Eduardo López-Huertas

Health effects of oleic acid and long chain omega-3 fatty acids (EPA and DHA) enriched milks. A review of intervention studies

Substitution of dietary saturated fat by oleic acid and/or polyunsaturated fatty acids (PUFA) has been described to reduce the cardiovascular risk by reducing blood lipids, mainly cholesterol. Additional benefits have been described for long chain omega-3 PUFA (eicosapentaenoic acid-EPA and docosahexaenoic acid-DHA) from fish oils. In recent years, food technology has been used to produce dairy drinks with a reduced content of saturated fat in favour of those fatty acids, most of them claiming cardiovascular benefits. This review summarises all the scientific evidence regarding the effects of milks enriched with long chain omega-3 PUFA (EPA+DHA) and/or oleic acid on cardiovascular health. Nine controlled intervention studies with enriched milks have reported effects on healthy volunteers, subjects with increased risk factors and cardiovascular patients. The main effects observed were reductions of blood lipids, mainly cholesterol, LDL-cholesterol and triglycerides.

Human milk probiotic Lactobacillus fermentum CECT5716 reduces the incidence of gastrointestinal and upper respiratory tract infections in infants.

Objectives: The aim of the study was to examine the effects of a follow-on formula containing Lactobacillus fermentum CECT5716 (L fermentum) on the incidence of infections in infants between the ages of 6 and 12 months. Patients and Methods: A randomized double-blinded controlled study including infants at the age of 6 months was conducted. Infants were assigned randomly to either follow-on formula supplemented with L fermentum plus galactooligosaccharide (experimental group, EG), or the same formula supplemented with only galactooligosaccharide (control group, CG). The main outcome was the incidence of infections for the 6-month duration of the study. Results: The EG showed a significant 46% reduction in the incidence rate (IR) of gastrointestinal infections (EG: 0.196 ± 0.51, CG: 0.363 ± 0.53, IR ratio 0.54, 95% confidence interval [CI] 0.307–0.950, P = 0.032), 27% reduction in the incidence of upper respiratory tract infections (EG: 0.969 ± 0.96, CG: 1.330 ± 1.23, IR ratio 0.729, 95% CI 0.46–1.38, P = 0.026), and 30% reduction in the total number of infections (EG: 1.464 ± 1.15, CG: 2.077 ± 1.59, IR ratio 0.70, 95% CI 0.46–1.38, P = 0.003), at the end of the study period compared with CG. Conclusions: Administration of a follow-on formula with L fermentum CECT5716 may be useful for the prevention of community-acquired gastrointestinal and upper respiratory infections.

Purification of Catalase from Pea Leaf Peroxisomes: Identification of Five Different Isoforms

Catalase activity was analyzed in seven organs of pea (Pisum sativum L.) plants: leaves, seeds, flowers, shoots, whole fruits, pods and roots. Leaves showed the highest activity followed by whole fruits and flowers. Catalase was purified from pea leaf peroxisomes. These organelles were isolated from leaves by differential and sucrose density-gradient centrifugation, and catalase was purified by two steps involving anion exchange and hydrophobic chromatography using a Fast Protein Liquid Chromatography system. Pure catalase had a specific activity of 953 mmol H2O2 min(-1) mg(-1) protein and was purified 1000-fold, with a yield of about 19 microg enzyme per kg of pea leaves. Analysis by SDS-PAGE and immunoblot showed that the pea catalase was composed of subunits of 57 kDa. Ultraviolet and visible absorption spectra of the enzyme showed two absorption maxima at 252 and 400 nm with molar extinction coefficients of 2.14 x 10(6) and 7.56 x 10(6) M(-1) cm(-1), respectively. By isoelectric focusing (pH 5-7), five different isoforms were identified and designated as CAT1-5, with isoelectric points of 6.41, 6.36, 6.16, 6.13 and 6.09, respectively. All the catalase isoforms contained a subunit of 57 kDa. Post-embedment, EM immunogold labelling of catalase showed a uniform distribution of the enzyme inside the matrix and core of pea leaf peroxisomes.

Superoxide radical generation in peroxisomal membranes: evidence for the participation of the 18 kDa integral membrane polypeptide.

Peroxisomes were isolated from pea (Pisum sativum L.) leaves and the peroxisomal membranes were purified by treatment with Na2CO3. The production of superoxide radicals (O2) induced by NADH was investigated in peroxisomal membranes from intact organelles incubated with proteases (pronase E and proteinase K). Under isoosmotic conditions, in the presence of pronase E, the production of O2-. radicals was inhibited by 80%. SDS-PAGE of peroxisomal membranes after protease treatment demonstrated a decrease in the 18-kDa PMP. This suggests that this polypeptide has a small fragment exposed to the cytosolic side of the peroxisomal membrane which is essential for O2-. production. The 18-kDa PMP was purified by preparative SDS-PAGE and in the reconstituted protein the NADH-driven production of O2-. radicals was investigated. The isolated polypeptide showed a high generation rate of superoxide (about 300 nmol O2-. x mg-1 protein x min-1) which was completely inhibited by 50 mM pyridine. The 18-kDa PMP was recognized by a polyclonal antibody against Cyt b5 from human erythrocytes. The presence of b-type cytochrome in peroxisomal membranes was demonstrated by difference spectroscopy. Results obtained show that in the NADH-dependent O2-. radical generating system of peroxisomal membranes, the 18-kDa integral membrane polypeptide, which appears to be Cyt b5, is clearly involved in superoxide radical production.

Human absorption of a supplement containing purified hydroxytyrosol, a natural antioxidant from olive oil, and evidence for its transient association with low-density lipoproteins

There is growing interest in the health effects of olive oil polyphenols, particularly hydroxytyrosol (HT), for their potential application in the treatment of inflammatory conditions such as cardiovascular disease (CVD). As oxidative modification of low-density lipoproteins (LDL) plays a central role in the development of CVD, natural antioxidants are a main target for the nutraceutical industry. In this study we firstly investigated the absorption of pure hydroxytyrosol (99.5%) administered as a supplement in an aqueous solution (2.5mg/kg BW) in the plasma and urine of healthy volunteers (n=10). Plasma C(max) for HT and homovanillic alcohol (HvOH) were detected at 13.0+/-1.5 and 16.7+/-2.4min, respectively. The HT and HvOH levels were undetectable 2-h after the administration. HT, HvOH, homovanillic acid and 3,4-dihydroxyphenylacetic acid were found as free forms (44%) or as glucuronide (34.4%) or sulphate (21.2%) conjugates in the 24-h urine samples of the subjects. In a second phase of the study, the same amounts of HT were administered to the subjects and the presence of HT in purified plasma lipoproteins was investigated in LDL fractions freshly isolated. 10min after the ingestion of the HT supplement, more than 50% of the total amount detected was present in the LDL-purified fractions and its concentration declined in accordance with its presence in plasma but no changes were found in total antioxidant capacity, malondialdehyde or LDL lag time. These results indicate that pure HT transiently associates with LDL lipoproteins in vivo.

IMMUNOCYTOCHEMICAL LOCALIZATION OF COPPER, ZINC SUPEROXIDE DISMUTASE IN PEROXISOMES FROM WATERMELON (CITRULLUS VULGARIS SCHRAD.) COTYLEDONS

In previous works using cell fractionation methods we demonstrated the presence of a Cu,Zn-containing superoxide dismutase in peroxisomes from watermelon cotyledons. In this work, this intracellular localization was evaluated by using western blot and EM immunocytochemical analysis with a polyclonal antibody against peroxisomal Cu,Zn-SOD II from watermelon cotyledons. In crude extracts from 6-day old cotyledons, analysis by western blot showed two cross-reactivity bands which belonged to the isozymes Cu,Zn-SOD I and Cu,Zn-SOD II. In peroxisomes purified by sucrose density-gradient centrifugation only one cross-reactivity band was found in the peroxisomal matrix which corresponded to the isozyme Cu,Zn-SOD II. When SOD activity was assayed in purified peroxisomes two isozymes were detected, Cu,Zn-SOD II in the matrix, and a Mn-SOD in the membrane fraction which was removed by sodium carbonate washing. EM immunocytochemistry of Cu,Zn-SOD on sections of 6-day old cotyledons, showed that gold label was mainly localized over plastids and also in peroxisomes and the cytosol, whereas mitochondria did not label for Cu,Zn-SOD.

Lactobacillus fermentum CECT 5716 is safe and well tolerated in infants of 1–6 months of age: A Randomized Controlled Trial

The objective of the study was to evaluate the safety and tolerance of an infant formula supplemented with Lactobacillus fermentum CECT5716, a probiotic strain isolated from breast milk, in infants of 1-6 months of age. A randomized double blinded controlled study including healthy infants was conducted. One month aged infants received a prebiotic infant formula supplemented with L. fermentum (experimental group) or the same formula without the probiotic strain (control group) for 5 months. The primary outcome of the study was average daily weight gain between baseline and 4 months of age. Secondary outcomes were other anthropometric data (length and head circumference), formula consumption, and tolerance. Incidence of infections was also recorded by pediatricians. No significant differences in weight gain were observed between both groups, neither at 4 months of age (29.0±7.8 vs 28.9±5.7g/day) nor at 6 months (25.1±6.1 vs 24.7±5.2g/day). There were no statistically significant differences in the consumption of the formulae or symptoms related to the tolerance of the formula. The incidence rate of gastrointestinal infections in infants of the control group was 3 times higher than in the probiotic group (p=0.018). Therefore, consumption of a prebiotic infant formula enriched with the human milk probiotic strain L. fermentum CECT5716 from 1 to 6 months of life is well tolerated and safe. Furthermore, the consumption of this formula may improve the health of the infants by reducing the incidence of gastrointestinal infections.

Milk enriched with “healthy fatty acids” improves cardiovascular risk markers and nutritional status in human volunteers

OBJECTIVE The main goal of the present study was to evaluate the effect of a commercially available milk containing small amounts of eicosapentaenoic acid and docosahexaenoic acid, oleic acid, and vitamins A, B6, D, E, and folic acid compared with semi-skimmed and skimmed milk in volunteers with moderate cardiovascular risk. METHODS Two hundred ninety-seven subjects 25 to 65 y of age with moderate cardiovascular risk were randomly allocated into three groups. In addition to their diets, one group consumed 500 mL/d of the enriched milk, another group consumed 500 mL/d of skimmed milk, and a control group consumed 500 mL/d of semi-skimmed milk. All groups consumed the dairy drinks for 1 y and blood samples were taken at 0 and 12 mo. RESULTS Consumption of enriched milk for 1 y produced significant (P < 0.05) increases in serum folate (58%) and high-density lipoprotein cholesterol (4%). Plasma triacylglycerols (10%), total cholesterol (4%), and low-density lipoprotein cholesterol (6%) were reduced significantly only in the supplemented group. Serum glucose, homocysteine, and C-reactive protein remained unchanged. In the skimmed milk and semi-skimmed milk groups, the only significant decreases were in serum folate (17% and 11%, respectively). CONCLUSION Daily intake of a milk enriched with fish oil, oleic acid, and vitamins improved the nutritional status and cardiovascular risk markers of volunteers, whereas skimmed milk and semi-skimmed milk did not.

Improvement of bone formation biomarkers after 1-year consumption with milk fortified with eicosapentaenoic acid, docosahexaenoic acid, oleic acid, and selected vitamins

The hypothesis of this study was that the replacement of regular milk with fortified milk in hyperlipidemic adults for 1 year would improve bone biomarkers. The fortified milk contained eicosapentaenoic acid and docosahexaenoic acid from fish oils, oleic acid, vitamins A, B(6), and E, as well as folic acid. We believe that the fortified milk will improve the blood fatty acid profile and vitamin status in subjects to benefit bone health biomarkers. From the 84 patients who accepted to participate, 11 of these were excluded for the presence of metabolic diseases and 1 was excluded for noncompliance with the protocol. Seventy-two hyperlipidemic patients (35-65 years) were randomly divided between 2 study groups. The supplement group (E; n = 39) consumed 0.5 L/d of fortified milk that contained fish oil, oleic acid, and vitamins. The control group (C; n = 33) consumed 0.5 L/d of semiskimmed milk containing the same amount of total fat. Blood samples were taken at T(0), T(3), T(6), and T(12) months to determine plasma fatty acids, vitamins B(6), E, and 25-hydroxyvitamin D and serum folate, calcium, soluble osteoprotegerin (OPG), soluble receptor activator of NF-kappaB ligand (RANKL), osteocalcin, parathormone, type I collagen carboxy-terminal telopeptide, and malondialdehyde. After 1 year, the E group showed a significant increase in plasma eicosapentaenoic acid (42%), docosahexaenoic acid (60%), vitamin B6 (38%), OPG (18%), RANKL (7%), OPG/RANKL (10%), red blood cell folate (21%), serum folate (53%), calcium (4%), vitamin D (11%), and osteocalcin (22%). Dietary supplementation with the fortified milk drink improved nutritional status and bone formation markers in adult hyperlipidemic patients.

Peroxisomes, Reactive Oxygen Metabolism, and Stress-Related Enzyme Activities

The main functions described for peroxisomes in plant cells are the oxidative photosynthetic carbon cycle of photorespiration, fatty acid β-oxidation, the glyoxylate cycle, and the metabolism of ureides (Huang et al, 1983; del Rio et al, 1992; Reumann, 2000). A characteristic property of peroxisomes is their metabolic plasticity since their enzymatic content can vary depending on the organism, cell/tissue-type and environmental conditions. An illustrative example of the inducible nature of peroxisomal metabolism is the light-induced transition of glyoxysomes, the specialized peroxisomes of oilseeds, to leaf-type peroxisomes (Huang et al, 1983; Mullen and Trelease, 1996). Likewise, during the senescence of leaves the reverse process is observed, that is the conversion of leaf peroxisomes into glyoxysomes (Vicentini and Matile, 1993; Nishimura et al, 1996; del Rio et al, 1998a). On the other hand, the cellular population of peroxisomes can proliferate in plants during senescence and under different abiotic stress conditions (del Rio et al, 1996, 1998a, 2001; Corpas et al, 2001). The induction of peroxisome biogenesis genes (PEX) by H2O2 has been demonstrated in both plant and animal cells, indicating that the signal molecule H2O2 is responsible for the proliferation of peroxisomes (Lopez-Huertas et al, 2000).