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Absorption rates and free radical scavenging values of vitamin C-lipid metabolites in human lymphoblastic cells

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In this study we investigated the cellular absorption rates, antioxidant and free radical scavenging activity of vitamin C-lipid metabolites. The absorption was measured in a human lymphoblastic cell line using a spectrophotometric technique. Cellular vitamin C levels in the human lymphoblastic H9 cell line were measured using the 2,4-dinitrophenylhydrazine spectrophotometric technique. Free radical scavenging activity of vitamin C-lipid metabolites was measured by the reduction of 1,1-diphenyl-2-picryl hydrazyl (DPPH) to 1,1-diphenyl-2-picryl hydrazine. Vitamin C-lipid metabolite scavenging of peroxyl radical oxygen reactive species (ORAC) was determined by fluorescence spectrophotometry. Compared to ascorbic acid (AA), calcium ascorbate (CaA), and calcium ascorbate-calcium threonate-dehydroascorbate (Ester-C), vitamin C-lipid metabolites (PureWay-C) were more rapidly absorbed by the H9 human T-lymphocytes. The vitamin C-lipid metabolites (PureWay-C) also reduced pesticide-induced T-lymphocyte aggregation by 84%, while calcium ascorbate-calcium threonate-dehydroascorbate (Ester-C) reduced aggregation by only 34%. The vitamin C-lipid metabolites (PureWay-C) demonstrated free radical scavenging activity of nearly 100% reduction of DPPH at 20 microg/ml and oxygen radical scavenging of over 1200 micro Trolox equivalents per gram. These data demonstrate that the vitamin C-lipid metabolites (PureWay-C) are more rapidly taken-up and absorbed by cells than other forms of vitamin C, including Ester-C. This increased rate of absorption correlates with an increased protection of the T-lymphocytes from pesticide toxicities. Further, vitamin C-lipid metabolites (PureWay-C) are a potent antioxidant and have significant free radical scavenging capabilities.
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Absorption rates and free radical scavenging values
of vitamin C-lipid metabolites in human lymphoblastic
cells
Benjamin S. Weeks
1
ABCDEF, Pedro P. Perez
2
ABCDEFG
1
Department of Biology and Environmental Sciences Program, Adelphi University, Garden City, NY, U.S.A.
2
Innovation Laboratories, Inc., Mount Sinai, NY, U.S.A.
Source of support: Innovation Laboratories, Inc., Mount Sinai, NY, U.S.A.
Summary
Background:
In this study we investigated the cellular absorption rates, antioxidant and free radical scavenging
activity of vitamin C-lipid metabolites. The absorption was measured in a human lymphoblastic cell
line using a spectrophotometric technique.
Material/Methods:
Cellular vitamin C levels in the human lymphoblastic H9 cell line were measured using the 2,4-di-
nitrophenylhydrazine spectrophotometric technique. Free radical scavenging activity of vitamin
C-lipid metabolites was measured by the reduction of 1,1-diphenyl-2-picryl hydrazyl (DPPH) to
1,1-diphenyl-2-picryl hydrazine. Vitamin C-lipid metabolite scavenging of peroxyl radical oxygen
reactive species (ORAC) was determined by fl uorescence spectrophotometry.
Results:
Compared to ascorbic acid (AA), calcium ascorbate (CaA), and calcium ascorbate-calcium thre-
onate-dehydroascorbate (Ester-C
®
), vitamin C-lipid metabolites (PureWay-C™) were more rapidly
absorbed by the H9 human T-lymphocytes. The vitamin C-lipid metabolites (PureWay-C™) also re-
duced pesticide-induced T-lymphocyte aggregation by 84%, while calcium ascorbate-calcium thre-
onate-dehydroascorbate (Ester-C
®
) reduced aggregation by only 34%. The vitamin C-lipid metab-
olites (PureWay-C™) demonstrated free radical scavenging activity of nearly 100% reduction of
DPPH at 20 μg/ml and oxygen radical scavenging of over 1200 μ Trolox
®
equivalents per gram.
Conclusions:
These data demonstrate that the vitamin C-lipid metabolites (PureWay-C™) are more rapidly tak-
en-up and absorbed by cells than other forms of vitamin C, including Ester-C
®
. This increased rate
of absorption correlates with an increased protection of the T-lymphocytes from pesticide toxici-
ties. Further, vitamin C-lipid metabolites (PureWay-C™) are a potent antioxidant and have signif-
icant free radical scavenging capabilities.
key words: absorption • T-lymphocyte • Vitamin C • DPPH • ORAC
Full-text PDF: http://www.medscimonit.com/fulltxt.php?IDMAN=10702
Word count: 2669
Tables: 3
Figures: 2
References: 29
Author’s address: Pedro P. Perez, PhD, 9 Sean Lane, Mount Sinai, NY 11766, U.S.A., e-mail: Innteam@aol.com
Authors’ Contribution:
A Study Design
B Data Collection
C Statistical Analysis
D Data Interpretation
E Manuscript Preparation
F Literature Search
G Funds Collection
Received: 2007.07.05
Accepted: 2007.08.07
Published: 2007.10.01
BR205
Basic Research
WWW.MEDSCIMONIT.COM
© Med Sci Monit, 2007; 13(10): BR205-210
PMID: 17901843
BR
Current Contents/Clinical Medicine • IF(2006)=1.595 • Index Medicus/MEDLINE • EMBASE/Excerpta Medica • Chemical Abstracts • Index Copernicus
BACKGROUND
Vitamin C is an important dietary component which is re-
quired for physiological and metabolic activities including
healthy neuronal development [1,2], prevention of neuro-
degenerative diseases [3,4], wound healing [1,5,6], and a
healthy immune system [1,7,10]. Therefore, the bioavail-
ability of vitamin C in the diet is the focus of intense re-
search interest. A calcium ascorbate preparation with small
amounts of dehydroascorbate, calcium threonate, xylonate
and lyxonate, has been shown to lead to the increased cel-
lular uptake of vitamin C [9,10], to provide increased pro-
tection from vitamin C defi ciency in rats [11], and to im-
prove uptake and circulating levels of vitamin C in humans
[12]. More recently a formulation of vitamin C-lipid me-
tabolites has been shown to more rapidly stimulate neurite
outgrowth, fi broblast adhesion and protect cells of the im-
mune system when compared to all other vitamin C formu-
lations [2]. This increased rate of bioactivity suggests an in-
crease in the bioavailability and rate of cellular uptake of
vitamin C-lipid metabolites when compared to other vita-
min C formulations.
Here, we measure the rate of cellular uptake of vitamin
C-lipid metabolites (PureWay-C™) when compared to cal-
cium ascorbate, ascorbic acid, and calcium ascorbate-calci-
um threonate-dehydroascorbate (Ester-C
®
). We also con-
rm that the vitamin C-lipid metabolites (PureWay-C™)
maintain and deliver both high and effective antioxidant
and free radical scavenging activity.
MATERIAL AND METHODS
Material
Dimethylsulfoxide was purchased from Sigma Chemical Co.,
St. Louis, Mo. and bifenthrin was purchased from Chem.
Services, West Chester, PA. Bifenthrin was brought to a stock
concentration of 10
–2
M in DMSO Phytohemagglutinin was
purchased from Sigma Chemical Co. St Louis, MO and sus-
pended in RPMI 1640 and stored at –20°C. Formulations
and certifi cates of analysis of ascorbic acid, calcium ascor-
bate, Ester-C
®
and PureWay-C™ were provided by Nature’s
Value, Coram, NY, from their respective suppliers, and were
dissolved in sterile, serum-free RPMI-1640 at a 50 μM. 2,4-di-
nitrophenylhydrazine was purchased from Sigma-Aldrich
(www.sigmaaldrich.com). 1,1-diphenyl-2-picryl hydrazine and
Trolox
®
were provided by ChromDex Analytics (Boulder,
CO) where the PDDH and ORAC assays were performed.
Cells culture and cell pellet preparation
The H9 human lymphoblastic cell line was obtained from
the NIH AIDS reagent program and cultured in a medium
of RPMI-1640 containing 10% fetal bovine serum and 0.01%
gentamycin and incubated in a CO
2
incubator at 37°C. Cell
viability and cell numbers for plating during experimenta-
tion was determined using trypan blue and a hemocytom-
eter counting slide.
Briefl y, 3.0×10
7
H9 cells were collected in into one pellet by
repeated centrifugation at 500 × g and the supernatant was
removed. In order to completely remove the serum compo-
nent from the culture medium and starve the cells for vita-
min C and nutrients that alter cellular vitamin C levels, the
pellet was washed three times by repeated resuspension in
50 ml of serum free RPMI and centrifuged at 500 × g. After
three washes with serum-free RPMI-1640, the pellet was re-
suspended in a fi nal 20 ml of serum-free RPMI, seeded in
a 150 cm
3
tissue culture fl ask and incubated in a CO
2
water-
jacketed incubator at 37.5°C. After 18 hours of starvation
the cells were collected into one pellet as described above
and counted for viability using trypan blue.
In order to test the uptake of the four various forms of vita-
min C, each vitamin C preparation was dissolved at 50 μM
in RPMI-1640. After determining cell viability as described
above, sets of eight 75 cm
3
tissue culture fl ask were seed-
ed with 6×10
5
cells in 4.0 ml of RPMI-1640 containing each
type of vitamin C preparation and these fl asks were placed
horizontally for incubation in a 37.5
o
C water-jacketed CO
2
incubator seeded in a and placed in the CO
2
water jacket
incubator at 37.5°C. Every 15 minutes, one fl ask containing
each of the different vitamin C preparations was removed
from the incubator and agitated to assure equal cell suspen-
sion which was divided into four 1.0 ml aliquots in micro-
centrifuge tubes and centrifuged at 500 × g. The superna-
tant was discarded and the cell pellets (1.5×10
5
cells/pellet)
were frozen at –20°C for further analysis. Three of the pellets
were used to determine Vitamin C levels and one was used
to determine protein content. This design permitted trip-
licate vitamin C readings at times ranging 15 through 120
minutes at 15 minute intervals for each of the four Vitamin
C formulations and the determination of protein content.
Therefore the cellular vitamin C concentration could be
determined as nmol/mg cellular protein.
Vitamin C assay
The cellular content of vitamin C was measured using the
method described by Lowry et al., 1947 [13]. Briefl y, to
the cell pellets, 40 μl of 5% trichloroacetic acid was added.
For adequate extraction of ascorbic acid the samples were
mixed well by vortex. Next, 13 μl of freshly prepared dini-
trophenylhydrazine reagent (2% dinitrophenylhydrazine,
0.25% thiourea, 0.03% CuS0
4
·5H
2
0 prepared in 9 N H
2
S0
4
)
was added to each tube. The samples were again mixed well
and incubated at 37.5°C for six hours. The samples were
then placed on ice and 50 μl of chilled (4°C) 65% H
2
S0
4
were added and mixed well by vortex. The optical densi-
ty of the samples was then read at 520nm in a Benchmark
plus Bio-Rad microplate reader and compared to a standard
curve (described below). Blanks were prepared by treating
400 μl of 5% trichloroacetic acid in the same manner as the
cell pellets. The standard curve was generated by dissolv-
ing various amounts of ascorbic acid (0.05–1.5 mg/ml) in
5% trichloroacetic acid and treated in the same manner as
the cell extracts.
Protein assay
The protein content of the cell pellets was determined us-
ing a BioRad Bradford Assay Kit. Briefl y, the cell pellets were
lysed and homogenized in 100 μl of RIPA buffer (150 mM
NaCl, 10 mM Tris, pH 7.2, 0.1% SDS, 1.0% Triton X-100,
1% Deoxycholate and 5 mM EDTA). Next, 400 μl of the the
BioRad Bradford Kit dye reagent concentrate was added di-
rectly to the samples. For the standard curve, fi ve dilutions
Basic Research Med Sci Monit, 2007; 13(10): BR205-210
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of a bovine serum albumin ranging from 0.1 to 0.5 mg/ml
were prepared and 400 μl of the the BioRad Bradford Kit
dye reagent concentrate was added directly to 100 μl of each
standard. Samples and standards were then vortexed and
allowed to sit on the bench-top for 10 minutes and the ab-
sorbance of 200 μl was then read at 595 nm in a Benchmark
plus Bio-Rad microplate reader.
T-cell homotypic aggregation assays
Cell were collected and 3.0 ml of 2×10
5
cells/ml were seed-
ed in wells of a six-well tissue cluster and then treated with
10 μg/ml of phytohemagglutinin and or 10
–5
M bifenthrin.
The stock bifenthrin was 10
–2
M in DMSO therefore a vehi-
cle control of 0.1% DMSO was used. Therefore the control
untreated cells for this experiment contained 1% DMSO.
The PHA was diluted in RPMI 1640 and so did not require
a vehicle control. Immediately after seeding the cells, trip-
licate wells were treated with the 0.5 μM ascorbic acid from
the various formulations and the cells were incubated in a
in a CO
2
incubator at 37°C for 30 minutes. After treatment
for 30 minutes the ability of vitamin C to inhibit homotyp-
ic aggregation was measured by counting aggregate size at
400× magnifi cation. The center fi eld of each well was as-
sessed by visual inspection and the number of cells in each
aggregate was counted and divided by the number of ag-
gregates to derive the number of cells per aggregate. Since
the treatments were done in triplicate, three fi elds were
counted for each treatment. While the number of aggre-
gates was noted, these data are not presented. The num-
ber of cells per aggregates is. The treatment with few cells
per aggregate also had very few aggregates to count. The
aggregate size was counted
DPPH and ORAC assays
The DPPH assay was carried out as described by Vani et
al., 1997 [14] with some modifi cations. Briefl y, 200 ml of a
1mg/ml ascorbic acid solution (1.0 mg PWC in 1.0 ml dis-
tilled water) was mixed with 50 μl of 0.659 mM 2,2-diphenyl-
1-picryl hydrazyl (DPPH) solution and incubated at 25°C for
20 minutes. The absorbance was then read at 510 nm.
The ORAC assay was performed as described by Cao et al.,
1993 [15]. Briefl y, 2,2’-Asobix (2-amidinopropane) dihy-
drochloride (AAPH) (0.414 g) was dissolved in 10 ml of 75
mM phosphate buffer to fi nal concentration of 153 mM and
kept on ice. The fl uorescein stock solution was prepared at
4.19×10
–3
mM in 75 mM phosphate buffer and kept at 4°C
in the dark. For the (±)-6-Hydroxy-2,5,7,8- tetramethylchro-
mane-2-carboxylic acid, (Trolox
®
) standard preparation, 0.25
g of Trolox
®
dissolved was in 50 ml of phosphate buffer to
yield 0.02 M stock. Next, PureWay-C™ (PWC) was dissolved
in acetone/water mixture (50–50) and subsequently dilut-
ed with 75 mM phosphate buffer (pH 7.4) to a varying ex-
tent to yield 200, 100, 50, 25, 12.5, and 6.25 μM for the test
reactions. The ORAC assay detects free radical damage to
the fl uorescein and a loss of fl uorescence. Antioxidants in-
hibit the free radical range damage to the fl uorescent com-
pound and prevent the reduction in fl uorescence. Reactions
containing the PureWay-C™ (PWC) and blanks (solvent)
were run in parallel using equivalent amounts of a gener-
ator of a radical oxygen species and the fl uoroscein and
the area under the curve from the experimental sample
was calculated. After subtracting the area under the curve
for the for the blank, the resultant difference is expressed
as antioxidant activity of the PureWay-C™ (PWC). Results
from different concentrations are compared with Trolox
®
and the ORAC results are expressed as Trolox
®
equivalents
(TE) per gram of sample.
RESULTS
The rate of vitamin C absorption in human T-cells was com-
pared between several different vitamin C formulations. We
found signifi cantly different rates of uptake depending on
the formulation of vitamin C. Over a two hour period, the
level of vitamin C-lipid metabolite uptake was consistently
higher than that observed with ascorbic acid, calcium ascor-
bate, and calcium ascorbate-calcium threonate-dehydroascor-
bate (Figure 1). At fi fteen minutes, cellular vitamin C levels
ranged from 7±1.4 nmols/mg cellular protein with ascor-
bic acid, to over double that amount (15±2.4nmol/mg) for
the vitamin C-lipid metabolites (Figure 1). The absorbed
vitamin C levels rose signifi cantly with time, peaking at ap-
proximately two hours with cellular levels ranging from
31 nmol/mg for ascorbic acid and 50 nmol/mg for vita-
min C-lipid metabolites (Figure 1).
Since ascorbic acid showed the least uptake at all time points
(Figure 1), the values for ascorbic acid were considered to
be 100% for all time point thus allowing a percent increase
comparison for the other formulations in Table 1. When com-
pared to ascorbic acid, calcium ascorbate-calcium threonate-
dehydroascorbate (Ester-C
®
), showed a 189% increase in up-
Time (minutes)
PWC
AA
CaA
Ester-C
Cellular Vitamin C (nmol/mg cellular protein)
60
30
0
0 30 60 90 120
Figure 1. Vitamin C absorption rates in the H9 human T-cell line.
Cells were starved of vitamin C for 18 hours in serum-free
RPMI-1640 and subsequently suspended in 50 μM of
vitamin C in various formulations: ascorbic acid (AA),
calcium ascorbate (CaA), calcium ascorbate-calcium
threonate-dehydroascorbate- Ester-C® (Ester-C), and
the vitamin C lipid metabolites-PureWay-C™ (PWC). At
the indicated times, cells were harvested and measured
for vitamin C and protein content as described in the
Materials and Methods section. An analysis of variance was
performed and the asterisks indicate a signi cant di erence
at 95% con dence (Sche e F test) between the vitamin C-
lipid metabolites (PWC) and calcium ascorbate-calcium
threonate-dehydroascorbate (Ester-C).
Med Sci Monit, 2007; 13(10): BR205-210 Weeks BS et al – Absorption rates and free radical scavenging values of vitamin…
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take, which is similar to earlier reports of 177% at 30 minutes.
The vitamin C-lipid metabolites (PureWay-C™) showed a
233% increase at both 30 and 45 minutes compared to ascor-
bic acid (Table 1) which is over a 120% improvement in ab-
sorption in the fi rst 45 when compared to calcium ascorbate-
calcium threonate-dehydroascorbate (Ester-C
®
).
The increase rate of absorption of the vitamin C-lipid metab-
olites correlated with a greater protective effect at after 30
minutes of exposure to pesticide (Table 2). When cells were
treated with the pesticide bifenthrin and various formula-
tions of vitamin C at time zero, the resultant pesticide-medi-
ated aggregation of the T-lymphocytes was reduced by 84%
with bifenthrin and only by 34% with calcium ascorbate-cal-
cium threonate-dehydroascorbate treatment (Table 2).
In order to confi rm that the vitamin C-lipid metabolite for-
mulation has potent antioxidant and have free radical scav-
enging capabilities, ORAC and PDDH evaluations were
conducted. Figure 2 shows that the vitamin C-lipid metab-
olites were able to scavenge 93% of the PDDH free radi-
cals at 20 μg/ml (Figure 2). Gallic acid, a known scavenger
was used as a positive control and also showed a maximal
93% scavenging. The vitamin C-lipid metabolites showed a
classic dose dependency scavenging of DPPH free radicals
(Figure 2) and by reaching 93% scavenging capability is in-
dicative of an excellent free radical scavenger [14].
In addition to free radical scavenging, the antioxidant ca-
pabilities of the vitamin C lipid metabolites were measured
by the ORAC method and the values obtained were com-
pared to other sources of antioxidants also measured by
the ORAC method (Table 3). The vitamin C lipid metab-
olites showed the stronger antioxidant activity on a gram
basis than common natural sources of antioxidants. For
example, PureWay-C™ has over 1300 units of antioxidant
activity per gram, in stark contract to freeze-dried Acai,
teas, some berries, and broccoli (Table 3). While these
comparisons are limited in value, it can be concluded that
PureWay-C™ is an excellent dietary supplement for it an-
tioxidant capabilities.
DISCUSSION
Vitamin C is an important component of the diet to en-
sure healthy physiological and metabolic activities such as
the development of a healthy nervous system [1,2]; preven-
tion of neurodegenerative diseases [3,4]; wound healing in
vitro [5], and in vivo [6]; and protection of the immune sys-
tem from xenobiotics [1,7,8]. For example, vitamin C has
been shown to enhance NGF-mediated neurite outgrowth
[1,2]. Further, fi broblast interaction with extracellular ma-
trix proteins and subsequent migration are indicators of
wound healing events [22–24], and, indeed, vitamin C has
been shown to enhance fi broblast adhesion to and interac-
tion with the extracellular matrix [1,25,26]. Further, leuko-
cyte cell-cell adhesion is associated with xenobiotic induced
hyperactivation and infl ammatory damage [1,7,8,27], and
Percent Increase
Compared to
ascorbic acid
CaA Ester-C® PureWay-C™
Time after
addition
15 min 128 171 214
30 min 122 189 233
45 min 133 192 233
Percent Increase
Compared to
Ester-C®
CaA Ester-C® PureWay-C™
Time after
addition
15 min 0 125
30 min 0 124
45 min 0 122
Table 1. Percent di erences in Vitamin C absorption rates in the H9
human T-lymphocyte line.
Calcium ascorbate (CaA), ascorbate-calcium threonate-
dehydroascorbate (Ester-C®), and vitamin C-lipid metabolites
(PureWay-C™) all showed better cellular absorption rates compared to
ascorbic acid (AA). In the top portion of the table, the levels of cellular
vitamin C are presented as the percent of that observed with ascorbic
acid (AA) at the indicated time points. In the lower portion of the
table, the cellular levels of PureWay-C™ are shown as the percent of
that observed with Ester-C®.
Activators of T-cell Aggregation
None PHA Bifenthrin
Vit. C Added
None 10±5 170±15 300±13
AA 9±4 75±12 120±5
CaA 12±4 110±10 137±8
EsterC 8±2 130±17 200±8
*PWC 11±6 20±9 50±10
Table 2. Vitamin C-lipid metabolites (PureWay-C™) inhibits xenobiotic
induced homotypic aggregation in human T-lymphocytes
more e ectively than calcium ascorbate-calcium threonate-
dehydroascorbate Ester-C®.
Human H9 T-lymphocytes were either untreated (None) or treated
with activators of T-lymphocyte aggregation phytohemagglutinin
(PHA) or bifenthrin and then either given no further vitamin C
treatments (None) (note, bifenthrin was dissolved in 0.1% DMSO,
so the no treatments contained 0.1% DMSO as a vehicle control) or
treated further with 50 μM ascorbic acid (AA), calcium ascorbate (CaA),
Ester-C® (EsterC) or PureWay-C™ (PWC). The cells were then incubated
for 30 minutes and the number of cells per aggregate was determined
as described in the Materials and Methods section. The error is
represented as ± the standard error of the mean with an n of 6.
* While all vitamin C preparations stimulated a statistically signi cant
reduction in aggregation, PWC (PureWay-C™), showed a statistically
signi cant greater reduction when compared to Ester-C®. PHA was
used as a positive control for the activation of aggregation.
Basic Research Med Sci Monit, 2007; 13(10): BR205-210
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vitamin C has been shown to prevent cigarette smoke-in-
duced leukocyte aggregation and attachment to vascular en-
dothelium [7,8]. Vitamin C has also been shown to reduce
pesticide mediated T-cell hyperactivation [1]. For each of
the above benefi cial activities, vitamin C-lipid metabolites
(PureWay-C™) have been shown to be more active and act
more quickly than calcium ascorbate-calcium threonate-de-
hydroascorbate (Ester-C
®
) [1].
In order for vitamin C to exert these benefi ts; it must be tak-
en into the cell. Therefore, the cellular uptake and reten-
tion of vitamin C have been an area of signifi cant study and
formulations of vitamin C have been examined for rates of
cellular uptake. To date, the most rapidly adsorbed vitamin
C formulation, calcium ascorbate-calcium threonate-dehy-
droascorbate (Ester-C
®
) was shown to have a 177% increased
absorption in the H9 human T-cell line [7]. The level of vi-
tamin C in human T-lymphocytes in fasting has been mea-
sured at approximately 12 nmol vitamin C per mg of cellu-
lar protein (12 nmol/mg) [28] and as high as 88 μg vitamin
C per 10
8
T-cells in people eating a healthy diet [29]. Here,
after starving human T-cell in vitro, we fi nd that the ascor-
bic acid uptake in these cells peaks at 31 nmol/mg cellu-
lar protein or 54 μg/10
8
cells while the vitamin C-lipid me-
tabolites peak at 51 nmol/mg cellular protein or 90 μg/10
8
cells. The more rapid cellular localization of vitamin C-lip-
id metabolites (PureWay-C™) compared to ascorbic acid
and calcium ascorbate-calcium threonate-dehydroascorbate
(Ester-C
®
) explains the ability of this formulation to better
enhance neurite formation and fi broblast adhesion. Here
we confi rm that the increase cellular absorption of the vi-
tamin C-lipid metabolites (PureWay-C™) is directly corre-
lated to an improved protection of human T-lymphocytes
from xenobiotic-induced hyperactivation.
Vitamin C is a chemical reducing agent (antioxidant) in
many intracellular and extracellular reactions such as oxi-
dative DNA and or protein damage, low-density lipoprotein
oxidation, lipid peroxidation, oxidants and nitrosamines in
gastric juice, extracellular oxidants from neutrophils and
endothelium-dependent vasodilation. Vitamin C-lipid me-
tabolites (PureWay-C™), which exhibited potent antioxi-
dant and free radical scavenging effect in vitro, can serve as
a good dietary for further evaluation of their bio-effi cacies
and molecular and biological mechanism in vitro as well as
in vivo on antioxidation effects, and may provide effi cient
antioxidant protection to humans and animals from all these
oxidation products or processes of oxidation that contrib-
ute to the pathogenesis of cancer, cardiovascular diseases,
and other age-related diseases by cytotoxic, genotoxic and
proinfl ammatory mechanism and atherosclerosis.
CONCLUSIONS
Vitamin C-lipid metabolites (PureWay-C™) are excellent anti-
oxidants and free radical scavengers and are more rapidly ab-
sorbed and lead to higher cellular vitamin C than do calcium
ascorbate-calcium threonate-dehydroascorbate or any other
tested vitamin C formulation. This rapid uptake explains the
ability of the vitamin C-lipid metabolites (PureWay-C™) to
better enhance neurite outgrowth and wound healing [1]
and to provide protection to the cells of the immune sys-
tem from pesticide exposure as shown here.
REFERENCES:
1. Weeks BS, Perez PP: A novel vitamin C preparation enhances neurite
formation and fi broblast adhesion and reduces xenobiotic-induced
T-cell hyperactivation. Med Sci Monit, 2007; 13(3): BR51–58
Free radical scevenging
(% inhibition of DPPH reductuin)
100
50
0
1 2.5 5 10 20
Lipid metabolized vitamin C (ug/ml)
Figure 2. Vitamin C lipid metabolite scavenging of free radicals. The
lipid metabolized vitamin C, at concentrations ranging from
1–20 μg/ml was mixed with 2,2-diphenyl-1-picryl hydrazyl
(PDDH) solution and incubated at 25°C for 20 minutes. The
absorbance was then read at 510 nm as described in the
Materials and Methods section.
Nutrient
source
ORAC
(μM TE/g)
Reference [Ref#]
PureWay-C™ 1343 Present study
trial #1 1062
trial #2 1394
trial #3 1402
trial #4 1440
Cinnamon 1243 Lan et al., 2007 [16]
Freeze-Dried
Acai
1027 Schauss et al., 2006 [17]
Green and
black teas
761.1
(235–1526)
Prior and Cao, 1999 [18]
Chokeberry 161 Wu et al., 2004 [19]
Broccoli 65.8 to 121.6 Kurilich et al., 2002 [20]
Soft wheat 32–48 Moore et al., 2005 [21]
Careless
gooseberry
21 Wu et al., 2004 [19]
Table 3. ORAC valuses comparing the antioxidant activity of vitamin
C lipid metabolites (PureWay-C™) with known dietary
antioxidants.
The ORAC values of the vitamin C-lipid metabolites was measured
four times (trial #1–#4) and the average is presented. Here the ORAC
values are presented in μM Trolox® Equivalents/gram of substance
tested. ORAC values can often be express as per serving, or per volume,
etc. Here a review of the literature for reports showing ORAC values
per gram was prepared so that the comparison with the vitamin
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Basic Research Med Sci Monit, 2007; 13(10): BR205-210
BR210
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... Selenium might be a cancer chemopreventive component in certain forms through the effect of antioxidant protection, enhanced carcinogen detoxification, enhanced immune surveillance, modulation of cell proliferation, inhibition of tumor cell invasion, and inhibition of angiogenesis (Zeng & Combs, 2008). Vitamin A, vitamin C, vitamin E, b-carotene, organic acid, and vegetable oil can reduce free radical accumulation, increase multi-enzyme activities, reduce GSH and ATP levels, and maintain lipids and calcium (Kim & Lee, 2004;Li et al., 2010;Porter, 2013;Tezcan, Gültekin-Özgüven, Diken, Özçelik, & Erim, 2009;Weeks & Perez, 2007). Recently, liposoluble vitamins, such as vitamins A and E, have attracted attention because of their effects on the mitochondrial membrane potential to stimulate cytochrome c release and caspase-3/and -7 activation in rat liver mitochondria (Forbes-Hernandez et al., 2014). ...
... Indeed, vitamins, in particular Vitamin C, and other antioxidants have been the most extensively studied nutrients for the ability of supplements to protect the immune system and prevent the infl ammation that results from the exposure to xenobiotics [12][13][14][15][16][17][18]. In this regard, a novel vitamin C formulation known as PureWay-C, has been shown to be more rapidly taken up by human T-cells when compared to other vitamin C formulations, such as Ester-C [19] and to provide the greatest protection against xenobiotic induced infl ammation [12]. ...
Article
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In vitro and in vivo studies demonstrate that nutritional supplementation reduces inflammation and inflammatory markers associated with T-cell adhesion mechanisms. Here, we investigate the effects of the nutritional supplements, Natramune (PDS-2865) and PureWay-C, on xenobiotic-induced alpha5beta1 integrin-mediated T-cell adhesion to fibronectin. The human CD4+ lymphoblastoid cell line CEM SS was treated with combinations of bifenthrin, blocking antibodies to human beta1 and alpha5 integrin, and nutrient supplements. After 30 minutes unattached cells were aspirated and the percent of attached cells was determined. Bifenthrin stimulated T-cell adhesion to fibronectin at concentrations between 1.0 and 100 microM with a maximal stimulation of 8.3-fold at 10 microM. At 500 microg/ml, Natramune reduced 100 microM bifenthrin-induced adhesion by nearly 90%. PureWay-C reduced by 1.5-fold the level of T-cell adhesion stimulated by bifenthrin concentrations of both 10 microM and 100 microM. The combination of Natramune and PureWay-C resulted in a 6.3 and 7.5-fold inhibition at 10 microM and 100 microM bifenthrin respectively. Antibody blocking studies demonstrated that bifenthrin induced CEM SS adhesion to fibronectin is mediated through alpha5beta1 integrin. Inhibition of T-cell adhesion achieved by anti-integrin antibodies was further reduced with 50 and 500 microg/ml Natramune treatment. Pretreatment of fibronectin with Natramune did not alter induced T-cell adhesion to fibronectin. These data demonstrate that xenobiotic-induced alpha5beta1 integrin mediated T-cell adhesion to fibronectin is reduced by nutritional supplementation with Natramune (PDS-2865) and PureWay-C. These data suggest the possibility that inflammatory responses associated with exposure to pollutants can be mitigated by nutritional supplementation.
... The antioxidant effects of vitamin C have been demonstrated in many experiments in vitro. 34 Because the free radical theory of aging posits that oxidative damage is at the root of many aspects of human aging Table 1 Selected cohort studies of ␤-carotene and cardiovascular disease (CVD) ...
Article
The free radical theory of aging posits that oxidative stress is among the major mechanisms in aging and age-related disease, including cardiovascular disease (CVD). Numerous in vitro and animal studies have supported the role of low-density lipoprotein (LDL) oxidation in atherosclerosis. This has led to the hypothesis that antioxidants could be used as an inexpensive means of prevention and possibly, treatment of coronary artery disease, stroke, peripheral vascular disease, and other CVD-related diseases. Epidemiologic cohort studies with large numbers of men, women, and diverse populations have been largely supportive of this hypothesis. However, interventional trials have been controversial, with some positive findings, many null findings, and some suggestion of harm in certain high-risk populations. Because of the mismatch between the epidemiologic studies and the interventional trials, some researchers have advocated ending antioxidant work. Others have questioned the validity of the LDL oxidative hypothesis itself. Clearly, further research is needed to understand the reasons for the mismatch between the epidemiologic and interventional work. Recent smaller interventional studies with carefully chosen populations, such as those under high levels of oxidative stress, have yielded largely positive results. This suggests that we need more hypothesis-driven and rigorous clinical trial designs. This should help clarify the true potential utility of antioxidants in CVD and may lead to a better understanding of the role of oxidative stress in atherosclerosis.
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Atherosclerotic cardiovascular diseases (CVD) are a major source of mortality and morbidity in the general population. Oxidative modification of low-density lipoprotein cholesterol (LDL-C) represents the most important determinant factor in the development and progression of atherosclerotic lesions. Oxidative damage and the production of free radicals (FRs) in the endothelium are some of the main factors involved in the pathogenesis of the atherosclerotic process that causes CVD. Appropriate nutritional practices are of central importance in managing risk and treatment of CVD; in fact, many current guidelines for a healthy general population contain nutritional recommendations to reduce the risk of these diseases. Observational studies of vitamins C and E, the most prevalent natural antioxidant vitamins, suggest that supplemental use of these vitamins may lower the risk for coronary events. Despite these data, several large, randomized controlled trials have failed to confirm the benefits of vitamin C and E in cardiovascular prevention. The aim of this review is to examine the published studies regarding the effect of vitamins (C and E) and beta-carotene supplementation in the prevention of CVD due to atherosclerosis.
Article
Previously, a novel formulation of vitamin C-lipid metabolites (PureWay-C) was shown to be more rapidly taken-up by human T-lymphocytes and more rapidly stimulate neurite outgrowth, fibroblast adhesion and inhibition of xenobiotic-induced T-cell hyperactivation. Here, PureWay-C serum levels were measured in healthy volunteers after oral supplementation. Plasma C-reactive protein and oxidized low density lipoprotein levels (LDL) were also measured. Healthy volunteers maintained a low vitamin C diet for 14 days and, following an overnight fast, received a single oral dose of (vitamin C) 1000 mg of either ascorbic acid (AA), calcium ascorbate (CaA), vitamin C-lipid metabolites (PureWay-C), or calcium ascorbate-calcium threonate-dehydroascorbate (Ester-C). Blood samples were collected immediately prior to the oral dose administration and at various times post ingestion. Twenty-four-hour urine collections were saved for oxalate and uric acid assays. PureWay-C supplementation leads to the highest absolute serum vitamin C levels when compared to AA, CaA and Ester-C. PureWay-C provides a statistically significant greater serum level than calcium ascorbate at 1, 2, 4, and 6 hours post oral supplementation whereas Ester-C shows a less but slightly statistically significant increase at only 1 and 4 hours. Oral supplementation with PureWay-C also led to a greater reduction in plasma C-reactive protein and oxidized LDL levels compared to the other vitamin C formulations. PureWay-C is more rapidly absorbed and leads to higher serum vitamin C levels and greater reduction of plasma levels of inflammatory and oxidative stress markers than other forms of vitamin C, including Ester-C.
Article
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Cellular functions involve the temporal and spatial interplay of ions, metabolites, macromolecules, and organelles. To define the mechanisms responsible for completing cellular functions, we used methods that can yield both temporal and spatial information on multiple physiological parameters and chemical components in the same cell. We demonstrated that the combined use of selected fluorescent probes, fluorescence microscopy, and imaging methods can yield information on at least five separate cellular parameters and components in the same living cell. Furthermore, the temporal and spatial dynamics of each of the parameters and/or components can be correlated with one or more of the others. Five parameters were investigated by spectrally isolating defined regions of the ultraviolet, visible, and near-infrared spectrum based on five distinct fluorescent probes. The parameters included nuclei (Hoechst 33342), mitochondria (diIC1-[5] ), endosomes (lissamine rhodamine B-dextran), actin (fluorescein), and the cell volume Cy7-dextran). Nonmotile, confluent Swiss 3T3 cells did not show any detectable polarity of cell shape, or distribution of nuclei, endosomes, or mitochondria. These cells also organized a large percentage of the actin into stress fibers. In contrast, cells migrating into an in vitro wound exhibited at least two stages of reorganization of organelles and cytoplasm. During the first 3 h after wounding, the cells along the edge of the wound assumed a polarized shape, carried the nuclei in the rear of the cells, excluded endosomes and mitochondria from the lamellipodia, and lost most of the highly organized stress fibers. The cell showed a dramatic change between 3 and 7 h after producing the wound. The cells became highly elongated and motile; both the endosomes and the mitochondria penetrated into the lamellipodia, while the nuclei remained in the rear and the actin remained in less organized structures. Defining the temporal and spatial dynamics and interplay of ions, contractile proteins, lipids, regulatory proteins, metabolites, and organelles should lead to an understanding of the molecular basis of cell migration, as well as other cellular functions.
Article
Background. Alzheimer dementia is a neurodegenerative disease characterised by loss of memory and other cognitive functions. Oxidative stress is a possible pathogenetic factor and the anti-oxidating vitamins C and E could therefore have a beneficial effect and reduce the damage caused by beta-amyloid. Materials and methods. The relevant literature (several observational studies and two clinical controlled trials) describing effects of vitamin C and E in Alzheimer dementia have been evaluated. Results. Several observational studies in mostly healthy, elderly individuals have indicated that vitamin C and E, mainly from food as well as the combination of high doses of the same vitamins, may have beneficial effect on the development of Alzheimer dementia. One clinical controlled trial in patients with manifest Alzheimer dementia, in which vitamin E 2000 mg/day was given as the only vitamin, has to a certain extent confirmed these results. Interpretation. A causal relationship between intake of the vitamins and Alzheimer dementia has not been clarified. The correct dosages are not known, but a diet rich in these vitamin could probably reduce the risk of dementia. With a high intake of vitamin E, the addition of vitamin C is necessary.
Article
Cellular functions involve the temporal and spatial interplay of ions, metabolites, macromolecules, and organelles. To define the mechanisms responsible for completing cellular functions, we used methods that can yield both temporal and spatial information on multiple physiological parameters and chemical components in the same cell. We demonstrated that the combined use of selected fluorescent probes, fluorescence microscopy, and imaging methods can yield information on at least five separate cellular parameters and components in the same living cell. Furthermore, the temporal and spatial dynamics of each of the parameters and/or components can be correlated with one or more of the others. Five parameters were investigated by spectrally isolating defined regions of the ultraviolet, visible, and near-infrared spectrum based on five distinct fluorescent probes. The parameters included nuclei (Hoechst 33342), mitochondria (diIC1-[5] ), endosomes (lissamine rhodamine B-dextran), actin (fluorescein), and the cell volume Cy7-dextran). Nonmotile, confluent Swiss 3T3 cells did not show any detectable polarity of cell shape, or distribution of nuclei, endosomes, or mitochondria. These cells also organized a large percentage of the actin into stress fibers. In contrast, cells migrating into an in vitro wound exhibited at least two stages of reorganization of organelles and cytoplasm. During the first 3 h after wounding, the cells along the edge of the wound assumed a polarized shape, carried the nuclei in the rear of the cells, excluded endosomes and mitochondria from the lamellipodia, and lost most of the highly organized stress fibers. The cell showed a dramatic change between 3 and 7 h after producing the wound. The cells became highly elongated and motile; both the endosomes and the mitochondria penetrated into the lamellipodia, while the nuclei remained in the rear and the actin remained in less organized structures. Defining the temporal and spatial dynamics and interplay of ions, contractile proteins, lipids, regulatory proteins, metabolites, and organelles should lead to an understanding of the molecular basis of cell migration, as well as other cellular functions.
Article
Abstract The in vitro antioxidant potential of Triphala and its constituents was tested with the following systems: radical scavenging activity measured by DPPH reduction, and superoxide radical and peroxy radical scavenging properties measured by riboflavin/light/NBT reduction and linoleic acid peroxidation, respectively. Alcohol extracts of Triphala and its constituents were studied comparatively and found to be strong anti-oxidants. Triphala was also effective in preventing superoxide-induced haemolysis of red blood cells. The extracts also prevented lipid peroxidation induced by Fe3+/ADP/Ascorbate system in rat liver mitochondria. The major phenolic compounds of the alcohol extracts were confirmed as tannins.
Article
The Oxygen Radical Absorbance Capacity (ORAC) assay was used to determine the total antioxidant capacity of tea. Green and black teas (n = 18) had a mean antioxidant capacity of 761.1 ± 85.3 μmol Trolox Equivalents (TE) per g dry matter. However, their antioxidant capacity varied from 235 μmol to over 1526 μmol Trolox equivalents (TE)/g dry matter, and total phenolics ranged from 32 to 147 mg/g in different commercial teas. One tea phenolics extract had an antioxidant capacity of 4796 μmol TE/g dry matter and 625 mg total phenolics/g. On a dry matter basis, an antioxidant capacity of 761 μmol TE/g is considerably higher than any of the other fruits and vegetables measured in our laboratory. However, since dry tea is not consumed directly, brewing conditions may influence the final antioxidant capacity in the tea as consumed. We tested both green and black teas by placing one tea bag (1.95 g) in 150 ml (5 oz.) of boiling water. In the first brewed cup, approximately 84% of the total antioxidant activity was solubilized within the first 5 min of brewing. An additional 13% of the antioxidant activity was extracted into the second glass of 150 ml with an additional 5 min of brewing. At the dilutions obtained after the first brewing, the tea as consumed would contain approximately 8.31 μmol TE per ml. This total antioxidant capacity compares to other drinks from fruits and vegetables that had antioxidant capacity values ranging from 1.6 to 15 μmol TE/ml. At these antioxidant levels, consumption of 150 ml of tea could make a significant contribution to the total daily antioxidant capacity intake. (−)-Epicatechin and (+)-catechin, two components from tea, had an antioxidant capacity of 2.36 and 2.49 μmol/μmol or 8.13 and 8.58 μmol/mg, respectively. In 16 tea samples we observed a mean of 10.0 ± 0.6 μmol TE/mg total phenolics. Tea can be an important source of what has been referred to as “non-nutrient” antioxidant phytochemicals. However, with the variation that exists in antioxidant capacity with various tea preparations, measures of antioxidant capacity intake are critical to the study of intake and health outcomes and/or biomarkers of health outcomes.
Article
Black peppercorn, nutmeg, rosehip, cinnamon and oregano leaf were extracted with 50% acetone and 80% methanol, and evaluated for their radical-scavenging activities against cation (ABTS+), DPPH, peroxyl (ORAC) and hydroxyl (HO) radicals. For each extract, total phenolic content (TPC) and chelating activity were also determined. The extracts of all botanical samples showed significant radical-scavenging capacities, TPC and chelating abilities. The 50% acetone extract of cinnamon had the highest ABTS+-scavenging capacity of 1243 μmol TE/g and the greatest ORAC value of 1256 μmol TE/g on a per weight basis. The 50% acetone extracts of black peppercorn and cinnamon showed higher ABTS+-scavenging, ORAC, Fe+2 chelating ability and TPC value, but lower DPPH value than the corresponding 80% methanol extracts. The 80% methanol extract of nutmeg had greater ABTS+, ORAC and TPC values than the 50% acetone extract. Electronic spin resonance (ESR) measurements demonstrated that cinnamon had the strongest HO-scavenging activities among all the tested botanical materials. These data indicate that black peppercorn, nutmeg, rosehip, cinnamon and oregano leaf may serve as potential dietary sources of natural antioxidants for improving human nutrition and health. The extracting solvent may alter the antioxidant activity measurement for selected botanicals, including spices and herbs.
Article
The Osteogenic Disorder Shionogi (ODS) rat, Clea Inc., Tokyo, Japan lacks the ability to synthesize L-ascorbic acid (AA). As with man, monkey and the guinea pig, this rat lacks L-gulonolactone oxidase necessary for the synthesis of AA from glucose. This study shows this animal to be an alternative to the guinea pig in AA studies. The anti-scorbutic potency of Ester C (EC), a calcium ascorbate and calcium threonate mixture, was compared with an AA dose of equal ascorbate activity equivalents (AAE) for anti-scorbutic activity in the ODS rat. The minimal anti-scorbutic dose of EC was determined to be 0.44 mg/kg/day (AAE), while an AA dose of 0.51 mg/kg/day (AAE) was not anti-scorbutic in a 24 day study. At 24 days EC rats gained 125% of initial body weight (BW) and the AA rats only 45% BW. Scorbutic signs at 24 days were scored on a 0 (min) to 3 (max) scale. The EC/AA ratio scores were: hemorrhage 0/1.4, behavior change 0/2.0, piloerection 0/2.2, mobility 0.4/2.2, dysbasia 0.6/2.8 and ataxia 0.4/1.0. Pearson's correlation coefficient for BW versus AAE was r = .34 for the AA group and r = .90 for the EC group. The morbidity index for EC was 0/5 and for the AA group 2/5. The AAE dose of AA which was 16% higher/day than the EC AAE dose was not anti-scorbutic, while the EC dose was anti-scorbutic. EC rats had 3.5X greater weight gain, a sensitive indicator of scurvy, than the AA rats. EC rats had 3-4 times less, if any, scorbutic signs than AA rats. The results clearly show that, based on ascorbate activity equivalents, EC has more available ascorbate activity/potency than AA. The mechanism of this increased potency is believed to be due to the facilitated transport of AAE into the cell by the threonate (a normal in vivo metabolite of AA) present in the EC product. In addition, previous studies have shown EC (AAE) to be higher in plasma and excreted less rapidly than the AAE derived from AA administered orally.
Article
The effects of preincubation of human T-lymphoma cells with increasing concentrations of calcium L-threonate on the uptake of L-[1-14C]ascorbic acid were examined. Calcium L-threonate (0-1,000 mg%) stimulated ascorbic acid (1.25 mg%) uptake in a dose-dependent manner. These results indicate that calcium threonate and possibly other ascorbic acid metabolites have biological activity and potential pharmacological applications.
Article
A serum factor that promotes migration of (mouse) 3T3 fibroblast cells is shown to be distinct from the growth-promoting and cell-survival factors of serum. The factor promotes migration of cells from the edge of a wound in confluent 3T3 cells, but cell migration under these conditions does not lead to the initiation of DNA synthesis. Subsequent addition of serum initiates DNA synthesis in the migrating cells. The results establish that breaking contacts between quiescent 3T3 cells is not sufficient to initiate DNA synthesis. The DNA synthesis observed in migrating 3T3 cells in the typical "woundhealing" experiment presumably results because the migrating cells have an increased ability to use serum factors. Serum-factor requirements for "wound healing" in cultured 3T3 cells are discussed.