Cite this asRocha Quintão EC (2023) Are measurements of non-cholesterol sterols in plasma useful in identifying susceptibility to atherosclerosis? Arch Prev Med 8(1): 001-007. DOI: 10.17352/apm.000031
Copyright© 2023 Rocha Quintão EC. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
This review discusses the validity of plasma non-cholesterol sterols precursors of cholesterol synthesis and phytosterols in the identification of human atherosclerosis. There is an insufficient demonstration that these sterols are valid methods of measurement of cholesterol metabolism. All markers, including cholestanol, that derive from cholesterol synthesis may only reflect body retention of sterols and not necessarily increased intestinal absorption. Also, in most studies, conventional risk factors of atherosclerosis, such as obesity, diabetes mellitus, gender, and age were not taken into account.
In recent years reviews related plasma non-cholesterol sterols as markers of atherosclerosis [1,2]. However, the role of these markers in atherosclerosis may be hampered by the interference of two factors, namely, doubts that these markers adequately identify alterations in cholesterol metabolism and the frequent exclusion in most studies of the interference of conventional risk factors on atherosclerosis. In one review on Cardiovascular Disease [CVD] risk, sitosterol and campesterol a generally increase or don’t vary in different metabolic disorders, however, data were not corrected for the interference of the conventional multiple independent risk factors for CVD . This was also the case in another review where cholesterol synthesis and intestinal absorption, in general, vary reciprocally as expected . These two points are addressed in the present review.
Since the early 1980s publications by TA Miettinen have presented plasma non-cholesterol sterol precursors of cholesterol as markers of cholesterol synthesis and phytosterols as intestinal cholesterol absorption markers [4-6]. Most papers correctly express the sterol results by plasma cholesterol concentration since non-cholesterol sterol values are dependent on the serum lipoprotein concentrations . For this reason, the present review excluded investigations expressing sterol values exclusively by plasma volume [8-12] whose indiscriminate use has previously been criticized . Therefore, this review analyzed only studies employing plasma sterol data properly corrected for plasma cholesterol.
Plasma phytosterols have been employed as markers of intestinal cholesterol absorption but their validity needs to be proven. One investigation supports their validity where the efficiency of intestinal cholesterol absorption is not related to apoE-LP phenotypes, but, as expected, plasma campesterol correlated with cholesterol absorption measured isotopically . Although the study included normal controls and hyperlipidemia cases, similar plasma phytosterol correlations were disclosed in both groups . Nonetheless, objection to the plasma phytosterol measurement as a method to measure cholesterol absorption was raised by the work of L Jakulj, et al , showing that in two groups that differed according to plasma campesterol concentrations the percent of dietary cholesterol absorbed did not differ when values were measured by combining labeled cholesterol administration with fecal sterol balance which is the gold standard procedure of intestinal cholesterol absorption measurement. Nonetheless, in their work, serum concentrations of campesterol and lathosterol varied reciprocally in the two groups as expected. Furthermore, their work improperly identified the absorption percentage with the total quantity of cholesterol absorbed. It follows from their completely different plasma lathosterol values in the two groups that substantially diverse amounts of endogenous cholesterol are excreted in bile and into the lumen of the intestine. Research has shown that the flow of cholesterol in bile is also modified when the amount of dietary cholesterol absorbed varies [6,15]. Because the marker of cholesterol synthesis [lathosterol] is 66% higher in the low campesterol as compared to the high campesterol group it is concluded that a greater mass of biliary cholesterol diminishes the absorption of dietary cholesterol and phytosterols present in the lumen of the intestine. Thus, the 25% dietary cholesterol absorption in both groups shown by L. Jakulj denotes very different total quantities of cholesterol absorbed, namely, the alimentary and biliary sources considered together as previously demonstrated [15,16]. In this sense, the work of L. Jakulk was critically evaluated by S. M Grundy  concluding that isotopic measurements of cholesterol absorption fail to quantify the amount absorbed due to the influence of biliary cholesterol secretion. Furthermore, their results might be biased by the interference of other factors in their cases such as a higher BMI in the low plasma campesterol group explaining greater cholesterol synthesis [6,14], and also increased plasma cholestanol likely derived from cholesterol synthesis .
Lathosterol, along with other markers, such as lanosterol and squalene, effectively measures cholesterol synthesis by the hepatic enzyme HMGCoA reductase activity that mirrors the simultaneously measured fecal cholesterol utilizing the balance technique . Support for this conclusion on lathosterol is afforded by the gold standard fecal balance procedure utilizing labeled cholesterol [6,19]. Also, according to research on humans treated with drugs that modify cholesterol metabolism, the activity of the hepatic enzyme HMGCoA reductase agrees with the results drawn from the sterol precursors of cholesterol synthesis such as squalene, lanosterol, and lathosterol . In a male population, cholesterol synthesis measured by the fecal sterol balance technique correlates with plasma concentrations of desmosterol and lathosterol . Plasma lathosterol increases because it measures the synthesis rate of cholesterol that simultaneously rises when the intestinal absorption of cholesterol is blocked by dietary plant sterols [21-23], sitostanol-supplemented margarine , or ezetimibe . Furthermore, as expected, drugs tailored to reduce cholesterol synthesis, such as statins, diminish the concentrations of cholesterol precursors in plasma [25,26]. According to several publications, plasma phytosterols vary inversely with non-cholesterol sterol precursor concentrations representing cholesterol synthesis, such as lathosterol [2,6,14,19,27-34]. This is due to the increased intake of phytosterols lowering plasma cholesterol by blocking the intestinal absorption of cholesterol thus raising the body’s cholesterol synthesis rate, a situation in which under no circumstance plasma phytosterol measurements represent increased absorption of cholesterol. In conclusion, in plasma increased phytosterols may mirror decreased absorption of cholesterol from food. Furthermore, decreased plasma phytosterol signifies decreased absorption of alimentary phytosterols and cholesterol, but does not identify the total amount of cholesterol absorbed from the intestinal lumen.
Cholestanol has been considered a marker of intestinal cholesterol absorption because often cholestanol behaves similarly to the phytosterols campesterol and sitosterol utilized as cholesterol absorption markers [6,14,30,31,35-37]. In another investigation, also utilizing oral administration of isotopic cholesterol in fecal cholesterol balance, a correlation of cholesterol absorption was found with plasma cholestanol in controls and postmenopausal women, and with sitosterol and campesterol in postmenopausal women alone although the percent cholesterol absorption was similar in both groups . However, considering that cholestanol is a metabolite of cholesterol, these results could be attributed to the body’s sterol retention which is indistinguishable from the amount of sterol absorbed from the gut. Retention means difficulty in excreting any sterols and not necessarily increased intestinal cholesterol uptake efficiency . A typical demonstration of the existence of retention mistakenly called increased absorption was demonstrated in a case of sitosterolemia a genetic disorder in which the sitosterol metabolic defect was largely corrected by liver transplantation . Consequently, sitosterolemia cannot be attributed to a defect in the intestinal absorption of phytosterols but to an impediment in their bodily excretion via bile. Also, retention of sterols in the body due to difficulty in fecal excretion of sterols has long been reported in other conditions such as familial hypercholesterolemia  and secondary hyperlipidemias such as in experimental nephrotic syndrome [41,42].
In the presence of retention, the finding in plasma of decreased values of precursors of cholesterol synthesis suggests that the latter would have been even lower if the retention process had not taken place. This metabolic problem is exemplified in sitosterolemia, a genetic disease in which the sterol retention process typically occurs [43-48]. Accordingly, the simultaneously elevated plasma concentrations of plant sterols and cholestanol together with cholesterol, serve to demonstrate that blockage in their biliary excretion [38,42-48] may also combine with increased cholestanol synthesis . In fact, in the genetic disease sitosterolemia, there is an increase in plasma cholestanol concentration  and in its synthesis by a different metabolic pathway than the regular one that occurs through the production of 7α-hydroxycholesterol .
Blockade of re-excretion of the alimentary cholesterol in bile as a major mechanism of plasma sterol elevation has been demonstrated in genetically hypercholesterolemic mice on a cholesterol-free diet  and when mice are subjected to a phytosterol-rich diet . Furthermore, a higher intake of phytosterols may hinder the reabsorption of cholestanol excreted in the bile resulting in a lower plasma concentration of the latter . Consequently, cholestanol may vary in the plasma due to changes in its synthesis, intestinal absorption, and excretion by bile, making it difficult to interpret its variation in plasma as a marker of intestinal absorption of cholesterol. In this regard, the presence of cholestanol in plasma was only assessed in mice and was attributed to a combination of excretion in the bile and efflux from the intestinal mucosa back into the lumen .
Conflicting interpretations of the populational data can occur due to bias attributed to the interference of several factors such as age, gender, dietary patterns, as well as clinical conditions like diabetes, hyperlipidemia of genetic origin, obesity, metabolic syndrome, hypertension, and smoking that are independently associated with atherosclerosis [52-58]. All these factors interfere with concentration markers of synthesis or absorption of cholesterol, and often with both types of markers simultaneously. In type 2 diabetes mellitus sitosterol, campesterol, and cholestanol are increased together with synthesis markers . In the latter review synthesis and absorption markers, as would be expected, differed from each other but were not corrected for BMI as required  on which they typically depend . In type 1 diabetes mellitus sitosterol, campesterol and cholestanol are not modified, whereas desmosterol is not modified and lathosterol is either not modified or diminishes . Therefore, utilizing these sterols’ non-cholesterol synthesis precursors as proper methods of cholesterol synthesis measurement to identify disturbances of cholesterol metabolism in human pathologies deserves to be analyzed in all publications dealing with CVD. This becomes clear when analyzing the results of seventeen publications on the subject summarized in Table [30,31,33,35,37,61-72]. Independent risk factors that may have influenced cholesterol metabolism were not reported in only one paper , adjustments were duly included in another regarding age, BMI, and plasma glucose  and adjustments for several conventional CV risk factors were done only in six reports [30,33,37,61,62,72]. Curiously, in one of them  CAD cases had elevated squalene, campesterol, sitosterol, and desmosterol, but lower lathosterol which is contradictory. Data on these sterols were similar in controls and the CAD cases investigated . In another study, the results of synthesis markers were discrepant: CAD-positive cases presented low desmosterol and high lathosterol levels although both are markers of cholesterol synthesis .
The problem becomes even more confusing with the demonstration that fecal excretion of endogenous cholesterol, an indication of cholesterol synthesis is negatively associated with carotid atherosclerosis . However, this study includes a high proportion of cases treated with statins and other medications that interfere with lipoprotein metabolism.
In conclusion, in all other investigations, the results of non-cholesterol sterol markers in plasma could have been influenced by one or more conventional independent risk factors of coronary heart disease. Furthermore, variable results were obtained regarding the synthesis and absorption markers, thus questioning the validity of their usefulness in atherosclerosis. Accordingly, synthesis markers were increased in four studies [33,37,62,67], diminished in five [31,64,69,70.72], not altered in seven [30,41,61-63,69,71], not reported in three [66-68] and incongruous in three [37,62,70]. Absorption markers were increased in atherosclerosis in nine investigations [31,33,35,37,61,64,67,69,72], diminished in three [30,63,68], not altered in five [62,66,69-71] and not reported in one , but incongruities did not occur.
It was possible to trust the results of plasma sterols as related to atherosclerosis only in eight publications because corrections for the various conventional risk factors were provided [30,33,35,37,61,62,67,72], even though synthesis markers in plasma have been validated, nevertheless, it was not possible to conclude on studies where synthesis markers did not vary [30,35,61,63,69,71] Table 1.
At present, measurements of these sterols in plasma do not reliably portray the degree of human atherosclerosis. Future investigations need to provide corrections for conventional cardiovascular risk factors hoping that systematically consistent results will emerge to validate plasma sterols as markers of atherosclerosis. Furthermore, the validity of the markers can be deduced provided that the role of their retention in plasma is accounted for which is currently not feasible due to the lack of adequate methods.
This review suggests that the presence of simultaneous plasma elevations of cholestanol, a product of cholesterol synthesis, and phytosterols may indicate an increase in sterol retention elicited by some conventional risk factors for atherosclerosis or drug action that need to be clarified in future experiments.
The author confirms sole responsibility for the following: study conception and design, data collection, analysis and interpretation of results, and manuscript preparation.
This work was supported by research grants from Fundação de Amparo a Pesquisa do Estado de São Paulo – FAPESP (2021/04366-6).
Data sharing is not applicable to this review because no new data were presented or analyzed in this study.
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