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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 rH2O in such subjects would be +0.47% which represents 15.46 g of water equivalents/day. This additional flux taken alone would cause rCO2 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 rCO2 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 rH2O and rCO2 for an average lactating mother as shown in Table 7.4. As with true sequestration resulting from protein synthesis the effect on rCO2 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 rH2O and rCO2 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 rCO2 will be larger (-0.9 to 1.2%).

The combined effect on rCO2 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 rCO2 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

Well nourished

Poorly nourished


Water equivalents in exchangeable groups (g/day) b



Error on rH2O



Error on rCO2




Water equivalents in exchangeable groups (g/day) c



Error on rH2O



Error on rCO2



Combined effect of export of exchangeable hydrogen

Error on rH2O



Error on rCO2



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 rCO2 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 rH2O and rCO2. 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 rH2O depends on the rate of sequestration/exchange and the true rH2O From the molar ratio of 2H flux in water to 18O flux in CO2 in humans it can be predicted that rCO2 is 3 to 5 times more sensitive to these processes than rH2O.

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 rCO2 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 rCO2.

6) During lactation the export of exchangeable hydrogen bound to solids in milk will result in an under-estimate of rCO2 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 rCO2 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 rCO2 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 rCO2 in 'normal' subjects.

7.7 References

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2. Jungas RL (1968) Fatty acid synthesis in adipose tissue incubated in tritiated water. Biochemistry; 7(10): 3708-3717.

3. Irving CS, Wong WW, Wong WM, Boutton TW, Shulman RJ, Lifschitz CL, Malphus EW, Helge H & Klein PD (1984) Rapid determination of whole-body kinetics by use of a digital infusion. Am J Physiol; 247: R709-804.

4. Coward WA (1988) The doubly-labelled water (2H218O) method: principles and practice. Proc Nutr Soc; 47 (3): 209-218.

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6. Haggarty P. Wahle KWJ, Reeds PJ & Fletcher JM (1987) Whole body fatty acid synthesis and fatty acid intake in young rats of the Zucker strain (Fa/- and Fa/Fa) Int J Obes; 11: 41-50.

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10. Bernhard K & Schoenheimer R (1940) The inertia of highly unsaturated fatty acids in the animal, investigated with deuterium. J Biol Chem; 133: 707-712.

11. Heffernan AG (1964) Fatty acid composition of adipose tissue in normal and abnormal subjects. Am J Clin Nutr; 15: 5-10.

12. Foster GL, Rittenberg D & Schoenheimer R (1938) Deuterium as an indicator in the study of intermediary metabolism. J Biol Chem; 125: 13-22.

13. Hilton MA, Frederick Jnr WB, Henry SS & Enns T (1953) Mechanisms in enzymatic transamination. J Biol Chem; 209: 743-754.

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16. Stetten D & Boxer GE (1944) Studies in carbohydrate metabolism. I. The rate of turnover of liver and carcass glycogen, studies with the aid of deuterium. J Biol Chem; 155: 231-242.

17. Eidinof ML, Perri GC, Knoll JE, Marano BJ & Arnheim J (1953) The fractionation of hydrogen isotopes in biological systems. J Am Chem Soc; 75: 240-248.

18. Haggarty P, Grieve EM & Christie SL (1988) Early developmental changes in 24 hr energy expenditure in obese and lean Zucker rats and its relationship to body composition. Proc Nutr Soc; 47: 128A.

19. Reeds PJ, Haggarty P. Wahle KWJ & Fletcher JM (1982) Tissue and whole-body protein synthesis in immature Zucker rats and their relationship to protein deposition. Biochem J; 204: 393-398.

20. Evans AG (1969) New dose estimates from chronic tritium exposures. Health Physics; 16: 57-63.

21. Moghissi AA, Carter MW & Bretthauer EW (1972) Further studies on the long term evaluation of the biological half-life of tritium. Health Physics; 23: 805-806.

22. Snyder WS, Fish BR, Bernard SR, Ford MR & Muir JR (1968) Urinary excretion of tritium following exposure of man to HTO - a two exponential model. Phys Med Biol; 13: 547-559.

23. Sanders SM & Reinig WC (1968) Assessment of tritium in man. In Diagnosis and Treatment of Deposited Radionuclides (eds Kornberg HA & Norwood WD) Amsterdam, Excerpta Medica, pp 535-542.

24. Cawley CN, Cannon LA & Moschellea JJ (1984) Models of tritium excretion following prolonged occupational exposure Health Physics; 47: 102-106.

25. Schoeller DA, Ravussin E, Schutz Y. Acheson KJ, Baertschi P & Jequier E (1986) Energy expenditure by doubly-labelled water: validation in humans and proposed calculation. Am J Physiol; 250: R823-830.

26. Haggarty P, Franklin MF & McGaw BA (1988) Advantages and limitations of the double labelled technique to measure energy expenditure. Obesity in Europe I; Eds P Bjorntorp & S Rossner, pp 365-370.

27. Norgan NG & Durnin JVGA (1980) The effect of six weeks of overfeeding on the body weight, composition and energy metabolism of young men. Am J Clin Nutr; 33: 978-988.

28. Garlick PJ, Clugston GA & Waterlow JC (1980) Influence of low-energy diets on whole-body protien turnover in obese subjects. AM J Physiol; 38: E235-E244.

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.

30. Haggarty P. McGaw BA & Franklin MF (1988) Measurement of fractionated water loss and CO2 production using triply labelled water. J Theor Biol; 134: 291-308.

31. Westerterp KR, Saris WHM, van Es M & ten Hoor F (1986) Use of the doubly-labelled water technique in humans during heavy sustained exercise. J Appl Physiol; 61: 2162-2167.

32. Prentice AM & Prentice A (1988) Energy costs of lactation. Ann Rev Nutr; 8: 63-79.

33. Schoeller DA, Leitch CA & Brown C (1986) Doubly labelled water method: in vivo oxygen and hydrogen isotope fractionation. Am J Physiol; 251: 1137-1143.

34. Coward WA, Cole TJ, Sawyer MB, Prentice AM & Orr-Ewing AK (1982) Breast-milk intake measurement in mixed-fed infants by administration of deuterium oxide to their mothers. Hum Nutr: Clin Nutr; 36C: 141-148.

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