Introduction

The Scandinavian simvastatin survival study (4S) showed that simvastatin clearly decreases serum cholesterol concentration and considerably reduces mortality from all causes and coronary events and major coronary events in patients with coronary heart disease.¹ As the reduction in the relative risk of major coronary events was not associated with the baseline serum lipid concentrations in the survival study,² however, we considered that reduction in relative risk may be related to baseline intestinal absorption or to endogenous synthesis of cholesterol. From among the serum non-cholesterol sterols, the cholesterol precursor sterolsthat is, lanosterol and other methyl sterols and demethylated cholestenol, desmosterol, and lathosterolare directly related to cholesterol synthesis,^3-9 especially in the liver,¹⁰ while cholestanol and plant sterolsthat is, campesterol and sitosterolare directly related to cholesterol absorption. ^{9 11} Accordingly, we would expect quantification of these sterols to reveal subgroups with high or low absorption or synthesis of cholesterol. To this end we examined whether measurement of baseline serum cholestanol concentration in the Finnish coronary subpopulation of the survival study predicts the extent to which simvastatin could reduce the risk of major coronary events.

	Patients and methods

The whole study population of the survival study and the methods used have been reported previously. ^{1 12} The present study population included the Finnish subgroup of 868 patients with coronary heart disease selected from 1374 candidates for the 4444 participants of the original study and were randomised to placebo (n=434) or simvastatin 20-40 mg/day (n=434) for 5 years and 3 months. Distributions of age, sex, and lipid concentrations were similar in the placebo and simvastatin subgroups and were comparable with those in the main study. The secondary end point of the survival studymajor coronary events, including coronary deaths, non-fatal myocardial infarctions, and revascularisation procedureswas used for analytical purposes.

Concentrations of total and low and high density lipoprotein cholesterol and triglycerides were analysed at the central laboratory from serum obtained after an overnight fast.¹² Concentrations of cholesterol and non-cholesterol sterol were measured in two saponified baseline serum samples by gas-liquid chromatography on a 50 m long SE-30 capillary column (polydimethylsiloxane), with 5-cholestane as an internal standard ^{13 14} by running the samples from a single patient in a single batch from frozen stored serum samples. The non-cholesterol sterols are transported in serum by lipoproteins, about 70% by low density lipoprotein, so that the decrease in concentration of low density lipoprotein cholesterol by simvastatin also changes the serum concentration of cholestanol and other non-cholesterol sterols. We have therefore expressed the values of these sterols in terms of mmol/mol of cholesterolthat is, as ratios to cholesterol. The values given are means of two baseline determinations.

	Results

In the Finnish subgroup the mean changes in lipid concentrations caused by simvastatinthat is, 28, 35, 8, and 15% for total, low density lipoprotein, and high density lipoprotein cholesterol and triglycerides, respectivelywere similar to those seen in the main study population.¹

The reduction in the risk of major coronary events in the whole subgroup studied (144 v 114 events in placebo v simvastatin group, respectively) was 21% (0.790; 95% confidence interval 0.642 to 0.971). When we analysed gradually increasing body mass index and serum lipid concentrations from the first to the fourth quarter only cholestanol:cholesterol ratio exhibited percentages of recurrent events, gradually increasing from 21% to 35% (values shown only for cholestanol) with the increasing baseline sterol quarters in the simvastatin but not in placebo group (footnote to table 1). The corresponding relative risk increased gradually from 0.623 (0.395 to 0.982) to 1.166 (0.791 to 1.720). Thus, the relative risk of major coronary events was increased by 16.6% (20.9% to 72.0%) in the highest quarter of the cholestanol ratio and significantly reduced by 37.7% (60.5% to 1.8%) in the lowest quarter. The concentrations of cholestanol showed inconsistent changes in the relative risk ratios. Age and the concentrations of total and low density lipoprotein cholesterol were similar in all the quarters separated according to the ratios of cholestanol (table 2). The serum concentrations of high density lipoprotein cholesterol increased whereas the serum concentrations of triglycerides and body mass index values decreased. The ratios of cholestanol to cholesterol were negatively related to those of cholesterol precursor sterols (cholestanol, desmosterol, and lathosterol) and strongly positively related to those of plant sterols (campesterol and sitosterol; data not shown).

As in stepwise logistic regression analysis the continuous cholestanol ratio was the only variable of table 1 significantly entered into the model (odds ratio 1.01; 1.00 to 1.02), the association between cholestanol and the occurrence of major coronary events was tested by using a fixed logistic model with three variables: treatment group, cholestanol as an ordinal scale variable based on quarters, and treatment group*cholestanol interaction. In this model both the goodness of fit (Hosmer-Lemeshow test, P value 0.983) and the consistency of logistic function (C C Brown test, P value 1.000) were good. For the treatment group the odds ratio was 3.29 (1.53 to 7.06) and the Wald's test value 3.05 (P<0.001). For cholestanol the odds ratio was 1.31 (1.07 to 1.61). The risk of recurrence of major coronary events increased 2.2-fold between the lowest and highest quarters of the distribution of cholestanol ratios by multiple logistic regression analysis (P<0.01; Wald's test value 2.63). For the interaction term the odds ratio was 0.73 (0.56 to 0.86) and Wald's test 2.29 (P<0.02).

	Discussion

Multivariate logistic regression analysis indicated that the baseline lipid concentrations did not contribute to the results shown by the cholestanol ratio. The higher the quarter of the cholestanol ratio the greater was the risk of major coronary events. The risk of recurrence of major coronary events was 2.2-fold between the lowest and highest quarters of cholestanol, a finding not applicable to total, low density lipoprotein, or high density lipoprotein cholesterol. Thus, simvastatin treatment of patients with a low baseline cholestanol ratio predicts a clear reduction in risk of major coronary events, this improvement not being seen in patients with high baseline cholestanol ratio. Similar findings were also observed when the cholestanol ratios were used as continuous or dichotomised variables in statistical analysis. Thus, quantification of baseline cholestanol in patients with coronary heart disease would give important new information to clinicians or practitioners for evaluation of the future success of the statin treatment. The ability to predict the ineffectiveness of the relatively expensive statins in the treatment of coronary patients supports the use of such measurements. Today, automated instrumentation allows comparatively rapid gas liquid chromatographic measurements, each analysis measuring concentrations of cholesterol and several non-cholesterol sterols; the most predictive sterol for evaluation of treatment seems to be cholestanol.

Why resistance to statin treatment?
Why then are the coronary subjects with high cholestanol ratios resistant to reduction in recurrence of major coronary events? Cholestanol itself is hardly atherogenic, even though simvastatin increases the ratios of cholestanol and plant sterols,¹⁷ phytosterolaemia is strongly atherogenic,¹⁸ and high plant sterol concentrations may be atherogenic.¹⁹ The incidence of major coronary events was, however, unrelated to the ratios of cholestanol (or plant sterols) in the placebo group. As already noted, the cholestanol ratios are positively related to absorption and negatively to synthesis of cholesterol, ^{9 11} measured either by the sterol balance technique or cholesterol precursor sterols in serum. ^{3-9 20 21} Thus, we suggest that patients with high baseline cholestanol ratios do not respond because of high baseline absorption and low synthesis of cholesterol. Preliminary studies showed lower responses induced by simvastatin in serum cholesterol concentrations of subjects with high rather than low baseline cholestanol ratios.

Suggested treatment
The present findings for the first time relate cholesterol metabolism to statin induced changes in the reduction of the risk of coronary events in secondary prevention. The discovery before treatment of a subgroup of patients who do not respond to statin treatment alone suggests that such patients should be treated by a combination of statin with hypolipidaemic agents increasing cholesterol synthesisnamely, bile acid binding resins or sitostanol ester induced cholesterol malabsorption.²² The latter combination decreased low density lipoprotein cholesterol further by about 10%²³ and even more when it was combined with statins in patients with high baseline ratios of cholestanol and plant sterols.²⁴