopic standards and using these to check the performance of preparation procedures and mass spectrometry. (Suitable standards are available from: Dr Robert Parr, Section of Nutritional and Health Related Studies, IAEA, Wagramerstrasse 5, P.O. Box 100, A-1400 Vienna, Austria). Laboratory working standards (i.e. gravimetric dilutions) can then be used to ensure accuracy on a day-to-day basis.
Precision of isotopic analyses on the other hand is an obligatory requirement. The degree of precision needed depends on the isotope dose, the isotope elimination rate, the metabolic period, and the number of points used in the calculation of the elimination rate and dilution space. For the two-point method, it is recommended that precision of the isotopic analysis be better than one six-hundredth of the initial isotopic enrichment 14. If adult doses are to be kept economically feasible (<US$300) then this translates into a required precision of 0.16 ‰ for 18O and 1.1 ‰ for 2H (where precision is defined as the standard error of multiple analyses obtained during the workup of samples for DLW studies). This will reduce the analytical error to less than 5% for metabolic periods of between 1 and 3 biological half-lives of 18O 14. Requirements can be relaxed in proportion to the enrichment above baseline for highly enriched samples. The multi-point method can tolerate a reduction in precision in proportion to the square root of the number of points, except in the case of the baseline sample which must meet the standards set out above.
The most practical method for
assessing adequacy of precision of isotopic analyses is to perform multiple analyses of a
single set of samples from a subject. These should be done on separate days and the carbon
dioxide production rate should be calculated independently for each set of analyses. For
the two-point: method, the coefficient of variation for carbon dioxide production should
be about 4%. It should be 2 to 3.5% for the multi-point method. This standard should be
relatively easily met in subjects whose 2H elimination rate is less than 75% of
their 18O elimination rate, but difficult to meet for those in whom it is
greater than 85%.
The 2H- and 18O-labelled water can be purchased from numerous stable isotope suppliers. Some of these suppliers are shown below:
Isotec Inc.
3858 Benner Road,
Miamisburg, Ohio 45342
USA.
CEA-ORIS,
Bureau de Stables Isotopes,
BP 21-91190,
Gof-sur-Yvette,
France.
Cambridge Isotope Laboratories
20 Commerce Way
Woburn, Massachusetts 01801
USA.
MSD Isotope
PO Box 899
Pointe Elaire-Dorval
Quebec
Canada H9R 4P7.
Isotope Department,
Weismann Institute of Science,
Rehovot,
Israel.
Delta Isotopes,
Wistaston Park,
Wistaston,
Crewe,
Cheshire, CW2 8JT,
UK.
Deuterium oxide is widely available from many sources with 2H enrichment of 99.8 atom percent and above. Oxygen-18 labelled water is available in either low (10 atom % 18O) or high enrichment (>95 atom % 18O). The oxygen-18 labelled water is usually normalised with respect to hydrogen. Therefore deuterium oxide and the normalised 18O labelled water can be purchased separately and then combined in the laboratory prior to the study. However, oxygen-18 labelled water (>95 atom % 18O) can be obtained without normalisation with respect to hydrogen. This labelled water usually has 2H enrichment of approximately 60 atom percent. An alternative is to purchase the 'un-normalised' 18O labelled water which has high enrichment of 2H.
Water with low enrichment of 18O
(10 atom %) is recommended for use with older children, adolescents, and adults because it
is less expensive and these subjects can tolerate larger volumes of the tracer water in a
doubly-labelled water study. With small infants, water with high enrichment of 18O
(>95 atom %) is preferable because infants are less tolerant to large volumes of the
tracer water.
The deuterium oxide and the 18O-labelled
water are not made for human consumption. The amount of deuterium oxide and 18O
labelled water used in a doubly-labelled water study will alter the natural abundances of 2H
and 18O in the body fluid by approximately 0.03 and 0.06 atom %, respectively.
Deuterium enrichment at this level is well below the toxicity level (10 atom %) reported
for deuterium oxide. Studies for mice and primates indicated that replacement of the
oxygen atoms in the body fluids and tissues with up to 60 atom % of 18O has no
physiological or pathological effects on these animals. However, in human studies
involving infants, children, and pregnant and lactating women, it is important to make
sure that the water tracer is bacteria and pyrogen free. Deuterium oxide is available
bacteria and pyrogen free from MSD Isotopes. Bacterial contamination can be removed by
filtration through sterile 0.2 mm filters. Pyrogens in the
tracer water can be removed by ultrafiltration.
To confirm the enrichments of
deuterium and 18O in the dose, a known amount of the dose must be diluted
gravimetrically with water of known 2H and 18O content in a
proportion similar to the dosage used in a doubly-labelled water study. To minimise
instrumental effects on the accuracy of the isotope ratio measurements, it is recommended
that the determination of the 2H and 18O enrichments of the dose and
the actual isotope ratio measurements of the samples be done using the same instruments
within the same time frame.
Gas-isotope-ratio mass spectrometers
are very accurate and precise instruments. With proper training in the operation of the
instrument and accessories, errors in isotope ratio measurements usually come from
improper sample collection and/or sample preparation. When working with small quantities
of physiological fluids, contamination of sample by moisture will dilute the enrichments
of 2H and 18O particularly in the post-dose samples. Evaporation
during storage or transit will also alter the isotopic enrichments of 2H and 18O
in the samples. The effect is most critical with 2H because of the large
isotope fractionation effect during evaporation and condensation of 2H2O.
Isotope fractionation can also occur during sample preparation when the water sample is
not converted quantitatively to H2 (uranium/zinc reduction) or to CO2 (guanidine
hydrochloride). Each laboratory or institute must evaluate each sample preparation
procedure which is to be adapted for preparation of physiological fluids for hydrogen and
oxygen isotope ratio measurements. Prior to actual sample analysis, daily calibration of
each instrument for optimal sensitivity and performance with laboratory working standards
is recommended. Prior to the purchase of an instrument, it is advisable for the laboratory
to consult current users to confirm instrument specifications and reliability.
Accessibility of service engineers and availability of replacement parts are important
factors in the final selection of instruments.
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