Simultaneous determination of plasmatic phytosterols and cholesterol precursors using gas chromatography-mass spectrometry (GC-MS) with selective ion monitoring (SIM)

Phytosterols (beta-sitosterol, cholestanol and campesterol) and cholesterol precursors (desmosterol and lathosterol), have been suggested as important biochemical markers of intestinal cholesterol absorption and liver biosynthesis, respectively, and as useful clinical parameters in the study of hypercholesterolemia, beta-sitosterolemia, atherosclerosis and cardiovascular disease, including pharmacological response to hypolipidemic agents. We developed an optimised analytical method for the simultaneous analysis of cholestanol, desmosterol, lathosterol, campesterol and beta-sitosterol in plasma using capillary gas chromatography coupled to mass spectrometry (GC-MS) with multiple selected ion monitoring (SIM). This method is based on the alkaline hydrolysis of sterol esters, extraction of free sterols and derivatization. The recovery of all sterols was in the range 76-101%. Within-day relative standard deviations (R.S.Ds.) and the between-day R.S.Ds. of cholestanol, desmosterol, lathosterol, campesterol and beta-sitosterol were less than 8%, and their plasma levels in 161 normal subjects were (mean+/-S.D.) 4.73+/-2.57, 2.37+/-1.04, 6.23+/-3.14, 3.67+/-1.95 and 5.92+/-3.62 micromol/l, respectively.     

Genetic analysis of indicators of cholesterol synthesis and absorption: lathosterol and phytosterols in Dutch twins and their parents.

Significant familial aggregation was observed for plasma levels of lathosterol (an indicator of whole-body cholesterol synthesis) and plant sterols campesterol and beta-sitosterol (indicators of cholesterol absorption) in 160 Dutch families consisting of adolescent mono- and dizygotic twin pairs and their parents. For lathosterol a moderate genetic heritability in parents and offspring (29%) was found. In addition, shared environment also contributed significantly (37%) to variation in plasma lathosterol concentrations in twin siblings. However, a model with different genetic heritabilities in the two generations (10% in parents and 68% in offspring) fitted the data almost as well. For plasma plant sterol concentrations high heritabilities were found. For campesterol heritability was 80% and for beta-sitosterol it was 73%, without evidence for differences in heritability between sexes or generations. No influence of common environmental influences shared by family members was seen for either campesterol or beta-sitosterol. Taken together, these results confirm and expand the hypothesis that individual differences in plasma levels of noncholesterol sterols are moderately (lathosterol) to highly (plant sterols) heritable.     

Decreased content,rate of synthesis and export of cholesterol in the brain of apoE knockout mice

Apolipoprotein E knockout (apoE-KO) mice present synaptic loss, cognitive dysfunction, and high plasma lipid levels that may affect brain function simulating Alzheimer disease. Plasma and brain sterols were measured in apoE-KO and in wild type control mice on a cholesterol-free, phytosterol-containing diet by gas chromatography coupled to a mass spectrometer. Plasma cholesterol and phytosterols (campesterol and sitosterol) were higher in apoE-KO compared to control mice. Cholesterol precursors (desmosterol and lathosterol) were not detected in plasma of control mice but were present in apoE-KO mice. In the brain amounts of cholesterol, desmosterol, campesterol and 24-hydroxycholesterol were significantly lower in apoE-KO than in controls. There is a tendency in apoE-KO for lower values of 7α-hydroxycholesterol and 7β-hydroxycholesterol. Cholesterol content, synthesis rates (desmosterol) and export of 24-hydroxycholesterol are reduced in the brain of the severe hypercholesterolemic apoE-KO mice.     

Alterations in cholesterol absorption/synthesis markers characterize Framingham Offspring Study participants with CHD

Data is limited on measures influencing cholesterol homeostasis in subjects at high risk of developing cardiovascular disease (CVD) relative to established risk factors. To address this, we quantified circulating indicators of cholesterol homeostasis (plasma phytosterols and cholesterol precursor concentrations as surrogate measures of cholesterol absorption and synthesis, respectively) in Framingham Offspring Study Cycle-6 participants diagnosed with established CVD and/or ≥50% carotid stenosis not taking lipid lowering medication (cases, N = 155) and matched controls (N = 414). Cases and controls had similar plasma LDL-cholesterol; HDL-cholesterol was significantly lower in males, while triglyceride concentrations were significantly higher in female cases relative to their respective controls. Cholesterol absorption markers were significantly higher (229 ± 7 vs. 196 ± 4, 169 ± 6 vs. 149 ± 3 and 144 ± 5 vs. 135 ± 3 for campesterol, sitosterol, and cholestanol, respectively), whereas cholesterol synthesis markers were significantly lower (116 ± 4 vs. 138 ± 3, 73 ± 3 vs. 75 ± 2 for lathosterol and desmosterol, respectively) in cases compared with controls, irrespective of sex. After controlling for standard risk factors, campesterol (2.47 [1.71-3.56]; P < 0.0001), sitosterol (1.86 [1.38-2.50]; P < 0.0001), cholestanol (1.57 [1.09-2.27]; P = 0.02), desmosterol (0.59 [0.42-0.84]; P = 0.003), and lathosterol (0.58 [0.43-0.77]; P = 0.0002) were significantly associated with CVD (odds ratio [95% confidence interval]). These data suggest that impaired cholesterol homeostasis, reflected by lower synthesis and higher absorption marker concentrations, are highly significant independent predictors of prevalent CVD in this study population.     

Sterols in the plasma and digestive gland-gonad complex of Biomphalaria glabrata snails, fed lettuce versus hen's egg yolk, as determined by GLC

1. Gas-liquid chromatography studies were done on sterols in plasma and the digestive gland-gonad (DGG) complex of Biomphalaria glabrata snails fed lettuce vs hen's egg yolk. 2. The major sterols present in the DGG of both populations were cholesterol, stigmasterol, beta-sitosterol, campesterol, and desmosterol. 3. The percentage composition of cholesterol in the DGG of yolk vs lettuce fed snails was 82 and 51, respectively. 4. The elution profiles of sterols in the plasma of yolk vs lettuce fed snails were similar; both contained desmosterol, campesterol, and stigmasterol, negligible amounts of cholesterol, and unidentified sterols. 5. The high lipid diet increased the level of cholesterol in the DGG but not in the plasma.     

Schistosoma mansoni: sterol and phospholipid composition of cercariae, schistosomula, and adults

The sterol and phospholipid composition of cercariae, schistosomula, and adult Schistosoma mansoni was analyzed by gas-liquid chromatography and high-performance liquid chromatography (HPLC). Cercariae and schistosomula contained cholesterol, desmosterol, campesterol, stigmasterol, and beta-sitosterol while adults contained only cholesterol. In all stages cholesterol comprised greater than 50% of the total sterols, and in cercariae and schistosomula desmosterol comprised 38 and 21% of the total sterols, respectively. The other three sterols, campesterol, stigmasterol, and beta-sitosterol, made up approximately 10% of the total. The same five sterols found in cercariae and schistosomula were present in the hepatopancreas of uninfected snails but with a much higher desmosterol concentration in the parasite, 38%, than in the snail, 2%. As in cercariae and schistosomula the three minor sterols comprised approximately 10%. Thus, the sterol composition of cercariae and schistosomula was similar but not identical to that of the snail host. Phosphatidylcholine was the major phospholipid of all three stages (50%) as determined by two HPLC procedures. The remaining phospholipids consisted of phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. In addition, in adults there were small quantities of sphingomyelin and lysophosphatidylcholine. The percentage of each phospholipid was similar among stages with the exception of a slight increase in phosphatidylserine in adults compared to cercariae and schistosomula. These results show that a characteristic lipid composition is found in cercariae, schistosomula, and adults.     

Profile of cholesterol-related sterols in aged amyloid precursor protein transgenic mouse brain

Cholesterol is implicated to play a role in Alzheimer disease pathology. Therefore, the concentrations of cholesterol, its precursors, and its degradation products in brain homogenates of aging wild-type and beta-amyloid precursor protein transgenic mice carrying the Swedish mutation (APP23) were analyzed. Among the sterols measured, lanosterol is the first common intermediate of two different pathways, which use either desmosterol or lathosterol as the predominant precursors for de novo synthesis of brain cholesterol. In young mice, cholesterol is mainly synthesized via the desmosterol pathway, while in aged mice, lathosterol is the major precursor. 24S-hydroxycholesterol (cerebrosterol), which plays a key role in the removal of cholesterol from the brain, modestly increased during aging. No differences in the levels of cholesterol, its precursors, or its metabolites were found between wild-type and APP23 transgenic mice. Moreover, the levels of the exogenous plant sterols campesterol and sitosterol were significantly elevated in the brains of APP23 animals at age 12 and 18 months. This time point coincides with abundant plaque formation.     

Polymorphisms in the ABCG5 and ABCG8 genes associate with cholesterol absorption and insulin sensitivity

The roles of polymorphisms of the sitosterolemia genes ABCG5 and ABCG8 in the regulation of cholesterol metabolism and insulin sensitivity were studied in mildly hypercholesterolemic noncoronary subjects (n = 263, 144 men and 119 women) divided into tertiles by baseline serum cholestanol-to-cholesterol ratio (< or = 118.3 and > or = 147.7 10(2) x mmol/mol cholesterol), a surrogate marker of cholesterol absorption efficiency. The lowest cholestanol tertile was associated with high body mass index (BMI), plasma glucose, serum insulin and triglycerides, and cholesterol synthesis markers (cholestenol, desmosterol, lathosterol) and low HDL cholesterol and cholesterol absorption markers (campesterol, sitosterol) (P < 0.01 for all). The 19H allele of the ABCG8 gene accumulated in the lowest cholestanol tertile (P < 0.001) and was associated with low total and LDL cholesterol and absorption markers and with high synthesis markers (P < 0.05 for all). The 604E allele of the ABCG5 gene in men was associated with high BMI, plasma insulin, low serum sitosterol, and high serum cholestenol levels (P < 0.05 for all). In a subgroup of 71 men, the 604E allele was associated with insulin resistance measured with the hyperinsulinemic euglycemic clamp. In conclusion, low cholesterol absorption efficiency was associated with characteristics of the metabolic syndrome. Low serum cholesterol and cholesterol absorption were linked to the D19H polymorphism of the ABCG8 gene, and characteristics of the insulin resistance syndrome in men were linked with the Q604E polymorphism of the ABCG5 gene.     

Cholesterol absorption and synthesis markers in individuals with and without a CHD event during pravastatin therapy: insights from the PROSPER trial

Cholesterol homeostasis, defined as the balance between absorption and synthesis, influences circulating cholesterol concentrations and subsequent coronary heart disease (CHD) risk. Statin therapy targets the rate-limiting enzyme in cholesterol biosynthesis and is efficacious in lowering CHD events and mortality. Nonetheless, CHD events still occur in some treated patients. To address differences in outcome during pravastatin therapy (40 mg/day), plasma markers of cholesterol synthesis (desmosterol, lathosterol) and fractional cholesterol absorption (campesterol, sitosterol) were measured, baseline and on treatment, in the Prospective Study of Pravastatin in the Elderly at Risk trial participants with (cases, n = 223) and without (controls, n = 257) a CHD event. Pravastatin therapy decreased plasma LDL-cholesterol and triglycerides and increased HDL-cholesterol concentrations to a similar extent in cases and controls. Decreased concentrations of the cholesterol synthesis markers desmosterol (−12% and −11%) and lathosterol (−50% and −56%) and increased concentrations of the cholesterol absorption markers campesterol (48% and 51%) and sitosterol (25% and 26%) were observed on treatment, but the magnitude of change was similar between cases and controls. These data suggest that decreases in cholesterol synthesis in response to pravastatin treatment were accompanied by modest compensatory increases in fractional cholesterol absorption. The magnitude of these alterations were similar between cases and controls and do not explain differences in outcomes with pravastatin treatment.     

High levels of plant sterols and cholesterol precursors in cerebrotendinous xanthomatosis

We measured the cholestanol, cholesterol precursor (lathosterol), and plant sterol (campesterol and sitosterol) concentrations of serum and bile in 11 patients with cerebrotendinous xanthomatosis. The mean values of serum cholestanol, lathosterol, campesterol, and sitosterol were, respectively, 8.4-, 2.5-, 2.7-, and 1.4-times higher in the patients than in normal control subjects (n = 26). Cholestanol (6.7-fold) and campesterol (3.7-fold) levels in bile (n = 4) were also elevated in the patients. There was no significant difference of serum sterol levels between patients with coronary artery disease and those without it. Chenodeoxycholic acid treatment for periods ranging from 6 months to 3 years and 4 months lowered serum lathosterol (57.7% reduction) and campesterol (57.8%) levels in parallel with cholestanol (70.8%) level, but the sitosterol level (19.7%) decreased less. Thus, increased levels of cholesterol precursor (lathosterol), plant sterols (campesterol and sitosterol), and cholestanol were found in the serum and bile in cerebrotendinous xanthomatosis. Chenodeoxycholic acid treatment effectively reduced the levels of these sterols, except for sitosterol.     

Plasma non-cholesterol sterols in patients with non-insulin dependent diabetes mellitus.

Plasma plant sterol concentrations (an index of cholesterol absorption efficiency) and plasma lathosterol concentration (an index of cholesterol synthesis rate) were measured in 52 patients with non-insulin dependent diabetes mellitus (NIDDM) and 36 non-diabetic controls. Plasma plant sterol concentrations were significantly (P less than 0.01) lower in diabetic patients (campesterol: men -36%, women -48%; betasitosterol: men -35%, women -42%). Fasting serum insulin levels were inversely correlated with plasma plant sterol concentrations in diabetic patients (campesterol: r = -0.347, P = 0.012; betasitosterol: r = -0.345, P = 0.012) and in non-diabetic men (campesterol: r = -0.578, P = 0.039; betasitosterol: r = -0.702, P = 0.008). Serum insulin levels were also correlated significantly with plasma lathosterol concentration in diabetic patients (r = 0.295, P = 0.034). The results of this study suggest that absorption of plant sterols and possibly cholesterol from the diet may be reduced in hyperinsulinemic diabetics.     

Effect of exogenous steroids on sterol synthesis in L-cell mouse fibroblasts

A number of steroids have been tested in an L-cell tissue culture system to determine their effects on cellular sterol biosynthesis and cellular growth. Cholesterol, desmosterol, lathosterol, 7-dehydrocholesterol, and cholestanone reduce de novo synthesis and produce only limited toxicity at high concentrations of exogenous sterol. Considerable cellular toxicity is observed when cells are grown in the presence of coprostanol and Delta(4)-cholestenone. No marked effect on either cell growth or sterol biosynthesis is produced by cholestanol, beta-sitosterol, stigmasterol, campesterol, ergosterol, cholesteryl oleate, or cholestane.     

The relationships of cholesterol metabolism and plasma plant sterols with the severity of coronary artery disease

Changes in the balance of cholesterol absorption and synthesis and moderately elevated plasma plant sterols have been suggested to be atherogenic. Measuring cholestanol, lathosterol, campesterol, and sitosterol, we investigated the relationships of cholesterol metabolism and plasma plant sterols with the severity of coronary artery disease (CAD) in 2,440 participants of the Ludwigshafen Risk and Cardiovascular health (LURIC) study. The coronary status was determined by angiography, and the severity of CAD was assessed by the Friesinger Score (FS). An increase in the ratio of cholestanol to cholesterol was associated with high FS (P = 0.006). In contrast, a high ratio of lathosterol to cholesterol went in parallel with low FS (P < 0.001). Whereas the campesterol to cholesterol ratio significantly correlated with the FS (P = 0.026), the relationship of the sitosterol to cholesterol ratio with the FS did not reach statistical significance in the whole group. Increased campesterol, sitosterol, and cholestanol to lathosterol ratios were associated high FS (P < 0.001). To conclude, there is a modest association of high cholesterol absorption and low cholesterol synthesis with an increased severity of CAD. An atherogenic role of plasma plant sterols themselves, however, seems unlikely in subjects without sitosterolaemia.     

Composition of plasma fatty acids and non-cholesterol sterols in anorexia nervosa

Anorexia nervosa is a model of simple starvation accompanied by secondary hyperlipoproteinemia. The pattern of plasma fatty acids influences the levels of plasma lipids and lipoproteins. The concentration of plasma lathosterol is a surrogate marker of cholesterol synthesis de novo, concentrations of campesterol and beta-sitosterol reflect resorption of exogenous cholesterol. The aim of the study was to evaluate fatty acids in plasma lipid classes and their relationship to plasma lipids, lipoproteins, cholesterol precursors and plant sterols. We examined 16 women with anorexia nervosa and 25 healthy ones. Patients with anorexia nervosa revealed increased concentrations of total cholesterol, triglycerides, HDL-cholesterol, campesterol and beta-sitosterol. Moreover, a decreased content of n-6 polyunsaturated fatty acids was found in all lipid classes. These changes were compensated by an increased content of monounsaturated fatty acids in cholesteryl esters, saturated fatty acids in triglycerides and both monounsaturated and saturated fatty acids in phosphatidylcholine. The most consistent finding in the fatty acid pattern concerned a decreased content of linoleic acid and a raised content of palmitoleic acid in all lipid classes. The changes of plasma lipids and lipoproteins in anorexia nervosa are the result of complex mechanisms including decreased catabolism of triglyceride-rich lipoproteins, normal rate of cholesterol synthesis and increased resorption of exogenous cholesterol.     

Effects of endurance exercise training on markers of cholesterol absorption and synthesis

Abnormal cholesterol metabolism, including low intestinal cholesterol absorption and elevated synthesis, is prevalent in diabetes, obesity, hyperlipidemia, and the metabolic syndrome. Diet-induced weight loss improves cholesterol absorption in these populations, but it is not known if endurance exercise training also improves cholesterol homeostasis. To examine this, we measured circulating levels of campesterol, sitosterol, and lathosterol in 65 sedentary subjects (average age 59 years; with at least one metabolic syndrome risk factor) before and after 6 months of endurance exercise training. Campesterol and sitosterol are plant sterols that correlate with intestinal cholesterol absorption, while lathosterol is a marker of whole body cholesterol synthesis. Following the intervention, plant sterol levels were increased by 10% (p<0.05), but there was no change in plasma lathosterol. In addition, total and LDL-cholesterol were reduced by 0.16 mmol and 0.10 mmol, respectively (p<0.05), while HDL-C levels increased by 0.09 mmol (p<0.05). Furthermore, the change in plant sterols was positively correlated with the change in VO2max (r=0.310, p=0.004), independent of other metabolic syndrome risk factors. These data indicate that exercise training reduces plasma cholesterol despite increasing cholesterol absorption in subjects with metabolic syndrome risk factors.     

Plasma noncholesterol sterols in male distance runners and sedentary men

Plasma lathosterol concentration is taken to be an index of the rate of cholesterol synthesis and plasma concentrations of plant sterols just as campesterol and betasitosterol are taken to be indeces of cholesterol absorption efficiency. These noncholesterol sterols were measured in plasma from 14 male distance runners and 10 sedentary men. Plasma lathosterol concentration was 30% lower (P less than 0.02) and plasma betasitosterol concentration was 33% higher (P less than 0.02) in the runners compared to the sedentary men. Plasma concentrations of lathosterol and plant sterols were inversely and significantly (P less than 0.05) correlated in both the runners and the sedentary men. Plasma plant sterol concentrations were correlated positively and significantly (P less than 0.01) with plasma high density lipoprotein cholesterol (HDL-C) concentrations in the runners and sedentary men combined. These findings suggest that more efficient cholesterol absorption may lead to higher plasma plant sterol concentrations and may contribute to lower cholesterol synthesis rates, reduced concentrations of plasma lathosterol and higher plasma HDL-C concentration in distance runners.     

Absorbability of plant sterols and their distribution in rabbit tissues

Rabbits were fed a low cholesterol diet containing 2% plant sterols for 10 weeks to determine the absorbability of these sterols and their deposition in the tissues. We found campesterol and beta-sitosterol in the blood and tissues. The plasma campesterol levels were 4.34--13.3 mg/100 ml, whereas, beta-sitosterol levels were 0.41--1 mg/100 ml. Stigmasterol was not detected. The total plasma plant sterol concentration was about 10% of the total plasma sterol. The mean terminal plasma cholesterol concentration averaged 60% higher (55 vs. 88 mg/100 ml, P less than 0.001) than the mean initial value. Campesterol was the preponderant sterol in all tissues studied, including the aorta. Sitosterol was found in small amounts in the tissues of the abdominal organs. Stigmasterol was not detected in any tissue studied. Esterified campesterol and sitosterol were detected in trace amounts in most tissues. Campesterol and sitosterol, particularly the former, accumulated in the tissues including the aorta.     

Injected phytosterols/stanols suppress plasma cholesterol levels in hamsters

Although plant sterols are known to suppress intestinal cholesterol absorption, whether plasma and hepatic lipid levels are influenced through non-gut related internal mechanisms has not been established. To examine this question 50 male hamsters were divided into 5 groups and fed semi-purified diets containing 20% energy as fat and 0.25% (w/w) cholesterol ad libitum for 60 days. The control group (i) received diet alone, while four additional groups consumed the diet plus one of four equivalent phytosterol mixtures (5 mg/kg/day) given either as (ii) tall oil phytosterols/stanols mixed with diet (oralSA), (iii) tall oil phytosterols/stanols subcutaneously injected (subSA), (iv) soybean oil phytosterols alone mixed with diet (oralSE), or (v) soybean oil subcutaneous injected phytosterols alone (subSE). The control group and both orally supplemented groups also received placebo subcutaneous sham injections. Neither food consumption, body weight, nor liver weight differed across treatment groups. Subcutaneous administration of SA and SE decreased plasma total cholesterol levels by 21% and 23% (p < 0.0001) and non-apolipoprotein-A cholesterol concentrations by 22% and 15% (p < 0.0002), respectively, compared to control. HDL cholesterol and TG concentrations remained unchanged across all groups, except for a decline of 25% (p < 0.0001) in HDL concentration in the subSE group versus control. Plasma campesterol levels were lower (p < 0.05) in the subSA group relative to all other groups. Plasma campesterol:cholesterol and campesterol:sitosterol ratios were, however, higher (p < 0.0001) for both the oral and subSE groups. Hepatic cholesterol levels were higher (p < 0.0001) in the oral and subSE phytosterol groups by 30% and 31%, respectively, relative to control. We conclude that low doses of subcutaneously administered plant sterols reduce circulating cholesterol levels through mechanisms other than inhibiting intestinal cholesterol absorption.     

Effect of phytosterols on cholesterol metabolism and MAP kinase in MDA-MB-231 human breast cancer cells

Epidemiological studies suggest that dietary phytosterols may offer protection form some types of cancer including breast cancer. In an attempt to investigate the mechanism by which phytosterols offer this protection, we investigated the effect of the two most common dietary phytosterols, beta-sitosterol and campesterol, on the mevalonate and MAP Kinase (MAPK) pathways in MDA-MB-231 cells. These pathways play a role in cell growth and apoptosis. MDA-MB-231 cell line was used in this study since it is a hormone-insensitive tumor cell line which represents the majority of advanced breast cancer cases. Cells grown in the presence of 16 microM beta-sitosterol or campesterol for 3 days exhibited a 70% and 6% reduction in cell growth, respectively, while cholesterol treatment had no effect on growth as compared to the control. Studies investigating the effect of sterol supplementation on the relative and total sterol composition of cells, showed that cells supplemented with cholesterol contained 23% more cholesterol than the control. Cells supplemented with campesterol had almost one-half the cholesterol of controls but accumulated campesterol to account for 40% of the total sterols. In the case of cells supplemented with beta-sitosterol, cells had only 25% of their sterols as cholesterol and the rest was in the form of beta-sitosterol. All sterols tested equally inhibited de novo cholesterol synthesis using 14C-acetate as substrate. beta-Sitosterol supplemented cells had reduced cholesterol synthesis when using 3H-mevalonolactone as substrate, which suggests that the inhibition in this pathway is downstream of mevalonate where processes such as isoprenylation of proteins may take place. Mevalonate supplementation to cells treated with beta-sitosterol did not completely correct the observed growth inhibition by beta-sitosterol. There was no effect of sterols on the concentrations of both low (21-26 kDa) or high (44-74 kDa) molecular weight isoprenylated proteins in these cells. On the other hand, both the quantity and activity of MAPK was elevated in the cells supplemented with beta-sitosterol. These data suggest that the down regulation of cholesterol synthesis from mevalonate and stimulation of the MAPK pathway may play roles in the inhibition of MDA-MB-231 cell growth by beta-sitosterol.     

Cholesterol esterification and atherogenic index of plasma correlate with lipoprotein size and findings on coronary angiography

We examined the association between rate of cholesterol esterification in plasma depleted of apolipoprotein B-containing lipoproteins (FER(HDL)), atherogenic index of plasma (AIP) [(log (TG/HDL-C)], concentrations, and size of lipoproteins and changes in coronary artery stenosis in participants in the HDL-Atherosclerosis Treatment Study. A total of 160 patients was treated with simvastatin (S), niacin (N), antioxidants (A) and placebo (P) in four regimens. FER(HDL) was measured using a radioassay; the size and concentration of lipoprotein subclasses were determined by nuclear magnetic resonance spectroscopy. The S+N and S+N+A therapy decreased AIP and FER(HDL), reduced total VLDL (mostly the large and medium size particles), decreased total LDL particles (mostly the small size), and increased total HDL particles (mostly the large size). FER(HDL) and AIP correlated negatively with particle sizes of HDL and LDL, positively with VLDL particle size, and closely with each other (r = 0.729). Changes in the proportions of small and large lipoprotein particles, which were reflected by FER(HDL) and AIP, corresponded with findings on coronary angiography. Logistic regression analysis of the changes in the coronary stenosis showed that probability of progression was best explained by FER(HDL) (P = 0.005). FER(HDL) and AIP reflect the actual composition of the lipoprotein spectrum and thus predict both the cardiovascular risk and effectiveness of therapy. AIP is already available for use in clinical practice as it can be readily calculated from the routine lipid profile.