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Cardiovascular disease risk and its associated complications correlate positively with circulating cholesterol levels. Plasma cholesterol levels are maintained by reciprocally related endogenous cholesterol synthesis and cholesterol absorption from dietary and biliary sources. Numerous in vivo clinical methods exist to quantify the absorption and synthesis of cholesterol in humans. This review summarizes these different methods available to study cholesterol absorption and synthesis, highlighting each method's strengths and weaknesses, as well as their applicability in different types of trials.
Elevated cholesterol, particularly low density lipoprotein (LDL) cholesterol, is a well defined risk factor for the development of atherosclerosis 1 through the formation of atherosclerotic lesions 2. Atherosclerosis is associated with cardiovascular disease (CVD), the leading cause of mortality and disability in developed countries. Cholesterol levels are maintained and regulated by cholesterol absorption and synthesis, which have a reciprocal relationship, and by cholesterol elimination into the bile. Therefore, the proper evaluation of absorption, synthesis and trafficking of cholesterol throughout the body is critical to health research.
Plasma cholesterol can be synthesized hepatically or extra-hepatically, or absorbed from the intestine, derived from dietary or biliary sources. Statins, a family of HMG-COA reductase inhibitors, have been shown to effectively reduce cholesterol synthesis, achieving plasma LDL cholesterol lowering of up to 60% 3 and a CVD risk reduction of one-third 4. Although efficacious in lowering LDL-C levels, statin use is associated adverse events including muscle cramping and rhabdomyolysis 5. Therapies which reduce cholesterol by inhibiting intestinal cholesterol or bile acid absorption are also available. Plant sterols/stanols, dietary fibre, and bile acid sequestrants have been shown to be effective in treating hyperlipidemia, with LDL cholesterol reductions of 10-15%, 8.5-13% and 5-30% respectively 6. Ezetimibe, a potent cholesterol absorption inhibitor, led to the discovery of the Niemann-Pick C1 Like 1 (NPC1L1) protein and its role in cholesterol absorption7. NPC1L1 is a novel sterol transporter is highly expressed in the jejunum, it is essential for the absorption of cholesterol. NPC1L1 null mice have reduced cholesterol absorption by upwards of 90%8. Ezetimibe has been demonstrated to lower LDL cholesterol levels by 16-19% 9. The wide range of cholesterol lowering response seen after each treatment is likely the product of genetic factors which modify cholesterol synthesis and absorption, as well as modulate the effectiveness of each intervention 10-12. Given this considerable genetic heterogeneity, a need exists for precise measurement of cholesterol synthesis and absorption as well as their response to different dietary, pharmaceutical and lifestyle interventions.
Methods used to elucidate the different facets of cholesterol trafficking have undergone significant evolution since their inception in the late 1950's. New technologies and instrumentation have been developed which have been adopted into techniques which are now available to measure cholesterol absorption and synthesis. The purpose of this review is to describe and systematically compare the different methods currently available to monitor cholesterol synthesis and absorption in humans. These comparisons will aid investigators in the selection of methods most appropriate for a particular project with respect to their advantages, drawbacks and assumptions.
Methods to assess cholesterol absorption
Direct methods of measuring cholesterol absorption measure cholesterol flux from gut to lymph. Such methods are so termed as the amount of cholesterol absorbed across a section of intestine is assessed directly, without using faecal or plasma levels of cholesterol to infer absorption. The first direct method measured cholesterol absorption using a duodenal canula for infusion of cholesterol, while lymph was sampled through cannulisation of the mesenteric or thoracic lymph ducts 13. The amount of cholesterol appearing in the lymph was taken as absorbed cholesterol. The use of radioactively labelled cholesterol in the duodenal infusion allowed for differentiation between exogenous and biliary cholesterol, enabling calculation of percent absorption of exogenous cholesterol. This method has been successfully used to assess cholesterol absorption in rats 14, but has limited feasibility in humans. An alternative direct intestinal perfusion method for use in humans was introduced by Grundy and Mok 15. This perfusion method required intubation with a triple-lumen tube. Liquid formula containing a known amount of cholesterol and ß-sitosterol is infused into the duodenum at the Ampulla of Vater through the first tube, while intestinal contents are aspirated from the 2nd and 3rd tube, positioned 10 and 100 cm distal to the first, respectively. Analysis of the aspirate allows for calculation of net hourly cholesterol absorption across a 100 cm gut segment. Discriminating between exogenous and endogenous sources of cholesterol is performed by infusing labelled cholesterol. A drawback is that cholesterol undergoes transformation into other sterol products by gut bacteria, this loss of cholesterol must be accounted for in this method. ß-sitosterol was used as a marker for cholesterol loss in the gut. ß-sitosterol was originally thought to be non-absorbable, unaffected by gut bacteria, and unable to affect cholesterol absorption. However, Grundy and Mok 15 noticed that even small increases in ß-sitosterol concentration used in this method were associated with noticeable reductions in measured cholesterol absorption. It has since been shown that ß-sitosterol and other plant sterols inhibit cholesterol absorption, even at the very low concentrations used in this method 16, 17. Plant sterols compete with cholesterol for absorption into gut enterocytes, then are largely exported back into gut and liver by ABCG5 and ABCG8 transporters 18. The use of an inhibitor of cholesterol absorption such as ß-sitosterol in a method used to assess cholesterol absorption therefore obviously leads to an underestimation of actual cholesterol absorption capacity. The largest limitation to use of direct measurement techniques is the substantial level of invasiveness involved. Notwithstanding, only direct methods yield objective measures of total, exogenous and endogenous cholesterol absorption across the intestine 13.
Cholesterol balance methods
Cholesterol balance methods assess absorption as the difference between dietary sterol intake and faecal cholesterol elimination, excluding cholesterol of endogenous origins 19. The technique has typically relied on labelled cholesterol to distinguish endogenous from exogenous sourced cholesterol within the faecal and plasma cholesterol pools measured. Four main balance methods exist, namely Method I, II, III and V , following the nomenclatures from Grundy and Arhens 20.
Method I consists of a single dose of [14C] or [3H] radioactively labelled cholesterol administered intravenously 20, 21. This radio-isotope labels endogenous cholesterol and its subsequent steroid products using the formulas:
Endogenous faecal neutral steroids are calculated by dividing the total radioactivity (dpm/day) in total faecal neutral steroids by the specific activity (dpm/mg) of plasma cholesterol 1-2 days prior, depending on gastrointestinal transit time.
Method II requires continuous oral labelling with radioactive cholesterol 20, 21. The radioactively labelled cholesterol is generally incorporated into a liquid diet to ensure a precise daily intake for many weeks. Cholesterol absorption is calculated using the formula:
The equations are solved for Y, with absorption measurements assessed at any time after four days of continuous radioisotope feeding. Isotopic steady state is not required. Results for cholesterol absorption measured by Methods I and II yield similar results 20. However, it has been shown that Method II provides more consistent estimates of cholesterol absorption than Method I. This improved precision of Method II has been attributed to i) Method II measuring unabsorbed dietary cholesterol directly, not indirectly as the difference between total faecal neutral steroids, and endogenous neutral steroids, both of which are much larger than unabsorbed dietary cholesterol, ii) colonic emptying time affecting Method I to a greater extent than Method II, this emptying time cannot always be calculated with precision, contributing to greater variability in Method I than II, and iii) Method II does not require isotope equilibration between plasma and intestinal cholesterol pools, unlike Method I. Given the advantages and improved precision of Method II, it should be chosen over Method I.
Method III, introduced by Wilson and Lindsey 22, required attaining of isotopic steady state. Continuous feeding with radioisotope labelled cholesterol for greater than 100 days is often required to reach an isotopic steady state. When an isotopic steady state is reached, daily cholesterol turnover is calculated, and daily absorbed dietary cholesterol is arrived at by the formula:
Failure to reach isotopic steady state will yield an underestimate of actual dietary cholesterol absorbed 13. The lengthy period required reaching isotopic steady state and the difficulty determining when this state has been attained renders Method III difficult and unreliable. Method III was shown to give an approximately 20 % lower value of cholesterol absorption than Method II, when compared directly. 21.
Method V 23 is a combination of methods I and II. Subjects are continuously fed radioactively labelled cholesterol as in Method I, then given a single bolus of different radioactively labelled cholesterol as required in Method II. The equations for Method II are used, with only the modification of the X *SA(X) term which is replaced by [(R)(SA(X oral) ) / (SA(X intravenous))], where R= daily faecal neutral steroid excretion of intravenous cholesterol (mg/day), SA(X oral) = plasma specific activity of oral isotope taken 1 day before R (dpm/mg), and SA(X intravenous) = plasma specific activity of intravenous isotope taken 1 day before R. Method V was designed for unusually high rates of cholesterol synthesis as a result of surgical or pharmacological interference with the enterohepatic circulation, which can lead to endogenous cholesterol being secreted into the gut prior to isotopic equilibration 13. High rates of synthesis result in a specific activity of plasma cholesterol that is greater than that of endogenous faecal neutral steroids. Method I and II both assume equivalent specific activity within endogenous faecal neutral steroids and plasma cholesterol. If plasma cholesterol specific activity exceeds that the specific activity of endogenous faecal neutral steroids Method I will lead to an underestimate and Method II lead to an overestimate of cholesterol absorption.
Methods I, II and V all require use of non absorbable markers to track gastrointestinal transit time, and for cholesterol loss in the gut. Chromic oxide is often used as such a marker of gastrointestinal transit time. Transit time must be accurately calculated in methods that measure faecal steroid activity to ensure that faeces sampled correspond with the timing of isotope administration. ß-sitosterol can be used as the control for cholesterol loss in the gut. It must again be mentioned that if ß-sitosterol is used, because of its inhibitory effects on cholesterol absorption, cholesterol absorption values obtained will underestimate true values. Loss of cholesterol due to bacterial degradation can be upwards of 25% 24. Therefore if the cholesterol loss in the gut is not accounted for by these methods, the calculated cholesterol absorption will be higher than its actual value 21.
Isotope ratio methods
Isotope ratio methods are capable of measuring percent, but not net, cholesterol absorption rates 25. Similar to balance methods, isotope ratio methods require accurate records of dietary cholesterol intake to estimate the mass of exogenous cholesterol consumed, then derive the proportion absorbed from the fractional data obtained 19.
Method IV, also termed faecal isotope ratio method, first introduced by Borgstrom 26, calculates cholesterol absorption as the percentage of a single oral dose of radioactively labelled cholesterol not recovered in the faeces. Single doses of both radioactively labelled cholesterol and ß-sitosterol are administered orally, after which faeces are collected for seven to eight days. The ratio of labelled cholesterol to labelled ß-sitosterol in a sample of the pooled faeces samples is compared to the ratio given orally. Cholesterol absorption is calculated using the formula:
This method has been modified to allow faecal sampling to be conducted only once. Sodhi et al. 27 administered a faecal flow marker, such as chromic oxide or carmine red, with the original test dose, then calculated the faecal isotopic ratio at the peak level of the marker in the faeces, rather than using a sample of pooled faeces. This single faecal sampling modification of Method IV has been compared to the original Method IV and Method I in baboons and found to yield consistently higher fractional absorption values, suggesting that this modification may lessen accuracy 28. Method IV, despite requiring a prolonged period of faecal collection, uses a far smaller dose of radioactivity than do balance methods, and is relatively straightforward and simplistic to execute 13. Method IV does still require ß-sitosterol as a marker of cholesterol loss in the gut, so it shares certain of the drawbacks previously discussed.
The plasma isotope ratio method (Method VI) was first introduced by Zilversmit 29 in rats and subsequently applied to humans 30. Method VI involves simultaneous oral and intravenous administration of [14C] and [3H] radio-labelled cholesterol and requires only a single blood sample 3-4 days afterward afterwards. The methodological principles are based on the measurement of drug absorption used in pharmacology 13. If the absorption of cholesterol was 100% then the specific activity curve of both radiolabelled cholesterol tags would converge, assuming equal doses of the different labels. If absorption is zero, none of the orally administered cholesterol, and therefore zero radioactivity from the oral radioisotope, would appear in the plasma. Since cholesterol absorption falls between zero and 100%, the ratio of the two plasma specific activities, after normalization for dose administered, is used to calculate absorption using the formula:
where % oral dose and % I.V. dose in plasma are the percentage of IV and oral tracer in the plasma sample, respectively 31 . The ratio is calculated 3-4 days after radioisotope administration to enable equilibration of each tracer, as the isotopes distribute across body sterol compartments and commence being lost at equivalent rates. Curiously, a delay of up to 48 h occurs in the peak appearance of oral cholesterol isotope in the blood of humans, likely related to the multiple sub-pools associated with the process of cholesterol absorption 13. The advantages of Method VI over alternative approaches include that: i) only a single blood sample is required, ii) a low dose of radioisotope is required, iii) faecal collections are avoided, and iv) the method does not depend on markers such as ß-sitosterol to correct for faecal losses. Method VI lends itself to repeated use because of its short duration and low level of labelling. Method VI allows for investigation of cholesterol absorption under different experimental parameters in the same individual within a comparatively short time frame. Method VI has been validated in humans numerous times against Method IV under different conditions yielding similar results. Samuel et al.30 comparing Method VI to Method IV in 12 hospitalized individuals showed that results from Method VI correlated with Method IV (r=0.83) and that results from both methods agreed within 5%. Samuel et al. 31 further validated Method VI against Method IV in an additional 8 individuals, demonstrating a level of accuracy of 3.5% at a 95% confidence level.
A third isotope ratio method, Method VIII, introduced by Crouse and Grundy 32 is similar to Method IV since average cholesterol absorption is calculated using the ratio administered cholesterol to ß-sitosterol measured in the faeces, but differs in method of isotope administration. [14C] Cholesterol and [3H] ß-sitosterol are administered orally three times daily over 10 days, with faeces collected from days 4-10 13, 19. Following day 3 of isotope administration the ratio of isotopes in the faeces becomes essentially constant and cholesterol absorption becomes calculable by the formula:
This method requires knowledge only of the ratio of radioactivity within a single faecal sample; faecal mass need not be calculated 32. Since ß-sitosterol, a cholesterol absorption inhibitor, is also administered with the labelled cholesterol, Method VIII underestimates actual cholesterol absorption.
Plasma cholesterol specific radioactivity following the consumption of a test meal containing radioactively labelled cholesterol (hereafter called Method VIIII) has also been investigated as a novel measure of cholesterol absorption. Lin et al. 33 assessed cholesterol absorption in 11 individuals with Smith-Lemli-Opitz syndrome (SLOS), a cholesterol synthesis disorder, and compared Method VIIII with cholesterol absorption measured by Method IV. These investigators sampled blood at 24 and 48 hours following radioisotope enriched tests meals and calculated the specific radioactivity of cholesterol in plasma. Values obtained correlated well with those calculated by Method IV (r=0.594, p=0.009, and r=0.474, p=0.047 over 24 and 48 hours, respectively). While Method VIIII fails to calculate cholesterol mass or percent absorption, it does allow for investigation of relative changes in cholesterol absorption within an individual following different interventions. Method VIIII requires only a single blood sample, and no stool collection, thus avoiding requirement for markers such as β-sitosterol to account for cholesterol losses in the gut. Method VIIII is very similar to the single isotope tracer method that will be discussed in the stable isotope methods section below 34. The robustness of the relationship between plasma radioactivity at 24 hours and cholesterol absorption, however, requires further validation, especially in healthy individuals.
While use of radio-isotopes has been invaluable in the investigation of cholesterol absorption, the advent of safer stable isotope laboratory techniques, and developments in isotope ratio mass spectrometry, has propelled the field much further 25. The switch to stable isotopes has reduced logistic difficulties related to containment, handling, disposal and overall safety associated with radioisotopes, allowing for investigation of cholesterol absorption in certain populations including children, pregnant and lactating mothers, which had previously been excluded due to ethical considerations around radio-isotope administration.
Stable isotope methods for cholesterol absorption
Cholesterol labelled with stable isotopes has been shown to possess identical kinetics as radio-labelled cholesterol 25, leading to the development of stable isotope techniques to investigate cholesterol absorption. Lutjohann et al. 35 introduced a stable isotope version of Crouse and Grundy 's 32 Method VIII discussed above. [13C] cholesterol and [2H] sitostanol were used in place of [14C] cholesterol and [3H] ß-sitosterol, quantified using gas-liquid chromatography combined with selected ion monitoring mass spectrometry. Cholesterol absorption was calculated as in Method VIII. The stable isotope method was twice compared to Method VIII, in six monkeys, yielding similar results. The stable isotope method produced an absorption range of 49-73% (mean of 60%), with coefficients of variation ranging from 3.9%-15.1% (mean 7.1%). The radioisotope produced a range of 51-69% (mean 61%) with coefficients of variation ranging from 1.9-13.6% (mean 5.1%) 35. The similar results demonstrated that the stable isotope Method VIII was as effective as the radio-isotope Method VIII, without the risk to radioactive exposure of subjects and research staff. This method used sitostanol, rather than β-sitosterol, as a marker for faecal losses of cholesterol. This, however, does not remedy the problem other methods suffer from when using β-sitosterol, as sitostanol is also an inhibitor of cholesterol absorption 36, 37.
The plasma isotope ratio method (Method VI) 29, 31 was also adapted to use stable isotopes. Bosner et al. 38 used 2H labelled oral and 13C labelled IV cholesterol to calculate the plasma stable isotope ratio and cholesterol absorption percentage. The Bosner et al. method uses gas-chromatography - mass spectrometry with selected ion monitoring (GC/MS-SIM) or GS/MS- chemical ionization mode (CI) to determine isotopic enrichment. Bosner et al. 39 further modified Method VI to a single isotope dual tracer method, using oral [2H]5 and IV [2H]6 cholesterol. Isotope detection in plasma cholesterol was done by GC/MS- selected mass monitoring. Jones et al. 40 were the first to use isotope ratio mass spectrometry to determine isotopic enrichment using the plasma isotope ratio method. Oral 13C and IV 3H cholesterol were administered to 15 hypercholesterolemic men, followed by blood sampling 2-3 days post administration. Free cholesterol from red blood cells was purified by thin layer chromatography and subsequently combusted to yield carbon dioxide and water. The CO2 was then measured for 13C enrichment against the international standard Pee Dee Belemetite (PDB) utilizing an isotope ratio mass spectrometer (IRMS). Water samples were also reduced to hydrogen gas via zinc reduction and 3H enrichment was measured against Standard Mean Ocean Water (SMOW) international standard by IRMS. The ratio of plasma enrichment of 13C to 2H cholesterol on day 3 following tracer administration was used to calculate cholesterol absorption. Recently, continuous flow gas chromatograph combustion IRMS (GC/C/IRMS) systems for 13C cholesterol and gas chromatograph pyrolysis IRMS (GC/P/IRMS) systems for 18O or 2H cholesterol have been used in calculating cholesterol absorption (Method VI)41, 42. The use of IRMS vs GC/MS-SIM has vastly increased the precision of this method. These plasma stable isotope ratio techniques, and the Zilversmit method using radioisotopes 29, 30, also estimate cholesterol turnover in the main M1 free cholesterol pool 43, 44 from the decay curve of the intravenously injected radio or stable isotope 40. The additional information regarding cholesterol turnover yielded by these methods make them desirable choices in situations when shifts in cholesterol turnover as well as absorption may be of interest.
Two single stable isotope methods for assessing cholesterol absorption have been introduced by Ostlund et al. 45 and Wang et al. 34. Ostlund et al. 45 administered 2H cholesterol to volunteers and measured the average oral cholesterol tracer in plasma ((mmol 2H cholesterol/mol natural cholesterol) in blood samples taken 4 and 5 days post tracer administration using GC/MS. Wang et al. 34 administered 13C cholesterol orally followed by blood sampling at 24, 48, 72 and 96 hours. 13C enrichment in plasma free cholesterol was measured using GC/C/IRMS. Average 13C enrichment from 24-96 hours and area under the curve (24-96 hours) of 13C enrichment were compared to cholesterol absorption percentage assessed using the stable isotope Method VI 38 in 2 studies. Both the average and area under the curve of 13C enrichment in plasma free cholesterol correlated with cholesterol absorption percentage measured by stable isotope method VI (r values ranging from r=0.81, p=0.0001 to r=0.88, p=0.0001)34. Both these single isotope methods can be employed to compare treatment effects, such as pharmaceutical or dietary interventions, relative to controls, on cholesterol absorption.
Absorption surrogate method
The use of serum plant sterol levels to predict cholesterol absorption was first proposed by Tilvis and Miettinen 46. These researchers showed that serum levels of ß-sitosterol and campesterol, when normalized for total serum cholesterol, correlated positively with cholesterol absorption as measured by Method VIII of Crouse and Grundy 32. This approach involves lipid extraction from a single blood sample followed by evaluation of serum plant sterol and cholesterol levels using GCFID, HPLC or GCMS methods. The use of the ratio of campesterol or ß-sitosterol to cholesterol has been subsequently employed numerous times to measure cholesterol absorption 47-51 . Nissinen et al. 48 showed that ß-sitosterol to cholesterol ratios were more strongly correlated with cholesterol absorption than were the ratios of campesterol to cholesterol, as measured by Method VIII, across three diets varying in both cholesterol and lipid levels in 29 healthy male volunteers. When using serum plant sterols as surrogates for cholesterol absorption it is imperative that factors which are known to change serum plant sterol levels, such as the dietary intake of plant sterols 52, be controlled for, so as not to identify changes in cholesterol absorption which may not exist 53. Serum plant sterol levels vary substantially within and across different populations 52, and may be severely elevated in certain individuals due to genetic disorders 54, 55. It is thus important that individuals with these genetic disorders not be included in studies using this method of estimating cholesterol absorption as serum plant sterol levels will not reflect cholesterol absorption in such individuals. Recently it has also been shown that serum plant sterol concentrations fail to accurately reflect cholesterol absorption in individuals with Smith-Lemli-Opitz syndrome 56, therefore. The use of serum plant sterols as surrogates for cholesterol absorption should be carefully verified and validated prior to its use within a particular population. Use of plant sterol surrogates does benefit from relative speed and simplicity compared to other previous discussed methods 19, 25. Furthermore this approach represents the only estimate of cholesterol absorption which can be done in large scale studies.
Methods of assessment of cholesterol synthesis
Cholesterol synthesis contributes substantially more to circulating cholesterol pools than cholesterol absorption, yielding approximately 700-900 mg a day 25, 57. This synthesis has been shown to undergo diurnal periodicity, maintaining cholesterol levels during fasting 58. Accurate assessment of cholesterol synthesis is essential to the field of cholesterol research.
Cholesterol synthesis can be estimated when intake of dietary cholesterol and excretion of total cholesterol are known during a metabolic steady state 20. The criteria needed for this metabolic steady state are constant plasma cholesterol and faecal cholesterol excretion levels during a period of constant weight. In this steady state cholesterol synthesis can be taken as the difference between cholesterol excretion, both faecal neutral sterols and bile acids, and assessed dietary intake. Dietary cholesterol intake must be accurately measured and faeces reliably collected over the experimental period for the method to yield accurate data. Faecal flow must also be monitored with oral administration of a marker such as Cr2O3, to assure faeces collected stems from the experimental periods. Faecal neutral sterols and bile acids are measured in faeces, typically by GC-MS, and cholesterol synthesis for a given period of time is calculated 59, 60. Although this method represents the gold standard for calculating cholesterol synthesis, it is vulnerable to errors in the estimation of both dietary cholesterol intake and faecal excretion, both of which can potentially cause significant errors. The balance method determines the actual mass of cholesterol synthesized during a given period. However, the technique does require metabolic steady state, as well as accurate measurement of cholesterol intake and a need for total faecal collection across the period of interest. Therefore, this method is not optimally suited for larger trials.
Fractional conversion of squalene
Daily cholesterol synthesis rate can be also estimated as the fraction of infused radiolabelled mevalonic acid converted to cholesterol 61, 62. Here, dosages of both [14C] mevalonic acid and [3H] cholesterol are administered intravenously. It is then assumed that the infusion of labelled mevalonic acid rapidly labels the plasma squalene pool, reaching a maximum enrichment within approximately 100 minutes. Cholesterol synthesis is subsequently estimated through measurement of squalene synthesis. Squalene synthesis is calculated by the percentage of mevalonic acid dose converted to cholesterol, divided by the area under the curve of plasma squalene specific activity. This method assumes that plasma squalene synthesis is equivalent to cholesterol synthesis, which may not always be the case. Cholesterol synthesis rates estimated using this method have been shown to agree with cholesterol synthesis calculated by the balance method within 8% 62. Benefits of this method are that the protocol requires only 1 hour of a participant's time, and can be repeated every 3 weeks 61. The method does, however, require the administration of intravenous radio-isotopes.
Cholesterol synthesis precursors
Concentrations of plasma intermediates along the synthesis pathway of cholesterol represent another approach to indirectly measuring cholesterol synthesis. Squalene 63, mevalonic acid 64, lanosterol, desmosterol and lathosterol have all been variously used as surrogates for cholesterol synthesis 65, 66. Such precursors have been shown to fluctuate with diurnal cholesterol synthesis patterns, as well as in conditions in which cholesterol synthesis is elevated or reduced 63. Levels of these precursors correlate closely with measured cholesterol synthesis when they are normalized for plasma cholesterol level, then when taken the absolute amount of precursor and are normally expressed in mmol/mol cholesterol 48, 66. When using cholesterol precursors as surrogates for cholesterol synthesis, dietary intakes of each precursor should be controlled, this is particularly important for squalene, which is abundant in olive oil and is at least partially absorbed into the blood 17. The most apparently reliable surrogate for cholesterol synthesis has been lathosterol 48, 60. Since cholesterol synthesis surrogates require only one blood sample they are ideal for estimating cholesterol synthesis in large cross sectional studies or in epidemiological trials.
Mass Isotopomer Distribution Analysis (MIDA)
Mass isotopomer distribution analysis (MIDA) measures the synthesis of several biological polymers in vivo 67 . The technique uses the relative abundance pattern or distribution of polymer species which differ only in mass of isotopomers produced during the administration of stable isotope labelled precursors. The distribution of the polymer species produced is compared to theoretical distribution patterns predicted by binomial and polynomial expansion. Using these theoretical distributions, parameters such as fractional synthesis rate can be calculated by combinatorial probability modelling. Since cholesterol is synthesized from subunits of acetyl-CoA, fractional synthesis of cholesterol can be calculated during the infusion of 13C labelled acetate 68. This method is invasive, requiring a 24-hr intravenous infusion and serial blood sampling from an indwelling catheter 69. Cholesterol in the blood samples is measured by GC-MS to determine the distribution pattern of isotopomers from which the rate of synthesis is obtained. The data analysis relies on more complex mathematical modelling constructs than do other methods for estimating cholesterol synthesis. The FSR measured by MIDA agrees well with FSR measured by deuterium incorporation, showing a significant correlation (r=0.84, p=0.0007) across both techniques in 12 subjects 69.
This method is based on the tritiated water uptake method by Dietchy and Spady 70 used originally in animals. Deuterium incorporation method uses water as a tracer to determine the synthesis of free cholesterol (FC). The fractional synthesis rate (in pools/day) of free cholesterol is calculated from the rate of incorporation of deuterated water into de novo synthesized plasma or erythrocyte cholesterol. Orally administered deuterated water equilibrates across the body water pool as well as with intracellular NADPH pools. Body water and NADPH exist as the precursors for 22 of the 46 hydrogens in synthesized cholesterol 71. Deuterium enrichment of the precursor pool, plasma water, and in erythrocyte or plasma cholesterol is measured by IRMS. Fractional synthesis rate of free cholesterol (FSR-FC) is calculated using the following formula:
Where δ refers to the change in deuterium enrichment over 24 hours and 0.478 is the ratio of cholesterol from body water and NADPH to total hydrogen in a cholesterol molecule, or the ratio of hydrogen which could be enriched by oral D2O administration 72. From the FSR-FC the ASR -FC (g/day) can be calculated by multiplying the FSR-FC by an estimate of the M1 pool size 44 and 0.33 the proportion of FC in total cholesterol. The ASR-FC approximates the daily production of newly synthesized cholesterol.
Three main assumptions required for deuterium incorporation include: i) that the fraction of hydrogen derived from plasma water (22/46, or 0.478) is constant in denovo synthesized cholesterol; ii) that de novo synthesized free cholesterol rapidly exchanges between the site of synthesis and the major free cholesterol (M1, plasma compartment) pool, and within this pool cholesterol migrates rapidly between cellular membranes and lipoproteins as well as between different classes of lipoproteins; iii) that deuterium uptake into free cholesterol of the major pool of cholesterol reflecting synthesis and iv) that synthesis of cholesterol in the major pool provides a reasonable measurement of total cholesterol synthesis as most sterol synthesis occurs in the gut and liver which contribute to the plasma cholesterol pool. It must be acknowledged that the major plasma pool of cholesterol is at equilibrium with two other slow turnover pools outside the plasma, and that the slow inter-pool cholesterol exchange could cause insignificant entry of labelled free cholesterol into the central pool within a 24 hour time period 70, 71. Although these assumptions are not without imperfection, the cholesterol synthesis estimates yielded by deuterium incorporation have been sensitive enough to show differences in cholesterol synthesis due to genetic factors, as well as dietary and pharmaceutical interventions. Deuterium incorporation has also been shown to correlate well with cholesterol synthesis measured by the balance method 59, MIDA 69 and cholesterol synthesis surrogate levels 65.
Combined Cholesterol absorption and synthesis technique
Advancements in quantification of cholesterol homeostasis must still be continued with the goal of finding more accurate methods to measure cholesterol synthesis and absorption simultaneously, which until now has been unfeasible. The ability to simultaneously measure changes in cholesterol absorption and synthesis could provide a valuable research tool, especially in investigating the reciprocal relationship between absorption and synthesis. At present, the plasma dual isotope method described by Bosner et al. 38 can be applied in concert with the deuterium incorporation approach 71 to measure cholesterol absorption, turnover and synthesis in a relatively short period of time (5 days). However in this method demonstrated by Jones et al. 40, cholesterol synthesis is measured directly after absorption and turnover, not at the same time. It is hypothetically possible to measure synthesis, turnover and absorption of cholesterol simultaneously using a triple stable isotope technique which combines the plasma dual isotope method and deuterium incorporation techniques, where 18O-cholesterol is administered intravenously, while 13C cholesterol and D2O is administered orally. Development of this method is currently being undertaken.
Methods used to quantify cholesterol absorption and synthesis reviewed in this article yield invaluable information, as well as provide effective means of measuring the experimental effects of various dietary, physiological and pharmacological interventions on whole body cholesterol trafficking and homeostasis. Methods have evolved substantially over the years, from radio-isotopes to stable isotopes, and from highly invasive to less invasive procedures. The advantages and drawbacks, as well as the type of information yielded by each approach, should be weighed carefully when selecting an appropriate method. The cost and available technical expertise and facilities will also limit which methods are available to each investigator. In sum, a veritable tool box of techniques that possess relative strengths and weaknesses are available to investigators, and from this array we must apply the most appropriate methods for each particular experimental question.