Antioxidant Effect of Standardized Extract of Propolis (EPP-AF®) in Healthy Volunteers: A "Before and After" Clinical Study.

BACKGROUND: Propolis is rich in polyphenols, especially flavonoids and phenolic acids, and has significant antioxidant activity, shown mainly in "in vitro" studies. OBJECTIVE: The aim of this study was to evaluate the antioxidant efficacy and safety of a standardized propolis extract in healthy volunteers. DESIGN: A two-phase sequential, open-label, nonrandomized, before and after clinical trial. METHODS: Healthy participants received two EPP-AF® doses (375 and 750 mg/d, P.O, tid) during 7 ± 2 days, starting with the lower doses. Immediately before starting EPP-AF® administration and at the end of each 7-day dosing schedule, blood and urine samples were collected for quantification of 8-OHDG (8-hydroxydeoxyguanosine) and 8-ISO (8-isoprostanes) in urine and GSH (reduced glutathione), GSSG (oxidized glutathione), SOD (superoxide dismutase), FRAP (Ferric Reducing Antioxidant Power), vitamin E, and MDA (malondialdehyde) in plasma. RESULTS: In our study, we had 34 healthy participants (67.7% women, 30 ± 8 years old, 97% white). The 8-ISO, a biomarker of lipid peroxidation, decreased with both doses of EPP-AF® compared to baseline (8-ISO, 1.1 (0.9-1.3) versus 0.85 (0.75-0.95) and 0.89 (0.74-1.0), ng/mg creatinine, P < 0.05, for 375 and 750 mg/d EPP-AF® doses versus baseline, mean and CI 95%, respectively). 8-OHDG, a biomarker of DNA oxidation, was also reduced compared to baseline with 750 mg/d doses (8-OHDG, 15.7 (13.2-18.1) versus 11.6 (10.2-13.0), baseline versus 750 mg/d, respectively, ng/mg creatinine, P < 0.05). Reduction of biomarkers of oxidative stress damage was accompanied by increased plasma SOD activity (68.8 (66.1-73.3) versus 78.2 (72.2-80.5) and 77.7 (74.1-82.6), %inhibition, P < 0.0001, 375 and 750 mg/d versus baseline, median and interquartile range 25-75%, respectively) and by increased GSH for 375 mg/d EPP-AF® doses (1.23 (1.06-1.34) versus 1.33 (1.06-1.47), µmol/L, P < 0.05). CONCLUSION: EPP-AF® reduced biomarkers of oxidative stress cell damage in healthy humans, with increased antioxidant enzymatic capacity, especially of SOD. This trial is registered with the Brazilian Registry of Clinical Trials (ReBEC, RBR-9zmfs9).

Human milk enriched with human milk lyophilisate for feeding very low birth weight preterm infants: A preclinical experimental study focusing on fatty acid profile.

BACKGROUND: Human milk, with essential nutrients and long chain polyunsaturated fatty acids (LC-PUFAs) such as the omega 3 and 6 fatty acids is important for development of the central nervous system and the retina in very low birth weight infants (<1,500 g). However, breast milk may not be sufficient to meet these needs. The possibility of supplementing breast milk with a lyophilisate of human milk was explored in this study. The objectives of this study were to determine the total lipid content and the lipid profile of the Human Milk on Baseline (HMB) and that of the Concentrates with the Human Milk + lyophilisate (with lyophilisate of milk in the immediate period (HMCI), at 3 months (HMC3m), and at 6 months (HMC6m) of storage). METHODS: Fifty donors from the Human Milk Bank of Children's Hospital provided consent, and donated milk samples. Macronutrient (including total lipids) quantification was performed using the MIRIS® Human Milk Analyzer, and the fatty acid profile was determined by gas chromatography (CG-FID, SHIMADZU®). RESULTS: There was a higher lipid concentration in HMCI relative to HMB. The concentrations of the main fatty acids (% of total) were as follows: palmitic acid (C16:0) HMB, 22.30%; HMCI, 21.46%; HMC3m, 21.54%; and HMC6m, 21.95% (p<0.01); oleic acid (C18:1n-9) HMB, 30.41%; HMCI, 30.47%; HMC3m, 30.55%; and HMC6m, 29.79% (p = 0.46); linoleic acid (C18:2n-6) HMB, 19.62%; HMCI, 19.88%; HMC3m, 19.49%; and HMC6m, 19.45% (p = 0.58); arachidonic acid (C20:4n-6) HMB, 0.35%; HMCI, 0.16%; HMC3m, 0.13%; and HMC6m, 0.15% (p<0.01); α-linolenic acid (C18:3n-3) HMB,1.32%; HMCI, 1.37%; HMC3m, 1.34%; and 1.34% HMC6m (p = 0.14); docosahexaenoic acid (C22:6n-3) HMB, 0.10%; HMCI, 0.06%; HMC3m, 0.05%; and HMC6m, 0.06% (p<0.01). There were no significant changes in the lipid profile when stored. There was no evidence of peroxidation during storage. CONCLUSIONS: Freeze-dried human milk fortified with a human milk concentrate brings potential benefits to newborns, mainly by preserving the essential nutrients present only in breast milk; however, further clinical studies are required to evaluate the safety and efficacy of the concentrate as a standard nutritional food option for very low birth weight infants.

Hepatic Osteodystrophy: The Mechanism of Bone Loss in Hepatocellular Disease and the Effects of Pamidronate Treatment.

OBJECTIVES:: The present study was designed to evaluate the bone phenotypes and mechanisms involved in bone disorders associated with hepatic osteodystrophy. Hepatocellular disease was induced by carbon tetrachloride (CCl4). In addition, the effects of disodium pamidronate on bone tissue were evaluated. METHODS:: The study included 4 groups of 15 mice: a) C = mice subjected to vehicle injections; b) C+P = mice subjected to vehicle and pamidronate injections; c) CCl4+V = mice subjected to CCl4 and vehicle injections; and d) CCl4+P = mice subjected to CCl4 and pamidronate injections. CCl4 or vehicle was administered for 8 weeks, while pamidronate or vehicle was injected at the end of the fourth week. Bone histomorphometry and biomechanical analysis were performed in tibiae, while femora were used for micro-computed tomography and gene expression. RESULTS:: CCl4 mice exhibited decreased bone volume/trabecular volume and trabecular numbers, as well as increased trabecular separation, as determined by bone histomorphometry and micro-computed tomography, but these changes were not detected in the group treated with pamidronate. CCl4 mice showed increased numbers of osteoclasts and resorption surface. High serum levels of receptor activator of nuclear factor-κB ligand and the increased expression of tartrate-resistant acid phosphatase in the bones of CCl4 mice supported the enhancement of bone resorption in these mice. CONCLUSION:: Taken together, these results suggest that bone resorption is the main mechanism of bone loss in chronic hepatocellular disease in mice.

Hepatic Osteodystrophy: The Mechanism of Bone Loss in Hepatocellular Disease and the Effects of Pamidronate Treatment

OBJECTIVES: The present study was designed to evaluate the bone phenotypes and mechanisms involved in bone disorders associated with hepatic osteodystrophy. Hepatocellular disease was induced by carbon tetrachloride (CCl4). In addition, the effects of disodium pamidronate on bone tissue were evaluated. METHODS: The study included 4 groups of 15 mice: a) C = mice subjected to vehicle injections; b) C+P = mice subjected to vehicle and pamidronate injections; c) CCl4+V = mice subjected to CCl4 and vehicle injections; and d) CCl4+P = mice subjected to CCl4 and pamidronate injections. CCl4 or vehicle was administered for 8 weeks, while pamidronate or vehicle was injected at the end of the fourth week. Bone histomorphometry and biomechanical analysis were performed in tibiae, while femora were used for micro-computed tomography and gene expression. RESULTS: CCl4 mice exhibited decreased bone volume/trabecular volume and trabecular numbers, as well as increased trabecular separation, as determined by bone histomorphometry and micro-computed tomography, but these changes were not detected in the group treated with pamidronate. CCl4 mice showed increased numbers of osteoclasts and resorption surface. High serum levels of receptor activator of nuclear factor-κB ligand and the increased expression of tartrate-resistant acid phosphatase in the bones of CCl4 mice supported the enhancement of bone resorption in these mice. CONCLUSION: Taken together, these results suggest that bone resorption is the main mechanism of bone loss in chronic hepatocellular disease in mice.

Dietary docosahexaenoic acid and eicosapentaenoic acid influence liver triacylglycerol and insulin resistance in rats fed a high-fructose diet.

This study aimed to examine the benefits of different amounts of omega-3 (n-3) polyunsaturated fatty acids from fish oil (FO) on lipid metabolism, insulin resistance and gene expression in rats fed a high-fructose diet. Male Wistar rats were separated into two groups: Control (C, n = 6) and Fructose (Fr, n = 32), the latter receiving a diet containing 63% by weight fructose for 60 days. After this period, 24 animals from Fr group were allocated to three groups: FrFO2 (n = 8) receiving 63% fructose and 2% FO plus 5% soybean oil; FrFO5 (n = 8) receiving 63% fructose and 5% FO plus 2% soybean oil; and FrFO7 (n = 8) receiving 63% fructose and 7% FO. Animals were fed these diets for 30 days. Fructose led to an increase in liver weight, hepatic and serum triacylglycerol, serum alanine aminotransferase and HOMA1-IR index. These alterations were reversed by 5% and 7% FO. FO had a dose-dependent effect on expression of genes related to hepatic ß-oxidation (increased) and hepatic lipogenesis (decreased). The group receiving the highest FO amount had increased markers of oxidative stress. It is concluded that n-3 fatty acids may be able to reverse the adverse metabolic effects induced by a high fructose diet.

Fish oil decreases hepatic lipogenic genes in rats fasted and refed on a high fructose diet.

Fasting and then refeeding on a high-carbohydrate diet increases serum and hepatic triacylglycerol (TAG) concentrations compared to standard diets. Fructose is a lipogenic monosaccharide which stimulates de novo fatty acid synthesis. Omega-3 (n-3) fatty acids stimulate hepatic ß-oxidation, partitioning fatty acids away from TAG synthesis. This study investigated whether dietary n-3 fatty acids from fish oil (FO) improve the hepatic lipid metabolic response seen in rats fasted and then refed on a high-fructose diet. During the post-prandial (fed) period, rats fed a FO rich diet showed an increase in hepatic peroxisome proliferator-activated receptor α (PPAR-α) gene expression and decreased expression of carbohydrate responsive element binding protein (ChREBP), fatty acid synthase (FAS) and microsomal triglyceride transfer protein (MTTP). Feeding a FO rich diet for 7 days prior to 48 h of fasting resulted in lower hepatic TAG, lower PPAR-α expression and maintenance of hepatic n-3 fatty acid content. Refeeding on a high fructose diet promoted an increase in hepatic and serum TAG and in hepatic PPAR-α, ChREBP and MTTP expression. FO did not prevent the increase in serum and hepatic TAG after fructose refeeding, but did decrease hepatic expression of lipogenic genes and increased the n-3 fatty acid content of the liver. n-3 Fatty acids can modify some components of the hepatic lipid metabolic response to later feeding with a high fructose diet.

Mercury exposure increases circulating net matrix metalloproteinase (MMP)-2 and MMP-9 activities.

Mercury (Hg) exposure causes health problems that may result from increased oxidative stress and matrix metalloproteinase (MMP) levels. We investigated whether there is an association between the circulating levels of MMP-2, MMP-9, their endogenous inhibitors (the tissue inhibitors of metalloproteinases; TIMPs) and the circulating Hg levels in 159 subjects environmentally exposed to Hg. Blood and plasma Hg were determined by inductively coupled plasma-mass spectrometry (ICP-MS). MMP and TIMP concentrations were measured in plasma samples by gelatin zymography and ELISA respectively. Thiobarbituric acid-reactive species (TBARS) were measured in plasma to assess oxidative stress. Selenium (Se) levels were determined by ICP-MS because it is an antioxidant. The relations between bioindicators of Hg and the metalloproteinases levels were examined using multivariate regression models. While we found no relation between blood or plasma Hg and MMP-9, plasma Hg levels were negatively associated with TIMP-1 and TIMP-2 levels, and thereby with increasing MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios, thus indicating a positive association between plasma Hg and circulating net MMP-9 and MMP-2 activities. These findings provide a new insight into the possible biological mechanisms of Hg toxicity, particularly in cardiovascular diseases.

Effect of NFkappaB inhibition by CAPE on skeletal muscle ischemia-reperfusion injury.

BACKGROUND/AIMS: Nuclear factor kappa B (NFkappaB) plays important role in the pathogenesis of skeletal muscle ischemia/reperfusion (I/R) injury. Caffeic acid phenyl ester (CAPE), a potent NFkappaB inhibitor, exhibits protective effects on I/R injury in some tissues. In this report, the effect of CAPE on skeletal muscle I/R injury in rats was studied. METHODS: Wistar rats were submitted to sham operation, 120-min hindlimb ischemia, or 120-min hindlimb ischemia plus saline or CAPE treatment followed by 4-h reperfusion. Gastrocnemius muscle injury was evaluated by serum aminotransferase levels, muscle edema, tissue glutathione and malondialdehyde measurement, and scoring of histological damage. Apoptotic nuclei were determined by a terminal uridine deoxynucleotidyl transferase dUTP nick end labeling assay. Muscle neutrophil and mast cell accumulation were also assessed. Lipoperoxidation products and NFkappaB were evaluated by 4-hydroxynonenal and NFkappaB p65 immunohistochemistry, respectively. RESULTS: Animals submitted to ischemia showed a marked increase in aminotransferases after reperfusion, but with lower levels in the CAPE group. Tissue glutathione levels declined gradually during ischemia to reperfusion, and were partially recovered with CAPE treatment. The histological damage score, muscle edema percentage, tissue malondialdehyde content, apoptosis index, and neutrophil and mast cell infiltration, as well as 4-hydroxynonenal and NFkappaB p65 labeling, were higher in animals submitted to I/R compared with the ischemia group. However, the CAPE treatment significantly reduced all of these alterations. CONCLUSIONS: CAPE was able to protect skeletal muscle against I/R injury in rats. This effect may be associated with the inhibition of the NFkappaB signaling pathway and decrease of the tissue inflammatory response following skeletal muscle I/R.

Recommended dose for repair of serum vitamin A levels in patients with HIV infection/AIDS may be insufficient because of high urinary losses.

OBJECTIVE: Retinol deficiency is quite frequent in the population of human immunodeficiency virus (HIV)-infected individuals. Serum retinol levels of less than 1.05 micromol/L determine a 3.5 to five times higher death risk. However, studies evaluating the efficacy of retinol supplementation in HIV-seropositive individuals have reported conflicting results. The World Health Organization recommends the treatment of vitamin A deficiency in seropositive individuals in the same manner as for seronegative individuals, but clinical studies proving the efficacy of this scheme are lacking. The proposal of the present study was to assess the efficacy of supplementation with high retinol doses in HIV-infected patients with vitamin A deficiency. METHODS: Twenty-five adult HIV-seropositive individuals were monitored over a period of 9 months, with determination of serum and urinary retinol every 3 months. The subjects received retinol palmitate doses ranging from 300,000 IU to 600,000 IU. Patients whose retinol levels were higher than 1.60 micromol/L were only observed. RESULTS: Eighteen patients received supplementation during clinical monitoring. The dose of 600,000 IU induced a significant mean increase in serum levels of 0.47 micromol/L (P = 0.049) within a period of three months. Those who received 300,000 IU presented a mean increase of 0.29 micromol/L. In contrast, the patients who did not receive replacement therapy presented a significant decrease (P = 0.017) in serum retinol levels, with initial and final values of 1.77 micromol/L and 1.55 micromol/L. The individuals with the worst response to supplementation presented a higher urinary loss of retinol at the beginning of the study. Even with a mean retinol supplementation of 771,428 IU during the study period, six patients had marginal serum retinol levels at the end of the study. CONCLUSION: We conclude that, in view of the high urinary loss of this nutrient, there is the need to redefine the ideal dose for the treatment of HIV-infected individuals.

DNA oxidative damage in patients with dialysis treatment.

BACKGROUND/AIMS: Chronic renal patients on hemodialysis (HD) and peritoneal dialysis (PD) treatment are exposed to oxidative stress and DNA damage. The objective of this study was to assess the oxidative damage to DNA in end-stage chronic renal failure, before and after vitamin E supplementation. METHODS: Patients on HD (n=29) and PD (n=22) received oral supplementation with 300 mg vitamin E three times a week for 4 weeks. A blood sample was collected at the beginning and at the end of the supplementation cycle for the determination of vitamin E levels (high-performance liquid chromatography), carbonyl groups, and DNA damage (8-hydroxy 2'-deoxyguanosine [8-OHdG] and comet assay). RESULTS: After supplementation, vitamin E concentration was increased by about 50%. Protein oxidation was initially observed in both groups, with a reduction after supplementation. DNA damage detected by the comet assay and by 8-OHdG analysis was significantly reduced (p<0.05) after supplementation in both groups. CONCLUSIONS: Vitamin E supplementation reduced oxidative DNA damage in both HD and PD patients. Treatments such as HD and PD induce oxidative stress and consequent DNA damage, and increased plasma vitamin E levels significantly contribute to the normalization of these events.