Isotopomer spectral analysis of intermediates of cholesterol
synthesis in human subjects and hepatic cells
B.
Lindenthal1,2,
T. A.
Aldaghlas1,
A. L.
Holleran1,
T.
Sudhop2,
H. K.
Berthold2,
K.
von Bergmann2, and
J. K.
Kelleher1
1 Department of Physiology, The George Washington University
School of Medical and Health Sciences, Washington, District of
Columbia 20037; and 2 Department of Clinical Pharmacology,
University of Bonn, 53105 Bonn, Germany
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ABSTRACT |
Steroid intermediates of the
cholesterol synthesis pathway are characterized by rapid turnover rates
relative to cholesterol due to their small pool size. Because the small
pools will label rapidly, these intermediates may provide valuable
information about the incorporation of isotopes in de novo synthesis of
cholesterol and related compounds. The labeling of cholesterol
synthesis intermediates from [1-13C]acetate was
investigated in human subjects and in liver cell models by means of
isotopomer spectral analysis (ISA). In human subjects, infusing
[1-13C]acetate into the duodenum for 12 h
demonstrated that ~50% of the plasma lathosterol pool was derived
from de novo synthesis during this interval. The lipogenic acetyl-CoA
precursor pool enrichment reached a constant value within 3 h of
the start of the infusion. In vitro studies indicated that liver cell
models decrease de novo lathosterol synthesis when cholesterol
synthesis is inhibited by statins or cholesterol-containing serum. We
propose a new calculation to increase the accuracy and precision of
cholesterol synthesis estimates in vivo combining the ISA of
lathosterol and cholesterol.
lathosterol; lanosterol; desmosterol 13C; tracers; theoretical models; experimental models; simvastatin; pravastatin; stable isotopes
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INTRODUCTION |
LANOSTEROL, THE FIRST
STEROL INTERMEDIATE in the cholesterol synthesis pathway, is
metabolized through numerous intermediates to form cholesterol
(21). The process involves three major steps, 1) reduction of the
24 double bond, 2)
demethylation on the positions 14, 4
, and 4
, and 3)
shifting the
8 double bond to
5, and may occur in parallel
pathways (Fig. 1). Only small amounts of the steroid intermediates of the cholesterol synthesis pathway are
normally detectable in human serum or tissues. However, highly coordinated intracellular trafficking of these sterols is involved in
the regulation of cholesterol synthesis (16). The
importance of these steroid intermediates has been highlighted recently
by the discovery that two 29-carbon (C) steroid intermediates of cholesterol synthesis serve as meiosis-activating factors
(2). Steroid cholesterol intermediates accumulate under
various pathophysiological conditions, including desmosterolosis,
cerebrotendinous xanthomatosis, and Smith-Lemli-Opitz syndrome, an
inherited defect in a late stage of cholesterol synthesis (3, 25,
27). The intracellular amounts of the sterol intermediates may
be modified by specific drugs and hormones (9, 17). Apart
from their pathophysiological importance, steroid cholesterol
intermediates in human serum, especially lathosterol, are used as
markers of cholesterol synthesis, serving as a surrogate for the
time-consuming and expensive direct measurements of cholesterol
synthesis by the fecal balance method. Serum lathosterol has been shown
to correlate well with the activity of the hydroxymethylglutaryl- CoA
reductase (HMG-CoA reductase), an important flux-controlling
step in cholesterol synthesis, and with direct measurements of
cholesterol synthesis, measured by the fecal balance method (1,
5, 13). To further evaluate the role of the sterol intermediates
and cholesterol in the estimation of cholesterol synthesis, this study
employed [1-13C]acetate to probe the labeling kinetics of
cholesterol.

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Fig. 1.
Late steps in cholesterol synthesis. The conversion of
lanosterol to cholesterol comprises the removal of the methyl groups at
positions 14, 4 , and 4 , the reduction of the 24 double bond,
and the shift of the double bond of 8 to 5.
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When multiple identical precursors condense to form a product,
13C-labeling studies have the potential to provide
estimates of the true rates of synthesis. The formation of sterols from
[13C]acetate is an example of this type of condensation
biosynthesis. Condensation biosynthesis considers cholesterol as a 27-C
polymer where 12 C are derived from C-1 of acetate and 15 C are derived from C-2 of acetate. Techniques for the analysis of mass spectrometry data to estimate biosynthesis of polymers have been described as mass
isotopomer distribution analysis (MIDA) (8) and isotopomer spectral analysis (ISA) (11, 12). Both ISA and MIDA
extract from the mass isotopomer profile of a product two parameters, one describing the fractional enrichment of the precursor (p in MIDA, D
in ISA) and one describing the fractional amount of the product formed
during the time the system was exposed to the tracer [f in MIDA,
g(time) in ISA]. Here, "precursor" refers to the subunit for the
polymerization, acetyl-CoA for cholesterol; "product" refers to the
polymerized molecule of interest. In this study, we examine both
cholesterol and sterol intermediates in the cholesterol synthesis
pathway, "sterol intermediates," as products of interest.
ISA differs from MIDA in that it uses as input the fractional abundance
of each measured isotopomer, whereas MIDA uses a pair of isotopomers.
Additionally, ISA uses nonlinear regression to find the best-fit
solution, simultaneously estimating both parameters from all available
isotopomers. ISA is especially useful in vitro, where the number of
detectable mass isotopomers of the product is greater than three. With
three or more isotopomers, the system becomes overdetermined, and the
nonlinear regression ISA procedure is designed to find the best-fit
solution for such systems. One of the difficulties in applying
isotope-labeling methods to cholesterol synthesis in vivo is the slow
turnover and large pool of cholesterol in humans, resulting in very low
labeling of plasma cholesterol. Compared with cholesterol, turnover is
faster and pool size smaller for the sterol cholesterol intermediates
appearing in plasma, suggesting that these molecules are promising
candidates for ISA applications. Here, we investigate labeling
properties of cholesterol sterol intermediates by use of ISA to gain
insights into the use of 13C tracer methods for cholesterol
synthesis. We propose novel uses of 13C labeling of
lathosterol to improve the estimates of cholesterol synthesis and to
evaluate the presence of lathosterol in plasma from sources other than
de novo synthesis.
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MATERIALS AND METHODS |
Cell culture.
HepG2 human hepatoma cells were cultured to confluence in 60-mm dishes
in phenol red-free Dulbecco's modified Eagle's medium (DMEM; Sigma,
St. Louis, MO) containing Pen-Strep (100 µg/ml), glucose (10 mM),
sodium bicarbonate (45 mM), and glutamine (4 mM) supplemented with 10%
fetal calf serum as described previously (11). With the
beginning of the experiment, the medium was changed to a serum-free
DMEM supplement with 10% lipid-free controlled processed serum
supplement (CPSR 1, Sigma). Drugs were added from ethanolic stock
solutions to final ethanol concentrations of 0.1-0.3% (vol/vol).
Controls received the same amount of ethanol. Slices of fresh rat liver
(25 µm; 50-80 mg wet wt, obtained from chow-fed Sprague-Dawley
rats) were incubated under the same conditions in a shaking water bath
(37°C). Solvents, chemicals, bezafibrate, and fenofibrate were
purchased from Sigma. Simvastatin was a kind gift from Dr. M. Stapff,
(MSD, Haar, Germany) and pravastatin from Dr. K. Hummel (Bristol-Myers
Squibb, Munich, Germany). In experiments for ISA, sodium
[1-13C]acetate (Cambridge Isotope Laboratories, Andover,
MA) was added in an aqueous solution to a final concentration of 1 mM.
Human studies.
The evening before the study, the volunteers were admitted to the
Metabolic Ward at the Department of Clinical Pharmacology, University
of Bonn, where they swallowed a plastic tube with a weight at the tip
of the tube. Overnight peristaltic transport carried the weighted tube
into the duodenum. The next morning, a liquid diet combined with
[1-13C]acetate was infused constantly into the duodenum
for the next 12 h. The liquid diet (1.42 kcal · kg body
wt
1 · h
1) contained 36% of
calories as fat, 16% as protein, and 48% as carbohydrates consisting
of maltose and saccharose (Nutrodip; Wander, Osthofen, Germany).
[1-13C]acetate (sodium salt, Isotec, Miamisburg, OH) was
administered at 0.12 mmol · kg body
wt
1 · h
1. The infusion of labeled
acetate into the duodenum was chosen to provide a high, constant level
of [13C]acetate to the liver via the portal vein. Blood
samples were obtained before the infusion was started and every hour
during the time of the study. The study was conducted according to the principles of the Helsinki Declaration, and the protocol had been approved by the local ethics committee of the Faculty of Medicine at
the University of Bonn. Each volunteer gave informed consent. Blood
samples were centrifuged after clotting, and serum samples were
immediately frozen at
20°C. Lathosterol was extracted from serum
samples (0.1 ml) after an alkaline hydrolysis and assessed for
13C incorporation as described in Sterol extraction
and analysis.
Sterol extraction and analysis.
Cells were washed three times with ice-cold PBS, pH 7.4 (once with 2 mg/ml BSA and twice with PBS alone). Sterols were extracted in
hexane-isopropanol (3:2 vol/vol; twice with 4 ml). One microgram of
epicoprostanol (stock solution 20 ng/µl in hexane) was added as
internal standard to each sample. After drying under nitrogen and an
alkaline hydrolysis (1 N NaOH in 80% ethanol for 1 h at 70°C),
the sterols were extracted in cyclohexane (two times with 3 ml). After
drying under nitrogen, sterols were converted to their trimethylsilyl
(TMS) derivatives by adding 100 µl of
bis(trimethylsilyl)trifluoroacetamide (Pierce)/n-decane (Sigma) (1:1;
vol/vol) for 1 h at 70°C in conical glass tubes. One to two
microliters were used for gas chromatography-mass spectrometry (GC-MS).
For the analysis of cholesterol, the final volume after the
derivatization was diluted 1:40 with n-decane.
GC-MS.
Analyses were performed on a Hewlett-Packard GC-MS system (5890 series
II GC combined with a 5971 mass selective detector) equipped with a
DB-XLB (30 m × 0.25 mm ID × 0.25 µm film) in the split/splitless mode. The temperature program was 150°C for 1 min,
followed by 20°C/min up to 260°C and 10°C/min up to 280°C (hold
for 15 min). After the elution of lathosterol, the electron multiplier
voltage (EMV) was raised by 300 for the measurement of lanosterol,
dihydrolanosterol, methylsterols, dimethylsterols, and plant sterols to
increase the sensitivity for these sterols. For the ISA analysis,
M + 0 to M + 10 ions were collected for cholesterol, lathosterol, lanosterol, and dihydrolanosterol by use of a
program with equal dwell time for each isotopomer. Single-ion monitoring was performed on ions specific for the TMS-derivatives of
the following sterols: cholesterol [458-468 mass-charge ratio (m/z)], lathosterol (458-468 m/z),
lanosterol (498-508 m/z), and dihydrolanosterol
(500-510 m/z). The internal standard epicoprostanol was
measured on 370 m/z. Cholesterol sterol intermediates and plant sterols in unlabeled experiments were monitored as follows: desmosterol (456 m/z; 441 and 351 m/z);
8-cholestenol (458 m/z); lathosterol (458 m/z); methylsterol (472 m/z); methylsterol-dien (470 m/z); dimethylsterol (486 m/z);
dimethylsterol-dien (484 m/z); dimethylsterol-trien (482 m/z); campesterol (472 m/z); sitosterol (486 and
396 m/z); lanosterol (498 and 393 m/z); and
dihydrolanosterol (500 and 393 m/z). Peak integration was
performed manually.
To obtain sufficient sensitivity, chromatograms were overloaded with
cholesterol, which does not disturb data collection of cholesterol
precursor eluting after cholesterol. It is optional to turn off data
collection from the mass spectrometer during the cholesterol elution.
To detect all the labeled ions of late-eluting peaks of lanosterol and
dihydrolanosterol, which are present in lower amounts, the EMV
was increased by 300 over the autotune setting to obtain sufficient
signal levels for HepG2 cells. In liver slices, only lathosterol was
present in sufficient amounts for analysis. Under our chromatographic
conditions, lanosterol has to be measured at 498 m/z,
because the plant sterol sitosterol is not well separated from
lanosterol. Because sitosterol shows an intense signal on 396 m/z, this signal might interfere with the signals of labeled
lanosterol measured on the fragment 393 m/z + 10 ions.
Therefore, lanosterol and dihydrolanosterol were measured on their
TMS-ether molecular ions 498 and 500 m/z, respectively, despite the lower sensitivity of these ions. It is notable that naturally labeled lanosterol as well as other cholesterol sterol intermediates can be measured at other, more favorable fragments.
ISA.
Calculations of the fraction of newly synthesized product
g(t) and precursor pool enrichments D by ISA were performed
as described previously (11, 12) using nonlinear
regression and analysis of all ions collected. The ISA parameters D and
g(t) represent fractions and may range from 0 to 1. D
relates to the immediate precursor pool for the polymerization
reaction, here acetyl-CoA. D represents the fraction of this precursor
pool derived from the 13C-enriched tracer. ISA calculations
require as input the fractional abundance at each C of the tracer added
to the system. D is not dependent on the actual enrichment of the
tracer; both singly and doubly labeled acetate may be used as tracers,
and the enrichment of the C atoms is not limited to 99%. When ISA and
MIDA are compared, it should be noted that the definitions of D and p,
precursor pool labeling in MIDA, differ slightly in that p is actual
labeling of the precursor pool and is a function of the isotopes used
(8). The second parameter, g(t), relates to the
product formation and varies with time. The parameter g(t)
is the fractional amount of the sampled product synthesized over the
time period of tracer exposure (t). The maximum value for
g(t) will be 1 if there are no sources of this compound
other than de novo synthesis using the pool labeled with the tracer.
This value will be observed when the entire pool has turned over.
The ISA program includes the additional C, H, and Si atoms from the
derivatization reagent in the molecular ions used for ISA calculations.
The effect of natural abundance of heavy isotopes of these other atoms
was included in the ISA calculations. However, instead of
"correcting" the raw data, the ISA model for cholesterol matched
the raw isotopomer fractions to a model of the 27-C moiety of
polymerized acetate units plus the natural abundance of the additional
atoms. In this way, ISA fully accounts for the effect of natural
abundance. Additionally, the three methyl groups present in lanosterol
and dihydrolanosterol, but not in cholesterol (derived from position 2 of acetate), were included in the ISA calculations. Results are given
as means ± SD. Statistical differences were assessed by
Student's t-test and considered significant with
P < 0.05.
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RESULTS |
Lathosterol synthesis in humans.
To test the hypothesis that ISA analysis of lathosterol synthesis is
feasible in human subjects, we infused [1-13C]acetate
into the duodenum. Blood samples were collected every hour for a period
of 12 h, and the isotope pattern of lathosterol was assessed by
GC-MS. The results of the ISA analysis in Fig. 2A show a rapid and constant
increase in the fractional de novo synthesis of lathosterol
[g(t)] in all three volunteers. However, the subjects
demonstrated some degree of variability. In two volunteers, the
g(t) value exceeded 60% after 12 h, whereas in one
volunteer this parameter was lower, but still >35%. For these rapidly
turning over pools, ISA analysis was reliable after only 2 h of
[1-13C]acetate infusion. The precursor pool enrichment D
(Fig. 2B) was constant by 4 h, with values for each
volunteer between 13 and 18%. The stability of the D value over time
demonstrates that the duodenal tracer administration protocol is
effective in delivering [13C]acetate that is utilized for
de novo synthesis.

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Fig. 2.
13C labeling and isotopomer spectral analysis
(ISA) parameters for lathosterol synthesis in 3 healthy human subjects
infused with [1-13C]acetate. A: fractional de
novo synthesis [g(t) values] over 12 h. B:
contribution of infused acetate to lipogenic acetyl-CoA pool. D,
precursor pool enrichment. Values are means ± SD;
n = 3 sample preparations.
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Kinetics of labeling and ISA analysis of lathosterol, lanosterol,
and dihydrolanosterol in HepG2 cells.
To further analyze the factors influencing the labeling of steroid
cholesterol intermediates, in vitro studies were conducted with HepG2
human hepatoma cells and with liver slices. Steroid cholesterol sterol
intermediates lathosterol, lanosterol, and dihydrolanosterol were
labeled in HepG2 cells by incubation with 1 mM
[1-13C]acetate for 12 h. The g(t) values
for lathosterol, lanosterol, and dihydrolanosterol after incubation
with 1 mM [1-13C]acetate in HepG2 cells are shown in Fig.
3A. For lathosterol and
lanosterol, g(t) values can be calculated as early as 3 h after the start of the incubation with [1-13C]acetate.
De novo synthesis of lanosterol and dihydrolanosterol increases rapidly
to >80% after 12 h, whereas lathosterol shows a slightly lower
fractional de novo synthesis. Because the labeling kinetics for
lathosterol were slower than for the other two intermediates, additional experiments, incubating cells in
[1-13C]acetate for 72 h, were conducted. These
studies revealed that the g(72 h) value for lathosterol was 0.9. The
finding that all compounds reached a g(t) value of 0.9 indicates that virtually all molecules of these intermediate pools are
progressively replaced by molecules derived from the biosynthetic
pathway. Notable also are the low standard deviations observed by
assessing g(t) and D values of these cholesterol sterol
intermediates. The precursor pool enrichments, D, measured at 12 h, were similar in steady state for all three intermediates, indicating
that 19-22% of lipogenic acetyl-CoA was derived from the 1 mM
[1-13C]acetate added to the incubation medium. Similar D
values were also calculated for cholesterol, even though the fractional
de novo synthesis was one order of magnitude lower (Table
1).

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Fig. 3.
Cholesterol steroid intermediates lathosterol,
lanosterol, and dihydrolanosterol labeling in HepG2 cells.
A: increases in fractional de novo synthesis with time
[g(t)] over the course of a 12-h incubation with 1 mM
[1-13C]acetate (n = 3). B: At
t = 0, tracer was removed after 12-h incubation and
decay of labeling plotted (n = 2).
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Table 1.
Influence of 10% human serum (5.1 mM cholesterol) on cholesterol and
lathosterol synthesis and on the L/C ratio compared with
cholesterol-free medium in HepG2 cells
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After 12 h of tracer incubation, the medium was then changed to
[12C]acetate, and the decay in labeling followed for
12 h. (Fig. 3B). The results are plotted as the ratio
of
(M + 1-10)/
(M + 0-10). Values slightly above 0.3 represent the natural abundances without 13C enrichment measured for the ions as described
in MATERIALS AND METHODS. The isotopomer-labeling profile
of the three sterol intermediates returned to the natural abundance
within 9 h with a half-life of <5 h. These results indicate that
molecules of these steroid intermediates were progressively replaced by
molecules derived from the biosynthetic pathway when the
13C incubation was terminated. Thus there is no evidence
for pools of these steroid intermediates that are not exchangeable with the biosynthetic pathway.
Effect of serum containing cholesterol on lathosterol and
cholesterol synthesis.
To examine the effects of cholesterol in the culture medium, we
incubated HepG2 cells with medium supplemented for 24 h with either 10% human serum containing 5.1 mM cholesterol or a lipid-free serum substitute (10% CPSR). This study indicated that the
cholesterol-containing serum decreased the fractional de novo synthesis
of lathosterol by 24% and of cholesterol by 35%, respectively (Table
1). Despite the pronounced difference in g(t) values for
lathosterol and cholesterol, there was no difference in the
contribution of acetate to the lipogenic acetyl-CoA pool (D values).
Thus the flux of [13C]acetate to lipogenic acetyl-CoA
relative to the flux of other compounds such as glucose to acetyl-CoA
was not affected by the addition of serum. The ratio of lathosterol to
cholesterol, frequently used as an indicator of cholesterol synthesis
in humans, was also significantly reduced in cholesterol-supplemented
medium compared with the incubations with cholesterol-free medium, as
expected if this ratio correlates with the cholesterol synthesis rate
(Table 1).
Effect of drugs on lathosterol synthesis in HepG2 cells and liver
slices.
Simvastatin and pravastatin are potent HMG-CoA-reductase inhibitors.
Simvastatin belongs to the more lipophilic statins including lovastatin, fluvastatin, cerievastatin, or atorvastatin, whereas pravastatin is more hydrophilic. Simvastatin reduced the fraction of
newly synthesized lathosterol in HepG2 cells and liver slices in a
dose-dependent manner (Fig. 4,
A and B). In contrast, pravastatin was
ineffective in HepG2 cells but was as potent as simvastatin in liver
slices to suppress lathosterol synthesis. In parallel with the reduced
fractional de novo synthesis of lathosterol, the ratio of lathosterol
to cholesterol was also significantly reduced by the same treatments
that reduced the fraction of new synthesis (Table
2). A comparison of this data
suggests that the decline in the lathosterol/cholesterol ratio is
largely the result of the effects of the statins on lathosterol
synthesis, because a 6-h exposure to statins affects lathosterol
synthesis (Fig. 4) and may have less effect on the larger cholesterol
pool, which will continue to receive influx from steroid intermediates. ISA analysis indicated that the precursor pool enrichments, D, for
simvastatin, but not for pravastatin, significantly increased (Fig.
5, A and B). A
similar increase in D was observed from the isotopomer distribution of
lathosterol at 3 h (data not shown).

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Fig. 4.
Effect of simvastatin (Sim.) and pravastatin (Prav.) on
g(t) of lathosterol in HepG2 cells (A) and liver
slices (B). Con., control. Values are means ± SD;
n = 3. n.d., Not detectable.
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Fig. 5.
Effect of simvastatin and pravastatin on acetate
precursor pool enrichments (D) of lathosterol in HepG2 cells
(A) and liver slices (B). Values are means ± SD; n = 3.
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Fibrates are inducers of peroxisome proliferation, and peroxisomes have
been shown to possess most of the enzymes of the cholesterol-synthetic pathway. To clarify the role of fibrates on cholesterol synthesis, HepG2 cells were preincubated with bezafibrate and fenofibrate (50 µM) for 24 h. Lathosterol synthesis was measured for 6 h
thereafter. As shown in Table 3, no
difference could be detected for g(t) and D values,
indicating that, unlike the statins, the fibrates do not affect the two
ISA parameters. Because ethanol was used as solvent for the drug
solutions, the effect of ethanol (0.1-1%, or 17-170 mM) on
ISA parameters was investigated in HepG2 cells over a 6-h period. No
effect of ethanol on D or g(t) was observed.
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DISCUSSION |
Isotopomer profiles of human serum lathosterol.
To investigate the labeling of lathosterol in human subjects, we
infused labeled acetate into the duodenum for 12 h. Because duodenal infusion provided a high, constant infusion of
[13C]acetate to the liver via the portal vein, it is a
more effective route for delivery of label to the liver than peripheral
infusion. This protocol readily labeled the hepatic sterol synthesis
pathway, as shown by the ISA analysis of plasma lathosterol (Fig. 2).
Although there have been a number of studies using
[13C]acetate to estimate cholesterol synthesis in vivo
using MIDA (6, 19), the present ISA analysis of
lathosterol from human plasma provides a view of the labeling of the
lipogenic acetyl-CoA pool at early time points that is more accurate
and precise than these estimates obtained by either MIDA or ISA of
plasma cholesterol. The reason for this is the large g(t)
values, from 0.2 to 0.8, for lathosterol, compared with those for
cholesterol, which are typically one order of magnitude lower at each
time point (Table 1). The larger g(t) values provide more
labeled isotopomers and improve the estimates of the parameters, as
discussed in more detail in A new calculation for estimating
cholesterol synthesis in vivo.
Figure 2B indicates that the acetyl-CoA precursor does not
reach a constant enrichment in the first 2 h. This result,
observed for lathosterol, must be true for cholesterol as well and
indicates that total synthesis rates based on stable isotopic labeling
will underestimate the rate of synthesis if the time to reach constant enrichment is not considered. Using ISA models for in vitro cholesterol synthesis, we also found evidence for a change in the precursor enrichment with time (11). In addition to this temporal
effect, evidence that the steady-state enrichment of lipogenic
acetyl-CoA across the liver is not constant has been reported by
Puchowicz et al. (20), who measured acetate
concentration and enrichment in arterial, portal, and hepatic venous
blood. As already stated, the ISA model used here with a single value
for D assumes a constant precursor enrichment. To evaluate rigorously
the question of whether zonation of precursor enrichment occurs in the
human model would require a comparison of the ISA statistics of the fit
of model to data for models with constant vs. varying values for D. Although this more sophisticated analysis was not performed in the
present study, it may provide additional insight into hepatic metabolic function. It should be noted that an overdetermined model having more
isotopomers than unknowns is required to perform this
comparison. Because lathosterol labels much more heavily than
cholesterol, such an analysis may be feasible in human subjects only
through the use of lathosterol as a surrogate for the precursor
enrichment profile of cholesterol. In summary, these data indicate that
human plasma lathosterol provides kinetic information about the
labeling pattern of the cholesterol synthesis pathway not easily
obtained from cholesterol itself.
Effect of drugs on lathosterol synthesis and labeling.
The amount and labeling of lathosterol were altered in vitro by
compounds known to alter cholesterol synthesis. HMG-CoA reductase inhibitors are widely used for the treatment of hypercholesterolemia and have been shown in numerous studies to be highly effective (10, 23). The two HMG-CoA reductase inhibitors simvastatin and pravastatin differ greatly with regard to their hydrophilicity. The
more hydrophilic drug pravastatin has been shown to be highly effective
in liver cells but showed much lower activity in extrahepatic cells
(22, 26). Several investigators (4, 30)
propose that an active transport process facilitates pravastatin
uptake, which is presumed to be mediated by a sodium-independent bile acid transporter. In contrast, the more lipophilic drug simvastatin may
traverse cell membranes by a passive process. Therefore, we tested the
effects of both drugs in HepG2 cells and liver slices on lathosterol
synthesis. HepG2 cells are known to have lost the sodium-dependent bile
acid transporter and therefore differ from freshly prepared hepatocytes
(14). The results in Fig. 4, A and
B, and Table 2 show that simvastatin decreased the
g(t) value of lathosterol as well as the
lathosterol/cholesterol ratio in HepG2 cells and liver slices and was
dose dependent. In contrast, pravastatin was not active in HepG2 cells
but was equally potent in liver slices. This finding is in accord with
previous reports that pravastatin is less active in HepG2 due to an
impaired uptake of pravastatin (28, 29). In contrast,
simvastatin is active in hepatocytes as well as in extrahepatic cells
(26). Another interesting finding is that simvastatin
increased the acetate precursor pool enrichment (D value) in HepG2
cells and tended to do so in liver slices (Fig. 5). This observation
suggests that the flux of acetate into the lipogenic acetyl-CoA pool
was not decreased proportionally with the decrease in cholesterol
synthesis. This finding contrasts with the effects of serum (Table 1).
It is also interesting to note, comparing Figs. 4A and
5A for simvastatin, that the ability to see a decrease in
the fractional amount of newly synthesized product, g(t), in
the presence of an increase in the tracer contribution to lipogenic
acetyl-CoA, D, illustrates an advantage of stable isotopes over
radioisotopes. If radioisotopes had been used here, measured by liquid
scintillation counting, the two opposing effects would have combined to
indicate that simvastatin had no effect on lathosterol synthesis.
However, with the use of ISA or MIDA, the two parameters may be
estimated, showing this interesting effect.
Controversial reports exist on the effects of fibrates on cholesterol
synthesis (25). Fibrates are known activators of
peroxisome proliferation, and peroxisomes have been linked to
cholesterol synthesis. Therefore, we preincubated HepG2 cells with
bezafibrate or fenofibrate (50 µM) for 24 h and applied ISA on
lathosterol synthesis for 6 h. No significant changes in either
g(t) or D value were found, suggesting that, under these
conditions, bezafibrate and fenofibrate have no effect on cholesterol
synthesis in HepG2 cells.
A new calculation for estimating cholesterol synthesis in vivo.
The challenges in estimating cholesterol synthesis in vivo provided the
rationale for this study of the labeling kinetics of steroid
cholesterol intermediates. Steroid cholesterol intermediates may have a
role here both as a surrogate marker that tracks cholesterol synthesis
and as a rich source of information about the labeling kinetics of the
cholesterol synthesis pathway. To illustrate the challenges in
measuring cholesterol synthesis in vivo using 13C tracers,
we simulated the mass isotopomer profiles for cholesterol of natural
enrichment and for cholesterol found in human serum after a 12-h
constant infusion of [1-13C]acetate (Fig.
6, A and B). We
simulated the cholesterol-labeling distribution reported by Empen et
al. (6), who found that [1-13C]acetate
infusion yielded an acetyl-CoA pool enriched to ~12% and a
fractional amount of new synthesis of ~4.5% of the plasma cholesterol over a 6-h period. These data are typical of MIDA studies
of cholesterol synthesis in human subjects; but lower values with
acetyl-CoA enrichment of 6% and fractional new synthesis of 3% have
also been used in MIDA calculations (19). Comparing isotopomer profiles A and B (Fig. 6)
reveals the small differences between natural enrichment and this in
vivo synthesis study. To estimate the two parameters characterizing the
biosynthesis, both MIDA and ISA calculations exploit these small
differences in the isotopomer profile. Clearly, estimating cholesterol
synthesis in vivo under these conditions requires careful measurement
of the mass isotopomer distribution. In contrast to cholesterol, the
labeling profile of lathosterol at 6 h differs dramatically from
the natural abundance profile. Figure 6C plots the labeling profile of a C27 steroid at the same acetyl-CoA enrichment
as Fig. 6B, 12%, but with a fractional de novo synthesis of
40%. Figure 6C is comparable to the profiles observed here
for human serum lathosterol synthesis in vivo at 6 h (Fig.
2A). In Fig. 6, B and C, the
contribution of de novo synthesis to each isotopomer is shown by the
open bar sections. Because ISA uses all of the mass isotopomers to
obtain the best fit estimate of the parameters, the plot in Fig.
6C provides a more robust estimate of the
parameters. Using masses 0-5 provides five independent
measurements to estimate the two parameters.

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Fig. 6.
Plots of mass isotopomer profiles for
C27 steroids as fractional abundances. For simplicity,
natural abundance of any derivatizing agent or atoms other than the 27 carbons is ignored. A: natural abundance profile
[13C natural abundance assumed to be 0.011 and mass + 0 = (1-13C)27]. B:
C27 steroid profile labeled with
[1-13C]acetate, with 4.5% g(t) = 0.045, and D = 0.12. C: similar to B except that
g(t) = 0.4.
|
|
Of what value is the accurate and precise estimate of ISA parameters D
and g(t) for lathosterol? Estimates of the fractional de
novo synthesis of lathosterol, g(t), provide information
only about lathosterol. However, the other ISA parameter, D, estimates the lipogenic acetyl-CoA precursor enrichment, D. This
parameter, when determined for lathosterol, is valid for the synthesis
of other sterols, including cholesterol. D is a property of the
acetyl-CoA pool used for all de novo steroid synthesis. Thus we propose
a method for improving the accuracy and precision of the measurement of
cholesterol synthesis in vivo by combining ISA analysis of lathosterol
and cholesterol. Lathosterol is readily available in the serum sample
used for GC-MS analysis of cholesterol; consequently, the experimental
requirements for ISA of lathosterol are easily accomplished. In
essence, we propose that the measurement of D provided by lathosterol
may be used to assist with the estimate of the parameters for
cholesterol synthesis. In a formal sense, this would be accomplished by
using an ISA model with three unknown parameters: D, lathosterol
g(t), and cholesterol g(t). ISA nonlinear regression would simultaneously estimate these three parameters from
the combined labeling profiles of lathosterol and cholesterol. Because
ISA uses simultaneously all of the available isotopomers of the two
compounds, it is particularly well suited for this task.
A variation of the method described is to use the lathosterol-labeling
profile to estimate D by ISA in combination with a precise method for
estimating the 13C enrichment of cholesterol. Two methods
that produce the accurate estimates of 13C enrichment of
organic molecules following gas chromatography are chemical reaction
interface for mass spectrometry (15) and combustion
isotope ratio mass spectrometry (7). In this variation, D
would be estimated by ISA analysis of lathosterol. Cholesterol would be
combusted to yield the total 13C enrichment. For example,
if 100% [1,2-13C]acetate is the tracer, the fractional
amount of new product synthesized, g(t), could be estimated
as follows
For simplicity, the equation ignores the correction for natural
abundance of any atoms other than the 27 sterol carbons. This procedure
could be used with any 13C tracer; however, the equation
would be slightly different for [1-13C]- or
[2-13C]acetate to account for the number of carbons in
cholesterol derived from these precursors.
A role for fractional amount of new synthesis, g(t) in developing
surrogate assays for estimating cholesterol synthesis in vivo.
The fractional amount of newly synthesized lathosterol g(t)
may itself be useful in estimating cholesterol synthesis. The well
established links between plasma lathosterol levels and cholesterol synthesis (1, 13) indicate that lathosterol levels may
serve as a surrogate for direct measurement of cholesterol synthesis by
the fecal balance method. Additionally, recent evidence indicates that
the amounts of cholesterol sterol intermediates including lathosterol
correlate with the fractional new synthesis rate determined by
deuterium incorporation (18). Here, we show that the
isotopically determined fractional amount of new synthesis of
lathosterol is altered in parallel with the lathosterol/cholesterol
ratio. Figure 4A demonstrates a specific pattern of changes
in the g(t) of lathosterol in the presence of simvastatin
and pravastatin. This pattern, that both compounds are effective in
liver but only simvastatin is effective in HepG2 cells, is also
observed by a comparison of the lathosterol/cholesterol ratio (Table
2). Thus, in addition to the amount of lathosterol, the fraction of new
synthesis of lathosterol may contribute to the development of surrogate
markers for total body cholesterol synthesis. However, because this
issue is complex, another application of lathosterol labeling may also be applicable. A recent study of the effects of a high-cholesterol diet
on serum lathosterol found that, although the high-cholesterol diet
reduced total body cholesterol synthesis, it did not decrease serum
lathosterol (5). The authors speculated that dietary lathosterol, from eggs in high-cholesterol diet, may have complicated the interpretation of the data. The demonstration here (Fig.
2A) that newly synthesized labeled lathosterol is readily
detected in serum samples provides a simple mechanism to separate de
novo synthesis of lathosterol from dietary sources. Thus lathosterol labeling provides a means of testing the hypothesis that plasma lathosterol derived from in vivo synthesis correlates with cholesterol synthesis. In summary, we conclude that the labeling of serum lathosterol may provide a useful tool for advancing the understanding of the cholesterol synthesis pathway in humans.
 |
ACKNOWLEDGEMENTS |
This study was supported by a grant from the Bundesministerium
für Bildung, Forschung, Wissenschaft und Technologie (01 EC 9402)
to B. Lindenthal and US Public Health Service Grant DK-45164 to J. K. Kelleher.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: J. K. Kelleher, Dept. of Physiology, The George Washington University School of Medicine and Health Sciences, 2300 Eye St. NW, Washington, DC
20037 (E-mail: jkk{at}mit.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 22, 2002;10.1152/ajpendo.00324.2001
Received 18 July 2001; accepted in final form 21 January 2002.
 |
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