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Comparative Bioavailability of Synthetic and Natural Vitamin C in Guinea Pigs

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Abstract

This study was undertaken to compare the bioavailabilities of synthetic ascorbic acid and a natural vitamin C which contains bioflavonoids. Adult male guinea pigs were orally dosed with 50 mg of ascorbate and the serum levels measured using a fluorometric method. The two forms of ascorbic acid gave similar peak concentrations of serum ascorbate but the natural vitamin C peaked later and remained in the serum for a longer time period. The bioavailability of the natural vitamin C was significantly greater (148%, p < 0.001) than that of the synthetic ascorbic acid.
Nutrition Reports International, 27, no.4, 1983.
Comparative Bioavailability of Synthetic and Natural Vitamin C in
Guinea Pigs
J.A. Vinson and P. Bose.
Department of Chemistry, University of Scranton, Scranton, PA 18510, USA.
Abstract
This study was undertaken to compare the bioavailabilities of synthetic ascorbic acid
and a natural vitamin C which contains bioflavonoids. Adult male guinea pigs were
orally dosed with 50 mg of ascorbate and the serum levels measured using a
fluorometric method. The two forms of ascorbic acid gave similar peak
concentrations of serum ascorbate but the natural vitamin C peaked later and
remained in the serum for a longer time period. The bioavailability of the natural
vitamin C was significantly greater (148%, p < 0.001) than that of the synthetic
ascorbic acid.
Introduction
The requirement of ascorbic acid (vitamin C) is a common property of living
organisms. Practically all animals except the guinea pig, monkey and man can
synthesise this vitamin. Vitamin C plays an important role in many metabolic
reactions including synthesis of collagen [1] and synthesis of proteins of the immune
system [2]. There is now considerable interest in ascorbate supplementation in
amounts that are relatively large when compared to the recommended dietary intake.
This interest is due to recent books by Linus Pauling on the efficacy of ascorbic acid
in the treatment of colds [3] and treatment of cancer [4].
Although natural and synthetic ascorbic acids are chemically identical, citrus fruits
and other natural sources of vitamin C contain other compounds including
bioflavonoids which could affect the bioavailability of ascorbic acid. In fact,
bioflavonoids have been shown to improve the utilisation of ascorbic acid and
increase its storage in guinea pigs [5, 6, 7]. However, human studies have shown
conflicting results. The comparison of serum and urine levels after oral intake of
comparable doses of natural and synthetic vitamin C have led to the conclusion that
natural vitamin C shows greater [8], equal [9], or less [10] availability than synthetic
ascorbic acid. Most recently, Nelson [11] by an intraluminal perfusion technique
found the two forms to be similarly absorbed.
Since the absorption of ascorbate into the plasma determines its metabolic availability
to the tissues [11], the plasma concentrations of the vitamin following an oral dose
may be used to determine the bioavailability of a supplement. Guinea pigs are an
appropriate animal model for vitamin C since the guinea pig, like man, has an active
transport system [12]. Also, the guinea pig minimises the large intersubject variation
in bioavailability which are often seen in human studies. The present study describes
the relative bioavailability of a synthetic and natural vitamin C after a single dosing of
guinea pigs.
Materials and Methods
Subjects: Ten adult male Hartley guinea pigs were assigned to one of two groups on
the basis of weight so that the average weight of the two groups was statistically
identical. They were fed a standard guinea pig chow for several weeks until the time
of the experiment.
Formulations: The two formulations were synthetic L-ascorbic acid (Fisher Scientific
Company, Pittsburgh, PA.) and Renatured vitamin C in Citrus Fruit Media. The
vitamin C in the latter was synthetic ascorbic acid added to natural proteins,
carbohydrates and bioflavonoids 18%, proteins 15% and carbohydrates 30%. The
product was a light brown, water soluble powder.
Dosage Schedule: The guinea pigs were fasted overnight preceding dosing. Each
guinea pig received by means of an analytical pipette 1 ml of a 50 mg/ml ascorbic
acid solution freshly prepared in distilled water. Blood samples (0.2 ml) were taken
from the heart during light ether anaesthesia before dosing and periodically after
dosing. Blood samples were collected with EDTA as an anticoagulant and
centrifuged at 4000 rpm for 10 minutes. Plasma (0.1 ml) was taken for ascorbic acid
analysis and assayed the same day or alternatively the proteins were precipitated with
metaphosphoric acid and the sample frozen at -20°C until analysis within 2 days.
Measurement of Ascorbate: Ascorbic acid was measured in plasma by fluorescence
[13] following precipitation of proteins with metaphosphoric acid and reaction with
1,2-naphthoquinone-4-sulphonic acid. A standard curve was determined using
freshly prepared aqueous standards. Quercetin, a representative bioflavonoid, gave
zero fluorescence at a concentration of 5 mg/100 ml. Thus, bioflavonoids which are
present in the natural vitamin C are not an interference in the assay procedure.
Results
Concentrations of ascorbate in plasma: The results for the determination of ascorbate
in plasma after an oral dosing of 50 mg of ascorbate in the form of synthetic ascorbate
and natural vitamin C are shown in Table 1. The two groups were statistically
compared by means of a student’s t-test. The pre-dose levels were not significantly
different for the two groups. Also, the maximum concentration of ascorbate were not
significantly different (0.221 mg/dl for the synthetic group and 0.214 mg/dl for the
natural group). The peak concentration was reached sooner for the synthetic group,
approximately 1.5 hours after dosing as compared with 2 hours for the natural vitamin
C. The natural vitamin C stayed in the plasma longer than the synthetic ascorbic acid
as it took more than 4 hours for the natural group to return to pre-dose level and less
than 3 hours for those receiving the synthetic material.
Table 1: Mean Plasma Concentrations of Ascorbate (mg/dl). After Oral Administration of Synthetic
and Natural Vitamin C to guinea pigs.
Time (hours)
Plasma Ascorbate Concentration (± SD)
Synthetic Group (n = 5) Natural Group (n = 5)
0 0.080 + 0.014 0.078 + 0.22
0.5 0.140 + 0.034 0.132 + 0.006
1.0 0.204 + 0.029 0.186 + 0.013*
1.5 0.221 + 0.013 0.199 + 0.047**
2.0 0.123 + 0.011 0.214 + 0.017**
3.0 0.076 + 0.018 0.140 + 0.013**
4.0 ----- 0.092 + 0.016
* p < 0.1, Natural vs. Synthetic
** p < 0.001, Natural vs. Synthetic.
Apparent biological half-lives were calculated by a least squares regression analysis
of the plot of log
e
vs. time over the last three sampling times where the concentration
of ascorbate was decreasing. The half-life of synthetic vitamin C was 1.0 hours and
of the natural material was 1.6 hours. These half-lives are not elimination half-lives
since they also reflect absorptive and distributive phases in ascorbate
pharmacokinetics.
Bioavailability of Ascorbate: The relative bioavailability of the two forms was
calculated by comparison of the area under the plasma concentration-time curve after
administration of each formulation. The areas were determined by means of a
planimeter and the results are shown in Table 2. The bioavailability of the natural
vitamin C was 148% that of the synthetic ascorbic acid and the difference was highly
significant (p < 0.001).
Table 2: Area (Arbitrary units) under the Plasma Ascorbate Concentration-time Curve after oral
administration of Synthetic and Natural Vitamin C to Guinea Pigs.
Synthetic Group Natural Group
Subject Weight (g) Plasma Area Subject Weight (g) Plasma Area
530 426 540 627
552 463 545 541
554 419 550 726
570 449 560 647
570 452 560 729
Mean ± S.D.
555 ± 16 442 ± 18 551 ± 9 654 ±78**
** p < 0.001, Natural vs. Synthetic.
Discussion
Previous guinea pig studies have been long term feeding experiments which have
compared the effects of synthetic ascorbic acid alone or mixed with bioflavonoids.
Parrot [5] in 1948 found that catechin, a bioflavonoid, when given with ascorbic acid
increased the ascorbate levels in the liver, spleen, kidney and adrenals of guinea pigs
and also prevented scourbutic lesion which were present when ascorbic acid was
given alone at a low dose. Blanc and von der Mühll [7] also found a synergistic
action between ascorbic acid and bioflavonoids with respect to the concentration of
ascorbate in the internal organs.
The best quantitative study was by Crampton and Lloyd [6] in 1950 who fed guinea
pigs daily sub-optimal doses of vitamin C (0.5-2.0 mg) in the form of synthetic
ascorbate or orange-grapefruit juice. This vitamin C was given alone or with 100 mg
of rutin, a bioflavonoid. After 42 days, the biological potencies were determined by
measuring the height of the odontoblast cells of the incisor teeth. There was no
difference between the biopotency of natural or synthetic vitamin C. However, rutin
increased the biopotency by an average of 56% and was most effective at low doses
of Vitamin C. However, the large excess of rutin/ascorbic acid of 200/1 to 50/1 is not
realistic for animal of human supplementation because isolated bioflavonoids are
much more expensive than ascorbic acid.
A biological action of bioflavonoids in animals and man was first suggested in 1936
by Szent-Gyorgi [14] who reported that these compounds prevent capillary fragility
and bleeding in scorbutic animals. A dietary role for bioflavonoids is suggested by
evidence of a widespread low-level blood cell aggregation in apparently healthy
human subjects which is inhibited in vitro by bioflavonoids [15].
Somogyi [16] first presented a mechanism for the effect of bioflavonoids on vitamin
C in physiological fluids. He hypothesised that flavonoids act as sparing factors in
slowing down the oxidation of Vitamin C. This antioxidant effect was shown by in
vitro studies with oxidants such as ascorbic acid oxidase, copper and peroxidase.
In the present study, a natural vitamin C product containing bioflavonoids was found
to be more readily absorbed by Guinea pigs than synthetic ascorbate. These results
indicate that human supplementation with natural vitamin C might prove efficacious.
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