The Journal of the American Nutraceutical Association

Vol. 10, No. 1, 2007 www.ana-jana.org

A Peer-Reviewed Journal on Nutraceuticals and Nutrition ISSN-1521-4524

IN THIS EDITION

• Folate: A Key to Optimal Health Throughout the Lifespan

• Lowering LDL and Total Serum Cholesterol: An Overview ofDrug and Dietary Therapies, Including the Portfolio Diet andIts Impact on Lipids and Hypertension

• Preventive Cardiology, Our Greatest Hope for EradicatingHeart Disease

• Migraine Prophylaxis: Comparable Effects or UnadjustedEffects That Could Be Misleading? Review of a Study byMaizels et al

• An Evaluation of the Immunological Activities ofCommercially Available β1, 3-Glucans

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Compound) by American BioSciences, Inc.• Vitamin C by Cognis• NSC 100™ by Nutritional Supply Corporation• Baker’s Yeast by Fleischman’s®• ViscoFiber™ by Cevena• Cell Forte/IP6/Inositol by Enzymatic Therapy • ASTRAGALUS by SmartBasic• Advanced Colostrom Plus™ by Symbiotics

These products produced less effect than saline, the negative control• Vitamin C by AIDP, Inc.• Ambrotose by Mannatech, Inc.• BioChoice® Immune 26 by Legacy for Life, Inc.• 4Life® Transfer Factor™ by 4Life Research• ACTIValoe™ by Aloecorp, Inc.

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Nutraceuticals & Medicine Conference, SPRING 2007CD-SET

Eight Keys for Preventing Osteoporosis and Building Bone Strength - Susan E. Brown, PhD, CNS, Director, The OsteoporosisEducation Project, East Syracuse, NY. Dr. Brown directs both the Nutrition Education and Consulting Service (NECS) and The OsteoporosisEducation Project in Syracuse, New York. NECS provides nutrition consulting, education and research services for the Central New York area.Dr. Brown is also a research associate, Anthropology Department, Syracuse University.

The Health Benefits of Probiotics and Their Importance in Disease Prevention: Guidelines for the Clinical Practice - Kelly A.Tappenden, PhD, RD, Associate Professor of Nutrition and GI Physiology, Department of Food Science and Human Nutrition, University ofIllinois at Urbana-Champaign, IL. Dr.Tappenden’s research program is directed at achieving a greater understanding of the regulation of smallintestinal function and health by various nutrients and gastrointestinal-specific peptides.

Integrative Approaches to the Management and Prevention of Diabetes Mellitus: Guidelines for the Clinical Practice - JayUdani, MD, CPI, Assistant Clinical Professor, UCLA /Geffen School of Medicine, Medical Director, Northridge Hospital Integrative MedicineProgram, and Medical Director, Medicus Research, Northridge, California. Jay Udani, MD, is the Medical Director of the Integrative MedicineProgram at Northridge Hospital and Assistant Clinical Professor at the UCLA /Geffen School of Medicine. Dr. Udani, a board certified Internist,was the Chief Resident of Internal Medicine at Cedars-Sinai Medical Center, and was the first Fellow in Integrative Medicine at Cedars Sinai.

Exploring Factors Related to Childhood and Adolescent Obesity: Guidelines for the Clinical Practice - Susan L. Johnson, PhD,Associate Professor, Department of Pediatrics, Director, The Children's Eating Laboratory, University of Colorado Health Sciences Center,Denver, CO. Dr. Johnson is an early childhood nutritionist in the Department of Pediatrics, UCHSC, and practices clinically at The Children’sHospital of Denver. She teaches courses in the UCDHSC School of Nursing and the UCDHSC School of Medicine regarding nutrition andfeeding of children, as well as nutrition and obesity in children and adults.

The ANA Free Radical Test Kit can be used to determine the level of free radical stress within thebody. Excess free radicals induce severe chronic damage to DNA, proteins, fats and other compounds result-ing in an increased risk of cancer, cardiovascular disease, arthritis and other diseases of aging. Healthcare pro-fessionals can use this simple urine screening test to determine if a patient needs greater antioxidant intake,and measure how well antioxidants are impacting free radical bio-activity.

The ANA Free Radical Test Kit provides a quick and inexpensive method to identify MDA(Malondialdhyde) in the urine of patients. When free radicals attack the walls of the body’s cells, oxidizedbyproducts such as MDA are produced. The test kit compares the color change in a urine sample with a colorchart provided in the test kit. The more oxidized fat byproducts, the darker the sample. Once the level ofexcess free radicals is identified, healthcare professionals can recommend a scientific nutrition and nutritionalsupplement treatment program designed to reduce free radical levels in the body and maintain them long-term.

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Now Available on CD

Contents – JANA Vol. 10, No.1, 2007

C O N V E R S A T I O N

C O N F E R E N C E R E P O R T

G L O B A L U P D A T E

To order reprints of articles, or additional copies of JANA:Allen Montgomery, RPh

5120 Selkirk Drive, Suite 100, Birmingham,AL 35242Phone 205-980-5710 Fax 205-991-9302

E-mail: allenm@ana-jana.org Website: www.ana-jana.org

Journal of the American Nutraceutical Association

EDITORIAL STAFF

EDITOR-IN-CHIEFMark Houston, MD

ASSOCIATE EDITORSBernd Wollschlaeger, MDBarry Fox, PhD

TECHNICAL EDITORJane LaelTerri Erickson

ART DIRECTOR Gary Bostany

EDITORIAL BOARDJordan R.Asher, MD, MsMM, SCHEthan Basch, MD, MPhilJan Basile, MDRussell Blaylock, MDHyla Cass, MDLisa Colodny, PharmD, BCNSPLoren Cordain, PhDJeanette Dunn, EdD, RN, CNSBrent Eagan, MDChristopher M. Foley, MDMichael Glade, PhDClare M. Hasler, PhD, MBARobert Krueger, PhDDaniel T. Lackland, PhDGarth L. Nicolson, PhDMark J.S. Miller, PhDRobert Rountree, MDDiana Schwarzbein, MDCatherine Ulbricht, PharmDWalter Willett, MD, DrPHBernd Wollschlaeger, MD

_____________________________

American Nutraceutical Association5120 Selkirk Drive, Suite 100Birmingham,AL 35242Phone: (205) 980-5710 Fax: (205) 991-9302Website: www.Ana-Jana.org

CEO & PUBLISHERAllen Montgomery, RPh

ANA is an alliance of individuals with interest innutraceutical science, technology, marketing andproduction. It was established to develop and pro-vide educational materials and continuing educa-tion programs for health care professionals onnutraceutical technology and science. ANA pub-lishes a E-newsletter, The Grapevine, and the Journalof the American Nutraceutical Association (JANA).

_____________________________

The Journal of the American Nutraceutical Association(ISSN-1521-4524) is published three times annuallyby the American Nutraceutical Association (ANA).Send all inquiries, letters, and submissions to theANA Editorial Department at 5120 Selkirk Drive,Suite 100, Birmingham, AL 35242. Contents ©2007 ANA,all rights reserved.Printed in the UnitedStates of America. Reproduction in whole or partis not permitted without written permission. It isthe responsibility of every practitioner to evaluatethe appropriateness of a particular opinion in thecontext of actual clinical situations. Authors, edi-tors, and the publisher cannot be held responsiblefor any typographical or other errors found in thisjournal. Neither the editors nor the publisherassume responsibility for the opinions expressedby the authors.

A Conversation with Seth Baum, MD,New Member of the ANA Medical Advisory Board ....................1

International Alliance of Dietary Supplement-FoodAssociations (IADSA) Sets Agenda for 2007 ............................ 4

N U T R A C E U T I C A L N E W S

ConsumerLab.com Survey Identifies Brands and MerchantsRated Highest by Dietary Supplement Users ............................ 5

Vitamin D Fortification Needs to be Considered........................ 6Wholegrain Breakfasts Linked to Lower Heart Failure Risk

Lowering LDL and Total Serum Cholesterol: An Overview ofDrug and Dietary Therapies, Including the Portfolio Diet and Its Impact on Lipids and Hypertension .............................15David J.A. Jenkins, MD, PhD, DSc

Folate: A Key to Optimal Health Throughout the Lifespan.............................................................. 7Lynn B. Bailey, PhD

P E R S P E C T I V E A R T I C L E

Preventive Cardiology, Our Greatest Hope for Eradicating Heart Disease ......................................................... 21Seth J. Baum, MD, FACC

O R I G I N A L R E S E A R C H

An Evaluation of the Immunological Activities of Commercially Available ββ1, 3-Glucans ..................................... 25Vaclav Vetvicka, PhD

O P I N I O N A R T I C L E

Migraine Prophylaxis: Comparable Effects or UnadjustedEffects That Could Be Misleading? Review of a Study by Maizels et al............................................ 23Yuxin Zhang, PhD

1 JANA Vol. 10, No. 1, 2007

C O N V E R S A T I O N

A Conversation with Seth Baum, MDNew Member of the ANA Medical

Advisory Board

Seth Baum, MD, FACC, a cardiologist in Boca Raton, Florida was recently appointed to the ANA Medical Advisory Board.

Barry Fox, PhD, JANA Associate Editor, conducted the following interview with Dr. Baum.

Q: Dr. Baum, how did you come to practice interventioncardiology?

My initial training and practice were quite conventional.I graduated from the Columbia College of Physicians andSurgeons, took a Residency in Internal Medicine at NYUMedical Center, then completed a Fellowship in Cardiology,Interventional Cardiology, and Electrophysiology at NewYork Medical College. In 1991, I opened a private practice inBoca Raton, Florida, specializing in clinical/interventionalcardiology.

For the first several years, I primarily did tertiary care.But after seeing patients come back for repeat procedures, Ibegan wondering if there wasn’t another approach. Insteadof repeatedly performing invasive techniques on peoplewho had already developed significant cardiovascular dis-ease, was it possible to help prevent the cardiovascularproblems from arising in the first place? Or, at least, toreduce the risk?

I began searching for ways to prevent disease, and tointervene in existing disease, without using catheters andinvasive means. One of the first areas I looked into wasnaturopathy. I read many of their texts, did distance train-ing in nutrition and supplementation, and over the next sev-eral years introduced alternative health strategies into mypractice. Overall, the results were positive and I felt I wasoffering my patients the best of both worlds: alternativemethods to help them reduce the risk of developing cardio-vascular disease, and standard medical interventional car-diology and electrophysiology for those who already hadadvanced disease.

From 2000 to 2003, I served asMedical Director for the John W.Henry Center for IntegrativeMedicine at the Boca RatonCommunity Hospital, as well asMedical Director of the Mind/BodyMedical Institute at Beth IsraelDeaconess Hospital, Boca RatonDivision. Today, I continue to haveone foot in the world of standardmedicine and one in the world of alternative medicine,working with my patients to reduce the risk of cardiovascu-lar disease, as well as lecturing and writing on PreventiveCardiology. I’m also Board Certified in Clinical Lipidologyand have achieved level 3 verification in Coronary CTAngiography. My dream is to integrate the best of standardand alternative approaches, and develop a center for pre-ventive cardiology, a concept that is unfortunately not aswidespread as it should be in this country.

Q: As a cardiologist practicing in integrative medicine,what do you think people should be doing to reduce theirrisk of developing heart disease?

I think it’s a good idea to eat a healthful diet, take appro-priate nutritional supplements, maintain an ideal body weight,exercise every day, learn to manage stress, and have theirblood lipids checked regularly. I believe that, in addition, peo-ple should ask their physicians to delve deeper into their lipidsand begin to look, for example, not just at the LDL cholesterollevel, but also at the number of LDL particles or LDL-P.

Seth Baum, MD, FACC

JANA Vol. 10, No. 1, 2007 2

Q:Which brings us right to the next issue. Most people arefamiliar with the standard “cholesterol numbers” – totalcholesterol, LDL “bad” cholesterol and HDL “good” cho-lesterol – and know that an elevated LDL is a risk factorfor cardiovascular heart disease. However, research con-ducted over the past ten or so years has suggested thatsimply keeping LDL cholesterol under control may not bethe best way to attack cardiovascular disease.

That’s true. This concept dates back to the 1960s, whenDr. Friedrickson, one of the fathers of lipidology, said thatour focus should be on the assessment of lipoproteins, ontheir number, size and characteristics, rather than simply onthe total amount of cholesterol carried by the LDL popula-tion. Unfortunately, back then we did not have the commer-cial capacity to measure lipoproteins, so a surrogate mark-er, LDL cholesterol, was chosen instead.

Let me step back a bit and remind you that cholesterolis transported through the body in different “vehicles,”including LDL – low density lipoprotein particles. LDLparticles come in different sizes and carry differingamounts of cholesterol, but we didn’t have an easy way ofmeasuring the number and sizes of those particles. What wecould do was measure the amount of cholesterol carried inthe population of LDL particles, so we did.

LDL-C, or the amount of cholesterol the population ofLDL particles contained, became the gold standard mea-surement by default, not by choice. It was assumed thatthere was a direct relationship between LCL-C and thenumber of LDL particles in the blood. All of the major tri-als were designed around measuring LDL-C. ReducingLDL-C became a major focus of treatment, and medicineswere designed to do that.

Unfortunately, we’ve found that no matter how low wedrive the LDL-C, we’re still missing a lot of people. We’reonly decreasing the risk by about 30%, which means we’remissing some 70% of cardiovascular events. Here’s anotheralarming statistic: about 50% of the people who have heartattacks have normal LDL-C levels.

Clearly, looking only at the LDL-C is not enough. It’sas if a traffic engineer were only counting the number ofpeople on the road, rather than the numbers of cars they’resitting in. There might be 1,000 people on a road right now,but the more important issue is how many cars they’re sit-ting in. If each person is in his own car, there are 1,000 carson the road and you have a traffic problem. But if those1,000 people are in 500 cars, traffic isn’t so bad. And ifthey’re sitting four to a car, there are only 250 cars on theroad, so there’s absolutely no traffic at all. If you were a traf-fic engineer trying to ensure that traffic flows smoothly, itwould certainly help to know the number of people on theroad. But it would be far better to know the number of vehi-cles on the road.

A number of researchers have looked into the LDLcholesterol versus LDL particle number issue. Major stud-ies that have looked at LDL particle information include theCardiovascular Health Study (Arterioscler Thromb VascBiol 2002), the Women’s Health Study (Circulation 2002),the Healthy Women’s Study (Am J Cardiol 2002), theVeterans Affairs HDL Intervention Trial (VA-HIT) (AmHeart Assoc 2002, Circulation 2006), the PravastatinLimitation of Atherosclerosis in Coronary Arteries (PLAC-1) (Am J Cardiol 2002), and the Framingham OffspringStudy (Am Heart Assoc 2004). These and other studies havefound that the LDL particle number is a vastly superiormeasure of cardiovascular risk and future events.

LDL particle number is a better measure of risk thanLDL cholesterol because the particles come in a variety ofsizes and vary in cholesterol content. This explains whycholesterol content and particle number fail to correlate.The more particles you have, the greater the chance that one– or more – will penetrate an arterial wall and begin the ath-erosclerotic process.

Q: How does the LDL cholesterol versus LDL particlenumber issue play out in your practice?

This issue arises in up to 70% of my patients. They’vebeen receiving treatment and their LDL cholesterol’s areonly moderately elevated, or perhaps even in the safe range.This suggests that their treatment has been successful, butwhen you measure their LDL particle number, you realizethat they are still at risk of cardiovascular disease and needmore therapy.

Q: Is the size of the LDL particles important? Shouldphysicians be measuring this as well?

The verdict is still out on particle size, but the numberof particles is clearly much more important than their size.

Q: You said earlier that it had been difficult to measureLDL particle number and size. Is that still a problem?

No. LipoScience Labs of Raleigh, North Carolina,offers the NMR LipoProfile, which uses nuclear magneticresonance spectroscopy to measure LDL particle concen-tration (or number) and size, as well as the LDL, HDL andVLDL subclass levels. The test is available nationwidethrough LabCorp. By way of disclosure, I should tell youthat I’m a consultant to LipoScience Labs.

Other labs, including Atherotech of Birmingham,Alabama, measure qualitative size and cholesterol content,although they do not count particles.

3 JANA Vol. 10, No. 1, 2007

Q: How are LDL particle results measured, and what arethe various levels?

LDL particle results are given as nmol/L. Anythingbelow 1,000 is considered optimal, from 1,000 to 1,300 isnear optimal, from 1,300 to 1,600 is borderline, above 1,600is high and over 2,000 is very high risk.

Q: How do you reduce an elevated LDL particle number?

The approach to reducing an elevated LDL particlecount is the same as for reducing elevated LDL cholesterol.The statin drugs can be helpful. Other drugs such as Zetia,which inhibits cholesterol absorption, and Niaspan, whichcan enlarge particle size and lower the particle number, often-times without affecting LDL cholesterol, are also helpful.

Non-pharmacologically, exercise can help lower the par-ticle number. Dietary interventions are also helpful. Transfatsare the biggest dietary offenders. Saturated fats should alsobe significantly limited, but not entirely eliminated.

Q: Not every researcher agrees that the LDL particlenumber should routinely be measured in cardiologypatients. Can you comment on the conflicting ideas?

It’s not unusual for doctors to disagree about the bestapproach to patient care; that happens in all areas of medicine.

A great deal of research has demonstrated that measur-ing the LDL particle number is better than simply lookingat the LDL cholesterol for identifying and managingpatients’ lipid abnormalities. Some researchers have arguedthat it’s not cost-effective to measure the LDL particle num-ber in large numbers of people over many years. I doubt thatthis is true, but we practicing physicians treat our patientsindividually – we care about the Mr. Jones or the Ms. Smithwho is in the office now, and deserves the best possible care.I want to be able to look all of my patients in the eye andtell them that I have given them the best possible care.

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4JANA Vol. 10, No. 1, 2007

Headway is expected on improved dietary supplementregulation this year as the International Alliance of DietarySupplement-Food Associations (IADSA) expands its planof action regionally and globally. This year will be a keyyear in the process of regional harmonisation in South EastAsia. IADSA continues to work closely with the AseanAlliance of Health Supplement Associations (AAHSA),which last December gained a place at the government tablefor negotiations on the future harmonisation of supplementregulation across the region.

Plans are also in the pipeline for regulation discussionswith China and Japan, and IADSA is organizing an Aprilworkshop in Yokohama where influential legislators fromAsia, Europe, and North America will address existing andfuture models of regulation.

In India work is underway on priority action followingthe country’s adoption of the Food Safety and Standards Act2006, which now classifies supplements as food. IADSA isalso in discussions with the Mexican authorities and theindustry association, Anipron, on the development of pro-posals for new regulation.

The Alliance’s global drive to develop strategy andimplement action on regulation and policy continues to playan important role particularly regarding CodexAlimentarius initiatives on additives, health claims, and thesafety of supplement ingredients.

"We continue to work towards regulatory systems wherethe guidelines are appropriate to the specific characteristicsof our products," said Randy Dennin, Chairman of IADSA."If we are able to achieve this, governments will havereduced healthcare costs and consumers will have wideraccess to safe and beneficial products. We will continue towork with national associations, the scientific community

G L O B A L U P D A T E

International Alliance of Dietary Supplement-Food Associations (IADSA)

Sets Agenda for 2007

and governments worldwide towards a positive result."

IADSA has more than doubled in size since its creationin 1998 as a voice for the worldwide dietary supplementmanufacturing industry, and the growing alliance now rep-resents 57 national trade associations.

On the research front, two groundbreaking publica-tions are being drafted by IADSA’s Scientific WorkingGroup –"Nutrition, Healthy Ageing, and Public Policy"–which addresses the value of dietary supplements in theageing population, and a second report on the safety ofbioactive substances in dietary supplements focusing onamino acids. IADSA’s reports are sent to regulators andother decision-makers in more than 100 countries, withsome translated into Spanish and Japanese.

For more information contact

David Pineda Ereño, IADSA Manager,

Regulatory Affairs, 50 Rue de l’Association,

1000 Brussels, Belgium.

Tel: +32 (0)2 209 1155, fax: +32 (0)2 223 3064

or email: davidpineda@iadsa.be

5 JANA Vol. 10, No. 1, 2007

ConsumerLab.com, White Plains, NY, has released theresults from their "Survey of Vitamin and SupplementUsers," which revealed that 77% of consumers surveyedreport being highly satisfied ("extremely" or "very" satis-fied) with the brands of dietary supplements that they use.An additional 21% were "somewhat" satisfied, and veryfew (2%) were dissatisfied. Satisfaction was also high withthe merchants from which supplements were purchased,with 71% of respondents being highly satisfied.

The most common place to purchase supplements wasonline, with 40% of respondents reporting an online pur-chase within the past year. This was followed by health foodstores (34%), vitamin stores (27%), pharmacies (25%),warehouse clubs (23%), catalogues (20%), supermarkets(18%), independent distributors (18%), mass merchants(18%), and health care practitioners (9%).

The following brands and merchants received the high-est overall satisfaction rating within their market segment.Rankings are based on the percent of respondents highlysatisfied.

Nutrilite, a brand marketed by independent distribu-tors, received the highest overall satisfaction rating both asa brand and merchant. However, sample bases for othercompanies within this category were not large enough to becomparatively ranked.

The survey was based on responses from 4,181 sub-scribers to ConsumerLab.com's e-newsletter. Respondentswere frequent users of supplements, with more than 90%reporting the use of two or more supplements each day.Twenty-four percent of respondents used 10 or more sup-plements per day. Ratings were given for several hundredbrands and merchants. Among these, 33 brands and 21 mer-

chants were included in the rankings for each havingreceived at least one hundred consumer ratings. The resultsare based entirely on consumer perceptions in November2006 and are separate from the laboratory findings ofConsumerLab.com.

N U T R A C E U T I C A L N E W S

ConsumerLab.com Survey Identifies Brands and Merchants Rated Highest

by Dietary Supplement Users

Survey ResultsTop-Rated Supplement Brands

Brand in Health Food Stores: Carlson

Brand in Mass Market Stores: Nature Made

Catalogue Brand: Puritan's Pride

Discount and Wholesale Club Brand: Kirkland (Costco)

Healthcare Practitioner Brand: Thorne Research

Pharmacy Brand: CVS

Vitamin Store Brand: Vitamin World

Top-Rated Supplement Merchants

Catalogue: Puritan's Pride

Discount and Warehouse Store: Costco

Grocery Store: Whole Foods

On-line Retailer: Vitacost.com

Pharmacy: Walgreens

Vitamin Store: Vitamin Shoppe

JANA Vol. 10, No. 1, 2007 6

N U T R A C E U T I C A L N E W S

Vitamin DFortification Needs to

be Considered

Wholegrain BreakfastsLinked to Lower

Heart Failure RiskResearchers are insisting that the upper limits for vitamin D

content in dietary supplements need to be increased, andthat foods need to be fortified with the nutrient.

A new University of Pittsburgh Schools of the HealthSciences study found 92.4 percent of African-American new-borns and 66.1 percent of white babies studied had insufficientvitamin D levels at birth. The study, which appears in theMarch issue of the Journal of Nutrition, evaluates data from200 black women and 200 white women, randomly selectedbetween 1997 and 2001.

The researchers found that more than 80 percent of theAfrican-American participants and nearly half of the whitewomen tested at delivery had levels of vitamin D that were toolow, despite the fact that over 90 percent of them had takenprenatal vitamins during pregnancy.

"Either more foods should be fortified, or we need toencourage supplementation of certain populations, especiallypregnant women," according to lead study author Dr. LisaBodnar. "The amount of vitamin D in supplements isn't nearlyenough," according to Dr. Robert Heaney, a professor atCreighton University School of Medicine who has conductednearly two decades worth of research on vitamin D. "Our bestestimate is that the body uses 4000 iu per day, and the dietaryreference intake for women up to the age of 50 is 200 iu perday," says Heaney. Now that vitamin D deficiency has becomebetter documented and researched, Heaney said there is nomore excuse to wait for fortification.

"The principle obstacle has been we haven't known howmuch vitamin D we have needed until the past two years," saidHeaney. "We're just starting to understand the implications."Dr. Heaney feels that securing widespread vitamin D fortifica-tion via the United States Food & Drug Administration is toolong of a process to wait for. Instead, the change will have tocome from industry itself.

"I think we'll see voluntary fortification," said Heaney."But the first thing that needs to be done is to raise public andprofessional awareness."

"We are working on a lot of research in the area of vitaminD," said Dr. Bodnar. "We are focused on understanding the con-sequences of vitamin D deficiency for mothers and their infants."

Heaney disagrees that significantly raising upper limits ofvitamin D, or widespread fortification could be accompaniedby health risks. "You have to go well above 10,000 iu per dayto get into unsafe levels," said Heaney. Heaney's voice on thesubject adds to several that have already been raised in favourof increasing vitamin D intake.

New report from Harvard shows that a bowl of wholegrain cereal daily could reduce the risk of heart

failure by up to 28 per cent.

In an epidemiological study of 10,469 cereal-eatingphysicians taking part in the Physicians' Health Study, thosewho ate two to six servings of wholegrain breakfast cerealsweekly reduced their risk of heart failure by 22 per cent. Theresearch, presented at the American Heart Association's 47thAnnual Conference on Cardiovascular Disease Epidemiologyand Prevention, adds to an already strong body of evidencelinking the consumption of wholegrain products to improve-ments in cardiovascular health.

"There are good and powerful arguments for eating awholegrain cereal for breakfast," said lead author Luc Djoussé,from Brigham & Women's Hospital and Harvard MedicalSchool. "The significant health benefits of wholegrain cerealare not just for kids, but also for adults. A wholegrain, high-fibre breakfast may lower blood pressure and bad cholesteroland prevent heart attacks."

Djoussé and Michael Gaziano calculated that eating sevenor more servings per week was associated with a 28 per centreduction in the risk of heart failure, while eating two to sixservings per week was associated with a 22 per cent risk reduc-tion. Eating only one serving per week reduced the risk ofheart failure by 14 per cent, they said.

Whole grains have received considerable attention in thelast year, especially in the US where the FDA permits foods con-taining at least 51 percent whole grains by weight and are low intotal fat, saturated fat, and cholesterol to carry a health claim link-ing them to a reduced risk of heart disease and certain cancers.

The term wholegrain is considered to be more consumer-friendly than the term fibre, which leads some manufacturersto favour it on product packaging since it is likely to strikemore of a chord of recognition for its healthy benefits.

The new study used food frequency questionnaires toassess the consumption of wholegrain and refined grain cere-als and related this to the incidence of heart failure from 1982to 2006. Of the 10,469 physicians (average age 53.7) whoreporting cereal consumption at baseline, 8,266 (79 per cent)ate wholegrain cereals compared to 2,203 (21 per cent) whoate refined cereals.

Further study is required to confirm these findings, withmechanistic studies needed to elucidate exactly how the grainsmay offer protection against heart failure.

JANA Vol. 10, No. 1, 20077

C O N F E R E N C E R E P O R T

Folate: A Key to Optimal Health Throughout the Lifespan

Lynn B. Bailey, PhD, Professor, Food Science and Human Nutrition Department, University of Florida,

Institute of Food and Agricultural Sciences

Proceedings Report from the American Nutraceutical Association’s Fall 2006Conference held in Memphis, Tennessee, October 21, 2006

Dr. Bailey’s research area of expertise is folate metab-olism, estimation of folate requirements and factors thatinfluence disease and birth defect risk, including geneticpolymorphisms. Dr. Bailey has conducted human metabol-ic studies over a period of 25 years, generating data that hasbeen instrumental in establishing new dietary intake recom-mendations for individuals throughout the lifecycle, includ-ing pregnant women and the elderly. Dr. Bailey has pub-lished more than 100 scientific journal articles and bookchapters and has edited a book entitled Folate in Health andDisease. She served as a member of the Institute ofMedicine’s Dietary Reference Intake committee for folateand other vitamins.

Dr. Bailey’s presentation at the conference examined thesources and function of folate and reviewed studies investi-gating folic acid effect’s on neural tube defects, vascular dis-ease, cancer, impaired mental function, and other ailments.The following report was prepared from her presentation andwritten by JANA Associate Editor, Barry Fox, PhD.

INTRODUCTION

Folate is a generic term that encompasses both the syn-thetic form of the vitamin found in supplements, and natu-

rally occurring food folate. Naturally-occurring concentrat-ed sources of food folate include orange juice, strawberries,vegetables such as spinach and asparagus, and legumessuch as black beans. As a general rule, the darker the greenleaf, the higher the folate content. The term “folate”includes the folic acid added to enriched food productssince 1998, as mandated by the FDA.

The chemical structure of folate from food sources issomewhat more complex than the structure of folic acidfrom supplements. Folate from food sources is a relativelycomplex structure with a polyglutamate side chain contain-ing molecules of the amino acid glutamate hooked together.When we consume foods with folate, enzymes in our intesti-nal tract cleave off this side chain, amino acid by amino acid.What we actually take up in our intestinal tract is the monog-lutamate form, with its single amino acid. Synthetic folate isthis monoglutamate form, which frees the intestinal tractfrom having to cleave to the simplified form.

We know that food folate is less bioavailable than syn-thetic folic acid. Does this mean that folic acid–the syn-thetic form–functions differently than folate, the foodform? The answer is that, once absorbed by the body, thetwo forms function the same.

8JANA Vol. 10, No. 1, 2007

Folate’s Function

Folate’s basic biochemical role is to serve as a coen-zyme for one-carbon transfer reactions. A key example ofthis is DNA synthesis. DNA methylation, which is verymuch involved with gene regulation, is dependent on folate,and a number of amino acids are dependent on folate forthese one-carbon transfer reactions (Figure 1).

Folate Requirements

How much folate is required? Or, how low can dietaryfolate fall before we develop hypomethylation? Dr. Baileyinvestigated the dosage issue in her laboratory at theUniversity of Florida in controlled metabolic studies inwhich human subjects were fed defined amounts of dietaryfolate. In the study represented in Figure 2, the volunteerswere fed a low-folate–but not severely folate–deficient–dietfor seven weeks. DNA methylation was measured at base-line, and again at seven weeks. These measurements weredone by giving the volunteers a radio-labeled methyl com-pound, which yields an inverse relationship with the incor-poration of the radioactive methyl group into the DNA. Inother words, the higher the tretiated-methyl-group accep-tance, the fewer native methyl groups are attached to theDNA. At baseline (Figure 2), there’s adequate, normal DNAmethylation. After seven weeks on the low-folate diet, therewas more radioactive methyl group incorporation. Thismeans that at seven weeks on a low-folate diet, the DNAwas hypomethylated. Research findings have linked folateto normal cell growth.

We know that red blood cell division is dependent onfolate, and that folate-dependent red blood cells provideoxygen and energy to the body. Folate also plays a key rolein immune system health by helping to maintain adequatenumbers of white blood cells. The lifespan of a white blood

cell is relatively brief (21 days), and leucopenia–reductionin white blood cells–can develop with a very short-termfolate-deficient diet. Indeed, Herbert’s 1962 study showedthat folate depletion can lead to leucopenia in just twenty-one days. The practical significance for clinicians is that ashort-term folate deficiency can lead to impairment ofimmune response.

Neural Tube Defects

One major public health finding is that folic acidreduces the risk of neural tube defects by up to 70%. Thisconclusion is based on a large body of scientific evidence,and is summarized in Figure 3.

The bottom half of the table shows the results of keyobservational studies in which the percent reduction in riskfell by 60–75% in women taking a multivitamin containingfolic acid during the periconceptual period–right before preg-nancy–through that first 28-day period. The upper half of thetable shows interventional studies in which a folic acid sup-plement was given. The gold-standard, placebo-controlledintervention trial is this MRC study, which resulted in a 72%reduction in risk with folic acid alone. This definitive studyled to the public health recommendations. However, becausea very high dose of folate was given in the MRC study–4,000mcg per day–we didn’t know the optimal dose.

China Study

A study conducted in China and published in 1999 gaveus the answer: 400 mcg, the amount found in most multivi-tamin supplements, is an effective dose for reducing neuraltube defect risk. The bar graph in Figure 4 shows the neuraltube defect rate per 1,000 births. The left side shows results

Figure 1.

9 JANA Vol. 10, No. 1, 2007

Figure 2.

Figure 3.

Rampersaud, Bailey et al. 2000

from northern China, where there is severe folate deficiency.Against this background of high folate deficiency, 400 mcgof folate produced a huge drop, an 85% reduction in neuraltube defects. The right side of the graph shows results fromthe south of China, which produces more vegetables and hasa longer growing season. There, 400 mcg folate reduced therisk of neural tube defects by 41%.

Intake Recommendation

Based on research such as this, the U.S. Public HealthService and the National Academy of Science published apublic health recommendation that all women capable ofbecoming pregnant should take 400 mcg of folic acid everyday, in addition to eating a healthy diet. Luckily, 400 mcg isthe amount found in most regular multi-vitamin supplements.

U.S. Public Health Service & Institute of Medicine, NAS

All women capable of becoming pregnant shouldtake 400 micrograms folic acid every day in additionto folate from a varied diet. However, in a consumerawareness and behavior study, only 26% of womenreported getting information about folic acid from

their health care providers.

JANA Vol. 10, No. 1, 2007 10

Do Women Consume Adequate Levels of Folic Acid?

About 30% of women of reproductive age take folicacid-containing vitamins, leaving 70% who do not. Whatwould motivate them to change their behavior? A Gallup Pollsurvey indicates that women would change their behavior iftheir health care provider recommended they do so. Whenwomen who had been informed about the value of takingfolic acid every day were questioned, only about 25% report-ed getting that information from their health care provider.

In addition to its public health recommendation, theFDA mandated that certain foods be fortified with folicacid; bread and other cereal grain products have been forti-fied since 1998. Has the fortification program done anygood? The scientific literature suggests a potential for up toa 70% reduction in neural tube defects, but we’ve only seena 26% reduction in the US. We have a ways to go, and weneed to rely on taking folic acid supplements (Figure 5).

FOLATE AND HEART DISEASE

We have data that both support and refute the hypothe-sis that folic acid is protective or preventive against chronicdisease. So we need to think of it as a scale and weigh theevidence to come up with a definitive answer. Knowing thatheart disease is the primary cause of death among women inthe US, Dr. Bailey is interested in homocysteine, and theability of folic acid to reduce homocysteine as a risk for vas-cular disease.

Figure 4.

Berry et al. 1999

Data from the Nurses Health Study found an inverseassociation between folate consumption and heart disease. Asthe graph in Figure 6 shows, the risk of heart disease in thehigher quintile of folate intake (that intake included supple-ments) was significantly less than in the lower intake groups.

Relative Risk for Coronary Heart Disease Associatedwith Elevated Homocysteine

The relationship between folate and homocysteine, anon-protein-forming amino acid, may help explain this asso-ciation. Homocysteine is associated with an elevated risk forheart disease. As homocysteine blood levels go up, the risk ofcoronary heart disease also increases. Figure 7 summarizesthe conclusions of cross-sectional, case control, and prospec-tive studies (Archives of Internal Medicine, 2000) looking atthe relative risk of coronary heart disease associated with ele-vated homocysteine. When the dot on the horizontal lines ishigher than one–that is, to the right of vertical line, the answeris yes, elevated homocysteine was associated with anincreased relative risk of coronary heart disease in that study.Study consensus is that the higher the homocysteine level, thegreater the risk for coronary heart disease.

Folate Needed for Normal Homocysteine Levels

What is the link between homocysteine and folate?Folate provides the methyl group that converts homocysteine

JANA Vol. 10, No. 1, 2007 11

to methionine. The only difference between homocysteineand methionine is the one methyl group that folate provides.Inadequate folate levels will prevent the conversion ofhomocysteine to methionine, and blood levels of homocys-teine will build up. Hundreds of studies have shown that thiselevation in homocysteine can be atherogenic.

Dose Required for Homocysteine Reduction

How much folate would have to be consumed to keephomocysteine blood levels in normal concentration? Dr.Bailey conducted a study with women between the ages of 65and 85, chosen because heart disease is the number one killerof women in the US today. The volunteers were fed controlled

Figure 5.

Figure 6.

Effect of Folic Acid Fortification on Neural Tube Defects in US

• Only ~ 26% in neural tube defects• Reduction much less than 50-70% with folic

acid supplements indicated in scientific literature

diets containing different amounts of folate over a 70-dayperiod. First they were given a folate-depletion diet, and thenthe amount of folate was increased to see how much wasrequired to normalize homocysteine levels. Two hundredmicrograms–an amount close to the old RDA of 180 mcg perday–was not enough to raise their homocysteine levels.However, 400 mcg, the amount in a typical multivitamin sup-plement, which is the current recommendation, brought thehomocysteine levels down significantly (Figure 8).

HOMOCYSTEINE AND VASCULAR DISEASE

The link between folate, homocysteine, and vasculardisease has led to the idea that folate might also help reduce

Nurses Health Study Rimm et at. JAMA 1998

JANA Vol. 10, No. 1, 2007 12

the risk of stroke. Unfortunately, several intervention stud-ies have shown that folic acid does not appear to reduce therisk of recurrence, once vascular disease has been estab-lished. But what is the effect of increased folic acid on vas-cular disease in the general population? An answer comesfrom the Centers for Disease Control, which evaluated theeffects of folic acid from fortification on a reduction in therate of mortality due to strokes in Canada.

The bar graph in Figure 9 shows the changes in serumfolate levels in men ages 40 to 59, men aged 60+, womenaged 49 to 59, and women aged 60+. Folic acid fortificationhas had a major impact on folate status in the US, across allage categories. As the bar graph in Figure 10 shows, folicacid fortification also brought down plasma homocysteinelevels. The drop is significant, as indicated by the asterisks,across all four groups. This increase in folic acid and fall inhomocysteine levels was accompanied by a reduction in therate of mortality due to strokes. The weight of the evidenceindicates that disease prevention–that is, consuming ade-quate folate over a long period of time–is the primary key.

Folate and Mental Function

Both low folate and high homocysteine are associatedwith impaired cognitive function, dementia, and Alzheimer’s.Figure 11 shows a data slide from the classic Nun’s Study

(Snowdon et al., 2000). As part of this study, Snowden andcolleagues looked at autopsy samples from nuns who haddied from Alzheimer’s disease, and for whom they had long-term blood level data. This figure compares serum folate lev-els to the severity of brain atrophy. When the blood levelswere low, there was severe atrophy; when the blood levelswere higher, there was none.

A controlled intervention trial conducted by Dr. J.Durger (who provided Dr. Bailey in-press data) examinedthe relationship between folate and cognitive function(Figure 12). Dr. Durger and her fellow investigators con-ducted a three-year, double-blind, placebo-controlled inter-vention trial with 818 men and women between 50 and 70years old. All had elevated homocysteine and normal B12

levels. Researchers assessed the volunteers’ mental functionat baseline and after three years of either 800 mcg of sup-plemental folic acid daily, or a placebo. They found a sig-nificant elevation in blood folate, and a 26% drop in homo-cysteine in the folic acid group. The more exciting result wasa significant improvement in memory, information process-ing speed, and sensorimotor speed in the same group.

On the other hand, other studies have not found posi-tive results. More research data is needed to arrive at adefinitive conclusion.

Figure 7. Figure 8.

Relative Risk Coronary Heart DiseaseAssociated with Elevated Homocysteine

Relative Risk (95% CI)Arch Intern Med 2000 Kauwell et al. 2000

13 JANA Vol. 10, No. 1, 2007

Figure 9.

Figure 10.

Yang et al. Circulation 113:1335, 2006

Yang et al. Circulation 113:1335, 2006

14JANA Vol. 10, No. 1, 2007

Intake Recommendations

How much folate is required to maintain good health?The bottom line is 400 mcg per day, the most recent DietaryReference Intake suggested by the Institute of Medicine.(This figure is given in dietary folate equivalents, a unit usedto convert the more bioavailable synthetic form to equivalentdietary folate.) The 400 mcg of dietary folate is the recom-mendation for an adult. A separate recommendation forwomen of childbearing age suggests they take a folic acidsupplement in addition to the 400 mcg per day dietary intake.

The Institute of Medicine determined that folic acid isnon-toxic, and set an upper limit of 1,000 mcg per day. Whyan upper limit, when folic acid is non-toxic? This issueaffects people who are deficient in vitamin B12, for whomfolic acid can mask megaloblastic anemia and delay itsdiagnosis, hence the basis of the 1,000-mcg upper limit.

CONCLUSION

Folate is a key to optimal health. We’ve seen evidenceof its benefits with regard to neural tube defect reduction.We’ve seen evidence, pro and con, relating to folic acid’seffects on vascular disease, cancer, and impaired mentalfunction. While there is conflicting data regarding its affectson chronic disease, data overall suggest that chronic pre-vention, not short-term intervention, is the key.

Healthcare professionals can modify behavior for thebetter by encouraging their patients to consume folate-densefoods during their entire lifespan. Women who could becomepregnant should be encouraged to take a folic–acid–contain-ing supplement, and women who drink alcohol shouldincrease folate intake to reduce breast cancer risk.

Figure 12.

Figure 11.

Effect of Folic Acid on Mental Function

• Folic acid intervention for 3 years– 800 mg/day or placebo (double-blind)

• Participants: 818 post-menopausal women(50-70 years) and men– homocysteine ≥13 µmol/L– serum B12 ≥200 pmol/L

• Mental function assessed at beginning andend of study

• Serum folate 5-fold in folic acid group• Plasma homocysteine 26% in folic acid

versus placebo group • Folic acid significantly improved several

cognitive functions that tend to decline withage : memory, information processingspeed, and sensorimotor speed

Durga J et al. In Press Lancet 2007

• Folate non-toxic• Folic acid at high doses may delay diagnosis of

vitamin B12 deficiency• Tolerable Upper Intake Level (UL) 1,000 mg/day

folic acid– "masking" B12 deficiency by correcting hematological abnormalities

Institute of Medicine, 1998

Snowdon et al. 2000

15 JANA Vol. 10, No. 1, 2007

C O N F E R E N C E R E P O R T

Lowering LDL and Total Serum Cholesterol: An Overview of Drug and Dietary Therapies,Including the Portfolio Diet and Its Impact on

Lipids and Hypertension

Proceedings Report From the American Nutraceutical Association’sFall 2006 Conference Held in Memphis, Tennessee, October 21, 2006

David J.A. Jenkins, MD, PhD, DSc. Professor, Dept. of Nutritional Sciences Director, Clinical Nutrition and Risk Factor Modification Center

St. Michael's Hospital, University of TorontoCanada Research Chair in Metabolism and Nutrition

Dr. Jenkins is credited with developing the concept ofthe glycemic index as a way of explaining how dietary car-bohydrate impacts blood sugar. His first paper on the sub-ject appeared in the March edition of the American Journalof Clinical Nutrition, 1981. His most recent publicationdeals with the Portfolio Diet. The results of his most recentstudy were published in the March 2006 edition of theAmerican Journal of Clinical Nutrition, and showed thatpeople who ate a diet rich in cholesterol-lowering foods fora year lowered their cholesterol levels by 20% or more, areduction comparable with that achieved by taking statins.Dr. Jenkins’ presentation at the ANA Conference examinedhis work in studying the role of diet and drug therapies inmanaging lipids and hypertension. JANA Associate Editor,Barry Fox, PhD, prepared this report on his talk.

INTRODUCTION

The statin intervention trials have had an immenseimpact on the practice of medicine. Perhaps the most suc-cessful class of drugs ever launched, statin sales commandan enormous share of the market. Statin trials are large, from6,000 participants up to the 20,000 randomized in the HeartProtection Study. Although the study results don’t entirelyagree with one another, their protocols tend to produce sim-

ilar reductions in LDL cholesterol. As the third column inFigure 1 indicates, the protocols yield 26 to 35% reductionsin LDL cholesterol. These studies were conducted with first-generation statins. Rosouvastatin and future generations ofstatins may produce a 60%+ drop in LDL cholesterol. Inexisting studies, we usually (but not always) see 26–30%fewer cardiovascular events as a result of statin intervention.

Dr. Jenkins felt we can’t get all the cardiovascular pro-tection we need in a nutraceutical poly-pill, so he decidedto see if he could gather all the good things possible fromthe plant kingdom into a risk management portfolio, simi-lar to a financial portfolio. We will return to this soon.

The ATP III Revision Committee has updated the guide-lines for cholesterol management (Figure 2). They conclud-ed, in light of recent statin trials, that people at very high riskshould have an LDL cholesterol of less than 70 mg/gL.

Rats tend to have cholesterol levels in this range, and,in general, rats don’t develop heart disease. If we were morelike rats, Dr. Jenkins noted, we’d be better off. When welook at animals genetically closest to us – the gibbon,orangutan, gorilla, and chimpanzee – we see that a lot oftheir metabolic machinery is similar to ours, including thedigestive tract. The gibbon, orangutan, and gorilla eat ahigh-fiber vegetarian diet, while the chimpanzee eats a

16JANA Vol. 10, No. 1, 2007

high-fiber omnivorous diet. The human is the odd “man”out. Humans eat a low-fiber omnivorous diet, tendingtoward a higher animal-produce diet. This change in thehuman diet is relatively recent, evolutionarily speaking, andwe have not evolved accordingly. In a sense, there is a dis-connect between our genetics and our dietary habits. That iswhy we need statins—or a modified diet.

Initial Study

Dr. Jenkins and his colleagues performed a study(Figure 3) to see how blood lipids would respond to dietscloser to what, presumably, we ate in the early stages ofhuman evolution For this study, a group of volunteers fol-lowed three different diets:

• a low-fat, therapeutic (NCEP Step II) diet, with five serv-ings a day of low-fat dairy, white rice, potato, fruit andvegetables.

• an early Stone Age, Neolithic, high-fiber starch-based diet,with 5 servings a day of low-fat yogurt, whole grains,lentils, fruit and vegetables.

• a Simian diet based on what humans presumably atebefore they mastered fire or developed stone implements,with 63 servings a day of fruit, vegetables and nuts(almonds and hazelnuts). Sixty-three servings wererequired for the subjects to maintain their weight. Thiswas not a low-protein diet: the total protein intake was 93grams. Total dietary fiber was 143 grams, with 1 gram ofphytosterols and about 70 grams of nuts per day.

The study was short in duration because it is difficult toget people to follow these diets for long periods of time.

The Step II diet produced a 5 to 10% reduction in LDLcholesterol, as seen in the first curve on the left side of Figure4. The second curve, representing the Neolithic Diet, quicklyfell to about a 23% reduction in LDL cholesterol. The thirdcurve, representing the Simian diet, produced almost a 35%reduction with no weight change. These results confirmed Dr.Jenkins’ belief that our biochemistry is out of sync, geneti-cally speaking, with our modern eating habits.

Health Claims Allowed by FDA for CHD Reduction

The FDA currently allows certain foods to make aclaim for CHD risk reduction: vegetable protein (soy), oatbran and other viscous fibers, nuts, and a provisional claimfor phytosterols (Figure 5). These same components wereparticularly notable in Dr. Jenkins’ Simian Diet, and theyhave different mechanisms of action. The viscous fiberstend to increase bile acid losses; soy protein tends to reducecholesterol synthesis and increase LDL receptor uptake;plant sterols reduce cholesterol absorption; while almondscontain antioxidants, monounsaturated fats, some plantsterols and vegetable protein, and work by multiple mecha-

Statin Intervention Trials:Cholesterol Lowering & CVD Prevention

Trial N % drop in Events VersusLDL-c Placebo

WOSCOPS 6,595 26% MI 30%pravastatin CHD-DEATH Fewer events

AFcaps/TEXcaps 6,605 25% MI 36%lovastatin CHD-DEATH Fewer events

PROSPER 5,804 34% CHD-DEATH 13%pravastatin MI Fewer events

CVA

HPS 20,536 35% Non-fatal MI 26%simvastatin CHD-DEATH Fewer events

Figure 1.

NCEP ATP III UpdateIn light of recent statin RCTs

People at Very High Risk:LDL-C < 70 mg/gL < 1.8 mmol/L

Grundy et al., Circulation 2004

Figure 2.

17 JANA Vol. 10, No. 1, 2007

nisms. This combination of foods effectively gives one acholestyramine analogue, a very mild sort of statin, an eze-timibe (simvastatin) type of product, and a sort of poly-pillin the form of a nut.

Portfolio Diet Study

The good results seen with the Simian Diet, plus the dif-ficulty of following such a diet in the modern world, led Dr.Jenkins to consider a portfolio approach, a diet containingmany foods readily available in the supermarket. Dr. Jenkinsand his colleagues did a series of studies on this portfolio diet.In the third study, Figure 6, they compared an older statin – 20mg lovastatin – to the Step II control diet and the Portfolio

Diet. They used lovastatin because the study was performedduring the Baycol scare. Baycol is the statin removed from themarket because it increased rhabdomyolysis; some study par-ticipants responded by being wary of statins.

Portfolio Diet Study Results

The results were as expected (see Figure 7). The 30%reduction in LDL cholesterol on the Portfolio diet was sim-ilar to the reduction on the statin drug, and superior to the9–10% reduction on the control diet. This happened quick-ly: within two weeks a physician should be able to seewhether the diet is working or not, assuming the patient issticking to the protocol.

Serum Lipid Response to a Diet Very High in Fiber fromVegetables and Fruit (Simian Diet)

• Low-fat therapeutic diet (NCEP step 2)low-fat dairy, white rice, potato, fruit & vegetables (5 servings/d)

• High-fiber starch-based (Neolithic)low-fat yogurt, whole grains, lentils, fruit & vegetables (5 servings/d)

• High-fiber vegetable-based (Simian)63 servings/d fruit 7 vegetables, nuts (almonds and hazelnuts)

Jenkins DJ, Kendall CW et al. Metabolism; 2001

Figure 3.

Figure 4.

Serum Lipid Response to a Diet Very High in Fiber fromVegetables and Fruit (Simian Diet)

Jenkins DJ, Kendall CW et al. Metabolism; 2001

18JANA Vol. 10, No. 1, 2007

The effects on C-reactive protein were surprising. Thestatin lowered C-reactive protein, but so did the PortfolioDiet, and to a similar extent (Figure 8). Dr. Jenkins foundthe same positive effects on C-reactive protein when he test-ed the diet in a larger series.

Portfolio Diet – Long Term Study

While the short–term studies were interesting, theyraised a question: What happens in the long run? With theshort-term diet, all food was prepared and packed in meta-bolic kitchens and shipped to the subjects, which made iteasier for them to follow the protocol. But can people livingin the “real world,” doing their own shopping and food

preparation, stick to the protocol over time? To find theanswer, Dr. Jenkins and his colleagues conducted anotherstudy – a year-long, open-label, non-randomized effective-ness study in which the participants were given dietaryadvice to follow the Portfolio Diet (Figure 9).

Figure 10 shows what happened to the volunteers’blood lipids during the study. The top line is the HDL cho-lesterol, which rose significantly over the year. The secondline is the LDL cholesterol, which fell dramatically in thefirst three or four weeks, rose, and then remained flat atabout a 15% reduction from baseline. Dr. Jenkins noted thatthe peak in the LDL line around week 16 was due to poor

Figure 5.

Current FDA Health Claimsfor CHD Risk Reduction

Vegetable Proteins• Soy

Viscous FibersPhytosterols• Oat β-glucan • Sterols• Barley β-glucan • Stanols• Psyllium

Nuts (almonds)

Figure 6.

Dietary PortfolioStudy Foods: Readily available in supermarkets.

Nuts: ~ 30 g/dalmonds

Viscous Fiber: ~ 20 g/doats, barley, psyllium, legumes, eggplant, okra

Vegetable Protein: ~ 80 g/d (50% soy)soy, beans, chick peas, lentils

Plant Sterols: ~ 2 g/dplant sterol margarine

Jenkins DJ, Kendall CW et al. Metabolism; 2002

Figure 7. Figure 8.

Dietary Portfolio Study #3: ResultsChanges in LDL-C

Dietary Portfolio Study #3: ResultsChanges in C-Reactive Protein

Jenkins DJ, Kendall CW et al. Metabolism; 2003 Jenkins DJ, Kendall CW et al. Metabolism; 2003

JANA Vol. 10, No. 1, 200719

eating habits at Christmas, which, curiously, didn’t makemuch difference in serum triglyceride levels, which fell overthe course of the study.

The net results of this study were about 50% of whatDr. Jenkins had seen in the studies performed under meta-bolic conditions. This means that about a third of the peopledid as well as they had done on a statin drug, a third did halfas well, and for the last third, nothing happened. Couldthese results be due to genetic differences in the volunteers?Half of the people in this year-long, “free living” study hadpreviously been on the metabolic diet, so Dr. Jenkins reex-amined their data and concluded that while genetics maymodulate the results, the really big issue is compliance.

When food is provided, people do well; when people areadvised how to prepare their own food and do so, theyrespond variously. On the positive side, a third of the peo-ple in this group managed the diet by themselves for a yearand did quite well. In fact, some of Dr. Jenkins’ volunteershave been on the diet for three years.

This long-term study also monitored the effect on bloodpressure, as well as C-reactive protein. Figure 11 shows theperiodic blood pressure readings for study volunteers whomaintained their weight, lost a modest amount of weight andhad a large weight loss. Systolic blood pressure tended to bereduced, and diastolic to some extent, with or without weightloss. The intermediate group – showing a modest weight loss

Figure 9.

Long Term Self–Selected PortfolioDietary Portfolio Study #4: Methods

Study Design:Non-randomized effectiveness study.

Duration:1 year plus.

Intervention:Dietary advice to follow the Portfolio Diet.

Jenkins DJ, Kendall CW et al. Am J Clin Nutr 2006

Figure 10. Figure 11.

Dietary Portfolio #4Blood Pressure Changes over

24 Weeks (n=65)

Long Term EffectivenessBlood Lipid Changes over

52 Weeks

Jenkins DJ, Kendall CW et al. Am J Clin Nutr 2006

20JANA Vol. 10, No. 1, 2007

over one year – had the same magnitude of blood pressurereduction as would be expected from the DASH diet.

SUMMARY

Dr. Jenkins concluded (Figure 12) that while statinshave their place, the use of cholesterol-lowering foods asrecommended by ATP III and to some extent, by AHA, mayin combination produce serum lipid reductions that bridge

the therapeutic gap between a generally good diet and statintherapy. There is a group of people who should work withdiet and lifestyle – plus exercise and nutraceuticals, a wholeportfolio of items – before going on drugs. Approximately30% of serious dieters can achieve a 20% LDL-C reductionin six months on a dietary portfolio of viscous fiber, plantsterols, and soy protein foods, together with almonds. Otherrisk factors for CHD may also be reduced, including bloodpressure and C-reactive protein.

Figure 12.

Summary from the Presentation Made by Dr. Jenkins

• Use of cholesterol-lowering foods as recommended by ATP III and AHA may in combination produce serum lipid reduction, which bridges the therapeutic gapbetween a generally good diet and statin therapy

• Approximately 30% of serious dieters can achieve a >20% LDL-C reduction in 6months on a dietary portfolio of viscous fiber, plant sterols, and soy protein foodstogether with almonds.

• Other risk factors for CHD may also be reduced, including blood pressure and C-reactive protein.

21 JANA Vol. 10, No. 1, 2007

P E R S P E C T I V E A R T I C L E

Preventive Cardiology, Our Greatest Hope for Eradicating Heart Disease

By Seth J. Baum, MD, FACCIntegrative Heart Care, Boca Raton, Florida

Member, ANA Medical Advisory Board

We physicians struggle daily to do what is best for ourpatients. We assiduously endeavor to stay current with theever-expanding volumes of medical literature, simultane-ously responding to our patients’ very real and immediatehealth issues. Journals multiply like viruses and studies fillthese journals. How do we digest all the data? How do wesift through divergent results and conclusions, and arrive atan approach that is reasonable and effective? How do wepractice evidence-based medicine when the evidence is soephemeral? On one hand, doctors tend to resist change,clutching their views like life preservers in a raging stormat sea. On the other hand, we can be fickle, latching on to asingle trial that jibes with our oft-times preconceivednotions and personal belief systems, the end result being adismissal of something that may be of immense value. Aperfect example of this latter phenomenon relates toHomocysteine. Elevated levels of Homocysteine haveclearly been associated with cardiovascular events – not tomention Alzheimer’s, osteoporotic fractures, maculardegeneration and stroke – yet a single negative trial in therealm of heart disease serves as a battle axe to theHomocysteine naysayers, which they weild to dismantle allprior literature. An example of the former phenomenonrelates to cholesterol. The powers that be, the doctors whoestablish guidelines such as NCEP and ATP 3, have heldtight to cholesterol as the answer to the cardiovascular

plague that afflicts the western world. Yet a thoroughreview of the major Statin trials (that utilize LDL-C, thecholesterol contained within LDL particles) teaches us thatcontrolling LDL-C eliminates only thirty or thirty-five per-cent of cardiovascular risk. What of the remaining sixty-five or seventy percent of events? If controlling LDL-C isTHE answer, how do we account for this massive remain-ing unmanaged risk? Clearly there is far more to this story.And this brings us to the concept of Prevention, anapproach to patient care that demands open-mindednessand at times, great inner strength.

The practice of Preventive Medicine, though a conceptthousands of years old, has recently enjoyed a resurgence.Perhaps it is the increasingly impersonal medical system orthe over-abundance of technologically-oriented aspects ofmedicine that has brought prevention to the fore in manycircles. Perhaps it is our patients’ clamoring for solutionsthat reach beyond the patchwork effects of medications andsurgery. Whatever the reason, prevention seems to be hereto stay. And when we look at some statistics as they relateto Cardiovascular Prevention – my particular area of inter-est – they reveal why prevention is so essential. Forty-twopercent of Americans still die from cardiovascular disease.That’s one American every thirty-three seconds. One and ahalf million heart attacks still occur annually in the UnitedStates. Sixty-four percent of us are overweight, while asolid thirty-three percent are, by definition, obese. A thirdof our country’s youth is now overweight and consequently,“Adult Onset” Diabetes Mellitus is becoming common-place in children. Without a concrete preventive approach,we will surely continue on the road to disaster. In theremainder of this article, I will address three tools that canbe incorporated in a clinical practice of PreventiveCardiology – LDL particle information, Coronary CTAngiography (CCTA), and assessment of the Carotid

* Correspondence:Seth J. Baum, MD, FACC2300 Glades Road, Suite 305EBoca Raton, FL 33431Phone: 561.367.8155 Fax 866.366.1269Email: sjbheart@vitalremedymd.com

22JANA Vol. 10, No. 1, 2007

Intima-Media Thickness (CIMT). Please do not infer thatthe absence of a discussion on Therapeutic LifestyleChanges (TLC) means that I undervalue exercise, dietaryinterventions, and stress modification. In fact, the oppositeis true. I believe wholeheartedly that TLC is the corner-stone of disease prevention. I also believe that in doingeverything humanly possible to try to stave off the numberone killer in the western world, we physicians should exam-ine and treat other important lipid components – HDL-Cand Triglyceride – as well as the “emerging cardiovascularrisk factors” such as CRP, Lp-PLA2, and Lp(a). My focuson the three aforementioned tools stems from an issue ofspace; it is just impractical to discuss all preventive tech-niques in this article. Also, follow-up case reports in thenext few issues of JANA will focus on the merits of thesethree tools. And so, let me now introduce these techniquesand their clinical applicability.

We have known for many decades that cholesterol playsa pivotal role in the genesis of atherosclerosis. In 1913,Anitshkow showed that cholesterol fed rabbits developedaortic atherosclerotic plaques, whereas sunflower oil fedrabbits did not. In the mid 1960s, Fredrickson, one of thefathers of Lipidology, emphasized that Lipoproteins (LDL,VLDL, HDL, IDL, and Chylomicrons), not the cholesterolcontained within them, should be the focus of our attention.Although he proclaimed that the preferred way to manageour patients would be to directly count these lipoproteins,the lack of a commercial means to do so led to the adopta-tion of LDL-C as a surrogate marker for LDL-P (the numberof Low Density Lipoprotein Particles), and thus a GoldStandard by default. Earlier I alluded to the inability of LDL-C to adequately predict CV events. Many studies have shownus this. Even the famed Framingham Trial found that half ofall heart attack victims have normal LDL-C levels, and eightypercent of premature CV events occur in individuals with anLDL-C under 125 mg/dl. From where does this LDL-C short-coming emanate, and why is LDL-P such a superior predic-tor? The answer to these questions lies in the nature of LDL(again, the particles that carry cholesterol).

It turns out that LDL particles vary greatly in both theirsize and the amount of cholesterol they contain.Consequently, there cannot possibly be a consistent anddirect relationship between LDL-C and LDL-P. One isunable to glean from LDL-C how many particles exist, andthe opposite holds true as well. This reality would be incon-sequential were it not for the fact that LDL particles arewhat penetrate arterial walls to cause vascular disease.These particles do not dump their cholesterol in the blood;they enter the intima-media as holoparticles and once insidethe arterial wall, they do their damage. In this circum-stance, as in many other aspects of medicine, gradientscome into play. The more particles there are in the blood-stream, the more likely they will be to invade the arteries.How much cholesterol they carry is not the issue; it’s howmany LDL particles there are that counts. This is why studyafter study has shown that LDL-P is far more effective atpredicting CV events than LDL-C. It is a better clinical toolby which we can manage our patients’ lipid abnormalitiesand prevent events. Though anecdotal, my management of

LDL-P has resulted in a dramatic decline in the number ofacute myocardial infarctions I see in my clinical practice.In fact, I cannot recall the last patient I saw who had anacute event with an optimally controlled LDL-P.

Coronary CT Angiography is a technology that fits wellin both medical camps – Preventive and Therapeutic. Ourability to image coronary arteries non-invasively throughhigh speed CT scanners has enabled us to detect early diseasein the vessel walls. The therapeutic implications abound, butfor now I’ll limit myself to the Preventive applications. Byseeing coronary artery disease directly, unequivocally, andfar sooner than either stress testing or cardiac catheterizationwould enable us to do, doctors can now aggressively attackrisk factors before the disease has progressed to a sympto-matic stage. We are all familiar with patients who resist ourattempts to manage their CV risks. The old adage, “A pictureis worth a thousand words” can be understood with crystalclarity when we present these resistant patients with imagesof their own diseased arteries. It is an unparalleled motivatorto see your own vessels clogging with plaque. Once thisalarming plaque accumulation is actually witnessed and theramifications of these findings fully explained (i.e., the veryreal risk and possibly imminent danger of stroke, MI, etc.), itis rare for patients to oppose making the appropriate health-ful changes in their lives.

Carotid Intima-Media Thickness can be utilized in afashion similar to the CCTA. By measuring the intima-media thickness, we can predict future cardiovascular andcerebrovascular events. In fact, in 2000, the AHA recog-nized IMT as an independent predictor of CV events, and in2006, the Screening for Heart Attack Prevention andEducation (SHAPE) Task Force recommended the use ofIMT in Low and Intermediate risk patients for improvedrisk categorization. The downside of CIMT is that it is onlya surrogate marker for coronary artery disease. UnlikeCCTA, it does not tell us with absolute certainty whether ornot a patient has CAD. However, because of the systemicnature of atherosclerosis, 70% of people with this disorderhave both coronary and carotid artery disease. This makesCIMT a very accurate predictor of the presence of coinci-dent coronary artery disease. The upside of CIMT is that itdoes not introduce radiation exposure or require the admin-istration of contrast. CIMT can be used not only as a meansof establishing a patient’s current CV risk (at times upgrad-ing low or intermediate risk patients to high risk), but alsoas a guide in managing our patients’ risk factors – whenintima-media thickness increases by more than 0.033 mmannually, we are alerted to the fact that much more needs tobe done from a preventive standpoint.

This article has been a brief introduction to three rela-tively recent advances in the world of preventive cardiology–LDL-P, CCTA, and CIMT. The next few issues of JANAwill include case reports that elucidate how internists, fam-ily practitioners, and cardiologists can utilize these tools tohelp prevent CV events. I am confident that by possessinggreater knowledge about our patients’ CV status, we will bemore likely to make real and permanent changes in ourpatients’ lives and by so doing, decidedly diminish theirrisks of succumbing to a CV event.

23 JANA Vol. 10, No. 1, 2007

O P I N I O N A R T I C L E

Migraine Prophylaxis: Comparable Effects orUnadjusted Effects That Could Be Misleading?

Review of a Study by Maizels et al. Published in

Headache: Journal of Head and Face Pain

Yuxin Zhang, PhD, XTiers Consulting, Inc., Potomac, Maryland

* Correspondence:Yuxin Zhang, PhDXTiers Consulting, Inc.9524 Woodington DrivePotomac, Maryland 20854Phone (240) 476-1784 Fax (301) 983-6307Email: yzhang@xtiers.com

Based on the findings from a randomized, double-blind,placebo-controlled trial (RCT) published in Headache:Journal of Head and Face Pain that evaluated the efficacyfor migraine prophylaxis, Morris Maizels et al. concludedthat Riboflavin 25 mg used in the trial as the placebo treat-ment showed an effect comparable to a combination of 400mg Riboflavin, 300 mg Magnesium and 100 mg Feverfew.1

Of the 49 patients who completed the 3-month trial, therewas no significant difference noted between the combina-tion drug group (n=24) and “placebo” group (n=25) for theprimary efficacy measure, a 50% or greater reduction frombaseline in monthly migraine frequencies, which wasachieved by 10 (42%) and 11 (44%) patients, respectively.With reference to the published data, Morris Maizels et al.concluded that the placebo response observed in this trialexceeded that of the placebo response found in any other tri-als of migraine prophylaxis, which was approximately 24%(95% CI: 18.3% to 28.8%), reported in a meta-analysis byVan der Kuy and Lohman.2

Were these really comparable effects or were theresome contributing factors Morris Maizels et al. did not con-sider? To the general public and research community, theconclusion provides very confusing information.3

This trial was originally designed to randomize 48patients per group for a statistical power of 80% to detect adifference of 30% in response rate at the significance levelof 0.05 (2-sided), based on an anticipated response rate of60% for the combination drug and 30% for the placebo.However, it did not employ a washout period prior to ran-domization for patients who might have been on migraineprophylaxis. Furthermore, the trial allowed patients to useTriptan medications, which are indicated for migraineheadache, throughout the study; and, patients could havechanged their Triptan doses over the course of the 3-monthtreatment. Considering the fact that Triptan medicationsreduce the migraine frequencies at a response rate rangingfrom 55% to 77%,4,5 the anticipated response rate of 60%was clearly a long shot for the combination drug in this trialthat actually had an add-on design; and consequently, thetrial was lacking adequate power.

As reported, patients in this trial used Triptan medica-tions on average 4.72 and 3.09 doses per month during thethird treatment month, respectively, for the “placebo” andcombination groups, whereas it was 4.2 and 3.3 doses permonth prior to randomization, respectively.1 In reference tothe baseline use, placebo-treated patients apparentlyincreased the monthly Triptan doses during the blinded

JANA Vol. 10, No. 1, 2007 24

treatment period, while patients on the combination treat-ment decreased the monthly Triptan doses. Relative to thepatients in the combination treatment, the use of Triptanmedications was approximately 53% more [i.e., (4.72-3.09)/3.09=0.53] at the third treatment month in the placebo-treated patients, which was obviously too large of a percent-age to be ignored.

To make a fair comparison, we calculated the adjustedresponse rate of the primary efficacy for the placebo group,relative to the combination group, for the use of Triptanmedications in the third treatment month. The calculationwas done in accordance with the following: subtract fromthe original response rate of the placebo group a portiondefined as the original response rate multiplied by the fac-tor of 0.53, which was attributable to the Triptan use. As aresult, the adjusted response rate was 21% [i.e., 44% (1-.58)=21%] for the placebo treatment, which fell well within therange of 18.3% to 28.8% for the placebo response in thepublished data reported by Van der Kuy and Lohman.2 Forthis trial, the primary efficacy response rate was therefore42% for the combination of 400 mg Riboflavin, 300 mgMagnesium, and 100 mg Feverfew; and 21% for the “place-bo” (or Riboflavin 25 mg) after adjusting the monthly use ofTriptan medications. Consequently, the p value (2-sided)associated with the difference in response rate after adjust-ment was 0.113 and 0.027, respectively, for both the actualsample size and the planned sample size, had the trial beencompleted as originally designed. Although the adjustmentto the response rate and p value calculation was performedad hoc and from a non-model-based approach, it wasunlikely that anyone who assessed the confounding factorof Triptan use would have reached the same conclusions asthe authors for this trial.

As mentioned before, this trial also allowed the on-going use of prophylactic drugs. However, it did not reportthe changes in the use of these prophylactic drugs in theplacebo and combination groups during the randomizedtreatment period, which could have impacted the efficacyfindings as well.

For researchers and healthcare professionals in a clini-cal practice, it is important to understand the limitations andweaknesses of a trial design and its conduct, to utilizeappropriate statistical methodologies, and take into consid-eration any possible factors that may confound the results.

ACKNOWLEDGMENTS

Supported by a grant from Concourse Health SciencesLLC, Encino, California.

REFERENCES

1. Maizels M, Blumenfled A, Burchette R. A combinationof riboflavin, magnesium, and feverfew for migraine pro-

phylaxis; a randomized trial. Headache: Journal of Headand Face Pain. 2004;44:885-890.

2. Van der Kuy P-HM, Lohman JJHM. A quantification ofplacebo response in migraine prophylaxis. Cephalalgia.2002;22:265-270.

3. Awang D VC. Combination of feverfew, magnesium, andriboflavin for migraine prevention. HerbalGram.2005;65:36-37.

4. Merck & Co. Inc. Maxalt® package insert accessed online,http://www.maxalt.com/rizatriptan_benzoate/maxalt/hcp/index.jsp, 2006 (June).

5. Ortho-McNeil Pharmaceuticals Inc. Axert® packageinsert, accessed online, http://www.axert.com/con-tent/Documents/AxertPI.pdf#zoom=100, 2006 (June).

25 JANA Vol. 10, No. 1, 2007

An Evaluation of the Immunological Activitiesof Commercially Available ββ1, 3-Glucans

Vaclav Vetvicka, PhD*, Jana Vetvickova, MSUniversity of Louisville, Department of Pathology, Louisville, Kentucky

O R I G I N A L R E S E A R C H

* Correspondence:Vaclav Vetvicka, PhDUniversity of Louisville511 South FloydLouisville, Kentucky 40202Phone: 502-852-1612 Fax: 502-852-1177E-mail: vaclav.vetvicka@louisville.edu

ABSTRACT

Introduction

β1,3-glucan’s role as a biologically activeimmunomodulator has been well documented for over 40years. Interest in the immunomodulatory properties ofpolysaccharides was initially raised after experiments show-ing that a crude yeast cell preparation stimulatedmacrophages via activation of the complement system.1

Further work identified the immunomodulatory active com-ponent as β1,3-glucan.2 Numerous studies (currently morethan 1,600 publications) have subsequently shown thatβ1,3-glucans, either particulate or soluble, exhibitimmunostimulating properties, including antibacterial andanti-tumor activities.3,4

Despite extensive investigations, no consensus on thesource, size and other biochemical or physicochemicalproperties of β1,3-glucan has been achieved. In addition,numerous concentrations and routes of administration havebeen tested – including oral, intraperitoneal, subcutaneousand intravenous applications.

This fact, together with the fact that there are probablymore than a hundred different samples on the US marketalone, leads to confusion about the quality, biologicaleffects, and overall efficiency of glucan. Therefore, wedecided to compare the basic immunological activities of agroup of glucans. The list of products chosen came fromthose heavily advertised, commonly available and easilyobtained in the US, Europe, Southeast Asia and Japan. Inorder to be certain that we are measuring the effects of glu-can only, we picked the commercial samples with glucan(either from one source or a mixture of different glucans) asthe only active ingredient.

The collection of tested biological reactions (phagocy-tosis, surface markers on splenocytes, cytokine synthesis,and stimulation of antibody response) represents both thehumoral and cellular branches of the immune reaction, thusoffering insight as to the immunological activities of stud-ied glucans.

MATERIAL AND METHODS

Animals

Female, 6–to 10–week–old BALB/c mice were pur-chased from the Jackson Laboratory (Bar Harbor, ME). Allanimal work was done according to the University ofLouisville IACUC protocol. Animals were sacrificed byCO2 asphyxiation.

Materials

RPMI 1640 medium, sodium citrate, dextran, Ficoll-Hypaque, antibiotics, sodium azide, bovine serum albumin(BSA), Wright stain, Limulus lysate test E-TOXATE,

26JANA Vol. 10, No. 1, 2007

Freund’s adjuvant and Concanavalin A were obtained fromSigma Chemical Co. (St. Louis, MO). Fetal calf serum(FCS) came from Hyclone Laboratories (Logan, UT).

ββ1,3-glucans

The glucans used in this study were purchased from thefollowing companies: Now BETA glucan from Now Foods(Bloomingdale, IL), IMMUTOL from Biotec (Tromso,Norway), Immune Builder and Maitake Gold 404 fromMushroom Science (Eugene, OR), Glucan #300 fromTransfer Point (Columbia, SC), Glucagel T from GraceLinc(Christchurch, New Zealand), and Senseiro from Sundory(Tokyo, Japan).

Antibodies

For fluorescence staining, the following antibodieshave been employed: anti-mouse CD4, CD8 and CD19,conjugated with FITC were purchased from Biosource(Camarillo, CA).

Flow cytometry

Cells were stained with monoclonal antibodies on icein 12x75-mm glass tubes using standard techniques. Pelletsof 5x105 cells were incubated with 10 µl of FITC-labeledantibodies (1 to 20 µg/ml in PBS) for 30 minutes on ice.After washing with cold PBS, the cells were re-suspendedin PBS containing 1% BSA and 10 mM sodium azide.Flow cytometry was performed with a FACScan (BectonDickinson, San Jose, CA) flow cytometer and the data fromover 10,000 cells/samples were analyzed.

Phagocytosis

The technique employing phagocytosis of syntheticpolymeric microspheres was described earlier.5,6 Briefly:peritoneal cells were incubated with 0.05 ml of 2-hydrox-yethyl methacrylate particles (HEMA; 5x108/ml). The testtubes were incubated at 37° C for 60 min. with intermittentshaking. Smears were stained with Wright stain. The cellswith three or more HEMA particles were considered positive.The same smears were also used for evaluation of cell types.

Evaluation of IL-2 production

Purified spleen cells (2x106/ml in RPMI 1640 mediumwith 5% FCS) were added into wells of a 24-well tissue cul-ture plate. After addition of 1 mg of Concanavalin A intopositive-control wells, cells were incubated for 72 hrs. in ahumidified incubator (37°C, 5% CO2). At the endpoint ofincubation, supernatants were collected, filtered through0.45 mm filters and tested for the presence of IL-2.7 Levelsof the IL-2 were measured using a Quantikine mouse IL-2kit (R&D Systems, Minneapolis, MN).

RESULTS

The number of individual glucans is almost as great asthe number of sources used for their isolation. The rationale

for this combination of glucan samples was not only theircommercial availability and success, but most importantly,we tried to include both soluble and insoluble glucans, andalso glucans from different sources, including yeast, mush-rooms and cereals (Table 1).

Glucagel barley β-glucan is a mixed link (13, 14)-ß-D-glucose polymer, in which cellotriosyl and cellotetraosylresidues occur in a ratio of ~3:1. The natural purificationprocess yields a reduced molecular weight ß-glucan (typi-cally ~130 kDa) that is more readily hydrated than otherconventionally purified β-glucans. The typical carbohy-drate content is 85–90%.

Senseiro is a soluble, high molecular weight glucanisolated from Agaricus blazei, consisting of approximately63% carbohydrate. Glucan #300 is a proprietary (13, 16)-ß-D-glucan purified from Saccharomyces cerevisiae byBiothera for Transfer Point and even when corresponding tothe glucan sold under WGP name, has much higher purity(app. over 96%).

β-glucans are generally considered to be potent stimu-lators of cellular immunity, with macrophages and neu-trophils being the most important targets. Not surprisingly,we started our evaluation of glucan activities by phagocyto-sis. We used the synthetic polymeric microspheres, HEMA,since their use, dose and timing are already well establishedin glucan studies.7-9 Results summarized in Figure 1 showsignificant effects of glucan samples on encapsulation ofsynthetic particles by peripheral blood neutrophils. The sig-nificant stimulation of phagocytic activity was found withfive glucans – Now Beta Glucan, Maitake Gold, ImmuneBuilder, IMMUTOL and Glucan #300. The other samples,with the exception of Glucagel T, also stimulated thephagocytosis, but at a much lower level and the results werenot significant.

Next, we compared the effects of tested glucans on theexpression of several membrane markers on splenocytes.Twenty-four hours after an ip. injection of 100 µg of indi-vidual glucan, spleen cells were isolated and the surfaceexpression of CD4, CD8 and CD19 was evaluated by flowcytometery. The results summarized in Figure 2 demon-strated that only three glucans –Now Beta Glucan, MaitakeGold, and Glucan #300–significantly increased the migra-tion of CD4- and CD8-positive T lymphocytes; none of theglucans had any significant effect on changes in the pres-ence of CD19-positive B lymphocytes.

Evidence of the immunomodulating activity was alsodemonstrated through effects on the production of IL-2 byspleen cells (Figure 3). The production of IL-2 was mea-sured after a 72 hr. in vitro incubation of spleen cells iso-lated from control and glucan-treated mice. Again, treat-ment of mice with Now Beta Glucan, Maitake Gold, andGlucan #300 showed the highest stimulation of IL-2 pro-duction. Immune Builder and IMMUTOL showed medium

27 JANA Vol. 10, No. 1, 2007

Figure 1.

Figure 2.

Effect of an ip. administration of 100 µg of different glucan samples on phagocytosis by peripheral blood granulocytes. Each value rep-resents the mean ± SD. *Represents significant differences between control (PBS) and glucan samples at P ≤0.05 level.

Effect of ip. injection of 100 µg of tested glucans on the expression of CD4, CD8 and CD19 markers by spleen cells. The cells from threedonors at each time interval were examined and the results given represent the means ± SD. *Represents significant differences betweencontrol (PBS) and samples at P ≤ 0.05 level.

28JANA Vol. 10, No. 1, 2007

level stimulation. As the secretion of IL-2 by non-stimulat-ed splenocytes (PBS group) is almost zero, even low stimu-lation by Glucagel T was significant. Another way to com-pare the effect on IL-2 formation and/or secretion is to com-pare it to the Con A stimulation. In this case, only Glucan#300 showed higher effects than Con A, whereas Now BetaGlucan and Maitake Gold were comparable, and the rest ofthe glucans showed much smaller effects.

We then focused on the use of glucan as an adjuvant.As an experimental model, we used immunization withovalbumin. Glucans were applied together with twointraperitoneal doses of antigen; a commonly used Freund’sadjuvant was used as additional positive control. The results(Figure 4) showed that only Immune Builder and Senseiroglucans had no effects on antibody response. All other glu-cans significantly supported the formation of specific anti-bodies. Glucans with the highest stimulation were GlucagelT and Glucan #300. It must be noted, however, that none ofthe glucans potentiated the humoral immunity to the levelof Freund’s adjuvant.

Table 2 summarizes the activities of individual glucansin all tested functions. Clearly, the most active samples wereGlucan #300, followed by Now Beta Glucan and MaitakeGold 404. Senseiro glucan was almost without measurableactivity.

DISCUSSION

Despite the extensive amount of scientific reports aboutglucans and their biological activities, most of the studiesare focused on the description of chemical and/or biologicalproperties of one particular glucan. Numerous types of glu-cans have been isolated from almost every species of yeastand fungi. For a long time, attention was focused mainly onglucans isolated from yeast and mushrooms. Recently, theexistence of a highly purified linear β1,3-glucan namedPhycarine, and subsequent study showing that Phycarineinduced a broad range of defense reactions in tobaccocells,10 brought new attention to seaweed-derived glucans.11-

13 More studies revealed that Phycarine significantly stimu-lated phagocytosis, synthesis and release of IL-1, IL-6 andTNF-α, and NK cell-mediated killing of tumor cells both invitro and in vivo.8 Similarly, recent clinical trials demon-strated the high activity of glucan isolated from barley.14 Itis clear, therefore, that the biological activities of glucansmight be related more to the purity and biochemical/physio-chemical characteristics than to the source.

Comprehensive reviews comparing several glucans arerare. However, in one of those studies, Yadomae reviewedhow the structural properties of glucans affected biologicalactivities and found that branched or linear 1,4–glucans

Figure 3.

Effects of glucans on Con A-stimulated secretion of IL-2 by spleen cells. *Represents significant differences between control (PBS) andsamples at P ≤ 0.05 level.

Glucan used in this study

Name Source Manufacturer/Distributor Solubility

β-1,3/1,6-D-glucan Saccharomyces cerevisiae Now Foods NoGrifola frondosa

MaitakeGold 404 Grifola frondosa MushroomScience Yes

Immune Builder Agaricus blazei MushroomScience NoCordyceps sinensisCoriolus versicolorGanoderma lucidumLentinula edodesGrifola frondosa

IMMUTOL Saccharomyces cerevisiae Biotec ASA No

Glucagel T Barley GraceLinc

Senseiro Agaricus blazei Sundory Yes

Glucan #300 Saccharomyces cerevisiae Transfer Point No

JANA Vol. 10, No. 1, 200729

have limited activity and β-glucans with a 1,3 configurationwith additional branching at the position 0-6 of the 1-3linked D-glucose residues have the highest immunostimu-lating activity.15 Readers seeking additional reviews mightsee Kogan16 or Vetvicka.17 However, it is important to keepin mind that these reviews are oriented towards comparingresults of numerous publications and none of them offers aface-to-face comparison of several glucans. At the sametime, with the high number of individual glucans and hugedifferences in their biological activities, it is imperative to

evaluate their biological properties before any suggestionsfor use of a particular glucan can be made.

In our paper, we compared seven commercially suc-cessful glucans, differing both in source (mushroom, yeastand barley) and solubility. At the same time, we used iden-tical amounts of glucans from each sample. In the case ofcomplex mixtures (such as Immune Builder), the totalamount of used sample corresponded to the ratio of indi-vidual glucans.

Table 1.

Table 2.

Comparison of individual glucans

Name Phagocytosis CD expression Il-2 production Antibody formation

Now Beta Glucan ++ +++ ++ ++

MaitakeGold 404 ++ +++ ++ ++

Immune Builder ++ + + -

IMMUTOL ++ + + +

Glucagel T - - +/- +++

Senseiro + + - -

Glucan #300 +++ +++ +++ +++

30JANA Vol. 10, No. 1, 2007

As various glucans are well known to stimulate phago-cytosis,18 one of the first tests of the immunological charac-teristics of any glucan is phagocytosis. We used the 2-hydroxyethyl methacrylate particles, which have only aslight negative charge and thus do not nonspecificallyadhere to the cell surface. This guarantees that only phago-cytosing cells will engulf these particles and significantlylowers the chance of false negativity.19 Our investigationshowed that while most of the tested glucans stimulatedphagocytosis of synthetic microspheres (with the exceptionof Glucagel T), the highest effects were obtained withGlucan #300.

As some of the glucans are known to regulate the influxof cells into individual lymphatic organs,8 we compared theeffects of a single injection on expression of the basic mem-brane markers present on splenocytes. Only three glu-cans–Now Beta Glucan, Maitake Gold, and Glucan #300 –changed the number of CD4- and CD8-positive lymphocytes.No glucan significantly changed the percentage of B lym-phocytes. The effects on CD4-positive cells corresponded tothe previously found effects of Phycarine8 or lentinan.20

In addition to the direct effect on various cells of theimmune system, the immunostimulating action of β-glu-cans is caused by potentiation of a synthesis and release ofseveral cytokines such as TNFα, IFNα, IL-1 and IL-2. Thiscytokine–stimulating activity is dependent on the triplehelix conformation.21 The only glucan without a trace of

pro-inflammatory cytokine stimulation is PGG-glucan.22

We focused on the stimulation of IL-2 production by spleencells in vitro and found that whereas all glucans (with theexception of Senseiro) stimulated production of IL-2, onlytwo of the samples (Maitake Gold and Glucan #300)showed stimulation comparable to the common stimulatorConcanavalin A. The activity of the most active glucan wascomparable to the previously published data.9,23

Glucans are usually considered stimulators or modula-tors of the cellular branch of immune reaction and very lit-tle attention has been focused on their potential effects onantibody response. We decided to take advantage of therecently published method of evaluating the use of glucanas an adjuvant.24 Our results rather surprisingly showed thatmost of the tested glucans revealed some level of stimula-tion of antibody response, the strongest being Glucagel Tand Glucan #300. In this case, however, the stimulationwas always significantly lower than in the case of combin-ing antigen and Freund’s adjuvant.

Data presented in this study and summarized in Table2 clearly demonstrated the differences in activities amongindividual types of glucans. Also, it is clear that individualglucans can be highly active in one particular part ofimmune reactions (e.g., Glucagel T on antibody produc-tion), and almost without any significant biological activityin other parts of defense reaction. Glucan #300 showed notonly a broad range of action, but in all tested reactions (with

Effects of two ip. injections of tested glucans on formation of antibodies against ovalbumin. Mice were injected twice (two weeks apart)and the serum was collected 7 days after last injection. Level of specific antibodies against ovalbumin was detected by ELISA. As positivecontrol, Freund’s adjuvant was used. *Represents significant differences between control (ovalbumin alone) and samples at P ≤ 0.05 level.

Figure 4.

31 JANA Vol. 10, No. 1, 2007

the exception of the antibody formation where it was the

second most active sample) was the biologically most rele-

vant immunomodulator.

Several conclusions can be made: 1) Not all glucans

are created equal; 2) some of the commercial glucans have

surprisingly low activity; 3) most glucans differ in biologi-

cal effects based on tested characteristics; and 4) for good

results in immunomodulation, it is more imperative to find

a glucan from a solid vendor who is able to back the claims

with solid scientific data. Thinking about the biological

source of glucan is much less important.

ACKNOWLEDGEMENT

The authors of this study have no significant financial

interest in any of the products or manufacturers mentioned in

this article. No external funding was provided for this study.

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