INTRODUCTION

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THE VOLUME OF BODY WATER IS physiologically well regulated although life threatening water changes are not uncommon. During surgery and in connection with trauma, fluid therapy and balance are vital for patient outcome. Although infusion therapy is equally important at all ages, it is at the extremes of age, i.e., in neonates and in the elderly, that fluid balance often is a difficult and delicate matter in clinical practice for both in-hospital treatment and prehospital care. It is therefore of great value to be able to accurately measure changes in body water content.

The latest development of gas isotope ratio mass spectrometers (GIRMS) using stable isotopes as tracers has opened up new possibilities for the clinical use of this highly technological device. Measurements with isotope tracers by using a dilution principle are well known, and deuterium is a widely used isotope for measurements of total body water (TBW). Analyses of deuterium/hydrogen (D/H) have previously been done by reducing water samples, with uranium or zinc, to H₂ and then analyzing for D/H (1). An alternative method is the use of equilibration (EQ) between samples of water and gaseous H₂ followed by analyses of D/H in the equilibrated H₂ (8). A promising new technique for measurement of hydrogen isotopes using chromium reduction (CR) has recently been developed (4). This new CR method also presents possibilities for measurements of small samples in all kinds of body liquids. The aim of this study was to evaluate the CR method and compare it to the EQ technique. Precision, accuracy, and variability for measurements of TBW were tested in a body compartment model.

	METHOD

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Experimental design. Different weights of ordinary tap water were contained in water tanks to cover a simulated range of total body water volumes from neonates to adults. The following "body water" volumes were used: 1.8 × 10³ ml, 3.0 × 10³ ml, 15 × 10³ ml, 30 × 10³ ml, 45 × 10³ ml, and 54 × 10³ ml. A high-precision digital scale (Stathmos, Vaxjo, Sweden) was used and had an exactitude of ±10 g in the weight range between 0 and 120 kg.

EQ technique. This system is a fully automatic EQ system (Thermo Finnigan MAT, Bremen, Germany). Glass bottles (20 ml) were filled with the water samples, and platinum catalysts (Hokko sticks) were placed in the bottles before they were connected to the analyzing rack. The rack was mounted on a sled that enabled the bottles to be immersed in a water bath. The sled was shaken using an eccentric drive. The temperature was maintained constant at 18°C ± 0.05°C. The temperature was chosen to minimize condensation inside the bottles. The sample bottles were connected via a short capillary to a manifold with a pneumatic valve. The line was evacuated by using a rotary vacuum pump. Then the bottles were filled with EQ gas. The EQ pressure (0.4-0.6 bar) gave an appropriate pressure in the inlet system after EQ. The EQ time was set to 120 min.

CR method. Chromium reduces water to hydrogen gas at >700°C according to the reaction
The reaction quartz tube was filled with ~50 g of chromium powder with a layer of quartz wool. The reaction furnace was connected on-line with the GIRMS. Samples are loaded into 0.75-ml vials in the tray of an auto sampler A200s. The sample, 2 µl, was injected by the auto injector into the reaction furnace at 900°C, by use of a gas-tight microsyringe. The sample quickly evaporated and was reduced almost instantly at the contact with chromium. The hydrogen gas flows into the inlet system because of the pressure gradient. Measurements start after pressure EQ between the reaction furnace and the variable volume bellows. Reaction and EQ times are 80 s from injection to start of measurement. In this study, an automatic H-device was used (ThermoQuest).

Apparatus. GIRMS is an analytical technique for accurate and precise measurements of stable isotopes. The mass spectrometer consists of an inlet system, an ion source, a magnet, and a collector. The entire analyzing process is completely controlled by a computer. Samples must be in a gaseous state before they are introduced into the ion source. For hydrogen isotope measurements, physiological fluids must be converted to hydrogen gas. This gas sample and a reference gas are usually introduced into the ion source through the inlet system. The inlet system matches the pressures between sample and reference gas. The gas molecules are presented sequentially through capillary leak lines into the ion source, which is under high vacuum. In the source, the gas molecules are ionized by electrons from a filament wire. Some of the molecules lose an outer electron and become positively charged. Repelling electrodes force these ions through a series of focusing lenses into the analyzing part of the mass spectrometer. A magnet is used to deflect the molecular ions according to their masses. Each mass has different ion beams and separated into specific collectors. A detectable electron current is discharged, and the ratio of these ion currents is measured and directly related to the isotope ratio.

Calculations. The dilution spaces were calculated from the equation
where N is dilution space, expressed in grams, W is the mass of water used to dilute the dose, A is the dose administered, a is the mass of the dose used in preparing the diluted dose, f is the fractionation factor for the physiological sample, _a is the measured value for the diluted dose, _t is the value for the tap water used in the dilution, _s is the value for the post-dose physiological sample, and _p is the value for the predose physiological sample (5).

Statistics. Mean volumes (± SD) were used, and results were evaluated by regression analysis and Mann-Whitney U-test. A P value <0.05 was considered to indicate statistical significance.

	RESULTS

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The average coefficient of variation within subjects was 0.49% for EQ and 0.79% for CR, indicating a high reproducibility with both techniques. Corresponding precisions were 0.98 and 1.58%, respectively.

Variabilities between calculated and measured weights. The highest variability when using EQ was measured at the lower volume range (Table 1, Fig. 1A). The variability when using CR was somewhat greater (Table 1, Fig. 1B). The differences between EQ and CR were, however, not statistically significant.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Differences between calculated and measured volumes, expressed in %, for various volume sizes. Individual values are presented using equilibration (EQ; A) and chromium reduction (CR; B).

Correlation between calculated and measured weights. The relationship between calculated and measured volumes, using EQ, was highly correlated (R² = 1.0) and could be expressed by the equation V_calculated = 0.433 + 1.004 × V_measured (r = 1.0). The same relationship, using CR, was described according to the expression V_calculated = 37.31 +1.01 × V_measured (r = 1.0), where V is volume. Regression lines for EQ and CR are presented in Fig. 2A and 2B.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Relationships between measured and calculated fluid volumes obtained by EQ (A) and CR (B) techniques are shown. The regression equation for EQ determinations was Vcalculated = 0.433 + 1.004 × Vmeasured (r = 1.0) and, for CR analysis, Vcalculated = 37.31 +1.01 × Vmeasured (r = 1.0), where V is volume.

	DISCUSSION

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The most important findings in this study were that GIRMS, using deuterium as a tracer, resulted in precise fluid volume determinations of small as well as large fluid spaces and that the reproducibility was 0.49% (mean) when using EQ and 0.79% when using CR. In addition, the CR technique requires samples of only 2 µl compared with the EQ technique in which 5 ml are needed. This opens up possibilities for the use of CR in neonates not only for single samples but also for serial tests and invites a more frequent clinical use of this technique whenever accurate determinations of body fluid volumes are needed.

The difference in reproducibility between EQ and CR techniques was small. It can therefore be argued that the techniques are interchangeable. The intercept, from regression analysis of 37 when using the CR technique compared with 0.43 using the EQ technique, may result in an unacceptable deviation between measured and calculated volumes when using CR. Thus the error caused by the intercept is insignificant for large volumes, which means that deviations from measured volumes are almost identical with the slope of the regression lines, i.e., 1.004 using EQ and 1.01 using the CR technique, which means a mean variability of 0.4% in the adult using EQ and of 1.0% using CR. On the basis of these results, the two techniques, EQ and CR, are both acceptable and fulfill high demands on precision and reproducibility in large fluid volumes. Even for small volumes of 1,000 ml, the CR technique results in a deviation of 3%, which is acceptable.

GIRMS is a technically advanced method, expensive to purchase, maintain, and run. Also, it requires a specially trained operator. It can be questioned whether this currently is an apparatus for clinical use. Alternatives may be more versatile, rapid, portable, and easy to use bedside. Bioimpedance (BIA) is one alternative. BIA measurements of TBW have between-measurement variations of 2-4 liters in an adult patient, i.e., a variability between 5 and 10% at a volume of 40 liters (6). BIA can therefore only detect relatively large intraindividual changes and may be best suited for quantification of group changes. Another alternative method is infrared spectrophotometric determinations of deuterium. This approach, however, is not as precise as GIRMS, with a mean precision of 2.5% using repeated measurements of the same sample (7). The stable isotope, ¹⁸O, could also be used for determinations of body fluid spaces. It has been shown that the deuterium dilution space is ~2% larger than that with ¹⁸O in adult patients and ~3% larger in premature infants (9). This is probably because deuterium participates in the nonaqueous exchange to a greater extent than does ¹⁸O (2). This is explained by the fact that the water-to-protein ratio is on the order of 3:1 in adults and 5:1 in neonates (3). Thus there are advantages with use of ¹⁸O determinations of body fluid volumes. These measurements of ¹⁸O require the same GIRMS technique as with deuterium, but ¹⁸O is more expensive. ¹⁸O is, however, an excellent alternative to deuterium for body fluid measurements. The difference between ¹⁸O and deuterium measurements is systematic, and deuterium is often the tracer used. In this in vitro study using deuterium and fluids that did not contain any proteins, there was a low variability of <1% and a good precision when the clinically more versatile CR technique was applied. Taken together, the deuterium approach for body fluid volume determinations is acceptable for studies in humans.

It was interesting to note that deviations from measured volumes were in most cases positive, indicating that the scale may underestimate volumes. Also the variability was somewhat greater when using CR compared with EQ. The differences were small, however, and most probably of minor clinical importance although all EQ determinations of small volumes were positive, indicating a systematic deviation that should be elucidated in future studies. Reduction of water by the use of chromium instead of zinc, uranium, and perhaps also manganese is advantageous because the other reduction reagents are known to give a larger variability (4). This is thought to be due to a less precise determination when analyzed water contains impurities, which always occur in biological samples. Hence, the CR technique seems to be acceptable for measurements of TBW in adult humans; it requires smaller sample volumes (2 µl) compared with EQ (5 ml), is faster, and may not be disturbed by fluid samples such as blood, plasma, urine, and spinal fluid that contain organic material (10).

In conclusion, both small and large fluid volume determinations using GIRMS with deuterium as a tracer were reproducible with low coefficients of variation; they are also precise and accurate. CR of water seems to be acceptable for clinical use compared with other reductive metals. The EQ technique requires more time and larger sample volumes. The potential for the use of stable isotopes such as deuterium and ¹⁸O as tracers in future studies of neonates and adults, focused on various body water spaces, body composition, and energy expenditure with relation to anesthesia, surgery, and intensive care, is promising.