A Validated Method for Quantification of Fatty Acids Incorporated in Human Plasma Phospholipids by Gas Chromatography-Triple Quadrupole Mass Spectrometry.

Fatty acids (FA) are important mediators of health maintenance and disease risk. Optimal quantification assays of FA in high and low abundance as well the identification of 13C-labeled tracers to monitor FA metabolism are of major interest. The article on hand reports about the development and validation of a gas chromatography (GC)-triple quadrupole mass selective detection (GC-TQMS) method for absolute quantification of FA in human plasma phospholipids (hpPL). The quantification of the calibration solution by GC-flame ionization detection (GC-FID), with the introduction of a correction factor, allows the direct comparison of individual FA concentrations in hpPL by GC-TQMS. Specificity, sensitivity, and reproducibility are achieved by optimized chromatographic separation and employment of GC-TQMS. The inter-method comparison between GC-FID and GC-TQMS concentrations revealed good comparability for 27 FA. A full validation has been performed with linearity over 4 magnitudes, a limit of detection of 0.18-38.3 fmol on column, a recovery of 83.6-109.6%, and intraday and interday precision data meeting the criteria of EMA and FDA guidelines. The method includes the absolute quantification of 58 positional and geometrical (cis/trans) isomeric FA in hpPL in the concentration range of 1-3000 nmol/mL, covering also low abundant positional cis/trans isomers. Results obtained from both methods are highly comparable, and selectivity and sensitivity are improved by using GC-TQMS. Additionally, we show here that calculation of 13C-labeled C16:0 tracer/tracee ratios in hpPL in human isotope enrichment studies is possible.

Low serum lathosterol levels associate with fatal cardiovascular disease and excess all-cause mortality: a prospective cohort study.

IMPORTANCE: A more precise identification of patients at "high cardiovascular risk" is preeminent in cardiovascular risk stratification. OBJECTIVE: To investigate the relationships between markers of cholesterol homeostasis, cardiovascular events and all-cause mortality. DESIGN, SETTING AND PARTICIPANTS: We quantified markers of cholesterol homeostasis by gas chromatography-mass spectrometry in 377 subjects with suspected coronary artery disease, who were not on lipid-lowering drugs at baseline. All patients were followed for occurrence of cardiovascular events and mortality over a period of 4.9 +/- 1.7 years. The standardized mortality ratio (SMR) was calculated as the ratio of the observed and the expected deaths based on the death rates of the Regional Databases Germany, and Poisson regression (rate ratio, RR) was used to compare subgroups. The SMR and RR were standardized for sex, age category and calendar period. In addition, Cox regression (Hazard ratio, HR) was used to determine the effect of co-variables on (cardiovascular) mortality within the cohort. MAIN OUTCOMES: Cardiovascular events, cardiovascular mortality and all-cause mortality. RESULTS: A total of 42 deaths were observed in 1818 person-years corresponding with an SMR of 0.99 (95% CI 0.71-1.33; p = 0.556). A fatal cardiovascular event occurred in 26 patients. Lower levels of lathosterol were associated with increased cardiovascular mortality (HR 1.59; 95% CI: 1.16-2.17; p = 0.004) and excess all-cause mortality (HR 1.41; 95% CI: 1.09-1.85; p = 0.011). Lower lathosterol tertile compared to the adjacent higher tertile was associated with 1.6 times higher all-cause mortality risk (RR 1.60; 95% CI 1.07-2.40; p for trend = 0.022). This corresponded with a 2.3 times higher mortality risk of a lathosterol-LDL ratio equal to or below the median (RR 2.29; 95% CI 1.19-4.43; p = 0.013). None of the other cholesterol homeostasis markers were associated with cardiovascular and all-cause mortality. CONCLUSIONS: In patients not on lipid-lowering agents, low serum lathosterol correlated with increased risk of cardiovascular events and excess all-cause mortality.

Plasma levels of the oxyphytosterol 7α-hydroxycampesterol are associated with cardiovascular events.

BACKGROUND AND AIMS: There are safety issues regarding plant sterol ester-enriched functional food. Oxidized plant sterols, also called oxyphytosterols, are supposed to contribute to plant sterol atherogenicity. This study aimed to analyze associations of plasma oxyphytosterol levels with cardiovascular events. METHODS: Plasma cholesterol was measured by gas chromatography-flame ionization detection. Plasma campesterol and sitosterol and their 7-oxygenated metabolites were analyzed by gas chromatography-mass selective detection. RESULTS: In 376 patients admitted for elective coronary angiography, who were not on lipid-lowering drugs, 82 cardiovascular events occurred during a follow-up period of 4.2 ±â€¯1.8 years. Patients with cardiovascular events had significantly higher 7α-hydroxycampesterol plasma levels (median, 0.46; [interquartile range (IQR) 0.22-0.81] nmol/L vs. median, 0.25 [IQR, 0.17-0.61] nmol/L; p = 0.003) and 7α-hydroxycampesterol-to-cholesterol ratios (median 0.08 [IQR, 0.04-0.14] nmol/mmol vs. median, 0.05 [IQR 0.03-0.11] nmol/mmol; p = 0.005) than controls without such events. Patients above the median were characterized by higher cumulative event rates in Kaplan-Meier-analysis (Logrank-test p = 0.084 and p = 0.025) for absolute and cholesterol corrected 7α-hydroxycampesterol, respectively. After adjustment for influencing factors and related lipids, the hazard ratios per one standard deviation of the log-transformed variables (HR) were 1.19 [95% confidence interval (CI), 0.95-1.48], p = 0.132 for 7α-hydroxycampesterol and HR, 1.18 [95% CI, 0.94-1.48], p = 0.154 for 7α-hydroxycampesterol-to-cholesterol ratio. None of the other investigated oxyphytosterols showed an association with cardiovascular events. CONCLUSIONS: In patients not on lipid-lowering drugs, absolute plasma levels of 7α-hydroxycampesterol and their ratios to cholesterol are associated with cardiovascular events. Further research is required to elucidate the role of OPS in cardiovascular diseases.

Vascular effects of oxysterols and oxyphytosterols in apoE -/- mice.

OBJECTIVES: The aim of our study was to investigate vascular effects of oxysterols and oxyphytosterols on reactive oxygen species (ROS), endothelial progenitor cells, endothelial function and atherogenesis. METHODS: Male apoE-/-mice were treated with cholesterol, sitosterol, 7-ß-OH-cholesterol, 7-ß-OH-sitosterol, or cyclodextrin by daily intraperitoneal application. The respective concentrations in the plasma and in the arterial wall were determined by gas chromatography-flame ionization or mass spectrometry. ROS production was assessed by electron-spin resonance spectroscopy in the aorta, endothelial function of aortic rings and atherosclerosis in the aortic sinus was quantitated after 4 weeks. RESULTS: Compared to vehicle, there was no difference in plasma cholesterol levels and arterial wall concentrations after i.p. application of cholesterol. 7-ß-OH-cholesterol concentrations were increased in the plasma (33.7±31.5 vs. 574.57.2±244.92 ng/ml) but not in the arterial wall (60.1±60.1 vs. 59.3±18.2 ng/mg). Sitosterol (3.39±0.96 vs. 8.16±4.11 mg/dL; 0.08±0.04 vs. 0.16±0.07 µg/mg, respectively) and 7-ß-OH-sitosterol concentrations (405.1±151.8 vs. 7497±3223 ng/ml; 0.24±0.13 vs. 16.82±11.58 ng/mg, respectively) increased in the plasma and in the aorta. The i.p-application of the non-oxidized cholesterol or sitosterol did not induce an increase of plasma oxysterols or oxyphytosterols concentrations. Oxidative stress in the aorta was increased in 7-ß-OH-sitosterol treated mice, but not in mice treated with cholesterol, sitosterol, or 7-ß-OH-cholesterol. Moreover, cholesterol, sitosterol, 7-ß-OH-cholesterol, and 7-ß-OH-sitosterol did not affect endothelial-dependent vasodilation, or early atherosclerosis. CONCLUSION: Increased oxyphytosterol concentrations in plasma and arterial wall were associated with increased ROS production in aortic tissue, but did not affect endothelial progenitor cells, endothelial function, or early atherosclerosis.

Postprandial plasma oxyphytosterol concentrations after consumption of plant sterol or stanol enriched mixed meals in healthy subjects.

Epidemiological studies have reported inconsistent results on the relationship between increased plant sterol concentrations with cardiovascular risk, which might be related to the formation of oxyphytosterols (plant sterol oxidation products) from plant sterols. However, determinants of oxyphytosterol formation and metabolism are largely unknown. It is known, however, that serum plant sterol concentrations increase after daily consumption of plant sterol enriched products, while concentrations decrease after plant stanol consumption. Still, we have earlier reported that fasting oxyphytosterol concentrations did not increase after consuming a plant sterol- or a plant stanol enriched margarine (3.0g/d of plant sterols or stanols) for 4weeks. Since humans are in a non-fasting state for most part of the day, we have now investigated effects on oxyphytosterol concentrations during the postprandial state. For this, subjects consumed a shake (50g of fat, 12g of protein, 67g of carbohydrates), containing no, or 3.0g of plant sterols or plant stanols. Blood samples were taken up to 8h and after 4h subjects received a second shake (without plant sterols or plant stanols). Serum oxyphytosterol concentrations were determined in BHT-enriched EDTA plasma via GC-MS/MS. 7ß-OH-campesterol and 7ß-OH-sitosterol concentrations were significantly higher after consumption of a mixed meal enriched with plant sterol esters compared to the control and plant stanol ester meal. These increases were seen only after consumption of the second shake, illustrative for a second meal effect. Non-oxidized campesterol and sitosterol concentrations also increased after plant sterol consumption, in parallel with 7ß-OH concentrations and again only after the second meal. Apparently, plant sterols and oxyphytosterols follow the same second meal effect as described for dietary cholesterol. However, the question remains whether the increase in oxyphytosterols in the postprandial phase is due to absorption or endogenous formation.