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Finalist
NutrAward
2012

The 4 th

generation
folate
Product overview
Gnosis S.p.A. Gnosis USA Inc.
Via Lavoratori Autobianchi, 1 169 N. Main Street
20832 Desio (MB) Italy Doylestown, PA 18901 - USA

www.gnosis-bio.com advanced biotech

 QUATREFOLIC® Index

Foods as source of folates, the first generation compounds 2

Quatrefolic®, the fourth generation folate 4
The new salt advantages 5
Stability 5
Solubility and Bioavailability 6
Safety 7
Dosage 7

Folates, folic acid and the link with Vitamin B12 8

Interactions with Drugs and Vitamin B12 8
Bioequivalence of 5-methyltetrahydrofolate and folic acid in single dose 9
Gene Variants 11

Health Benefits 11
Pregnancy 11
Plasma folate levels and risk of spontaneous abortion 13
Effects on female and male fertility 13
Anemia 14
Hyperhomocysteinemia 14
Cardiovascular Disease (CVD) 15

References 17

Release M_03
1
 ®

Foods as source of folates,

the first generation compounds
Folate is a generic name for a naturally occurring family of B-group vitamins.
Folates are widely distributed in nature and are essential for the maintenance of cellular functions and health.
As humans (and other mammals) cannot synthesize folates, they must be obtained via diet.

However, natural folates (the first generation) are susceptible to oxidation, they rapidly lose activity in foods and
have a bioavailability range of 25-50%, depending on kind of food.
On the contrary, folic acid, the synthetic form of monoglutamyl folate, is almost completely stable for months or even
years and can be considered as a “second generation of folate, the stable one”.

The absorption of monoglutamyl folate occurs in the jejunum by a saturable, carrier-mediated, process at physiological
(micromolar) concentration of intraluminal folates (1, 2)*.
In order to diffuse all cells into body through the circulatory system, the folate monoglutamate must be transformed in the
5-methyltetrahydrofolate form, which passes by diffusion from blood into all body cells.
Oral supplementation with folic acid increases the body’s pool of 5-methyltetrahydrofolate in healthy and diseased
individuals.

Folate-requiring reactions, collectively referred to as “one-carbon metabolism”, include those involved in

• amino acid metabolism
• purine and pyrimidine synthesis
• formation of the primary methylating agent, S-adenosyl-methionine (SAM).

Total folate content in some common foods

The principal function of folate coenzymes is to accept or donate one-carbon units in key metabolic pathways.
The conversion of tetrahydrofolate (THF) to 5,10-methylene-THF is a crucial first step in the cycle that employs the
3-carbon of serine as a major carbon source.

*References are each numbered and described at the end of the document.

2
 A portion of the 5,10-methylene-tetrahydrofolate thus produced undergoes irreversible enzymatic reduction to
5-methyltetrahydrofolate, 5-methyltetrahydrofolate by methylene-tetrahydrofolate reductase (MTHFR). The N-5 methyl group of 5-methylte-
a central role in AA trahydrofolate is removed and transferred by vitamin B12 coenzyme to homocysteine, thus forming methionine.
metabolism, DNA synthesis In addition to protein synthesis, methionine serves as a methyl group donor through conversion to S-adenosyl-
and SAM formation methionine (SAM), a key biological methylating agent involved in over 100 methyltransferase reactions with a wide
variety of acceptor molecules.
The methionine synthase reaction also regenerates THF required for the formation of 5,10-methylene-THF and
10-formyl-THF used directly in thymidylate and purine synthesis, respectively (3).

Principal Components of the Folate Biochemical Cycle.

Folic acid (supplements and fortified foods)

3
S-Adenosyl- Dihydrofolate
Methylation methionine
reactions
5
DHFR
S-Adenosyl-
homocysteine Choline Methionine
Tetrahydrofolate

Betaine Serine
2 Glycine
Homocysteine B12
5,10-Methylene-
tetrahydrofolate

4 Purine
MTHFR biosynthesis
1
Pyrimidine
5-methylfolate
methyltetrahydrofolate biosynthesis

Principal Components of the Folate Biochemical Cycle. Abbreviations: DHFR = dihydrofolate reductase; MTHFR =
methylenetetrahydrofolate reductase. Reactions: 1 - Biosynthesis of nucleotides for incorporation into DNA and RNA; 2 -
Remethylation of homocysteine to form methionine (vitamin B12 serves as a coenzyme in this reaction); 3 - Methylation of
substrates, including DNA, RNA, phospholipids, and proteins; 4 - MTHFR, which catalyzes the formation of 5-methyltetrahydrofolate
needed for methylation reactions; 5 - Dihydrofolate reductase enzyme.

Dietary or genetically determined folate deficiency leads to mild hyperhomocysteinemia, which has been associa-
ted with various pathologies. Molecular mechanisms of homocysteine-induced cellular dysfunction include increa-
sed inflammatory cytokine expression, altered nitric oxide bioavailability, induction of oxidative stress, activation of
apoptosis and defective methylation.
Moreover, the involvement of folate and homocysteine metabolism has been documented in ageing-related dise-
ases, in several developmental abnormalities, in pregnancy complications and in male and female subfertility (4).

At repeated doses, Lamers in 2006 demonstrated that the administration of (6S )-5-methyltetrahydrofolate is more
effective than folic acid in improving folate status.

Healthy women (n=144) aged 19–33 y received 400 μg folic acid, the equimolar amount of (6S )-5-methylte-
trahydrofolate (416 μg), 208 μg (6S )-5-methyltetrahydrofolate, or placebo as a daily supplement for 24 wk.
Red blood cell and plasma folate concentrations were measured at baseline and at 4-wk intervals (5).

3
 ®

®
Quatrefolic , the fourth
generation folate
Until now, 5-methyltetrahydrofolate calcium salt (the third generation) was the only folic acid derivative available
on the market, and able to penetrate the body cells without further metabolism.
Gnosis R&D objective was to develop an innovative salt form able to overcome the existing limits related to stability
and slight solubility.

In February 2008, Gnosis patented a new generation of folate derivative, namely (6S)-5-methyltetrahydrofolate
glucosamine salt (Quatrefolic®), endowed with a long lasting stability and a peculiarly high water solubility as well
as an improved bioavailability and a well established safety. (U.S. Patent No. 7,947,662 - Patents Pending PCT/
EP2008/052037 and Patents Pending PCT/EP2008/052034).

In 2010, after a deep review of the extensive safety information and the uniqueness of the Quatrefolic®
compound, FDA has accepted the New Dietary Ingredient (NDI) notification for the ingredient concluding that
FDA has accepted a dietary supplement containing the new dietary ingredient is expected to be safe under the conditions of use
the NDI notification recommended or suggested on the label.

The generations of folate

4
 The new salt advantages
With Quatrefolic® Gnosis has managed to develop an innovative product with significant advantages over previous
folate generations. This remarkable step forward is attributed to the development of the glucosamine salt of
QuatrefolicTM, through two main steps:

• Improving solubility in water of 5-methyltetrahydrofolate, Quatrefolic® is defined as water soluble, a significant

Quatrefolic® overcomes opportunity for improving bioavailability, where as the calcium salt version is defined as sparingly soluble.
5-methyltetrahydrofolate • Guaranteeing a safety and high-quality stable profile by choosing glucosamine as natural compound naturally
calcium salt limits present in the body.

The result of this project, Quatrefolic®, claims key relevant benefits as

• Long lasting stability • Improved bioavailability

• High water solubility • Established safety

Structural formula of Quatrefolic®

HO
O
O-
O HO
O
CH3 O-
O N
N H O HO OH
N N
+
H NH3
H2N N N 2
H H

Stability
Quatrefolic® shows an extraordinary long lasting chemical stability guaranteeing a purity quite unaltered even after
Easy handling and several months, and an assay reduction in 1 year less than 1%, allowing easy handling and storage.
storage
The stability of Quatrefolic® powder form was tested according to ICH (international Conference on Harmonisation
of Technical Requirements for Registration of Pharmaceuticals for Human Use) guidelines both at room temperature
and other conditions keeping samples in airtight containers, protected from light, and measuring purity and assay
at different points.

Stability of Quatrefolic® at room temperature

5
 ®

Solubility and Bioavailability

In addition to the high chemical stability, Quatrefolic® demonstrates a surprisingly high solubility in water – greater
than 1 g/ml – compared with the slight solubility of the reference compound, (6S)-5-methyltetrahydrofolate calcium
salt (1.1 g/100 ml). Quatrefolic® has showed to be about 100 times more soluble than calcium salt.
Quatrefolic™ is 100
times more soluble High water solubility means the product may be better absorbed by mucosal cells which may facilitate access to
than calcium salt the blood and circulation with the potential for improved bioavailability. The first test was a direct bioavailability
comparison between Quatrefolic®, (6S)-5-methyltetrahydrofolate calcium salt and folic acid. After single oral dosing
in rats, Quatrefolic® showed peak plasma levels about 20% higher than those reached after a corresponding dose
of (6S)-5-methyltetrahydrofolate calcium salt.

Bioavailability of Quatrefolic® and (6S)-5-methyltetrahydrofolate calcium salt after oral dosing in rats

Pre-clinical study

The bioequivalence study of Quatrefolic® and (6S)-5-methyltetrahydrofolate calcium salt in healthy volunteers
Once the animal studies were completed, a human clinical trial was performed on 24 healthly volunteers of both
sexes in order to verify the superior bioavailability of Quatrefolic®.
It was a single dose, balanced, two sequences, two periods, two treatments randomized crossover study, with a 7
day wash out between two consecutive treatments.

Quatrefolic® and 5-methyltetrahydrofolate: pharmacokinetic comparison

Clinical study

Results

The study confirmed the experimental findings in the rat: Quatrefolic® has better bioavailability than (6S)-5-methyl-
tetrahydrofolate calcium salt. Mean Cmax for Quatrefolic® was 94.56 nmoles/l vs 85.29 nmoles/l for (6S)- 5-methyl-
tetrahydrofolate (+10.9%) and mean AUC0-24 was 594.2 nmoles/l (+9.6%) and 501.1 nmoles/l for Quatrefolic®
and (6S)-5-methyltetrahydrofolate respectively. Quatrefolic® can be considered 10% more bioavailable than (6S)-
5-methyltetrahydrofolate calcium salt.

6
 Safety
In a number of published and unpublished experimental human clinical trials and animal studies, the safety of (6S)-
GRAS (generally 5-methyltetrahydrofolate and Quatrefolic® has been extensively investigated. Based on a critical evaluation of the
recognized as safe) available data an independent expert panel confirms that Quatrefolic® is "generally recognized as safe" ("GRAS")
for use as a source of folate in conventional and medical foods.

Toxicological studies
Bacterial mutation in S.typhimurium and E.coli
The bacterial mutation assay was performed in order to assess the compound’s ability to induce gene
IN VITRO mutations in S.typhimurium and E.coli. The reverse mutation assay was run in bacterial strains already
mutant at a locus whose phenotypic effects are easily detected, and, since many chemicals can demonstrate
mutagenic activity only after metabolism to some reactive forms, the test was performed in presence and
in absence of a rat liver metabolic system ( S9 microsomal fraction).

The test concluded that Quatrefolic® does not induce reverse mutation in S.thyphimurium and E.coli at doses up to
5,000 μg/plate.

Mutation in L5178YTK+/- mouse lymphoma cells.

The assay was done in order to confirm the inability of Quatrefolic® to induce mutations in L5178YTK+/- mouse lym-
phoma cells cultured after in vitro treatment, and in absence or presence of a rat liver microsomal system.
The test concluded that Quatrefolic® does not induce mutations at concentrations up to 5,000 μg/ml.

Chromosome aberrations in Chinese hamster ovary cells (CHO) in vitro.

The assay was made in order to demonstrate the inability of Quatrefolic® to induce any chromosomal aberration in
presence or absence of a S9 liver microsomal fraction.
No chromosomal aberrations were observed in CHO after in vitro treatment with concentrations of Quatrefolic® up
to 5,000 μg/ml.

Single dose oral toxicity

The acute toxicity of Quatrefolic® was assessed in rats of both sexes, dosing the product by gavage at 500 mg/kg
IN VIVO
level. After dosing, animals were observed for a 7 day period and finally sacrificed.
No mortality occurred at this dose and during the observation time, and no clinical significant signs were observed
in any animal. Changes in body weight were not relevant, and no anomalies were recorded at the autopsy perfor-
med after the observation time. The lack of mortality points out that the maximum tolerated dose is greater than
500 mg/kg body weight, a dose some thousand times the one suggested for human use.

Dosage
The intended uses of Quatrefolic® and use levels will be same as that of folic acid, expressed on the basis of the
"Recommended Dietary Allowances for Folate for Children and Adults".

Dosages

7
 ®

Folates, folic acid and the link

with Vitamin B12
Interactions with Drugs and Vitamin B12
A number of drugs have been shown to affect the normal metabolism of folate and may cause folate deficiency.

Anti-folate drugs-drugs needed for certain conditions that adversely affect folate status
Methotrexate, used to treat neoplastic disease and rheumatoid arthritis, works as a folate antagonist by targeting
a key enzyme in folate metabolism, the dihydrofolate reductase. Many of the side effects mimic folate deficiency
and include gastrointestinal problems (nausea, diarrhea), stomatitis, headache, vertigo, pneumonitis, leucopenia,
thrombopenia, hair loss and infections (6, 7).

Anti-convulsant drugs
Low folate levels have consistently been reported in epileptic patients on phenytoin, phenobarbital or pirimidone,
Anti-epileptic drugs while data on valproate are conflicting (8, 9).
reduce folate levels On the contrary, it has been demonstrated that supplementation with folic acid can prevent folate deficiency and
improve phenytoin pharmacokinetics (10).

Anti-inflammatory drugs
NSAIDs inhibit the Commonly used non-steroidal anti-inflammatory drugs (NSAIDs) have anti-folate activity via their action as
inhibitors of enzymes involved in folate metabolism. Although the dose-response relationships with respect to an-
enzymes of folate
tagonistic effects on folate metabolism have not been established, folic acid food fortification has not been reported
metabolism
to cause any interference in the treatment actions of these drugs (11).

Oral contraceptives
It is suggested that oral contraceptives impair folate metabolism and tend to slightly, but significantly, reduce the
Oral contraceptives serum and erythrocyte levels of folate and to increase the urinary excretion of formiminoglutamic acid, an interme-
may interfere with diary product of histidine that requires THF to be further metabolized. This reduction is probably due to an increase
folate status in folate coenzyme utilization (12, 13).

Alcohol
Chronic alcoholism is probably the leading cause of folate deficiency in the Western world, with an incidence of
folate deficiency in chronic alcoholic patients as high as 87%, if low serum levels are used as the criterion of folate
deficiency, and an incidence of megaloblastic anemia up to a 61% (14, 15).
Alcohol and Possible causes of folate deficiency in alcoholic patients include:
megaloblastic • inadequate diet with resultant decreased body storage
anemia • intestinal malabsorption
• altered serum protein binding and tissue affinity
• altered hepatobiliary metabolism.

Vitamin B12
The potential adverse effects of high doses of folic acid supplements in relation to vitamin B12 deficiency relate to
intakes above the safe upper intake level of 1,000 μg/day.

8
Since the metabolism of folate and vitamin B12 are linked, in the case of vitamin B12 deficiency, conversion of
5-methyltetrahydrofolate to THF declines and eventually ceases. The synthesis of 5-methyltetrahydrofolate in
cells by the enzyme 5,10 methylene-THF reductase is irreversible. Thus, once formed, 5-methyltetrahydrofolate
can only be used by a single enzyme – namely B12-dependent methionine synthase. If vitamin B12 is deficient,
the enzyme methionine synthase ceases to function and, as a consequence, the folate present in cells becomes
“metabolically trapped” as 5-methyltetrahydrofolate. This situation produces a “pseudo folate deficiency”, because
although the cells have adequate levels of folate, it is trapped in the 5-methyltetrahydrofolate form not acting as a
co-factor for purine and pyrimidine biosynthesis.

If folic acid is consumed in very high doses (>1,000 μg per day), it can enter cells in “free” form and is converted
directly to THF and THF-polyglutamates, through pathways that are not dependent on vitamin B12.
In this way, ”free” folic acid can restart DNA biosynthesis, and correct anemia without affecting the methylation
cycle, which needs intervention of the vitamin B12.

Thus, while the anemia will be treated by folic acid, the neuropathy seen in vitamin B12 deficiency due to interrup-
tion of the methylation cycle will not, and some evidence exists to suggest it may become worse.
This masking of vitamin B12 anemia by taking folic acid makes the presence of B12 deficiency more difficult to
diagnose, allowing the neuropathy associated B12 deficiency to progress. Therefore, the main risk of exposure to
large doses of folic acid (>1,000 μg) is the masking of megaloblastic anemia, a diagnostic symptom of vitamin B12
deficiency.

Bioequivalence of 5-methyltetrahydrofolate and folic acid in single dose

The short term bioequivalence of folic acid and (6S)-5-methyltetrahydrofolate has been demonstrated in humans
by Pentieva and co-workers in the laboratory setting and by Venn and co-workers in the clinical practice. The im-
plications are that the natural folate derivative could have all the beneficial effects associated with folic acid, but
without the potential disadvantage of masking the anemia of vitamin B12 deficiency. Importantly, (6S)-5-methyl-
tetrahydrofolate is a natural folate derivative, a normal constituent of the body, and safety and tolerance of high
doses are not issues of concern (16, 17).

The short term bioequivalence of folic acid and (6S)-5-methyltetrahydrofolate

Plasma folate response (corrected for baseline values) in men after treatment with 500 μg (6S)-5-methyltetrahydrofolate,
500 μg folic acid, or placebo administered in random order at weekly intervals. Values are means ± SEM (Standard Error Mean),
n=13. Means without a common letter at a time differ p<0.05 (16).

At repeated doses, Lamers in 2006 demonstrated that the administration of (6S )-5-methyltetrahydrofolate is more
effective than folic acid in improving folate status.
Healthy women (n=144) aged 19–33 y received 400 μg folic acid, the equimolar amount of (6S )-5-methylte-
trahydrofolate (416 μg), 208 μg (6S )-5-methyltetrahydrofolate, or placebo as a daily supplement for 24 wk.
Red blood cell and plasma folate concentrations were measured at baseline and at 4-wk intervals.

9
 ®

The increase observed in red blood cell folate over time was significantly higher in the group receiving 416
μg (6S)-5-methyltetrahydrofolate/day than in the groups receiving 400 μg folic acid/day or 208 μg (6S)-
5-methyltetrahydrofolate/day (p<0.001). No plateau was reached in red blood cell folate concentration in
the 3 treatment groups during 24 wk of intervention; however, plasma folate plateaued after 12 wk (5).

Plasma folate

Geometric mean of plasma folate concentrations over time after 24 wk of supplementation with 400 μg folic acid/d (OE, n =34),
416 μg (6S)-5-methyltetrahydrofolate/d (D, n=35), 208 μg (6S)-5-methyltetrahydrofolate/d (n =33), or placebo (F, n =34). Bars
represent 95% CIs. A significant interaction was observed between time and intervention (p<0.001, repeated-measures ANOVA).
(5).

Willems et al. showed that after administration of (6R,S)-5-methyltetrahydrofolate to patients with established
coronary artery disease (CAD) the peak plasma concentration of (6S)-5-methyltetrahydrofolate is more than 7 times
higher compared to that after folic acid administration.
This difference is independent from the patients’ MTHFR genotype (18).

Pharmacokinetic properties of orally administered (6R,S)-5-methyltetrahydrofolate versus folic acid in

patients with coronary artery disease.

Genotype and treatment: (6S)-5-methyltetrahydrofolate plasma concentration (ng/ml) in patients with MTHFR CC genotype or TT
genotype following the administration of (6R,S)-5-methyltetrahydrofolate or folic acid, 5 mg each (18).

10
Gene Variants
Normal MTHFR activity may help maintain the pool of circulating folate and methionine, preventing the build-up of
homocysteine. MTHFR catalyzes the conversion of 5,10-methylene-tetrahydrofolate into 5-methyltetrahydrofolate
and its gene is located on chromosome 1 at 1p36.3.
It has been shown that two common MTHFR alleles (C677T and A1298C) are associated with congenital anomalies.
MTHFR activity among C677T homozygotes is 50-60% lower than in normal subjects. People homozygous for the
C677T allele tend to have mildly increased blood homocysteine levels if their folate intake is insufficient, but normal
blood levels if their folate intake is adequate.

The activity of the encoded enzyme A1298C allele is decreased, although less than in the case with the C677T
allele. People homozygous for the A1298C allele do not appear to have higher serum homocysteine levels
than controls. However, people who are heterozygous for the A1298C and C677T alleles (i.e., people with the
A1298CIC677T genotype) tend to have a biochemical profile similar to that seen among C677T homozygotes, with
increased serum homocysteine levels and decreased serum folate levels.

The population frequency of the C677T allele showed regional and ethnic variations. For example, the allele fre-
quency was high in Italy and among Hispanics living in California and low among U.S. Blacks and in some areas of
sub-Saharan Africa, while the population frequency of the A1298C allele is less documented (19).

Population frequency of homozygosity by geographic area and ethnicity

Population frequency of homozygosity for the C677T allele of 5,10-methylene-tetrahydrofolate reductase (MTHFR), by geographic
area and ethnicity, 1995-1999 (19).

Health Benefits
Pregnancy
Low dietary intake of folic acid increases the risk for delivery of a child with a neural tube defect (NTD). Before
conception and in the first part of gestation, folic acid supplementation significantly reduces the occurrence of NTD.
Spina bifida and anencephaly are the most common neural tube defects (NTDs) and, in randomized controlled trials,
folic acid supplementation before conception and during the first trimester has been shown to reduce the recurrence
of NTDs by 72% in women with a previous NTD affected pregnancy.

11
 ®

Data from U.S. birth certificates indicate a 19% decline in the birth prevalence of NTDs and a 23% de-
cline in spina bifida prevalence among births conceived after mandatory folic acid fortification
(October 1998 through December 1999) compared with the NTD prevalence before folic acid fortification (October
1995 through December 1996) (20).

Trend in spina bifida

Birth

Trends in Spina Bifida Among All Births, National Center for Health Statistics Vital Statistics Data, 1990-1999, for 45 U.S. States
and Washington, D.C.
Arrows indicate statistically significant increases and decreases by the exponential weighted moving average analyses with
parameters of p=0.01 and weight=0.075 (20).

In Canada, the overall prevalence of neural-tube defects at birth decreased from 1.58 per 1,000 births before forti-
fication mandatory (November 1998) to 0.86 per 1,000 births during the full-fortification period, a 46% reduction
(95% confidence interval, 40 to 51). The magnitude of the decrease was higher for spina bifida (53%) than for
either anencephaly (38%, p=0.02) or encephalocele (31%, p=0.03).

A greater reduction in rates was found in regions with a higher baseline prevalence of neural tube defects than in
regions with a lower prevalence (21).

Prevalence of Neural-Tube Defects

Prevalence of Neural-Tube Defects, According to Diagnostic Category, in Seven Canadian Provinces from 1993 through 2002. NOS
denotes not otherwise specified (21).

12
 Plasma folate levels and risk of spontaneous abortion
It has been suggested that the rapidly developing cells in the embryo may suffer by lack of adequate folate. Failure
Low plasma to produce sufficient DNA and to regulate DNA function could lead to spontaneous abortion. In their case-control
levels of folate study, George and co-workers evaluated the relationship between plasma folate levels and the risk of spontaneous
increase the risk abortion.
of spontaneous Compared with women with normal plasma folate levels (2.20-3.95 ng/ml), women with low (<2.19 ng/ml) fo-
abortion late levels were at increased risk of spontaneous abortion (adjusted odds ratio 1.47), whereas women with higher
folate levels (3.96-6.16 ng/ml) showed no increased risk of spontaneous abortion (22).

Effects on female and male fertility

In 2008, Chavarro and co-workers reported the prospective analysis of incident ovulatory infertility among partici-
pants to the Nurses’ Health Study II, a 9 years prospective cohort study designed to investigate the role of diet and
other lifestyle factors in common chronic diseases, run in 18,555 married, pre-menopausal women who attempted
to become pregnant.
Objective of the study was to examine whether the use of multivitamins and intake of specific nutrients in multivi-
tamins was associated with ovulatory infertility.

A first analysis concluded that multivitamin users had approximately one-third lower risk of developing ovulatory
Multivitamin infertility when compared with non-users (p<0.001). After a further data adjustment for known and suspected risk
users had factors for infertility, only the intake of folic acid was associated with a reduced risk of ovulatory infertility (23).
approximately
one-third lower Relative risk of ovulatory infertility
risk of develo-
ping ovulatory
infertility

Q1 Q2 Q3 Q4 Q5

Folic acid daily median intake (μg)

Multivariate-adjusted relative risk of ovulatory infertility and intake of folic acid (23).

13
 ®

It has been shown that the administration of a daily dose of 15 mg folic acid for 3 months to 65 infertile men with
A folic acid
an excessive round cell count in their ejaculate induced statistically significant modifications of all analysed sperm
supplement parameters, particularly it was observed an increase in sperm density (from 15 to 22.6x106/ml) and motility (from
increases male 17.7% to 27.8%) as well as a decrease in round cell count (from 9.7 to 6.4x106/ml) (4).
sperm density
and improves Anemia
conceptus
Folic acid has a long history of use in conjunction with vitamin B12 for the treatment of macrocytic anemia. De-
pending on the clinical status of the patient, the dose of folic acid or 5-methyltetrahydrofolate required to reverse
macrocytic anemia varies, but the therapeutic dose is usually 800-1,000 μg daily. Duration of therapy to reverse
macrocytic anemia can be as short as 15 days after initiation of supplementation, or it may require prolonged sup-
plementation.

Hyperhomocysteinemia
Homocysteine is widely accepted as independent risk factor for coronary, cerebral, and peripheral vascular diseases
(24). This has been observed even when presupplementation plasma folate concentrations were well within the
range of values currently accepted as reflecting adequate status. In several studies, daily folic acid administration
in high (pharmacologic) doses of 0.4 to 10 mg resulted in significant reductions in plasma total homocysteine (25).
In a placebo-controlled study on 100 men with hyperhomocysteinemia randomly assigned to 5 groups of daily treatment
In men, only with placebo, folic acid (0.65 mg), vitamin B12 (0.4 mg), vitamin B6 (10 mg) or a combination of the three vitamins
for 6 wk, folic acid supplementation reduced plasma homocysteine concentrations by 41.7% (p<0.001), whereas
folic acid reduces
the daily vitamin B12 supplement lowered homocysteine concentrations by 14.8% (p<0.01). The daily pyridoxine
hyperhomocysteinemia
dose did not reduce significantly plasma homocysteine concentrations. The combination of the three vitamins re-
duced circulating homocysteine concentrations by 49.8%, which was not significantly different (p=0.48) from the
reduction achieved by folate supplementation alone (26).

Effects of different vitamin supplements on plasma concentration of homocysteine

Effects of different vitamin supplements on plasma concentration of homocysteine in men with hyperhomocysteinemia (26).

Reducing elevated plasma homocysteine means lowering not only an independent risk factor for neural
tube defects and other birth defect and altered male and female fertility, but also for cardiovascular
disease, Alzheimer’s disease, cognitive decline, osteoporosis, rheumatoid arthritis, kidney failure, and cancer.

14
 Cardiovascular Disease (CVD)
Interestingly, a large population-based cohort study of men and women, 40 to 42 and 65 to 67 years old, showed
that in the older age group total homocysteine levels are strong predictor for CVD hospitalization in the following 5
years. The relationship observed among the elderly was graded, independent of other measured CVD risk factors,
and applied to all of the major categories of CVD.
The association was strongest among people with preexisting CVD and/or antihypertensive treatment (27).

Time to first hospitalization with cardiovascular disease (CVD)

Reducing
hyperhomocysteinemia
reduces the rate of
hospitalization for CVD

Time to first hospitalization with cardiovascular disease (CVD) as the main discharge diagnosis by baseline plasma total
homocysteine levels (27).

In another prospective study on patients with coronary artery disease (CAD) angiographicaly documented, a strong
dose-response relation between the total homocysteine level and overall mortality was observed (28).

Chronic exposure of vascular endothelium to homocysteine compromises the production of adequate amounts of
nitric oxide (NO) leading to injury of the endothelial lining and the initiation of atherosclerosis. 5-methyltetrahydro-
folate improves NO synthesis by:
5-methyltetrahydrofolate
• reducing plasma homocysteine levels
acts also directly on the
• enhancing the availability of key endothelial NO cofactors, such as tetra-hydrobiopterin
vessel wall • reducing the production of superoxide anions
• substituting for tetrahydrobiopterin as a cofactor in the enzyme nitric oxide synthesis, the net effect of which is
improvement of peripheral blood flow.

The effects of 5-methyltetrahydrofolate were documented ex vivo by incubating vessels with 5-methyltetrahydrofolate
(1 to 100 μmol/l) and in vivo by intravenous infusion of 5-methyltetrahydrofolate or placebo before vessel harvest.
5-methyltetrahydrofolate improved NO-mediated endothelium-dependent vasomotor responses and reduced va-
scular superoxide, both ex vivo and in vivo (29).

15
 ®

Superoxide and peroxynitrite production

5-methyltetrahydrofolate
decreases superoxide
production in vessels of
CAD patients

Superoxide and peroxynitrite production were significantly decreased after incubation with increasing concentrations of
5-methyltetrahydrofolate for 45 minutes in both SVs (A, n=32, and B, n=6) and IMAs (C, n=23 and D, n=6) compared with control
vessels (incubated with buffer) from the same patients. Values are expressed as median (horizontal line), 25th to 75th percentile
(box), and range (whiskers). *P<0.01 vs control. RLU/sec/mg indicates relative light units per second per milligram (29).

In a double-blind, placebo-controlled, crossover study on individuals with coronary artery disease, supplementation
with high-dose folic acid (30 mg per day) improved blood flow in coronary arteries.
Folate was associated with a reduction in mean arterial pressure (100 ± 12 mmHg vs 96 ± 11 mmHg, placebo vs folate,
p<0.03) (30).

Acute effects of folic acid administration on coronary flow in CAD patients

Acute folate Mean Arterial Pressure ± ±

administration
Myocardial Blood Flow ± ±
improves coronary
flow parameters in ± ±
CAD patients

Effect of high-dose folate on coronary dilator reserve

Folate increased dilator reserve by ~83% in abnormal segments (0.72 ± 0.60 ml/min/g vs 1.31 ± 1.08 ml/min/g, mean ± SD,
placebo vs folate, p<0.05), whereas dilator reserve in normal segments remained unchanged

16 (2.00 ± 0.61 ml/min/g vs 2.12 ± 0.69 ml/min/g, placebo vs folate, p=NS). (NS: Not Significant) (30).

16
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Finalist
NutrAward
2012

The 4 th

generation
folate
Product overview
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