247
Human Cholesterol Synthesis Measurement
Using Deuterated Water
Theoretical and Procedural Considerations
P.J.H. Jones, C.A. Leitch, Z.-C. Li, and W.E. Connor
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Human cholesterogenesis is measurable as the rate of Incorporation of deuterium derived from deuterium
oxide (D2O) within the body water pool into plasma or erythrocyte cholesterol pools. Oral D2O equilibrates
across body water, thus enabling extracellular sampling of pools (such as urine) to serve as accurate
indicators of intracellular deuterium enrichments at the point of synthesis. Required doses of D2O fall
below the threshold associated with negative side effects. Deuterium/carbon incorporation ratios into
cholesterol during biosynthesis have been established that are applicable in humans. Models using
unconstrained and constrained curve fitting permit improved flexibility in interpretation of deuteriumuptake kinetics. However, sample-size restrictions presently limit the ability of the technique to examine
the kinetics within individual lipoprotein species. Correction of enrichment data for proton exchange
during the combustion and reduction phases of sample preparation is an additional important procedural
concern. In summary, the deuterated-water procedure is a useful tool in studies of human cholesterol
synthesis that offers the advantages of short measurement interval, relative noninvasiveness, and
provision of a direct index of synthesis in comparison with other available techniques. (Arteriosclerosis and
Thrombosis 1993;13:247-253)
KEYWORDS • cholesterol • deuterium • synthesis • methodology • erythrocytes • cholesterol
ester • humans
C
holesterol synthesis contributes significantly to
total body input in humans1 and thus exists as
an important factor in determining body cholesterol balance. Cholesterol formation rates in humans
are measurable using several techniques. These techniques include input-output analysis,2'3 kinetic approaches using labeled cholesterol,4-3 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity
measured directly in the liver6 or indirectly in skin
fibroblasts7 and mononuclear leukocytes,8 and plasma
sterol precursor-level quantification.910 Steady-state requirements vary across methods. With input-output
analysis or kinetic approaches, cholesterol pool steadystate conditions must exist for the measurements to be
valid. With other methods, cholesterogenesis can be
measured without steady-state cholesterol pools. Moreover, the time window of measurement varies from 7
days or more for input-output analysis to a few hours
for sterol precursors. Although wide-ranging, such
methods are limited in three ways: they either are
accurate but require extended measurement periods,
are immediate but overly invasive, or measure synthesis
indirectly.
From the Division of Human Nutrition (PJ.HJ., C.A.L., Z.-C.L),
University of British Columbia, Vancouver, Canada, and the Division
of Endocrinology, Metabolism and Clinical Nutrition (W.E.C.), The
Oregon Health Sciences University, Portland.
Supported by a grant from the British Columbia and Yukon
Heart and Stroke Foundation.
Address for reprints: Dr. Peter J.H. Jones, Division of Human
Nutrition, 2205 East Mall, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4.
Received March 11, 1992; revision accepted November 4, 1992.
In contrast, the deuterium-uptake procedure uses an
intermediate time window of measurement (6-24
hours), is not overly invasive, and measures synthesis
directly. Cholesterogenesis is measured as the increase
in deuterium abundance in circulatory-pool cholesterol
over time as a function of body water deuterium oxide
(D2O) enrichment. The method offers various attractive
features that lend themselves to use in in vivo human
studies. The goal of the present review was to examine
various theoretical issues related to the method and to
present experimental results concerning related
assumptions.
History of Deuterium's Use in
Cholesterogenesis Measurement
The use of deuterium as a tracer to measure lipogenesis was pioneered by Rittenberg and Schoenheimer in
1937.n Mice maintained at constant body water D2O
enrichment incorporated deuterium label into lipids to
a plateau of approximately one half that of body water.
Later, in humans, London and Schwartz12 showed a
parallel rise in plasma and erythrocyte cholesterol deuterium enrichment over several weeks in two subjects
given regular doses of D2O. This study was the first
reported use of a stable-isotope tracer in the study of
human lipid metabolism; at that time, radiolabeled
nuclides were tracers of choice in studying cholesterogenesis.13 Taylor et aJ14 later refined the deuteriumuptake technique; as a result of improved precision of
measurement using isotope ratio mass spectrometry,
they were able to reduce the D2O dose required.
Nevertheless, sensitivity limitations of this early instru-
248
Arteriosclerosis and Thrombosis
Vol 13, No 2 February 1993
mentation necessitated doses of D2O large enough to
result in vertigo in some subjects. More recently, improvements in isotope ratio mass spectrometry have
enabled measurement of deuterium enrichment
changes in cholesterol over comparatively short intervals using D2O dosages that, almost without exception,
produce no side effects.15 In addition, ongoing improvements in our understanding of human cholesterol turnover have enabled refinements in modeling and
interpretation.
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Theoretical Considerations
Water as a Tracer
The deuterium-uptake method shares many of its
theoretical underpinnings with a similar technique that
uses tritiated water (3H2O) developed for in vitro16 and
in vivo17 application in animals. In both approaches,
tracer water equilibrates between the precise intracellular site of synthesis and the extracellular physiological
fluid, such as urine or plasma, that is used to determine
the level of labeling. A primary advantage of either
method is that the tracer that is taken up into de novo
synthesized sterol originates from a pool of known
enrichment. Potential errors arising from differential
labeling of intracellular and extracellular precursor enrichments, such as occur with the use of labeled metabolites other than water, are thus obviated. For instance,
incorporation rates of acetyl CoA units into identical rat
tissue slices differ when the incubation contains 14Clabeled glucose, acetate, or octanoate. Such differences
reflect not synthetic rate changes, but variations in the
amount of dilution of the labeled acetyl CoA pool
derived from each metabolite. Certain of these problems have been overcome with the use of modern
xenobiotic-probe approaches.18 Thus, body water serves
as an ideal precursor pool for whole-body studies of
lipogenesis.
Although water equilibrates across body pools, intercompartment transmission is not instantaneous. In animals, the interval required for plasma water to reach
the plateau tracer concentration after intravenous injection of a bolus of labeled water is approximately 30
minutes.17'19 Previous data in human subjects have
shown that equilibration of plasma water deuterium
enrichment after a single oral dose of D2O occurs
between 3-6 hours.20 Data from a similar experiment
using earlier time points are presented in Figure 1. Two
fasted subjects drank a loading dose of D2O at time
zero, after which saliva and urine samples were collected at hourly intervals for 4 hours. Values expressed
as percent plateau enrichment show equilibration occurring by 1-2 hours after dosing. Changing enrichment
during the first postlabeling hour would produce a
measurement error in experiments of very short duration; however, for experiments of even a few hours, the
relatively short equilibration interval would be expected
to result in a minimal error. To avoid the problem of
equilibration of the precursor, the baseline cholesterolenrichment sample could be obtained 1-2 hours after as
opposed to before administration of the D2O dose.
An additional advantage of the deuterium-uptake
method is the absence of cholesterol proton exchange
with aqueous proton pools. When commercial free
cholesterol at natural abundance was incubated with
1
2
Time (hr)
Urine
3
• Saliva
FIGURE 1. Line graph shows equilibration of urinary (m
and solid line) and salivary (n and dashed line) water
deuterium enrichment over time before and after a single dose
ofdeuterated water (D2O) in two subjects. Plotted is the mean
percent deuterium enrichment in each pool relative to the
mean of 1-4-hour time points in subjects fasted overnight and
then given 0.06 g/kg body wt D/D followed by a 50-ml rinse of
tap water. Subjects consumed no food or water over the
measurement period and provided hourly samples for 4 hours
after dosing.
3
H2O19 or with 99.8% D2O and shaken at 37°C overnight
(P.J.H. Jones, unpublished data), incorporation into the
isolated sterol was nil. Although it cannot be ruled out
that deuterium exchange does not occur under any
physiological situation, data strongly indicate that deuterium uptake must represent synthetic activity.
Fractional Uptake Rate for Label
To accurately measure net cholesterogenesis using
D2O uptake, the ratio of incorporation of deuterium
versus protium into cholesterol during biosynthesis must
be established, as has been determined for fatty acids.21
Not all of the various precursors along the cholesterol
biosynthetic pathway incorporate the label at a level
proportional to that of body water. Thus, knowledge of
the fraction of the 46 protons incorporated per de novo
synthesized cholesterol molecule that originates from
labeled precursors is needed. This area has been thoroughly considered for the 3H2O method.17 In brief,
hydrogens bonded to the growing cholesterol molecule
are derived from three sources. Seven are obtained
directly from body water, reflecting the exact tracer
concentration therein. Fifteen originate from reduced
nicotinamide adenine dinucleotide phosphate (NADPH)
during the synthetic cascade, and the remaining 24 derive
from cytosolic acetyl CoA. Previous studies comparing
uptake rates from 3H2O and 14C-acetate have shown that
protons derived directly from H2O and NADPH equilibrate with body water.16 In contrast, those from acetyl
CoA are unlabeled, unless the period of measurement is
sufficiently prolonged to permit recycling of labeled
acetate into acetyl CoA pools. Assuming negligible acetate recycling, the ratio of 0.81 labeled protons per
carbon atom is accepted as most representative of synthesis.17 This value corresponds to 22 deuterium atoms
per 27 carbon atoms within the cholesterol molecule.
Correcting for the ratio of hydrogen to carbon atoms in
cholesterol, the fraction of deuterium incorporated at the
Jones et al D2O in Cholesterol Synthesis Measurement
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level of whole-body enrichment per total hydrogen atoms
(deuterium/total hydrogen) is 0.478.
Potential limitations exist in the estimation of deuterium/carbon when using either 3H2O or D2O. While the
theoretical assumptions concerning the enrichment of
protons from water and acetate are reasonably well
defined, there is less certainty regarding those derived
from NADPH. Protons used in cholesterol biosynthesis
from NADPH originate from two sources. The first is
the pentose phosphate pathway, which does not exchange protons with cellular water and thus does not
contribute to enrichment of sterol during synthesis. The
second, the malic enzyme system, may result in exchange of NADPH with water protons at cellular concentrations.17 A number of in vivo and in vitro studies
using tritium suggest small but potentially significant
variations in the deuterium/carbon ratio, suggesting
significant contributions of pentose phosphate pathwayderived NADPH or some other change in NADPH
equilibration.22-25 A range from 21 to 25 3H atoms per
27 carbon atoms within the cholesterol molecule has
been reported, which translates into potential errors of
about 10% in apparent measures of cholesterogenesis
using labeled water. Although some studies have shown
incorporation ratios to be consistent across changing
metabolic conditions and acetyl CoA substrate supply,22-23 it has also been suggested that ethanol use as a
substrate results in a reduction in the 3H/carbon ratio.26
Disparity between cholesterol synthesis rates determined by 3H2O and sterol-balance methods previously
reported in guinea pigs fed various dietary fats has been
suggested as being caused by variability in the source of
NADPH protons, secondary to differences in macronutrient oxidation rates.27 Moreover, it cannot be ruled out
that with D2O, deuterium/carbon ratios may differ
across various tissues. Collectively, these findings suggest that the use of deuterium uptake to measure
cholesterol synthesis may be unreliable under certain
conditions that result in shifts in NADPH equilibration.
Modeling Assumptions
In addition to selecting the correct deuterium/carbon
ratio for the system under study, choosing an appropriate model to accurately describe the pattern of change
in cholesterol deuterium enrichment is central to successful data interpretation. The simplest, constrained
model describing the deuterium-uptake rate assumes
that monoexponential enrichment increases to an asymptotic value at the maximum theoretical plasma
cholesterol enrichment, which is represented by the
plasma water enrichment and can be corrected by using
the appropriate deuterium/carbon enrichment ratio
(Figure 2). For cholesterol, this plateau corresponds to
the fractional uptake rate (0.478 x deuterium enrichment of body water). As a consequence of the low
turnover rate relative to pool size, reaching this plateau
requires considerable time. Indeed, in a single subject
given continual deuterium doses, plasma total cholesterol deuterium enrichment continued to increase after
100 days.12
The initial 1-2-day period after deuterium dosing is
considered to yield the most interpretable synthesis data
for several reasons. First, during the immediate postdose interval, movement of labeled cholesterol out of
the rapid-turnover compartment is small; thus, uptake
50
100 150 200
Time (days)
250
249
300
FIGURE 2. Graph of model used to calculate cholesterol
fractional synthesis rates by using constrained and unconstrained monoexponential approaches. Theoretical enrichment plateau is 0.478 that of plasma water (line a). Enrichment curves with identical turnover rates plateauing at 75%
(line b) and 50% (line c) of theoretical value are also shown.
Note that when plateau values below the theoretical level are
achieved, turnover rate represents that of the overall pool, not
only that of cholesterol synthesis.
rates are relatively linear. Considering the extended
time required to reach the asymptote, this linear segment of the curve extends for many hours. In subjects
consuming three meals per day, deuterium uptake measured every 4 hours was, with diurnal exception, relatively linear over 48 hours.28 Second, during the shortterm postdose period, recycling of the label
incorporated into cholesterol from other esters or free
body pools was also small. Accounting for label reentry
requires more complex kinetic analysis. Third, during
earlier postdose periods there is minimal recycling of
the label into acetate pools that can be expected to alter
the deuterium/carbon incorporation ratio.
In contrast, deuterium incorporation kinetics followed over longer periods follow an exponential curve,
as has been demonstrated in pigs29 and humans.12 In
pigs, exponential inflection in deuterium enrichment in
erythrocyte cholesterol is detectable at 48 hours and
pronounced at 96 hours.29 In groups of pigs fed both
cholesterol-free and 0.5% cholesterol-containing diets,
monoexponential fits using the corrected water-enrichment level as the predicted plateau resulted in poor
curve fits for deuterium enrichment. In each case the
asymptote to the plateau value was well below that
predicted by the simple model. Long-term enrichment
data in humans from London and Schwarz12 have also
shown a tendency toward a plateau below the theoretical prediction when modeled with a monoexponential
fit.
There are several possible reasons for the departure
of actual data from the predicted model over periods
exceeding 24-48 hours. First, the longer term deuterium-enrichment curve may not be definable by a single
monoexponential function. Goodman et al30 demonstrated the multiexponential nature of the kinetic behavior of elimination for 14C-cholesterol injected into
humans, reflecting interpool mixing. Second, incoming
dietary and endogenously recycled cholesterol continuously dilutes the rapid-turnover pool. Dietary cholesterol represents a sizable fraction of overall body input.
250
Arteriosclerosis and Thrombosis Vol 13, No 2 February 1993
4 8 12 16 20 24 2 8 3 2 3 6 4 0 4 4 4 8
Time (hr)
- Obxrved Enrichment -
- C o n * r * » d Modal
—
Uncoratr^cwj Modd
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FIGURE 3. Line graph shows application of constrained and
unconstrained models to deuterium enrichment data (authors'
unpublished observations) obtained from six subjects over 48
hours. Subjects consumed prepared diets as three meals per day
and were given 0.7 g-kg estimated body water'1 99.8% Dfi at
time zero (7:30 AM). Blood samples were collected before and at
4-hour intervals after the initial dose. Subjects consumed
drinking water containing Dfi over the 48-hour blood-sampling period to maintain body-water enrichment at the plateau
level. Plasma free-cholesterol enrichment was determined as
described.28 Monoexponential function fitting was performed
with the SYSTAT curve-fitting program (SYSTAT, Evanston,
III). Shown are mean observed enrichment values expressed as
percent body-water enrichment and best-fit curves for each
model
Recycled cholesterol also contributes significantly to
body input, as is suggested from the observation of
negative deuterium-uptake rates in subjects during
short-term fasting.28-31-32 Negative uptake rates reflect
entry of unlabeled cholesterol to a level that exceeds
that of labeled, synthesized sterol. In a case in which
dietary and recycled sterol enter the central pool at an
identical rate as de novo synthesized cholesterol, the
deuterium enrichment of plasma cholesterol would
reach one half the predicted plateau value (Figure 2,
line c). Last, as previously mentioned, the deuterium/
carbon incorporation ratio is altered as the label accrues
within body metabolites over time. For these reasons
interpretation of cholesterol deuterium-uptake data
over longer periods is more complex than during the
relatively linear immediate postdose period.
For situations in which the theoretical plateau level is
achieved, the deuterium incorporation rate is equal to
the synthesis rate because there is no diluting source of
unlabeled cholesterol. In cases in which true enrichment plateaus below theoretical predictions are
achieved, the turnover rate for cholesterol, as determined from the rate of deuterium incorporation, reflects turnover of the total free-cholesterol pool, not
only that of de novo labeled sterol. In such situations
knowledge of the plateau value as a fraction of the
theoretical maximum may be useful in determining the
proportion of total pool turnover that can be attributed
to de novo synthesis. The plateau value can be determined by extrapolating early time points or, preferably,
by experiments of longer duration. The derived turnover
rate can be corrected for the amount of unlabeled sterol
entering the pool (Figure 3). Here, six healthy male
subjects fed liquid diets as three meals per day consumed a loading dose of D2O followed by maintenance
doses for 48 hours. Plasma free cholesterol deuterium
enrichment was measured every 4 hours. Cholesterol
deuterium-uptake curves were modeled using constrained curve fitting, in which plateau enrichment was
assumed to be 0.478 that of plasma water, and using
unconstrained curve fitting, in which the curve was
extrapolated to define its own plateau. With the constrained model, fractional synthetic rates (FSRs) were
calculated as 0.075±0.005 pool • day"1. With the unconstrained model, the mean plateau value obtained
through extrapolation was 67.8±3.6% of the theoretical
plateau. FSR values obtained directly from the curve fit
averaged 0.1191±0.011 pool-day" 1 . With the unconstrained model, the cholesterol synthesis rate, calculated as the product of the fraction of pool turnover
attributable to synthesis and the turnover rate, was
0.0807 pool • day"1. Assuming a rapid-turnover pool size
of approximately 25 g, one half of which is esterified and
therefore turning over more slowly, net cholesterol
synthesis rates were 0.94 and 1.00 g • day"1 for constrained and unconstrained approaches, respectively.
The major disadvantage of the unconstrained model is
its inaccuracy in predicting the plateau value from early
time points. Optimally, derivation of the turnover rate
from early time points and the plateau value from
additional samples collected after deuterium dosing
provides true synthetic FSR.
Even over durations of <24 hours, significant movement of label occurs into the esterified cholesterol
pool.28 Such loss of label from the primary pool of
measurement will result in some underestimation of
synthetic rate with either the constrained or unconstrained approaches. With appropriate modeling,fluxof
the label into the ester pool should permit calculation of
the esterification rate.
Procedural Considerations
Sample extraction, purification, and combustion/reduction procedures associated with the deuterium-uptake
methodology have been previously described.28-29-31-32
Once extracted from tissue, cholesterol may be purified by
thin-layer chromatography28-31-32 or high-pressure liquid
chromatography.29 Combustion and reduction steps are
typically performed manually, off-line from the mass spectrometer. Regardless of the methodology used, the analysis is multistage, thus presenting numerous opportunities
for sample proton contamination. Chief contamination
sources are entry into the preparation system of unlabeled
protons from solvent impurities during extraction and
from water vapor during distillation. To minimize contamination artifacts, samples are analyzed in replicate. Given
a typical intrareplicate coefficient of variation of 2-4%o,
the minimal detectable difference in FSR is 0.003
pool • day"1, assuming a plasma water deuterium enrichment of 3,500%o.
Sampling Size and Site Considerations
Both sample size and site limitations exist as constraints to studies of the deuterium-uptake method in
humans, in contrast with animal experimentation. For
humans, relatively large initial plasma volumes are
required for cholesterol deuterium-enrichment analyses. In nonnolipidemic subjects, 2 ml plasma yields
Jones et al
200
12
18
Time (hr)
- Ptoonw
24
D2O in Cholesterol Synthesis Measurement
251
synthesis should thus be made with caution over the
initial postdose period.
Within the plasma compartment, although deuterium
uptake occurs into both free and ester pools, incorporation into the total free compartment exceeds that into
ester by twofold.28 The more rapid uptake into free
sterol indicates its role as the primary compartment to
receive newly synthesized sterol. Of the available options, free plasma cholesterol is thus likely to be most
sensitive to perturbations in cholesterogenesis. For instance, diurnal rhythmicity observed in the free-cholesterol compartment28 has not been reported in either the
erythrocyte or ester compartment.
- Ejythrocyta
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FIGURE 4. Plasma free (m and solid line) and erythrocyte (a
and dashed line) cholesterol deuterium enrichment (%o) over
baseline (hour zero) and at 6, 12, and 24 hours after loading
dose (0.7g/kg estimated body water) and maintenance doses of
DjO. Isotopic enrichments over baseline are expressed as per
mil (%c) versus standard mean ocean water (SMOW), where
per mil is defined as [(R^vit/Rau^mi)-l]xl,000,
where R is
the ratio of heavy-to-light isotope and R^doi is SMOW. Data
are mean±SD of six subjects.
approximately 1 mg free cholesterol, the amount required per replicate to produce the 1 )i\ combustion
water necessary for isotope ratio mass spectrometric
analysis. The blood volume requirement can be reduced
if free and esterified cholesterol are combined and
considered as a unified pool. Alternatively, erythrocyte
cholesterol, found solely in free form, enables use of
smaller blood samples. Sample-size limitations have
hampered studies in infants and of plasma lipoprotein
subfractions. At present, plasma free,14'28-31-32 esterified,28 and erythrocyte29 cholesterol deuterium uptakes
have been investigated.
Circulatory cholesterol is selected for sampling since
it is both readily accessible and is a part of the rapidturnover pool that also comprises hepatic and intestinal
sterol.33 The free-cholesterol component of this pool
equilibrates between plasma and other subpools12-33
with a turnover rate between the liver and plasma of
0.34 pool-hr"1 in the rat.34 Erythrocytes do not synthesize cholesterol'2; thus, sterol in this subcompartment is
obtained from the plasma. Erythrocyte cholesterol exists in the free form; however, the rate of equilibration
between the plasma and erythrocytes is unknown. To
examine this question six subjects consumed fixed diets
for 4 weeks. During the final week subjects drank
priming (0.7 g D2O/kg estimated body water) and
maintenance doses of D2O over 24 hours. Plasma was
obtained before the priming dose and at 6, 12, and 24
hours thereafter. Plasma free and erythrocyte cholesterol deuterium enrichments were determined at these
time points. Results (Figure 4) indicate that deuterium
incorporation was initially slower into erythrocytes compared with plasma due to interpool mixing, probably
resulting from a delay in equilibration between the two
compartments. These findings indicate that over the
initial 6-12-hour postdose interval, use of erythrocyte
deuterium uptake results in an underestimation of
sterol synthesis into the rapidly exchangeable pool. Use
of erythrocyte deuterium enrichments to assess sterol
Selection of Combustion/Reduction Tube Material
Combustion and reduction procedures for converting
sterol to hydrogen gas for mass spectrometric analysis
have used quartz28'29 and Pyrex glass tubes.31-32 Pyrex
tubing is less costly and more readily available, although
combustion temperatures for Pyrex are of necessity
lower than those with quartz. Regardless of the tubing
material selected, during both combustion and reduction stages cholesterol-derived protons exchange with
the inner surface of the tube, resulting in isotopic
dilution. The amount of exchange varies, depending on
variations in the amount and isotopic composition of
exchangeable hydrogen present on the inner wall of the
tube.
Representative effects of deuterium exchange during
the combustion process are shown in Figure 5. Replicate 1-mg lipid samples were prepared with copper
oxide and silver wire in preannealed 20-cm x 5-mm
quartz tubes and combusted at 650°C for 4 hours. Tubes
were of fixed length to equalize the interior surface
area. The exchange associated with the use of quartz
tubing resulted in a 12% offset in enrichment relative to
theoretical predictions (Figure 5A).
In a second experiment, replicate cholesterol samples
of different deuterium enrichments were prepared in
fixed lengths of both preannealed quartz and glass
tubing and combusted at 650°C and 510°C, respectively,
for 4 hours. An 18% offset in enrichment of samples
combusted in glass relative to theoretical predictions
(Figure 5B) was observed. These offsets appeared to be
linear over the range of working standards in the range
of the sample enrichments.
Reduction procedures provide an additional opportunity for exchange of protons and attendant dilution of
enrichment. To examine the extent of exchange, 2-fi\
samples of four representative water standards differing
in deuterium isotopic enrichment were prepared in
12-cm x 5-mm glass or quartz preannealed tubes cut to
standardized lengths. Tubes were reduced at 510°C for
30 minutes. Results (Table 1) showed a reduction tube
effect on the water-standard enrichments. Regression
analysis demonstrated linearity across the four standards with both glass and quartz tubes. However, data
for each tube type fit different lines, necessitating the
use of separate regression equations to produce calibration curves for the two tube types. Once calibrated,
water samples prepared in either tube type yielded
comparable enrichments. In sets of water samples compared by using each tube type with standards analyzed
in the tubing of the appropriate material, the average
difference in enrichment was <3%.
252
Arteriosclerosis and Thrombosis
Vol 13, No 2 February 1993
1500
70O
-100
100
300
500
Actual Enrichment (o/oo)
700
-300
0
300 600 900 1200 1500
Quartz-based Enrichment (o/oo)
FIGURE 5. Line graphs show effect of combustion-tube composition on observed net enrichment measurements. Panel A shows
theoretical (u and solid line) versus observed (a and dashed line) deuterium enrichments for a [U-D]palmitate dilution series when
combusted in quartz tubing. Panel B shows cholesterol samples of differing deuterium enrichments when combusted in quartz (m
and solid line) and Pyrex (a and dashed line) tubes. Isotopic enrichments are expressed as net enrichments per mil (%o) versus
standard mean ocean water.
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At both combustion and reduction stages of deuterium-enrichment analysis, proton exchange between the
sample and the tubing surface may result in underestimation of cholesterol deuterium enrichment. To address this problem, such dilution must be corrected for
at each stage by using standards of known enrichment.
Conclusion
A need exists for methods that are capable of direct,
short-term measurement of human de novo cholesterol
synthesis. D2O offers certain advantages over other
available methods. Deuterium/carbon incorporation
factors have been established; however, whether and to
what degree such factors are alterable by changes in
metabolic conditions within the organism remain to be
fully defined. The theoretical cholesterol deuteriumenrichment plateau is considered to be about one half
that of the body-water enrichment. Available data show
that a time period of several months is required for
cholesterol enrichment to reach its plateau value. Because of the increased complexity of modeling assumpTABLE 1. Comparison of Glass and Quartz Reduction-Tube
Material on Observed Versus Theoretical Deuterium
Enrichments of FOOT Water Standards
Sample
GISP
CTWS
SMOW
SDHS
Tubing type
Glass
Quartz
Glass
Quartz
Glass
Quartz
Glass
Quartz
Mean
enrichment
(%»)*
-192.0±2.8
-195.0±3.3
-44.2±4.1
-50.3±3.9
-1.5±3.9
5.7±2.2
393.1 ±6.3
394.5 ±10.8
n
7
8
8
6
8
6
6
6
Theoretical
enrichment
(%»)
-189.0
-49.0
0.0
394.0
Data are mean±SD.
GISP, Greenland Island standard precipitate; CTWS, Chicago
tap water standard; SMOW, standard mean ocean water; SDHS,
stock dilution heavy standard.
'Isotopic enrichments are expressed as per mil (%o) versus
SMOW, where per mil is defined as [(R« ? pt/R mn d^)-l]x 1,000,
where R is the ratio of heavy-to-light isotope and R,,,,^ is
SMOW.
tions over longer periods, the initial 6-24-hour interval
of deuterium uptake is considered to be a relatively
linear portion of the overall incorporation curve, representing the optimal period for measuring synthesis and
turnover. Using periods shorter than 6 hours will result
in artifacts, because of both the time lag required for
equilibration of D2O across the body-water compartment and the mixing of deuterated cholesterol within
erythrocytes and plasma cholesterol pools. Extending
the period beyond 24 hours leads to more complex
curve fitting as labeled sterol mixes among various
pools, as well as allowing the precursor incorporation
ratio to increase as acetate begins to accrue the label.
However, over periods of longer duration, cross-compartmental mixing and entry into pools of exogenous
and recycled cholesterol result in enrichment curves
that tend toward a lower than predicted plateau value.
Thus, cholesterol turnover rates determined by using
deuterium uptake require correction for flux of unlabeled sterol through the cholesterol pool. Such correction can be obtained from the value of the final plateau
relative to the predicted value. More advanced models
will be needed to better delineate the kinetics of
cholesterol synthesis using D2O in vivo. In summary,
although in certain circumstances not all related assumptions have been proven, D2O uptake is a potentially useful tool in studies of human cholesterol synthesis, provided that theoretical and practical issues are
considered.
Acknowledgments
The excellent technical assistance of Gayle Wickens and
Qement Fung is acknowledged. The superior assistance of
Lauren Hatcher and the staff of the Clinical Research Center,
Oregon Health Sciences University, is also appreciated.
References
1. Dietschy JM: Regulation of cholesterol metabolism in man and
other species. Klin Wochenschr 1984;62:338-345
2. Samuel P, lieberman S, Ahrens EH: Comparison of cholesterol
turnover by sterol balance and input-output analysis, and a shortened way to estimate total exchangeable mass of cholesterol by the
combination of the two methods. J Upid Res 1978;19:94-102
3. Grundy SM, Ahrens EH, Davignon J: The interaction of cholesterol absorption and cholesterol synthesis in man. J Upid Ra
1969;10:3O4-315
Jones et al
Downloaded from http://atvb.ahajournals.org/ by guest on June 15, 2017
4. Goodman DS, Noble RP: Turnover of plasma cholesterol in man.
J Clin Invest 1968;47:231-241
5. Ferezou J, Rautureau J, Coste T, Gouffier E, Chevalier F: Cholesterol turnover in human plasma lipoproteins: Studies with stable
and radioactive isotopes. Am J Clin Nutr 1983;36:235-244
6. Bjorkhem I, Miettinen T, Reihner E, Ewerth S, Angelin B, Einarsson K: Correlation between serum levels of some cholesterol
precursors and activity of HMG CoA reductase in human liver.
J Lipid Res 1987;28:1137-1143
7. Chait A, Bierman EL, Albers JJ: Low-density lipoprotein receptor
activity in cultured human skin fibroblasts. J Clin Invest 1979;64:
1309-1319
8. Nguyen L, Salen G, Shefer S, Tint GS, Shore V, Ness GC:
Decreased cholesterol biosynthesis in sitosterolemia with xanthomatosis: Diminished mononuclear leukocyte 3-hydroxy-3methylglutaryl coenzyme A reductase activity and enzyme protein
associated with increased low density lipoprotein receptor function. Metabolism 1990^9:436-443
9. Popjack G, Boehm G, Parker TS, Edmond J, Ewards PA, Fogelman AM: Determination of mevalonate in blood plasma in man
and rat: Mevalonate "tolerance" tests in man. J Lipid Res 1979;20:
716-728
10. Parker TS, McNamara DJ, Brown CD, Kolb R, Ahren EH, Alberts
AW, Tobert J, Chen J, Schepper PJD: Plasma mevalonate as a
measure of cholesterol synthesis in man. / Clin Invest 1982;74:
3037-3041
11. Rittenberg D, Schoenheimer R: Deuterium as an indicator in the
study of intermediary metabolism: XI. Further studies on the
biological uptake of deuterium into organic substances with special
reference to fat and cholesterol formation. / Biol Chan 1937;121:
235-253
12. London IM, Schwartz H: Erythrocyte cholesterol: The metabolic
behavior of the cholesterol of human erythrocytes. / Clin Invest
1953^2:1248-1252
13. Gould RG, LeRoy GV, Okita GT, Kabara JJ, Keegan P, Bergenstal DM: The use of Cl4-labeled acetate to study cholesterol metabolism in man. J Lab Clin Med 1955;46:372-384
14. Taylor CB, Mikkelson B, Anderson JA, Forman DT: Human
serum cholesterol synthesis measured with the deuterium label.
Arch Pathol 1966;81:213-231
15. Jones PJH, Leatherdale ST: Stable isotopes in clinical research:
Safety reaffirmed. Clin Sa 1991;80:277-280
16. Lakshmanan MR, Veech RL: Measurement of the rate of rat liver
sterol synthesis in vivo using tritiated water. J Biol Chem 1977;252:
4667-4673
17. Dietschy JM, Spady DK: Measurement of rates of cholesterol
synthesis using tritiated water. J Lipid Res 1984;25:1469-1476
18. Hellerstein MK, Christiansen M, Kaempfer S, Kletke C, Wu C,
Reid JS, Mulligan K, Hellerstein NS, Shackleton CHL: Measure-
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
D2O in Cholesterol Synthesis Measurement
253
ment of de novo lipogenesis in humans using stable isotopes. J Clin
Invest 1991;87:1841-1852
Jeske DJ, Dietschy JM: Regulation of rates of cholesterol synthesis
in vivo in the liver and carcass of the rat measured using ['HJwater.
J Lipid Res 198O;21:364-376
Schoeller DA, Dietz W, van Santen E, Klein PD: Validation of
saliva sampling for total body water determination by H2I8O dilution. Am J Clin Nutr 1982;35:591-594
Jungas RL: Fatty acid synthesis in adipose tissue incubated in
tritiated water. Biochemistry 1968;7:3708-3717
Andersen JM, Dietschy JM: Absolute rates of cholesterol synthesis
in extrahepatic tissues measured with 3H-labeled water and 14Clabeled Substrates. J Lipid Res 1979;20:740-752
Dietschy JM, McGarry JD: Limitations of acetate as a substrate for
measuring cholesterol synthesis in liver. J Biol Chem 1974;249:
52-58
Loud AV, Bucher NLR: The turnover of squalene in relation to
the biosynthesis of cholesterol. J Biol Chem 1957;233:37-41
Pullinger CR, Gibbons GF: The relationship between the rate of
hepatic sterol synthesis and the incorporation of [3H]water. J Lipid
Res 1983;24:1321-1328
Selmer J, Grunnet N: Ethanol metabolism and lipid synthesis by
isolated liver cells from fed rats. Biochim Biophys Ada 1976;428:
123-147
Fernandez ML, Yount NY, McNamara DJ: Whole body and
hepatic cholesterol synthesis rates in the guinea pig: Effect of
dietary fat quality. Biochim Biophys Ada 1990; 1044:340-348
Jones PJH, Schoeller DA: Evidence for diurnal periodicity in
human cholesterol synthesis. J Lipid Res 1990;31:667-673
Wong WW, Hachey DL, Feste A, Leggitt J, Clarke LL, Pond WG,
Klein PD: Measurement of in vivo cholesterol synthesis from ^ O :
A rapid procedure for the isolation, combustion, and isotopic assay
of erythrocyte cholesterol. / Lipid Res 1991^32:1049-1056
Goodman DS, Smith FR, Seplowitz AH, Ramakrishnan R, Dell
RB: Prediction of the parameters of whole body cholesterol metabolism in humans. J Lipid Res 1980;21:699-713
Roe RP, Jones PJH, Frohlich JJ, Schoeller DA: Association
between apolipoprotein E phenotype and endogenous cholesterol
synthesis as measured by deuterium uptake. Cardiovasc Ra 1991;
25:249-255
Jones PJH, Dendy SM, Frohlich JJ, Leitch CA, Schoeller DA:
Cholesterol and triglyceride fatty acid synthesis in apolipoprotein
E2-associated hyperlipidemia. Arteriosclerosis 1992;12:106-113
Norum KR, Berg T, Helgerud P, Drevon CA: Transport of cholesterol. Physiol Rev 1983;63:1343-1419
Magot T, Frein Y, Champarnaud G, Cheruy A, Lutton C: Origin
and fate of rat plasma cholesterol in vivo: Modelling of cholesterol
movements between plasma and organs. Biochim Biophys Ada
1987;921:587-597
Downloaded from http://atvb.ahajournals.org/ by guest on June 15, 2017
Human cholesterol synthesis measurement using deuterated water. Theoretical and
procedural considerations.
P J Jones, C A Leitch, Z C Li and W E Connor
Arterioscler Thromb Vasc Biol. 1993;13:247-253
doi: 10.1161/01.ATV.13.2.247
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