PUFA NEWSLETTER. April 2010 Contents. Volume 15 Issue 1. EDITORIAL Omega-3 Fatty Acid Research on a Roll...2

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1 PUFA NEWSLETTER Volume 15 Issue 1 April 2010 Contents EDITORIAL Omega-3 Fatty Acid Research on a Roll...2 CARDIOVASCULAR HEALTH Mixed Results on Pre-Surgical Long-Chain Omega-3s and Post-Operative Atrial Fibrillation...3 Red Blood Cell or Plasma PUFAs Strongly Correlated with Cardiac Levels...5 MATERNAL AND INFANT HEALTH Dose-Response Study Examines Relationship Between DHA-Supplemented Formula and Visual Acuity...6 High Dietary Linoleic Acid Linked to Lower Omega-3 PUFAs in Maternal Red Blood Cells...8 Gestation Time Not Increased with Omega-3s in Women at Risk of Preterm Delivery BRAIN FUNCTION DHA Promotes Neurite and Synapse Development and Function in Cells and Animals...12 Long-Chain PUFAs Increase Complex Neurite Outgrowth in Cells from Young and Old Supplementary DHA Linked to Greater Cortex Activity During Attention Task in Boys MENTAL HEALTH AND COGNITION Modest Dose Long-Chain Omega-3s Lower Risk of Progression to Psychosis Blood DHA Associated with Several Dimensions of Cognition in Healthy Adults VISUAL FUNCTION DHA with Epithelial Growth Factor Enhance Nerve Regeneration After Corneal Surgery CLINICAL CONDITIONS Type 2 diabetes Long-Chain Omega-3 Fatty Acids Improve Microvascular Function in Type 2 Diabetes...22 FRONTIERS Omega-3 PUFAs Might Regulate Cell Aging by Slowing Telomere Shortening...24 PUFA NEWSLETTER STAFF Editor Joyce A. Nettleton, DSc Communications Manager Angela Dansby Sponsor DSM Nutritional Products, Kaiseraugst, Switzerland, SCIENCE ADVISORY BOARD J. Thomas Brenna, PhD Cornell University Ithaca, NY, USA Stefan Endres, MD University of Munich Munich, Germany Marlene P. Freeman, MD Massachusetts General Hospital Boston, MA, USA William S. Harris, PhD University of Missouri Kansas City, MO, USA Maria Makrides, PhD Women s and Children Hospital Adelaide, Australia Letters and editorial comments should be submitted to joyce@fatsoflife.com and technical comments to angela@fatsoflife.com. Subscribe to the PUFA Newsletter at

2 EDITORIAL Omega-3 Fatty Acid Research on a Roll With this issue of the PUFA Newsletter and its companion for consumers, Fats of Life, we launch a new publication schedule of three issues per year. The newsletters will appear in April, August and December. One consequence of this change is a greater number of research articles we cannot include. To draw your attention to some of those, we will list a few at the end of each section under the heading, Worth Noting. Three articles in the April issue may be milestones. One is a tightly controlled study among young patients at ultra-high risk of developing a psychotic disorder. After 3 months of consuming just over 1 gram of long-chain omega-3 fatty acids or a placebo daily, only 2 out of 41 patients in the omega-3 group progressed to psychosis. In the placebo group, 11 of the 40 participants developed a psychotic disorder. These significant differences persisted for a year after the treatment ended. The second highlight is the effect of DHA plus pigment epithelial-derived growth factor on the regeneration of corneal nerves following corneal surgery. When administered together, there was up to a 3-fold increase in corneal nerve regeneration. Neither substance alone affected nerve growth. These results suggest enhanced recovery from corneal surgery and the possible preservation of healthy tear production. Shedding a tear could become something to celebrate. A third candidate mile-marker is the observation that people with stable heart disease who had higher levels of docosahexaenoic acid (DHA) in their blood experienced less shortening of their telomeres than those with low DHA levels. Telomeres are the protective ends of the chromosomes that gradually shorten during normal cell division and aging. Telomeres preserve faithful cell reproduction, but when they become too short, the cell enters senescence. Evidence suggests that individuals with shorter telomeres have higher cardiac mortality and other unwanted consequences of aging. How DHA affects telomere shortening or the enzyme that lengthens telomeres is an unfolding chapter in understanding aging processes. Other noteworthy research describes the potential effect of diets high in linoleic acid on the content of DHA in the red blood cells of pregnant women. Are linoleic acid-rich diets packing a double whammy for women whose consumption of long-chain omega-3s is already low? Advanced medical imaging techniques may one day make it possible to quantify the DHA content in different brain regions. A study in this issue describes an association between neural activity in the cortex during an attention task and the consumption of supplementary DHA. Other reports in the Brain Function section describe the critical importance of DHA in neurite and synapse development. The newsletter team welcomes your suggestions and comments. We hope to meet many readers at the biennial meeting of the International Society for the Study of Fatty Acids and Lipids in Maastricht, the Netherlands, May 29 to June 2, Joyce A. Nettleton, DSc Editor, PUFA Newsletter and Fats of Life joyce@fatsoflife.com 2

3 CARDIOVASCULAR HEALTH Mixed Results on Pre-Surgical Long-Chain Omega-3s and Post-Operative Atrial Fibrillation One mechanism by which longchain omega-3 Atrial fibrillation (irregular rapid heartbeats) often poly unsaturated follows cardiac surgery, fatty acids (n-3 increasing the risk of LC-PUFAs) might stroke and heart failure. reduce the risk Long-chain omega-3 fatty of cardiovascular acids, with anti-arrhythmic disease and mortality is by effects, might reduce improving the the occurrence of this electrophysical condition. So far, the properties of the data are inconsistent. heart. Consumption Three new studies leave of fish or n-3 LCthe question undecided. PUFAs is associated with a significant reduction in sudden cardiac death in primary and secondary prevention trials, which are almost always caused by arrhythmias. One of the most common types of arrhythmia is atrial fibrillation, the irregular trembling of the upper chambers of the heart. When this happens the synchrony between the upper and lower chambers of the heart is lost and the heart s pumping function may be compromised. Atrial fibrillation can increase the risk of stroke and heart failure. The condition is relatively common in the immediate post-operative period among patients undergoing coronary artery bypass surgery. A recent report from Texas described a 29% higher risk of long-term mortality in patients who developed atrial fibrillation after such surgery compared with patients who did not develop the condition. Several investigators have explored the effects of fish or n-3 LC-PUFA consumption on the chance of developing atrial fibrillation in people with and without cardio vascular disease, but findings have been inconsistent. A Finnish study of more than 2,000 men, some with cardio vascular disease, reported that serum docosa hexaenoic acid (DHA) levels were associated with a significant 38% Figure. Illustration of atrial fibrillation. Image reproduced with permission from the Heart Rhythm Society. lower chance of atrial fibrillation over nearly 18 years. Italian investigators reported a significant reduction in hospitalization for atrial fibrillation and all-cause mortality in the first year following a myocardial infarction among patients who consumed n-3 LC-PUFAs. In a different study, patients undergoing coronary artery bypass surgery who were infused with fish oil upon admission to the hospital experienced a significantly lower incidence of atrial fibrillation following surgery compared with control patients (17% vs 31%). Similarly, post-operative atrial fibrillation occurred in significantly fewer coronary artery bypass patients who consumed 2 g/day of n-3 LC-PUFAs for 5 days or more before surgery (15% vs 33%) compared with controls. However, in the prospective Rotterdam study of more than 5,000 participants who did not have atrial fibrillation at enrolment, dietary consumption of n-3 LC-PUFAs was not associated with the risk of atrial fibrillation over 6 years. A Danish prospective cohort study of more than 47,000 participants also reported that consumption of fish or n-3 LC-PUFAs was not associated with a reduced risk of atrial fibrillation over nearly 6 years. On the other hand, a 12-year prospective study in older adults reported a significant 28% lower chance of developing atrial fibrillation in participants who ate tuna 1 to 4 times a week. It would appear that n-3 LC-PUFAs might be linked to lower risk of atrial fibrillation in the high-risk period following surgery, but might have little effect in community-living populations in the long-term. Moreover, the doses of n-3 LC-PUFAs used in the short-term studies, about 2 g/day, are likely higher than those in free-living populations. This article describes the findings of a randomized, double-blind, placebo-controlled trial with n-3 LC-PUFAs in 108 patients undergoing coronary artery bypass graft surgery. The study focused on the immediate post-operative period of 2 to 3 days when the chance of atrial fibrillation is highest. Eligible participants had no current or previous history of atrial fibrillation, were not taking anti-arrhythmic drugs and had not taken fish oil supplements within the previous 3 months. Treatment consisted of 2 g/day of n-3 LC-PUFAs as ethyl esters or olive oil control capsules for at least 5 days prior to surgery (median 17 days) along with standard cardiac care. Treatment continued until hospital discharge when the study ended. Patients were monitored using a heart rhythm monitor for 5 days post-operatively. Presence of atrial fibrillation was determined by analyzing the peaks and troughs from the heart monitor during each 24-hour period. Equivocal results 3

4 were resolved by analyzing the 24-hour recordings for atrial fibrillation episodes of 30 seconds or more. The primary outcome was any atrial fibrillation lasting 30 seconds or longer. Figure. Post-operative incidence of atrial and fibrillation and length of hospital stay in 103 patients who underwent coronary artery bypass surgery and consumed 2 g/day of n-3 LC-PUFAs or olive oil for at least 5 days prior to surgery. Of the 103 patients completing the study, 50% experienced at least one episode of atrial fibrillation lasting 30 seconds or more. This incidence is comparable to the usual occurrence observed in the authors medical institution. Other studies have reported incidences of atrial fibrillation ranging from 16% to 88%. Atrial fibrillation occurred in 56% of the n-3 LC-PUFA patients compared with 43% of placebo patients (Figure). The difference, however, was not statistically significant. Both groups stayed in the intensive care unit for a median of 1 day and the total hospital stay was 7 and 8 days for the placebo and n-3 LC-PUFA groups, respectively. The observed incidence of atrial fibrillation was higher than usually reported and the authors suggested that this might have been due to the use of continuous heart rhythm monitoring for 5 days. Patient medications may have contributed to the differences among studies as well. The presence of subclinical inflammation has been linked to a higher risk of atrial fibrillation, but in this study, levels of C-reactive protein did not differ between the 2 groups. Thus, the authors concluded that 2 g/day of n-3 LC-PUFAs given to coronary artery bypass patients did not reduce the risk of atrial fibrillation in the first 5 days after surgery. Another study from Germany (Heidt et al.) examined the effect of providing n-3 LC-PUFAs or soybean oil intravenously to 52 coronary artery bypass patients from hospital admission until discharge from the intensive care unit. The source of the n-3 LC-PUFAs was Omegaven, an intravenous emulsion containing fish oil. According to the package insert, this product contains 1.3 to 2.8 g of eicosapentaenoic acid (EPA) and 1.4 to 3.1 g of docosahexaenoic acid (DHA) per 100 ml. For participants at the mean body weight of 84 kg, the daily dose of n-3 PUFAs would be 1 to 2.3 g of EPA and 1.2 to 2.6 g of DHA. Atrial fibrillation was determined from continuous rhythm monitoring while the patients were in the intensive care unit. The definition of atrial fibrillation any confirmed episode of irregular rapid heartbeats longer than 15 minutes differed substantially from the foregoing study, which counted occurrences of 30 seconds or longer. Patients infused with n-3 LC-PUFAs experienced a lower rate of postoperative atrial fibrillation in the first 3 days after surgery compared with the control group (days 2 and 3, 7% vs 12% for n-3 LC-PUFA and control groups, respectively), differences that were statistically significant (P < 0.05). Control patients also spent significantly more time in intensive care than the n-3 LC-PUFA group. A third study addressed the same question in Of 3 new studies patients under going on the effects of open heart surgery n-3 LC-PUFAs on (Heidarsdottir et al.). The atrial fibrillation investigators compared after cardiac surgery, the incidence of atrial 2 reported no fibrillation in patients significant reduction receiving n-3 LC-PUFAs in the occurrence (1.2 and 1.0 g EPA and of atrial fibrillation DHA, respectively) or compared with olive oil capsules for 5 to controls. Results 7 days prior to surgery. Atrial fibrillation was from epidemiological defined as an episode and controlled lasting longer than intervention studies 5 minutes and was remain inconsistent. documented until hospital discharge. These investigators observed no difference in the incidence of post-operative atrial fibrillation between the n-3 LC-PUFA-treated patients and controls. As in the first paper, Saravanan et al., these investigators observed an incidence of post-operative atrial fibrillation in both groups of patients of 54%. These 3 studies add to the list of reports on the effectiveness (or lack thereof) of n-3 LC-PUFAs on postsurgical atrial fibrillation in patients with cardiovascular disease, but do not resolve the inconsistencies. Thus, the usefulness of n-3 LC-PUFAs in patients undergoing 4

5 coronary artery bypass or open heart surgery remains questionable. Although another study on this issue is underway, the question of how helpful n-3 LC-PUFAs may be in reducing the incidence of post-surgical atrial fibrillation remains unresolved. In addition, a different study on the efficacy of n-3 LC-PUFAs for the maintenance of normal sinus rhythms in patients with a history of atrial fibrillation is now recruiting participants. Saravanan P, Bridgewater B, West AL, O Neill SC, Calder PC, Davidson NC. Omega-3 fatty acid supplementation does not reduce risk of atrial fibrillation following coronary artery bypass surgery: a randomized, double-blind, placebo controlled clinical trial. Circ Arrhythm Electrophysiol 2010;3: Heidt MC, Vician M, Stracke SK, Stadlbauer T, Grebe MT, Boening A, Vogt PR, Erdogan A. Beneficial effects of intravenously administered N-3 fatty acids for the prevention of atrial fibrillation after coronary artery bypass surgery: a prospective randomized study. Thorac Cardiovasc Surg 2009;57: Heidarsdottir R, Arnar DO, Skuladottir GV, Torfason B, Edvardsson V, Gottskalksson G, Palsson R, Indridason OS. Does treatment with n-3 polyunsaturated fatty acids prevent atrial fibrillation after open heart surgery? Europace 2010;12: Worth Noting Harris WS. Omega-3 and atrial fibrillation post-cabg. [Letter] Thorac Cardiovasc Surg 2010;58:60. org/ /s Red Blood Cell or Plasma PUFAs Strongly Correlated with Cardiac Levels The dietary consumption of long-chain omega-3 Data from patients polyunsaturated fatty undergoing heart acids (n-3 LC-PUFAs) surgery show strong is reflected in the phospholipid fatty acid correlations between content of these PUFAs red blood cell n-3 in plasma and red blood and n-6 PUFAs and cell membranes. These trans fatty acids biomarkers are believed and those in atrial to be more useful muscle. Red cell and reliable than the PUFA concentrations assessment of dietary accurately reflect fatty acid intake, which cardiac PUFA levels. is subject to errors of measurement and recall. Red blood cell PUFA concentrations may also provide a useful estimate of tissue content and have been correlated with levels in brain, heart and other tissues. A study in 20 heart transplant patients reported a highly significant correlation between red blood cell eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA) and cardiac concentrations of these fatty acids. Australian investigators reported the rapid uptake of supplemental EPA and DHA into the atrial muscle and red blood cells of patients undergoing cardiopulmonary bypass surgery. Incorporation into atrial muscle was most rapid in the first 10 days after n-3 LC-PUFA consumption, reaching a plateau after 30 days. The uptake of DHA was more than twice that of EPA. In this follow-up analysis of the Australian study, the investigators describe the correlations between the concentrations of n-3 LC-PUFAs in red blood cells and plasma phospholipids and their content in atrial muscle. Tissue samples came from patients undergoing coronary artery bypass or valve repair or replacement surgery. Participants had consumed olive, flaxseed or no oil supplements prior to surgery, but had not consumed fish oil supplements, except as part of their usual dietary habits. n-3 LC-PUFA intakes were believed to represent those of the general population. Samples of atrial muscle were obtained at surgery. Phospholipid fatty acids were measured in atrial muscle, red blood cells and plasma. Table. Correlation coefficients for univariate associations between atrial and red blood cell or plasma phospholipid fatty acid concentrations in 61 patients undergoing heart surgery. All correlations were significant at P < except for arachidonic acid in red blood cells and plasma (P= and 0.002, respectively). Fatty acid Red blood cell Plasma n-6 PUFAs Linoleic Arachidonic n-3 PUFAs Alpha-linolenic Eicosapentaenoic Docosapentaenoic Docosahexaenoic EPA+DHA Trans 18:1 (total) The key findings were the strong correlations between red blood cell or plasma phospholipid n-3, n-6 and trans fatty acids and their corresponding concentrations in atrial muscle (Table). Significant correlations were observed for alpha-linolenic acid, EPA, docosapentaenoic acid, DHA and EPA+DHA. The correlations were slightly 5

6 stronger for red blood cell fatty acids compared with those in plasma. Multivariate analysis revealed an inverse association with female sex and atrial DHA, EPA+DHA and total n-3 LC-PUFAs, but not with EPA or docosapentaenoic acid. Similarly, red blood cell and plasma linoleic and arachidonic acid concentrations were correlated with their corresponding atrial levels, but the relationships were not as strong as for the n-3 PUFAs. The correlation coefficient for red blood cell linoleic acid was stronger than for arachidonic acid, e.g., 0.69 vs Multivariate analysis did not uncover other variables significantly related to n-6 PUFAs in atrial tissue. Red blood cell or plasma concentrations of total trans 18:1 (two isomers), trans 18:2 and total trans fatty acids were significantly correlated with their counterparts in atrial tissue, although levels of trans 18:2 were very low. Age was positively linked to total trans 18:1 and inversely to atrial docosapentaenoic acid. These observations To determine the PUFA provide concrete and trans fatty acids evidence that red blood cell or plasma in the heart, this study phospholipid n-3, suggests looking at n-6 and trans fatty their concentrations acids are appropriate in red blood cell or surrogates for their plasma phospholipids. concentrations in Strong correlations cardiac atrial between blood and muscle. The strong atrial fatty acids mean that heart fatty acids could be predicted from blood values. correlation between red blood cell and atrial tissue EPA+DHA concentrations provides additional evidence that the omega-3 index measured in red blood cells is a reliable reflection of the EPA+DHA content in atrial muscle. It is worth noting that red blood cell trans fatty acids and less strongly those in plasma were correlated with atrial trans fatty acids. Whether atrial trans fatty acids are involved in the increased risk of coronary heart disease, acute coronary syndrome, primary cardiac arrest, systemic inflammation and other abnormalities linked to cardiovascular disease is not known. However, high levels of these fatty acids in red cells have been associated with a greater risk of cardiovascular disease and cardiac arrest. This report indicates that red blood cell PUFA concentrations and less strongly those in plasma phospholipids accurately reflect the corresponding concentrations in atrial muscle. These findings favor the Visual acuity at 12 months of age was significantly poorer in infants consuming formula without DHA or arachidonic acid supplementation. At one of the 2 study sites significantly better visual acuity maturation was achieved with 0.64% DHA supplementation. DHA supplementation up to 0.96% was without adverse effects. use of red blood cell fatty acids as biomarkers of n-3 and n-6 PUFA status in the heart and would be preferable to dietary assessment as a reflection of exposure to these fatty acids. Metcalf RG, Cleland LG, Gibson RA, Roberts-Thomson KC, Edwards JR, Sanders P, Stuklis R, James MJ, Young GD. Relation between blood and atrial fatty acids in patients undergoing cardiac bypass surgery. Am J Clin Nutr 2010;91: Worth Noting Bjerregaard LJ, Joensen AM, Dethlefsen C, Jensen MK, Johnsen SP, Tjonneland A, Rasmussen LH, Overvad K, Schmidt EB. Fish intake and acute coronary syndrome. Eur Heart J 2010;31: Vanden Heuvel JP. Cardiovascular disease-related genes and regulation by diet. Curr Atheroscler Rep 2009;11: MATERNAL AND INFANT HEALTH Dose-Response Study Examines Relationship Between DHA-Supplemented Formula and Visual Acuity Randomized placebo-controlled trials in human infants have shown that breast-fed infants and those fed infant formula supplemented with long-chain omega-3 polyunsaturated fatty acids (n-3 LC-PUFAs) and arachidonic acid (ARA) have higher scores on various visual function tests compared with infants fed unsupplemented formula. Differences have been more pronounced for preterm than term infants. For example, term infants of mothers who consumed 400 mg/day of docosahexaenoic acid (DHA) from 16 weeks gestation until delivery had significantly higher visual acuity scores at 60 days of age compared with infants whose mothers consumed a placebo and whose usual diets were low in DHA. These findings suggest a beneficial effect of having DHA intakes above the recommended or required amounts. However, as is often the case, not all studies in term infants have reported improved visual function with DHA supplementation. 6

7 Evidence of improved visual and Supplementation of cognitive function infant formula with DHA in infants fed higher and arachidonic acid is amounts of DHA in associated with improved visual function compared with unsupplemented formula. Current DHA human milk compared with those fed formula with no additional DHA supplementation may not provided support legally exceed 0.32% of for the addition of DHA and ARA total fatty acids in the to infant formula. U.S., but is this the Other supporting optimum amount? This factors included dose-response study the wide variation explored that question. in breast milk DHA content, the high concentration of DHA in the retina, which contributes to retinal function, and the need in neonatal baboons for DHA contents higher than currently permitted to achieve tissue saturation of the cerebral cortex. As a result, most commercial infant formulas in the U.S. contain approximately 0.3% DHA and 0.6% ARA. Although there is some evidence that consumption of DHA in amounts above 0.3% is associated with higher concentrations in some tissues (e.g., cerebral cortex), whether there are benefits from higher intakes is unknown. In a recent report, Lisa Smithers and colleagues reported that feeding preterm infants milk containing 1% DHA, which is 3 times the standard amount, was not associated with any clinically meaningful changes in language development or behavior. However, visual acuity measured at 4 months corrected age was higher in the high-dha group compared with those fed 0.3% DHA-supplemented milk. This level of consumption did not saturate red blood cell phospholipid with DHA and levels remained below those observed in term infants. In preterm infants, at least, amounts of DHA greater than 0.3% supplementation, e.g., 1% DHA, might benefit their development. Data on the effects of different amounts of DHA added to infant formula are surprisingly sparse. Moreover, PUFA-supplemented infant formula differs in its content of ARA, linoleic acid and alpha-linolenic acid so comparisons among different trials can be confounded. In the paper described here, Eileen Birch and colleagues at the Retina Foundation of the Southwest, USA, other universities in the U.S. and Canada and Mead Johnson Nutrition collaborated on a prospective, randomized, double-masked and controlled trial of 4 different levels of DHA supplementation of healthy term infants fed cow milk-based formula for the first 12 months of life. Infants from Dallas and Kansas City participated. All DHA-supplemented formulas contained 0.64% ARA and either no DHA or ARA (control), 0.32%, 0.64% or 0.96% DHA. PUFAs came from single-cell algae (DHA) and fungal oils (ARA). The contents of linoleic acid (16.9% to 17.5%) and alpha-linolenic acid (1.61% to 1.68%) were similar in all formulas. The primary outcome was the maturation of visual acuity at 12 months of age. Other objectives included visual acuity assessment at 1.5, 4 and 9 months of age and red blood cell fatty acids at 4 and 12 months. Infants consumed formula exclusively for the first 4 months of life with additional foods thereafter as specified by the infants physicians. Experienced electrophysiologists assessed visual acuity using protocols for sweep visual evoked potentials (VEP) developed by Norcia et al. All VEP records were scored by the same trained expert in one study location and expressed as log minimum angle of resolution (MAR). Lower values represent better visual acuity. A total of 343 infants were enrolled in the 12-month study, of whom 244 (71%) completed the protocol. Data from each study site and formula group were analyzed separately because of the significant interaction between study site and formula. VEP visual acuity at 12 months of age and at each of the preceding periods (Figure) was significantly poorer in the control infants compared with each of the DHAsupplemented groups and the combined DHA groups (P < 0.001). There were no significant differences in visual acuity among the DHA-supplemented groups at either site or at any age. Control group VEP scores were better in the Kansas City than the Dallas infants, but were significantly poorer in the 0.32% DHAsupplemented formula infants. The Kansas City infants had statistically significant improvements in visual acuity with the 0.64% DHA-supplemented formula, but not below that amount. No additional improvement in acuity occurred in the 0.96% DHA group. The reason for the difference in response to the 0.32% DHA formula between the two sites is not evident, but might be linked to population differences or genetic variation. When the VEP scores were analyzed by sex, both sexes in the Dallas group had improved visual acuity with the DHA-supplemented formulas (combined data), whereas, only males showed improved scores in the Kansas City group. Control infants in Kansas City, especially among females, had more mature visual acuity at 12 months of age than the Dallas control infants. Although the Kansas 7

8 Figure. Mean sweep VEP visual acuity measures for infants consuming formula supplemented with 0 to 0.96% DHA from birth until 12 months of age. At each age, visual acuity in the control group was significantly poorer (higher) than in each of the DHA-supplemented groups. Scores among the DHA-supplemented groups did not differ significantly. Figure American Society for Nutrition. Reproduced with permission from Am J Clin Nutr 2010;91: City participants came from lower socioeconomic status families and differed from the Dallas participants in race, ethnicity and parents education, it could not be determined whether these factors accounted for the differences in the control groups at each site. The authors concluded that supplementation of infant formula containing 0.64% ARA with 0.32% or 0.64% DHA is associated with significantly more mature visual acuity at 12 months of age compared with unsupplemented formula. There was no evidence that 0.96% DHA was associated with better visual acuity, but there was suggestive evidence from exploratory analysis that some infants might respond more favorably to DHA supplementation at 0.64%. The study also provides evidence that DHA supplementation up to 0.96% of fatty acids was well tolerated without adverse effects. This report assures those concerned about infant DHA intakes above 0.5%. The observations in the unsupplemented infants also imply that DHA from maternal transfer during pregnancy is insufficient for optimum visual acuity maturation in the first months of life. Birch EE, Carlson SE, Hoffman DR, Fitzgerald-Gustafson KM, Fu VL, Drover JR, Castañeda YS, Minns L, Wheaton DK, Mundy D, Marunycz J, Diersen-Schade DA. The DIAMOND (DHA Intake And Measurement Of Neural Development) Study: a double-masked, randomized controlled clinical trial of the maturation of infant visual acuity as a function of the dietary level of docosahexaenoic acid. Am J Clin Nutr 2010;91: High Dietary Linoleic Acid Linked to Lower Omega-3 PUFAs in Maternal Red Blood Cells Consumption of sufficient long-chain omega-3 polyunsaturated fatty acids (n-3 LC-PUFAs) in pregnancy is important to ensure sufficient docosahexaenoic acid (DHA) for fetal brain growth and development. Intakes of these fatty acids in many western countries are well below recommended amounts, in part Consumption of n-3, because fish but not n-6, LC-PUFAs consumption tends affects the red blood cell to be low. For content of these fatty example, in the acids. Whether the high U.S., the average intake of linoleic acid consumption of seen in most western n-3 LC-PUFAs by diets affects the red cell women of childbearing age is content of DHA needed for fetal development is about 100 mg/ not known. These studies day, whereas the suggest that linoleic acid internationally recommended consumption is linked intake of DHA is a to lower red cell DHA. minimum of 200 mg/day. Maternal diets short in DHA provide less of this fatty acid for transfer to the fetal brain in the third trimester. Low DHA intakes may limit optimal brain development as implied by the improved neurodevelopmental outcomes of infants whose mothers consumed n-3 LC-PUFAs or DHA supplements. Infants of low DHA-status mothers had even poorer DHA status at birth than their mothers, suggesting limited DHA transfer to the infant. Red blood cells are a primary transfer vehicle for LC-PUFAs to the placenta and brain. Their membrane fatty acid composition is directly affected by PUFA consumption, so they are useful biomarkers of maternal and fetal exposure to LC-PUFAs. The effect of current western fatty acid intake patterns high linoleic acid and low long-chain n-3 LC-PUFAs on red blood cell membrane fatty acid composition has not been fully explored, especially in pregnant women. Sheila Innis and colleagues at the University of British Columbia, Canada, have investigated this topic in 8

9 several studies. They published evidence that diets high in n-6 PUFAs and low in DHA lead to increased uptake of n-6 LC-PUFAs and lower accretion of DHA in animal brains. Infants of mothers with low fish intakes who did not consume n-3 LC-PUFA supplements were 3 times more likely to have visual acuity scores below the mean compared with infants whose mothers consumed n-3 LC-PUFA supplements. These findings suggest that infant brain (and eye) development may be compromised by low maternal DHA consumption in pregnancy. In the studies described here, Innis group reports that higher intakes of n-3 LC-PUFAs were associated with greater concentrations of these PUFAs in red cell phosphoglycerides. In contrast, consumption of arachidonic acid, the major n-6 LC-PUFA also needed for brain development, was not related to red cell membrane arachidonic acid content. The same was true for linoleic acid. The investigators collected blood from 105 pregnant women at 36 weeks gestation. The participants had median intakes of DHA and eicosapentaenoic acid (EPA) of 100 and 60 mg/day, respectively. Median arachidonic acid consumption was 108 mg/day, while linoleic acid was nearly 11 g/day, with some women consuming as much as 28 g/day. As expected, dietary intakes of DHA and EPA were correlated with red blood cell phospholipid concentrations of these fatty acids and were inversely associated with the concentrations of the n-6 LC-PUFA derivatives, docosatrienoic acid (22:3n-6) and docosapentaenoic acid (22:5n-6). The latter accumulates when DHA intakes are low or deficient. Figure. Scatter plots of the inverse relationship between red blood cell (RBC) phospholipid linoleic acid (LA) concentrations and phospholipid DHA and arachidonic acid (ARA) in 105 Canadian women at 36 weeks' gestation. Abbreviations: PC, phosphatidylcholine; EPG, ethanolamine phosphoglyceride. Image reproduced with permission from: Am J Clin Nutr 2010;91: American Society for Nutrition. 9

10 The most striking observation was the inverse relationship between the red cell phospholipid concentrations of DHA, EPA and arachidonic acid and the membrane linoleic acid concentration (Figure). This observation suggests that high levels of dietary linoleic acid reduce the incorporation of n-3 LC-PUFAs and arachidonic acid into red cell membranes, probably through competition for acylation as the investigators mentioned. The implication is that high linoleic acid consumption can lower red cell n-3 LC-PUFA levels, especially of DHA, and may worsen the risks to the fetus of maternal diets already low in DHA. Red cell phospholipid arachidonic acid was not correlated with dietary EPA, DHA or arachidonic acid. This observation supports other reports that erythrocyte membrane arachidonic acid content is relatively insensitive to dietary intake. However, there are reports of increased red cell arachidonic acid concentrations with greater arachidonic acid consumption or increased biosynthesis. Concentrations of DHA, EPA and arachidonic acid in red blood cell membranes were inversely related to the concentration of linoleic acid in the membrane. Low DHA and EPA membrane content was also linked to high dietary linoleic acid. Higher intakes of linoleic acid were unrelated to red cell membrane arachidonic acid concentrations, indicating that linoleic acid converted to arachidonic acid did not influence red cell membrane composition. It should be noted that relationships between dietary and red cell membrane fatty acid concentrations were similar for both phosphatidylcholine and ethanolamine phosphoglycerides, although the LC-PUFA concentrations in phosphatidylethanolamine were higher. A similar effect on membrane phospholipid DHA and EPA content as seen with linoleic acid was observed with dietary alpha-linolenic acid, but because this fatty acid is highly correlated with linoleic acid consumption, the authors suggest that the effect of alphalinolenic acid was likely due to linoleic acid. It is also noteworthy that LC-PUFA intakes were highly skewed, with individual variation of 2,000 percent or more. Thus, median intakes fail to reflect the breadth of LC-PUFA intakes, especially for n-3 LC-PUFAs. Both reports document the low and highly variable intakes of DHA in healthy pregnant Canadian women. Consumption of DHA is half the recommended amount of 200 mg per day during pregnancy and lactation. The second study listed below substantiates the effect of consuming 1 to 2 fish meals a week in raising red blood cell membrane DHA and EPA concentrations and thereby, reducing the ratio of arachidonic acid to DHA. These data confirm previous observations that dietary arachidonic acid is unrelated to red cell membrane content of the fatty acid. In addition, Innis group provides evidence that contemporary high intakes of linoleic acid in the presence of low n-3 LC-PUFA or fish consumption may contribute to the low levels of n-3 LC-PUFAs in red cell membrane phospholipids. Friesen RW, Innis SM. Linoleic acid is associated with lower long-chain n-6 and n-3 fatty acids in red blood cell lipids of Canadian pregnant women. Am J Clin Nutr 2010;91: Friesen RW, Innis SM. Dietary arachidonic acid to EPA and DHA balance is increased among Canadian pregnant women with low fish intake. J Nutr 2009;139: Gestation Time Not Increased with Omega-3s in Women at Risk of Preterm Delivery The consumption of long-chain omega-3 polyunsaturated fatty acids (n-3 LC-PUFAs) during pregnancy is necessary to assure adequate availability of docosahexaenoic acid (DHA) for fetal Evidence that long-chain omega-3s consumed during pregnancy prolongs gestation and might reduce the chance of preterm delivery remains inconsistent. In this study, high-risk pregnant women treated with progesterone and long-chain omega-3s did not experience prolonged gestation time compared with control women. brain growth and function, as well as visual and cognitive development. It is now recommended that pregnant and lactating women consume at least 200 mg of DHA per day. Besides the benefits of n-3 LC- PUFAs to fetal and infant development, there is evidence that these fatty acids may prolong gestation, although the evidence has been inconsistent. Prolonging gestation, especially in women at high risk of preterm delivery, could reduce preterm births and the health risks to such infants. Sjurdur Olsen and colleagues reported that in pregnant women with previous pregnancy complications, fish oil 10

11 supplementation delayed delivery in low and middle, but not high fish consumers. In a US study, seafood or n-3 LC-PUFA intake was not associated with the length of gestation or the risk of preterm birth. Others reported increased gestation time in women consuming 133 mg of DHA per day in the last trimester of pregnancy. In a Cochrane review of marine oil supplementation during pregnancy, the authors observed that supplemented mothers experienced 2.5 days longer gestation compared with unsupplemented mothers, but that delivery before 37 weeks was not statistically different between the two groups. However, risk of delivery before 34 weeks gestation was significantly lower in the marine oil group. These reports make it clear why the issue of n-3 LC-PUFAs during pregnancy and risk of preterm delivery remains controversial. Added to the literature on this subject is a randomized controlled study among women with at least one spontaneous preterm delivery who consumed n-3 LC-PUFAs and had weekly injections of 17-α hydroxyprogesterone caproate (progesterone). The study s purpose was to see whether the addition of n-3 LC-PUFAs to progesterone treatment in pregnant women who had experienced at least one preterm delivery between 20 and 36 weeks gestation might have additional effects. Figure. Pregnancy outcomes of mothers at high risk of preterm delivery treated with progesterone with or without 2 g n-3 LC-PUFAs from weeks gestation until delivery. The n-3 LC-PUFA and placebo groups had 434 and 418 participants, respectively, except for the respiratory distress outcomes (n=425 and 403, respectively). None of the group differences were statistically significant except for respiratory distress. Eligible participants included 852 women with at least one preterm delivery who did not have a major fetal anomaly, fish oil consumption greater than 500 mg per week in the preceding month, or allergy to fish or other clinical involvements. After a run-in period of one week to assure compliance, participants were randomized to consume either 2,000 mg per day of n-3 LC-PUFAs (1,200 mg of eicosapentaenoic acid, EPA plus 800 mg of DHA) or a placebo of mineral oil. Assignment to treatment group occurred at 20 weeks gestation. All women received weekly injections of 250 mg of progesterone. Because progesterone has been shown to reduce the recurrence of preterm birth, including a treatment group without progesterone was considered unethical. Treatments continued until delivery or 36 weeks gestation, whichever came first. Fish consumption was assessed using a food frequency questionnaire with 4 categories of fish. The primary outcome was delivery prior to 37 weeks gestation (259 days) with secondary outcomes of delivery less than 35 weeks or 32 weeks, spontaneous preterm delivery, medically indicated preterm delivery or delivery after 40 weeks gestation. Additional fetal and neonatal outcomes were also specified in advance. Delivery prior to 37 weeks gestation occurred in 42% of the placebo women and 38% of the n-3 LC-PUFA group, a difference that was not statistically significant (Figure). Spontaneous delivery before 37 weeks also did not differ between the groups (36% and 33% for placebo and n-3 LC-PUFA groups, respectively). The gestational age of the n-3 LC-PUFA group was 2 days longer than the placebo group on average, but the difference was not statistically significant. Median birthweights and the proportions of infants weighing less than 1,500 or 2,500 g also did not differ significantly between the groups, although the number of infants born weighing less than 2,500 g or 1,500 g was lower in the n-3 LC-PUFA group. The authors reported that 30% of the participants ate no fish or less than 1 serving per month, with the remaining eating at least one fish meal per month. This study is the first to report respiratory distress in n-3 LC-PUFA-supplemented preterm infants. Of the 828 infants admitted to the nursery, respiratory distress syndrome was significantly higher in the n-3 LC-PUFA group than the placebo group (59 vs 35 cases). The literature suggests that in adults and a group of pediatric burn patients with acute respiratory distress, enteral nutrition with fish oil, antioxidants and arginine actually improved oxygenation, pulmonary compliance and clinical outcomes. Clearly, there are large differences between these study infants and other patient groups for whom data are available. 11

12 Of interest is a published report that Women at high risk of preterm infants preterm delivery treated weighing between with progesterone with 1,000 and 1,999 g or without n-3 LC-PUFAs who developed had no significant respiratory distress differences between had significantly groups in gestation lower cord blood time, rates of preterm arachidonic acid delivery or low birthweight infants. Infants levels than infants who did not develop the condition. In of the n-3 LC-PUFAsupplemented mothers this study, maternal plasma arachidonic were at significantly acid concentrations higher risk of were significantly respiratory distress. lower in the n-3 LC- PUFA-supplemented mothers compared with the placebo group, but there was no correlation between plasma arachidonic acid levels and the incidence of respiratory distress. A different report suggested that extremely preterm infants with bronchopulmonary dysplasia had significantly lower PUFAs in their tracheal aspirates than healthy infants, which might contribute to the lower risk of this condition in the first few days of postnatal life. n-3 LC-PUFAs have been associated with the increased production and secretion of lung surfactant in preterm baboons and mice and reduce the production of inflammatory cytokines in the lungs. Thus, they would be expected to have beneficial effects in pulmonary function. This study on the effect of n-3 LC-PUFAs among progesterone-treated pregnant women with a history of preterm delivery found no statistically significant differences in the rate of preterm delivery or spontaneous preterm birth, low birthweights or length of gestation with n-3 LC-PUFA supplementation of 2,000 mg/day. There were trends toward improved outcomes with n-3 LC-PUFA supplementation, but none reached statistical significance. The study also reported an increased occurrence of respiratory distress syndrome in the infants of the n-3 LC-PUFA supplemented mothers compared with the placebo group (14% vs 9%). Although there were hints of possible benefits of n-3 LC-PUFAs on preterm delivery, this study does not support their use in pregnancy specifically for that purpose. The observation of the increased risk of respiratory distress in the n-3 LC-PUFA group warrants careful follow-up. Harper M, Thom E, Klebanoff MA, Thorp J Jr, Sorokin Y, Varner MW, Wapner RJ, Caritis SN, Iams JD, Carpenter MW, Peaceman AM, Mercer BM, Sciscione A, Rouse DJ, Ramin SM, Anderson GD; Eunice Kennedy Shriver National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network. Omega-3 fatty acid supplementation to prevent recurrent preterm birth: a randomized controlled trial. Obstet Gynecol 2010;115(2 Pt 1): Worth Noting Smithers LG, Collins CT, Simmonds LA, Gibson RA, McPhee A, Makrides M. Feeding preterm infants milk with a higher dose of docosahexaenoic acid than that used in current practice does not influence language or behavior in early childhood: a follow-up study of a randomized controlled trial. Am J Clin Nutr 2010;91: Beyerlein A, Hadders-Algra M, Kennedy K, Fewtrell M, Singhal A, Rosenfeld E, Lucas A, Bouwstra H, Koletzko B, von Kries R. Infant formula supplementation with long-chain polyunsaturated fatty acids has no effect on Bayley developmental scores at 18 months of age--ipd meta-analysis of 4 large clinical trials. J Pediatr Gastroenterol Nutr 2010;50: BRAIN FUNCTION DHA Promotes Neurite and Synapse Development and Function in Cells and Animals The details of how long-chain omega-3 DHA deficiency impairs the growth and development polyunsaturated fatty acids (n-3 LC-PUFAs) affect brain growth of neurons in the and function have hippocampus, the become better brain s learning and understood, thanks to increasingly memory center. Here, sophisticated technical the effects of DHA and equipment for observation and analysis in arachidonic acid on hippocampal neurons animal and cell studies. from DHA-fed and We now have more deficient animals were precise insights into compared. Only DHA n-3 LC-PUFA function in showed significant cell signaling, growth and beneficial and the activation developmental effects. or inactivation of specific brain regions in response to stimuli or disease. Particular attention has been given to docosahexaenoic acid (DHA) because of its high concentration in neural membranes, its sensitivity to dietary intake and its importance as a precursor of cell-protective neuro protectins. DHA is essential for brain and retina 12

13 development, but also for maintaining visual function, cognition and cell survival and protection against cell injury. Infants fed diets low in DHA have less DHA in their brains than their breastfed counterparts. DHAdeficient animals have smaller neurons, cognitive shortfalls, decreased dopaminergic proteins, delayed cell migration to the CA1 subregion of the hippocampus and dentate gyrus areas of the hippocampus, impaired learning and memory, and reduced neurite outgrowth. Provision of DHA to deficient animals and cultured neurons overcomes many of these abnormalities, stimulates neurite growth and may continue to do so in aged tissue. DHA supplementation in adult animals was associated with a greater than 30 percent increase in the number of dendritic spines (protrusions from a dendrite), which may promote neuronal membrane synthesis. Further, supplementation with DHA and uridine reportedly increased the number of synapses and dendritic spines and improved cognitive function in normal rodents. Arachidonic acid had no such effect and might inhibit the growth of secondary neurites. Some have suggested that neurite growth promotion is specific to DHA but other data contradict that assertion. Two recent papers examined the effects of DHA and LC-PUFAs on neuronal development, synaptic function and neurite growth in experimental animals and cultured neurons. In the first study, the investigators fed mice diets adequate (2.5% alpha-linolenic acid and 0.9% DHA) or deficient (0.09% alpha-linolenic acid) in n-3 PUFAs during pregnancy and lactation and then examined the hippocampi of the offspring at 18 days gestation or 18 days after birth. Embryonic hippocampal cells were cultured with or without DHA, oleic acid, arachidonic acid or docosapentaenoic acid (22:5n-6) from the second day after seeding. Neurite outgrowth and synapsin puncta (points of immunoreactivity equivalent to a synapse) were determined after incubating the cells with antibodies against microtubule-associated protein and several structures associated with developing synapses. Incubation was followed by staining and photography, with measurement of neurite length and synapsin puncta in 20 unclustered neurons from triplicate wells of cells. The number of synapsin puncta was determined per 10 μm neurite length at 60x magnification. Analysis of the neurons cultured for 10 days from 18-day embryonic hippocampal tissue revealed significant growth and branching in the cells cultivated with DHA, but not those grown with any other fatty acids or the unsupplemented controls. Only the DHA-treated neurons showed a significant increase in the number of synapsin puncta per neuron. These neuron-specific phosphoproteins are associated with the membranes of synaptic vesicles. This observation suggests improved synaptogenesis associated with the availability of DHA. Figure. Synaptic activity electrical currents in hippocampal neurons cultured with DHA or ARA or no fatty acids as reflected by the means of current amplitude and frequency. Amplitudes for DHA or ARA cells differe significantly from controls, P<0.05 and <0.01, respectively. Synaptic current frequency in DHA-treated cells differed significantly from controls and ARA-treated cells, P<0.001 and <0.01, respectively. Using electrophysiological techniques, the investigators examined synaptic function by measuring the postsynaptic currents in whole-cell preparations of gammaaminobutyric acid-producing neurons. Importantly, the frequency of the post-synaptic currents, which reflects the number of active synapses and the relative levels of pre-synaptic release, was significantly greater in the DHA-treated cells compared with the control and ARA-treated neurons (Figure). In addition, the researchers determined which of two types of receptors those producing gammaaminobutyric acid or glutamine was most active in each culture by using antagonists for each one. Control cells treated with the gamma-aminobutyric acid antagonist exhibited significantly reduced post-synaptic current frequencies, suggesting that the synaptic activity in these cells came mainly from receptors producing gamma-aminobutyric acid. On the other hand, DHA-treated cells had a significantly lower synaptic current frequencies in response to the glutamine antagonist. These findings suggest that DHAtreated neurons promote the expression of glutamineproducing receptors. Further, DHA-treated neurons 13

14 expressed higher levels of synapsin 1, a protein involved in synaptogenesis that is specific to neurons. The investigators also evaluated the effect of maternal n-3 PUFA deficiency during pregnancy on the composition, growth and development of hippocampal neurons. Compared with animals fed the n-3 PUFAcontaining control diet, hippocampal neurons in the offspring of the deficient mothers contained significantly less DHA and had shorter neurites, fewer branches and less synapsin-positive puncta. These defects occurred in spite of the increased production of docosapentaenoic acid (22:5n-6). Culturing the deficient neurons with DHA restored neurite growth and synaptogenesis. DHA, but not docosapentaenoic acid (n-6) or alpha-linolenic acid promoted hippocampal neurite growth, synapse formation, synaptic transmission and long-term potentiation, effects that enhance neuronal function. Deficits resulting from DHA deficiency were overcome in cultured neurons with the addition of DHA. It is thought, but not certain, that the alterations in hippocampal neurons with DHA deficiency are related to the impaired cognition observed in n-3 PUFAdeficient animals. To test this hypothesis, the researchers evaluated the longterm potentiation of hippocampal synaptic transmission, an indication of the ability of synapses to change the strength of their responses. This flexibility or plasticity is believed to underly learning and memory. Long-term potentiation in the hippo campal slices from mice fed n-3 PUFA deficient or control diets from pregnancy through lactation was evaluated using 2 successive high-frequency stiumulus bursts. DHA deficiency inhibited the induction of longterm potentiation in hippocampal neurons compared with controls and high-frequency stimulation induced the phenomenon only in the neurons from DHA-fed animals. These experiments demonstrated that DHA supplementation promotes neurite growth and synapse formation and improves synaptic transmission and long-term potentiation. Longer neurites and more extensive neurite branching are directly related to synaptic function during memory. Only DHA, not DPA (22:5n-6) or arachdonic acid, affected neurite growth and function. DHA supplementation promoted neurotransmitter release and the expression of glutamate receptors without affecting gamma-aminobutyric acid receptors. There are reports that in neuro degenerative conditions, such as aging, glutamate receptor expression is reduced and long-term potentiation impaired, changes which can be reversed by n-3 PUFA supplementation. The investigators also showed that maternal DHA deficiency during pregnancy adversely affects synaptic plasticity in the offspring, but that impaired neurite growth and synaptogenesis could be reversed by early DHA supplementation in the offspring. Whether these effects occur in human infants experiencing DHA shortfalls during fetal development or early infancy is not clear. It will be important to know whether DHA supplementation in early infancy, especially in preterm infants, improves neurite growth and development and ultimately cognition. This remains a difficult challenge given the limitations of currently available methods for studying human infants. However, these studies strengthen the links between DHA adequacy in fetal life and early infancy as well as subsequent neuro development and cognition. Cao D, Kevala K, Kim J, Moon HS, Jun SB, Lovinger D, Kim HY. Docosahexaenoic acid promotes hippocampal neuronal development and synaptic function. J Neurochem 2009;111: Long-Chain PUFAs Increase Complex Neurite Outgrowth in Cells from Young and Old As the preceding Long-chain PUFAs promote the growth and complexity of embryonic neurons grown in cell culture. DHA has the strongest effects. Whether DHA and other LC-PUFAs enhance neuronal growth in adult and aged animal cells was not known until this study s findings. article showed, docosahexaenoic acid (DHA) promotes neurite outgrowth and synaptogenesis in embryonic hippocampal neurons and is required for the normal development of these cells. Offspring of DHAdeficient animals exhibited deficits in neuronal development compared with omega-3 (n-3)-fed animals, but these were overcome by supplying DHA in the cell culture medium. These observations have implications for humanneuronal development in fetal life and infancy, neurodegenerative diseases, tissue injury and aging. The authors of the paper described here explored the effects of 3 long-chain polyunsaturated fatty acids 14

15 Figure. Structure of a dorsal root ganglion and its location in the human spine. Image reproduced with permission from the Huntington s Outreach Project for Education, at Stanford University (HOPES) 2009, (LC-PUFAs), including DHA, eicosapentaenoic acid (EPA) and arachidonic acid (ARA), an n-6 LC-PUFA, in cultured neurons isolated from young, adult and old rats. Neurons were obtained from dorsal root ganglia (Figure) in male and female animals and cultured with the 2 concentrations of PUFAs or nerve growth factor (NGF) or all-trans-retinoic acid (ATRA), substances known to promote neuron growth. Cells were obtained from animals at postnatal days 3 and 9 and those aged 2 to 4 (adult) and 18 to 20 (old) months. In the youngest age groups, the investigators pooled the cells from male and female animals. The investigators conducted their studies in neurons from male and female animals to assess whether sex influenced neuronal growth. This research group had previously shown that DHA provided from diet and intravenous infusion conferred significant neuroprotection, reduced cell death and improved recovery in animals with spinal cord injury. The researchers evaluated all neuronal cells from 3 coverslips of cultured cells and categorized neurite growth into none, simple (no dendrite branching), complex (with dendrite branching) or the presence of growth cones, the tips of growing dendrites. The number of cells did not differ with age or fatty acid. In neurons from 3-day-old samples, ARA stimulated the growth of complex neurons and single growth cones compared with controls, while reducing the percentage of cells with no outgrowths. In 9-day-old, adult and older cells, single outgrowths were more numerous than complex ones and those lacking outgrowths. The percentage of cells with complex outgrowths continued to fall with cell age reaching half as many in the adult and old cells compared with those observed in the 9-day cells. With EPA, neurites in 3-day-old samples had significantly more cells with single or complex outgrowths compared with controls and significantly fewer with no outgrowths. These effects were markedly decreased in 9-day-old and adult samples, but were observed again in the cells from the old animals. DHA, on the other hand, markedly ARA, EPA and DHA increased number of promoted neuronal complex neurons and growth and complexity those with growth in cultured cells cones in cells from from young and 9-day old, adult and old rat tissues, with old animals compared DHA having the with the controls. strongest effects. DHA treatment also significantly reduced the percentage of cells without outgrowths at all ages. The DHA treatment was concentration-dependent in tissues from male 9-dayold, adult and old animals with greater concentration significantly increasing proportion of neurons having greater complexity. These responses were greater than those observed with EPA. Thus, physiologic concentrations of LC-PUFAs enhanced or altered neurite growth in favor of more complex development at the expense of neurons with no outgrowths in tissue from male animals. The effects of DHA were markedly stronger than EPA and especially ARA at all ages from day 9 through old age. 15

16 In neurons from female animals, all LC-PUFAs reduced the number of cells with no outgrowths and increased the percentage of cells with simple or complex outgrowths compared with controls. But because there was greater variability in the effects compared with male animals, the differences did not reach statistical significance. Similarly, n-3 LC-PUFAs increased the percentage of cells with complex outgrowths and growth cones, but the large variability precluded statistical significance, except for intermediate concentrations of DHA and the reduction in cells with no outgrowths and the increase in cells with complex outgrowths or growth cones, which were significant, P < When the effect of the 2 growth factors was examined, the maximum length of neurite outgrowths was significantly increased only with NGF, ATRA and DHA, while only DHA increased the total neurite length significantly. This study demon- These findings suggest strated the in that the effects of n-3 vitro growthpromoting effects LC-PUFAs, especially of physiological DHA, on neurite growth, concentrations complexity and plasticity, of ARA, EPA and are not restricted to early DHA in immature, development, but are mature and aged effective in aging. Although animal dorsal root weaker than DHA, ARA ganglia. In agreement with the also enhanced neurite growth at all ages. preceding report, the strongest effects occurred with DHA and were comparable to those seen with 2 established neuronal growth factors. DHAassociated growth effects persisted into relative old age. In contrast with the preceding study in hippocampal neurons and a previous report, in which ARA did not enhance neuronal growth, this fatty acid promoted neurite growth and complexity in these dorsal root ganglia. Several other reports have documented enhanced neurite outgrowth with ARA and the fatty acid is involved in the effectiveness of growth factors and cell adhesion molecules. The differences among reports may relate partly to the nature of in vitro versus in vivo studies. Responses in adult and aged neurons from female animals were more variable than those from comparably aged males. This study suggests that the effects of n-3 LC-PUFAs, especially DHA, are not restricted to early development, but contribute to neurite growth, complexity and plasticity during aging. There is substantial evidence that DHA is involved in the synthesis of synapses, synaptic proteins and increased dendritic spine density and that it has neuroprotective properties in an animal model of Alzheimer s disease. This report emphasizes the importance of adequate brain DHA for healthy neuronal growth and protection against injury throughout life. Robson LG, Dyall S, Sidloff D, Michael-Titus AT. Omega-3 polyunsaturated fatty acids increase the neurite outgrowth of rat sensory neurones throughout development and in aged animals. Neurobiol Aging 2010;31: Supplementary DHA Linked to Greater Cortex Activity During Attention Task in Boys Docosahexaenoic acid (DHA), the The high DHA content in principal longchain omega-3 the brain cortex may be related to neural activity polyunsaturated in the frontal cortex. fatty acid (n-3 LC-PUFA) in the This brain imaging study brain, accumulates was designed to find rapidly during the out whether increased last trimester of consumption of DHA was pregnancy and related to cortical neural the first 2 years activity in 8-year old boys, of life. It may be whose brains are still less appreciated accumulating DHA. that DHA continues to increase in the cerebral cortex through age 18, a time of neuronal maturation, synaptogenesis and gray matter expansion. After that, cortex DHA and arachidonic acid decline. The concentration of DHA is especially high in the frontal cortex (Figure), the brain region where appropriate responses to internal and external stimuli are planned and complex perceptual information is integrated, activities often called executive function. It would appear that maintaining or increasing cortex DHA content throughout life might help maintain or improve cortical function. However, little is known about whether dietary DHA can increase cortical DHA content, especially after the first 2 years of life. There is evidence that higher dietary DHA increases cortical DHA in neonatal baboons. In the study described here, investigators at the University of Cincinnati College of Medicine, USA, used functional magnetic resonance imaging to assess neural activity during sustained attention in healthy 8-year-old boys. This technique permits the investigators to detect the brain areas involved in a mental task and map the changes in brain blood flow that correspond to neural activity. This activity is assessed by signals dependent upon blood oxygen levels. It is not known whether 16

17 changes in cortex neural activity are related to cortex DHA in humans. Evidence from animal studies suggests that increasing the availability of DHA improves spatial memory and learning in n-3 PUFA-deficient animals. Brain DHA concentration was significantly associated with the cerebral regions having intense energy utilization according to data in 2 primate species. Indirect evidence for an effect of DHA on emotionrelated neural activity in boys with attention-deficit hyperactivity disorder suggests a positive effect in the expression of happiness over sadness or fear. Figure. Illustration of the frontal lobes of the human brain showing the prefrontal cortex (yellow). Numbers correspond to Brodmann areas. Source: Wikipedia Thirty-eight boys aged 8 to 10 years were randomly assigned to treatment and control groups. Of those enrolled, 33 completed the 8-week study. Participants consumed capsules containing 400 or 1,200 mg of DHA or a corn oil placebo for 8 weeks. All capsules were the same color and contained orange flavoring. The boys were right-handed, had no iron-magnetic material in their bodies, had normal intelligence, were not taking nor had taken psychoactive medications and had no history of seizures, major illness or brain injury. The sustained attention task required each child to respond by pressing a button when they saw a single number twice sequentially. A control task required the participant to press the response button 5 times when he saw the number 1 and was alternated with the attention task. Control and attention tasks were given in epochs of 30 seconds each for a total of 40 numbers per epoch. Five blocks of one epoch each were obtained along with an additional control epoch at the beginning. Performance was evaluated according to the percentage of correct selections, errors of commission, discrimination and reaction time. The attention and control tasks were performed during magnetic resonance imaging using an audiovisual goggle system and were performed at baseline and after 8 weeks DHA supplementation. The investigators measured the concentration of DHA and arachidonic acid in red blood cell membranes at baseline and 8 weeks. At the end of the supplementation period, there were no differences among groups for any of the performance measures. Although the outcomes on the attention task were not affected by DHA consumption, both DHA doses gave rise to increased neural activation during the attention task in the right dorsolateral prefrontal cortex (corresponding to Brodmann s area 9). There was decreased activation of the bilateral occipital cortex compared with the placebo group. In the boys consuming the high dose of DHA, even greater increases in the activation of the left dorsolateral prefrontal cortex were observed, along with greater decreases in the activation of the bilateral cerebellum compared with the placebo group. These changes were not attributable to differences in performance on the attention task. Consumption of Consumption of DHA DHA significantly increased prefrontal increased its concentration in red blood cortex neural activation cell membranes in in specific regions and a dose-dependent decreased it in other manner, 2-fold and areas without affecting 3-fold for the low the boys performance and high DHA doses, on a sustained attention respectively. Only task. Increased neural consumption of the activity was correlated high-dose DHA was with increased red blood associated with a cell DHA content. significantly lower (-13%) concentration of arachidonic acid. Red blood cell DHA was positively associated with dorsolateral prefrontal cortex activity and inversely with reaction time at baseline and after 8 weeks. This correlation suggests a relationship between DHA and functional cortical activity. The study also observed increased activity in the right dorsolateral prefrontal cortex of participants who consumed the low-dose DHA, but greater activity in the left side with highdose supplementation. Lateralization and asymmetry has been reported with respect to glucose metabolism and plasma DHA composition in patients with major depressive symptoms and in rotation behavior and cholinergic neuron density in n-3 PUFA-deficient animals. The authors speculate that brain DHA composition may be involved in communication and functional connectivity between the brain s two hemispheres. This report appears to be the first controlled neuroimaging study linking the consumption of DHA with 17

18 functional cortical activity in humans. It was designed to feature healthy, pre-adolescent participants whose brains are continuing to accrue DHA. The association between red blood cell DHA concentrations and neural activity increase the plausibility that the observed patterns of neural activity were attributable to the DHA intervention. As the authors suggest, this imaging technique might further elucidate the mechanisms by which reduced DHA affects patients with conditions such as attention-deficit hyperactivity disorder and major depression. The evidence and rationale for how DHA deficiency might be involved in affective disorders, especially in pediatric and adolescent groups and in aging, is discussed in the second article by McNamara cited below. McNamara RK, Able J, Jandacek R, Rider T, Tso P, Eliassen JC, Alfieri D, Weber W, Jarvis K, Delbello MP, Strakowski SM, Adler CM. Docosahexaenoic acid supplementation increases prefrontal cortex activation during sustained attention in healthy boys: a placebo-controlled, dose-ranging, functional magnetic resonance imaging study. Am J Clin Nutr 2010;91: McNamara RK. Docosahexaenoic acid deficiency and prefrontal cortex neuropathology in recurrent affective disorders. J Nutr 2010;140: MENTAL HEALTH AND COGNITION Modest Dose Long-Chain Omega-3s Lower Risk of Progression to Psychosis Psychotic illness can be viewed as Interventions to prevent individuals at high risk of a continuum along which individuals developing a psychotic with subclinical symptoms may disorder have achieved progress to frank only modest results, psychosis. Not all often with unwanted side experts subscribe effects. In this controlled to this view, with trial in young patients at some holding that risk of psychosis, longchain omega-3 fatty acids to diagnostic psychosis belongs significantly reduced the categories. Most conversion to psychosis. agree, however, that high-risk individuals can be identified and their progress to psychotic disorder prevented or delayed. Antipsychotic medications, cognitive therapy or both are used to reduce the likelihood of conversion to psychosis or relapse, with patient evaluation and specialist programs contributing to an intervention s effectiveness. Current treatments have achieved only modest success. A relatively recent addition to the treatment and prevention approach for individuals with psychotic disorders, mainly schizophrenia, is treatment with long-chain polyunsaturated fatty acids (LC-PUFAs), particularly those of the omega-3 (n-3) family. A Cochrane Database review concluded that results of controlled trials to date are inconclusive. Findings from small, short trials suggested that the consumption of eicosapentaenoic acid (EPA) might reduce the use of antipsychotic drugs and improve an individual s mental state. Another review suggested that n-3 LC-PUFAs were protective in mood disorders, uni- and bipolar depression and potentially in major depressive disorder, but there was less evidence of benefit in schizo phrenia. There are many plausible mechanisms by which these fatty acids might affect mental function and the fact that they are without adverse effects makes them worthy of full exploration. In this study, investigators at the University of Vienna, Austria, and collaborating institutions in Australia and Switzerland explored whether n-3 LC-PUFAs could prevent a first episode of a psychotic disorder, reduce psychiatric symptoms and improve functioning in young individuals with subthreshold indications of psychosis. To be eligible, participants had to have met one or more of 3 validated risk factors for psychosis. These were: attenuated positive psychotic symptoms with predetermined scores on several Positive and Negative Syndromes of Schizophrenia Scales (PANSS), transient psychosis as determined by symptom scores on the PANSS scales, or genetic risk plus a decrease in functioning according to pre-set criteria. Of the 81 individuals who met the inclusion criteria, 76 completed the 1-year study including a 12-week intervention period. Study participants ranged in age from 13 to 25 years (mean 16 years) and were considered at ultra-high risk of psychotic disorder. Approximately onethird of participants were male. Individuals were ineligible if they had a history of previous psychotic disorder or manic episode, substance-induced psychosis, acute suicidal or aggressive behavior, diagnosed substance dependence, neurological disorders, IQ below 70 or other relevant conditions. After enrolment, participants were 18

19 randomly assigned to consume either 1.2 grams of n-3 LC-PUFAs per day (700 mg EPA and 480 mg docosahexaenoic acid) or a coconut oil placebo masked with 1% fish oil for 12 weeks. Researchers monitored the participants weekly for 4 weeks and thereafter at 4 and 12 weeks, then 6 and 12 months. The study protocol did not permit antipsychotic medications or mood stabilizers, but offered 9 sessions of need-based psychological and psychosocial interventions with other personal or crisis assistance available as appropriate. The primary endpoint of the study was conversion to psychotic disorder, defined by threshold criteria on 3 dimensions of the PANSS scales ( 4 on hallucinations, 4 on delusions or 5 on conceptual disorganization), which had to be sustained for at least 1 week. Nonproject psychiatrists independently evaluated patients who progressed to a first episode of a psychosis to confirm the diagnosis. Secondary outcomes included other measures on the PANSS scales, and changes in scores on the Montgomery Asberg Depression Rating Scale, the Global Assessment of Functioning and the Structured Clinical Interview for Axis I Disorders. The investigators also measured red blood cell fatty acids at baseline and after 12 weeks. Individuals at high risk of psychosis who consumed 1.2 grams of long-chain omega-3s per day for 12 weeks had significantly less progression to psychosis (5%) compared with those consuming placebo capsules (28%). The omega-3 group also exhibited significantly higher functioning than the control group. Group differences persisted for 1 year. After the 12-week intervention, 4.9% of the participants c o n s u m i n g n-3 LC-PUFAs (2 of 41) progressed to psychosis, whereas 27.5% of the placebo patients did so (11 of 40), a statistically significant difference in the primary outcome, (P = by logrank test). For the secondary endpoints, the n-3 LC-PUFA group had significantly lower PANSS positive, negative, general and total scores at 12 weeks, 6 and 12 months compared with the placebo group. Only 1 patient in the n-3 LC-PUFA group developed psychosis during the post-treatment period and symptom severity did not increase over the 12 months. None of the other measures were significantly different between the groups. The n-3 LC-PUFA group also exhibited significantly higher functioning than the control group, as shown by the lower score on the Global Assessment of Functioning at 12 weeks compared with the baseline. The change from baseline in the n-3 LC-PUFA group was 17.7 compared with 7.2 in the placebo group (P = 0.002). There were no statistically significant differences between the groups in adverse events. This controlled clinical trial in young individuals at high risk for psychosis is a milestone demonstration that a moderate dose of n-3 LC-PUFAs, 1.2 grams per day for 12 weeks, is effective in significantly reducing the progression to a psychotic disorder and improving patient function. Moreover, the reduced progression and improved function persisted after treatment cessation for up to 1 year. Whether this persistence was related to the treatment cannot be deduced from this study, in part because red blood cell fatty acids were measured only at the baseline and 12 weeks. There is no reason to think that the likely uptake of docosahexaenoic acid in the brain would have been washed out once n-3 LC- PUFA consumption stopped. As the authors noted, the transition to psychosis may have been delayed, but not prevented by this intervention. When compared with antipsychotic drugs, for which effectiveness is controversial, these findings suggest that n-3 LC-PUFAs may be more effective with substantially higher patient acceptance and virtually no unwanted side effects. Additional trials are needed to confirm and expand these results. Amminger GP, Schäfer MR, Papageorgiou K, Klier CM, Cotton SM, Harrigan SM, Mackinnon A, McGorry PD, Berger GE. Long-chain omega-3 fatty acids for indicated prevention of psychotic disorders: A randomized, placebo-controlled trial. Arch Gen Psychiatr 2010;67: Blood DHA Associated with Several Dimensions of Cognition in Healthy Adults The importance of having sufficient docosahexaenoic acid (DHA) available throughout life is becoming increasingly clear. DHA is the principal long-chain omega-3 polyunsaturated fatty acid (n-3 LC-PUFA) in brain and functions in cell signaling, the production of neuroprotectin D1 and the growth and regeneration of neurons. From the standpoint of brain function and behavior alone, DHA sufficiency has been associated with improved cognition, milder symptom severity in various mental illnesses, e.g., major depression, aggressive behavior and increased attention to name a few conditions. Not surprisingly, the literature is inconsistent in several of these examples and stronger evidence is needed. Indirect evidence suggests that increased consumption of DHA during infancy and childhood, at least until 19

20 DHA sufficiency is essential for healthy brain and retina development and function from infancy through old age. Whether it can maintain cognition or prevent dementia is an unresolved question, particularly once cognition has declined. This study examined the effect of blood n-3 PUFA levels and several dimensions of cognition in healthy adults. about age 18, increases brain content of DHA and improves neural activity in specific regions of the brain. Neuroimaging techniques have increased the options for human studies seeking to explore the relationships between dietary intake and changes in brain volume and neural activity. To understand better the effects of DHA and n-3 LC-PUFAs on cognitive performance at different ages, it would help to resolve some of the contradictory findings on this topic. It appears that these fatty acids may slow the decline in cognition in older adults whose mental faculties are normal or nearly so, but those with advanced cognitive decline or Alzheimer s disease are unresponsive to supplementary n-3 LC-PUFAs. It is possible that the impaired cognition in Alzheimer s disease differs substantially from the cognitive decline observed before or without the onset of this disease. Two epidemiologic studies in adults reported that cognitive performance was related to fish or n-3 LC-PUFA consumption, particularly in regard to psycho motor speed and mental efficiency, but not general memory. Few studies have examined multiple dimensions of cognition, which would be important for learning which types of mental function might be related to n-3 LC-PUFAs. Matthew Muldoon and colleagues at the University of Pittsburgh, USA, examined the relationship between blood levels of n-3 LC-PUFAs and 5 major dimensions of cognitive performance in healthy middle-aged adults recruited from southwestern Pennsylvania. Participants were free of major heart, kidney or liver disease, neurological disorders and psychotic illnesses, pregnancy or perimenopausal menstrual irregularities, several other illnesses and did not consume fish oil. Participants included 280 men and women aged 30 to 54 years (mean age 45 years) who provided blood samples for serum phospholipid fatty acid analysis and underwent several neuropsychological tests, including several subtests from the Wechsler Memory Scale and the Wechsler Abbreviated Intelligence Scale. The assessments evaluated the following cognitive dimensions: nonverbal reasoning and mental flexibility, attention and concentration, general memory, working memory and verbal knowledge and processing. The investigators evaluated the relationships between Table. Linear regression coefficients for the relationships between serum n-3 PUFAs and selected cognitive function scores in healthy middle-aged adults COGNITIVE MEASURE DHA P EPA P ALA P Nonverbal reasoning Matrix reasoning 0.20 < General memory Logical memory Working memory Composite Letter-number sequence Spatial span Backward digit span Verbal knowledge Vocabulary Verbal fluency

21 blood fatty acid levels and cognitive scores using 2-step multiple regression analysis, controlling for age, sex and race. Additional analyses included blood pressure and education level as confounding variables. Of the three n-3 PUFAs, DHA was most strongly related to cognitive function. Eicosapentaenoic acid (EPA) was significantly related only to 3 subset scores of working memory, while alpha-linolenic acid (ALA) was unrelated to any cognitive score. These observations confirm that DHA is the n-3 LC-PUFA most strongly associated with cognition. DHA was the main n-3 PUFA related to cognitive function with higher levels being linked to improved matrix reasoning, logical memory, working memory and vocabulary in healthy adults. As shown in the Table, DHA was significantly related to non verbal reasoning (matrix reasoning), logical memory, 4 dimensions of working memory and vocabulary. When blood pressure was included in the analysis, all findings related to DHA remained significant. When e d u c a t i o n a l attainment and vocabulary were taken into account, matrix reasoning, composite working memory and most other tests remained significantly related to phospholipid DHA concentrations. Responses for matrix reasoning and composite working memory scores were dose-dependent, with the highest scores generally occurring in the third DHA quintile and higher. This observational study of cognition in healthy, middleaged adults reported many significant associations between higher serum phospholipid DHA and 4 dimensions of cognition. These findings agree with other observational data reporting better cognition in those with diets high in fish compared with those who seldom consume fish. This study is useful because it documents the association between DHA and working memory and other cognitive functions and addresses one of the age gaps in the relationship between DHA and cognition. Muldoon MF, Ryan CM, Sheu L, Yao JK, Conklin SM, Manuck SB. Serum phospholipid docosahexaenoic acid is associated with cognitive functioning during middle adulthood. J Nutr 2010;140: Worth Noting Conklin SM, Runyan CA, Leonard S, Reddy RD, Muldoon MF, Yao JK. Age-related changes of n-3 and n-6 polyunsaturated fatty acids in the anterior cingulate cortex of individuals with major depressive disorder. Prostaglandins Leukot Essent Fatty Acids 2010;82: Bountziouka V, Polychronopoulos E, Zeimbekis A, Papavenetiou E, Ladoukaki E, Papairakleous N, Gotsis E, Metallinos G, Lionis C, Panagiotakos D. Long-term fish intake is associated with less severe depressive symptoms among elderly men and women: the MEDIS (MEDiterranean ISlands Elderly) epidemiological study. J Aging Health 2009;21: VISUAL FUNCTION DHA with Epithelial Growth Factor Enhances Nerve Regeneration After Corneal Surgery One of the primary ways to correct visual acuity problems and corneal defects is refractive surgery, such as photo refractive keratectomy Widely performed corrective and laserassisted in situ corneal surgeries, including keratomileusis, LASIK operations, damage popularly known corneal nerves and often as LASIK surgery. lead to dry eye. This study While usually examined treatments to correcting the enhance nerve regeneration underlying visual and improve corneal health. problems, these surgeries damage the corneal sensory nerves, impair lacrimal gland function and can cause dry eye syndrome (dry eye). The latter complication occurs in more than half of patients undergoing LASIK surgery and can persist up to 6 months and beyond. It may also be accompanied by pain, soreness and eyelid-sticking. The most common treatments rely on the application of artificial tear fluids and ointments, which relieve symptoms, but do not affect the underlying condition. To alleviate and correct the underlying nerve damage, the investigators in this report by Soledad Cortina and colleagues targeted the regeneration of corneal nerves and explored the conditions and agents that facilitated such nerve regeneration. The researchers conducted their studies in rabbits that underwent lamellar keratectomy in the left eye and subsequent removal of the cornea at the end of the study. For the study of corneal nerve regeneration, animals were treated with pigment epithelial-derived factor (PEDF), a potent nerve growth agent and inhibitor of angiogenesis, docosahexaenoic acid (DHA) or both. DHA has been associated with dendrite formation and complexity, synaptogenesis in the brain and corneal nerve 21

22 regeneration in experimental animals. To examine potential mechanisms of action, the study measured corneal neuroprotectin D1 (NPD1), a product of DHA metabolism with strong anti-inflammatory and neuroprotective actions. Using lamellar dissection, the investigators transected all stromal nerves in the cornea and then monitored nerve regeneration. Exposure to PEDF and DHA was carried out using collagen shields soaked in the test substance or carrier vehicle for 5 minutes and then secured on the rabbit cornea for periods up to 6 weeks of treatment (8 weeks after surgery). After treatment, the corneas were examined in the anesthetized animals using confocal microscopy. This imaging technique achieves higher resolution images by using a pinhole aperture and eliminating out-of-focus light. Images were taken in the center of the cornea for nerve area analysis and quantification using an image analysis program. Whole corneas were extracted and a portion analyzed for lipids, NPD1 and immunochemistry. The corneas were incubated with anti-βiii-tubulin antibody to identify neurons and quantify nerve damage using immunofluorescence. After treatment with PEDF plus DHA for 2 or 4 weeks, there was a significant increase in stromal nerve regeneration of more than 3 times the regeneration observed with the treatment vehicle alone. Subsequent treatments were carried out for 6 weeks. Treatment with PEDF+DHA increased the subepithelial nerve area to 29% compared with 10% in the vehicle-only group. Similarly, stromal nerves increased by 19% compared with 7% in the control group. With PEDF or DHA alone, corneal nerve regeneration did not increase. Increased nerve regeneration observed with the two substances together suggests synergism between them. To explore whether surgically treated corneas produced NPD1, the investigators examined the NPD1 content of corneas treated with DHA for 1 or 2 weeks and compared the results to treatment with PEDF+DHA. The investigators found that NPD1 increased after surgery in corneas treated with DHA for 1 week and that levels were greater with PEDF+DHA treatment. Levels observed at 2 weeks were lower, suggesting that NPD1 synthesis occurs early after surgery. In the PEDF+DHA-treated corneas 1 week after surgery, there were no nerves in the epithelial and subepithelial surgical areas, whereas nerves were observed in the stroma. After 2 weeks, epithelial nerves appeared. After 6 weeks of treatment with both agents, the number of superficial epithelial cells increased significantly. This observation was interpreted as consistent with a healthier corneal epithelium. The authors noted that in dry eye, the number of superficial epithelial cells declines. The most striking observation in Post-surgical treatment this study was of nerve-damaged the enhanced corneas with PEDF+DHA regeneration up to enhanced nerve 3-fold of surgically regeneration by up to damaged corneal 3-fold. DHA treatment nerves treated with enhanced the production PEDF plus DHA of NPD1 in the corneas, compared with untreated corneas. suggesting one Both substances possible mechanism were required for the improved health together, as neither of treated corneas. alone affected nerve regeneration. These experiments suggest that DHA might be active through the production of NPD1, as the concentration of the latter increased in corneas treated with PEDF+DHA. Usually, the DHA content of cornea is very low, but NPD1 production was increased in mice fed DHA-rich diets. Implications from this research suggest that nerve regeneration following photorefractive keratectomy or LASIK surgery can be enhanced with the application of PEDF+DHA and that the development of post-surgical dry eye could be reduced and possibly prevented. Cortina MS, He J, Li N, Bazan NG, Bazan HE. Neuroprotectin D1 synthesis and corneal nerve regeneration after experimental surgery and treatment with PEDF plus DHA. Invest Ophthalmol Vis Sci 2010;51: He J, Bazan HE. Omega-3 fatty acids in dry eye and corneal nerve regeneration after refractive surgery. Prostaglandins Leukot Essent Fatty Acids 2010; doi: /j.plefa CLINICAL CONDITIONS Type 2 diabetes Long-Chain Omega-3 Fatty Acids Improve Microvascular Function in Type 2 Diabetes Patients with type 2 diabetes face increased risks of cardiovascular disease, stroke, kidney disease, visual problems, foot complications, nerve damage and others. Arterial dysfunction, especially in the vascular endothelial cells, increases the risk of atherosclerosis and cardiovascular complications. For these reasons, preventing vascular disease is one of the main goals of therapy in type 2 diabetes. Long-chain omega-3 22

23 Impaired vascular function in people with type 2 diabetes contributes to their greater risk of cardiovascular disease. This study examined whether long-chain omega-3s might improve vascular function in such patients during the highrisk period following a meal. p o l y u n - saturated fatty acids (n-3 LC- PUFAs) have been associated with increased forearm blood flow in response to acetylcholine in patients with chronic heart failure and diabetes, suggesting enhanced endothelium-dependent vasodilation. Fish oil was also associated with improved arterial compliance in patients with type 2 diabetes, providing additional evidence that n-3 LC-PUFAs improve vascular and endothelial cell function. In this study, investigators at Ruhr-University in Bochum, Germany, and the Mount Sinai Medical Center in New York City examined macro- and microvascular function in individuals with type 2 diabetes who consumed approximately 1,680 mg per day of n-3 LC-PUFAs for 6 weeks. To assess macrovascular function, the research team measured flow-mediated dilatation in the right brachial artery using a 2-dimensional ultrasound imaging system to measure arterial diameter. To evaluate the microcirculation, the investigators used laser-doppler flowmetry of the right hand. This technique uses the velocity of moving red blood cells relative to blood flow during reactive hyperemia, such as follows the release of a tourniquet or brief ischemia. The study focused on the postprandial period 2, 4 and 6 hours following a high-fat meal containing 40 grams of fat. The postprandial period poses higher risks for patients with type 2 diabetes because blood glucose and then insulin levels rise sharply, increasing endothelial and cardiac dysfunction. Improvements in glucose control after a meal have been associated with better myocardial and vascular function in patients with type 2 diabetes. Participants with type 2 diabetes and total cholesterol levels above 270 mg/dl, but who had no history of cardiovascular events or a surgical intervention in the past 6 months and who were free of arterial hypertension, hypertriglyceridemia and advanced diabetic complications or nephropathy, were recruited from the hospital inpatient population undergoing diabetes therapy. The study was designed as a randomized, controlled, double-blind crossover trial. The 34 participants were randomized to consume 1.7 grams/day of n-3 LC-PUFAs (920 mg of eicosapentaenoic acid and 760 mg docosahexaenoic acid per day) or an olive oil placebo for 6 weeks. After a 6-week washout period, participants were given the intervention not consumed in the first 6 weeks. At baseline and each 6-week patient visit, the investigators obtained fasting and post prandial vascular measurements and blood samples. Over the 6-hour course of postprandial blood flowmediated dilatation measurements, the decrease in dilatation from fasting values was significantly less for patients who consumed the n-3 LC-PUFAs compared with those taking the placebo (Table). The difference in area-under-the-curve measurements, -8.2 versus -2.3 for placebo and controls, respectively, was Table. Postprandial changes in macro- and microvascular measures, including flow-mediated dilatation (FMD) and reactive hyperemia (RH), in patients with type 2 diabetes who consumed n-3 LC-PUFAs or a placebo for 6 weeks Vascular measurement Fasting 2 hr 4 hr 6 hr FMD Placebo 5.5 ± ± ± ± 0.6 FMD, area under curve Reactive Hyperemia (RH) n-3 LC-PUFAs 4.9 ± ± ± ± 0.6 Placebo -8.2 ± 2.2 n-3 LC-PUFAs -2.3 ± 1.8* Placebo 7282 ± ± ± ± 666 n-3 LC-PUFAs 5656 ± ± ± ± 683 RH, area Placebo -705 ± 2636 under curve n-3 LC-PUFAs 5013 ± 2793* *Significantly different from placebo, P <

24 significant (P < 0.05). These differences suggest that the consumption of n-3 LC-PUFAs partially prevented the deterioration in macrovascular function observed in the placebo group. Similarly, the estimates of microvascular circulation after brief ischemia were significantly increased in the n-3 LC-PUFA participants, but not in the controls. There were no significant changes in blood glucose or insulin over the 6-hour course of measurements, except that blood glucose and insulin increased significantly from baseline after 2 hours, but subsided to fasting levels after 6 hours. Area-under-the-curve measurements for blood triglycerides were significantly less in the n-3 LC-PUFA participants compared with the placebo group. Although the study Six weeks consumption did not investigate of n-3 LC-PUFAs by the potential patients with type 2 mechanisms underlying changes in diabetes was associated vascular function, with less impaired macrovascular function following the authors suggested their a high-fat meal and with observations are improved small-vessel consistent with circulation after an episode the view that of ischemia. These macrovascular observations suggest changes reflected that n-3 LC-PUFAs might in flow-mediated improve vascular dilatation reflect function in this disease. changes in nitric oxide release, whereas those in the microvascular circulation are regulated by changes in prostaglandins and other nonendothelium-dependent pathways. These findings suggest improved postprandial microvascular function in patients with type 2 diabetes who consumed modest amounts of n-3 LC-PUFAs for 6 weeks. The treatment attenuated the reduction in flow-mediated dilatation without affecting fasting measurements of vascular function. These observations provide additional evidence that n-3 LC-PUFAs contribute to improved vascular function and lower risk of cardiovascular disease in type 2 diabetes. Stirban A, Nandrean S, Götting C, Tamler R, Pop A, Negrean M, Gawlowski T, Stratmann B, Tschoepe D. Effects of n-3 fatty acids on macro- and microvascular function in subjects with type 2 diabetes mellitus. Am J Clin Nutr 2010;91: Worth Noting Wong C-Y, Yiu K-H, Li S-W, Lee S, Tam S, Lau C-P, Tse H-F. Fish-oil supplement has neutral effects on vascular and metabolic function but improves renal function in patients with Type 2 diabetes mellitus. Diabetic Medicine 2010; 27: Ko GD, Nowacki NB, Arseneau L, Eitel M, Hum A. Omega-3 fatty acids for neuropathic pain. Clin J Pain 2010;26: Iwasaki M, Yoshihara A, Moynihan P, Watanabe R, Taylor GW, Miyazaki H. Longitudinal relationship between dietary ω-3 fatty acids and periodontal disease. Nutrition 2010; doi: /j.nut Miyake Y, Sasaki S, Tanaka K, Fukushima W, Kiyohara C, Tsuboi Y, Yamada T, Oeda T, Miki T, Kawamura N, Sakae N, Fukuyama H, Hirota Y, Nagai M; Fukuoka Kinki Parkinson s Disease Study Group. Dietary fat intake and risk of Parkinson s disease: a casecontrol study in Japan. J Neurol Sci 2010;288: FRONTIERS Omega-3 PUFAs Might Regulate Cell Aging by Slowing Telomere Shortening The effects of polyunsaturated fatty acids (PUFAs) in regulating gene expression help explain how dietary PUFAs contribute to the reduction of cardiovascular disease risk and mortality. A recent review describes these effects in detail. Fatty acid regulation of gene expression has been demonstrated in cardiovascular disease, Alzheimer disease and retinal pigment epithelial cells, insulin signaling in the kidney, cholesterol absorption, human macrophages, endothelial cells and many others. It is now emerging that long-chain omega-3 polyunsaturated fatty acids (n-3 LC-PUFAs) might be involved in the regulation of the telomeres repetitive DNA sequences at the ends of the chromosomes (Illus) in the chromosomes of germ, stem and white blood cells, such as leukocytes. In 2009, the Nobel Prize Assembly awarded its Prize in Physiology and Medicine to three scientists for their work on telomeres and telomerase in protecting chromosomes. To refresh some basic genetics, chromosomes are structures of DNA (deoxyribonucleic acid) and protein found in all cells capable of reproducing themselves. In each cell division, chromo somes are duplicated almost to their ends, with each new chromosome passing on to the new cells. Telomeres protect the chromosome from incomplete replication. They also prevent the ends of the 24