Fast determination of virgin olive oil phenolic metabolites in human high-density lipoproteins.

In recent years it has been confirmed that the consumption of olive oil prevents the oxidation of biomolecules owing to its monounsaturated fatty acids (MUFA) and phenolic content. The main objective of the study was to develop an ultra-high-performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) method for the determination of phenolic compounds in human high-density lipoprotein (HDL) samples. At the same time, the influence of olive oil consumption on the phenolic metabolite levels was evaluated in a European population. The participants were 51 healthy men, aged 20-60. They were randomized to two consecutive intervention periods with the administration of raw olive oil with low and high polyphenolic content. The UHPLC-MS/MS analytical method has been validated for hydroxytyrosol and homovanillic acid in terms of linearity (r(2) = 0.99 and 1.00), repeatability (5.7 and 6.5%) reproducibility (6.2 and 7%), recovery (98 to 97%), limits of detection (1.7 to 1.8 ppb) and quantification (5.8 and 6.3 ppb).The levels of the studied metabolites increased significantly after high polyphenolic content virgin olive oil ingestion (p <0.05) compared with lowpolyphenolic content olive oil. Virgin olive oil consumption increases the levels of phenolic metabolites in HDL and thus provides human HDL with more efficient antioxidant protection.

Fatty-acid composition of maternal and umbilical cord plasma and early childhood atopic eczema in a Spanish cohort.

BACKGROUND/OBJECTIVES: Fatty-acid status during in-utero development might influence the risk of atopic diseases in early childhood. The aim of this work was to identify the relationship between maternal plasma and cord blood fatty acid (FA) composition and the risk of atopic eczema in the offspring at 14 months of age. SUBJECTS/METHODS: Two hundred and eleven non-atopic mothers and their children were studied. Mothers were recruited in their first trimester of gestation and children were monitored until 14 months of age. Samples of maternal plasma and cord blood plasma were analyzed to determine the FA profile of total lipids. Presence of atopic eczema in the infants was documented through questionnaires at 6 and 14 months of age. RESULTS: Higher concentrations of total long-chain polyunsaturated FA (LC-PUFA) were found in maternal plasma of non-atopic children in relation to atopic group. Moreover, this maternal plasma LC-PUFA content was negatively correlated with the atopic eczema (odds ratios (OR)=0.83, P=0.04) in infants. Regarding cord blood samples, docosahexaenoic acid (DHA C22:6n3) and the sum of total n-3 and of LC-PUFA n-3 showed a negative correlation with the prevalence of the disease (OR=0.50, 0.49 and 0.49, respectively). CONCLUSIONS: Our results show that the fatty-acid status of the fetus during pregnancy has an important role in the development of atopic eczema in early childhood. The prevalence of this atopic disorder is related to lower cord blood plasma levels of FA belonging to n-3 series, especially DHA.

Determination of conjugated linoleic acid in human plasma by fast gas chromatography.

A new method for the determination of the main isomers of conjugated linoleic acid (CLA) in human and animal plasma was developed by gas chromatography coupled to flame ionization detection (GC-FID). The new method introduces three main advantages in comparison to the current available methodologies: firstly it does not require previous lipid extraction, secondly the chromatographic separation of CLA isomers was performed on an Rtx-2330 column significantly shorter and thinner than the typical long highly polar capillary columns in use that allows a faster analysis than in current methodologies, and thirdly the amount of sample needed to perform the analyses was substantially lower than the amount used in current routine methodologies. Its application to human plasma and rat plasma showed to be robust and reliable for quick and correct identification of the main CLA isomers in particular, and the total fatty acid profile in general, in routine analysis.

Compliance with the European and national nutritional objectives in a Mediterranean population.

OBJECTIVE: To analyze compliance with the current European and Spanish nutritional objectives in a representative sample from Catalonia, a Spanish Mediterranean region; and to examine relationships between diet and plasma fatty acid composition. DESIGN: Cross-sectional nutritional survey. SETTING: Population based random sample derived from the Catalan Nutrition Survey. SUBJECTS: A total of 516 healthy adult men (n=203) and women (n=313). METHODS: Dietary habits were assessed by means of a quantitative food frequency questionnaire. A physical exam included height, weight, waist and hip circumferences, and a fasting blood draw. RESULTS: Gender differences were observed in nutrient and energy intakes. Women showed a better compliance with the nutritional recommendations for monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) than did men. Men showed a better compliance for saturated fatty acid (SFA) and carbohydrate than did women. However, the SFA:MUFA:PUFA ratio was similar in both gender (1.6:2.3:1.0 for men; 1.7:2.5:1.0 for women). The highest compliance was observed for nutritional goals of sodium, calcium and fruit and vegetable intakes for both genders. In addition, the present study showed that levels of certain fatty acids in plasma are clearly associated with dietary intake of foods rich in these components. The highest correlations were found for n-3 long chain polyunsaturated fatty acids with blue fish intake in both men and women (r (men)=0.36 and r (women)=0.42; P<0.001). CONCLUSIONS: The diet followed in Catalonia seems to ensure compliance with most of the intermediate nutritional objectives for the Spanish population. However, a reduction in the SFA intake and an increase in the carbohydrate intake could be recommended in order to reduce the current prevalence of overweight and obesity in this Mediterranean region. SPONSORSHIP: This study was supported by the Catalan Department of Health, the Nutrition Catalan Centre of the Institute of Catalan Studies, and Mercadona SA.

Determination of gamma- and alpha-tocopherols in human milk by a direct high-performance liquid chromatographic method with UV-vis detection and comparison with evaporative light scattering detection.

A rapid direct method (Method I) for measuring gamma- and alpha-tocopherols in human milk was developed and validated using reversed-phase high-performance liquid chromatography with ultraviolet/visible (UV-vis) detection. Human milk, with an internal standard (alpha-tocopherol acetate) added, was diluted in hexane. The chromatographic system consisted of a short column (50 mm x 2.1mm I.D., 3 microm particle size) that allowed the separation of the gamma- and alpha-tocopherols in less than 6 min. The new direct method (Method I) was compared with other methods. Method II (saponification with ultraviolet/visible detection) determined 24% and 22% less gamma- and alpha-tocopherols, respectively. Method III (saponification with evaporative light scattering detection) gave the same values for alpha-tocopherol content as Method II. However, the amount of sample used in the application of Method III was higher than that used in Method II. Furthermore, Method I uses smaller amounts of solvents, and it is simpler and faster than Methods II or III. Only a small volume of sample is needed, which is an additional advantage for biological assays.

Determination of phospholipid fatty acids in biological samples by solid-phase extraction and fast gas chromatography.

A new method for the determination of phospholipid fatty acids in biological samples, combination of solid-phase extraction (SPE) and fast gas chromatography (GC) was developed. Its application to human plasma and human erythrocytes showed to be robust and reliable for quick and correct identification in routine analysis.

Influence of dietary source of docosahexaenoic and arachidonic acids on their incorporation into membrane phospholipids of red blood cells in term infants.

Here we studied whether the chemical structure of dietary arachidonic and docosahexaenoic acids in full-term infant diets affects their incorporation into erythrocyte membrane phospholipids. From birth to 3 months, infants were fed breast milk (n = 9) or formula milk containing arachidonic acid and docosahexaenoic acid provided by egg phospholipids (n = 10) or by low-eicosapentaenoic acid fish oil and fungal triglycerides (n = 10). We compared the fatty acid composition of erythrocyte phosphatidylethanolamine, phosphatidylcholine and sphingomyelin before and after administration of the experimental diet. At 3 months, infants on formula milk showed lower concentrations of docosahexaenoic acid (in phosphatidylcholine and in phosphatidylethanolamine) and arachidonic acid (in phosphatidylcholine) than those receiving breast milk. We conclude the incorporation of the two fatty acids into erythrocyte phospholipids depends mainly on the lipid composition of the diet received rather than the chemical form in which they are delivered.

Rapid high-performance liquid chromatographic method for Vitamin C determination in human milk versus an enzymatic method.

Vitamin C is an antioxidant that can be considered a possible biomarker of oxidative stability in human milk. A high-performance liquid chromatographic method was developed and validated for determining the total Vitamin C (ascorbic acid and dehydroascorbic acid) and ascorbic acid levels in human milk. This method was then compared with an enzymatic method (a Colorimetric technique) for quantifying ascorbic acid levels. Repeatability and reproducibility were acceptable for all methods. However, the high-performance liquid chromatography (HPLC) technique provided more satisfactory results than the enzymatic method due to this last method detected 37% less ascorbic acid and does not determine the total Vitamin C because of the enzymatic method cannot reduce the dehydroascorbic acid (DHA) to ascorbic acid. Furthermore, the HPLC method has the added advantages that it requires less reagents and material, and is simpler and less time consuming than the enzymatic method. In conclusion, the drawbacks of this enzymatic method would justify its substitution for a HPLC method.

Headspace gas chromatographic method for determining volatile compounds in infant formulas.

Powder infant milk formula quality deterioration and consequently the termination of shelf life results in the appearance of off-flavors mainly determined by a composite effect of spoilage volatiles. A headspace gas chromatographic method to determine propanal, pentanal and hexanal as the main volatiles present in the headspace of powder infant formula oxidation is described as a rapid indication of oxidative status. Under optimum conditions the limits of detection for propanal, pentanal and hexanal were 17.19, 16.87 and 19.60 ng and the limits of quantification were 37.37, 31.96 and 35.97, respectively. The calibration graphs of the method were linear from 25 to 1500, 20 to 3500 and 30 to 8500 ng for propanal, pentanal and hexanal, respectively, with determination coefficients exceeding 0.99. The precision results showed that the relative standard deviations of the repeatability and reproducibility were between 2.2 and 5.5%. The analytical method was simple, rapid, and reliable and permitted the analysis of a large number of formulas using small sample volumes.

Triacylglycerol markers of mature human milk.

OBJECTIVES: To determine which triacylglycerol (TAG) species in mature human milk are less affected by external factors and may thus be considered as TAG markers, as well as to determine which species are most influenced by these external conditions. Furthermore, we examine the correlation between the TAG markers and their fatty acids (FAs). SETTING AND SUBJECTS: Six healthy women from Barcelona (Catalonia, Spain). DESIGN AND INTERVENTIONS: In order to obtain the maximum variability of sampling conditions, 40 mature human milk samples were collected from different mothers, on different days, at different times of the day, and from different breasts during and after both the baby's and mother's meal. TAG and FA profiles were determined and correlated. The TAG composition was determined by high-performance liquid chromatography with an evaporative light-scattering detector, and also with atmospheric pressure chemical ionisation mass spectrometry. FAs compositions were determined by gas chromatography. RESULTS: The results were analysed using the SPSS statistical package and proved to be more variable than might have been found in a more restrictive sample design. Nevertheless, despite these conditions, some TAG species were found in relatively constant levels in mature human milk, and could thus be considered as markers of the mature milk TAG profile. TAG species that we can classify in this group were: LaMO, CaPO, LaCaO, LaPCa, LaOL, MPLn, LLO, LaOO, MPL, and MOL. The names do not indicate the location of fatty acids in the glycerol molecule. On the other hand, concentrations of other TAG species vary considerably between samples and consequently these may be understood to be especially affected by the external factors. TAGs like PaLS, MPO, PaOO, PPP, MPS, SPP, LOO, PPO, MOS, SSP, POL, and SOS are in this second group. Correlation between the TAG markers and their FAs was examined by Pearson's test and a significant correlation was found for some FAs. CONCLUSIONS: The TAG species present in mature human milk are affected in different ways by external factors such as dietary intake, nutritional status, length of lactation, time of the day, etc. Some TAGs may be considered as markers of mature human milk as they are relatively constant under a wide range of sampling conditions and do not depend on the factors mentioned. SPONSORSHIP: This study was supported by the Fundació Mestres Jané.

Effect of ingestion of virgin olive oil on human low-density lipoprotein composition.

OBJECTIVE: To measure the incorporation of oleic acid and antioxidants (phenols and vitamin E) to low density lipoprotein (LDL) after acute and short-term ingestion of virgin olive oil. To study whether this incorporation contributes to an increase in LDL resistance to oxidation. SETTING: Department of Food and Nutrition, University of Barcelona, Spain and Department of Lipids and Cardiovascular Epidemiology, IMIM, Barcelona, Spain. SUBJECTS: Sixteen healthy volunteers aged 25-65 y. DESIGN AND INTERVENTIONS: To observe the change in the fatty acid profile, vitamin E, phenolic compounds and LDL oxidation-related variables after the postprandial phase and after daily ingestion of olive oil for one week. RESULTS: Few changes were observed in the postprandial phase. However, after a week of olive oil consumption there was an increase in oleic acid (P=0.015), vitamin E (P=0.047), phenolics (P=0.021) and lag time (P=0.000), and a decrease in the maximum amount of dienes (P=0.045) and oxidation rate (P=0.05). CONCLUSION: After ingestion of virgin olive oil, an increase in antioxidants and oleic acid in LDL was observed as well as an improvement of LDL resistance to oxidation. Our results support the idea that daily ingestion of virgin olive oil could protect LDL from oxidation. SPONSORSHIP: This study was supported by a research grant from Spain (ALI 97-1607-C02-02).

Rapid high-performance liquid chromatographic method for the simultaneous determination of retinol, alpha-tocopherol and beta-carotene in human plasma and low-density lipoproteins.

A reversed-phase HPLC method with diode-array detection was used to simultaneously determine retinol, alpha-tocopherol and beta-carotene in human plasma and low-density lipoproteins. An aliquot of sample was de-proteinized with ethanol containing beta-tocopherol acetate as internal standard, and the analytes were extracted twice with hexane. The solvent was evaporated to dryness under a stream of nitrogen and the residue was redissolved in methanol to be injected directly into the HPLC system. A multiple solvent system based on methanol, butanol and water at a flow-rate of 2 ml/min and held at 45 degrees C provided clear separation of these compounds in only 8 min. The method showed good linearity, precision and accuracy for all compounds. Owing to its simplicity, this method may be useful in routine clinical and epidemiological work.

Rapid determination of vitamin E in vegetable oils by reversed-phase high-performance liquid chromatography.

A quick and direct method for measuring tocopherols (alpha, beta+gamma and delta) in vegetable oils has been developed using RP-HPLC with UV detection. Previous extraction of tocopherols is not required. The oil is diluted in hexane and an aliquot is mixed with ethanol containing an internal standard (alpha-tocopherol acetate). The chromatographic system consists of an ODS-2 column with a methanol-water mobile phase. Tocopherols are detected at 292 nm in less than 5 min after injection. The method is precise (RSD=2.69%) and has a high mean recovery (98.14%).

Simultaneous determination of alpha-tocopherol and beta-carotene in olive oil by reversed-phase high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic method was developed for the determination, in one run, of alpha-tocopherol and beta-carotene in virgin olive oil. The method involved a rapid saponification and a later extraction with a mixture of hexane-ethyl acetate. The chromatographic system consists of an ODS-2 column with a mobile phase of methanol-water-butanol and a diode-array detector. Linearity, precision, recovery and sensitivity were satisfactory. The main advantage of the proposed method is the speed and simultaneous determination of both compounds at the same time.

Determination of plasma fatty acid composition in neonates by gas chromatography.

Total fatty acids in plasma of neonates have been analysed as their methyl esters by gas chromatography. They were separated on a capillary column coated with a SP-2380 stationary phase. As little as 100 microliters of plasma is used for the analysis. The extraction procedure was performed with dichloromethane-methanol (2:1) and fatty acids were methylated with boron trifluoride-methanol. The quantification of fatty acids is based on an internal standard method. Absolute values (micrograms fatty acid per 100 microliters plasma) are given together with relative values (%). At a signal-to-noise ratio of 3, the detection limits for flame ionisation detection are between 0.08 to 0.51 ng. The high sensitivity and precision permits the effective determination of the fatty acids in neonate plasma.

High-performance liquid chromatographic determination of plasma triglyceride type composition in a normal population of Barcelona. Relationship with age, sex and other plasma lipid parameters.

A coupled TLC-HPLC procedure is proposed for the separation and determination of plasma triglycerides. The method was tested by application to plasma samples corresponding to a normal population of Barcelona (Spain). Eighteen different triglyceride types were identified and their relative proportions were established, in order to give a "normal profile" for men and women. Sex-related differences (p < 0.05) were only found for dioleostearin and palmitodilinolein + linoleooleopalmitolein (LLP+LOPa). A correlation study showed that palmitodiolein and total cholesterol levels increase with age, whereas LLP-LOPa decreases in men and palmitolinoleoolein + palmitooleopalmitolein in women.