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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Delta 24 double bond, 2) demethylation on the positions 14, 4alpha , and 4beta , and 3) shifting the Delta 8 double bond to Delta 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, 4alpha , and 4beta , the reduction of the Delta 24 double bond, and the shift of the double bond of Delta 8 to Delta 5.

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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); Delta 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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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

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 Sigma (M + 1-10)/Sigma (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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Table 2.   Influence of simvastatin and pravastatin on the L/C ratio in HepG2 cells and liver slices



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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.

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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Table 3.   Influence of bezafibrate and fenofibrate on lathosterol synthesis in HepG2 cells


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
g(<IT>t</IT>) for cholesterol<IT>=</IT><SUP>13</SUP>C<IT>/</IT>[(<SUP>13</SUP>C<IT>+</IT><SUP>12</SUP>C) ·1/D(lathosterol)]
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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bjorkhem, I, Miettinen T, Reihner E, Ewerth S, Angelin B, and Einarsson K. Correlation between serum levels of some cholesterol precursors and activity of HMG-CoA reductase in human liver. J Lipid Res 28: 1137-1143, 1987[Abstract].

2.   Byskov, AG, Andersen CY, Nordholm L, Thogersen H, Xia G, Wassmann O, Andersen JV, Guddal E, and Roed T. Chemical structure of sterols that activate oocyte meiosis. Nature 374: 559-562, 1995[ISI][Medline].

3.   Clayton, P, Mills K, Keeling J, and FitzPatrick D. Desmosterolosis: a new inborn error of cholesterol biosynthesis. Lancet 348: 404, 1996[ISI][Medline].

4.   Cohen, LH, van Vliet A, Roodenburg L, Jansen LM, and Griffioen M. Pravastatin inhibited the cholesterol synthesis in human hepatoma cell line Hep G2 less than simvastatin and lovastatin, which is reflected in the upregulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and squalene synthase. Biochem Pharmacol 45: 2203-2208, 1993[ISI][Medline].

5.   Duane, WC. Serum lathosterol levels in human subjects reflect changes in whole body cholesterol synthesis induced by lovastatin but not dietary cholesterol. J Lipid Res 36: 343-348, 1995[Abstract].

6.   Empen, K, Lange K, Stange EF, and Scheibner J. Newly synthesized cholesterol in human bile and plasma: quantitation by mass isotopomer distribution analysis. Am J Physiol Gastrointest Liver Physiol 272: G367-G373, 1997[Abstract/Free Full Text].

7.   Goodman, KJ, and Brenna JT. High-precision gas chromatography-combustion isotope ratio mass spectrometry at low signal levels. J Chromatogr A 689: 63-68, 1995[ISI][Medline].

8.   Hellerstein, MK, and Neese RA. Mass isotopomer distribution analysis at eight years: theoretical, analytic, and experimental considerations. Am J Physiol Endocrinol Metab 276: E1146-E1170, 1999[Abstract/Free Full Text].

9.   Holleran, AL, Lindenthal B, Aldaghlas TA, and Kelleher JK. Effect of tamoxifen on cholesterol synthesis in HepG2 cells and cultured rat hepatocytes. Metabolism 47: 1504-1513, 1998[ISI][Medline].

10.   Jones, P, Kafonek S, Laurora I, and Hunninghake D. Comparative dose efficacy study of atorvastatin versus simvastatin, pravastatin, lovastatin, and fluvastatin in patients with hypercholesterolemia (the CURVES study). Am J Cardiol 81: 582-587, 1998[ISI][Medline].

11.   Kelleher, JK, Kharroubi AT, Aldaghlas TA, Shambat IB, Kennedy KA, Holleran AL, and Masterson TM. Isotopomer spectral analysis of cholesterol synthesis: applications in human hepatoma cells. Am J Physiol Endocrinol Metab 266: E384-E395, 1994[Abstract/Free Full Text].

12.   Kelleher, JK, and Masterson TM. Model equations for condensation biosynthesis using stable isotopes and radioisotopes. Am J Physiol Endocrinol Metab 262: E118-E125, 1992[Abstract/Free Full Text].

13.   Kempen, HJ, Glatz JF, Gevers-Leuven JA, Van der Voort HA, and Katan MB. Serum lathosterol concentration is an indicator of whole-body cholesterol synthesis in humans. J Lipid Res 29: 1149-1155, 1988[Abstract].

14.   Kullak-Ublick, GA, Beuers U, and Paumgartner G. Molecular and functional characterization of bile acid transport in human hepatoblastoma HepG2 cells. Hepatology 23: 1053-1060, 1996[ISI][Medline].

15.   Kusmierz, JJ, and Abramson FP. Improved measurement of stable isotope ratios in gas chromatography/mass spectrometry using the microwave-powered chemical reaction interface for mass spectrometry. Biol Mass Spectrom 22: 537-543, 1993[ISI][Medline].

16.   Lange, Y, and Steck TL. The role of intracellular cholesterol transport in cholesterol homeostasis. Trends Cell Biol 6: 205-208, 1996[ISI].

17.   Lindenthal, B, Holleran AL, Aldaghlas TA, Ruan B, Schroepfer GJ, Jr, Wilson WK, and Kelleher JK. Progestins block cholesterol synthesis to produce meiosis-activating sterols. FASEB J 15: 775-784, 2001[Abstract/Free Full Text].

18.   Matthan, NR, Raeini-Sarjaz M, Lichtenstein AH, Ausman LM, and Jones PJ. Deuterium uptake and plasma cholesterol precursor levels correspond as methods for measurement of endogenous cholesterol synthesis in hypercholesterolemic women. Lipids 35: 1037-1044, 2000[ISI][Medline].

19.   Neese, RA, Faix D, Kletke C, Wu K, Wang AC, Shackleton CHL, and Hellerstein MK. Measurement of endogenous synthesis of plasma cholesterol in rats and humans using MIDA. Am J Physiol Endocrinol Metab 264: E136-E147, 1993[Abstract/Free Full Text].

20.   Puchowicz, MA, Bederman IR, Comte B, Yang D, David F, Stone E, Jabbour K, Wasserman DH, and Brunengraber H. Zonation of acetate labeling across the liver: implications for studies of lipogenesis by MIDA. Am J Physiol Endocrinol Metab 277: E1022-E1027, 1999[Abstract/Free Full Text].

21.   Schroepfer, GJ. Sterol biosynthesis. Annu Rev Biochem 51: 555-585, 1982[ISI][Medline].

22.   Sirtori, CR. Tissue selectivity of hydroxymethylglutaryl coenzyme A (HMG CoA) reductase inhibitors. Pharmacol Ther 60: 431-459, 1993[ISI][Medline].

23.   Smith, SC. Review of recent clinical trials of lipid lowering in coronary artery disease. Am J Cardiol 80: 10H-13H, 1997[Medline].

24.   Stahlberg, D, Reihner E, Ewerth S, Einarsson K, and Angelin B. Effects of bezafibrate on hepatic cholesterol metabolism. Eur J Clin Pharmacol 40, Suppl1: S33-S36, 1991[ISI][Medline].

25.   Tint, GS, Irons M, Elias ER, Batta AK, Frieden R, Chen TS, and Salen G. Defective cholesterol biosynthesis associated with the Smith-Lemli-Opitz syndrome. N Engl J Med 330: 107-113, 1994[Abstract/Free Full Text].

26.   Van Vliet, AK, van Thiel GC, Huisman RH, Moshage H, Yap SH, and Cohen LH. Different effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors on sterol synthesis in various human cell types. Biochim Biophys Acta 1254: 105-111, 1995[ISI][Medline].

27.   Wolthers, BG, Walrecht HT, van der Molen JC, Nagel GT, van Doormaal JJ, and Wijnandts PN. Use of determinations of 7-lathosterol (5 alpha-cholest-7-en-3 beta-ol) and other cholesterol precursors in serum in the study and treatment of disturbances of sterol metabolism, particularly cerebrotendinous xanthomatosis. J Lipid Res 32: 603-612, 1991[Abstract].

28.   Yamazaki, M, Suzuki H, Hanano M, Tokui T, Komai T, and Sugiyama Y. Na(+)-independent multispecific anion transporter mediates active transport of pravastatin into rat liver. Am J Physiol Gastrointest Liver Physiol 264: G36-G44, 1993[Abstract/Free Full Text].

29.   Ziegler, K, Blumrich M, and Hummelsiep S. The transporter for the HMG-CoA reductase inhibitor pravastatin is not present in Hep G2 cells. Evidence for the nonidentity of the carrier for pravastatin and certain transport systems for BSP. Biochim Biophys Acta 1223: 195-201, 1994[ISI][Medline].

30.   Ziegler, K, and Hummelsiep S. Hepatoselective carrier-mediated sodium-independent uptake of pravastatin and pravastatin-lactone. Biochim Biophys Acta 1153: 23-33, 1993[ISI][Medline].


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