JANA Vol 8 #3 - European Nutraceutical Association

JANA Vol 8 #3 - European Nutraceutical Association

The Journal of the American Nutraceutical Association
Vol. 8, No. 3, 2005
www.ana-jana.org
Exercise
Nutraceuticals
Good Nutrition
Essentials for Healthy Aging
IN THIS EDITION
• The Fructose Controversy: Separating Fact from Fiction
• Cholesterol and Triglyceride-Lowering Effect of Two Spice
Preparations
• Reduction in the Frequency of Arrhythmic Episodes in
Patients with Paroxysmal Atrial Arrhythmia with a
Vitamin/Essential Nutrient Supplementation Program
• Flax and Soy Phytoestrogen Effects on Renal Injury and
Lipid Content in Experimental Polycystic Kidney Disease
• Immunostimulating Properties of Two different β-glucans
Isolated from Maitake Mushrooms (Grifola frondosa)
• The Effects of Australian Tea Tree and Jojoba Essential Oils
with Minerals for Treatment of Nail Fungus
A Peer-Reviewed Journal on Nutraceuticals and Nutrition
ISSN-1521-4524
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Nutritional and Nutraceutical Therapies for the Metabolic Syndrome: Guidelines for the Clinical Practice.
____ Mark C. Houston, MD, MSc, FACP, FAHA, - Clinical Professor of Medicine,Vanderbilt University School of Medicine,
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Specialist in Clinical Hypertension, Director, Hypertension Institute and Vascular Biology, Clinical Research Staff Physician,Vascular Institute,
Saint Thomas Hospital, Nashville,Tennessee.
Health Implications for Dietary Fiber: Guidelines for the Clinical Practice on the Role of Fiber in the
____ Prevention of Cardiovascular Disease. Joanne R. Lupton, PhD. - Regent’s Professor and University Faculty Fellow at Texas
A&M University and holder of the William W. Allen Endowed Chair in Human Nutrition. She chaired the Macronutrients Panel for the
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The Role of Dietary Fatty Acids in the Prevention and Treatment of Human Inflammatory Diseases:
____ Guidelines for the Clinical Practice. Floyd (Ski) H. Chilton III, PhD. - Director,Wake Forest and Harvard Center for
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focuses on the roles that particular types of fat, arachidonic acid, arachidonate-containing phospholipids and arachidonic acid metabolites
play in regulating inflammatory and proliferative responses. His work over the past 10 years has examined the effect of diet on inflammation
associated with major diseases such as asthma, arthritis and cancer.
____ The Effects of Nutraceutical Antioxidants as a Prevention Strategy and Treatment of Muscle Damage. Allan
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Contents – JANA Vol. 8, No. 3, 2005
P
Journal of the
American
Nutraceutical
Association
E R S P E C T I V E
A
R T I C L E
The Fructose Controversy:
Separating Fact from Fiction ...................................................... 1
Jeffrey S. Bland, PhD, FACN
EDITORIAL STAFF
EDITOR-IN-CHIEF
Mark Houston, MD
ASSOCIATE EDITORS
Bernd Wollschlaeger, MD
Lisa Colodny, PharmD
TECHNICAL EDITOR
Jane Lael
Terri Erickson
ART DIRECTOR
Gary Bostany
EDITORIAL BOARD
Jordan R. Asher, MD, MsMM, SCH
Ethan Basch, MD, MPhil
Jan Basile, MD
Russell Blaylock, MD
Hyla Cass, MD
Lisa Colodny, PharmD, BCNSP
Loren Cordain, PhD
Jeanette Dunn, EdD, RN, CNS
Brent Eagan, MD
Christopher M. Foley, MD
Michael Glade, PhD
Clare M. Hasler, PhD
Ralph Hawkins, MD
Robert Krueger, PhD
Daniel T. Lackland, PhD
Garth L. Nicolson, PhD
Mark J.S. Miller, PhD
Robert Rountree, MD
Diana Schwarzbein, MD
Catherine Ulbricht, PharmD
Walter Willett, MD, DrPH
Bernd Wollschlaeger, MD
_____________________________
P
I L O T
S
T U D Y
Cholesterol and Triglyceride-Lowering Effect
of Two Spice Preparations ..........................................................12
Eva Lydeking-Olsen, NP, BSc, Janne Springborg Clewlow, MSc,
Vita Damsoe, BSc, An-Mari Mey Hensen, HP, BSc
O
R I G I N A L
R
E S E A R C H
Reduction in the Frequency of Arrhythmic Episodes in
Patients with Paroxysmal Atrial Arrhythmia with a
Vitamin/Essential Nutrient Supplementation Program ...........19
Matthias Rath, MD, Tatiana Kalinovsky, MSRN,
Aleksandra Niedzwiecki, PhD
Flax and Soy Phytoestrogen Effects on
Renal Injury and Lipid Content in Experimental
Polycystic Kidney Disease ...................................................... 26
Malcolm R. Ogborn, MBBS, FRACP, FRCPC,
Evan Nitschmann, MSc, Neda Bankovic-Calic, MD, PhD,
Hope A. Weiler, RD, PhD, Harold Aukema, PhD
Immunostimulating Properties of Two Different β-glucans
Isolated from Maitake Mushrooms (Grifola frondosa) ............ 33
Vaclav Vetvicka, PhD, Jana Vetvickova, MS
American Nutraceutical Association
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CEO & PUBLISHER
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ANA is an alliance of individuals with interest in
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The Journal of the American Nutraceutical Association
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C
L I N I C A L
C
A S E
R
E P O R T S
The Effects of Australian Tea Tree and
Jojoba Essential Oils with Minerals for
Treatment of Nail Fungus .......................................................... 40
Leigh Erin Connealy, MD
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P
E R S P E C T I V E
A
R T I C L E
The Fructose Controversy:
Separating Fact from Fiction
Jeffrey S. Bland, PhD, FACN*
President and Chief Science Officer
Metagenics, Inc., Gig Harbor, Washington
ABSTRACT
Fructose is a monosaccharide found in many natural
foods, and its modest consumption has been demonstrated
to have beneficial effects on blood sugar and lipid parameters. Paradoxically, several recent studies have reported that
fructose consumption is associated with adverse physiological effects including hypertriglyceridemia, reduced HDL
cholesterol, increased uric acid levels, and increased blood
postprandial insulin and glucose, which has led to considerable misunderstanding and confusion among clinical nutrition and dietetics professionals. Many factors influence the
response to fructose, such as glycemic load, caloric percent
of sugars, the delivery form of the sugar and, most notably
the actual amount of intake, which has increased considerably over the past few decades. This increase has primarily
resulted from the increased consumption of high fructose
corn syrup-containing beverages like soft drinks and sweetened fruit drinks. Unfortunately, these specifics are often
not included in the evaluation process, and in many cases,
are not even reported so that study results can be kept in
context. This lack of consistent reporting about fructose has
led to the labeling of fructose as detrimental. This paper
* Correspondence:
Jeffrey S. Bland, PhD, FACN
Metagenics, Inc.
9770 44th Ave., NW, Suite 100
Gig Harbor, WA 98332
Phone: 253-851-3943; Fax: 253-851-9749
Email: JeffBland@metagenics.com
1
reviews the metabolism of fructose, and discusses some of
the recent data in order to place this debate in perspective.
Key Words: fructose, HFCS, triglycerides, cholesterol,
metabolic syndrome, diabetes
INTRODUCTION
Fructose: A Common Dietary Monosaccharide
Fructose, a 6-carbon sugar found in fruit, vegetables
and honey is the most common naturally occurring monosaccharide.1 Because early data indicated that fructose does
not induce an insulin response but attenuates the blood glucose peak when taken with glucose, and that fructose is
known to produce the same sweetness for less energy,
researchers and diabetes care organizations initially recommended fructose as a preferred sweetener for individuals
with diabetes.2,3 Over the past three decades, however, the
consumption of fructose has increased 120-fold–from an
estimated 0.5 lb/year per person in 1970 to 62.4 lb/year per
person.4 This increase is correlated with increased incidences of obesity, diabetes, and metabolic syndrome; therefore, some researchers have proposed that fructose is a factor in promoting these conditions.5,6 Taken together with
controversial data suggesting that fructose can lead to
hypertriglyceridemia,7 in the mid-1990s, the American
Dietetic Association (ADA) indicated that the use of added
fructose as a sweetening agent may have "no overall advantage over other nutritive sweeteners," even though they
went on to state that there is no reason to avoid naturally
occurring fructose in fruit, vegetables and other foods.8
JANA Vol. 8, No. 3, 2005
Developers of nutraceuticals, supplements, and medical and specialty food products have limited sweetener
choices. Glucose has inherent concerns of leading to
increases in blood insulin and glucose levels; whereas,
sucrose (table sugar) also contains glucose and can increase
insulin and glucose responses. Synthetic sweeteners are
available, but not acceptable for a natural food-based product. And while some natural products may be allowable in a
supplement, they may not be generally recognized as safe,
(GRAS), and thus are not allowable in a food product (e.g.,
stevia). Fructose is a preferred choice in some cases
because it can be used at lower amounts than other natural
sweeteners to achieve the same sweetness level, and to help
attenuate blood glucose and insulin levels. Therefore, this
debate on the benefits and adverse effects of fructose is particularly relevant to the natural foods market.
In an attempt to tease apart fact from fiction within this
controversy, and to better understand the effects of fructose
in the diet, it is important to review some of the basic physiological and nutritional aspects of fructose-containing
sweeteners. This paper briefly reviews the basic biochemistry and physiology of fructose and discusses the controversial fructose data with respect to human physiology and
medical relevance.
Metabolic Fate of Fructose
Many types of sugars are found in food, and different
terms have been identified to describe them (Table 1). The
most common monosaccharides are fructose, glucose and
galactose. Disaccharides that are also common sweeteners
in foods include sucrose (glucose + fructose), the milk
sugar lactose (glucose + galactose), and maltose (glucose +
glucose). Once consumed, these disaccharides are broken
down into their respective monosaccharides by specific
enzymes (disaccharidases). Glucose, if present in the disaccharide, is released and absorbed across the intestinal
mucosa by way of a sodium-glucose transport system.
Similarly, when fructose is present in the disaccharide, it is
released and available for absorption. In contrast to glucose,
however, fructose is transported across the intestinal barrier of the duodenum and jejunum by a sodium-independent
facilitated diffusion process.9 Studies indicate that fructose
is absorbed more slowly than glucose, but its absorption is
enhanced when glucose is present.10
The metabolism of fructose has been extensively
reviewed.4,6,10-12 Briefly, it has been noted that its metabolism differs from that of glucose in several ways. Glucose
enters the body’s cells by way of the glucose transporter
GLUT-4, which requires insulin in all tissues except the
eyes, nerves and kidneys.6 In this process, insulin activates
the insulin receptor, which then results in an increased
number of GLUT-4 transporters on the cell surface. In contrast, fructose enters cells via the GLUT-5 transporter,
which does not depend on insulin.10 The beta cells of the
pancreas lack the GLUT-5 transporter, and therefore, fructose does not directly stimulate insulin secretion.6 It is
interesting to note that the GLUT-5 transporter is also
absent from cells in the brain, which may be the reason why
fructose does not produce the same type of satiety signal as
does glucose.
In humans, at least half of the absorbed fructose is
rapidly taken into the liver, where it is phosphorylated to
form fructose-1-phosphate by the enzyme fructokinase
(Figure 1).11,12 Fructose-1-phosphate (a 6-carbon sugar) is
readily cleaved by the enzyme aldolase B to form two triose
(3-carbon) molecules. These 3-carbon molecules become
the backbone for triglyceride synthesis, and are also used
for the generation of the phospholipids that are necessary
for healthy cell membranes. Fructose can also be converted
into glucose-6 phosphate via the phosphofructokinase pathway, which leads to an increase in liver glycogen levels.
Although the data are somewhat equivocal, the balance of
Table 1. Definitions of Sweeteners.
Sugar
free monosaccharide or disaccharide present in a food
Added sugar
any sugar that is added to a food, including sweeteners such as sucrose,
high fructose corn syrup (HFCS), honey, molasses, and other syrups
Naturally occurring sugar
sugar naturally occurring in food and not added in processing, preparation,
or at ingestion
Total sugars
total amount of sugars present in a food, including naturally
occurring and added sugars
Free Fructose
fructose that exists in food as a monosaccharide
Total Fructose
both the monosaccharide fructose, and the fructose found in other sugars,
such as the disaccharide sucrose, which is composed of equal amounts
of glucose and fructose
High fructose corn syrup (HFCS) composed of approximately half fructose and half glucose with other
oligosaccharides
JANA Vol. 8, No. 3, 2005
2
Figure 1. Fructose utilization in the liver showing its interrelationship with glucose and fatty acid metabolism. Pase, phosphatase; P,
phosphate. Reproduced from Mayes PW. (1993) Intermediary
metabolism of fructose. Am J Clin Nutr 58(suppl):754S-65S with
permission by the American Journal of Clinical Nutrition.© Am J
Clin Nutr. American Society for Clinical Nutrition.
Figure 2. The relationship between fructose metabolism and uric
acid formation in the liver. P, phosphate; AMP, adenosine
monophosphate; ADP, adenosine diphosphate; ATP, adenosine
triphosphate; Pi, organic phosphate; GMP, guanosine monophosphate; PRPP, 5-phosphoribosyl-1-pyrophosphate; IMP, inosine
monophosphate. Reproduced from Mayes PW. (1993) Intermediary
metabolism of fructose. Am J Clin Nutr. 58(suppl):754S-65S with
permission by the American Journal of Clinical Nutrition.© Am J
Clin Nutr. American Society for Clinical Nutrition.
studies indicate that fructose together with glucose promotes glycogenesis better than glucose alone.11
over the past several decades.14 Furthermore, he noted that
these advancements, coupled with the mass amount of corn
grown in the United States, have resulted in increased availability of inexpensive corn-based industrial products, the
major uses of which are as food ingredients for human consumption. The sweetener high fructose corn syrup (HFCS)
accounts for about five percent of these processed corn
products in the United States.
The increase in blood glucose and insulin is much
smaller after a dose of fructose than when a similar amount
of glucose is consumed.12 For this reason, fructose has been
considered valuable as a glucose substitute in diets for individuals with diabetes. Clinical studies have demonstrated
that a small amount of fructose, when ingested with glucose, also reduces the glycemic response and increases liver
synthesis of glycogen in patients with type 2 diabetes.4,12,13
Glucose metabolism is under the control of phosphofructokinase, an enzyme that is regulated by ATP and glucose.12
In contrast, fructose can bypass the phosphofructokinase
regulatory step; therefore, excessive fructose can continue
to be metabolized (Figure 2), which ultimately produces an
increase in lactate with only a small increase in diet-induced
thermogenesis.4,11,12
High Fructose Corn Syrup: the "Other" Fructose
In a 2004 Time magazine article entitled "The Corn
Connection," Eric Roston pointed out that technological
advancements in the processing of corn have resulted in a
substantial decrease in the cost of goods for corn products
3
The production of HFCS begins with the removal of
protein and fiber from cornstarch, after which the purified
starch is hydrolyzed to form glucose, and then some of this
glucose is isomerized to yield fructose. The most common
forms of HFCS are HFS-42 and HFS-55. Although the
name suggests this is "fructose," in fact, fructose comprises only around half of these products: HFS-42 is 42% fructose and 53% glucose; HFS-55 is 55% fructose and 42%
glucose.15 The final HFCS product also contains negligible
amounts of other sugars, such as various oligosaccharides.
HFCS is cheaper than many other sweeteners, is sweeter
than sucrose and glucose on an equal weight basis (e.g.,
some reports indicate HFS-42 and HFS-55 are 1.16 and
1.28 times as sweet as sucrose, respectively),6 and is easily
formulated into beverages.6,15
JANA Vol. 8, No. 3, 2005
In its pure form, fructose (i.e., crystalline fructose) is
not the same as HFCS; it is not a combination of glucose
and fructose, nor does it contain other sugars.15 Crystalline
fructose can be purified from HFCS using a process by
which the non-fructose substances are removed.6,15 In contrast to HFCS, crystalline fructose is not a primary source
of fructose in processed food, but is mainly used in powdered drinks and specialty food products.16
HFCS and Fructose Consumption
Because fructose is the most common monosaccharide
in fruit, vegetables, and other foods, it has been consumed
at moderate levels for thousands of years. It is estimated
that, prior to the 20th century, the average intake of fructose
was around 16 to 20 g per person per day.4 Table 2 summarizes the fructose that is delivered in commonly consumed
fruit. The dietary fructose intake from one serving of fruit
can range from ~ 4 g (e.g., one peach) to ~10 g (e.g.,
banana). Traditional dietetic and nutritional wisdom would
suggest that the ingestion of two or three portions of fruit
per day is considered valuable as part of an overall dietary
program. This intake of fruit would be expected to deliver
from 10 to 35 g of fructose daily. Natural sweeteners such
as honey and molasses are also natural sources of fructose.
For example, taking into account the free fructose and the
fructose liberated after digestion of the disaccharides in
these foods, 100 kcal of honey (~30 g serving) delivers 13.6
g fructose, and 100 kcal of blackstrap molasses (~47 g serving) delivers 11.8 g fructose.17
The availability of a commercial process for making
inexpensive HFCS has led to a considerable shift in the food
production system. At one time, sucrose derived from sugar
cane or beets was the primary sweetener. Currently, HFCS
sweeteners are often used in the absence of sucrose. The
extra sweetness of HFCS and its ability to be used in beverages such as soft drinks have resulted in HFCS accounting for approximately 50% of sweetener intake,12 and 20%
of total dietary carbohydrate intake.4 As pointed out by Dr.
George Bray and his colleagues at the Louisiana State
University’s Pennington Biomedical Research Center, the
development of the inexpensive, sweet, corn-based syrup
made it profitable to replace sucrose (table sugar) and simple sugars with HFCS in the American food supply so that
HFCS now represents a major source of added sugars in the
diet.6 The primary factor in this increase in daily fructose
intake has come from the increased consumption of HFCS in
such products as processed drinks (e.g., colas). In fact, the
sugars from processed drinks and desserts total about twothirds of all caloric sweeteners consumed in the United
States.6 As stated by Basciano and colleagues: "In 1992, the
USDA recommended that only 40 g of extra sugar should be
added to a standard 2000 calorie a day diet. The amount of
HFCS found in only one 12-oz soft drink equals this total proportion of daily intake."4 This increase of sweeteners not only
represents empty carbohydrate calories, but also accounts for
the increase to a typical consumption of 50 g to 100 g fructose per day per person in the United States in 2002.2,4
In June, 2005, the Center for Science in the Public
Interest (CSPI) published the second edition of the report
entitled "Liquid Candy: How Soft Drinks are Harming
Americans’ Health." In this report, it was noted that carbonated beverages are the single biggest source of refined
sugars in the American diet. Youths and young adults
appear affected the most: among 12- to 19-year-olds, carbonated soft drinks provide 9% and 8% of boys’ and girls’
Table 2. Fructose content in 100 g (3.5 oz) portions of some commonly consumed fruit. Values are an average and may vary
somewhat during different growing seasons and with specific subspecies of fruit.
Sugar Content
Total
Fruit
kcal/serve
Composition (%)
Glucose
Fructose
Amount per Serving (g)
Sucrose
Total Sugar
Fructose*
Banana
85
26.5
30.1
43.4
19.6
10.2
Figs, dried
274
50.2
34.4
15.4
64.2
27.0
American
Grapes
69
51.6
46.2
2.2
9.3
4.9
Orange
49
29.4
21.2
49.4
8.5
3.9
Peach
38
13.3
13.3
73.3
7.5
3.7
Pear
61
28.7
57.4
13.8
8.7
5.6
*Sum of free fructose and the fructose present in sucrose
Data are compiled from Ensminger AH, Ensminger ME, Konlande JE, Robson JRK. (1994) Foods & Nutrition
Encyclopedia, 2nd Edition. Boca Raton: CRC Press; 1994;1:341-2.
JANA Vol. 8, No. 3, 2005
4
daily caloric intake, respectively.18 Teenage boys alone get
44% of their daily refined sugar intake from soft drinks.
These intakes should take into account that a considerable
number of individuals consume no soft drinks, and therefore, a specific individual’s consumption could be much
higher than the average value. Moreover, as noted by Barry
Popkin of the University of North Carolina-Chapel Hill,
who has investigated food intake patterns among the United
States population, "Sugar-loaded beverages are really just
empty calories that block out healthy foods."18,19
Therefore, there are different sources of fructose in the
American diet, the most common being HFS-42, HFS-55,
and sucrose, with crystalline fructose, fruit, and natural
sweeteners like honey and molasses also moderately contributing to fructose intake. Because each of these sources
have different compositions, they may have differing
effects; therefore, it is important to separate the data
obtained with HFCS from the data obtained with other
sources of fructose. Moreover, it is important to critically
evaluate whether HFCS-containing products are replacing
nutrients in the diet, and to consider the amount of total
sugar consumption in various studies with HFCS to understand if the effects seen following its consumption can be
extrapolated to the consumption of fructose alone before
conclusions are drawn. Unfortunately, many of the studies
that have been published have either failed to clearly define
the form of fructose that was studied, or have generalized
their conclusions from one form of sweetener to all fructose-containing foods.
HEALTH EFFECTS OF FRUCTOSE
Effect of Fructose on Glucose Tolerance and Insulin
Secretion
Daly and colleagues have provided extensive, in-depth
reviews of data collected on the influence of fructose on glucose and insulin metabolism and note that, although animal
studies have provided convincing evidence for an effect of
high-fructose diets in decreasing insulin sensitivity, research
in humans has produced very conflicting results, with limited evidence for an effect.20,21 Much of the data accumulated in animal studies have represented diets in which fructose
comprised at least 35% of energy, or have used sucrose at
>50% of energy.21 In an average 2,000 kcal human diet, for
example, this would constitute 125 g to 175 g fructose per
day. With the high fructose content of products such as soft
drinks, this data may represent some intake levels, but is
higher than most individuals consume.
Fructose is known to have a low glycemic index, to
attenuate the glucose peak, and does not increase insulin
secretion after acute doses. For example, Moore et al. reported that consumption of 7.5 g fructose dose with 75 g glucose
solution reduced the glycemic response in both healthy and
type 2 diabetic subjects.22,23 Heacock et al. confirmed this
5
effect on glycemic peak, reporting that a small (10 g) dose
of fructose taken 30 or 60 minutes before a starch challenge
(potatoes providing 50 g available carbohydrate) significantly attenuated the glucose peak by 27% and 25%, respectively, but they saw no change in triglycerides when the fructose
was given with the starch.24 As shown in Table 3, studies in
humans have provided conflicting results with no clear
demonstration of an adverse effect of fructose on blood glucose and insulin levels. In discussing this disparity in the
human research, Daly summarizes by saying, "Overall,
despite the wealth of interest that sucrose has attracted,
research has failed to show a consistent effect of dietary
sucrose or fructose on insulin sensitivity."21
Tournian et al. investigated the effects of fructose infusion on hepatic insulin and glucose metabolism, and proposed that stimulation of hepatic glycogen synthesis and
inhibition of net glycogenolysis by fructose are involved in
maintaining a constant glucose production.25 In a followup study, these researchers found that acute fructose infusion induced hepatic insulin resistance in humans.26 It is
important to point out that the infusion was performed in
the presence of exogenous insulin to provide a consistent
blood glucose level of ~144 mg/dL, and to stimulate an
exaggerated metabolic response. As noted by the authors,
it remains to be determined whether fructose ingestion as
part of the diet will exert the same result.25,26
These observations have left dietitians wondering if
fructose should be advised as a sweetener choice for the
insulin-resistance/glucose-intolerant patient. In a 2003
review, McGuinness and Cherrington address this issue and
note that catalytic doses of fructose (defined as <10% of
total carbohydrate flux) in a glucose-containing meal lead
to improved glucose tolerance.27 They conclude this review
stating: "This improvement [in glucose tolerance] is primarily mediated by the activation of hepatic glucokinase
and consequent facilitation of liver glucose uptake. The
improvement in glucose tolerance is most evident in insulin
resistant settings (e.g., type 2 diabetes and infection).
…Thus, activating glucokinase during a carbohydrate meal
may serve to minimize the hyperglycemia seen in insulin
resistant settings where underlying defects in hepatic glucose disposal are present."27 This mechanism suggests that
the effect of fructose on glucose tolerance requires first pass
metabolism in the liver, and would not be seen from intravenous delivery of the fructose.
Effect of Fructose on Triglyceride Synthesis
The effect of fructose consumption on blood triglyceride levels is probably the most discussed and controversial area of fructose research today. This area has been
extensively reviewed, and studies have shown conclusively
that diets high in fructose (>25% energy) increase serum
triglycerides.4,12,20,21,28,29 The mechanism leading to this
JANA Vol. 8, No. 3, 2005
Table 3. Human studies – effects of sugars compared with starch on insulin sensitivity.
Dietary intervention
Trial design
Time
period
Subject group
Change in basal
insulin concentrations
Insulin sensitivity
Isoenergetic exchange of
sucrose for starch at 30% of
energy
Crossovera
6 wk
Healthy, with
hypertriglyceridemia
subgroup (n = 19)
Greater in sucrose group
__
Sucrose at 5%, 18%, and 33%
of energy
Crossoverb
6 wk
Hyperinsulinemia
(n = 24)
Increased as sucrose
content rose
__
Fructose at 0%, 7% and 15%
of energy
Crossoverc
5 wk
Healthy with
hyperinsulinemia
subgroup (n = 23)
NS, but increased
postprandial insulin
__
Addition of 250 g fructose or
glucose
Case controld
1 wk
Healthy (n = 7)
No change from basal
Fructose substituted for 24% of
total carbohydrate (i.e.,
13.2%)
Single factor e
2 wk
NIDDM (n = 7)
NS
__
Fructose substituted for 20% of
carbohydrate during 45% or
85%
Crossoverf
>2 wk
Hypertriglyceridemia
with or without
NIDDM (n = 6)
NS
__
Fructose substituted for 20%
starch
Crossoverg
4 wk
NIDDM (n = 10)
Fructose at 13%
Single factorh
3 mo
NIDDM (n = 6)
NS
Sucrose (32%) for starch at
70% of energy
Corssoveri
4 wk
Healthy (n = 9)
NS
__
Replacement of 45 g starch
with sucrose
Crossoverj
6 wk
NIDDM or IDDM (n
= 12 of each)
NS
__
Exchange of starch and
sucrose at 23% of energy
Crossoverk
14 d
Healthy (n = 9)
NS
__
Low glycemic compared with
high glycemic (25% sucrose
compared with 1% sucrose)
Crossoverl
28 d
Healthy men (n = 7)
NS
25% fall in fructose group
as assessed by IVITT
(intravenous-insulintolerance test)
Increased sensitivity in
fructose group as assessed
by euglycemic clamp
No change during
euglycemic clamp
Decreased insulin
sensitivity with lowglycemic diet
Table taken from Daly ME, Vale C, Walker M, Alberti KGMM, Mathers JC. Dietary carbohydrates and insulin sensitivity: a review of the
evidence and clinical implications. Am J Clin Nutr. 1997;66:1072-85. Reproduced with permission by the American Journal of Clinical
Nutrition.© Am J Clin Nutr. American Society for Clinical Nutrition.
a. Am J Clin Nutr. 1979;32:2206-16.
b. Am J Clin Nutr. 1981;34:2348-58.
c. J Nutr. 1983;1131819-26.
d. Am J Clin Nutr. 1980 ;33:273-8.
e. Diabetes Care. 1986 ;9:111-9.
f. Am J Clin Nutr. 1979;32 :1043-50.
g. J Intern Med. 1993 ;233:145-53.
h. Metabolism .1990 ;39:58-63.
i. Clin Sci. 1970;38:1-9.
j. Diabetologia. 1986;29:216-20.
k. Br J Nutr. 1972;27:395-405.
l. Am J Clin Nutr. 1996;63:47-53.
increase is proposed to involve the increase in triglyceride
production with excess fructose (Figure 1) and may also be
influenced by a decrease in turnover due to a shift in VLDL
production and, hence, decreased turnover of triglycerides.4
That excess dietary fructose can contribute to triglyceride production is not controversial. The controversial
aspects of this discussion relate to whether this is a clinically relevant observation. For example, although some
researchers have reported increases in triglycerides in clinical studies using diets with 20% of energy as fructose, others have reported no change in triglycerides with 20% of
JANA Vol. 8, No. 3, 2005
energy as fructose.28,29 Moreover, Reiser reported dosedependent increases in blood triglyceride levels in male but
not female subjects using diets with 5%, 18% and 33%
energy as sucrose,30 and Osei reported no change in triglycerides with a diet of approximately 12% energy as fructose.31 Several studies have indicated that males and
females respond differently as well. For example, after a
diet containing 17% fructose (as added free fructose) was
provided to healthy men and women, only the men showed
an increase of plasma triglyceride levels; no increase was
seen in the women.29 Therefore, the influence of fructose on
6
triglycerides at moderate or low intakes is equivocal.
The early human studies suggesting an effect of fructose on hypertriglyceridemia, such as those performed by
the group at the USDA Research Center in Beltsville,
Maryland, used diets high in fats (e.g., 42% fat, 43% carbohydrate, 15% protein).20,21,32 Frayn and his colleagues at
the Burnleet General Hospital, UK, have investigated the
effect of a bolus fructose dose (0.75 g per kg body weight)
with a high-fat meal (1 g fat per kg body weight) and found
that the elevation of triglycerides was positively related to
fasting insulin concentration, suggesting that insulin-resistant individuals might be more sensitive to this effect.33
Daly, who has extensively reviewed this literature, commented that reproducible effects of fructose on postprandial hypertriglyceridemia have only been shown in the context
of a significant fat load.21 He further added, "We should not
ignore the possibility that sucrose may affect insulin sensitivity only with a high fat intake, particularly given the high
fat intake in the positive studies by the Beltsville group.
Furthermore, these studies should be done in different subject groups [patients with a body mass index >25 kg/m2 but
without diabetes, patients with hypertriglyceridemia, and
patients with diabetes (type 1 and type 2 separately)].
Results should be interpreted not only as results of the
dietary intervention itself but also as changes from the subjects’ habitual diet."
The dietary context of fructose is exemplified by a
study from Yves Rayssiguier and colleagues at the Centre
de Recherche en Nutrition Humaine d’Auvergne in
France.34 Although the title of this report indicates that the
effect of honey was compared to that of a fructose diet on
triglyceride levels ["Substituting honey for refined carbohydrates protects rats from hypertriglyceridemic and prooxidant effect of fructose"], the comparator diets consisted of
the "fructose diet" that delivered 34 g fructose and 31 g glucose per 100 g dry matter, and the "honey" diet of 65 g
honey (34 g fructose, ~27 g glucose, and ~4 g other components) per 100 g dry matter. Both diets provided 68% of
energy as simple sugars (as the fructose + glucose, or the
honey), with 21% and 12% of energy as protein and fat,
respectively. These researchers reported that the hypertriglyceridemic effect of fructose was not observed when
the fructose was provided in the honey diet, but was
observed with the "fructose" diet, which consisted of the
refined carbohydrates.34 These observations indicate that
the total diet appears to influence this response.
Furthermore, the hypertriglyceridemic effect reported for
fructose appears to be individualized and may not be seen
in all patients.
that these monosaccharides have on central nervous system
function and, therefore, on appetite. Fructose, unlike glucose, does not stimulate insulin secretion. For example, in
rhesus monkeys, an 8-hour intravenous fructose infusion
resulted in markedly reduced insulin secretion and no
increase in circulating leptin concentrations; however, infusion of the same amount of glucose increased plasma leptin
levels by more than 50% above baseline fasting levels.35
Results from the laboratory of Dr. Karen Teff and her
colleagues from the Monell Chemical Senses Center and
Department of Medicine at the University of Pennsylvania
support this finding. Teff et al. studied women who were
administered different isocaloric diets containing 30% of
energy as either free fructose or glucose and report a significant decrease of ~20% to 30% in secretion of leptin and
the orexigenic gastroenteric hormone ghrelin.36 The investigators concluded that the changes with the fructose-containing diet could promote overeating through the reduction
of the satiety signals, thereby contributing to obesity.
In an August 2004 report in JAMA, Walter Willett, MD,
DrPH, (Department of Nutrition, Harvard School of Public
Health) and his colleagues report the findings of an analysis on consumption of sugar-sweetened beverages and type
2 diabetes.5 In this study, a higher consumption of sugarsweetened beverages was associated with higher body mass
index (BMI) and an increased risk of type 2 diabetes.
Interestingly, fruit juice consumption was not associated
with diabetes risk, whereas fruit punch consumption was.
These researchers commented that, "…vitamins, mineral,
soluble fiber and phytochemicals in fruit juices may have
beneficial effects counterbalancing potential adverse effects
of sugars." Moreover, they noted that women who increased
their sugar-sweetened soft drink consumption also
increased energy intake from other foods. They suggest that
consumption of these beverages either induces hunger and
food intake, or may reflect detrimental accompanying
dietary and lifestyle changes.
The Effects of Fructose as Part of a Low Glycemic Load
Diet
Effect of Dietary Fructose on Appetite
In a year-long study comparing a low-carbohydrate diet
with a conventional low-fat diet that had no specific restriction of sugars and refined starch, participants on the low-carbohydrate diet were seen to fare better with respect to changes
in atherogenic dyslipidemia values and glycemic control even
though caloric intake and weight loss were similar between
the two groups.37 Willett commented on this finding in an editorial, in which he discussed how nutritionists and dietitians
have disparaged the very-low-carbohydrate ("Atkins") diet
because it requires a high saturated fat content and the fact
that its purported benefits had not been tested.
One of the more intriguing differences between fructose and glucose metabolism may be the diverse influences
He pointed out, however, that four randomized trials in
adults have compared a very-low-carbohydrate diet with a
7
JANA Vol. 8, No. 3, 2005
low-fat diet and all of these trials have supported the conclusion that the low-carbohydrate diet is associated with
improved insulin response and lowered triglycerides.
Therefore, Dr. Willett has stated that “we can no longer dismiss very-low-carbohydrate diets.” 38 Diets that are called
"low-carbohydrate" represent a low glycemic load and,
therefore, provide for a low glycemic index.
Studies in humans are difficult to fully control, however, and, as noted by Wood and colleagues in a recent review
on the nutrigenomics of insulin resistance and type 2 diabetes, diets are only vaguely described in many of the studies on metabolic syndrome.39 The diets are often termed
"high-fat" or "high-sugar," yet often the specific dietary
constituents are not precisely defined. The composition of
the total diet includes not only the relative amount of the
simple carbohydrate (e.g., sucrose, glucose, and fructose),
but also the amount and type of complex carbohydrates like
starch (e.g., amylose and amylopectin) and fiber. The type
and amount of phytonutrients, vitamins and minerals, as
well as the proportion of protein and fat, are also important
factors in the effect of sugars on metabolic function.40
Rigden and Lerman have recently described a series of
case histories of complicated, high-risk, obese patients with
insulin resistance/metabolic syndrome who consumed a low
glycemic load diet with a fructose-sweetened supplement
delivering 30 grams of fructose daily.41 In this report, the
individuals who consumed the fructose-sweetened soy protein supplement had reduced triglycerides and stabilized
postprandial blood sugar levels. Additionally, these patients
lost weight without feeling overly hungry.
A randomized, controlled prospective trial compared a
similar fructose-sweetened soy protein and phytosterol beverage and a low glycemic index diet to the American Heart
Association (AHA) Step 1 diet in postmenopausal women
with hypercholesterolemia.42 Individuals who consumed the
fructose-containing beverage showed significantly reduced
triglycerides, LDL and total cholesterol, and increased HDL
cholesterol. Both programs incorporated weight management by setting caloric intake goals to result in weight loss,
but a significantly greater improvement was observed in cardiovascular disease risk factors with the low glycemic index
diet and fructose-sweetened soy beverage [29% protein,
43% carbohydrates, 29% fat] as compared to the AHA Step
1 diet [20% en protein, 54% en carbohydrates, 27% fat].42 A
significantly greater improvement was also seen in the
triglyceride-to-HDL cholesterol ratio, a known marker for
insulin sensitivity.43 These data show that clinical programs
with modest intakes of fructose in the context of a low
glycemic index diet are effective for the management of
lipids, insulin sensitivity, and excess body fat in postmenopausal women with hypercholesterolemia and/or obesity, and do not appear to promote increased triglycerides.
On the contrary, this approach has been demonstrated in
these studies to support improved blood lipids.
JANA Vol. 8, No. 3, 2005
Other Benefits of Fructose
Fruit consumption is associated with lowered risk of
cardiovascular disease. A commonly accepted hypothesis
to explain this observation is that the flavonoids in fruit provide beneficial, protective antioxidant support.44,45
Recently, however, Balz Frei and Silvina Lotito from the
Linus Pauling Institute at Oregon State University have questioned whether flavonoids are the main substances responsible for the benefit of fruit consumption, especially given that
many flavonoids are poorly absorbed and require much higher doses for efficacy than may be predicted from average fruit
consumption.46 These researchers tested the antioxidant
capacity in plasma of subjects after consumption of various
test foods and concluded that fructose mimicked the effects
of fruit (apples) with respect to supporting plasma antioxidant reserve. It was suggested that the effect of fructose on
urate promotes this higher antioxidant capacity.46
Excessive Consumption of Fructose
Intakes of fructose beyond that of 30% of calories can
promote undesirable metabolic consequences. For example,
because fructose can bypass the phosphofructokinase regulatory step that is the major controlling step in the intermediary metabolism of glucose in the liver, excessive fructose
can continue to be metabolized into substances such as lactate and triglycerides, and can also result in elevations of
uric acid.7,11,47 The pathway showing the influence of a high
fructose intake on uric acid is provided in Figure 2, and
involves the depletion of ATP and the increased catabolism
of adenosine to uric acid. Because elevated uric acid and
triglyceride levels have been associated with insulin resistance and metabolic syndrome (syndrome X), the suggestion has been made that high fructose intake may precipitate insulin resistance and hyperinsulinemia.4,12,20,21
Fructose and Gastrointestinal Symptoms
Consumption of large amounts of fructose in some
individuals may be associated with symptoms of Irritable
Bowel Syndrome (IBS), such as bloating, flatulence and
diarrhea. For example, Johlin et al. have investigated fructose intolerance in 256 patients referred to their clinic
because of the severity and idiopathic nature of their
abdominal pain and found evidence that 47 of these patients
had small bowel overgrowth (SBO), and 75.6% of those
without SBO (158 patients of the 256 referred) had an
intolerance to fructose as the major contributor to their
symptoms.48 The mechanism of this intolerance relates to
the amount of unabsorbed fructose that then traverses
through the intestinal tract, which can serve as an osmotic
load and draw fluid into the intestinal lumen and, after
reaching the lower intestine, be fermented by intestinal bacteria, producing excessive amounts of hydrogen, methane,
carbon dioxide and other gases.48-50 The resulting symp-
8
toms may include bloating, pain and discomfort, distention
of the small intestine, flatus and diarrhea. Choi et al. report
similar findings, in which 73% of 183 patients with unexplained gastrointestinal symptoms had evidence of unabsorbed fructose.49
Upon reviewing the literature on the contribution of
dietary fructose to gastrointestinal symptoms, Skoog and
Bharucha indicate that incomplete fructose absorption can
be seen in many healthy individuals at high doses of fructose. For example, incomplete absorption of fructose after a
50 g load (delivered as a 10% solution) was observed in
37.5% of healthy subjects.50 A higher percent of people
show unabsorbed fructose when more concentrated fructose
solutions are used, such as 20% solutions, even at the same
total load, but a 10% solution closely approximates most
carbonated beverages. Fructose malabsorption is considered higher in patients with functional bowel disorders,
although the prevalence is not well established.50 Hereditary
fructose intolerance has also been recognized as a cause for
some cases of fructose intolerance.51,52
In her book, Breaking the Vicious Cycle, Intestinal
Health Through Diet, Elaine Gottschall counsels that a specific diet restricted in fruits and certain vegetables and
increased in animal products is very successful in reducing
the problems associated with IBS for many patients.53 Other
authors have noted that, because glucose enhances the
absorption of fructose, when glucose and fructose are taken
at equal amounts, the facilitation of fructose’s absorption by
the glucose can prevent the intolerance symptoms. Skoog
and Bharucha, in particular, have investigated this facilitation process and advocate that the fructose intolerant patient
should consume products that contain glucose at an equal or
higher amount to the fructose to avoid symptoms.50 Certain
amino acids also appear to facilitate the absorption of fructose. It is important to note that sorbitol, a sugar alcohol
found naturally in foods, has the opposite effects and
inhibits fructose absorption.50 Sorbitol and fructose occur
together in many fruits, including apples, cherries, peaches
and pears. Interestingly, bananas and strawberries do not
contain sorbitol. Understanding these subtleties of fructose
absorption may allow for more choices for patients with
functional bowel disorders like IBS.
SUMMARY
The USDA has recommended that, depending on calorie intake, people consume no more than 6% to 10% of their
calories from added sugars, and the World Health
Organization has recommended that people limit their intake
of added sugars to 10% or less of total calories. The Institute
of Medicine, which provides nutritional advice to the United
States Food and Drug Administration, has advised that people get 25% or fewer of their calories from added sugars. For
a 2,000 Kcal/day diet, these guidelines constitute anywhere
9
from 30 g to 125 g added sugars. It is interesting to note that
one soft drink alone delivers from 40 g to 52 g of added
sugar, while sweetened fruit beverages may have even more
added sugar. Studies should identify what is actually contributing to the substantial health concerns of today – that is,
whether it is the specific sugar, fructose, or whether it is the
impact of processed foods with high amounts of added sugars and no nutritional value–, as well as provide clear details
on the context within which the fructose is consumed.
Given the evidence presented over the past several
years, it is apparent that a principal issue in this debate is
the displacement of "quality calories" by empty calories
coming from such products as HFCS-sweetened beverages–where added sugars in one beverage constitute the
entire amount of added sugar recommended by world
health organizations. Modest intake (<20%) of fructose has
not been demonstrated to have adverse effects on either
serum lipids or insulin sensitivity. Quite the contrary, modest intake of fructose as part of a low glycemic load diet has
been associated with improved hepatic glycogen storage,
reduced postprandial insulin and glucose, and a lower
triglyceride-to-HDL cholesterol ratio, all of which indicate
improved insulin sensitivity rather than reduced insulin sensitivity.
Much of the difficulty of making conclusions about the
effect of fructose relates to the varying compositions of the
diets in different studies, the differing magnitudes of fructose doses and the specific source of fructose. These differences are often extraordinary, leading to difficulty in a
direct interpretation of the adverse metabolic effects of
fructose. Furthermore, the lack of standardization of a test
diet with respect to glycemic load complicates analysis considerably. While it is clear that excessive fructose consumption can have adverse metabolic effects in some individuals,
fructose is considered an important part of the overall
dietary intake. Fructose is a low-glycemic index alternative
sweetener source and may provide benefit as an antioxidant.
When consumed at reasonable dietary levels, fructose does
not lead to significant adverse events.
As has been pointed out by Fried and Rao from the
Department of Nutritional Science at Rutgers University,
the magnitude of the effect that sugars have on triglyceride
concentrations depends on aspects of the total diet.54 Too
often, studies take one principal out of context, study it in
an exaggerated intervention trial, and then form general
conclusions from it. Such is often the case with regard to
studies that have been done with fructose-enriched diets,
where the amount of fructose that is added is well beyond
that which an individual normally consumes. In order to
truly understand how a specific diet containing fructose
influences metabolic function, an appropriate intervention
trial against a control needs to be accomplished. It is only
through this type of clinical evidence that reasonable con-
JANA Vol. 8, No. 3, 2005
clusions can be drawn. Until then, clinicians might consider these guidelines with their patients:
1. Minimize the intake of total sugars.
2. Remove sweetened fruit drinks and soft drinks, and do
not use table sugar to sweeten food.
3. Promote a diet with a low glycemic load, moderate fat
intake, and high in complex carbohydrates.
4. Allow for moderate intake of sugars, including fructose,
when included as part of a complex food.
In conclusion, modest consumption of fructose has been
demonstrated to have beneficial effects on blood sugar and
lipid parameters. High intakes of fructose, however, have
resulted from the increased consumption of processed sweeteners like HFCS over the past few decades. This increase in
fructose consumption is much greater than what the human
body has experienced and managed over the past centuries.
It appears that the availability of high processed foods is a
primary issue in this supranormal fructose intake.
Therefore, focusing on the quality of the diet, not just the
removal of a specific sugar that has value in modes amounts,
should be considered during nutritional counseling.
ACKNOWLEDGEMENTS
I thank Kathy Sawyer for editorial assistance, DeAnn
Liska, PhD, for medical writing, and Deanna Minich, PhD,
CN, and Bhaskar Shenai, PhD, for technical comments
and review.
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26. Dirlewanger M, Schneiter P, Jequier L, Tappy L. Effects of
fructose on hepatic glucose metabolism in humans. Am J
Physiol Endocrinol Metab. 2000;279:E907-11.
27. McGuinness OP, Cherrington AD. Effects of fructose on
hepatic glucose metabolism. Curr Opin Clin Nutr Metab
Care. 2003;6:441-8.
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28. Havel PJ. Dietary fructose: implications for dysregulation of
energy homeostasis and lipid/carbohydrate metabolism. Nutr
Rev. 2005;63(5):133-57.
29. Bantle JP, Raatz SK, Thomas W. Effects of dietary fructose on
plasma lipids in healthy subjects. Am J Clin Nutr.
2002;72:1128-34.
30. Reiser S, Powell AS, Scholfield DJ, Panda P, Fields M, Canary
JJ. Day-long glucose, insulin, and fructose responses of hyperinsulinemic and nonhyperinsulinemic men adapted to diets
containing either fructose or high-amylose cornstarch. Am J
Clin Nutr. 1989;50:1008-14.
31. Osei K, Falko J, Bossetti BM, Holland GC. Metabolic effects
of fructose as a natural sweetener in the physiologic meals of
ambulatory obese patients with type II diabetes. Am J Med.
1987;83249-55.
32. Van den Berghe G. Fructose: metabolism and short-term
effects on carbohydrate and purine metabolic pathways. In:
Macdonald I, Vrana A, eds. Metabolic effects of dietary carbohydrates. Prog Biochem Pharmacol. 1986;21:1-32.
33. Abraha A, Humphreys SM, Clark ML, Metthews DR, Frayn
KN. Acute effect of fructose on postprandial lipademia in diabetic and non-diabetic subjects. Br J Nutr. 1998;80(2):169-75.
34. Busserolies J, Gueux E, Rock E, Mazur A, Rayssiguier Y.
Substituting honey for refined carbohydrates protects rats from
hypertriglyceridemic and prooxidant effects of fructose. J
Nutr. 2002;132:3379-82.
35. Havel P. Glucose but not fructose infusion increases circulating leptin in proportion to adipose stores in rhesus monkeys.
Exp Clin Endocrinol Diabetes. 1997;105:37-8.
36. Teff KL, Elliott SS, Tschöp TJ, et al. Dietary fructose reduces
circulating insulin and leptin, attenuates postprandial suppression of ghrelin, and increases triglycerides in women. J Clin
Endocrinol Metab. 2004;89(6):2963-72.
44. Kris-Ehterton PM, Hecker KD, Bonanome A, Coval SM,
Binkoski AE, Hilpert KF, Griel AE, Etherton TD. Bioactive
compounds in foods: their role in the prevention of cardiovascular disease and cancer. Am J Med. 2002;113 (supplº
9B:71S-88S.
45. Bazzano LA, Serdula MK, Liu S. Dietary intake of fruits and
vegetables and risk of cardiovascular disease. Curr
Atheroscler Rep. 2003;5:492-9.
46. Lotito SB, Frei B. The increase in human plasma antioxidant
capacity after apple consumption is due to the metabolic effect
of fructose on urate, not apple-derived antioxidant flavonoids.
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47. Dills Jr WL. Protein fructosylation: fructose and the Maillard
reaction. Am J Clin Nutr. 1993;58(suppl):779S-87S.
48. Johlin FC Jr, Panther M, Kraft N. Dietary fructose intolerance:
diet modification can impact self-rated health and symptom
control. Nutr Clin Care. 2004;7(3):92-7.
49. Choi YK, Johlin FC Jr, Summers RW, Jackson M, Rao SS.
Fructose intolerance: an under-recognized problem. Am J
Gastroenterol. 2003;98:1348-53.
50. Skoog SM, Bharucha AE. Dietary fructose and gastrointestinal
symptoms: a review. Am J Gastroenterol. 2004;99(10):2046-50.
51. Hommes FA. Inborn errors of fructose metabolism. Am J Clin
Nutr. 1993;58(5 suppl):788S-95S.
52. Ali M, Rellos P, Cox TM. Hereditary fructose intolerance. J
Med Genet. 1998;35:353-65.
53. Gottshall E. Breaking the Vicious Cycle: Intestinal Health
through Diet. Baltimore, MD: Kirkton Press Ltd. 1994.
54. Fried SK, Rao SP. Sugars, hypertriglyceridemia, and cardiovascular disease. Am J Clin Nutr. 2003;78(suppl):873S-80S.
37. Stern L, Iqbal N, Prakash S, Chicano KL, et al. The effects of
low carbohydrate versus conventional weight loss diets in
severely obese adults: one-year follow-up of a randomized
trial. Ann Intern Med. 2004;140:778-85.
38. Willett WC. Editorial: Reduced-carbohydrate diets: No roll in
weight management? Ann Intern Med. 2004;140(10):836-37.
39. Wood PA. Genetically modified mouse models for disorders of
fatty acid metabolism: pursuing the nutrigenomics of insulin
resistance and Type 2 diabetes. Nutrition. 2004;20:121-26.
40. Kelley DE. Sugars and starch in the nutritional management of
diabetes mellitus. Am J Clin Nutr.2003; 78(suppl):858S-64S.
41. Rigden S, Lerman RH. Successful management of complicated high-risk obese patients using a comprehensive program
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42. Lukaczer D, Liska DJ, Lerman RH, Darland G, Schiltz B,
Tripp M, Bland JS. Effect of a low-glycemic-index-diet with
soy protein and phytosterols on CVD risk factors in postmenopausal women. Nutrition. 2005; in press.
43. McLaughlin T, Abbasi F, Cheal K, Chu J, Lamendola C,
Reaven G. Use of metabolic markers to identify overweight
individuals who are insulin resistant. Ann Intern Med.
2003;139:802-9.
11
JANA Vol. 8, No. 3, 2005
P
I L O T
S
T U D Y
Cholesterol and Triglyceride-Lowering Effect
of Two Spice Preparations
Eva Lydeking-Olsen, NP, BSc,*
Janne Springborg Clewlow, MSc, Vita Damsoe, BSc,
An-Mari Mey Hansen, NP, BSc
Institute for Optimum Nutrition, Copenhagen, Denmark
ABSTRACT
Background
The use of non-pharmacologic dietary supplements is
of potential benefit in addressing hypercholesterolemia, a
well-established risk factor for ischemic heart disease.
decrease of 13% on total cholesterol and a borderline significant effect with a decrease of 16% on LDL cholesterol.
Ratios between the atherosclerotic (total and LDL) cholesterol and the protective HDL-cholesterol improved by 11%
(TC:HDL ratio) and 14% (LDL:HDL ratio). The U85-2
showed no effects.
Objective
Two herbal products were investigated for their cholesterol-lowering effects.
Design
This investigation was a 24-week, double-blind study
with 20 participants. U85-2 contained tannin, cayenne pepper and vanillin; U100-3 contained turmeric, ensian root,
hot paprika and vanillin. At inclusion, plasma cholesterol
was > 6 mmol/l, or with one or more risk factors: > 5 or 4.5
mmol/l respectively. Previous self-reported dietary patterns
(mean fat/energy %: 34) and physical activity levels were
maintained as estimated by indirect methods: no changes
were seen in weight or hip/waist measure.
Results
U100-3 had a significant effect with a decrease of 16%
on non-HDL cholesterol, a close to significant effect with a
* Correspondence:
Eva Lydeking-Olsen, NP
Director, Institute for Optimum Nutrition
Teglgårdstræde 4, 1st floor
DK-1452
Copenhagen, Denmark
Tel: 45 3332 4480 Fax: 45 3332 4481
E-mail: forskning@optinut.dk
JANA Vol. 8, No. 3, 2005
CONCLUSION
The U100-3 preparation has a beneficial and clinically
relevant long-term effect and should be investigated further
for treatment of mild to moderate hypercholesterolemic
individuals.
Key Words: hypercholesterolemia, spice preparation,
paprika, turmeric, vanilla, gentian.
BACKGROUND
Cardiovascular disease is one of the most significant
health problems in the western world, causing loss of both
quality of life and life years as well as being a socio-economic burden. Cardiovascular disease develops as an interaction between a number of factors – including heredity,
lifestyle, diet and physical activity.
Hypercholesterolemia is a known risk factor for
ischemic heart disease. Approximately half the Danish population has plasma cholesterol levels over 5 to 6 mmol/l.
Mild to moderate hypercholesterolemia is presumably due,
for the most part, to dietary and lifestyle factors.
The therapeutic targets most often suggested are a total
plasma cholesterol level of ≤ 5.0 or 5.2 mmol/l, LDL cholesterol (low-density lipoprotein cholesterol) ≤ 3.5 mmol/l,
12
HDL cholesterol (high-density lipoprotein cholesterol) ≥
1.0 mmol/l, and plasma triglyceride ≤ 2.0 mmol/l
(www.americanheart.org). For each 1% reduction in LDL
cholesterol, the risk of a blood clot in the heart is reduced
by at least 2%, and for each 1% increase in HDL cholesterol, the risk is reduced by 3% to 4%.1,2,3
Red rice (rice fermented with a special yeast, Monascus
purpureus) acts at an earlier stage of cholesterol metabolism
by inhibiting cholesterol synthesis. This cholesterol synthesis inhibition has produced reductions in total cholesterol of
11% to 32%, and in LDL cholesterol of about 22%.13 Red
rice has a mechanism of action identical with that of statins,
a group of pharmaceutical products approved to treat hypercholesterolemia. In fact, it has been shown that one of its
active ingredients is lovastatin, a selective inhibitor of
HMG-CoA reductase, a rate-limiting enzyme in cholesterol
biosynthesis. This being the case, there have been disputes
about whether red rice should be approved as a dietary supplement or a pharmaceutical product.
An important risk factor of thromboembolism related
to cholesterol levels is the ratio between total or LDL cholesterol and HDL cholesterol – a ratio reflecting whether
cholesterol is deposited (LDL) or transported for decomposition and elimination (HDL).4,5 It is desirable to achieve a
ratio between total cholesterol and HDL ≤ 4.2 and a corresponding ratio between LDL and HDL cholesterol of < 2.5.6
The reduction in cholesterol levels and an enhanced ratio
between the different types are best achieved by adjustments in diet and lifestyle combined with dietary supplements, and in severe cases where these measures prove
inadequate, with medication. In addition, prevention of
ischemic heart disease requires modification of other risk
factors such as smoking, diabetes, overweight, inactivity,
hyperhomocysteinemia, hypertension, and the elevation of
plasma lipoprotein (a), depending on the individual risk
profile. Various dietary supplements have exhibited a cholesterol-lowering effect by different mechanisms of action.
These supplements include guar gum, psyllium, red rice
and garlic. Diet alone has been found to reduce blood cholesterol, and from a health-economic point of view, it compares well with medication.3,7,8 However, a strict diet can be
difficult to maintain, and in those individuals who are
unable or unwilling to fully comply, the effect on cholesterol levels is inadequate.
The mechanism of action in garlic preparations is
unknown, but presumed to be due to allicin, which is
formed by conversion of the compound alliin.14 Results
from human clinical trials show considerable inconsistency
regarding garlic and its cholesterol-lowering effect. Some
short term clinical studies of up to 3 months duration15,16
show a reduction in total cholesterol of about 10%, and in
LDL cholesterol of 10% to 16%. Contrary to these results,
the last three years have produced six well-designed studies
that failed to prove a beneficiary effect of garlic after three
to six months of treatment.17,18
U85-2 and U100-3 are two different spice preparations
with potential cholesterol-lowering effects. U85-2 contains
tannin, vanillin and cayenne pepper, while U100-3 contains
turmeric, gentian root, hot paprika, and vanillin. Table 1
specifies the composition of the two preparations. Tannin is
available from grapefruit seeds, tea and wine, while turmeric, cayenne, vanilla and paprika are well-known spices and
gentian is an ancient medicinal plant.
Guar gum and psyllium are rich in fibers and bind to
water in the intestinal contents, thereby inhibiting intestinal
cholesterol uptake and enhancing fecal excretion of dietary
cholesterol.9 Psyllium also upregulates cholesterol turnover
by increasing enzyme activities related to both cholesterol
synthesis and catabolism.10 In clinical trials with humans,
guar gum has been shown to reduce total cholesterol by
10% to 15% and LDL cholesterol by 10% to 20%,11 while
psyllium may lower total cholesterol by about 4% and LDL
cholesterol by about 7%.12
Animal trials have shown that both cayenne and tannin
significantly lower blood levels of cholesterol and triglycerides and improve the HDL:LDL ratio.19,20,21 Conversion
into bile salts is the body's primary route of eliminating
cholesterol,22,23 and the mechanisms of action of cayenne,
vanilla and turmeric work by stimulating the formation and
secretion of bile salts from the liver. Furthermore, cayenne
enhances hepatic metabolism of lipids by increasing the
enzyme levels, thereby reducing fatty deposits in the liver.19
Table 1. Composition of the two tested herbal/spice mixtures (mg per daily dosage, 4 tablets)
U85-2
Cayenne (Capsicum spp)*
Tannin
Hot paprika (Fructus Capsici aedelsuss sharp)
Gentian root (Radix Gentiana)
Turmeric** (Rhizoma Curcumae javan.)
Vanillin
U100-3
80
120
20
100
300
500
20
* Capsaicin content 0.4%
** Cucurmin 1.0% to 1.3% (double determinations), essential oils 4% to 5% (double determination)
13
JANA Vol. 8, No. 3, 2005
Tannin has a different mechanism of action and acts by
binding to cholesterol in the intestine. In this way, it reduces
cholesterol absorption significantly and increases the
amount of cholesterol excreted with the feces.24
supplied (Soehnle Domino) and subsequently analysed
electronically in Dankost 3000 on the basis of the national
food-composition tables.
ANALYSIS
MATERIALS AND METHODS
Objectives and Design
The objective of this study was to investigate U85-2
and U100-3 for their potential cholesterol-and triglyceridelowering effects in a 24-week double-blind, prospective
study. The project was approved by the Danish Data
Protection Agency (File no. 1999-1200-343) and the regional Ethics Committee (File no. (KF) 07-00-043/02).
Participants were informed, verbally and in writing, and
signed informed consent documents.
Inclusion Criteria
Included in the study were adult individuals, at least 18
years of age and with no upper age limit, of both sexes with
total cholesterol values:
Analysis of blood lipids was performed on freshly
drawn blood at weeks 0 and 24 following overnight fasting.
Total cholesterol, HDL cholesterol and triglycerides were
measured by a standard enzymatic technique at a precision
of 2%, 6% and 3%, respectively, at Nova Medical Medilab
(Copenhagen, Denmark), which has GLP status. LDL cholesterol values were calculated by the Friedewald equation
LDL = (TC-HDL-(TG/5)).25 Data were entered into Excel®
and analysed by the SAS® 8.2 statistics package (SAS INC,
Cary, NC) and StatXact 5. The effect of dietary supplements on cholesterol values was evaluated by a non-parametric test, the Wilcoxon test for paired differences, as data
were not normally distributed. A two-sided p value < 0.05
was considered significant. The randomisation code was not
broken until after the final analysis.
• > 6 mmol/l without any other risk factors
• > 5 mmol/l with one other risk factor
RESULTS
• > 4.5 mmol/l with at least two risk factors
Twenty subjects completed the study, and one of these
subjects was excluded from the analysis because of a
change in existing medication (hormone replacement therapy) during the study. Two subjects received antihypertensives and continued with their regimens. One individual
with known multiple allergies was an early drop-out
because of a potential hypersensitivity reaction.
Risk factors were smoking, overweight/obesity, hypertension, diabetes, hereditary disposition and vascular disorders/atherosclerosis.
Exclusion Criteria
• known, familial, severe hypercholesterolemia
• treatment with cholesterol-lowering medication or cholesterol-lowering food supplements
• children, pregnant women and alcohol and drug abusers
Recruitment took place in the clinic and in general
practices in the local area of Copenhagen.
A total of 21 individuals were randomised, double-blind,
to one of two treatments. Randomisation lists were generated by the use of a table with random numbers, by a pharmacist trained in these procedures who was not dealing with the
data acquisition, thus neither the subjects nor the staff handling data acquisition knew which group subjects were
included. The food supplements to be tested were of similar
shape, color and smell (vanilla), rendering the contents
indistinguishable. The dosage of the dietary supplements
was 2 tablets bid (morning and evening), with the possibility of reduction in case of adverse reactions. Dispensing more
tablets than needed for each period and counting the number
of tablets left over assessed compliance.
Subjects were asked not to change their diets and physical activities, or their administration of dietary supplements and existing medication throughout the study.
Dietary intake (food) was recorded/weighed daily at
the start of the study for seven days on the electronic scales
JANA Vol. 8, No. 3, 2005
Table 2 shows baseline characteristics for the two
analysis groups. As seen from table 2, the two analysis
groups were comparable with regards to baseline characteristics (age, height, weight, BMI, number of current or previous smokers, use of antihypertensive medication and
dietary energy intake). The fat intake was quite low because
the subjects in both groups had been previously diagnosed
with hypercholesterolemia years prior to the study. In
response to this diagnosis, most of them had already
changed to a more low-fat diet, but without adequate effect.
Adverse reactions were few and mild. In the U85-2
group, one subject became nauseous from the tablets and
one suffered mild colic for a few weeks at the start, which
abated without dose reduction or interruption. In the U1003 group, one subject suffered colicky pain, which ceased
after dose reduction to 2-3 tablets a day. One subject reported dizziness, headache, flushing of the face, impaired concentration, fatigue and joint stiffness. These reactions
appeared within two weeks from the start in an individual
with known multiple allergies, were dose-dependent and
tapered off a few days after the dietary supplement was discontinued without any sequelae. The capsaicin content of
hot paprika has a vasodilatatory effect, which may lead to
the reactions described in sensitive individuals. The subject
14
Table 2. Baseline characteristics1
Treatment group
U85-2
(n = 10)
U100-3
(n = 9)
Age (years)
55.5 ± 14.5
(33 - 74)
64.0 ± 8.8
(47 - 77)
Height (m)
1.71 ± 0.1
1.74 ± 0.07
Weight (kg)
78.2 ± 11.2
77.7 ± 14.6
BMI (kg/m2)
26.9 ± 3.4
(22.4 - 32.8)
25.6 ± 3.4
21.1 - 30.5)
1
3
2
1
6
1
8946 ± 3077
(5,340 – 14,562)
9902 ± 2403
(7,470 – 13,814)
33.9
15.8
46.2
4.1
33.8
15.6
41.6
9.0
Variable
Current smokers (number)
Previous smokers (number)
Antihypertensive therapy (number)
Energy consumption (KJ/day)
Energy %
• Fat
• Protein
• Carbohydrate
• Alcohol
1. mean + SD, range in parenthesis. The two groups showed no statistically significant difference between the variables.
in question dropped out of the study, and no attempts were
made to resume therapy in order to establish causality.
Compliance was similar in the two groups: 93.2% in
the U85-2 group and 94.5% in the U100-3 group.
One subject in each group took more tablets than
scheduled (137% and 129%, respectively), with no adverse
reactions. The absence of any major discomfort or adverse
reactions, apart from those mentioned during the six months
of the study, indicates that both preparations are non-toxic
and well tolerated.
Table 3 shows the results of the lipid analyses at the
start of the study (week 0) and after 24 weeks.
U85-2: A non-significant decrease was seen in all parameters except for HDL, where a non-significant increase
was observed.
U100-3: A statistically significant decrease was seen
for the non-HDL cholesterol of 1.64 mmol/L (p=0.035).
The decrease in non-HDL cholesterol corresponds to 16%.
For total cholesterol, a non-significant decrease of 1.69
mmol/L (p=0.055) was observed. The decrease in total cholesterol corresponds to 13%.For LDL cholesterol, a borderline significant decrease of 0.95 mmol/L (p=0.051) was
observed. The decrease in LDL cholesterol corresponds to
16%. For HDL cholesterol, no change was observed during
the study period.For Triglyceride, a non-significant decrease
of 0.27 mmol/L (p=0.24) was observed. The decrease in
Triglyceride corresponds to 16%. No study subjects had
15
excessively elevated triglyceride values, but the highest values tended to decrease the most (by 40% to 60%).For the
ratio between total cholesterol and HDL cholesterol, a nonsignificant decrease of 0.52 (p=0.098) was observed. The
decrease in ratio corresponds to 11%.
For the ratio between LDL and HDL cholesterol, a
non-significant decrease of 0.52 (p=0.074) was observed.
The decrease in ratio corresponds to 14%.
The effects of the preparations bore no relation to body
weight, age or compliance, which supports the choice of
dosage. There were no clinical or statistically significant
changes in blood pressure, body weight, BMI, waist or hip
circumference and waist:hip ratio during the study (data not
shown), indicating that the subjects maintained their dietary
patterns and physical activity levels. Consequently, it is less
likely that the differences observed in lipid values are due to
changes in diet or physical activity levels during the study.
DISCUSSION
The ingredients in U100-3 inhibit intestinal absorption
of cholesterol. In addition, they act by direct stimulation of
hepatic formation and secretion of bile salts from cholesterol, which means that they inhibit cholesterol absorption
while increasing elimination (McCaleb 1993, Sambaiah
1980, Srinivasan 1992 and 1993).
A review of literature shows that a change in diet alone
can reduce serum total cholesterol by 10−15% and LDL
JANA Vol. 8, No. 3, 2005
Table 3. Lipid concentrations in subjects treated with the spice preparations.
Parameter
Start
Mean SEM
Week 24
Mean SEM
Mean SEM
U-100-3, n= 9
Total Cholesterol
HDL Cholesterol
LDL Cholesterol1
Non-HDL Cholesterol
Triglyceride
Total Cholesterol : HDL ratio
LDL1 -HDL ratio
7.77
1.66
5.77
6.11
1.70
4.86
3.64
0.54
0.13
0.51
0.51
0.17
0.40
0.39
6.77
1.66
4.82
5.11
1.43
4.34
3.12
0.43
0.13
0.45
0.44
0.21
0.50
0.45
-1.00
0.00
-0.95
-1.00
-0.27
-0.52
-0.52
0.56
0.04
0.54
0.55
0.19
0.30
0.28
-13
-0.0
-16
-16
-16
-11
-14
-16.5
-1.00
-16.5
-17.5
-10.5
-14.5
-15.5
0.055
1.000
0.051
0.035
0.238
0.098
0.074
U-85-2, n=10
Total Cholesterol
HDL Cholesterol
LDL Cholesterol 1)
Non-HDL Cholesterol
Triglyceride
Total Cholesterol : HDL ratio
LDL1 - HDL ratio
6.83
1.60
4.89
5.23
1.70
4.56
3.31
0.23
0.14
0.26
0.27
0.28
0.43
0.38
6.64
1.66
4.69
4.98
1.45
4.44
3.07
0.26
0.15
0.30
0.31
0.24
0.41
0.33
-0.19
0.06
-0.20
-0.25
-0.25
-0.12
-0.24
0.30
0.07
0.29
0.29
0.23
0.20
0.18
-2.8
3.8
-4.1
-4.8
-15
-2.7
-7
-5.50
7.50
-6.50
-8.00
-6.50
-6.00
-6.50
0.541
0.480
0.539
0.443
0.477
0.574
0.557
Difference from start to week 24
Pct change Test
Two-sided exact
p value2
in means statistic2
Units are mmol/L.
1. LDL cholesterol calculated using the Friedwald equation: LDL = TC - HDL - TG/5.
2. Test used: Wilcoxon Signed Rank Test for paired differences on the difference from start to week 24.
cholesterol 10–16%.7, 26 In studies of dietary supplements,
it is important that the subjects do not change their dietary
patterns during the study period; therefore, we instructed
the subjects in the present study to strictly maintain their
dietary patterns and physical activity level.
U100-3 therapy led to a reduction in total cholesterol of
13% and in LDL cholesterol of 16%. This reduction corresponds to the levels obtained by guar gum11 and in the most
optimistic studies, by garlic.15,16 However, guar gum has
rarely been investigated in studies of similar long duration,
which is relevant in evaluation of cholesterol-lowering food
supplements and medications where a sustained effect is
highly important. A comprehensive meta-analysis of studies
investigating the cholesterol-lowering effect of garlic has
shown that the studies found a reduction in total cholesterol
at one and three months, but not at six months.17,18 It is possible that the body adjusts to a cholesterol-lowering therapy
so that the effect may be transient.
This study is somewhat limited due to the small number of subjects; however, the results are statistically significant and of a clinically interesting magnitude.
It can be argued that the initial group differences in the
total and LDL cholesterol values is of clinical importance,
but these differences arise from one subject with high initial
values in the U100-3 group and one subject with rather low
initial values in the U85-2 group. As the distribution of subjects was due to chance because of the randomisation, it is
not appropriate to exclude any of these subjects from the
JANA Vol. 8, No. 3, 2005
analysis and the use of a non-parametric test minimizes the
impact of outliers.
It could also be stated that other factors were in play,
such as dietary changes. As we did not undertake a second
dietary registration at the end of study, this factor cannot
entirely be ruled out. If the diet had changed, some of the
indirect indicators of dietary changes, such as weight, BMI
or anthropometric measures of waist and waist:hip ratio,
would most likely have changed. However, these statistics
remained unchanged. Additionally, the subjects had dietary
counselling and had changed their diets long before the
onset of this study, and it is unlikely that dietary changes
would have occurred in the U100-3 group only.
It was not possible to examine other potential physiological effects of the products such as the effect on the cell
membranes and platelet aggregation, or the anti-oxidative
effect. Animal trials have demonstrated that both turmeric
and capsaicin (present in cayenne and paprika) stabilize cell
membranes and reduce their cholesterol contents,27 and capsaicin also inhibits platelet aggregation and displays antioxidative properties.28 Turmeric has been fairly thoroughly
investigated for cancer prophylaxis, although not yet in
humans,29 and both turmeric and capsaicin inhibit cyclooxygenase-2, an enzyme involved in various inflammatory
disorders such as arthritis, migraine and pain disorders.30
These findings from in-vitro and animal research suggest
that spice preparations may have other favourable long-term
effects apart from those investigated here.
16
The adverse reaction profile of U100-3 was also positive,
with only two subjects experiencing symptoms. One of them
had known multiple allergies with general hyperreactivity,
while the other developed abdominal pain that ceased at dose
reduction. Side effects for other preparations used for the
same purpose such as garlic preparations, would in all cases
give a strong and characteristic odor, while colicky pain and
flatulence could accompany guar gum and psyllium intake.
CONCLUSION
The study has shown that U100-3, a dietary supplement
containing the spices hot paprika, vanillin, turmeric and
gentian, has a beneficial and clinically relevant long-term
effect in humans with hypercholesterolemia. U100-3 lowers
cholesterol levels by affecting both total and LDL cholesterol, but not HDL cholesterol. It is a favorable aspect that
the HDL cholesterol level is maintained, thereby improving
the ratios between the atherosclerotic cholesterol forms (TC
or LDL) and the protective HDL cholesterol. A larger controlled clinical trial is warranted.
ACKNOWLEDGEMENTS
The authors would like to thank the subjects enrolled for
having invested their time and energy in this study, nutritionist Caritas Locher for having performed the dietary analysis ,
and statistician Martin Eeg for statistical supervision.
This study was supported by a grant from MB Pharmos
A/S, Copenhagen, Denmark, which has the patent rights in
U100-3. U100-3 is marketed under the brand name of
Lippital.®
CONTRIBUTIONS AND INTERESTS:
Eva Lydeking-Olsen designed the study, analysed the
data and wrote the paper together with Janne Springborg
Clewlow. Vita Damsoe and An-Mari Mey Hansen took care
of data accrual and contributed to the writing of the paper.
Institute for Optimum Nutrition, Holistic Therapy and
Research is a private non-profit institute, independent of
MB Pharmos, and none of the authors are affiliated with
MB Pharmos. The sponsor and supplier of the study
approved the study protocol, but had no role in data analysis, data interpretation, or writing of the report.
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Kinosian B. Cholesterol measures to identify and treat
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of incremental programmes for lowering serum cholesterol concentration: is individual intervention worthwhile? BMJ. 1991;302:1119-1122.
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10. Romero AL, West KL, Zern T, Fernandez ML.The seeds
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11. Todd PA, Benfield P, Goa KL. Guar gum, a review of its
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12. Anderson J, Allgood L, Lawrence A et. al. Cholesterollowering effects of psyllium intake adjunctive to diet
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13. Heber D, Yip I, Ashley JM, Elashoff DA, Elashoff RM,
Go VLW. Cholesterol-lowering effects of a propietary
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use of alliinase. Pharmazie. 1999 Apr;54(4):289-293.
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preparations on blood lipids, blood pressure and wellbeing. Br J Clin Res. 1995;6:91-100.
17. Kerckhoffs DAJM, Brouns F, Hornstra G, Mensink RP.
Effects on the human serum lipoprotein profile of βGlucan, soy protein and isoflavones, plant sterols and
stanols, garlic and tocotrienols. J Nutr. 2002;132:24942505.
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18.Ackermann RT, Mulrow CD, Ramirez G, Gardner CD,
Morbidoni L, Lawrence VA. Garlic shows promise for
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18
O
R I G I N A L
R
E S E A R C H
Reduction in the Frequency of Arrhythmic
Episodes in Patients with Paroxysmal Atrial
Arrhythmia with a Vitamin/Essential Nutrient
Supplementation Program
Matthias Rath, MD, Tatiana Kalinovsky, MSRN,* Aleksandra Niedzwiecki, PhD
Matthias Rath Research Institute, Santa Clara, California
ABSTRACT
Chronic deficiency of vitamins and other essential
nutrients impairs cellular bio-energy production and can
lead to disturbances in the generation and conduction of
electrical impulses in the myocardium. We investigated the
effect of long-term supplementation with a combination of
vitamins and other essential nutrients on the number of clinically apparent episodes in patients with paroxysmal atrial
arrhythmia. A randomized, double-blind, placebo-controlled, multi-center study in Germany was undertaken on
131 patients (ITT), aged 18 to 70 years, diagnosed with
paroxysmal atrial arrhythmia, who were receiving antiarrhythmic medication for at least three months, and who
reported at least one paroxysmal cardiac episode per month.
Study participants were advised to continue their prescribed
medication and were treated with either an essential nutrient formulation or placebo during the 24-week study.
Analysis of data demonstrated a significant decrease in the
frequency of clinically apparent arrhythmic episodes with
vitamin/essential nutrient supplementation (ITT analysis:
* Corresondence:
Tatiana Kalinovsky, MSRN
13980 W. Bell Rd, Ste11-125
Surprise, AZ 85374
Phone: 623-266-3618 Fax: 623-266-3618
Email: t.kalinovsky@drrath.com
19
p=0.0221; PP analysis: p=0.0160) that improved with time
(45.5% of the supplemented group experienced frequent
arrhythmic episodes at three months, in contrast to only
27.3% at six months). By addressing the underlying cause
of arrhythmia, a deficiency in nutrients that generate bioenergy in the heart muscle cells, a vitamin/essential nutrient supplementation program provides a safe and effective
reduction of arrhythmic episodes.
Key Words: atrial paroxysmal arrhythmia, nutrient synergy,
carnitine, coenzyme Q10, bioenergy, lysine, vitamin C, B
vitamins
INTRODUCTION
Chronic deficiency of vitamins and other essential
nutrients impairs cellular bio-energy production, and can
lead to disturbances in the generation and conduction of
electrical impulses in the myocardium. This disturbance of
electrical impulses due to chronic vitamin and nutrient deficiency can be an underlying cause of the majority of
arrhythmic episodes of unknown origins, according to the
Rath concept.1,2 There is an accumulating body of evidence
supporting the beneficial effects of optimal levels of vitamins and other essential nutrients on the metabolic, nutritional and functional status of the myocardium.3 Our previous study on the synergistic effect of a combined vita-
JANA Vol. 8, No. 3, 2005
min/essential nutrient formula on progression of coronary
artery calcification in patients with documented coronary
artery disease, using Ultrafast CT, observed a decrease from
a 44% calcification rate prior to intervention to 15% after
the course of one year of nutritional supplementation.4
To generate electricity, the “electrical cells” of the heart
need large amounts of bio-energy. Therefore, they need a constant supply of nutrients that facilitate the conversion of food
into cellular energy. The most critical among them are: coenzyme Q10, carnitine, the B vitamins, lysine, and vitamin C,
together with magnesium, calcium and potassium.2,4 McCarty
suggests the use of meaningful doses of the mitochondrial
megavitamins as protection from cardiovascular disease.5
Carnitine, an often deficient non-essential amino acid,
is essential for cardiac energy production. It is produced
from lysine, an essential amino acid, with the participation
of vitamin C. Since both lysine and vitamin C are not produced in the human body, their deficiency is likely to impair
endogenous carnitine levels. The carnitine molecule is necessary to translocate fatty acids through the outer mitochondrial membrane for conversion to ATP in order to sustain heart function. A hospital-based, double-blind clinical
study of patients admitted after myocardial infarction
demonstrated that intake of 2 grams of carnitine per day for
four weeks cut the number of complications from arrhythmia, angina and heart failure in half.6
Like carnitine, coenzyme Q10 is essential for ATP production by the mitochondria. Low levels of coenzyme Q10
have been reported to be associated with increased severity
of heart failure.7 Three months of adjunctive treatment of
congestive heart patients with coenzyme Q10 resulted in
reduction of arrhythmias in 62% of the treatment group in
contrast to the placebo group.8 Furthermore, a one-month
coenzyme Q10 treatment of patients with acute myocardial
infarction, reduced angina pectoris and total arrhythmias,
and improved ventricular function.9
In addition to carnitine and coenzyme Q10, other nutrients such as magnesium, the B vitamins, vitamin C and vitamin E help optimize the pumping performance of the
heart.10,11 These nutrients optimize the function of the
heart’s electrical cells, as well as the myocardial smooth
muscle cells of the blood vessel walls, supporting regular
heart contractions. A deficiency of these nutrients leads to
an imbalance in cellular energy that can cause irregular
heartbeat. Many studies have been done on the therapeutic
effect of individual nutrients on cardiovascular health.
However, for optimal biological effect, these nutrients must
complement and support each other in synergy.
The objective of this Phase II clinical study was to
investigate whether long-term administration of a combination of vitamins, amino acids and other essential nutrients,
individually shown to be effective in improving cardiac
health, in addition to conventional basic therapy, could lead
JANA Vol. 8, No. 3, 2005
to a reduction in the number of clinically apparent episodes
in patients with paroxysmal atrial arrhythmia.
MATERIALS AND METHODS
A randomized, double-blind, placebo-controlled multicenter study was undertaken to evaluate the effect of vitamin/essential nutrient supplementation on arrhythmia.
Internists and general practitioners at 35 clinics in Germany
conducted this multi-center study on 131 patients diagnosed with paroxysmal atrial arrhythmia. Of these, 90
patients (44 in the supplemented group and 46 in the placebo group) strictly adhered to the study protocol and completed the six-month study. (See Figure 1–STARD diagram.) Although the number of patients in the ITT (n=131)
and the PP (n=90) populations differed, the results from
both groups shared the same trend.
Inclusion criteria for selection were: males/females
from 18 to 70 years of age, and patients with paroxysmal
atrial arrhythmia receiving anti-arrhythmic treatment for at
least three months and reporting at least one paroxysmal
cardiac episode per month. All participants met the
entrance criteria and were diagnosed with long-lasting or
chronic arrhythmia. There was no statistical difference in
the distribution of males and females in each group, and the
average age in the supplemented group was 58 years and in
the placebo group, 56 years. (See Table 1 for baseline
demographics and characteristics.)
The study was conducted according to the recommendations of the Declaration of Helsinki as amended in South
Africa and Edinburgh, Scotland (1996 and 2000), and AMG,
particularly sections 40 and 41 of the Tenth Amendment to
the Drugs Act, the Principles of Proper Implementation of
Clinical Studies and the ICH-GCP Note for Guidance.
Table I. Baseline Patient Demographics and Treatment
Characteristics.
Supplemented Group
Placebo Group
63 patients
68 patients
Age (mean)
57.7years
55.9 years
Females
37.7%
42.3%
Males
62.3%
57.7%
Race —Caucasian
100%
100%
Body Weight
78 kg/171.83 lbs
76 kg/167.43 lbs
Body Height (cm)
169.4
168.8
Treated with Beta-blockers
64%
66%
Treated with Calcium
20%
20%
20%
21%
channel blockers
Cardiac Therapy
20
Figure 1. STARD Flow Diagram
Methods of determining efficacy
The primary target parameter was defined as the number
of clinically symptomatic episodes in six months compared to
the placebo group. Since the patient’s clinical benefit was the
most important therapeutic aspect, participants were instructed to document, in a diary, all clinical arrhythmic symptoms
(rapid heart rate, palpitations, chest pain, dyspnea, dizziness,
weakness, tiredness, and perspiration outbreaks), time of
occurrence, episode duration, and episode severity, and return
for monthly follow-up. Since this study was designed as a first
“proof of concept,” sophisticated technologies such as remote
EKG telephonic transmission were not used.
Statistical Methods/Issues
For confirmatory analysis of the main target parameter,
with α = 0.05, the Mantel-Haenszel test was used. Patient
classification for this purpose was: no episodes, 1-3, 4-6, 710, and over 10 episodes during the period. Analysis of the
21
secondary parameters was done in an exploratory sense with
α = 0.05 without α-adjustment. The secondary parameters,
related to episode frequency, were analyzed analogously to
the main target parameter, with class boundary adjustment.
The time to first episode after the fifth week was statistically compared for the groups using Kaplan-Meier analysis and
a log-rank test. No interim analysis was performed.
Intervention
All patients were advised to continue taking their prescribed conventional medications during the study period,
and were provided conventional standard treatment for
paroxysmal atrial arrhythmia. (Approximately 60% of the
patients in both treatment groups were being treated with
beta-blockers and 20% by calcium blockers.) In addition,
patients allocated to the supplement group were each provided with blister packages containing vitamin/essential
nutrients labeled with a lot # (to ensure the double-blind
JANA Vol. 8, No. 3, 2005
character of the study) and the appropriate week. (See Table 3
for the nutrient composition.) The placebo group patients
received identical blister packs of placebo tablets containing
material of no medical significance such as cellulose, fructose,
etc., but physically indistinguishable from the two types of
nutrient tablets. Patients were instructed to take the prescribed
nutrient/placebo tablets provided for 24 weeks.
Efficacy Parameters
Frequency of symptomatic episodes of arrhythmia was
the primary target parameter for determining efficacy of
treatment. Secondary parameters included: the number of
clinically apparent episodes in each group during study
months 1-3 and during months 4-6, time elapse before first
occurrence of clinically apparent arrhythmic symptoms,
pre-and post-study 24-hour Holter monitoring for assessment of arrhythmia-specific changes, and pre-and posttreatment scores on the SF–36 (Short Form 36 Healthy
Survey,12 a standard Quality of Life Questionnaire) to evaluate how vitamin intake affected the patients’ perceived
general well-being and quality of life. The questionnaire
evaluated 36 parameters describing physical functions, role
functions from the emotional perspective, social functionality, level of pain, psychological status, vitality, and perception of general health and other aspects.
RESULTS
For the primary efficacy parameter (clinically apparent
arrhythmic episodes during months 1-6), a statistically significant effect of vitamin supplementation on the reduction
of clinically apparent arrhythmic episodes was observed in
both analysis sets (p=0.0221 for ITT analysis set, p=0.0160
for PP analysis set) (Figure 2). Only 47.8% of the supplemented patients reported seven or more arrhythmic
episodes during the treatment study, in contrast to 73.9%
reported in the placebo group (PP analysis). The number of
patients with less than seven episodes in the supplemented
group (52.7%) was almost twice that in the placebo group
(26.1%). Furthermore, the number of patients with more
than ten episodes was significantly less in the supplemented group (45.5%) than in the placebo group (69.6%). In
addition, the elapse of time prior to the first arrhythmic
episode was shorter in the placebo group than in the supplemented group (Log Rank Test: p=0.3797 for ITT analysis set and p=0.0332 for PP analysis set).
The data was also analysed to determine the effect of
supplementation on arrhythmic episodes with time at three
months vs. six months. At three months, 45% of the supplemented patients experienced seven or more arrhythmia
attacks in contrast to 27.3% at six months (Figure 3).
Approximately 22.7% of the supplemented patients reported no arrhythmic episodes at three months in contrast to
43.2% at six months (Figure 4).
For all dimensions of the SF-36, the differences
between the post-and pre-study values of the supplemented
group demonstrated a stronger perceived quality of life than
did those of the placebo group (Table 2). Baseline values
were comparable between treatment groups. This questionnaire evaluates 36 different parameters that relate to the
physical functioning of patients, including pain, emotional
Table 2. Effect of Supplementation on General Well-Being of Patients
SF-36 -Changes from baseline values
(ITT Analysis Set: Supplemented n=63, Placebo n=68)
Scores on SF-36
Mean - Supplemented Group
Mean - Placebo Group
General Health
+ 4.0
- 0.3
Vitality
+ 9.5
+ 2.8
Mental Health
+ 7.4
- 1.6
Physical Functioning
+ 5.9
+ 4.3
Bodily Pain
+ 11.4
+ 7.7
Social Functioning
+ 10.4
+ 2.3
Role – Physical Functioning
+ 22.5
+ 18.0
Role – Emotional Functioning
+ 16.7
+ 13.5
Total Score
+ 90
+ 47
Two-sided Wilcoxon Rank Sum Test (Normal Approximation): p=0.0118
JANA Vol. 8, No. 3, 2005
22
Table 3. Nutrient Composition
Bottle 1A (Serving size - three tablets)
Vitamin A (from 7.5% Betatene (Henkel))
Vitamin C (as Ascorbic Acid, Ascorbyl Palmitate, Calcium
Ascorbate, Magnesium Ascorbate)
Vitamin D3 (as Cholecalciferol)
Vitamin E (Mixed Covitol)
Vitamin B1 (from Thiamine Mononitrate
Vitamin B2 (as Riboflavin)
Niacin (as from Niacinamide)
Vitamin B6 (from Pyridoxine HCl)
Folic Acid
Vitamin B12 (as Cyanocobalamin)
Biotin
Pantothenic Acid (from D-Calcium Pantothenate)
Calcium (from Glycinate, Ascorbate)
Phosphorus (from Dicalcium Phosphate)
Magnesium (from Magnesium Glycinate, Magnesium
Ascorbate)
Zinc (from Zinc Glycinate)
Selenium (from L-Selenomethionine)
Copper (from Copper Glycinate)
Manganese (from Amino Acid Chelate)
Chromium (from Chromium Glycanate)
Molybdenum (from Molybdenum Glycinate)
Potassium (from Potassium Proteinate)
L-Lysine (from L-Lysine HCl)
L-Proline
Citrus Fruit Peel Bioflavanoids
L-Arginine (from L-Arginine HCl)
L-Cysteine (from L-Cysteine Monohydrate HCl)
Inositol
L-Carnitine (from L-Carnitine Tartrate
CoEnzyme Q10
Pycnogenol
1665 IU
600 mg
130 IU
130 IU
7 mg
7 mg
45 mg
10 mcg
90 mcg
20mcg
65 mcg
40mcg
35 mg
15 mg
40 mg
7 mg
20mcg
330 mcg
1.3 mg
10 mcg
4 mcg
20 mg
110 mg
110 mg
100 mg
40 mg
35 mg
35 mg
35 mg
7 mg
7mg
Bottle 1B (Serving size - 2 tablets)
Vitamin C (from Calcium Ascorbate, Magnesium
Ascorbate)
Vitamin E (as d-Alpha Tocopheryl Succinate)
Vitamin B1 (from Thiamine Mononitrate)
Vitamin B2 (as Riboflavin)
Niacin (as Niacinamide)
Vitamin B6 (from Pyridoxine HCl)
Vitamin B12 (as Cyanocobalamin)
Biotin
Pantothenic Acid (from Calcium D-Pantothenate)
Calcium (from Calcium Ascorbate)
Taurine
L-Carnitine (from L-Carnitine Tartrate)
CoEnzyme Q10
23
Figure 2. Arrhythmic episode frequency over six-month
study of nutrient supplemented (n=44) and placebo (n=46)
patients (PP analysis set) Exact Mantel-Haenszel ChiSquare Test: p=0.0160.
700 mg
Figure 3. Percentage of supplemented (n=44) patients
experiencing frequent (>7) arrhythmic episodes during
treatment intervals 1-3 months and 4-6 months.
Figure 4. Percentage of supplemented (n=44) patients
experiencing no arrhythmic episodes during treatment
intervals 1-3 months and 4-6 months (PP analysis set).
70 IU
15 mg
15 mg
30 mg
4 mg
7 mcg
130 mcg
40mcg
13 mg
200 mg
160 mg
20 mg
JANA Vol. 8, No. 3, 2005
status, vitality, general health perception, and other aspects
of health. Evaluation of the “mental health” measure
results, for example, showed an improvement of 7.4 in the
supplemented group and a decrease of –1.6 in the placebo
group (Two-sided Wilcoxon rank sum test was statistically
significant for ITT analysis: p=0.0118). For PP analysis,
statistical significance was almost achieved (two-sided
Wilcoxon rank sum test p=0.0506).
DISCUSSION
Overall, the results of this randomized double-blind,
placebo-controlled trial conclusively documented the effectiveness of supplementation using a combined vitamin/essential nutrient supplementation program in controlling arrhythmia, a condition for which conventional medicine does not
provide a solution, and instead attempts to control symptomatically. Active treatment with the multivitamin/essential
nutrient combination over a six-month period was significantly more effective in reducing clinically apparent symptoms of arrhythmia in patients than standard drug regimens
alone. Furthermore, the reduction in arrhythmic episodes was
more pronounced at six months than at three months of supplementation, suggesting increased benefits with a longer
duration of supplementation. Thus, with the adjunctive use of
the vitamin/essential nutrient supplement, the likelihood of
being free from arrhythmia doubled (15.9% in the supplemented group vs. 6.5% in the placebo group).
These health benefits were achieved by addressing the
underlying cause of arrhythmia, the deficiency of bio-energy-generating nutrients. The electrical cell cluster that triggers the heartbeat sends electrical impulses approximately
once every 830 milliseconds. To generate electricity, these
electrical cells of the heart need large amounts of bio-energy. Therefore, they need a constant supply of nutrients that
facilitate the conversion of food into cellular energy. The
most critical among them are coenzyme Q10, carnitine, the
B vitamins, lysine and vitamin C, together with magnesium,
calcium and potassium. For optimal biological effect, these
nutrients must complement and support each other in synergy. These nutrients optimize the function of the heart’s
electrical cells, as well as the cells building the heart muscle, blood vessels and other organs. When these nutrients
are lacking, the heart cells fail to generate electrical energy
and electrical impulses are sent in a chaotic manner.
This study sheds a new light on conventional medicine’s
approach to arrhythmia, which has relied on mechanical regulation of the heart rhythm using catherization, or drugs
such as beta-blockers and calcium channel blockers which
can have severe side effects, the most important of these
being the generation of even more irregular heartbeats and
not infrequently, sudden cardiac death. Large clinical trials
have revealed that anti-arrhythmic drugs, which are used by
more than 1.5 million Americans and many more people in
JANA Vol. 8, No. 3, 2005
European and other countries, do not offer health benefits
and increase the risk of serious complications, including
death. In 1989, a study using anti-arrhythmic drugs in
patients who had experienced heart attacks was prematurely
stopped when preliminary results showed the risk of death
was two-and-a-half times (2.5) greater in patients taking
drugs.13 In 2002, two large studies, one conducted in Canada
and the other in the Netherlands, provided similar evidence.14 The six-year study, conducted with more than 4,000
patients, showed higher death and hospitalization rates
among patients on anti-arrhythmic drugs. These drugs
included those that affect heart rate, such as dioxin, betablockers, and calcium channel blockers. The European study
came to the same conclusion, and also found that women
taking anti-arrhythmic medications faced a higher risk of
heart failure, stroke and other medical events than men.
Of significance, the results of this study indicate that
combining conventional drug treatment with a
vitamin/nutrient supplement program to treat arrhythmia is
an effective, safe, therapeutic approach that provides
enhanced improvement with long-term use. Due to the synergistic effect of specific vitamin/essential nutrients, therapeutic effect is achieved with moderate levels of these nutrients in contrast to single nutrient megadose approaches. In
addition to improvement in arrhythmic episode frequency,
the vitamin/essential nutrient program used by the supplemented group significantly improved their perceived quality of life, especially in the area of mental health. This is an
important benefit of supplementation, as arrhythmia
patients not only suffer from depression and fear of experiencing heart dysfunction, but also from deteriorating health
and a gradual diminishing of their quality of life. To a large
extent, these adverse mental and emotional consequences
are associated with drug side effects and the belief that a
cure for paroxysmal atrial arrhythmia is not available.
SUMMARY
Although further clinical studies are warranted to better determine its effectiveness for treatment of arrhythmia,
this nutrient combination offers great potential as an
adjunctive treatment for arrhythmia patients and as a preventative measure for patients with a predisposition for
developing arrhythmia.
ACKNOWLEDGMENTS
The study was conducted by internists in 35 health centers in Germany and supervised by Dr. Ute Engelmann and
Frank Koch.
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Med. 2002; 347:834-40.
25
JANA Vol. 8, No. 3, 2005
O
R I G I N A L
R
E S E A R C H
Flax and Soy Phytoestrogen Effects on
Renal Injury and Lipid Content in
Experimental Polycystic Kidney Disease
Malcolm R. Ogborn, MBBS, FRACP, FRCPC,*1,2 Evan Nitschmann, MSc,1
Neda Bankovic-Calic, MD, PhD,1 Hope A. Weiler, RD, PhD,1,2
Harold Aukema, PhD1,2
1. Department of Pediatrics and Child Health
2. Department of Human Nutritional Sciences
University of Manitoba, Winnipeg, Manitoba, Canada
ABSTRACT
Soy protein and dietary flaxseed, interventions rich in
phytoestrogens, reduce renal injury in animal models. We
studied the flax lignan, secoisolaricoresinol dyglycoside
(SDG) and the soy isoflavone genistein to assess their contributions to this effect in animals with polycystic kidney
disease (PKD). Male animals heterozygous for the
Han:SPRD-cy gene and their healthy littermates were fed a
diet with SDG, 20 mg/kg diet, or genistein, 75 mg/kg diet
for 8 weeks from weaning. Renal histology was studied in
animals with PKD. Renal function and fatty acid composition, which have been linked to dietary amelioration of
renal injury in other studies, were then measured in both
PKD animals and unaffected litter mates to differentiate
physiologic and metabolic consequences of diet versus
renal dysfunction. SDG reduced all indices of histologic
injury. Genistein, however, only reduced macrophage infiltration and staining for oxidized LDL. There were weak
dietary effects upon renal linoleic acid and docosahexanoic
* Correspondence:
Dr. Malcolm Ogborn
JBRC513-715 McDermot Avenue
Winnipeg, MB R3E 3P4, Canada
Phone: 204-789-3754 Fax: 204-789-3915
Email: mogborn@mich.ca
JANA Vol. 8, No. 3, 2005
acid in healthy and PKD animals, but no difference in
arachidonic acid proportion. SDG and genistein have measurable effects upon early pathology in rat PKD. They may
prove to be a practical way of obtaining renal health benefit. They may not, however, reproduce all of the effects of
a change in dietary practice.
Key Words: phytoestrogen, kidney, morphometry, renal
failure, polycystic, soy, flaxseed, pathology.
INTRODUCTION
Flaxseed or flax oil ingestion has demonstrated possible benefits in the reduction of inflammation in rheumatic
disease, reduction of atherosclerosis and modification of
behavior of hormone-dependent tumors.1-3 Flaxseed is the
richest natural source of mammalian lignans, present in the
husk of the seed as an ester secoisolaricoresinol dyglycoside (SDG) which is hydrolysed in the intestinal lumen to
enterodiol and enterolactone.4 SDG or its hydrolyzed products may influence health or disease through estrogenic
pathways,5 an antioxidant effect,6-8 or through antagonism
of platelet activating factor by specific receptor blockade.9
Flaxseed and SDG have been shown to reduce plasma levels of insulin-like growth factor 1 (IGF-1) in a rat mammary tumor model through an as yet unknown mechanism.10
Recent studies have identified potential benefits of flaxseed
26
or flax oil in the 5/6 nephrectomy model11 and murine or
human lupus erythematosus.12-14 Recently SDG has also
been shown to ameliorate murine lupus.9 We have previously demonstrated that partial dietary substitution of ground
flaxseed into standard rat chow or the use of flax oil as the
lipid source in a synthetic diet modifies renal injury, particularly interstitial inflammation and fibrosis, in the
Han:SPRD-cy rat.15-17 The Han:SPRD-cy is a model of
polycystic kidney disease, characterized by autosomal dominant inheritance with marked sexual dimorphism in expression, and an excellent system in which to explore the modification of chronic renal injury.18,19 The disease is characterized by progressive dilatation of nephrons in young animals,
and associated with marked interstitial inflammation and
fibrosis with associated nephron loss in older animals.15,16
We have previously demonstrated that soy protein feeding of the Han:SPRD-cy rat20 and pcy mouse21 reduced renal
cyst formation, renal epithelial cell proliferation, and renal
interstitial inflammation and fibrosis, and is associated with
significant indirect effects on renal polyunsaturated fatty
acid content, possibly through ∆6-desaturase inhibition.22
The soy saponin Bb has been linked to at least part of the
benefit observed in the pcy mouse.23 Dietary soy protein is
associated with reduced expression of IGF-1 in conjunction
with reduced disease severity.24 Knight et al., using data
from a large longitudinal study of health outcomes in nurses
recruited at random in the New England area, have shown
that protein intake from non-animal sources is associated
with a slower decline in renal function in the subset of the
study population that had mild renal insufficiency.25 Other
than alteration in the availability of specific amino acids, soy
protein may also contain other biologically active compounds such as saponins and phytoestrogens of which genistein is the most abundant.26,27 Genistein is known to have
estrogenic effects in mammals28 and is also a potent in vitro
inhibitor of tyrosine kinase, an enzyme intrinsic to many
sub-cellular processes29 and induces the activity of antioxidant enzymes.30 The success of both soy and flaxseed based
interventions in the amelioration of renal injury has led to
speculation that dietary phytoestrogens may have a role to
play in slowing progression of renal injury.31,32
As major alteration in dietary practice is difficult to
achieve at an individual patient level, we undertook studies
to determine if genistein and SDG, both of which can be
now be produced as concentrated micro-nutrients, contributed to the observed benefits of soy protein and flaxseed
feeding in the Han:SPRD-cy rat.
MATERIALS AND METHODS
Animals
Han:SPRD-cy rats were obtained from our own breeding colony that is derived from animals that were kindly
provided to us by Dr. Benjamin Cowley, University of
27
Kansas Medical Center, Kansas City, Kansas. All animal
procedures and care were examined by the University of
Manitoba Committee on Animal Use and certified to be
within the guidelines of the Canadian Council on Animal
Care. Surviving male offspring of known Han:SPRD-cy
heterozygotes were used in this study. Two thirds of these
animals would be expected to be heterozygous, as homozygotes in our colony rarely survive beyond weaning.
Male animals were randomly assigned to treatment or
control groups at weaning. Control animals in both experiments received a synthetic powdered AIN 76 diet using
casein as the protein source and 7% corn oil as the lipid
source. Experimental diets consisted of either supplementation with SDG at a level of 20 mg/kg diet (a gift of Dr. N.
Westcott and Dr. A. Muir, Agriculture Canada, Saskatoon)
or genistein 75 mg/kg (Toronto Research Chemical Inc.,
North York, Ont). Doses were based upon estimates of content of a soy and flaxseed based diet in previous studies and
doses of SDG and genistein used in other studies in vascular, neoplastic and renal disease.1, 9, 10, 15, 16, 21, 22, 33 Animals
on experimental diets were pair-fed with animals on a control diet, thus producing two separate sets of experiments but
permitting the exclusion of the amount of food eaten as a
significant variable in the modification of disease. Animals
were euthanized after 8 weeks on the diet, and kidney and
liver tissue as well as serum were collected for analysis.
Histology
Tissue from the left kidney was used for histologic and
immunohistochemical analysis.15, 17, 20, 22 This tissue was
fixed in 10% formalin for 120 minutes prior to embedding
in paraffin and sectioning at 5 microns. Sections for measurement of cystic volume and qualitative study of renal
histology were stained with hematoxylin and eosin.
Sections for quantitative analysis of fibrosis were stained
using aniline blue alone in adaptation of Masson’s
trichrome stain. This adaptation demonstrates a perfect concordance of staining with an immunofluorescent detection
of collagen type III.15 The aniline blue staining permits
image analysis measurement using a standard incandescent
microscope light source. Animals were classified as affected if a single longitudinal cross section of the kidney contained at least 10 areas of tubular dilatation with associated
increase in extracellular matrix. Classification was confirmed by 2 experienced observers (NBC, MRO).
Immunohistochemistry
Cell proliferation was studied using immunohistochemical detection of proliferating cell nuclear antigen
(PCNA)15, 17, 20, 22 using a monoclonal anti-PCNA antibody
(Dako M 0879, Dako A/S, Glostrup, Denmark) at a dilution
of 1:50 for 90 minutes. Secondary detection was achieved
with a Vectastain Elite rat adsorbed anti-mouse IgG kit
(Vector Laboratories, Burlingame, CA) with peroxidasediaminobenzidine color development.
JANA Vol. 8, No. 3, 2005
Macrophages were identified using a monoclonal antibody against the rat equivalent of the human CD68 antigen
(Chemicon MAB1435, Chemicon International Inc.,
Temecula, CA) at a dilution of 1:100 for 90 minutes.
Secondary detection was achieved in the same way as for
PCNA.
using a modified Folch extraction procedure.15,17,22
Chromatographs were integrated using Varian Saturn software, version 5.51. Fatty acid methyl esters were identified
by comparison to retention times of Supelco37 component
FAME mix and expressed as percent total lipid.
Oxidized LDL (ox-LDL) was identified using a rabbit
antibody against copper-oxidized LDL (AB3230,
Chemicon International, Inc, Temecula, CA) at a dilution of
1:100 incubated for 2 hours, with subsequent visualization
using the DAKO Envision secondary detection system
(DAKO Corporation, Carpinteria, CA).
Data were analyzed using a general linear model
ANOVA to permit study of both disease and intervention
effects using Minitab release 13 (Minitab Inc., PA) with the
Tukey test applied for post hoc comparisons.
Image Analysis
RESULTS
Image analysis of histologic and immunohistologic
sections was performed on a minimum of 50 randomly
selected digitally captured image fields.15,17,20-22,34
Macrophage numbers and PCNA positive cells were quantitated by counting, using module 2500 of the
Imagemeasure software package. The counts were reported
as a mean per high power image field (40X microscope
objective) with at least 50 fields counted. Results were corrected for the extent of cystic change in each kidney and
thus represent counts per solid tissue area.
A total of 135 animals were studied, of which 87 had
PKD and 48 were normal. The distribution of normal vs.
PKD animals did not differ significantly between treatment
groups (Chi-square test). Results from control-diet-fed animals in the two feeding trials were compared in all categories and then pooled to a single control group when that
analysis revealed no effect due to experimental order.
Animal weight was significantly reduced and serum creatinine increased by PKD compared to healthy animals
(p<0.001, Table 1), but no dietary effect was observed. Post
hoc comparisons confirmed significant differences between
each group of normal animals and all groups of PKD animals. A very weak dietary effect upon cholesterol was
detected (p=0.049), but no significant post hoc effects were
noted (Table 1). No disease or dietary effect on serum
triglyceride concentration was seen (Table 1).
Chemistry
Serum creatinine and cholesterol were determined in
serum by spectrophotometric methods using Sigma kits
(Sigma Chemical Co., St. Louis, MO) adapted to a 96-well
plate reader.
Gas Chromatography
Lipids were extracted for gas chromatographic analysis
Statistical analysis
Cystic change was significantly reduced by SDG
(p<0.001 vs. control and p=0.006 vs. genistein; Figure 1).
Table 1. Animal weights and serum biochemistry in Han:SPRD-cy rats fed control, genistein-supplemented or SDG-supplemented diets.
Diet
Disease
status
N
Weighta
(gm)
creatinine
(µmol/l)
cholesterolb
(mmol/l)
triglyceride
(mmol/l)
Control
normal
Control
PKD
Genistein
normal
Genistein
PKD
SDG
normal
SDG
PKD
15
374 (6)
47
348 (4)
11
381 (7)
23
356 (5)
12
374 (7)
17
334 (6)
75 (6.9)
115 (4.1)
76 (8.0)
125 (5.6)
78 (7.7)
128 (6.5)
2.8 (0.5)
2.8 (0.3)
1.8 (0.5)
2.2 (0.4)
2.5 (0.5)
3.7 (0.4)
0.52
(0.05)
0.55
(0.05)
0.50
(0.06)
0.53
(0.03)
0.41
(0.05)
0.57
(0.04)
Results are expressed as mean with SEM in parentheses.
a = significant disease effect, p<0.001, with significant pair-wise differences in post hoc testing between normal and PKD animals in
each group, p<0.01.
b = significant dietary effect, p<0.05
JANA Vol. 8, No. 3, 2005
28
SDG also significantly reduced interstitial fibrosis (p<0.001
vs. control and genistein; Figure 2) and epithelial proliferation (p<0.001 vs. control and genistein; Figure 3). Both
genistein (p<0.001 vs. control) and SDG (p=0.002 vs. control) reduced macrophage infiltration (Figure 4), with no
difference detected between genistein and SDG. A very
similar effect was seen with detection of ox-LDL (Figure 5)
with both SDG (p=0.009) and genistein (p=0.001) demonstrating reduced staining compared to control, with no difference detected between the 2 treatments.
GC analysis of renal tissue revealed modest dietary
effects on linoleic acid (LA) in SDG treated animals,
although pair-wise comparisons did not detect any significant differences (Table 2). The n-3 PUFAs, α-linolenic acid
and eicosapentanoic acid were not consistently detected in
most renal tissue samples and are not reported. This result
is consistent with our previous studies using corn oil as a
lipid source.20,22 There was a small but statistically significant effect of diet on the longer chain n-3 PUFAs, docosahexanoic acid, with genistein-treated animals demonstrating
slightly higher levels compared to SDG-treated animals,
although neither group differed from control in pair-wise
comparisons. LA:arachidonic acid ratio did not differ
between experimental groups, suggesting no diet or disease
effect on ∆6-desaturase, as was demonstrated in our previous study with soy protein22 and flax oil.17
crude dietary substitutions in the form of whole flaxseed or soy
protein concentrate.15-17, 20-22 It also seems that SDG may have
a broader range of action, influencing fibrosis and disregulated
epithelial proliferation, in addition to sharing anti-inflammatory and anti-oxidative effects with genistein.
A number of previous studies with SDG suggest that it
may have multiple actions, both through enterodiol and
enterolactone that are produced by hydrolysis of the SDG,
and through actions of the intact molecule.4,35,36 Following
studies that had shown evidence of benefit of flaxseed feeding in murine and human lupus,12,13 Clark et al. demonstrated significant absorption of intact SDG in the MRL/lpr
mouse model of lupus nephritis.9 A dose as low as 0.6 mg
was sufficient to block the biological effects of a lethal dose
Figure 1. Renal cystic change in animals fed control,
SDG or genistein-supplemented diets.
a
p<0.001
m
i
p<0.001
f
o
n
o
i
t
c
a
r
f
DISCUSSION
Our results indicate that dietary phytoestrogens may contribute to the effects of nutritional strategies on the evolution of
renal injury in the Han:SPRD-cy rat. The results also suggest,
however, that these compounds are unlikely to be responsible
for all of the observed benefits seen in studies with relatively
0.5
0.4
0.3
0.2
0.1
0.0
Values are mean proportion of image area of 5 micron sections
occupied by cysts. Error bars represent SEM.
Table 2. Selected renal polyunsaturated fatty acid proportions in Han:SPRD-cy rats fed control, genistein-supplemented
or SDG-supplemented diets.
Disease
status
LA a
(18:2 ω6)
AA
(20:4 ω6)
DHAb
(22:6 ω3)
LA:AA
ratio
Control
normal
Control
PKD
Genistein
normal
Genistein
PKD
SDG
normal
SDG
PKD
15.9 (1.2)
16.7 (0.7)
17.7 (1.3)
16.0 (0.9)
12.9 (1.2)
14.0 (1.1)
19.5 (2.0)
18.6 (1.2)
19.1 (2.4)
22.9 (1.6)
24.6 (2.0)
20.6 (1.9)
0.71
(0.08)
1.33
(0.33)
0.65
(0.05)
1.57
(0.19)
0.86
(0.09)
1.13
(0.38)
0.96
(0.06)
0.89
(0.26)
0.66
(0.08)
0.67
(0.33)
0.44
(0.08)
0.85
(0.31)
Results are expressed as mean % total lipids with SEM in parentheses.
a = significant dietary effect, p=0.005, but no significance pair-wise differences in post hoc testing.
b = significant dietary effect, p<0.001, with genistein-fed animals having higher values than SDG-fed animals but not controls, p<0.01.
29
JANA Vol. 8, No. 3, 2005
Figure 2. Renal fibrosis in animals fed control, SDG or
genistein supplemented diets.
i
f
o
n
o
i
t
c
a
r
f
p<0.001
0.075
p<0.001
0.050
0.025
Figure 3. Renal epithelial proliferation in animals fed
control, SDG or genistein supplemented diets.
f
p<0.001
e
p<0.001
g 30
a
m
i
/ 20
s
l
l
10
e
c
0.000
0
Values are mean proportion of image area of 5 micron sections
occupied by aniline blue positive tissue, corresponding to collagen. Error bars represent SEM.
Values are mean number of nuclei per high power video field in
tubular epithelium demonstrating positive staining for PCNA in
5 micron sections. Error bars represent SEM.
Figure 4. Renal macrophage infiltration in animals fed
control, SDG or genistein supplemented diets.
Figure 5. Renal ox-LDL staining in animals fed control,
SDG or genistein supplemented diets.
e
g 30
a
m
i
/ 20
s
l
l
10
e
c
p=0.002
p<0.001
0
f
o
0.100
n
o
i
t
c
a
r
f
0.075
p=0.009
p=0.001
0.050
0.025
0.000
Values are mean number of nuclei per high power video field
demonstrating positive staining for rat CD68 equivalent antigen
in 5 micron sections. Error bars represent SEM.
Values are mean proportion of image area of 5 micron sections
staining positive for ox-LDL. Error bars represent SEM.
of platelet activating factor, and time and dose dependent
benefits on renal function and proteinuria were observed.
The extent to which the platelet activating factor receptorblocking function of SDG can be generalized to other peptide
growth or cell activation factors is not yet known. Rickard et
al. have shown that SDG depresses plasma levels of IGF-1
through a mechanism that is yet to be identified.10 Aukema
et al. have recently demonstrated that IGF-1 levels correlate
with disease expression and its modification by soy protein
feeding and gender in the Han:SPRD-cy rat.24 IGF-1 is a
known participant in regulation of renal epithelial proliferation and interstitial fibrosis37,38 and may be relevant to the
anti-proliferative and anti-fibrotic actions of SDG.
SDG and its metabolites are known to be antioxidants,6-8,39 a finding supported by our results. Other studies have implicated oxidant injury in the evolution of
Han:SPRD-cy rat renal disease.40-42 The use of α-tocopherol as antioxidant therapy in the Han:SPRD-cy rat was
not associated with clinical or histologic improvement,
although renal content of a-tocopherol was higher in female
animals with lesser disease.40 Although SDG did have significant effects upon renal histology that may certainly warrant investigation with respect to modification of the final
common pathway to renal failure, these effects were not
associated with a change in decline of renal function, unlike
our prior studies with both intact flaxseed and lignan-poor
JANA Vol. 8, No. 3, 2005
30
flax oil.17, 20 It would seem that SDG might contribute to,
but is not solely responsible for, the benefits of flaxseed.
The detection of an in vivo renal anti-inflammatory role
for genistein is novel. An antioxidant effect of soy protein has
been proposed as a basis for an experimental anti-atherogenic
effect.43 Genistein is reported to upregulate the activity of
antioxidant enzymes, thus reducing another potent stimulus
for macrophage infiltration.44 A specific anti-inflammatory
effect of genistein has been demonstrated in experimental
ileitis, and has been correlated with inhibition of expression of
inducible nitric oxide synthase.45 Our ox-LDL staining studies support a possible role for genistein as a renal antioxidant,
an area where it demonstrated an effect similar to that of SDG.
Genistein is widely used in in vitro studies as an
inhibitor of tyrosine kinases that are an important part of
peptide-growth-factor mediated proliferative responses in
renal epithelium, including an organ explant model of cystic disease.46 The concentration of genistein that produces
this effect in vitro is higher than that likely to be achieved at
a tissue level in in vivo studies.47 In this study, genistein
alone failed to reproduce the cyst reduction and anti-proliferative effect of soy protein substitution containing genistein, suggesting that it is not the active factor in this part of
the benefit of soy. The failure of genistein to change cyst
formation or indices of epithelial proliferation both in this
study and that by Tomobe et al. in the pcy mouse,21 in which
inflammation and fibrosis are far less prominent than the
Han:SPRD-cy rat, would suggest that genistein has no
inhibitory effect on the proliferative growth factors that
have been implicated in cyst formation, such as epidermal
growth factor.46 It also suggests that there is no role for the
observed inhibitory effect of genistein on DNA topoisomerase with subsequent interruption of the cell cycle, an
effect also possibly limited to high concentrations.5
The failure of genistein to reproduce the effects of soy
protein does not exclude a micronutrient effect altogether. In
addition to isoflavones, soy protein contains saponins that
have been subject to recent interest.26,28 Philbrick et al. have
recently reported treatment of pcy mice by supplementation
with purified saponin preparation, supplementation with alcoholic extract of soy containing both saponins or isoflavones, or
by replacement of dietary protein with an isolate containing
saponins and isoflavones.23 Feeding of the alcoholic extract
reduced kidney weights and water, and was associated with
preservation of renal function. This finding was reproduced
by soy protein isolate and purified soyasaponin Bb.
Another potential shared effect of SDG and genistein
might be an estrogenic inhibition of macrophage infiltration.
Disease expression in the Han:SPRD-cy and its modification
by diet have been linked to alteration in expression of monocyte chemoattractant protein 1 (MCP-1) by our group and
others.34,48 MCP-1 expression is reduced in the presence of
estrogen or estrogen agonists and such reduction has been
associated with anti-inflammatory and antifibrotic effects in
31
experimental arthritis.49-51 SDG and genistein may, in part,
reproduce the gender dimorphism that is characteristic of
this model and has been proposed in human disease.52-54
Our study indicates that these phytoestrogens have biologic activity that may be relevant to the management of the
progression of chronic renal injury, but are unlikely to be
responsible for the benefits observed with soy or flaxseed
feeding alone. Both flaxseed and soy protein substitution
are complex interventions and should not be overinterpreted
as evidence of biologic effects of a single micronutrient.
Although the possibility exists that pharmacologic dosing
with these agents may be useful, recent enthusiasm for these
agents as potential broad spectrum therapies for renal disease should be tempered until longer-term studies of efficacy and dose response relationship have been performed.
ABBREVIATIONS USED
IGF-1
Insulin-like growth factor 1
LA l
Linoleic acid
ox-LDL
oxidized low density lipoprotein
MCP-1
monocyte chemoattractant protein 1
PCNA
proliferating cell nuclear antigen
PKD
polycystic kidney disease
PUFA
polyunsaturated fatty acid
SDG
secoisolaricoresinol diglycoside
ACKNOWLEDGMENTS
This work was supported by operating grants from the
Children’s Hospital Foundation of Manitoba, Inc., the
Manitoba Health Research Council and the Flax Council of
Canada. The work was performed in the laboratory facilities of the Manitoba Institute of Child Health in the John
Buhler Research Centre, Winnipeg, Manitoba. Dr. Hope
Weiler is the recipient of a New Investigator Award of the
Canadian Institutes of Health Research.
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31. Velasquez MT, Bhathena SJ. Dietary phytoestrogens: a possible role in renal disease protection. Am J Kidney Dis.
2001;37:1056-1068.
32. Ranich T, Bhathena SJ, Velasquez MT. Protective effects of
dietary phytoestrogens in chronic renal disease. J Ren Nutr.
2001;11:183-193.
JANA Vol. 8, No. 3, 2005
33. Prasad K, et al. Protective effect of secoisolariciresinol diglucoside against streptozotocin-induced diabetes and its mechanism. Mol Cell Biochem. 2000;206:141-149.
34. Bankovic-Calic N, et al. Renal remodeling in dietary protein
modified rat polycystic kidney disease. Pediatr Nephrol.
1999;13:567-570.
35. Rickard SE, Thompson LU. Chronic exposure to secoisolariciresinol diglycoside alters lignan disposition in rats. J Nutr.
1998;128:615-623.
36. Rickard SE, Thompson LU. Urinary composition and postprandial blood changes in H-secoisolariciresinol diglycoside
(SDG) metabolites in rats do not differ between acute and
chronic SDG treatments. J Nutr. 2000;30:2299-2305.
37. Rossert JC, Terraz-Durasnel G, et al. Growth factors,
cytokines, and renal fibrosis during the course of diabetic
nephropathy. Diabetes Metab. 2000;26 (suppl 4):S16-S24.
38. Weston CE, et al. Effect of oxidant stress on growth factor
stimulation of proliferation in cultured human proximal
tubule cells. Kidney Int. 1999;56:1274-1276.
39. Prasad K. antioxidant activity of secoisolariciresinol diglucosidederived metabolites, secoisolariciresinol, enterodiol, and enterolactone. Intl J Angiology. 2000;9:220-225.
40. Torres VE, et al. Renal concentration of alpha-tocopherol:
dependence on gender and lack of effect on polycystic kidney
disease in Han:SPRD rats. Am J Kidney Dis. 1998;31:687-693.
41. Qian Q, Harris PC, Torres VE. Treatment prospects for autosomal-dominant polycystic kidney disease. Kidney Int.
2001;59:2005-2022.
42. Maser RL, et al. Oxidant stress and reduced antioxidant
enzyme protection in polycystic kidney disease. J Am Soc
Nephrol. 2002;13991-13999.
43. Damasceno NR, et al. Soy protein isolate reduces the oxidizability of LDL and the generation of oxidized LDL autoantibodies in rabbits with diet-induced atherosclerosis. J Nutr.
2000;130:2641-2647.
44. Cai QH. Effect of dietary genistein on antioxidant enzyme
activities in SENCAR mice. Nutr Cancer. 1996;25:1-7.
45. Sadowska-Krowicka H, et al. Genistein and gut inflammation: role
of nitric oxide. Proc Soc Exp Biol Med. 1998; 217:351-357.
46. Pugh JL, Sweeney WE Jr, Avner ED. Tyrosine kinase activity
of the EGF receptor in murine metanephric organ culture.
Kidney Int. 1995;47:774-781.
47. Kim H, Peterson TG, Barnes S. Mechanisms of action of the
soy isoflavone genistein: emerging role for its effects via
transforming growth factor beta signaling pathways. Am J
Clin Nutr. 1998;68:1418S-1425S.
48. Cowley BD Jr, et al. Increased renal expression of monocyte
chemoattractant protein-1 and osteopontin in ADPKD in rats.
Kidney Int. 2001;60:2087-2096.
49. Frazier-Jessen MR, et al. Estrogen suppression of connective
tissue deposition in a murine model of peritoneal adhesion
formation. J Immunol. 1996;156:3036-3042.
50. Kovacs EJ, et al. Estrogen regulation of JE/MCP-1 mRNA
expression in fibroblasts. J Leukoc Biol. 1996;59:562-568.
51. Frazier-Jessen MR, Kovacs EJ. Estrogen modulation of
JE/monocyte chemoattractant protein-1 mRNA expression in
murine macrophages. J Immunol.1995;154:1838-1845.
52. Magistroni R, et al. Genotype-renal function correlation in
type 2 autosomal dominant polycystic kidney disease. J Am
Soc Nephrol. 2003;14:1164-1174.
53. Gabow P A, et al. Factors affecting the progression of renal
disease in autosomal-dominant polycystic kidney disease.
Kidney Int. 1992;41:1311-1319.
54. Cowley BD Jr, et al. Gender and the effect of gonadal hormones on the progression of inherited polycystic kidney disease in rats. Am J Kidney Dis. 1997;29:265-272.
32
O
R I G I N A L
R
E S E A R C H
Immunostimulating Properties of Two Different
β−glucans Isolated from Maitake Mushrooms
(Grifola frondosa)
Vaclav Vetvicka, PhD,* Jana Vetvickova, MS
University of Louisville, Department of Pathology
Louisville, Kentucky
ABSTRACT
β-glucans have been extensively studied for their
immunological and pharmacological effects. The number of
individual glucans is almost as great as the number of
sources used for isolation. Not surprisingly, mushrooms are
one of the prime sources of (1-3)-β-D-glucans, but their
activities are not always described consistently. In addition,
the most commonly used route of administration is injection,
which is less convenient for clinical practice.
In this paper, we compared the immunostimulating
properties of two different glucans from Maitake mushrooms with lentinan, a standard, well-researched mushroomderived glucan. A number of major immunological parameters were tested – phagocytosis, NK cell activity, expression
of surface markers, cytokine secretion and apoptosis. Our
study showed not only have these glucans significantly
increased all tested characteristics, but they also have similar, and in some tests even higher activity than lentinan, are
active at lower doses and can be administered orally with no
loss of activity. Therefore, this report represents evidence
* Correspondence:
Vaclav Vetvicka, PhD
University of Louisville
Department of Pathology and Laboratory Medicine
511 S. Floyd St., MDR Bldg., Rm. 224
Louisville, KY 40202
Phone: 502-852-1612 FAX: 502-852-1177
Email: vetvickavaclav@netscape.net
33
that Maitake-derived supplements taken orally can stimulate
the defense systems.
Key Words: glucan, phagocytosis, cytokines, mushrooms.
INTRODUCTION
Polysaccharides in general have a long history as
immunomodulators, and interest in them rose particularly
after experiments showed that zymosan stimulates
macrophages via activation of the complement system.1
Various types of β-glucans can be isolated from numerous
sources, the major ones being yeast, mushrooms and seaweed. Despite the fact that the number of individual β−glucans is almost as great as the number of sources used for its
isolation, and despite the enormous numbers of studies performed all over the world, it is still impossible to say that
only one particular glucan is the optimal immunomodulator.
As with all natural products, there are considerable variations not only among individual β−glucans, but also among
individual batches. In view of the ever increasing popularity
of glucans as immunomodulators, the functional comparison of new glucans is therefore more important than ever.
Thus far, the immunostimulating effects of β−glucans
have been demonstrated in every single animal species tested,
from earthworms2 to humans. This supports not only the conclusion that β−glucans are active over the broad spectrum of
biological species, but also that they represent one of the first
immunostimulants active across the evolutionary spectrum.
JANA Vol. 8, No. 3, 2005
Soluble fungal β−glucans such as schizophyllan and
lentinan have been used for tumor immunotherapy in Japan
for the past 25 years.3,4 These polysaccharides promote natural host defense mechanisms and boost specific tumor
immunity. For some time, investigators have failed to define
the exact mechanisms of action. Only after identification of
CR3 (CD11b/CD18) molecule as the principal receptor for
β−glucans of leukocytes5 was it possible to determine the
cellular basis for their action and to design more rational
approaches for their potential use in immunotherapy.
However, not all cellular and molecular mechanisms
responsible for glucan effects on the immune system are
completely understood.
In addition to the well-studied mushroom-derived glucans
such as lentinan,6 numerous additional glucans have been isolated from various mushrooms. Some glucans, such as β−glucans from Phytophthora parasitica, were merely described
without any biological tests.7 Many of them, however, have
very interesting properties, including the strong antitumor
activity of β−glucan from Glomerella cingulata8 or potentiation of TNF by scleroglucan.9 As some of the published reports
described rather confusing data, we decided to evaluate the
immunostimulating effects of two mushroom-derived glucans
and to compared them to the standard glucan, lentinan.
MATERIAL AND METHODS
Animals
Female, 6-to-10-week old BALB/c mice were purchased from the Jackson Laboratory (Bar Harbor, ME). All
animal work was done according to the University of
Louisville IACUC protocol. Animals were sacrificed by
CO2 asphyxiation.
Materials
RPMI 1640 medium, sodium citrate, dextran, FicollHypaque, antibiotics, sodium azide, bovine serum albumine
(BSA), Wright stain, Limulus lysate test E-Toxate, and
Concanavalin A were obtained from Sigma Chemical Co.
(St. Louis, MO). Fetal calf serum (FCS) was purchased
from Hyclone Laboratories (Logan, UT), and CytoTox 96
Non-Radioactive Cytotoxicity Assay from Promega
(Promega, Madison, WI).
After washing with cold PBS, the cells were resuspended in
PBS containing 1% BSA and 10 mM sodium azide. Flow
cytometry was performed with a FACScan (Becton
Dickinson, San Jose, CA) flow cytometer and the data from
over 10,000 cell samples were analyzed.
Cell lines
Human cell lines ZR-75-1, PC-3, SW900 and K562
were purchased from ATCC. The cells were maintained in
RMPI 1640 medium supplemented with 10% FCS, 2 mM
glutamine, and antibiotics.
β -1,3 glucans
Two different soluble β−glucans were isolated from
Maitake mushrooms (Grifola frondosa) according to manufacturer’s specification. Briefly, MaitakeGold 404 was produced under a patented method (US Patent 5,854,404). The
product, a glucan/protein complex, is derived by thermally
extracting the fruit body of Maitake with water under pressure
at 100°C or more for 30 minutes to an hour. After that, alcohol is added to the extract at a final concentration of 20% to
60% by volume to remove floating material by filtration. The
resulting extract is concentrated under heating to remove
residual alcohol. The product, a hygroscopic powder in shades
of brown is soluble in water, alkaline solutions, and dimethyl
sulphoxide, and has a molecular weight around 1,000 kD.
Maitake Pro D Fraction is prepared as follows: Fruit
bodies of Maitake are treated with hot water, and the watersoluble fraction is then saturated with ethyl alcohol. The
resulting precipitate is then treated with acetic acid and
alkalic material. The extract is acid-insoluble, alkali-soluble, with a molecular weight around 1 ,000 kD.10
MDF glucan is a highly-purified β−glucan.11 MTG glucan is a β−glucan-protein complex.12 MDF used in our
experiments was Grifton-Pro Maitake D Fraction manufactured by Maitake Products, Inc. The MTG used in our
experiments was MaitakeGold 404 purchased from
Tradeworks Group, Inc. (Brattleboro, VT).
The soluble mushroom-derived β−glucan, lentinan
(MW app. 1,000 kD), was obtained from the Developmental
Therapeutic Program, Division of Cancer Treatment, Drug
Synthesis and Chemistry Branch, National Cancer Institute
(Bethesda, MD), and served as a control.
For fluorescence staining, the following antibodies
have been employed: anti-mouse CD4, CD8, and CD19,
conjugated with FITC were purchased from Biosource
(Camarillo, CA).
All media and buffers were tested for endotoxin contaminations and shown to contain >0.1 ng/ml of endotoxin
using the Limulus lysate test (E-Toxate). The glucans were
administered either by ip. injection or via intragastric tube
(100 µg/day).
Flow cytometry
Phagocytosis
Cells were stained with monoclonal antibodies on ice
in 12x75 mm glass tubes using standard techniques. Pellets
of 5x105 cells were incubated with 10 µl of FITC-labeled
antibodies (1 to 20 µg/ml in PBS) for 30 minutes on ice.
The technique employing phagocytosis of synthetic
polymeric microspheres was described earlier.13 Briefly:
peritoneal cells were incubated with 0.05 ml of 2-hydroxyethyl methacrylate particles (HEMA; 5x108/ml). The test
tubes were incubated at 37° C for 60 min with intermittent
Antibodies
JANA Vol. 8, No. 3, 2005
34
shaking. Smears were stained with Wright stain. The cells
with three and more HEMA particles were considered positive. The same smears were also used for evaluation of cell
types.
ELISA reader at 450 nm with a correction at 570 nm. Data
were calculated from the standard curve prepared by the
automated data reduction using linear regression analysis.
A standard curve was run with each assay.
In vitro cytotoxicity assay
For evaluation of IL-2, we incubated purified spleen
cells (2x106/ml in RPMI 1640 medium with 5% FCS) in
wells of a 24-well tissue culture plate. After addition of 1 µg
of Concanavalin A into positive-control wells, cells were
incubated for 72 hr in a humidified incubator (37°C, 5%
CO2). At the endpoint of incubation, supernatants were collected, filtered through 0.45 µm filter and tested for the
presence of IL-2 (Vetvicka et al., 2002). Levels of the IL-2
were measured using a Quantikine mouse IL-2 kit (R&D
Systems, Minneapolis, MN).
Spleen cells were isolated from the spleens of mice by
standard methods. Cell suspension was generated by pressing minced spleen against the bottom of a petri dish containing PBS. After elimination of erythrocytes by 10-second
incubation in distilled water and five washes in cold PBS,
the cells were resuspended in PBS and counted. The viability was determined by trypan blue exclusion, and only cells
with viability better than 95% were used in subsequent
experiments. Splenocytes (106/ml; 0.1 ml/well) in V-shaped
96-well microplates were incubated with various glucans (2
µg/ml) for 30 min at 37° C and then washed three times
with RPMI 1640 medium. After washing, 50 µl of target
cell line K562 (three different concentrations of target cells
were used: the final effector-target ratio was 10:1, 50:1, and
100:1) were added. After spinning the plates at 250x g for 5
min, the plates were incubated for 4 hrs at 37°C. The cytotoxic activity of cells was determined by the use of CytoTox
96 Non-Radioactive Cytotoxicity Assay according to the
manufacturer’s instructions. Briefly, 10 µl of lysis solution
was added into appropriate control wells 45 min before the
end of incubation. The next step was to spin the plates at
250x g for 5 min., followed by transferring 50 µl of supernatant into flat-bottomed, 96-well microplates. After 50 µl
of reconstituted substrate was added into each well, plates
were covered and incubated for 30 min. at room temperature in the dark. The optical density was determined by
using a STL ELISA reader (Tecan U.S., Research Triangle
Park, NC) at 492 nm. Specific cell-mediated cytotoxicity
was calculated using the formula:
Percent-specific killing (% cytotoxicity) =100x [(OD492
experimental - OD492 spontaneous) divided (OD492 maximum - OD492 spontaneous)] as described in the manufacturer’s instructions, where spontaneous release was target
cells incubated with medium alone, and maximum release
was that obtained from target cells lysed with the solution
provided in the kit.
Apoptosis
Apoptosis was evaluated using the APO-BRDU kit
(BD Biosciences, San Diego, CA) according to the manufacturer’s instruction with the use of a flow cytometer
equipped with a 488 nm laser.
Statistics
Student’s t-test was used to statistically analyze the data.
RESULTS
Phagocytosis
It is well established that β−glucans strongly influence
the phagocytic activity of professional phagocytes. At the
same time, however, it is always important to start with
phagocytosis when a new glucan is being evaluated. First we
compared the effects of MTG and MDF β−glucans on
phagocytosis of synthetic HEMA microspheres by peripheral macrophages (Figure 1). When the glucans were administered intraperitoneally, both tested glucans stimulated the
phagocytosis more than lentinan used as a control. On the
Figure 1. Effect of an administration of different glucan (100
µg/mouse) samples on peritoneal macrophage phagocytosis. Each
value represents the mean ± SD. *Represents significant differences between control (PBS) and glucan samples at P ≤0.05 level.
Cytokine evaluation
BALB/c mice were intraperitoneally injected with various doses of tested glucans. Control mice obtained PBS
only. After various time intervals, the mice were sacrificed
and blood was collected in Eppendorf tubes. Subsequently,
the serum was prepared, collected and stored at –80°C for
no more than 1 week.
The levels of TNF-α and IL-1 in serum samples were
evaluated using a commercial kit OptEIA Mouse TNF-?
(Mono/Mono) and OptEIA Mouse IL-1 Sets (Pharmingen,
San Diego, CA) according to the manufacturer’s instructions. The optical density was determined using a STL
35
JANA Vol. 8, No. 3, 2005
other hand, after oral administration (14 days of intragastric
delivery of (100 µg of glucan/mouse) we found no differences between lentinan and our β−glucans, but all three types
of glucan showed significant stimulation of phagocytosis.
In the next step we compared the effects of these glucans on phagocytosis by peripheral blood leukocytes.
Results summarized in Figure 2 showed significant stimulation of monocytes by both MTG and MDF, whereas significant stimulation of neutrophils was achieved only after
injection with lentinan (similar level of stimulation by MTG
was not significant due to higher variation among samples).
These experiments were followed up by evaluation of dosedependence. Six different doses (from 1.8 to 250 µg/mouse)
were used. Lentinan was active only in three higher doses,
MTG caused increase in numbers of phagocytosing mono-
cytes even at low 2.6 µg concentration, whereas MDF was
effective only at the two higher concentrations (Figure 3).
MEMBRANE MARKERS
We then evaluated the effects of MTG and MDF on
expression of several membrane markers. Twenty four
hours after an ip. injection of either MTG, MDF, or lentinan, spleen cells were isolated and the surface expression of
CD4, CD8, and CD19 was evaluated by flow cytometer.
The results summarized in Figure 4 show that the application of both MTG and MDF significantly increase the number of CD4-positive T helper cells in the spleen.
Cytokines
Next, we compared the effects of a single intraperitoneal injection of MTG, MDF, or lentinan on systemic in
Figure 2. Effect of an ip. administration of different glucan (100
µg/mouse) samples on phagocytosis by peripheral blood granulocytes and monocytes. Each value represents the mean ± SD.
*Represents significant differences between control (PBS) and
glucan samples at P ≤0.05 level.
Figure 4. 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 three donors at each time interval were examined and the
results given represent the means ± SD. *Represents significant
differences between control (PBS and samples) at P ≤ 0.05 level.
Figure 3. Potentiation of phagocytosis of synthetic microspheres
by different doses of ip-injected glucans. Monocytes with three
and more HEMA particles were considered positive. Each value
represents the mean ± SD. *Represents significant differences
between control (PBS) and glucan samples at P ≤0.05 level.
Figure 5. Stimulation of IL-1β secretion in peripheral blood by
different types of glucan. Each value represents the mean ± SD. As
the control values (PBS) were always zero, each value represents
significant differences at P ≤0.05 level.
JANA Vol. 8, No. 3, 2005
36
vivo release of two different cytokines, IL-1β and TNF-α.
Peripheral blood was isolated at three different intervals after
the injection and the serum obtained was stored at –80°C for
no more than 1 week. The data summarized in Figure 5
shows that MTG and MDF caused significant elevation in
levels of IL-1β in every tested interval. These elevated levels
were demonstrated as early as 30 minutes and as late as 24
hours after application. Lentinan-caused increase could be
observed only after 24 hrs. For both MTG and MDF glucans,
the highest concentration of IL-1β was found 60 minutes
after injection. There is a possibility that contamination of
samples with LPS is causing the measured effects, but we
also used LPS-free samples with virtually identical results.
A different situation was observed in the case of TNF-α,
where only the MTG showed significant effects at the 60minute interval. And again, LPS-free samples yielded similar
data as normal samples (Figure 6). Several individual doses
of all three glucans have been used, but the most pronounced
effects were found with 100 µg doses.
Evidence of the immunomodulating activity was also
Figure 6. Stimulation of TNF-α secretion in peripheral blood by
different types of glucan. Each value represents the mean ± SD. As
the control values (PBS) were always zero, each value represents
significant differences at P ≤0.05 level.
demonstrated through effects on the production of IL-2 by
spleen cells (Figure 7). The production of IL-2 was measured after a 72 hr in vitro incubation of spleen cells isolated from control and glucan-administered animals. Injection
of lentinan and MDF resulted in strong stimulation of IL-2
production, which was even significantly higher than control stimulation by Concanavalin A. The effects of MTG
were slightly lower than control Con-A-stimulated levels,
but still almost 60 times higher than the level where only
PBS was used (63 pg of IL2/ml).
Cytotoxicity
Next, for evaluation of the effects on NK cells, human
K562 cells were incubated with mouse spleen cells stimulated by either by our glucan samples or lentinan (Figure 8).
A short treatment of glucans was adequate to cause significant enhancement of cytotoxicity at the higher E:T ratio.
Apoptosis
We also measured the effects of our samples on induction of apoptosis. We decided to use three different and
well-established human cancer cell lines: ZR-75-1 (breast
cancer), PC-3 (prostate cancer), and SW900 (lung cancer).
We used these lines to represent three different types of
human cancer. However, using the APO/BRDU test, no
apoptotic cells were observed (data not shown).
DISCUSSION
β−glucans, originally recognized as potent stimulants
of the reticuloendothelial system,1 are commonly considered
typical representatives of substances that achieve their resistance-enhancing effects by stimulation of macrophages.14
More recent discoveries of the interaction of immunocytes
with β−glucans have demonstrated that complement recep-
Figure 7. Stimulation of IL-2 secretion by splenocytes by different types of glucan. *Represents significant differences between
control (PBS and samples) at P ≤ 0.05 level.
37
Figure 8. Effects of tested glucans on NK cell cytotoxicity of
K562 cells. Different ratios of NK cells to target cells were tested
for cytotoxicity in the presence or absence of β-glucans for 30
minutes at 37°C. The data points shown are mean values from
three experiments. The differences were significant at P≤0.05 level
at all three effector to target cell ratios.
JANA Vol. 8, No. 3, 2005
tor type 2 (CR3; CD11b/CD18) is primarily responsible for
both the binding and biological effects of β−glucans.5,15,16
Subsequent experiments helped to show that glucan used
orally has similar biological effects to injected glucans,17 and
that the mechanisms responsible for glucan-mediated killing
of tumor targets is via interaction of anti-tumor antibodies
with glucan-activated cells.18 The results of these experiments changed our view of glucans from merely nonspecific immunostimulators to natural agents specifically affecting
immune reactions.
Despite our knowledge of the mechanisms of interactions between glucan and mammalian cells, and despite a
vast number of individually described β−glucans, consensus on which glucan is best suited for stimulation of immunity remains lacking.
Most β−glucans are derived from fungi and have a
backbone structure of linear β−1,3-linked D-glucose molecules (β1,3-D-glucan) with β-1,6-linked side chains of
β1,3-D-glucan of varying sizes occurring at different intervals.19 However, contradictory data and conclusions exist on
the effects of molecular weight, degree of branching, conformation and polymer charge on biological activities.9,19
Therefore, the quest for the biologically most active β−glucan continues.
Most of the β−glucans with high activity have been isolated from the Basidiomycetes, and not surprisingly, the two
mushroom glucans most often studied, lentinan and schizophyllan, both came from the Basidiomycete family. The
comparison of the biological and antitumor properties of
these β−glucans can be found in Borchers et al.20
Due to the significant differences in activities among
various glucans isolated from numerous sources, it is
imperative to evaluate the biological properties of any glucan before making suggestions for use in clinical practice.
This investigation focused on the biological activities of two
individual glucans, MTG and MDF, isolated from Maitake
mushrooms (Grifola frondosa).
In the present study we demonstrated that the Maitakederived glucans are functionally similar not only to lentinan,
often considered a standard β−glucan, but also to other glucans.21 The evaluation of the biological activities started, as
usual, by effects on phagocytosis. Using the synthetic
microspheres as prey, we took advantage of the unique
properties of these microspheres, which have an extremely
low negative charge and thus no nonspecific adherence to
the cell membrane.22 First we measured the phagocytosis of
peripheral blood cells, and later the peritoneal cells. In both
cases, we found a significant increase in the number of
phagocytosis monocytes and macrophages after both MDF
and MTG. When we compared the dose-dependence of the
increased phagocytosis, the surprising result was that MTG
was effective even in very small doses (2.6 and 3.7
µg/mouse). More importantly, the oral administration of our
JANA Vol. 8, No. 3, 2005
samples showed similar stimulation of phagocytosis.
The evaluation of the effects of Maitake glucans on
expression of cell surface markers showed that both glucans
caused a significant increase in the number of CD4-positive
cells after the 24 hr interval. The rest, i.e., the numbers of
CD8 and CD19 cells, were unchanged. The data on lentinan
did not differ from PBS control, which is in agreement with
previously described findings.21
In addition to the direct stimulation of cells involved in
immune reactions, the immunostimulating effects of natural immunomodulators such as glucans are indirectly caused
by potentiation of synthesis and subsequent release of
numerous cytokines. Individual glucans significantly differ
in their effects upon cytokine synthesis.23-26 The only glucan found so far with either in vivo or in vitro stimulation
of cytokines, is betafectin.27
We found only minor effects of tested glucans on TNFα production, with only MTG having significant effects.
When IL-1β was tested, both glucans showed significant
stimulation even 30 minutes after injection, and these effects
lasted for the whole tested interval. On the other hand, all
samples strongly stimulated the production of IL-2, which is
comparable to the earlier finding using yeast-derived glucans.28 As cytokine production is extremely sensitive to the
presence of LPS, contamination with LPS might mask or
even overcome the real effects of any immunomodulator.
Therefore we used LPS-free samples (depleted by addition
of 10 µg/ml of polymixin B) in parallel to the regular glucan
samples. In both cases, there were no differences between
normal and LPS-free samples. Due to these results, we used
only regular samples in all other experiments.
As the last set of experiments we evaluated the effects
of glucan injection on NK cell activity. Using a human
K562 cell as a model, we found that in higher E:T ratios,
the 4 hr incubation of spleen cells with glucan samples
caused significant potentiation of natural killer cells. When
compared to lentinan, MTG was effective at lower ratio.
The sporadic data about glucan and apoptosis are confusing. While some authors found induction of apoptosis,10,29 others found no effects.30 Our data using breast,
lung and prostate cancer cells showed absolutely no effects
of tested samples (data not shown).
Our current paper clearly demonstrates that Maitakederived glucans act via the same mechanisms as yeastderived glucans, and that they are, in many cases, even
more biologically and immunologically active. When compared to lentinan, Maitake-derived glucans (MDF and
MTG in particular) showed a higher stimulation of defense
reactions. Another important conclusion is the fact that
these glucans do not lose their biological activities when
delivered orally. And finally, this report represents a proof
that orally-taken Maitake-derived supplements can stimulate the defense systems.
38
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JANA Vol. 8, No. 3, 2005
C
L I N I C A L
C
A S E
R
E P O R T S
The Effects of Australian Tea Tree and
Jojoba Essential Oils with Minerals for
Treatment of Nail Fungus
Leigh Erin Connealy, MD*
South Coast Center for New Medicine
Tustin, California
OVERVIEW
Two case studies were performed over the course of
ninety days to assess the effects and time of a natural antiinfective formula** on eliminating severe nail fungus of
toes and fingers.
INTRODUCTION
Nail fungus is a common dermatologic disorder affecting more than 30 million Americans.5 It requires proper
diagnosis and must be identified by microscopic examination and/or culture before being treated.1,4 Most cases are
seen in men and women between the ages of 40 and 65.2,5
Distal subungual onychomycosis (DSO) is by far the most
common pattern of the infection.1,3 Initiated by fungal
invasion of the distal nail bed, the underside of the nail plate
is subsequently infected. The infection spreads proximally
and can involve the entire nail unit. An inflammatory
response generally occurs within the nail bed, resulting in
* Correspondence:
Leigh Erin Connealy, MD
South Coast Medical Center for New Medicine
14642 Newport Avenue, Suite 200
Tustin, CA 92750
Phone: 714-669-4447 Fax: 714-669-4448
Email: connealymd@earthlink.net
JANA Vol. 8, No. 3, 2005
the accumulation of debris underneath the nail plate (subungual hyperkeratosis) which sometimes leads to the separation of the nail plate from the nail bed (onycholysis).5
Subungual hyperkeratosis and onycholyis are very common
findings in DSO. As the nail plate is destroyed, it becomes
progressively more discolored, thickened and brittle.
Trichophyton rubrum is the most common cause of both
DSO and proximal subungual onchomycosis (PSO). PSO
has similar pathogenic mechanisms to DSO, except that
PSO is initiated through assault at the proximal nail fold in
patients with compromised immune systems.
Antimicrobial activities of one natural oil in the formula tested in this study, tea tree oil, obtained from
Melaleuca alternifolia has been previously attributable to
its hydrocarbon and terpine constituents, including terpinen-40l, a-terpineol and linalool.
TREATMENT
In the two case reports, a topically-used essential oil
formulation was developed from a blend of Australian tea
tree oil and jojoba essential oils with the minerals copper,
chromium oxide green tea, and manganese dioxide.
Patients were advised to soak their nails in warm water for
five minutes to prepare the nail, then apply several drops of
the liquid twice a day to the affected area. They were
instructed to gently massage the liquid in, over and around
the nail, nail bed and skin to maximize deep penetration.
The subjects were observed over the course of ninety days.
40
CASE REPORT 1
A 55-year-old man came to the clinic to be treated for
his left thumbnail, which was white in color around the nail.
He complained of pain and dryness and there was a visible
yellow cast to the surface. He explained that application of
various otions and creams had no positive effect. The
patient was advised to use test essential oil formula twice
per day on the affected areas. After thirty days of treatment,
the patient showed significant improvement. In ninety days,
the patient’s nail was pink with no abnormal discoloration,
and there was no pain in the surrounding area.
** The test formula used in this study (Nature’s Oil for
Nails) was acquired from Perfectly Healthy of Santa Ana,
California. Leigh Erin Connealy, MD, has a personal financial interest in Perfectly Healthy.
REFERENCES
1. Elewski EB. Onychomycosis: pathogenesis, diagnosis, and
management. Clinical Microbiology Reviews.1998;11:415429.
2. Scherer WP, McCreary JP, Hayes WW. The diagnosis of
onychomycosis in a geriatric population. J Am Podiatr
Med Assoc. 2001;91:456-464.
3. Rich P, Harkless LB, Atillasoy ES. Dermatophyte test
medium culture for evaluating toenail infections in
patients. Diabetes Care. 2003;26:1480-1484.
4. Gupta AK, Cooper EA, MacDonald P, Summerbell RC.
Utility of inoculum counting (Walshe and English) critical
in clinical diagnosis of onychomycosis caused by non dermatophytic filamentous fungi. Journal of Clinical
Microbiology. 2001;39:2115-2121.
SUPERFICIAL WHITE
ONYCHOMYCOSIS
BEFORE TREATMENT
SUPERFICIAL WHITE
ONYCHOMYCOSIS
AFTER TREATMENT
5. Habif TP, Campbell JL, Quitadamo MI, Zug KA. Skin
disease diagnosis and treatment. A Color Guide to
Diagnosis and Therapy of Clinical Dermatology.
2001;190-195.
CASE REPORT 2
A 47-year-old woman came to the clinic to be treated
for nail fungus on her right foot. She complained of discoloration, thickening, brittleness and dryness. There was a
visible yellow and brown coloration to her nails. Initial
inspection revealed a grossly abnormal large nail on her
right big toe. The patient was treated with the test essential
oil formula, twice per day on all affected areas. After sixty
days, there was marked improvement in pain reduction;
after ninety days, the nails were significantly cleared and the
fungal spread was arrested.
ONYCHOMYCOSIS
BEFORE TREATMENT
41
ONYCHOMYCOSIS
AFTER TREATMENT
JANA Vol. 8, No. 3, 2005