Biomedical subjects
Publications and source records attributed to M C López-Sabater.
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.
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.
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.
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.
The Mediterranean diet, in which olive oil is the main source of fat, has been associated with a reduced incidence of coronary heart disease (CHD) and low blood pressure levels. Virgin olive oil (VOO), besides containing monounsaturated fat, is rich in phenolic compounds (PC) with antioxidant properties. The aim of this study was to examine the antioxidant and anti-hypertensive effect of two similar olive oils, but with differences in their PC (refined: 14.7 mg/kg versus virgin: 161.0 mg/kg), in 40 males with stable CHD. The study was a placebo controlled, crossover, randomized trial. A raw daily dose of 50 mL of VOO and refined olive oil (ROO) were sequentially administered over two periods of 3 weeks, preceded by 2-week washout periods in which ROO was used. Lower plasma oxidized LDL (p < 0.001) and lipid peroxide levels (p = 0.003), together with higher activities of glutathione peroxidase (p = 0.033), were observed after VOO intervention. Systolic blood pressure decreased after intake of VOO (p = 0.001) in hypertensive patients. No changes were observed in diastolic blood pressure, glucose, lipids, and antibodies against oxidized LDL. Consumption of VOO, rich in PC, could provide beneficial effects in CHD patients as an additional and complementary intervention to the pharmacological treatment.
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.
A rapid, sensitive method has been developed for the simultaneous determination of retinol acetate, delta-, gamma-, alpha-tocopherol and alpha-tocopherol acetate. We compare two experimental procedures for simultaneous direct solvent extraction of these vitamins without previous saponification. Method I: the fat milk sample was extracted with ethanol-hexane and injected directly into the chromatographic column. Method II: the power milk sample was extracted with ethanol-hexane and also injected directly into the column. Under optimum conditions the limits of detection for retinol acetate, delta-, gamma-, alpha-tocopherol and alpha-tocopherol acetate were 0.33, 21.2, 32.9, 32.5 and 3.2 ng and the limits of quantification were 0.42, 25.3, 37.9, 36.8 and 6.3 ng, respectively. The precision results showed that the relative standard deviations of repeatability and reproducibility were between 0.74 and 5.7%.
We developed and validated a new high-performance liquid chromatographic method for the separation of phospholipid classes in human milk, infant formulas and phospholipidic sources of long-chain polyunsaturated fatty acids (LC-PUFAs) used in paediatric nutrition. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and sphingomyelin were separated in less than 25 min using an Extrasil silica column (150 x 4.0 mm I.D., 3-microm particle size) by isocratic elution with a mixture of isopropanol-hexane-water. Phospholipids were determined by an evaporative light-scattering detector. Several chromatographic conditions were assayed to optimise the method, whose suitability is shown by the detection limits, linearity ranges and precision rates obtained. The main advantages of the proposed method are its speed and the direct determination of the main phospholipids present in human milk, infant formulas and the phospholipid sources of LC-PUFAs used in paediatric nutrition.
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.
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).
OBJECTIVE: To investigate differences in fatty acid and sn-2 fatty acid composition in colostrum, transitional and mature human milk, and in term infant formulas. SETTING: Departament de Nutrició i Bromatologia, University of Barcelona, Spain and University Hospital of Granada, Spain. SUBJECTS: One-hundred and twenty mothers and 11 available types of infant formulas for term infants. DESIGN: We analysed the fatty acid composition of colostrum (n=40), transitional milk (n=40), mature milk (n=40) and 11 infant formulas. We also analysed the fatty acid composition at sn-2 position in colostrum (n=12), transitional milk (n=12), mature milk (n=12), and the 11 infant formulas. RESULTS: Human milk in Spain had low saturated fatty acids, high monounsaturated fatty acids and high linolenic acid. Infant formulas and mature human milk had similar fatty acid composition. In mature milk, palmitic acid was preferentially esterified at the sn-2 position (86.25%), and oleic and linoleic acids were predominantly esterified at the sn-1,3 positions (12.22 and 22.27%, respectively, in the sn-2 position). In infant formulas, palmitic acid was preferentially esterified at the sn-1,3 positions and oleic and linoleic acids had higher percentages at the sn-2 position than they do in human milk. CONCLUSION: Fatty acid composition of human milk in Spain seems to reflect the Mediterranean dietary habits of mothers. Infant formulas resemble the fatty acid profile of human milk, but the distribution of fatty acids at the sn-2 position is markedly different.
An alternative method to determine the sn-2 monopalmitin in infant formulas was developed and validated. This method offers many advantages over the traditional methods. It follows the official method in the first steps, purification of the fat or oil through an alumina column, and subsequently the triglycerides are incubated with pancreatic lipase in order to obtain the sn-2 monoglycerides. In traditional methods the sn-2 monoglycerides are separated by preparative thin-layer chromatography and then, the 2-monoglycerides are converted into the corresponding fatty acid methyl esters and analysed by gas chromatography. In our method, separation, quantification and identification of the sn-2 monoglycerides were achieved by high-performance liquid chromatography with evaporative light-scattering detection. The detection limit (0.19 microg), quantification limit (0.38 microg), linearity range (r=0.999, range 1-200 microg) and precision (SD=1.10) show the suitability of the proposed method. This method is faster, cheaper and simple and does not consume large quantities of reagents and materials.
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.
The distribution of long-chain saturated fatty acids in triglycerides is different in infant formulas to that in human milk. In human milk, palmitic acid is predominantly esterified in the sn-2 position (beta-position) of the triglycerides, whereas in infant formulas, it is esterified mainly in the sn-1,3 positions (alpha,alpha'-positions). The specific distribution of the fatty acids in the triglyceride plays a key role in lipid digestion and absorption. We studied fatty-acid, calcium and magnesium composition in the faeces of three groups of at term newborn infants fed different diets: Group A (n=12) was fed from birth to 2 months with human milk (66% palmitic acid in beta-position), Group B (n=12) was fed with formula alpha (19% palmitic acid esterified in beta-position) for 2 months, and Group C (n=12) was fed with formula alpha during the first month and with formula beta (44.5% palmitic acid in beta-position) during the second month. Samples were taken at the end of the first month (t0) and at the end of the second month (t1). Groups A and C presented significantly lower contents of palmitic acid in faeces at t1 than at t0, whereas in Group B, amounts remained similar. Faecal calcium in Groups A and C decreased in the second month (t1), although the fall was no statistically significant. In Group B, calcium amounts showed no change. We found that infant formula beta when compared with infant formula alpha reduced significantly the contents of total fatty acids and palmitic acid in faeces. We conclude that palmitic acid in beta-position is, therefore, beneficial for term infants.
We have validated and compared two direct methods for the determination of fatty acids in feces by capillary gas chromatography. Method I consisted of esterification of fatty acids using acetyl chloride. Method II used boron trifluoride-methanol as esterification reagent. The two methods were assayed with and without previous freeze-drying of the fecal sample. We found that the two methods could be carried out without sample freeze-drying. Precision and recovery rates were determined and the results were satisfactory. Both methods gave similar results, but Method II has certain advantages over Method I, such as speed, safety, and better recovery rates.
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%).
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.
Inulin is a naturally occurring carbohydrate with beneficial nutritional and technological properties. A high-performance liquid chromatographic (HPLC) method was developed for the quantitative determination of these beta-fructans in meat products, containing this type of additive. The method includes extraction of inulin with hot water, followed by hydrolysis with inulinase enzyme, and determination of the released fructose by HPLC with refractive index detection. An internal standard of rhamnose was used to quantify fructose. The method incorporates a sample blank (without inulinase hydrolysis) for each specimen to subtract contributions of free fructose and fructose from sucrose. The results showed good precision with average RSDs of 2.4% for repeatability and 5.2% for reproducibility. Analytical recovery ranged from 102 to 106%. Satisfactory linearity (r=0.999) was obtained.