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7.4 Export of exchangeable hydrogens

As already pointed out the exchange of water hydrogen and oxygen with material which is subsequently exported from the body will for all practical purposes appear as sequestration and should be dealt with as such.

7.4.1 Urine and faeces

Schoeller et al ³³ have estimated isotope losses in urine and faeces in adult man to be 0.47% of ²H flux and 0.34% of ¹⁸O flux. Note that these authors found that no more than 10% of isotope losses in urine and faeces were exchangeable suggesting that most are the result of true sequestration and not just due to export of exchangeable hydrogen. The resulting error on r_H2O in such subjects would be +0.47% which represents 15.46 g of water equivalents/day. This additional flux taken alone would cause r_CO2 to be under-estimated by 1.7%. However, because of the simultaneous loss of 0.34% of the ¹⁸O flux these additional losses will largely cancel out resulting in a small error on r_CO2 of -0.75%.

7.4.2 Milk

There are no exchangeable hydrogens in milk fat if we assume that it is all in the form of triglyceride.

The number of exchangeable hydrogens on protein can be calculated using the procedure described by Culebras & Moore ¹. The main proteins in milk are casein and lactalbumin and from their amino acid composition it can be calculated that they contain respectively, 1.665 and 1.789 moles exchangeable hydrogen per 100g protein. Taking an average value of 1.727 moles it can be estimated that 100 g of milk protein will exchange with 0.8634 moles of water equivalents (0.1556 g water equivalents/g protein). From this we can calculate the error on r_H2O and r_CO2 for an average lactating mother as shown in Table 7.4. As with true sequestration resulting from protein synthesis the effect on r_CO2 of export of exchangeable hydrogen in protein is very small at less than 0.15% (Table 7.4).

Using the same procedure it is possible to calculate the potential error on r_H2O and r_CO2 introduced by export of exchangeable hydrogen in lactose which has 8 hydroxyl groups and therefore 8 exchangeable hydrogens (Table 7.4). The number of exchangeable hydrogens per gram of protein and lactose is very similar but since lactose production greatly exceeds that of protein in milk the effect on r_CO2 will be larger (-0.9 to 1.2%).

The combined effect on r_CO2 of export of exchangeable hydrogen on protein and lactose is -1.0 to -1.3%. Since exchange is known to occur and since it can be estimated relatively accurately from the chemical formulae of compounds this value is likely to be a true estimate of the effect on r_CO2 and is not subject to the same qualifications as the estimation of sequestration in fat.

Table 7.4. The effect on the DLW method of export of exchangeable hydrogen in human milk ^a

^a See Footnote A in Table 7.3.

^b Molecular weight of lactose = 342 a.m.u. Eight exchangeable hydrogens per molecule of lactose.

^b 1.727 moles exchangeable hydrogen/100 g protein.

7.5 The effect of sequestration/exchange on related techniques

There are two techniques related to the DLW method which would also be subject to errors introduced by H isotope incorporation into products other than water. These are the triply-labelled water (TLW) method of measuring fractionated water loss and r_CO2 ³⁰ and the milk transfer technique ³⁴.

TLW depends on the different fractionation factors for evaporative ³H₂O and ²H₂O loss but the mass effects which give rise to the differential fractionation also cause isotope discrimination during sequestration. Preferential incorporation of ²H over ³H into non-labile positions would appear as fractionated water loss and would therefore over-estimate the TLW-derived value for fractionated evaporative water loss.

In the milk transfer method one of the assumptions is that ²H is transfered from mother to baby only in the form of water. However, ²H can also be transfered from mother to baby in sequestered positions and on exchangeable groups in the components of milk. Taking a nominal milk water transfer of 600 ml/day we can predict from the values presented in Table 7.4 that export of exchangeable hydrogen to the baby will result in an over-estimate of milk transfer of 1.3 to 1.7%. Transfer of H in sequestered positions in lactose will further increase this overestimate to 2.7 to 3.2%. When H sequestered into fat is included the final error on estimated milk transfer is 3.5 to 3.7%.

7.6 Summary and conclusions

Any loss of ²H or ¹⁸O as products other than CO₂ and water will introduce an error into the calculation of r_H2O and r_CO2. In addition to ordinary water flux, hydrogen can be lost from the body water as sequestered hydrogen bound to carbon in fat, protein and carbohydrate or as hydrogen in the exchangeable positions of compounds which are subsequently exported from the body. To a limited extent ¹⁸O may also be sequestered and exported as exchangeable ¹⁸O and this will partially offset any error due to ²H loss. The proportional effect of these processes on the isotopically-derived values for r_H2O depends on the rate of sequestration/exchange and the true r_H2O From the molar ratio of ²H flux in water to ¹⁸O flux in CO₂ in humans it can be predicted that r_CO2 is 3 to 5 times more sensitive to these processes than r_H2O.

Experimental constraints make it very difficult to quantify the effect of sequestration in human subjects. There are however some things that we can slate with some degree of certainty:

1) The effect of protein synthesis can be ignored when calculating the effects of sequestration.

2) Glygogen turnover is unlikely to introduce significant bias into the DLW technique although cycles of sequestration and release could introduce additional variability into ²H decay.

3) The largest sequestration effect is likely to occur during fat synthesis simply because the fatty acids are the most reduced class of compound in the body.

4) Even in weight-stable subjects there is constant turnover of the body constituents (notably fat) which will result in sequestration. However, we cannot as yet assess the magnitude of this effect.

5) Loss of isotope in urine and faeces will introduce an error into r_CO2 corresponding to approximately -4 1/day in 'normal' adult male subjects therefore workers may wish to add a 'correction factor' of +4 1/day to their estimate of r_CO2.

6) During lactation the export of exchangeable hydrogen bound to solids in milk will result in an under-estimate of r_CO2 of 1.0 - 1.3%. Isotope sequestration may further increase this value to 1.5 - 3.4%.

7) Under extreme anabolic conditions and using pessimistic assumptions regarding de novo fat synthesis the maximum error on r_CO2 due to ²H sequestration was estimated at 5%. Although there are a number of uncertainties inherent in this calculation it seems unlikely that the error would be as high as this under many circumstances.

The relatively small effects which can be quantified should not be dismissed too lightly since all the processes outlined here cause r_CO2 to be under-estimated and will therefore be additive perhaps resulting in measurable consequences for the DLW technique. However, until more information is obtained on turnover of body constituents we must conclude from the circumstantial evidence of the validation studies that, within the limits of our knowledge of the other factors which affect the DLW technique (fractionated water loss for example), ²H sequestration does not have a measurable effect on the calculation of r_CO2 in 'normal' subjects.

7.7 References

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29. Haggarty P, McGaw B. Fuller MF, Smith JS & Christie SL (1988) Long term validation (during tissue deposition) of the doubly labelled water technique for measuring energy expenditure (EE). Proceedings of the First European Congress on Obesity; p310.

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