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3
* School of Dietetics and Human Nutrition, McGill University, Sainte Anne de Bellevue, QC, Canada H9X 3V9;
Lady Davis Institute for Medical Research, Jewish General Hospital, Montréal, QC, Canada H3T 1E2; and
** The Biomedical Mass Spectrometry Unit, McGill University, Montréal, QC, Canada H3A 1A3
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • humans • extracellular water volume • corrected bromide space • sulfate space • body composition • stable isotope
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Bauer 1976
,
Kragelund and Dyrbye 1967
, Lacroix et al. 1965
, Malpartida and Moncloa 1967
, Omvik et al. 1979
, Pierson et al. 1982
, Ryan et al. 1956
, Waki et al. 1991
, Walser et al. 1953
). The tracer is well absorbed and may be administered
orally (Bauer 1976
, Omvik et al. 1979
).
However, the radioactivity and short half-life of 35S limit
its usefulness. We have developed a method to measure serum sulfate
concentrations and 34SO4 enrichments using
electrospray tandem mass spectrometry (ESI-MS/MS). In this paper, we
describe the use of this methodology to measure the SS of normal
humans. This study was designed to select the appropriate dose of orally administered sodium [34S]sulfate to determine the SS and to select the optimum sampling interval. The ECW volume was calculated from extrapolation to time zero of serum 34SO4 concentrations using the natural logarithm (ln) of serum 34SO4 between h 2 and 4 and h 3 and 5 of their 34SO4 decay slopes to identify the optimum sampling interval. We compared the best estimate of the SS with simultaneously measured corrected bromide space (CBS), a common alternative for estimating ECW.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Eight healthy nonobese men with normal blood biochemistries were
studied at the Clinical Research Unit of the Jewish General Hospital of
Montreal (Table 1
).
All gave written consent for the study, which was approved by the
Research Ethics Committee.
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Na234SO4 was purchased from Icon
Services, Mt. Marion, NY (93% 34S) and from Isoflex USA,
San Francisco, CA (99% 34S). Using the methods described
in this paper, the isotope from Icon Services was determined to be 94%
34S. NaBr was purchased from A&C American Chemicals,
Montreal, QC, Canada. All of the water used was Type 1 or ultrapure
water (resistivity of 18.2 M
/cm), purified by treatment with
Milli-RO Plus and Milli-Q UF systems (Millipore, Bedford, MA).
OnGuard-Ag cartridges (#39637) were obtained from Dionex, Oakville, ON,
Canada, MPS micropartition cartridges (YMT 10,000-Da molecular weight
cut-off membrane filter) from Amicon, MA, and 0.22-µm syringe filters
from Chromatographic Specialties, Brockville, ON, Canada. To remove
traces of sulfate before use, the OnGuard-Ag cartridges were rinsed
with 30 mL water and the Amicon filter membranes were soaked in water
for 1 h with three water changes.
Study design.
Subjects were studied at 0700 h in the postabsorptive state. Their
body weight was recorded and total body water (TBW) measured by
bioimpedance analysis (RJL Systems BIA-101A, Mt. Clemens, MI)
(Kushner and Schoeller 1986
).
Experimental protocol.
Each of the first four subjects consumed 50 mg Na234SO4 (0.58–0.78 mg Na234SO4/kg body weight) and 30 mg NaBr/kg body weight dissolved in 160 mL of deionized water. Because this resulted in more than adequate serum enrichments, the tracer sulfate dose was reduced to 0.5 mg Na234SO4/kg body weight for the final four subjects. Blood samples were drawn before and 30, 60, 90, 120, 180, 240 and 300 min after tracer administration. Volunteers refrained from drinking water before and throughout this period. The blood sampling catheter was kept patent by infusing 4.5 g/L NaCl at a rate of 20–24 mL/h. Urine was collected over 5 h.
Analytical methods.
Clotted blood was centrifuged at 1400 x g for 30 min;
the serum was transferred into screw cap vials and stored at -30°C.
Serum sulfate was analyzed by ESI-MS/MS for sulfate concentration and
34SO4/32SO4 enrichment,
as described elsewhere (Boismenu et al. 1998
). The
sensitivity of this method is such that at 2 µmol/L, the 97 and 99
ions are easily detected at signal-to-noise ratios of 100 and 20,
respectively. As a consequence, serum sulfate concentrations <200
µmol/L and tracer/tracee ratios <0.01 can be quantified with
excellent precision. Briefly, to measure
34SO4/32SO4 enrichment,
1.0 mL of serum was mixed with 0.5 mL of water and 5.0 mL of ice-cold
methanol, kept on ice for 10 min, then centrifuged at 1400 x
g for 10 min at 4°C. The supernatant was acidified with
0.1 mL of 1 mol/L HCl to remove bicarbonate anions, then passed through
an OnGuard-Ag cartridge to remove inorganic anions other than sulfate.
The first 3 mL of filtrate were discarded and the rest saved for
analysis. To measure 32SO4 concentration, 0.5
mL of 600 µmol/L Na234SO4 (0.3
µmol) internal standard replaced the 0.5 mL of water above.
ESI-MS/MS analyses were performed using a Quattro II triple quadrupole (Micromass, Manchester, UK) configured for negative ion analysis. Samples were introduced directly into the electrospray probe at 40 µL/min with a 1-mL disposable syringe under the following conditions: neutral loss mode, 17 Da; range, 92–102 Da; cone voltage, 25 V; source temperature, 120°C; sample infusion pump, 40 µL/min; nitrogen bath gas flow rate, 300 L/h; nebulizer gas flow rate, 18 L/h; collision cell energy, 23 eV; and argon pressure in the collision cell, 1.3 x 10-1 Pa. Acquisitions of 2 min each were made in triplicate in multichannel acquisition mode. The following ions were monitored: H32SO4- (m/z97) and H34SO4- (m/z 99).
To measure serum sulfate enrichment due to the administered tracer, a
calibration curve was constructed by preparing varying mole ratios of
34SO4/32SO4, as
described by Tserng and Kalhan (1983)
. The tracer to tracee ratio (TTR)
was calculated by subtracting the baseline mole ratio from the sample
mole ratio as follows:
 |
where M is the signal intensity of m/z 97 and M+2 the signal intensity of m/z 99.
To measure serum 32SO4 concentration, 0.3 µmol of 34SO4 internal standard was added to tubes containing concentrations of Na232SO4 ranging from 0 to 600 µmol/L and an areas ratio standard curve was constructed. Serum 32SO4 concentrations were then determined using this standard curve after subtracting the contribution at mass 99 due to the tracer as determined in a sample to which no internal standard was added. Serum 34SO4 concentration was calculated as the product of sample TTR and 32SO4 concentration.
Bromide was measured by ion exchange high performance chromatography with conductivity detection (IEC-CD) using a Dionex 2110i chromatography system (Dionex, Sunnyvale, CA) under the following conditions: carbonate-bicarbonate buffer mobile phase flow rate, 2.0 mL/min; suppressor regenerant, 25 mmol/L H2SO4; regenerant flow rate, 2 mL/min; background conductivity, 17 µSi; conductivity detector output range, 10 µSi; injection volume, 25 µL; 4 mm AMMS-II anion micromembrane suppressor; ion exchange Ionpac AG4A-SC precolumn; and AS4A-SC analytical column. Peak integration was performed with a Waters 740 Data Module (Milford, MA). Before injection onto the column, serum was diluted 40-fold in water and passed through an MPS micropartition cartridge. Urine was diluted 100-fold and filtered through 0.22-µm syringe filters. Samples were injected into the IEC-CD with a 1-mL disposable syringe (Becton Dickinson, Franklin Lakes, NJ). All samples were analyzed in duplicate.
Calculations.
The SS was calculated as SS = [(dose of
34SO4)/(zero-time serum
34SO4 concentration)] x 0.95 x 0.94,
where SS is the sulfate space in liters, the dose of
34SO4 is expressed in µmol, zero-time serum
34SO4 concentration is in µmol/L, 0.95 is the
correction factor for the Donnan equilibrium and 0.94 is the correction
factor for the water content of serum (Bell et al. 1984
,
Forbes 1987
). Zero-time 34SO4
concentration was calculated by submitting the natural logarithm of
34SO4 concentration to a linear function and
extrapolating to time zero, as described by Bauer (1976)
and Ryan et al. (1956)
, using values either between h 2
and 4 (ln[34SO4]h2–4) or between h 3 and 5
(ln[34SO4]h3–5). Zero-time
34SO4 concentration is the theoretical
34SO4 concentration value that would be
obtained if the 34SO4 dose distributed
instantaneously in the ECW.
The corrected bromide space (CBS) was calculated as described by
Bell et al. (1984)
as follows:
 |
where CBS is in L, Br dose in mmol, Br excreted is the mmol of
Br excreted over 5 h, [serum Br]h 5 is the serum Br
concentration at h 5 in mmol/L, [serum Br]baseline is the
baseline serum Br concentration in mmol/L, 0.90 is the correction
factor for the nonextracellular distribution of Br, 0.95 is the
correction factor for the Donnan equilibrium (Forbes 1987
) and 0.94 is the correction factor for the water content
of serum (Bell et al. 1984
, Forbes 1987
,
Leth and Binder 1970
, Vaisman et al. 1987
).
Statistical analysis.
The paired t test was used to compare SS calculated using
ln[34SO4]h2–4 and
ln[34SO4]h3–5. The F-test was
used to compare the variability in data fits for individual
determinations of SS as well as between-subject variability for SS
determined as ln[34SO4]h2–4 and
ln[34SO4]h3–5. The paired t test
was used to compare SS by ln[34SO4]h3–5 with
CBS. Differences between data were considered significant at
P 
0.05. Results are presented as mean ± SD.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)but there was variability among the subjects. The time courses of
individual serum 34SO4 concentrations are shown
in Figures 2
(higher tracer dose) and
3(lower tracer dose). Results with the lower tracer dose were comparable
to those with the higher one. Figure 1
also shows that average serum
32SO4 concentrations decreased by 18% (P< 0.0001) over the 5-h study period.
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, the
log-linear reduction in 34SO4 concentration was
better described using concentrations at h 3, 4 and 5 than
concentrations at h 2, 3 and 4. Thus the variance for the average
coefficient of determination (a measure of goodness of data fit for a
linear function) based on h 2–4 was 0.1901, but only 0.0002 for values
between h 3 and 5 (P < 0.0001, F-test). In
addition, the use of the later sampling times resulted in a
significantly smaller between-subject SD of the calculated
SS (P = 0.04) and a smaller estimate of its size
(Table 2
).
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,it was consistently less than CBS with all but one value lying to the
right of the line of identity. SS determined in this way was 79.7 ± 5.7% of CBS (P = 0.008).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mean enrichment 5 h after oral administration of Na234SO4 was TTR = 0.027 ± 0.006, well within the sensitivity of the method. Indeed it is likely that a dose one half to one fourth the lowest dose used in this study would provide excellent precision.
Although, on average, 34SO4 enrichment reached
a maximum 2 h after oral tracer administration, there was
variability among the subjects, with some reaching a maximum at h 3
(Figs. 1–3)
. In this respect, our data differ from those of
Bauer (1976)
who obtained the maximum
35SO4 concentration 60–105 min after an oral
dose of radiosulfate. This could be explained by slower
gastrointestinal absorption of a 30- to 50-mg dose of
34SO4 than the much smaller mass (2–3 MBq, or
60–80 µCi) of a dose of radioactive 35SO4.
In our study, serum concentrations that included h 2 were variably
influenced by the tracer absorption phase, whereas those between h 3
and 5 consistently represented the elimination phase of serum
34SO4 (Figs. 2 and 3)
. SS calculated using h
3–5 was associated with a better data fit for individual studies and a
smaller between-subject variance than SS calculated using h 2–4. We
conclude that using data points before h 3 after oral tracer
administration may result in greater variability and the overestimation
of SS.
It is noteworthy that the serum 32SO4 concentration decreased gradually over the course of the SS measurement. This has not been noted in previous studies in which serum radioactivity rather than sulfate was measured, but it is to be expected because sulfate is cleared into the urine and sulfur intake is zero during this measurement. This does not invalidate an isotope dilution technique based on zero-time extrapolation. In fact, if SS is calculated by extrapolation of ln serum TTR rather than 34SO4 concentration, the resulting SS is insignificantly different: 182 ± 46 mL/kg for extrapolation of tracer enrichment vs. 187 ± 47 mL/kg for tracer concentration (P = 0.26).
As shown in Table 3
,the SS determined in this study is similar to values obtained by other
researchers using oral or intravenous radiosulfate. The CBS values
obtained are also similar to ones reported by other researchers for
normal persons. The SS was 79.7 ± 5.7% of CBS, confirming
results obtained by Lacroix et al. (1965)
in men and
women (85 ± 10%), Yu et al. (1996)
in men
(83.9%) and Barratt and Walser (1969)
in rats (80 ± 0.4%) when
SS and CBS were compared directly. The reason for this constant
difference between the SS and the CBS is unknown.
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We conclude that the oral administration of 34SO4 provides a practical alternative for measuring SS, with the advantage of avoiding oral administration of a radioactive tracer. Serum samples are best obtained at h 3, 4 and 5 after tracer ingestion. Our results confirm that SS is 20% smaller than CBS in adults of normal body composition even after standard correction for Br penetration into erythrocytes.
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FOOTNOTES |
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1 Supported by Medical Research Council of Canada
Operating Grant MT 8725 (L.J.H.) and MA-6712 supporting the Biomedical
Mass Spectrometry Unit. P.H. was supported by the University Staff
Development Project of the Ministry of University Affairs, Thailand.
2 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
4 Abbreviations used: CBS, corrected bromide
space; ECW, extracellular water; ESI-MS/MS, electrospray tandem mass
spectrometry; IEC-CD, ion exchange chromatography with conductivity
detection; SS, sulfate space; TBW, total body water; TTR, tracer to
tracee ratio.
Manuscript received July 28, 1998. Initial review completed September 25, 1998. Revision accepted December 8, 1998.
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REFERENCES |
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