高效液相色谱测定血清胆固醇酯n-3脂肪酸指数方法的建立

目的 建立一种HPLC测定血清胆固醇酯n-3脂肪酸指数的方法.方法 血清标本来自于70名北京医院健康志愿者和36例冠心病患者,用NaOH乙醇溶液水解血清三酰甘油,乙酸终止水解反应,正己烷提取胆固醇和胆固醇酯,HPLC分离脂质,UV(205 nm)检测.20:5-CE和22:6-CE用液相色谱串联质谱鉴定,各胆固醇酯峰面积修正因子用测定相应组分胆固醇含量的方法确定,归一化法定量,计算n-3脂肪酸胆固醇酯的百分比(即胆固醇酯n-3指数).结果 色谱分析在6 min内完成;影响胆固醇酯n-3指数测定的三酰甘油用4 mol/L NaOH乙醇溶液水解30 s可被去除;测定血清胆固醇酯n-3指数的批内和总CV为0.66%和0.90%,106名试验对象血清胆固醇酯n-3指数略呈正偏态分布(偏度值=1.25,峰度值=1.70),全体中位数为0.98%(0.37%～2.40%).n-3指数对数基本呈正态分布,(x)±s为0.003 7%±0.15%.性别组n-3指数分布与全体类似,女性和男性中位数(范围)分别为1.08%(0.60%～2.40%)和0.95%(0.37%～2.11%),不同性别间差异具有统计学意义(t=-3.021,P=0.003).结论 成功建立了HPLC测定血清胆固醇酯n-3指数的方法,有望在营养监测和心血管病危险分析中发挥作用.     

Fatty Acid Composition of Serum Lipid Fractions in Relation to Gender and Quality of Dietary Fat

The fatty acid compositions of serum lipid fractions are believed to reflect the quality of dietary fat, but only a few cross-sectional studies have examined these relationships in a representative free-living population. We related the composition of dietary fat obtained by 7-day food records from 84 free-living middle-aged married couples, on their habitual diets, to gas chromatographic analyses of the percentage compositions of fatty acids in three lipid fractions of fasting sera. Dietary polyunsaturated fat had Pearson's correlation coefficients of 0.63, 0.73 and 0.44 with n-6 polyunsaturated fatty acids in serum cholesteryl esters, triglycerides and phospholipids, respectively. Intake of fish showed correlations of 0.60, 0.36 and 0.52 with the percentage of eicosapentaenoate in the respective fractions. Dietary saturated fat had correlations of 0.57 and 0.54 with saturated fatty acids in serum cholesteryl esters and triglycerides, respectively. Dietary monounsaturated fat did not correlate positively with mono-unsaturated fatty acids in any serum lipid fraction. There were some small but significant gender differences in the serum fatty acid compositions. Conclusions: of the three serum lipid fractions, triglycerides appear to be the best reflectors of dietary polyunsaturated and saturated fat, but the intake of monounsaturated fat is poorly reflected in all serum lipid fractions. Intake of fish is mirrored in all serum lipid fractions, best in cholesteryl esters and phospholipids.     

Threonine allele in codon 54 of the fatty acid binding protein 2 gene does not modify the fatty acid composition of serum lipids in obese subjects

Intestinal fatty acid binding protein (I-FABP) participates in the metabolism of fatty acids in the intestinal enterocytes. Threonine encoding allele in codon 54 of the I-FABP gene has been suggested as regulating the absorption of long-chain fatty acids. We examined the fatty acid composition of serum lipid fractions and the concentration of serum free fatty acids after an overnight fast in obese subjects, aged 24–56 years, on their habitual diet. The body mass index of the subjects ranged from 29.7 to 43.3 kg m⁻². Six subjects were homozygous for the Thr-54 allele of the I-FABP gene, 37 subjects were heterozygous for the Thr-54/Ala-54 allele and 24 subjects were homozygous for the Ala-54 allele. We did not find any consistent differences in the proportions of long-chain fatty acids in serum triglycerides, cholesterol esters or phospholipids, but the concentration of serum free fatty acids tended to be higher in subjects who were homozygous for the Thr-54 allele ( P  = 0.13, for trend). In conclusion, our findings suggest that a polymorphism at codon 54 of the I-FABP2 gene does not substantially modify the fatty acid composition of serum lipids in obese Finns.     

Omega-3 polyunsaturated fatty acids and cardiovascular disease: an emphasis on omega-3-acid ethyl esters 90 for the treatment of hypertriglyceridemia

A number of epidemiological/observational studies, as well as large-scale randomized intervention studies, have been conducted to provide evidence for the efficacy of ω-3 fatty acids against atherosclerotic diseases. Currently, ω-3 fatty acids are commercially available in many parts of the world containing the same active ingredients as Lotriga^® (ω-3-acid ethyl esters 90 [O3AE highly concentrated ω-3 fatty acid ethyl esters, consisting of eicosapentaenoic acid-ethyl ester and docosahexaenoic acid-ethyl ester [EPA-E/DHA-E]). A recent head-to-head comparative study of O3AEE90 versus EPA-E demonstrated that O3AEE90 4g/day led to a significantly greater reduction in triglycerides (TG) than EPA-E 1.8g/day and that O3AEE90 2g/day produced comparable effects on TG to those with EPA-E 1.8g/day. While both agents were shown to be useful in lowering TG, the hallmark feature of O3AEE90, that is, the presence of the DHA-E component versus its absence in EPA-E, needs to be further examined for its clinical implications.     

Gas-chromatographic analysis of patterns of fatty acids of cholesteryl esters and phosphatidylcholine.

We have developed a quick, reliable method for compositional analysis of the C14 to C20 fatty acids of cholesteryl esters, based on the use of a double extraction-solvent system and whisker-walled capillary columns with a new polar phase. Fatty acids of phosphatidylcholine, after separation by thin-layer chromatography, were determined on the same column. Our normal values are in good agreement with those published previously. We found marked changes in the fatty acid composition of both cholesteryl esters and phosphatidylcholine in cord blood serum, in various disease states, and in post-mortem specimens. The previously reported correlation between oleic (18:1) and linoleic (18:2) acids in serum cholesteryl esters was confirmed for normal adults, but no such correlation exists in cord blood specimens. We also found a significant correlation between the relative amounts of fatty acids in cholesteryl esters and phosphatidylcholine for all unsaturated fatty acids and for palmitate in all groups of subjects.     

HPLC测定血清中七种结合胆汁酸

ＳＥＰ－ＰＡＫＣ１８小柱预处理血清后，采用反相ＨＰＬＣ结合紫外检测，定量测定脂肪肝患者与对照组血清中七种结合胆汁酸，结果显示，血清结合胆汁酸为一种敏感的实验检测指标，为临床检验提供了简便、快速的方法。     

Partial conservation of the sn-2 position of dietary triglycerides in fasting plasma lipids in humans

The authors investigated the effect of the position of fatty acids within dietary triglycerides on the composition of plasma lipids. Sixty volunteers consumed two diets of equal fatty acid compositions for 3 weeks each. In the palm oil diet 82% of palmitic acid was attached to the sn -1 and sn -3, and 18% to the sn -2 position of glycerol. In the diet rich in a palm oil analogue, Betapol, these figures were 35% and 65% respectively. Oleic and linoleic acid in palm oil were mainly in the sn -2 position, and in Betapol mainly in the sn -1 and sn -3 position. The proportion of palmitic acid in the 2 position of fasting plasma triglycerides was 10.2 g 100 g⁻¹ on palm oil and 12.3 on Betapol; that of oleic acid was 46.9 vs. 43.6 ( P  < 0.0001). Overall oleic acid content of triglycerides was 1.4 g 100 g⁻¹ lower on Betapol than on palm oil ( P  = 0.002). Betapol increased palmitic and palmitoleic acid in cholesteryl esters by 1 g 100 g⁻¹ ( P  < 0.0001) at the expense of oleic and linoleic acid. The positional configuration of dietary fatty acids has small but consistent effects on lipid metabolism that persist beyond fat absorption and chylomicron clearance.     

Correlation of serum fatty acid composition with dietary intake data in children and young adults.

The percentage compositions of fatty acids in serum cholesteryl esters and phospholipids were analysed with gas chromatography in 759 nine- to 24-year-old Finns. The correlations of serum fatty acids with dietary fat intake data were calculated from a 48-h recall survey. The dietary P/S ratio had correlations of r = 0.50 and r = 0.40 with linoleate (18:2) in serum cholesteryl esters and phospholipids, respectively. The intake of fish and fish products correlated positively with the percentages of eicosapentaenoate (20:5 n-3) and docosahexaenoate (22:6 n-3) in both cholesteryl esters and phospholipids. Dietary intake of saturated fat correlated positively with the percentages of all saturated fatty acids in cholesteryl esters and with myristate (14:0) in phospholipids. The intake of monounsaturated fats did not correlate positively with serum monoenes. In conclusion, the dietary P/S ratio is well reflected in the fatty acid composition of serum cholesteryl esters and phospholipids, the intake of saturated fats less well, and the intake of monounsaturated fats not at all.     

Effect of acute hyperinsulinemia on fatty acid composition of serum lipids in non-insulin-dependent diabetics and healthy men.

The fatty acid pattern of serum phospholipids, cholesteryl esters, triglycerides and free fatty acids was measured before and after a 5-h two-step euglycemic hyperinsulinemic clamp (75 and 1400 microU/ml) in 21 non-insulin-dependent diabetics and 14 age-, weight-, and sex-matched healthy controls. Acute hyperinsulinemia was associated with a statistically significant increase in essential fatty acid and a decrease in non-essential fatty acid contents in triglycerides while the levels of serum triglycerides and free fatty acids dropped in both groups. The fatty acid composition of phospholipids and cholesteryl esters remained unchanged as did the levels of serum phospholipids, total cholesterol and HDL cholesterol.     

Phytanic acid and very long chain fatty acids in genetic peroxisomal disorders

1. Phytanic acid, phytanyl-triacylglycerols, and very long chain fatty acids were analysed by gas chromatography or thin-layer chromatography in blood and tissues of patients with different genetic peroxisomal disorders (Refsum's disease, X-linked adrenoleukodystrophy, neonatal adrenoleukodystrophy, Zellweger syndrome). 2. We evaluated these analyses in the detection of patients with Refsum's disease, X-linked adrenoleukodystrophy, neonatal adrenoleukodystrophy, and Zellweger syndrome, and of carriers of X-linked adrenoleukodystrophy. In particular, the analysis of phytanyl-triacylglycerols by thin-layer chromatography proved to be a rapid and reliable method for the detection of patients and the monitoring of their dietary treatment in Refsum's disease. In X-linked adrenoleukodystrophy, carrier detection may depend on very long chain fatty acid analysis in more than one material (e.g. plasma and fibroblasts). 3. Analysis of phytanic acid showed that in patients with multiple impairments of peroxisomal functions (Zellweger syndrome, neonatal adrenoleukodystrophy) phytanic acid levels may be increased not only in serum, but also in the tissue (e.g. brain, adrenals, kidney). 4. Analysis of very long chain fatty acids in cholesterol esters from the brain, adrenals, kidney, and liver of patients with peroxisomal disorders revealed four different types of very long chain fatty acid patterns according to the behaviour of C 26:0 and of other saturated and monounsaturated very long chain fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improved staining characteristics of serum lipids after halogenation and esterification of thin-layer chromatograms.

We describe a procedure for treatment of thin-layer chromatographic serum lipid patterns so that they may be stained by dyes for evaluation by densitometry. After development of the chromatogram [Clin. Chem. 18, 384 (1972)] the plates are dried and sprayed with butyryl chloride. This esterifies the free cholesterol. After drying, the plate is treated with iodine monobromide, to add iodline to the double bonds. The triacylglycerols (triglycerides), cholesterol esters, free cholesterol, and phospholipids are now all in the form of esters with no unsaturated double bonds. The free fatty acids are also now saturated. The chromatogram is now stained with basic fuchsine in acetate buffer (0.1 mol/liter, pH 5.0). Excess stain is removed with a buffered solution of guanidine hydrochloride. Erythrosine B may also be used. With basic fuchsine the background will be a uniform pink. With erythrosine B the background is white, but the stain tends to be washed out of the free fatty acids. The chromatograms are evaluated by densitometry, with use of a 540-nm filter for basic fuchsine and a 520-nm filter for erythrosine B. The stained chromatograms and densitometric scans accurately represent the relative concentration of the various lipid fractions as compared to that of an internal standard, and correlate with the nature of the disease being explored.     

Novel sustained-release dosage forms of proteins using polyglycerol esters of fatty acids.

In order to develop a novel delivery system for proteins based on polyglycerol esters of fatty acids (PGEFs), we studied a model system using interferon-alpha (IFN-alpha) as the test protein. A cylindrical matrix was prepared by a heat extrusion technique using a lyophilized powder of the protein and 11 different types of synthetic PGEFs, which varied in degree of glycerol polymerization (di- and tetra-), chain length of fatty acids (myristate, palmitate and stearate) and degree of fatty acid esterification (mono-, di- and tri-). In an in-vitro release study using an enzyme-linked immunosorbent assay (ELISA) as a detection method, the matrices prepared from a monoglyceride (used for comparison) and from diglycerol esters exhibited a biphasic release pattern with a large initial burst followed by slow release. In contrast, the matrices prepared from tetraglycerol esters showed a steady rate of release without a large initial burst. In an in vivo release study, initial bursts of IFN-alpha release were, also, dramatically reduced when the matrices were prepared from the tetraglycerol esters of palmitate and stearate, and the mean residence time (MRT) of IFN-alpha was prolonged, whereas the matrices prepared from monoglyceride and from diglycerol esters showed large initial bursts of IFN-alpha release. Since the release rates from the matrices prepared from the tetraglycerol esters of palmitate and stearate were governed by Jander's equation modified for a cylindrical matrix, the release from those matrices was concluded to be a diffusion-controlled process. The bioavailability of IFN-alpha after implantation of the matrix formulation prepared using all types of PGEFs, except for tetraglycerol triesters, was almost equivalent to that after injection of IFN-alpha solution; consequently, IFN-alpha in these matrices appears to remain stable during the release period.     

Trans-fatty acids and cardiovascular risk: does origin matter?

Several studies have aimed to unravel the contribution of different types of dietary fatty acids to human health and disease. Investigations have consistently shown that high consumption of industrially produced trans-fatty acids from partially hydrogenated vegetable oils is harmful to human health, in particular cardiovascular health. Therefore, the U.S. Food and Drug Administration announced that partially hydrogenated oils are no longer ‘generally recognized as safe’, and trans-fatty acids are not permitted in the U.S. food supply. On the other hand, recent studies analyzing the association between circulating trans-fatty acids and disease have revealed that some ruminant-specific trans-fatty acids are associated with a reduction in incidence of disease. In this special report, we highlight recent findings and point out perspectives for future studies on this topic.     

Platelet fatty acid composition in relation to fatty acid composition in plasma and to serum lipoprotein lipids in healthy subjects with special reference to the linoleic acid pathway

The fatty acid composition in platelet phospholipids and in the plasma lipid esters as well as the serum lipoprotein lipid concentrations were determined in 67 healthy male subjects in order to establish the relationships between blood lipids and platelets. A positive correlation was found between the concentrations of the triglyceride rich serum lipoprotein lipids and the relative percentage of saturated and monounsaturated fatty acids in plasma. The correlations were also positive between the serum high density lipoprotein-cholesterol concentration and the relative content of linoleic acid in the plasma cholesterol esters and phospholipids. Negative correlations were found between the relative percentage of saturated and monounsaturated fatty acids in the plasma lipid esters versus linoleic acid in plasma and in the platelets. On the other hand there were positive correlations between linoleic acid in the plasma lipid esters and in the platelet phospholipids. These results indicate a direct dietary influence on the platelet phospholipid fatty acid composition. The correlations between the fatty acids of the n-6 series within plasma and platelets as well as between plasma and platelets indicate that a high linoleic acid content is not associated with an increased arachidonic acid concentration. The results also indicate that the limiting metabolic step in the conversion of linoleic acid into arachidonic acid may be located at different levels in plasma and in the platelets.     

Enzymatic preparation of phytosterol esters with fatty acids from high-oleic sunflower seed oil using response surface methodology

Phytosterol esters are functional compounds that can effectively reduce plasma cholesterol concentration, and have wide applications in the food industry. In this study, a simple and efficient enzymatic method was successfully applied to synthesize phytosterol oleic acid esters with fatty acids from high-oleic sunflower seed oil. Among the tested lipases, Candida rugosa lipase (CRL) exhibited higher catalytic activity in the esterification of phytosterols with fatty acids (oleic acid 84%) from high-oleic sunflower seed oil. Box–Behnken design and response surface methodology were used to investigate the influence of reaction factors on the conversion of phytosterols. The maximum conversion of phytosterols (96.8%) and yield of phytosterol esters (92%) could be obtained under optimal conditions: reaction temperature 50 °C, a molar ratio of phytosterols to fatty acids at 1 : 2.3, enzyme loading of 5.8%, isooctane volume of 2 mL and reaction time of 2 h. It was noteworthy that this enzymatic esterification method indeed expended a much shorter reaction time (2 h) than that observed in previous reports. In general, the enzymatic preparation of phytosterol oleic acid esters with fatty acids from high-oleic sunflower seed oil will be a simple and rapid method for producing unsaturated fatty acid esters of phytosterol with both higher oil solubility and oxidative stability, which is beneficial as functional food ingredients.

Enzymatic preparation of phytosterol esters with fatty acids from high-oleic sunflower seed oil has been established with very mild reaction conditions.     

Bile acids in peroxisomal disorders

We examined serum bile acids in patients with different peroxisomal disorders. Patients with Zellweger syndrome (n = 23), infantile form of Refsum disease (n = 6) and neonatal adrenoleukodystrophy (n = 4) consistently had increased levels of bile acid precursors. Patients with X-linked adrenoleukodystrophy, (n = 5) classical Refsum disease (n = 3), hyperpipecolic acidaemia (n = 4) and rhizomelic chondrodysplasia punctata (n = 9) did not have increased bile acid precursor levels. Total serum bile acids (41 micrograms ml-1) and the percentage of bile acid precursors (80%) were highest in typical Zellweger patients who died young. Long-living Zellweger patients, neonatal adrenoleukodystrophy patients and infantile Refsum disease patients had, on average, less cholestasis and a lower percentage of bile acid precursors. We also observed that total serum bile acids and the percentage of bile acid precursors decreased with age in long-living Zellweger patients. Screening for bile acid precursors, combined with very long chain fatty acids analysis is, in our experience, an easy and reliable first-line approach to the detection of peroxisomal disorders.     

Dietary Fats and Platelet Function among Finnish Men

The aim of this study was to elucidate the role of platelet aggregation as a risk factor for ischemic heart disease (IHD) and the relationship between fatty acids and platelet function. Platelet aggregation upon adenosine diphosphate (ADP), adrenaline and thrombin were measured in middle aged men in east and west, two regions of Finland with a nearly twofold difference in IHD mortality. Platelet aggregation results were correlated with the fatty acid compositions of plasma lipid fractions, adipose tissue triglycerides and platelet phospholipids.

There was no significant east-west difference in platelet reactivity to ADP, adrenaline and thrombin. ADP-induced platelet aggregation showed significant negative correlations with all the platelet C20-C22 n-3 polyunsaturated fatty acids (PL)FA), but significant positive correlations with the percentage of 18:2n-6 in adipose tissue and plasma cholesterol esters (CE) and triglycerides (TG). Adrenaline-induced aggregation correlated negatively with the percentage of 20:5n-3 in plasma CE and TG, and positively with the total percentage of saturated fatty acids in platelets. Aggregation upon thrombin had a negative correlation with the 20:3n-6/20:4n-6 ratio in plasma CE and a positive correlation with 18: 2n-6 in adipose tissue.

The percentages of the major PUFA in platelets correlated sgnificantly with the same fatty acids in plasma CE and phospholipids PL. Platelet 20: 5n-3 had a highly significant negative correlation with the percentage of 18:2n-6 in plasma and adipose tissue lipids. Platelet 20: 4n-6 was unrelated to its precursors in plasma. The percentage of 18: 2n-6 in plasma and adipose tissue lipids was consistently lower in men from eastern than western Finland, whereas the percentage of platelet 20: 5n-3 was higher in the eastern men.

The results indicate, that (1) platelet function may not play a role in the regional gradient in IHD rate in Finland, (2) in a free-living population, relatively small changes in plasma and platelet fatty acids may have significant influence on platelet aggregation and on thrombosis tendency and (3) an increase in dietary PUFA content may not affect platelet function favourably, unless attention is paid to the maintaining of the n-3/n-6 ratio in the diet.     

Antimicrobial Action of Esters of Polyhydric Alcohols

A broth dilution method was used to determine the minimal inhibitory concentration of a series of fatty acid esters of polyhydric alcohols against gram-negative and gram-positive organisms. Gram-negative organisms were not affected. Gram-positive organisms were inhibited as follows. Of the monoglycerol esters, monoglycerol laurate was the most active. Esters of polyglycerols (tri-, hexa-, and decaglycerol esters) were generally active when the fatty acid had chain lengths of 8 to 12 carbon atoms. Sucrose esters, when active, except for laurate, are more active than the free fatty acid. The spectrum of antimicrobial action of esters of polyhydric alcohols is narrower when compared with the free acids.     

Mitochondrial dysfunction induced by fatty acid ethyl esters, myocardial metabolites of ethanol.

Mechanisms responsible for alcohol-induced heart muscle disease have been difficult to elucidate partly because of previously obscure, demonstrable cardiac metabolism of ethanol. Recently, fatty acid ethyl esters were identified in our laboratory and found to be myocardial metabolites of ethanol. In the present study, they have been shown to induce mitochondrial dysfunction. Incubation of isolated myocardial mitochondria with fatty acid ethyl esters led to a concentration-dependent reduction of the respiratory control ratio index of coupling of oxidative phosphorylation and decrement of maximal rate of oxygen consumption. Furthermore, fatty acid ethyl esters were demonstrated to bind to mitochondria in vitro, and, importantly, 72% of intracellularly synthesized ethyl esters were found to bind to mitochondria isolated from intact tissue incubated with ethanol. Protein binding of fatty acid ethyl esters was markedly less than that of fatty acids. Because uncoupling of mitochondrial oxidative phosphorylation correlated with the cleavage of fatty acid ethyl ester shown to be initially bound to mitochondria, with resultant generation of fatty acid, a potent uncoupler, in a locus in or near the mitochondrial membrane, fatty acid ethyl esters may contribute to a potentially toxic shuttle for fatty acid with transport from physiological intracellular binding sites to the mitochondrial membrane; direct effects of fatty acid ethyl esters may also be deleterious. Operation of this shuttle as a result of ethanol ingestion and subsequent accumulation of fatty acid ethyl esters may account for the impaired mitochondrial function and inefficient energy production associated with toxic effects of ethanol on the heart.