peake ijsnem 2003

27
J.M. Peake is with the School of Human Movement Studies at the University of Queensland, St Lucia QLD 4072 Australia. 125 Vitamin C: Effects of Exercise and Requirements With Training Jonathan M. Peake Ascorbic acid or vitamin C is involved in a number of biochemical pathways that are important to exercise metabolism and the health of exercising individu- als. This review reports the results of studies investigating the requirement for vitamin C with exercise on the basis of dietary vitamin C intakes, the response to supplementation and alterations in plasma, serum, and leukocyte ascorbic acid concentration following both acute exercise and regular training. The possible physiological significance of changes in ascorbic acid with exercise is also addressed. Exercise generally causes a transient increase in circulating ascorbic acid in the hours following exercise, but a decline below pre-exercise levels occurs in the days after prolonged exercise. These changes could be associated with increased exercise-induced oxidative stress. On the basis of alterations in the concentration of ascorbic acid within the blood, it remains unclear if regular exercise increases the metabolism of vitamin C. However, the similar dietary intakes and responses to supplementation between athletes and nonathletes suggest that regular exercise does not increase the requirement for vitamin C in athletes. Two novel hypotheses are put forward to explain recent findings of attenuated levels of cortisol postexercise following supplementation with high doses of vitamin C. Key Words: ascorbic acid, antioxidant, cortisol, adrenal gland, exercise, oxida- tive stress, gluconeogenesis Introduction Ascorbic acid, or vitamin C, is a six-carbon compound similar in structure to glu- cose. It exists in two active forms: the reduced form known as ascorbic acid and the oxidized form, dehydroascorbic acid. The molecular structure contains two ioniz- able enolic hydrogen atoms that give the compound its acidic character (Figure 1; 54). Ascorbic acid is considered to be an outstanding antioxidant. Chemically, ascorbic acid exhibits redox characteristics as a reducing agent. Physiologically, ascorbic acid provides electrons for enzymes, for chemical compounds that are oxidants, or other electron acceptors (91). The intermediate free radical formed in International Journal of Sport Nutrition and Exercise Metabolism, 2003, 13, 125-151 © 2003 Human Kinetics Publishers, Inc. ORIGINAL RESEARCH

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Page 1: Peake Ijsnem 2003

Vitamin C and Exercise / 125

J.M. Peake is with the School of Human Movement Studies at the University ofQueensland, St Lucia QLD 4072 Australia.

125

Vitamin C: Effects of Exerciseand Requirements With Training

Jonathan M. Peake

Ascorbic acid or vitamin C is involved in a number of biochemical pathwaysthat are important to exercise metabolism and the health of exercising individu-als. This review reports the results of studies investigating the requirement forvitamin C with exercise on the basis of dietary vitamin C intakes, the response tosupplementation and alterations in plasma, serum, and leukocyte ascorbic acidconcentration following both acute exercise and regular training. The possiblephysiological significance of changes in ascorbic acid with exercise is alsoaddressed. Exercise generally causes a transient increase in circulating ascorbicacid in the hours following exercise, but a decline below pre-exercise levelsoccurs in the days after prolonged exercise. These changes could be associatedwith increased exercise-induced oxidative stress. On the basis of alterations inthe concentration of ascorbic acid within the blood, it remains unclear if regularexercise increases the metabolism of vitamin C. However, the similar dietaryintakes and responses to supplementation between athletes and nonathletessuggest that regular exercise does not increase the requirement for vitamin C inathletes. Two novel hypotheses are put forward to explain recent findings ofattenuated levels of cortisol postexercise following supplementation with highdoses of vitamin C.

Key Words: ascorbic acid, antioxidant, cortisol, adrenal gland, exercise, oxida-tive stress, gluconeogenesis

Introduction

Ascorbic acid, or vitamin C, is a six-carbon compound similar in structure to glu-cose. It exists in two active forms: the reduced form known as ascorbic acid and theoxidized form, dehydroascorbic acid. The molecular structure contains two ioniz-able enolic hydrogen atoms that give the compound its acidic character (Figure 1;54). Ascorbic acid is considered to be an outstanding antioxidant. Chemically,ascorbic acid exhibits redox characteristics as a reducing agent. Physiologically,ascorbic acid provides electrons for enzymes, for chemical compounds that areoxidants, or other electron acceptors (91). The intermediate free radical formed in

International Journal of Sport Nutrition and Exercise Metabolism, 2003, 13, 125-151© 2003 Human Kinetics Publishers, Inc.

ORIGINAL RESEARCH

Page 2: Peake Ijsnem 2003

126 / Peake

the oxidation of ascorbic acid to dehydroascorbic acid is relatively non-reactive,particularly with oxygen (14). Furthermore, dehydroascorbic acid is reduced bycells back to ascorbate, which can then be reutilized (107).

Ascorbic acid is distributed in varying concentrations throughout the bodyand is involved in a variety of metabolic reactions relating to exercise, such as thesynthesis and activation of neuropeptides, collagen, carnitine, and protection againstthe harmful effects of reactive oxidant species (79). In light of this involvement, it isconceivable that exercise may enhance the utilization, metabolism, and excretion ofascorbic acid, thereby increasing the dietary requirement for the vitamin. The firstevidence of a possible link between vitamin C and exercise came rather unexpect-edly from the observation of scurvy symptoms exhibited by explorers on early polarexpeditions (78). The diets consumed by these explorers were likely to be seriouslylacking in fresh foods containing vitamin C. However, it is also plausible that such astressful existence could have exacerbated the effects of a vitamin C–deficient dietin such a way that the turnover and requirement for ascorbic acid may have in-creased. While vitamin C does not appear to improve athletic performance (39),athletes may have increased dietary vitamin C requirements, and supplementationmay have important effects on athletes that are more subtle than improvements inaerobic capacity or performance measures (30).

The purpose of this review is to: (a) discuss the role of ascorbic acid in exercisemetabolism, (b) summarize the results of studies examining dietary intakes of vita-min C and the responses to supplementation, and (c) address exercise-inducedalterations in the distribution of ascorbic acid within the body, with reference to themechanisms and physiological significance of such changes.

The Distribution, Transport, Function,and Metabolism of Ascorbic Acid

The distribution of ascorbic acid within the human body is outlined in Table 1.Exercise studies have been limited to examining changes in ascorbic acid concen-trations within plasma/serum and leukocytes. Saturated plasma ascorbic acid con-centrations are in the range of 70 to 80 �mol/L, whereas saturated lymphocyte,monocyte, and neutrophil ascorbic acid concentrations occur at 3.4, 3.2, and 1.3�mol/L, respectively (64). Leukocytes store ascorbate against very high concentra-tion gradients (43), probably for protection against intracellular reactive oxidants(91). Ascorbic acid and its oxidized form, dehydroascorbic acid, are transportedindependently. Whereas dehydroascorbic acid is transported into cells by GLUT 1

Figure 1 — Redox properties of ascorbic acid (modified with permission from 36).

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Vitamin C and Exercise / 127

and GLUT 3, the transport protein for ascorbic acid is yet to be identified. Substrateavailability is likely to be critical in determining which molecule (i.e., ascorbic acidor dehydroascorbic acid) is transported at any particular time. Under non-oxidizingconditions, ascorbate is present in greater amounts and is therefore more likely to betransported. In contrast, when oxidants are present, ascorbate is oxidized, resultingin the transient formation of dehydroascorbic acid (91). Now available to surround-ing cells, dehydroascorbic acid is preferentially transported for intracellular reduc-tion (91) either by enzymatic reduction (80) or glutathione (109). This recycling ofascorbic acid is likely to be important under conditions of oxidative stress (91).

The redox properties of ascorbic acid (Figure 1) make it an effective reducingagent in several important enzymatic reactions within the body, all of which requirethat enzyme-bound metal ions are maintained in their reduced state (79). In theadrenal gland ascorbic acid is stored in high concentrations where it is utilized by theenzyme dopamine �-monooxygenase within chromaffin granules to synthesizenoradrenaline (Figure 2A; 24). In the pituitary gland and the brain, ascorbic acid isinvolved with the enzyme peptidyl-glycine �-amidating monooxygenase (72) forthe activation of a variety of neuropeptides and hormone releasing-factors (Figure2D; 79). The vitamin is required by the prolyl- and lysyl-hydroxylase enzymes forthe hydroxylation of proline and lysine residues, respectively, in the production ofcollagen (Figure 2B; 12), and also by the enzymes 6-N-trimethyllysine hydroxylaseand �-butyrobetaine hydroxylase in carnitine synthesis (Figure 2C; 74). This latterrole could possibly account for the ascorbic acid content of both cardiac and skeletalmuscle (74). In the liver, ascorbic acid is possibly involved in glucose (11), choles-terol, and lipid metabolism (54). A variety of immune functions are enhanced byascorbic acid (2), which could explain the concentration of the vitamin in the spleen.

Table 1 Distribution of Ascorbic Acid Within the Organs and Fluidsof the Human Body (49)

Ascorbic acid concentrationOrgan/fluid (mg/100 g)

Pituitary gland 40–50Adrenal gland 30–40Eye lens 25–31Liver 10–16Pancreas 10–15Spleen 10–15Kidney 5–15Cardiac muscle 5–15Brain 3–15Lungs 7Skeletal muscle 3–4Plasma 0.4–1

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128 / Peake

It is currently unclear exactly how ascorbic acid influences pancreatic or renalfunction. It has been suggested that high doses of vitamin C might influence insulinsecretion and blood glucose concentrations (8, 10), possibly through competitionbetween dehydroascorbic acid and glucose for GLUT 1 and GLUT 3 transporters(92). However, this possible regulatory influence seems unlikely to account for thelevels of ascorbic acid found within these tissues. It is the antioxidant activity ofascorbic acid which is important in many tissues such as the lungs (68), the eye (23),the cardiovascular system (67), and plasma (37). This antioxidant activity is dis-cussed in more detail in a later section.

In light of these exercise-related metabolic requirements for ascorbic acid, itis plausible that periods of regular intense exercise, musculoskeletal growth, andrepair may promote the need for vitamin C in the diets of athletes. However, owingto obvious limitations inherent to the biopsy of internal tissue in humans, any directassessment of the actual utilization of ascorbic acid in these reactions as a result ofeither acute or chronic exercise is difficult. Given that ascorbic acid is an antioxidantin plasma (37), changes in the plasma content of ascorbate may provide indirectevidence of its utilization in neutralizing reactive oxidant species; nevertheless, nostudies of exercise-induced oxidative stress have reported changes in the circulatinglevels of ascorbic acid derivatives, such as dehydroascorbic acid, which would offersome indication of the metabolism of ascorbic acid itself. Two studies compared theurinary output of ascorbic acid between sedentary individuals and athletes andfound no difference (32, 89). Hence, although there is in vitro evidence that ascorbicacid is likely utilized in several exercise-related metabolic pathways, measurementsof ascorbic acid concentrations in blood and urine do not reflect its consumptionwithin these reactions.

An alternative approach to determining the physiological requirement forascorbic acid has been to compare the dietary intake of vitamin C between athletes

Figure 2 — Role of ascorbic acid in the synthesis of (A) norepinephrine, (B) collagen, (C)carnitine, and (D) the �-amidation of neuropeptides (modified with permission from79).

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Vitamin C and Exercise / 129

and nonathletes, in addition to assessing the response of athletes to vitamin Csupplementation. If athletes exhibit low plasma/serum ascorbic acid concentrationspre-supplementation, then it may be anticipated that these values would increaseupon the addition of extra vitamin C within the diet.

Vitamin C Status

The vitamin C status of individuals has been assessed using several different meth-ods. Dietary intakes of ascorbic acid have been used for the general assessment andprediction of vitamin C status. The correlation between plasma/serum ascorbic acidconcentration and dietary ascorbic acid intake is only modest (r = 0.32–0.55; 89).One group failed to find any relationship (44). The normal ranges for plasma andleukocyte ascorbic acid concentrations are listed in Table 2. There has been somedebate as to which component of whole blood provides the most reliable index ofvitamin C status and whether whole blood does in fact reflect the true tissue stores ofthe vitamin (18, 29, 42, 53, 65). In two studies, the responses to supplementationwith vitamin C were similar between plasma and leukocytes (9, 55). Furthermore,plasma and leukocyte ascorbic acid concentrations are correlated with each other(15). In view of the inherent difficulties associated with measuring the ascorbic acidcontent of internal organs in humans, a combination of measurement of dietaryintakes, plasma/serum, and leukocyte ascorbic acid concentrations appears to be thebest available indication of vitamin C status.

Vitamin C Intakes of Exercisingand Non-exercising Individuals

The recommended daily allowance for vitamin C varies among countries. In GreatBritain and Canada, it is 30 mg, in New Zealand and Australia it is 40 mg, in theUnited States it is 60 mg, whereas in Germany it is 75 mg (108). Vitamin C con-sumption is related to total energy intake (17). Some investigators have assessed thedietary vitamin C intake of athletes on the basis of the recommended daily allow-ance (25, 75, 96, 106). With the exception of a small number of athletes in twostudies (25, 96), the majority of data on vitamin C intakes in athletes have indicatedthat most athletes are receiving adequate amounts of vitamin C in their diets. Mean

Table 2 Normal Range for Whole-Body Ascorbic Acid Content, Plasma,and Leucocyte Concentrations (54)

Plasma Mixed leucocytes MononuclearBody pool �mol/L �g/108 cells leucocytes �g/108 cells(mg) (mg/dL) (nmol/108 cells) (nmol/108 cells)

500–1500 23–84 114–301 142–250(10–22 mg/kg) (0.4–1.5) (20–53) (25–44)

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130 / Peake

intakes in athletes are reported to range from 90 to 140 mg per day. Intakes wereeven higher in those athletes taking vitamin C supplements, which in one study wasfound to be the most commonly used supplement among a large number of athletes(34). Others have compared the intakes of exercising individuals with those ofsedentary controls and have generally observed higher intakes within the athleticpopulation (33, 34, 44, 81, 89).

Several studies have examined vitamin C intakes in athletes from a variety ofsports (25, 44, 89, 96). The results from a 24-h dietary recall suggested wide varia-tion among sports, with some indication of seasonal variation, but no consistenttrends emerged (96). Low intakes have been observed in male gymnasts and wres-tlers (96), and also in female athletes (25). In contrast to these findings, the resultsfrom another study indicated very little difference in 1- or 7-day vitamin C intakesamong athletes from different sports, including wrestlers (96). Lower intakes insome athletes may reflect dietary interventions directed at weight control in theseathletes, as it has been demonstrated that vitamin C intakes are related to total energyintakes (34). Therefore, the amount of vitamin C in the diets of most athletes appearsto be sufficient, based on the recommended daily allowance. Another approach tomeasuring vitamin C status has been to examine the response of plasma or serumascorbic acid concentrations to supplementation with varying doses of vitamin C.

Supplementation Studies

Athletes have been given vitamin C supplements ranging from 85 to 1500 mgvitamin C, for periods of 1 day up to 8 months (5, 44, 58, 69, 76, 81, 83, 98, 106). Theeffects of supplementation on both resting and exercise-induced changes in ascor-bic acid concentration have been investigated (Table 3). There was little or no effectof supplementation on plasma or serum ascorbic acid concentrations in severalstudies (48, 58, 95, 98), whereas others have demonstrated increases in the concen-tration of ascorbic acid in blood following supplementation (5, 44, 69, 90, 106).However, there are several issues to consider when interpreting these results.

The dose of vitamin C in the supplement, the regular dietary intakes of vitaminC, and the concentration of ascorbic acid within plasma/serum and leukocytes priorto supplementation would all appear to be important determinants of the response tosupplementation. Plasma ascorbic acid concentrations in one study plateaued atvitamin C intakes of 250 mg and above per day, whereas the ascorbic acid content ofneutrophil, monocytes, and lymphocytes plateaued at lower intakes of 200 mg perday (64). In another study, the optimal intake for absorption capacity was reported tobe 3 mg vitamin C per kilogram of body weight per day (89). The athletes in many ofthe studies in Table 3 were receiving ≥ 200 mg of vitamin C per day in total.Therefore, plasma/serum levels were likely close to saturation. Himmelstein et al.(48) have reported that the athletes in their study had higher total dietary intakes ofvitamin C than sedentary individuals and consequently showed a smaller responseto supplementation. Similarly, Thompson et al. (101) observed a modest but signifi-cant increase in plasma ascorbic acid concentration but no change in the ascorbicacid of lymphocytes following 2 weeks of supplementation with 400 mg vitamin Cper day. The mean (±SD) dietary intake of vitamin C prior to supplementation was147 (±70) mg per day, and this level of intake was offered in explanation for the lackof change in lymphocyte ascorbic acid content with supplementation.

Page 7: Peake Ijsnem 2003

Vitamin C and Exercise / 131T

able

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Page 8: Peake Ijsnem 2003

132 / Peake

Pre-supplementation levels of plasma/serum ascorbic also seem to affect theresponse to supplementation. In one study (106), the athletes were seemingly ran-domly assigned to either a supplement or placebo group with no attempt to match thegroups for pre-supplementation whole blood ascorbic acid concentrations. Consid-ering also that these pre-supplementation values were not reported, the true efficacyof supplementation in this study is difficult to evaluate. Among the studies listed inTable 3, serum ascorbic acid concentration increased by 40–50% in those individu-als with serum levels in the middle of the normal range (44, 69, 90). A much moredramatic increase was reported among individuals with plasma ascorbic acid con-centrations below the normal range; administration of an acute 500-mg dose ofvitamin C increased plasma ascorbic acid significantly from 26.3 (±18.2) to 117(±28.3) �mol/L (5). This latter result may indicate that athletes with low circulatinglevels of ascorbic acid are more likely to exhibit a greater response to supplementa-tion. Alternatively, it has been suggested that there may be an initial transientincrease in plasma ascorbic acid followed by a decrease during prolonged supple-mentation (98). However, data from Levine et al. (64) indicate that with a 60-mgdose of vitamin C, plasma levels of ascorbic acid were saturated after 3 weeks andremained stable thereafter. Hence, although no data are available on the saturationof cellular stores of ascorbic acid, based on the plasma response, a minimum periodof 3 weeks of vitamin C supplementation may be necessary for the saturation ofcellular stores of ascorbic acid.

In several of the studies listed in Table 3, the participants were given a combi-nation of vitamins and minerals, which may have influenced the absorption ofascorbic acid and, consequently, the response to supplementation (98). The assess-ment of the effectiveness of supplementation is also likely to be influenced by thetype of vitamin C supplement and the timing of supplementation and blood sam-pling. In the study by Levine et al. (64), intravenous injection of vitamin C predict-ably caused a very rapid increase in plasma ascorbic acid concentration when com-pared to oral ingestion of the same dose. Furthermore, the appearance of ascorbicacid in plasma occurred at a two-fold faster rate following the oral ingestion of 1250mg compared to 250 mg vitamin C. Plasma ascorbic acid concentrations also re-turned to baseline twice as rapidly following the higher dose. Following the 200-mgdose, plasma ascorbic acid remained elevated up to 10 hours after ingestion (64). Incontrast, Alessio et al. (1) found no difference in the effects of supplementation with1000 mg for either 1 day or 2 weeks on postexercise changes in thiobarbituric acidreactive substances or oxygen radical absorbance capacity. These factors should beborne in mind when evaluating the alterations in blood measures following supple-mentation.

Several groups have attempted to address the issue of ascorbic acid require-ments of athletes and have reported conflicting findings. One group observed thatathletes had intakes of vitamin C that were, on average, around 40% higher than thecontrols (34). Together with the finding that plasma ascorbic acid concentrationswere in fact similar between the athletes and controls, this difference in intakeswould suggest that either athletes do have increased requirements for ascorbic acid,and/or they retain more. In contrast, in the study by Guilland et al. (44), althoughathletes demonstrated a marked increase in serum ascorbic acid concentration aftersupplementation, this increase was of similar magnitude to that of sedentary controls.

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Vitamin C and Exercise / 133

The dietary intakes were also similar between the two groups. The fact that theathletes did not exhibit a greater response to supplementation is suggestive thatregular exercise does not actually increase the requirement for ascorbic acid. Never-theless, it is reasonable to contend that through its utilization in metabolic pathwayssuch as carnitine, noradrenaline, and collagen synthesis and antioxidant reactions,athletes may require more vitamin C than sedentary individuals. At similar dietaryintakes, athletes may use a greater proportion of the vitamin C consumed in their dietthan nonathletes. Nonathletes may not need all of the vitamin C they consume andmay excrete the excess. In such a way, athletes would actually have increasedrequirements. However, two studies of the ascorbic acid content in urine revealed nodifference between athletes and sedentary controls (32, 89).

In conclusion, the majority of existing data from dietary and supplementationstudies do not support the concept that athletes have increased requirements forvitamin C for the following reasons. First, the dietary intake of athletes and seden-tary controls is similar, as is the response to supplementation with vitamin C. Sec-ond, there does not appear to be a strong association between dietary intakes ofvitamin C and the concentration of ascorbic acid within the blood. Last, theexcretion of ascorbic acid in urine—which may be taken as an assessment ofthe utilization of vitamin C within the body—does not differ between athletesand nonathletes.

Changes in Plasma, Serum, and Lymphocyte Ascorbic AcidConcentrations Following Acute Exercise

Transient alterations in the concentration of ascorbic acid in plasma and lympho-cytes have been reported in plasma and lymphocytes following acute exercise.These changes are summarized in Table 4. Some studies have observed a rise ofvariable magnitude in circulating ascorbic acid levels following exercise (28, 41,42, 69, 84, 90), some have found no change (40, 66, 76, 82, 83, 101, 104), whileothers have reported a reduction (16). Meydani et al. (70) observed that after 45 minof downhill running, when the increase in plasma ascorbic acid was corrected forchanges in plasma volume, no significant change was seen. Several studies havealso demonstrated significant increases in lymphocyte ascorbic acid concentration(41, 42, 100, 101).

It is worthy of note that in several of the studies listed in Table 4, plasmaascorbic acid concentrations in the days following exercise were actually belowbaseline (42, 66, 76, 83, 90). In the study by Gleeson et al. (42) post-race haemodilutionaccounted for the reduced plasma ascorbic acid concentration at days 2 and 3;however, it could not account for the significant decrement seen after 24 hours. Thisdecrement could possibly reflect alterations in the antioxidant demands of tissues inthe days following exercise. In the study by Liu et al. (66), commensurate withreduced plasma ascorbic acid concentrations 4 days postexercise, there was also asignificant diminution of trapping antioxidant capacity of plasma and a significantincrease in the susceptibility of low-density lipoprotein (LDL) to oxidation. Muscledamage resulting from strenuous exercise such as a marathon is likely to cause localinflammatory reactions (97), and phagocytes involved with tissue damage are asource of reactive oxidants (47). Although ascorbic acid is a water-soluble antioxidant

Page 10: Peake Ijsnem 2003

134 / Peake

Tab

le 4

Mea

n ( ±

SD)

Cha

nges

in

Asc

orbi

c A

cid

Con

cent

rati

on I

mm

edia

tely

Pos

t-ex

erci

se

Asc

orbi

c A

cid

Con

cent

ratio

n (�

mol

· L

–1)

Blo

odC

orre

cted

Subj

ects

Act

ivity

com

part

men

tPr

e-ex

erci

sePo

st-e

xerc

ise

for

HC

Ref

.

Unt

rain

ed (

n =

24)

60 m

in b

ox s

tepp

ing

Seru

m53

.3(±

12.

4)p

59.6

(± 2

1.2)

p√

(69)

112.

5(±

20.

9)c

140.

4(±

20.

6)c ‡

99.9

(± 4

6.4)

e11

0.4

(± 5

6.6)

e *U

ntra

ined

(n

= 8

)35

min

uph

ill (

+5%

) w

alki

ngPl

asm

a21

.0(±

2.2

7)19

.3(±

2.2

7)√

(16)

at 6

0% V

O2m

ax

35 m

in d

ownh

ill (

–20%

)18

.2(±

2.9

5)10

.8(±

2.2

7)*

runn

ing

at 6

0% V

O2m

ax

Cyc

lists

(n

= 1

1)90

min

at 6

5% V

O2m

axPl

asm

a41

.1(±

25.

2)48

.1(±

29.

5)√

(104

)

Tri

athl

etes

(n

= 3

9)Ir

onm

an tr

iath

lon

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ma

96.0

(± 2

6.7)

e95

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23.

3)e

√98

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35.

8)94

.8(±

35.

8)

Run

ners

(n

= 8

)M

arat

hon

Plas

ma

54.7

(± 2

6.6)

79.0

(± 3

4.7)

*√

(41)

Lym

phoc

ytes

17.4

(± 3

.96)

a21

.3(±

7.3

5)a ‡

Run

ners

(n

= 9

)H

alf-

mar

atho

nPl

asm

a52

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12.

3)67

.0(±

15.

9) ‡

√(4

2)L

ymph

ocyt

es15

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1.8

0)a

19.9

(± 2

.70)

a †

Run

ners

(n

= 7

)H

alf-

mar

atho

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asm

a38

.4(±

15.

3)51

.5(±

26.

2)*

√(2

8)

(con

tinu

ed)

Page 11: Peake Ijsnem 2003

Vitamin C and Exercise / 135

Run

ners

(n

= 1

6)M

arat

hon

Seru

m41

.9( ±

25.

2)p

56.8

(± 5

2.0)

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(90)

72.8

(± 5

5.2)

c,e

105.

6(±

117

) ‡c,

e

Run

ners

(n

= 1

1)M

arat

hon

Plas

ma

86.0

(± 3

3.2)

103

(± 3

3.2)

√(6

6)

Run

ners

(n

= 2

9)U

ltram

arat

hon

Seru

m82

.9(±

10.

8)p

134

(± 1

9.9)

pX

(76)

128

(± 1

0.2)

c15

0(±

14.

8)c

153

(± 1

0.2)

c16

1(±

6.8

1)c

Run

ners

(n

= 2

0)90

min

run

ning

at 7

5% V

O2m

axPl

asm

a35

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16.

0)p

42.0

(± 2

1.4)

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39.

1)c,

e ‡

Run

ners

(n

= 2

9)U

ltram

arat

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Seru

m82

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0.0)

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0)p

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2)13

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0)c

145

(± 3

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150

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Run

ners

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= 1

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ltram

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m11

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116

(± 1

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(83)

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(± 1

1.9)

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11.

9)p

Run

ners

(n

= 2

8)U

ltram

arat

hon

Plas

ma

69.8

(± 8

.50)

c18

3(±

14.

2)c §

√(7

7)39

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6.8

)p73

.8(±

8.5

)p

Not

e. N

D =

no

avai

labl

e da

ta, H

C =

hae

moc

once

ntra

tion.

*Si

gnif

ican

t (p

< .0

5); †

sign

ific

ant (

p <

.01)

; ‡si

gnif

ican

t (p

< .0

01);

§si

gnif

ican

t (p

< .0

5)ve

rsus

pla

cebo

. a Con

cent

ratio

n ex

pres

sed

as �

mol

· g–1

of

prot

ein;

c vita

min

C–s

uppl

emen

ted

grou

p; e v

itam

in E

–sup

plem

ente

d gr

oup;

p pla

cebo

gro

up.

Tab

le 4

(con

tinu

ed)

Asc

orbi

c A

cid

Con

cent

ratio

n (�

mol

· L

–1)

Blo

odC

orre

cted

Subj

ects

Act

ivity

com

part

men

tPr

e-ex

erci

sePo

st-e

xerc

ise

for

HC

Ref

.

Page 12: Peake Ijsnem 2003

136 / Peake

and is therefore not directly involved in the protection of LDL against oxidation, itdoes neutralize free radicals that can lead to oxidation of LDL (36) through mecha-nisms explained below. Hence, the decline in plasma ascorbic acid content observedin the days following the marathon (66) may be attributed to the consumption ofascorbic acid in oxidative reactions contributing to LDL oxidation. Alternatively, itcould also reflect the involvement of ascorbic acid in maintaining �-tocopherol in areduced state (57)—the plasma concentration of which was unchanged 4 days afterthe marathon (66). Further research is warranted to confirm these concepts.

The reasons for differences with regard to exercise-induced changes betweenthese studies are not immediately clear, but some suggestions may be offered.Differences in assay techniques to measure plasma ascorbic acid concentrationsmay have contributed to the variability of findings between studies. Alternatively,the validity of measuring ascorbic acid in the plasma or serum compared to leuko-cyte concentrations has been questioned (70, 88).

Differences in the extent of oxidative stress caused by exercise could alsohave been a factor. Many of the studies in Table 4 involved prolonged enduranceexercise, which is likely to cause an increase in oxidative stress (28, 40, 66, 90), andit has been suggested that oxidative stress is a stimulus for the release of ascorbicacid from the adrenal gland (82). During exercise, it has been proposed that a pro-oxidant-antioxidant equilibrium is in operation, and that the intensity of exercise isthe critical factor in determining alterations in this equilibrium (110). It is possiblethat differences in the degree of oxidative stress induced by exercise among thestudies might have caused some variation in the amount of ascorbic acid released. Ifthis concept is in fact true, then it follows logically that pre-exercise plasma/serumascorbic acid concentrations would also influence the extent of exercise-inducedoxidative stress and consequently, the magnitude of ascorbic acid release duringexercise in response to any oxidative challenge. Among the studies in Table 4, thereis partial support for this idea. When pre-exercise plasma/serum ascorbic acid con-centrations were in the middle of the range (i.e., 40–60 �mol/L), there was anincrease in plasma/serum ascorbic acid (28, 41, 42, 69, 77, 90). In contrast,when pre-exercise plasma/serum ascorbic acid concentrations were at the high endof the range (i.e., ≥80 �mol/L), less ascorbic acid was released (40, 66, 69). This wasparticularly apparent in those studies involving supplementation (76, 82, 83).

There is also tentative evidence for an exercise mode effect on the magnitudeof changes in plasma ascorbic acid concentration. Among cyclists and triathletes theincrease was modest (40, 104), whereas among runners plasma ascorbic acid levelsappear to be elevated to a greater extent (41, 42, 66, 76, 82, 83, 90). Considering thatthere are no existing data with respect to the possible effect(s) of exercise mode onthe degree of oxidative stress, the differences above cannot be reconciled on thebasis of differences in exercise-induced oxidative stress. Differences in tissue dam-age resulting from each form of exercise also seem an unlikely influence, in light ofthe recent findings that supplementation with vitamin C did not influence inflamma-tory reactions to exercise-induced muscle damage (77, 84). Therefore, it remains tobe determined what exercise factors (i.e., intensity, duration, mode) affect changesin plasma ascorbic acid during exercise. Future exercise studies could investigatepossible differences between different forms of exercise with respect to alterationsin antioxidant capacity and oxidative stress.

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Vitamin C and Exercise / 137

Chronic Effects of Exercise on Resting Plasma, Serum, andLeukocyte Ascorbic Acid Concentrations

Despite evidence from comparisons of exercising and non-exercising individualsindicating similar dietary intakes of vitamin C and similar responses to supplemen-tation, the limited existing data on the effect of training or chronic exercise oncirculating ascorbic acid concentrations are unclear. A marginal deficiency in plasmaor serum ascorbic acid concentration has been reported in only a small number ofathletes (34, 90, 98), although the deficiency criteria varied among these studies.Contrasting results have been obtained from cross-sectional comparisons of plasmaascorbic acid concentrations between athletes and sedentary controls. In their studyof a variety of athletes, Fogelholm et al. (34) reported similar levels. The percentageof both controls and athletes classified as ascorbic acid deficient (<22.7 �mol/L)was similar (0–1%). There was also no effect of weight or energy expenditure.Brites et al. (13) found higher plasma ascorbic acid levels in soccer players com-pared to sedentary controls. Eight weeks of endurance training did not alter theserum ascorbic acid concentration of athletes (32), yet the levels of ascorbic acidwere still higher than those of sedentary controls. In contrast, 3 months of training inmarathon runners caused a decline in all circulating antioxidants, except serumascorbic acid, which increased from a mean (±SEM) serum concentration of 107(±5) to 180 (±22) �mol/L with training (7).

The concentration of ascorbic acid within plasma may be a less reliable indexof vitamin C status than that within leukocytes. The relationship between regularphysical training and circulating ascorbic acid concentrations was investigated in 6sedentary subjects and 12 runners (88). The runners were divided into two groups of6 based on training status. Whereas the plasma concentrations were not related totraining status, the lymphocyte concentrations in the highly trained runners weresignificantly higher compared to the controls. In cyclists, measurements of leuko-cyte ascorbic acid concentration were made in the 3rd year of a 4-yr training cycleand immediately prior to the Olympics (31). A significant reduction in the ascorbicacid content of both lymphocytes and neutrophils was observed over the course ofthe study. Compared to the pre-Olympic values, after the Olympics the mean (±SD)ascorbic acid concentration within lymphocytes had declined from 326 (±86) to 24(±2) nmol per 108 cells, whereas neutrophil ascorbic acid concentration fell from 94(±22) to 69 (±17) nmol per 108 cells. The decline in the ascorbic acid content of boththese cell types could be due to the consumption of ascorbic acid in neutralizingreactive oxidants (3), particularly if the athletes in this study suffered any infections(52).

For several reasons, few definitive conclusions can be drawn regarding theeffects of regular exercise on vitamin C status in athletes, as indicated by the levelsof ascorbic acid in serum, plasma, and leukocytes. First, the magnitude and directionof changes in circulating ascorbic acid concentrations among the studies reportedabove are variable. Second, in the studies reported above, it was not indicated if anyattempt was made to control the effect of exercise in the days prior to blood samplingon circulating ascorbic acid concentrations, which fluctuate in the period followingexercise (42, 66, 76, 83, 90). Last, it is unclear whether comparisons can reasonablybe made for changes in ascorbic acid within different blood compartments.

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138 / Peake

Ascorbic Acid and Cortisol

In spite of available data from several studies, the precise relationship betweenchanges in ascorbic acid and cortisol with exercise is yet to be elucidated. It wasoriginally suggested that ascorbic acid and cortisol are released simultaneously aspart of the general stress response to exercise (42). More recently, it has beenreported that when rats (a species capable of synthesizing ascorbic acid endog-enously) performed one bout of intense exercise, there was a significant increase inplasma ascorbic acid concentration 2 hrs postexercise that also corresponded to asignificant decline in the ascorbic acid content of the adrenal glands. These authorssuggested that the responses were indicative of a stress response (102). Unfortu-nately, no data were available on changes in cortisol. Others have proffered thatascorbic acid and cortisol release during exercise are linked through oxidative stressand inflammatory reactions (82). The evidence for and against these concepts isdiscussed below.

The biosynthesis of cortisol in the adrenal gland begins with the conversion ofcholesterol to pregnenolone (see Figure 2). This is the rate-limiting step and is themajor site of action for ACTH. This step involves two hydroxylation reactions,followed by the side-chain cleavage of cholesterol. The process requires molecularoxygen and a pair of electrons and is regulated by a single enzyme, CYP11A1. Theelectrons are donated by NADPH first to adrenodoxin reductase, a flavoprotein,then to adrenodoxin, an iron-sulphur protein, and lastly to CYP11A1. Pregnenoloneis then transported out of the mitochondria before further synthesis of steroidsoccurs. Both adrenodoxin reductase and adrenodoxin are also involved in the actionof CYP11B1 (11-�-hydroxylase) (4).

It was originally suggested that ascorbic acid must first be released from theadrenal gland in order to permit the synthesis of cortisol, as ascorbic acid wasbelieved to impede electron transport required for the side-chain cleavage of choles-terol (61). In theory, this concept could explain several experimental observations.First, it may be responsible for the apparent depletion of ascorbic acid within theadrenal gland following ACTH treatment or conditions of stress (27, 51). Second, inyoung chickens it has been shown that vitamin C administered in doses of 1000 mgper kg of body weight ameliorated the immunosuppressive effects of exogenouscortisol and high environmental temperatures, in addition to reducing the mortalityrates associated with such stress (99). Last, this concept may also account for theobservation of Gleeson et al. (42) that following exercise, increases in plasmaascorbic acid correlated with a rise in plasma cortisol concentration (r = 0.89, p <.01). They interpreted this finding as possible evidence that ascorbic acid and corti-sol release from the adrenal gland could be coupled as part of the stress response toexercise (42).

There is evidence against this coupling, however. Kipp and Rivers (60) dem-onstrated that the injection of ACTH in guinea pigs caused an increase in plasmacortisol concentrations, in addition to a significant reduction in the ascorbate con-tent within the adrenal gland. Moreover, ACTH significantly enhanced the uptakeof radio-labeled ascorbic acid into the adrenal gland. Nevertheless, despite therelease of cortisol, there was no change in the concentration of ascorbic acid withinplasma or any other tissues (60). Although Kipp and Rivers did not measure dehy-droascorbic acid, the oxidation of ascorbic acid to dehydroascorbic acid during thesynthesis of cortisol (62) could account for its apparent depletion within the adrenal

Page 15: Peake Ijsnem 2003

Vitamin C and Exercise / 139

gland upon ACTH stimulation. Freeman (35) has reported that 1 hour after theinjection of ACTH, relative to chickens not supplemented with vitamin C, there wasa greater increase in plasma corticosteroid concentrations in those chickens receiv-ing additional vitamin C in their diet.

In another study by the same group, the vitamin C content of diets fed toguinea pigs was manipulated, injections of ACTH were administered to the animals,and the resultant alterations in adrenal ascorbic acid and plasma cortisol concentra-tions were measured (63). Although dietary vitamin C strongly influenced the con-centration of ascorbic acid in the adrenal glands, it was without effect on basal orACTH-stimulated changes in plasma cortisol. Furthermore, among the animalstreated with ACTH, only a weak association was found between adrenal ascorbicacid and plasma cortisol concentrations. Last, similarly to earlier findings (60),ACTH treatment did not influence plasma ascorbic acid concentrations. Takentogether these data indicate that the absolute concentration of ascorbic acid withinthe adrenal gland is not critical for the synthesis of cortisol (63). While ascorbic acidis required for the synthesis of cortisol, ascorbic acid does not appear to be releasedsimultaneously with cortisol as a stress response.

In 1962, Jenkins (56) demonstrated a role for ascorbic acid in the synthesis ofcortisol, specifically in the terminal step of the conversion of 11-deoxycortisol tocortisol (Figure 3). This finding was supported more recently by Moser (71) who

Figure 3 — Biosynthesis of cortisol in the adrenal gland (4).

Page 16: Peake Ijsnem 2003

140 / Peake

cultured porcine adrenal cells for one week with and without 50 �mol/L ascorbicacid and stimulated the cells on days 2, 3, and 4 with ACTH. While the release ofcortisol on day 2 was not dependent on ascorbic acid, on days 3 and 4, the amount ofcortisol produced was significantly higher in those cells cultured with ascorbic acid.In contrast, there was a marked decline in cortisol release after repetitive stimulationin the cells cultured without ascorbic acid. Hence, ascorbic seems to be a criticalfactor in the synthesis of cortisol. It has since been shown that ascorbic acid exerts itsinfluence in this process by acting as an antioxidant in this process, which stands toreason considering the high concentration of ascorbic acid within the adrenal gland(49). The transfer of electrons between NADPH, adrenodoxin reductase, adrenodoxinand CYP11A1, produces superoxide radicals (46), which may impair the activity ofCYP11B1 (11-�-hydroxylase). Hornsby et al. (50) demonstrated that when primarybovine adrenocortical cells were cultured in a serum-free medium, the addition ofcortisol as a pseudosubstrate caused a decline in 11-�-hydroxylase activity. How-ever, in the presence of 50 �mol/L cortisol, the addition of 5 mmol/L ascorbic acidalmost completely prevented this loss of 11-�-hydroxylase activity. This effect wassynergistic with low oxygen in the culture medium. Hence, rather than stimulatingsteroidogenesis per se, ascorbic acid appears to act by protecting cytochromes, suchas CYP11B1.

Several other groups have subsequently investigated the possible relationshipbetween the release of ascorbic acid and cortisol during exercise by supplementingathletes with 500–1500 mg vitamin C in the week before, and on the day of, anultramarathon (76, 82, 83). In each of these studies, vitamin C caused a significantincrease in pre-race plasma/serum ascorbic acid concentrations and significantlyattenuated the post-exercise serum cortisol responses, relative to the placebo group.In contrast to the work of Gleeson et al. (42), modest negative correlations have beenreported between changes in plasma/serum ascorbic acid and cortisol postexercise(76, 77, 83). These findings are supported by other studies not involving athletes.Supplementation with 1000 mg vitamin C for 16 weeks caused a significant reduc-tion in resting serum cortisol concentrations in aged women with coronary heartdisease, but not in healthy age-matched controls (22). Patients who were given 1000mg vitamin C while under anesthetic demonstrated a transient suppression of bloodACTH and cortisol concentrations (73).

In accounting for these findings, it has been suggested that ascorbic acid isutilized during exercise to counter oxidative stress, while cortisol is released tocounter inflammatory reactions resulting from exercise-induced muscle damage(82). Oxidative stress has been implicated in chronic inflammation. The findings ofDe la Fuente et al. (22) outlined above, are supportive of a link between oxidativestress and inflammation. Under some conditions and in certain cell lines, a shift inthe glutathione redox state can activate NF�B (26). It is unknown whether there is asignal transduction pathway directly linking alterations in the glutathione redoxstate with the release of cortisol, or if increases in ascorbic acid and cortisol withexercise are incidental. Certainly, it is tempting to support the concept that oxidativestress is a stimulus for the release of cortisol during exercise, particularly on thebasis that elevated plasma ascorbic acid concentrations—which presumably sig-naled adequate antioxidant defense capacity—attenuated the cortisol response (76,82, 83). Nevertheless, it must be questioned whether there is in fact a link betweenoxidative stress reactions and the inflammatory response to exercise-induced muscledamage (77, 84).

Page 17: Peake Ijsnem 2003

Vitamin C and Exercise / 141

Despite the attenuation of cortisol release following vitamin C supplementa-tion, this effect may actually have promoted the inflammatory response (see Figure4), as indicated by significantly higher levels of acute phase reactants such as C-reactive protein and creatine kinase (83), and significantly reduced concentrationsof anti-inflammatory cytokines such as interleukin-1 receptor antagonist andinterleukin-10 (76, 82). Another group demonstrated that supplementation with asmaller dose of vitamin C (400 mg) for 2 weeks prior to exercise did not alter thecortisol response, yet the postexercise plasma concentration of the anti-inflamma-tory cytokine interleukin-6 was significantly reduced (101). Therefore, it seemsunlikely that exercise-induced oxidative stress causes the release of cortisol fromthe adrenal gland, at least not in order to counter inflammation. However, the effectsof vitamin C could depend on the supplementation dosage.

Two novel, yet largely speculative hypotheses are presented below to accountfor the observation of attenuated postexercise cortisol levels following vitamin Csupplementation (76, 82, 83). High doses of vitamin C may inhibit the synthesis ofcortisol, possibly through pro-oxidant effects. Under certain conditions in vitro,ascorbic acid may work as a pro-oxidant rather than as an antioxidant by reducingtransition metal ions (reaction 1), which in turn drives the Fenton reaction (reactions2 and 3), potentially resulting in oxidative stress (45).

AH– + Fe3+ A∑– + Fe2+ + H+ (reaction 1)

H2O2 + Fe2+ HO∑ + Fe3+ + OH– (reaction 2)

LOOH + Fe2+ LO∑ + Fe3+ + OH– (reaction 3)

There is also strong evidence against a pro-oxidant effect of ascorbic acid,however (6). A series of in vitro trials were performed in which iron was added toplasma in amounts exceeding the latent iron-binding capacity. While this causedappreciable oxidation of ascorbic acid and an increase in bleomycin detectable iron,which is potentially catalytic for free radical reactions, ascorbic acid maintained itsantioxidant activity, and there was no detectable alteration in the concentration ofF

2-isoprostane and protein carbonyls as markers of oxidative stress (6).

In spite of this evidence, data from a more recent study do lend support to theconcept that ascorbic acid can act as a pro-oxidant under some conditions (21).Individuals were given either a placebo, or 12.5 mg vitamin C and 10 mg of the

Figure 4 — Proposed relationship between exercise-induced oxidative stress and in-flammatory responses to muscle damage.

(1)

(2)

(3)

Page 18: Peake Ijsnem 2003

142 / Peake

antioxidant N-acetylcysteine per kg body weight for 7 days prior to a brief, intensesession of eccentric resistance exercise. The plasma concentration of serum bleomycindetectable iron was elevated in both groups in the days after exercise but wassignificantly higher in the supplement group. Supplementation also significantlyincreased plasma total antioxidant status. Importantly, there was evidence of oxida-tive stress. In the supplemented group, the plasma concentrations of 8-isoprostaglandin F

2� tended to be higher (p = .07), whereas lipid hydroperoxide

levels were significantly higher (p < .001). Although no correlations were reported,it is tempting to speculate that the combination of increases in serum-free iron,plasma antioxidant capacity, and lipid peroxidation markers were indicative of pro-oxidant effects of ascorbic acid during exercise. Unfortunately, plasma ascorbicacid concentrations were not measured, as this may have provided further insightinto the mechanisms in operation. The interaction between Fe3+ and ascorbic acidmay depend on the ratio of ascorbic acid to dehydroascorbic acid (87). It is alsounknown if there was any effect of the N-acetylcysteine on plasma ascorbic acidconcentration (21).

Of interest to the current discussion is whether interactions between ascorbicacid and iron could also influence the synthesis of cortisol in the adrenal glandduring exercise. There is evidence suggesting that oxidative conditions impair theactivity of the enzyme responsible for cortisol synthesis, 11-�-hydroxylase (50).The production of superoxide anions by polymorphonuclear cells during exercise(47) could possibly induce the release of catalytic iron from ferritin (86) and/ormyoglobin (85). The combined effects of elevated concentrations of serum-freeiron and ascorbic acid in the circulation during exercise could promote the forma-tion of reactive oxidants (93), leading in turn to impaired activity of 11-�-hydroxy-lase and less synthesis of cortisol, which could possibly explain the recent findingsof Peters et al. (82, 83). In evidence against this novel hypothesis, Nieman et al. (77)recently reported that vitamin C supplementation had no effect on plasma F

2-

isoprostane and lipid hydroperoxide concentrations following an ultramarathonrace, yet postexercise serum cortisol correlated negatively with plasma ascorbicacid (r = 0.50, p = .0006).

The second hypothesis offered presently is related to a possible role for ascor-bic acid in glucose metabolism during exercise. Although ascorbic acid within theadrenal gland may not influence ACTH activity and cortisol synthesis directly (63),high concentrations of ascorbic acid in other tissues such as the liver could possiblyhave a regulatory effect on the release of cortisol. The evidence for this conceptcomes from a study by Kodama et al. (62) who investigated the effect of infusing aglucose-electrolyte solution containing 200 mmol/L ascorbic acid over 1.5 h into amale volunteer. The concentration of ascorbic acid in plasma rose above 1000�mol/L within 0.5 h, and began to decrease thereafter, reaching baseline 3 h later.Interestingly, the decline in plasma ascorbic acid corresponded to a rapid and dra-matic increase in plasma cortisol and ACTH concentrations. Kodama et al. pro-posed that the liver acts as a “vitamin C/cortisol absorber” for two reasons: First, theliver contains high concentrations of ascorbic acid (49) and, second, both cortisol(59) and possibly even ascorbic acid are involved in the regulation of carbohydratemetabolism within the liver (11).

Using human erythrocytes as a model of peripheral tissues, Braun et al. (11)demonstrated that the oxidized form of ascorbic acid, dehydroascorbic acid, isconverted to glucose-6-phosphate through the pentose phosphate pathway and/or

Page 19: Peake Ijsnem 2003

Vitamin C and Exercise / 143

gluconeogenesis. They proposed that through glycolysis, glucose-6-phosphate ismetabolized to form lactate, which is then transported to the liver to be convertedback to glucose-6-phosphate and eventually, glucose (11). Interestingly, it has alsobeen demonstrated that glucose can inhibit the cellular uptake of dehydroascorbicacid (105), and that high concentrations of plasma ascorbic acid inhibit the glucose-induced secretion of insulin from the pancreas (8). These factors could influence therole of ascorbic acid in gluconeogenesis described above. Recently, cortisol wasalso shown to enhance gluconeogenesis in humans. A high dose cortisol infusionwas administered to fasted individuals, and this caused a significant 10% increase ingluconeogenesis as a proportion of hepatic glucose production (59). Therefore, itmay be hypothesized that high plasma concentrations of ascorbic acid followingsupplementation with large doses of vitamin C could possibly compete with cortisolby maintaining blood glucose concentrations via the stimulation of gluconeogen-esis during prolonged exercise. There is support for this concept in the study byPeters et al. (82) because although supplementation with 1500 mg vitamin C attenu-ated the cortisol response to exercise relative to those athletes taking 500 mg vitaminC or a placebo, the postexercise alterations in plasma glucose concentrations weresimilar between the three groups. These are novel concepts that warrant furtherinvestigation.

In summary, ascorbic acid is necessary for the synthesis of cortisol within theadrenal gland. Increases in the plasma concentration of ascorbic acid during exer-cise likely occur to counter exercise-induced oxidative stress, but do not appear tocause the release of cortisol, either as a general stress response or to counter inflam-matory reactions. It is possible, yet unproven, that the synthesis of cortisol duringexercise may be influenced by interactions between ascorbic acid and free iron and/or that ascorbic acid may have a role in glucose metabolism during prolongedexercise.

Ascorbic Acid and Exercise-Induced Oxidative Stress

If ascorbic acid is released during exercise in response to oxidative stress (82), thenwhat exactly is the stimulus for its release? A comprehensive review of free radicalsin their regulation of physiological functions was published recently (26). Theconcentration of reactive oxygen species depends on the balance between their rateof production and their rate of clearance by various antioxidant compounds andenzymes. Cells or tissues are in a stable state if the rates of oxidant formation andantioxidant scavenging capacity are constant and in balance. The intracellular thiol/dilsulfide redox state is the key determinant of this redox homeostasis. An alterationof this redox state, which may occur either by an increase in the concentration ofreactive oxidant species or a decline in the activity of one or more antioxidantsystems, induces redox-sensitive signal cascades that lead to enhanced expressionof antioxidant enzymes, elevated concentrations of nonenzymatic antioxidants oran increase in the cysteine transport system. This latter response may in turn facili-tate an increase in intracellular glutathione in certain cell types (26). Within thecontext of exercise, the thiol/disulphide ratio may represent the concept of the pro-oxidant-antioxidant equilibrium, as discussed by Zembron-Lacny and Szyszka (110).

If the production of reactive oxidants during exercise disturbs the intracellularredox state, what role does ascorbic acid play in restoring the balance? Ascorbic acidrepresents the first line of antioxidant defense in human plasma, and also effectively

Page 20: Peake Ijsnem 2003

144 / Peake

protects low-density lipoprotein against oxidative stress reactions (37, 38). Duringexercise, ascorbic acid likely exerts its antioxidant activity both directly and also inconcert with alpha-tocopherol and glutathione (Figure 5). The hydrogen atomsproduced in the oxidation of ascorbic acid first to ascorbyl radical and then todehydroascorbate (see Figure 1), react with a lipid peroxyl radical (LOO•) to formthe non-radical product, LOOH. Trapping of LOO• prevents this radical from at-tacking polyunsaturated fatty acid side chains (L’H) and other lipoproteins in plasma,and thereby halts the chain reaction of lipid peroxidation (reaction 4) (36).

LOO• + L'H LOOH + L'• (reaction 4)

Dehydroascorbic acid is reduced back to ascorbic acid by glutathione (109) andenzymatic reduction (80). Ascorbic acid may assist alpha-tocopherol in trappingaqueous radicals. Alpha-tocopherol also traps peroxyl radicals before they propa-gate radical chain reactions leading to lipid peroxidation (36). By donating a hydro-gen atom, ascorbic acid may reduce the alpha-tocopherol radical produced in thistrapping reaction back to alpha-tocopherol (57) (Figure 5).

In view of this role for ascorbic acid, it has been suggested that oxidative stressinduced by regular exercise could increase vitamin C requirements (13, 31, 95).However, the evidence for this postulate is conflicting. Some studies have notedchanges in biomarkers of oxidative stress without any change in circulating levels ofascorbic acid (28, 40, 90), some have demonstrated increases in ascorbic acid with-out a change in indices of oxidative stress (7, 77, 95, 104), and others have reportedcommensurate increases in both ascorbic acid and oxidative stress indicators (58,101). While an increase in indices of oxidative stress following exercise may notnecessarily correspond to an elevation of ascorbic acid levels per se, there is someevidence supporting more generalized antioxidant responses to oxidative stressduring exercise. Following a half-marathon, plasma malondialdehyde increasedafter exercise, in conjunction with a dramatic increase in the total antioxidant capac-ity of blood (19, 20). The majority of data have indicated that the vitamin C supple-mentation does not completely prevent, but attenuates, exercise-induced oxidativestress as indicated by increases in the levels of serum diene conjugation (103),

Figure 5 — Antioxidant activity of ascorbic acid in neutralizing aqueous radicals andregenerating alpha-tocopherol. See text for further explanation. AscH–, ascorbate; Asc•–,ascorbyl radical; DHA, dehydroascorbic acid; GSSG, oxidized glutathione; GSH, re-duced glutatione; LOO•, lipid peroxyl radical; LOOH, non-radical product, TocH,alphatocopherol; Toc•, alphatocopherol radical.

(4)

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thiobarbituric acid–reactive substances (90), malondialdehyde and exhaled pentane(5, 58, 101), the oxidation of low-density lipoproteins (5, 94), and electron spinresonance (5). Another group (1) demonstrated that supplementation with 1000 mgvitamin C attenuated the increase in thiobarbituric acid–reactive substances follow-ing submaximal exercise, but there was no effect on oxygen radical absorbancecapacity.

The results from two other groups contrast with the findings above. Relativeto a placebo group, supplementation with 1000 mg vitamin C pre-exercise waswithout effect on plasma malondialdehyde concentrations after 90 min of high-intensity shuttle running (100). This suggests that supplementation was ineffectivebecause endogenous antioxidant defenses were adequate prior to supplementation.However, in apparent contradiction to this suggestion, they also contended thatantioxidant defenses had been overwhelmed as indicated by increased levels ofmalondialdehyde after exercise. One month of supplementation with 1000 mg vita-min C was also without effect on serum ascorbic acid concentrations, yet it reducedresting levels of plasma lipoperoxide. The placebo group displayed a decline inserum ascorbic acid concentration but no change in plasma lipoperoxide (95). It wasnot stated whether the effects of recent exercise had been controlled for at the time ofsampling, as this factor could have influenced the results.

The disparity among these studies could be due to the fact that ascorbic acid isa water-soluble antioxidant, and indices of oxidative stress are based on lipidperoxidation. Ascorbic acid is effective in preventing the initiation of lipidperoxidation. Aside from the chain-propagating reaction (reaction 4), lipid peroxylradicals may also be converted to a cyclic peroxide, which then degrades to otherbreakdown products, such as malondialdehyde (36). Ascorbic acid is not effectivein preventing the formation of these products, and this could explain some of thediscrepancies between changes in plasma/serum ascorbic acid concentration andmarkers of oxidative stress in the studies above.

Conclusions

Ascorbic acid is known to be involved in a number of metabolic pathways that areimportant to exercise. Nevertheless, reports of alterations in the concentration ofascorbic acid within plasma/serum or urine following vitamin C supplementationand exercise do not support the concept that athletes have a greater requirement forvitamin C in their diets. During acute exercise, it is commonly contended that themajor role of ascorbic acid is in counteracting oxidative stress. However, whetherascorbic acid is oxidized to dehydroascorbic acid in these reactions during exercisehas not been investigated to date. Future exercise studies should investigate measur-ing the concentration of ascorbic acid derivatives, such as dehydroascorbic acidfollowing exercise. It is conceivable that ascorbic acid is efficiently recycled byexercise, thereby obviating the need for supplementation beyond regular dietaryintakes. Two novel hypotheses have been put forward in this review. First, in addi-tion to causing lipid peroxidation, the products of prooxidant reactions that poten-tially follow supplementation with high doses of vitamin C may have more wide-ranging effects on other aspects of physiological function. Second, although it hasbeen questioned whether cortisol is a factor in stimulating gluconeogenesis, there isrecent evidence that lends tentative support to the idea that both cortisol and ascorbicacid have a regulatory influence on gluconeogenesis. These concepts await further

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investigation before they can be applied to the control of physiological systemsduring exercise.

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