Effects of Functional Foods Consumption on the Lipid Profile and Nutritional Status of Elderly

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1 400 Internacional Journal of Cardiovascular Sciences. 2015;28(5): ORIGINAL MANUSCRIPT Effects of Functional Foods Consumption on the Lipid Profile and Nutritional Status of Elderly Manuela Dolinsky, Renata Moreira da Silva Corrêa de Oliveira, Gabrielle de Souza Rocha, Sergio Girão Barroso, Silvia Maria Custodio das Dores, Vilma Blondet Azeredo Universidade Federal Fluminense Faculdade de Nutrição Departamento de Nutrição e Dietética Niterói, RJ Brazil Abstract Background: The consumption of functional foods (FF), as oat and linseed, is a safe and efficient strategy for reducing the risk of non-transmissible chronic diseases (NTCD). Objective: Analyzing the effect of food supplementation with brown linseed flour or oat in flakes on the body mass index (BMI), on the waist circumference (WC) and on the lipid profile of elderly. Methods: Longitudinal controlled, randomized study, with duration of eight weeks, made with 60 senior individuals (age > 60 years), evaluated at three moments: baseline (T0), four weeks (T1) and eight weeks (T2) after the study started. Weight, height and WC were measured and blood samples were collected. Volunteers were randomly divided into control group (CG), linseed group (LG) and oat group (OG). Results: BMI and WC have not changed significantly throughout the study in any group. OG showed reduction of 18.4% in the total cholesterol (TC) levels in the plasma compared to LG in the end of the study period (p<0.05). OG showed also, at T2, values 30.4% lower for LDL cholesterol than LG and CG (p<0.05). On the other hand, LG showed increase of 12.0% in the triglycerides levels from T0 to T2 (p<0.05). Conclusion: The supplementation with functional foods was beneficial for reducing the TC and LDL cholesterol. Keywords: Functional food; Elderly; Flax; Avena sativa; Nutritional status Introduction Many countries have nowadays a low, or even negative/ neutral, demographic growth rate, with the number of older inhabitants increasing in a higher pace than that of newly born. Such population aging results, naturally, in a higher incidence of diseases 1. Meanwhile, the growing modernization and urbanization in the latest decades generate changes in the populations food standards, which has also collaborated significantly to the increase in the prevalence of non-transmissible chronic diseases (NTCD) 1. Aging causes important alterations in the individual s body composition, with changes in muscle mass and in the body fat distribution standard 2. The body mass index (BMI) has shown to be useful for diagnosing obesity, but it is limited regarding body composition data 2. Thus, the waist circumference (WC) has been considered as an efficient manner of estimating volume and distribution of that fat. Moreover, the fat central distribution standard, revealed by WC, is correlated to some infirmities, like diabetes mellitus (DM) and cardiovascular diseases (CVD) 3. Corresponding author: Manuela Dolinsky Av. Epitácio Pessoa, 2330 ap. 105 Ipanema Rio de Janeiro, RJ Brazil manudolinsky@gmail.com DOI: / Manuscript received on September 24, 2015; approved on November 15, 2015; revised on November 24, 2015.

2 Int J Cardiovasc Sci. 2015;28(5): In this context, the consumption of functional foods is emphasized as a safe and efficient strategy for reducing the risk of NTCD 4 for the senior population. Among the functional foods, oat and linseed have been used due to their potentially beneficial effects on health. RJ, Brazil. The elderly were recruited from social projects managed by a Non- Governmental Organization (NGO) in Niterói. The participants signed the Informed Consent Form. ABBREVIATIONS AND ACRONYMS AC abdominal circumference The Food and Drug Administration (FDA), from the United States, recognizes oat as a cholesterol reducing agent. This statement is based on several studies showing the consumption of oat leading to the reduction in the serum cholesterol due to its soluble fiber contents, beta glucan 5 in special. Those components have the relevant property of resisting to digestion, making viscous solutions reducing the cholesterol absorption 6. Linseed is considered as a functional food for the presence of bioactive compounds, like lignans, alphalinolenic acid (ALA) (omega 3) and dietary fibers 7. Lignans are phytoestrogens whose potential benefits to health, such as hypocholesterolemic properties due to the modulation of enzymes involved from cholesterol metabolism, have been shown in researches 7,8. Such properties for reducing cholesterol have also been attributed to ALA, through mechanisms including favorable changes to the levels of plasmatic lipids due to hepatic alterations in the cholesterol metabolism. Supplementation with linseed flour has been associated to beneficial effects on the cardiovascular risk factors 7. This way, the use of functional foods has increased, as an attempt to keep the quality of life of the growing population of elderly, contributing to the maintenance of health and prevention of diseases. The objective of this study was assessing the impact of supplementation with brown linseed flour or oat in flakes on the BMI, WC and the lipid profile of elderly residing in the city of Niterói, RJ, Brazil. Methods The protocols applied to this study are under the ethical principles included in the Helsinki Statement and were approved by the Ethics Committee on Research of the institution under no. CEP CMM / HUAP 252/08; CAAE no An eight-week longitudinal controlled and randomized clinical essay was performed, with individuals aged 60 years, of both genders, residing in the city of Niterói, The inclusion criteria used were: elderly 60 years, not using drugs for dyslipidemia, nutritional supplements or not presenting any serious disease. The exclusion criteria adopted were: diagnosis of chronic diseases (cancer, chronic renal disease under substitutive renal therapy, unbalanced cardiovascular disease etc.); contraindication to the consumption of any food used in the study; changes in the lipid profile during the study, and the patients were sent to medical care. The sample calculation was performed by using the G*Power software (3.1.2 version) and the means and standard deviations of a pilot sample. Considering a significance level of 5% (α=0.05) and test power of 90% (1-β = 0.90), the number of 18 patients for each group was considered as adequate. Twenty patients were recruited per group for ensuring this minimum number after any loss 6. BMI body mass index CG control group CVD cardiovascular diseases DM diabetes mellitus FF functional foods LG linseed group NTCD non-transmissible chronic diseases OG oat group T0 baseline evaluation T1 four-week evaluation T2 eight-week evaluation TC total cholesterol WC waist circumference WHO World Health Organization The 60 participants were monitored and evaluated every day for eight weeks, specifically at start (T0), in the fourth (T1) and in the eighth week (T2) of the study, being randomly distributed in three groups with 20 individuals/group: Control group (CG), not subject to any intervention; Linseed group (LG), supplemented with 30 g of ground brown linseed flour per day; and Oat group (OG), supplemented with 60 g of oat in flakes per day. The supplements were distributed in T0 and T1. GL and GA received oral and written instructions about the consumption of their supplements. No other intervention in the diet of the participants was made in addition to the inclusion of brown linseed or oat in flakes, according to the group. The quantity of brown linseed flour and oat in flakes supplied was established based

3 402 Int J Cardiovasc Sci. 2015;28(5): on the total fiber content of each supplement, so as to standardize the quantity of fiber ingested. The brown linseed flour and oat in flakes were purchased in the local market and properly stored under refrigeration, until they were distributed to the participants. Data were collected between June 2010 and February 2011, in the Experimental Nutrition Laboratory, Nutrition and Dietary Department, Universidade Federal Fluminense. For the anthropometric evaluation, weight and height were assessed for calculation of the BMI and the rating of the anthropometric nutritional status, based on the World Health Organization (WHO) parameters 9. AC was used for evaluating the metabolic risk, under WHO 10. All measurements were performed in each of the three phases of the study (T0, T1 and T3), under the following protocol: body mass determined on anthropometric scale Caumaq 102 PL (Santa Catarina, Brazil), with 100 g divisions; height measured with 0.1 cm accuracy, by using a stadiometer coupled to the scale; AC measured with inelastic tape at the medium point between the lower rib and the iliac crest, being rounded to the nearest centimeter. Blood samples (10 ml) were collected after an overnight fast of 12 hours. Lipid profile (TC, HDL and triacylglycerol - TG) was determined by colorimetric enzyme essay, using BIOCLIN commercial kits (Química Básica Ltda, Belo Horizonte, MG). LDL and VLDL (mg/dl) were calculated under the formulas of Friedewald et al. 11. The data regularity was tested by using the Kolmogorov- Smirnov test with correction of Lilliefors for the significance. When the data met the regularity criterion, the one-way variance analysis (one-way ANOVA) was used for comparing the data group independent from numeric variables; in the other cases, the nonparametric test of Kruskal-Wallis was used. Tukey tests or Dunnet s T3 test were used as post-test for multiple comparisons intended to find distinct groups. In the case of the paired data groups, the Friedman test was used for comparing more than two groups and the Dunn test acted as post-test. The reduction found in the clinic parameters was expressed as percentage. Software SPSS 10.0 was used for the analysis of data and the statistic decisions were made when p<0.05. Numeric data were expressed as mean and standard deviation. Results The mean age of participants was of 68.92±7.75 years; with predominance of women (85.2%); most of them (59.0%) with overweight or obesity; high risk or very high risk of metabolic complications (82.0%). No significant change in BMI was found throughout the study. As to the risk intensity values for metabolic complications under the AC, no significant change was found between the proportions of cases without risk or with (higher or very higher) risk in the evaluations made in T0 and T2 (Table 1). Figures 1 and 2 present the TC and TG serum concentrations, respectively. It was found that, in average, all groups presented TC above the desired level (200 mg/dl) at the beginning of the study. CG kept TC constant throughout the study, while OG presented reduction of 12.8% (p<0.05). The linseed consumption has not changed this parameter. TG serum concentration of all groups was within the parameters wanted (<150 mg/dl). CG and LG presented significant increase (p<0.05) of 21.0% and 12.0%, respectively, in the end of the study. Figure 3 presents LDL values for the three groups studied, at T0, T1 and T2, showing no significant difference among the groups at T0 and T1. However, at T2, OG presented LDL values 30.4% lower in average (p<0.05) than those of CG and LG. There was no significant difference of LDL during the evaluations made both to CG and LG. The HDL concentration has not changed significantly among the groups studied at the three moments of the study (Figure 4). However, the HDL levels of the individuals in CG presented higher reduction in that parameter (28.0%), when compared to those of OG and LG (Figure 4).

4 Int J Cardiovasc Sci. 2015;28(5): Table 1 Characteristics of the population studied, per groups Variables CG LG OG Total n (%) n (%) n (%) n (%) Gender Female Male Age group (in years) BMI Light thinness Eutrophy Pre-obesity Obesity grade Obesity grade Abdominal circumference Without Risk High risk Very high risk CG control group; LG linseed group; OG oat group; BMI body mass index Figure 1 Concentration of serum total cholesterol in the groups studied Superscript letters indicate significant difference among groups; * indicates significant difference among periods. CG control group; LG linseed group; OG oat group T0 baseline evaluation; T1 four-week evaluation; T2 eight-week evaluation

5 404 Int J Cardiovasc Sci. 2015;28(5): Figure 2 Concentration of serum cholesterol in the groups studied Superscript letters indicate significant difference among groups; * indicates significant difference among periods. CG control group; LG linseed group; OG oat group T0 baseline evaluation; T1 four-week evaluation; T2 eight-week evaluation Figure 3 LDL values of the groups studied (CG, LG and OG) at the three moments (T0, T1 and T2) Superscript letters indicate significant difference among groups; * indicates significant difference among periods. LDL low-density lipoprotein; CG control group; LG linseed group; OG oat group T0 baseline evaluation; T1 four-week evaluation; T2 eight-week evaluation

6 Int J Cardiovasc Sci. 2015;28(5): Figure 4 HDL values of the groups studied (CG, LG and OG) at the three moments (T0, T1 and T2) Superscript letters indicate significant difference among groups; * indicates significant difference among periods. HDL high-density lipoprotein; CG control group; LG linseed group; OG oat group T0 baseline evaluation; T1 four-week evaluation; T2 eight-week evaluation Discussion According to BMI, 59.0% of the participants presented excessive weight and obesity, and 21.3% out of those were obese. The results found in the study are confirmed by national epidemiologic inquiries, showing that the prevalence of overweight/obesity in the adult population of Brazil is already reaching 64.9% of women and 62.5% of men 12. No significant change was observed in the BMI values for the period of evaluations, similarly to the observations of other researchers, who have reported the absence of weight gain even in the supplemented groups 7,13. This result may be related to the consumption of dietary fibers by the members of the supplemented groups, once these are associated to the weight control by regulating the appetite and increasing the satiety 14. Epidemiologic studies suggest an inverse relation between BMI and body weight with the ingestion of dietary fibers 15. Since, as discussed, BMI has limitations regarding the body composition data, AC measures can provide further information as to the distribution of body fat 2. In this study, approximately half of the sample (47.5%) presented very high risk of metabolic complications, according to AC values at T0, which was not affected by the treatments. While considering any kind of risk of metabolic complications, this number increases to 82.0%, confirming other literature data 16. As to the lipid profile, the linseed supplementation has not reduced the TC levels of the volunteers studied, and these results agree to those found by Stuglin and Pasad 17, in which adult men ingested 32 g of linseed per day for four weeks and their lipid profile remained unaltered. Other experiment, that gave 40 g of linseed per day for 12 months to women in menopause, found reduction of only 0.1% in TC 7. Different results were found in the study by Patade et al. 4, in which post-menopause women received 30 g of linseed per day during 12 weeks and presented reduction of 7.0% in the TC levels. In study with rabbits, secoisolariciresinol diglycoside (SDG), linseed s main lignan, was associated to reductions in TC 18. Arjmandi et al. 13 suggest that linseed lignans can reduce plasma cholesterol by modulating the 7-α-hydroxylase and acyl-coa cholesterol transferase, involved in the metabolism of cholesterol.

7 406 Int J Cardiovasc Sci. 2015;28(5): The absence of significant results in this study can be justified by the lipid rancidification or oxidation of the linseed, supplementation time, quantity supplemented and means of supplementation. Although the linseed offered has been correctly stored, the lipid oxidation may have occurred, as the type of linseed used in the study was the flour ready to be used. However, even if the lipid sources had oxidized, an effect of the fibers present in the supplement on the TC levels was expected. Some studies suggest that a larger period of supplementation is necessary for finding significant changes to the lipid profile 19. Regarding the quantity of linseed flour to be supplemented, some authors say that 50 g/day would be enough to achieve significant results 20 and that 30 g/day would be a low dose for reducing the lipid profile 21. The positive results by Patade et al. 4 are possibly due to the fact that linseed being included in breads and muffins, once the baking can increase the bioavailability of linseed s photochemical. In this study, the supplementation with oat reduced the TC levels in 18.04%, between the first and the last evaluation. Similar results were found by Liatis et al. 22, in which the bread enriched with beta glucans (3 g/day) reduced the TC levels of patients with type II diabetes in 12.8%. Cholesterol reduction with beta glucans occurs through the formation of a viscous layer in the small intestine, which would inhibit the absorption of cholesterol from the diet and the new absorption of biliary acids 23. Other mechanism proposed would be increasing the connection of biliary acids in the intestinal light, leading to the reduction of the cholesterol and biliary acids enterohepatic circulation 23. Soluble fibers are also able to reduce total cholesterol and LDL through the effects on the postprandial glucose. They reduce the glucose absorption rate, by reducing the production of insulin by the pancreas. Once the insulin is responsible for the activation of the HMG CoA reductase enzyme, its reduction is likely to induce a diminished synthesis of cholesterol 24. According to some studies, soluble fibers would be able to increase the activity of the cholesterol 7-α-hydroxylase enzyme, the main responsible for regulating the cholesterol hepatic conversion into biliary acids, contributing to a higher depletion of hepatic cholesterol 25. Finally, soluble fibers have been recognized as able to increase the bacterial fermentation in the colon by producing short chain fatty acids. These, propionate overall, would indirectly cause reduction in blood cholesterol by inhibiting the hepatic synthesis of cholesterol 26. The reduction of the TC levels found in OG is very relevant, once, for each 10% of reduction in that lipid, the total mortality reduces in 11.0%, and the mortality for heart coronary disease, in 15.0% 27. Moreover, it is observed that for each 1% of reduction in the TC serum levels, the risk of developing CAD is reduced in 2-3% 28. The supplementation with brown linseed flour has not reduced TG, on the contrary, LG presented increase of their levels after the supplementation. The linseed consumption has been associated to TG elevations in studies with human beings 17 and animals 18. Similar results were found in a study supplementing the same quantity of linseed (30 g/day) for three months in the diet of American post-menopause women, with the increase in the TG levels of 3% 4. The supplementation with oat in flakes has not changed the serum TG levels in any evaluation made in this study. Similar results were found in a study where 6 g/day of oat with concentration of beta glucans have not caused alterations to the TG levels in the treatment group 26. The supplementation with brown linseed flour has not resulted in LDL reduction, against the literature data 7. That distinction may be attributed to methodological differences, as the quantity and/or time of supplementation. Linseed is the richest source of lignans, one of the main groups of phytoestrogens 7. Studies suggest that phytoestrogens act as selective modulators for the estrogen receptor, able to reduce the LDL plasma concentrations in human beings and animals 8,29. On the other hand, in OG, significant reductions were found in the LDL values. These results are similar to those found in the literature, when the supplementation of 3 g/day of beta glucans for three weeks reduced the LDL in approximately 16% 22. The results of this study are similar to those by Zani 30, which has supplemented elderly women with 30 g/day of oat for 12 weeks and has also observed reduction in the LDL levels. In this research, the supplementation with oat in flakes has not improved the HDL levels, as well as the results

8 Int J Cardiovasc Sci. 2015;28(5): found by Zani 30, where elderly women supplemented with oat have not presented significant changes to the HDL levels. In other study, providing 38 g/day of linseed to post-menopause women, the supplementation has not made significant changes to the blood HDL levels 13. HDL levels <35 mg/dl have been related to the increase of 2.5 times in the risk of mortality for heart coronary disease 31. Conclusions The daily consumption of oat in flakes is able to reduce total cholesterol and LDL-c in elderly. On the other hand, linseed has not shown any positive effect in reducing the participants serum lipids. New studies with an increased number of volunteers and study period are suggested for assessing the impact of supplementing oat and linseed on the NTCD risk factors. Potential Conflicts of Interest This study has no relevant conflicts of interest. Sources of Funding This study was partially funded by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Pró-Reitoria de Pesquisa, Pós-graduação e Inovação da Universidade Federal Fluminense (PROPPI-UFF) Academic Association This study is not associated with any graduate programs. References 1. World Health Organization. [Internet]. Noncommunicable diseases country profiles WHO Global Report [2015 jan. 13]. Available from: < publications/ncd_profiles_report.pdf> 2. Zamboni M, Mazzali G, Zoico E, Harris TB, Meigs JB, Di Francesco V, et al. Health consequences of obesity in the elderly: a review of four unresolved questions. Int J Obes (Lond). 2005;29(9): Janssen I, Mark AE. Elevated body mass index and mortality risk in the elderly. Obes Rev. 2007;8(1): Patade A, Devareddy L, Lucas EA, Korlagunta K, Daggy BP, Arjmandi BH. Flaxseed reduces total and LDL cholesterol concentrations in Native American postmenopausal women. J Womens Health (Larchmt). 2008;17(3): US Food and Drug Administration. [Internet]. Final Rule: Food labeling: Health claims; Oats and coronary heart disease [2015 jan. 22]. Available from: < IngredientsPackagingLabeling/LabelingNutrition/ucm htm> 6. Gunness P, Gidley MJ. Mechanisms underlying the cholesterollowering properties of soluble dietary fibre polysaccharides. Food Funct. 2010;1(2): Dodin S, Cunnane SC, Mâsse B, Lemay A, Jacques H, Asselin G, et al. Flaxseed on cardiovascular disease markers in healthy menopausal women: a randomized, double-blind, placebo-controlled trial. Nutrition. 2008;24(1): Zhang W, Wang X, Liu Y, Tian H, Flickinger B, Empie MW, et al. Dietary flaxseed lignan extract lowers plasma cholesterol and glucose concentrations in hypercholesterolaemic subjects. Br J Nutr. 2008;99(6): WHO Expert Consultation. Appropriate body-mass index for Asian populations and its implications for policy and intervention strategies. Lancet. 2004;363(9403): Erratum in: Lancet. 2004;363(9412): World Health Organization. [Internet]. Obesity: preventing and managing the global epidemic. Report of a WHO Consultation on Obesity; WHO Technical Report Series n [cited 2015 abr. 9]. Available from: < publications/obesity/who_trs_894/en> 11. Friedewald W, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 1972;18(6): Instituto Brasileiro de Geografia e Estatística. Pesquisa nacional sobre despesas familiares Rio de Janeiro: IBGE; Arjmandi BH, Khan DA, Juma S, Drum ML, Venkatesh S, Sohn E, et al. Whole flaxseed consumption lowers serum LDLcholesterol and lipoprotein(a) concentrations in postmenopausal women. Nutr Res. 1998;18(7): Papathanasopoulos A, Camilleri M. Dietary fiber supplements: effects in obesity and metabolic syndrome and relationship to gastrointestinal functions. Gastroenterology. 2010;138(1): Ben Slama F, Jebali N, Chemli R, Ben Rayana C, Achour A, Najar MF, et al. Dietary fiber in the diets of urban Tunisian women: association of fiber intake with BMI, waist circumference and blood chemistry: preliminary study. Clin Res Hepatol Gastroenterol. 2011;35(11): Hauner H, Bramlage P, Lösch C, Steinhagen-Thiessen E, Schunkert H, Wasem J, et al. Prevalence of obesity in primary care using different anthropometric measures results of the German Metabolic and Cardiovascular Risk Project (GEMCAS). BMC Public Health. 2008;8: Stuglin C, Prasad K. Effect of flaxseed consumption on blood pressure, serum lipids, hemopoietic system and liver and kidney enzymes in healthy humans. J Cardiovasc Pharmacol Ther. 2005;10(1):23-7.

9 408 Int J Cardiovasc Sci. 2015;28(5): Prasad K. Reduction of serum cholesterol and hypercholesterolemic atherosclerosis in rabbits by secoisolariciresinol diglucoside isolated from flaxseed. Circulation. 1999;99(10): Bloedon LT, Balikai S, Chittams J, Cunnane SC, Berlin JA, Rader DJ, et al. Flaxseed and cardiovascular risk factors: results from a double blind, randomized, controlled clinical trial. J Am Coll Nutr. 2008;27(1): Cunnane SC, Ganguli S, Menard C, Liede AC, Hamadeh MJ, Chen ZY, et al. High alpha-linolenic acid flaxseed (Linum usitatissimum): some nutritional properties in humans. Br J Nutr. 1993;69(2): Austria JA, Richard MN, Chahine MN, Edel AL, Malcolmson LJ, Dupasquier CM, et al. Bioavailability of alpha-linolenic acid in subjects after ingestion of three different forms of flaxseed. J Am Coll Nutr. 2008;27(2): Liatis S, Tsapogas P, Chala E, Dimosthenopoulos C, Kyriakopoulos K, Kapantais E, et al. The consumption of bread enriched with betaglucan reduces LDL-cholesterol and improves insulin resistance in patients with type 2 diabetes. Diabetes Metab. 2009;35(2): Othman RA, Moghadasian MH, Jones PJ. Cholesterol-lowering effects of oat β-glucan. Nutr Rev. 2011;69(6): Erkkilä AT, Lichtenstein AH. Fiber and cardiovascular disease risk: how strong is the evidence? J Cardiovasc Nurs. 2006;21(1): Babio N, Balanza R, Basulto J, Bulló M, Salas-Salvadó J. Dietary fibre: influence on body weight, glycemic control and plasma cholesterol profile. Nutr Hosp. 2010;25(3): Queenan KM, Stewart ML, Smith KN, Thomas W, Fulcher RG, Slavin JL. Concentrated oat beta-glucan, a fermentable fiber, lowers serum cholesterol in hypercholesterolemic adults in a randomized controlled trial. Nutr J. 2007;6: Gould AL, Rossouw JE, Santanello NC, Heyse JF, Furberg CD. Cholesterol reduction yields clinical benefit: impact of statin trials. Circulation. 1998;97(10): Charlton KE, Tapsell LC, Batterham MJ, O Shea J, Thorne R, Beck E, et al. Effect of 6 weeks consumption of beta-glucan-rich oat products on cholesterol levels in mildly hypercholesterolaemic overweight adults. Br J Nutr. 2012;107(7): Brzezinski A, Debi A. Phytoestrogens: the natural selective estrogen receptor modulators? Eur J Obstet Gynecol Reprod Biol. 1999;85(1): Zani VT. Efeito da intervençao dietética com aveia em mulheres idosas com syndrome metabólica. [Tese de Doutorado]. Porto Alegre: Pontifícia Universidade Católica do Rio Grande do Sul; Corti MC, Guralnik JM, Salive ME, Harris T, Field TS, Wallace RB, et al. HDL Cholesterol predicts coronary heart disease mortality in older persons. JAMA. 1995;274(7):