Ascorbic acid safety: analysis of factors affecting iron absorption

Toxicology Lefters, 5 1 ( 1990) 189-20

I

189

Elsevier

TOXLET

02306

Ascorbic acid safety: analysis of factors affecting iron absorption

Adrianne

Bendich and Marvin Cohen

Hoffmann-LaRoche Inc., Nutley, NJ (U.S.A.) (Received

23 July 1989)

(Accepted

29 October

Key words: Ascorbic

1989) acid safety; Iron absorption;

Ircn overload

SUMMARY The potential mended

for excessive iron absorption

dietary

allowance

jects) in which ascorbic parameters response

with iron absorption

curve relating

iron absorbers

ascorbic

acid dosage

acid on the absorption

iron absorption

and plasma

transferrin

(> 2 SD from population

> 100 mg/d indicated

ingesting

by examining

ascorbic

published

acid was part of a test meal given to determine

associated

effect of ascorbic

by subjects

(60 mg) was evaluated

no change

were identified:

acid doses above the recom-

literature

(24 studies,

1412 sub-

effects on iron absorption.

(1) a relatively

(1llOOO mg) and percent

shallow

iron absorption;

Three

slope for the dose(2) no significant

of high (60 mg) iron doses: and (3) an inverse relationship

between

saturation.

of ‘high’

Ascorbic

acid did not increase

mean) above control

in the distribution

the incidence

levels; limited data for ascorbic

of iron absorption

acid doses

values.

INTRODUCTION

The recommended

dietary

allowance

(RDA)

of iron for adults ranges from 10 mg

in men and menopausal women to 18 mg in premenopausal women [ 11. The second National Health and Nutrition Examination Survey (NHANES II) examined iron status using 5 criteria: mean corpuscular volume, transferrin saturation, erythrocyte protoporphyrin, serum ferritin, and hemoglobin levels. The prevalence of impaired iron status for adult men (2&74 years) ranged from 0.8 to 2.9%, while adult women showed a prevalence of 2.7-9.6s [2]. These findings suggest that, for many indtviduals, the diet is either not supplying sufficient iron or that available iron is not being absorbed to the extent needed for maintenance of adequate iron stores. The relatively Address.for correspondence: Dr. A. Bendich. Nutley.

Department

of Clinical Nutrition,

Hoffmann-LaRoche,

NJ 071 IO, U.S.A.

0378-4274/90/S

3.50 @ 1990 Elsevier Science Publishers

B.V. (Biomedical

Division)

Inc.,

poor absorption of non-heme iron from plant products [3] and the ability of many dietary components (e.g. tannins, egg albumin) to interfere with iron absorption [4, 51 are additional factors that may contribute to low iron status. The treatment of iron deficiency has included fortification of food with iron and ingestion of iron supplements [6]. However, increased iron intake can produce adverse gastrointestinal effects [7] and has been associated with an increased risk of hemochromatosis [8]. A recent epidemiological study suggested that high iron stores may be associated with an increased risk of cancer [9]. Ingestion of supplemental ascorbic acid with meals has been proposed as a means of increasing iron absorption [6, lo]. The ability of ascorbic acid to enhance the absorption of non-heme iron has been known for many years [l I], and several studies over the past 334 decades have generally confirmed this effect in different populations. The mechanism of the interaction is believed to be the formation of a complex that is more soluble and more readily absorbed than iron alone [12, 131. The ability of ascorbic acid to inhibit the oxidation of ferrous (Fe2+) to the less soluble ferric (Fe3+) form of iron is also believed to play a role [14]. Clinical data on ironascorbic acid interactions have not been examined to assess the risk, if any, of iron overload in subjects ingesting ascorbic acid at doses above the U.S. RDA (60 mg). This issue has been raised because of isolated case-reports in which high ascorbic acid doses (SOCrlOOO mg) appeared to exacerbate iron toxicity in patients with hemochromatosis or thalassemia major [15-171. In addition, evidence of iron overload (increased ferritin levels and transferrin saturation) was reported by Haubenstock et al. [I81 in a patient on chronic hemodialysis who received 1000 mg ascorbic acid weekly. The above reports suggest that there may be an undesirable interaction of ascorbic acid and iron under certain circumstances, such as the presence of a subclinical form of hemochromatosis or related disorder. The incidence of hemochromatosis is approximately 3/1000 [ 191, and thalassemias of varying severity may have an incidence of 7.5510% in some ethnic groups [20]. Edwards et al. [21] surveyed 11065 apparently healthy blood donors in an attempt to detect subjects in the general population at risk for hemochromatosis. Transferrin saturation of 50% or more (normal value is - 25%) was found in 112 subjects; 42 of these had 62% or greater transferrin saturation. Iron overload can be a consequence of chronic liver disease and frequent blood transfusions used in the treatment of conditions such as renal insufficiency or leukemia [22]. Since a study of ascorbic acid safety as it may apply to iron status has not been reported, a critical analysis of available data was undertaken. Clinical data on iron absorption over a wide range of ascorbic acid intakes were used to calculate doseresponse relationships and to estimate the potential for iron toxicity. The data also permitted an examination of the influence of iron dosage and degree of transferrin saturation on ascorbic acid-iron interactions. The results of these analyses were evaluated only from a toxicological point of view; the nutritional effects of iron-ascorbic

191

acid combination ascorbic

on iron status are not discussed.

acid supplementation

The question

addressed

can increase iron stores above recommended

is whether levels.

METHODS

A comprehensive literature search was made for studies in which iron absorption was measured in healthy subjects under conditions where the ascorbic acid content of the food items in a test meal was determined for a known amount of vitamin C added. Data were obtained from 24 studies comprising 1412 subjects. Ascorbic acid intake ranged from 1 to 1000 mg, with the 10-100 mg dose range having the largest representation. In order to determine the consistency of any patterns that emerged over the dose-response curve; the U.S. RDA of ascorbic acid (60 mg/d) was used as a reference point. The database was divided into two subsets: (a) studies in which the control population (no ascorbic acid) and treatment population (ascorbic acid) were different subjects (intergroup comparisons); and (b) studies in which the control and treatment groups were the same individuals (crossover comparisons). Table I summarizes the characteristics of each study used, including the number of subjects, doses of ascorbic acid and iron, and the availability of individual subject data. Data on percent iron absorbed were compiled in relation to increasing doses of ascorbic acid and, where available, increasing levels of transferrin saturation. The latter parameter was further divided into 3 groups comprising 15% or less transferrin saturation (iron-deficient), 15.1-24.9s saturation (marginal iron reserves), and 25% saturation or greater (normal iron reserves) according to the guidelines suggested by Layrisse et al. [25]. Since iron dosage generally remained within a relatively narrow range (see Table I), this parameter was considered to be constant. One study which specifically related iron dosage, iron absorption, and ascorbic acid dosage was treated separately. Statistical treatment of the data consisted of: (a) the use of weighted the number of subjects in any given cluster of groups varied by a factor (b) calculation of standard deviation and standard error of the mean cases where 3 or more groups were available for evaluation; (c) use of

means when of 2 or more; (SEM) in all the Student’s

t-test to determine statistical significance of changes in iron absortion. All data used were reported by the authors of the 24 studies cited; in a few cases, values were easily derived from the available information. RESULTS

Effect of ascorbic acid dosage on iron absorption Table II summarizes the relationship between ascorbic acid dosage and percent iron absorption. Significant increases in iron absorption in subjects ingesting a wide range of ascorbic acid doses were found for both intergroup and crossover compari-

__

_ _ + _

+

+ + _

f +

+ +

24 25 26 27 2x 29

30 30

31 32 33

34 35

36 37 3x

ships III Table II.

‘Women only. “Not reported.

_

.~

17 82 8 30 92 50 24 96 47 242 30 39 13 II 18 57

+ + + + _ +

+ _

_ _ +

_ _

+ _ _

_ + t + + +

30 263

+ +

Dose of

4 4 2.8 3.1

100 50 70

2 1.3-8.6

1

3.6-5.3 2.6-3.5 2.1 4.3

1.3-9.4

4 _h

500 50-1000

50 250 50~100 50 1000 50 100~500 8 50

20dOd 24.8 4.3%10.7

3 3

100&600 2-14 50-65

20 20-80

I.0 75 123.8 12.5 200 500 20-40

4.1 1.6-5.1 9.8 28.8 5 2-3 2.3-5.2

5.4-7.8 0.44.5

35-100 12.5-500

IO00

0.3 5. IA.6

(mg)

irOIl

90 35-100

Dose of ascorbic acid (mg)

ACID INTERACTIONS

+” +” +” + +* _ _

+ + +’ _ _

+ _ _

_

+ + + + + _

+ +” +* _

Individual subject data

+ ,1

+”

_ _ _

_

+

+’

_

_ + +

f”

_ + +*

_ i_ _

Group (pooled) data only

tron was not added to food. ‘Men only. dOnly data with lower iron dose (20 mg) were used for doseeresponse

38 36 31 6 6 27 82

30 21

Number of subjects

IRON-ASCORBIC

_

Intergroup (crossover) comparison

hut supplcmcntal

_

IO 23

+ +

+ +

5 IO

39 40 40 31 42 43 44

Intergroup comparison

OF S’T’UDIES USED TO EVALUATE

Reference

PROFILES

TABLE I

relation-

193

TABLE

II

DOSE-RESPONSE

RELATIONSHIPS

FOR ASCORBIC

Dose range of ingested

% Iron absorption”

ascorbic

(mean

acid per meal

ACID EFFECTS

ON IRON ABSORPTION

i SEM)

(mg) Intergroup

Crossover

comparisons

comparisons

11.6&3.7*


9.6+3.8*

(7; 118)b 13-25

(4; 40)

13.3k7.9

7.8f

10.9i

2650

1.8*

11.2* 1.2’

(12; 96) 51-100’

501-1000

dPooled baseline 257 subjects. comprising hNumber

baseline

13.4* 1.6*

(15; 134)

(7; 188)

17.3*2.4*

20.9*3.9*

(6; 68)

(8; 74)

13.0*7.9*

17.1**

(3; 29)

(2; 23)

iron absorption

Pooled

for intergroup iron absorption

comparisons for crossover

was 3.9*0.5% comparisons

using 27 groups

was 4.6+0.6%

comprising

using 60 groups

688 subjects. of groups

and total number

means. The total number U.S.

(14; 252)

9.7* 1.6*

101-500

1.6

(9; 192)

(5; 55)

of subjects.

of subjects is provided

Statistical

evaluation

only for information

of data was performed

on group

purposes.

RDA is 60 mg/d.

*Significant

difference

**Statistical

significance

(PcO.05)

from respective

control

group (no ascorbic

acid).

was not determined.

sons. An apparent dose-response relationship was noted over the range of 51-500 mg ascorbic acid in the intergroup comparisons, and a similar pattern over a somewhat wider dose range (133500 mg ascorbic acid) occurred in the crossover comparisons. Ascorbic acid enhancement of iron absorption appeared to level off or decrease at the highest doses (>500 mg ascorbic acid), but the relatively small sample size does not allow a firm conclusion to be made. Effect qf transferrin saturation Since the data in Table II were derived from populations with varying levels of iron reserves (normal, marginal or deficient), an attempt was made to clarify the ef-

194

TABLE

III

RELATIONSHIP

BETWEEN

ASCORBIC

ACID

INTAKE,

TRANSFERRIN

SATURATION

AND

IRON ABSORPTION Dose range of ingested ascorbic

Z

Iron absorption

(mean i SEM)

% Transferrin

(mg)

saturation 15. I f 24.9


_

acid/meal

13

9. I

3.1 (I)h

13 -25

9.4*

12.8k2.2

tions with deficient. “Number

ll.4&

1.3*

(X(l)

31.6+X.7

17.0*6.7

10.6*4.x*

(23)

(21)

(26)

X.5&2.5

6.7& 1.2* (17)

(11)

was 7.9kO.796

marginal

3

(65)

(9) aBaseline iron absorption

fJ.2+ 1.0* (25)

14.2&I

I.9

31.7*11.8

_501- 1000

1.4

(20)

(60) 101 500

2.4*0.3* (32)

(3)

(31) 2o.oi

(9)h

lO.Oi4.2

9.3 f 2.2

51 IO@

0.9kO.2

(2)h

(7) 26 50

225

(n=99),

and adequate

5.2&0.7%

transferrin

(n=84),

and 3.8kO.5~

(n= 155) for popula-

levels. respectively.

of subjects in group.

‘U.S. RDA is 60 mg. *Statistically absorption

significant

(PiO.05)

from corresponding

at the I3 25 mg dose range of ascorbic

deficient

transferrin

group

(e.g. 2.4 vs 9.3% iron

acid).

feet of ascorbic acid dosage on iron absorption by examining data from groups with different levels of transferrin saturation using this measurement as an indicator of iron status (Table III). All 3 groups showed a clear dose-related effect of ascorbic acid on iron absorption. The most marked effect was found in iron-deficient subjects ( < 15% transferrin saturation), where ascorbic acid in doses of 5&100 mg increased iron absorption approximately 2.5 times above baseline levels. An apparent inability of the highest doses of ascorbic acid (> 500 mg) to further increase iron absorption above that found in the lOlL500 mg dose range was a consistent pattern at all 3 levels of transferrin saturation. A consistent inverse relationship was found between transferrin saturation and iron absorption. This effect was statistically significant at all ascorbic acid dose ranges where there were sufficient data. The most marked ‘inhibi-

195

TABLE

IV

EFFECT

OF HIGH

IRON DOSAGE

ON INTERACTION

y%Iron absorption

Dose of ascorbic

(mean

WITH ASCORBIC

ACID”

+ SEM)b

acid (mg) Dose of iron (mg)

Dose of iron (mg)

(as ferrous

(as ferric ammonium

sulfate)

5.8_+ 2.6

100

6.4i

(8)

6.7k2.0

(11)

(13)

12.0_+2.5

600

1.1

(25)

6.6k2.0

300

20

60

20

7.0_+ 1.0

(13)

(22)

citrate) 60

3.4_+ 1.3

3.9_+0.9

(8)’

(25)

7.6k2.1

5.1_+ 1.6

(11)

(13)

8.7_+ 1.7*

3.3kO.5

(13)

(10)

dData derived from Grebe et al. [31]. bBaseline iron absorption mg dose; the respective cNumber

in brackets

*Significant

for ferrous

sulfate was 7.8 If: 1.8% (n = 15) for 20 mg and 6.4 k 1.6% for the 60

values for ferric ammonium

indicate

citrate were 1.5 k 0.3% (n = 15) and 1.8 k 0.4% (n = 12).

group size.

(P< 0.05) difference

from 4g iron absorption

after 100 mg dose of ascorbic

acid.

tory’ effect of transferrin saturation on iron absorption appeared to occur at the highest doses (> 500 mg) of ascorbic acid, where iron absorption was approximately 5-fold less in subjects with normal transferrin levels than in the group with iron deficiency. At lower ascorbic acid doses, subjects with normal transferrin saturation showed approximately a 224-fold reduction in iron absorption relative to deficient subjects. EfSrct of iron dosage

The influence of different iron-ascorbic acid combinations on iron absorption was specifically examined by only one of the studies reviewed [31]. The absorption of 20 or 60 mg iron (as ferrous sulfate or ferric ammonium citrate) was determined after administration in combination with ascorbic acid doses ranging from 100 to 600 mg. The data from this study are summarized in Table IV. When 20 mg of iron was administered, 100-600 mg ascorbic acid did not significantly increase the absorption of ferrous iron, but 600 mg ascorbic acid significantly increased ferric iron absorption when compared to the 100 mg ascorbic acid dose. When iron dosage was increased to 60 mg, 300 or 600 mg ascorbic acid did not significantly increase iron absorption above the values found with 100 mg ascorbic acid. In addition, 3 of the 6 comparisons showed that subjects ingesting 60 mg iron had a lower percentage iron absorption than when 20 mg iron was given.

TABLI’

V

‘HIGH’

IRON

ABSORPTION

IN SUBJECTS

(k2Sy;)

AFTER

IN<;ESTION

OF ASCORBIC‘

Incidence

of values

> 2 standard

deviations

WITH

NORMAL

TRANSFERRIN

SATURATION

ACID

from mean” Incidence

Dose range of ascorbic

(Ing)

aad

0

7, I55 (4.57;) 0:“)

< 13

I’32 (3.1%)

13 25 26 so

I ,3s (4.0g)

5l-IOV

4:6X (5.9yq

,‘Thc data \verc baaed on the distribution of’normal’

iron absorption

(2 standard

of individual deviations

7; Iron absorption

values for I55 subjects.

The limit

from the mean of 3.X%) was 12.Xy6

hU.S. RDA 1s 60 mg’d.

Estinwtrs qj’risk fbr it-m to.*ic.itl-

Individual data for subjects with a transferrin saturation of at least 25% were used to evaluate whether ingestion of ascorbic acid at higher than RDA levels increased the number of subjects at risk for iron toxicity. Individuals with ‘high’ iron absorption despite normal levels of transferrin saturation may be most likely to develop adverse effects. Those subjects with values more than two standard deviations above the control iron absorption group mean were considered to be in the ‘high’ iron absorption group. Table V summarizes the data treated in this manner for doses of ascorbic acid up to 100 mg. A baseline incidence of 4.5% ‘high’ iron absorbers was obtained from a population sample of 155 subjects in which iron absorption was > 12.8% (2 standard deviations from the mean of 3.8%). Intake of ascorbic acid up to 100 mg/d did not affect the baseline incidence. The data on ascorbic acid doses above 100 mg were treated differently because of the small sample size (n = 36) available

over the ascorbic

acid dose range of 25f~lOOO

mg. Table VI represents these data as individual transferrin saturation values for each of the 36 subjects. No pattern was noted between ascorbic acid dosage and the degree of transferrin saturation that was associated with ‘high’ iron absorption. Thus, there was no clear association between ‘high’ iron absorption and high ascorbic acid intake. DISCUSSION

Three factors-the shape of the doseeresponse curve, degree of transferrin saturation, and iron dosage-had marked influences on iron absorption in subjects ingesting ascorbic acid. A clear dose-related effect of ascorbic acid on iron absorption was

197

TABLE

VI

‘HIGH’

IRON ABSORPTION

Ascorbic

IN SUBJECTS

SATURATION

% Transferrin

saturation

acid (mg)

250-300

26, 21, 30, 34b, 41, 44b, 45,46

500.-600

25, 25, 25b, 26, 26, 27h, 30, 30, 30, 31b, 32

1000

25b, 27, 29b. 3S’, 36, 3gb, 38, 55, 58

“Data obtained %ubjects

>25%

acid doses above RDA rangea

Dose of ingested ascorbic

WITH TRANSFERRIN

from Cook et al. [24, 281, Grebe et al. [3 I], Kuhn et al. [34], Sayers et al. [39].

absorbing

> 12.8% of an iron dose.

noted over a portion of the dose-response curve (- 10-100 mg in the crossover studies). The effect of ascorbic acid on iron absorption appeared to plateau or decrease at doses of approximately 500 mg or more ascorbic acid. Since the sample size at the high end of the ascorbic dose-response curve was relatively small, additional studies are needed to clarify the ascorbic acid-iron interaction at high ascorbic acid intake levels. Another factor supporting the limited ability of ascorbic acid to enhance iron absorption in individuals with adequate iron stores was the observation that the overall dose-response relationship between ascorbic acid and iron absorption showed a relatively shallow slope. Even in the most responsive subjects (iron deficiency as indicated by transferrin levels < 15%), iron absorption was increased to a maximum of approximately 4 times baseline over approximately a 40-fold ascorbic acid dosage range. Subjects with higher levels of transferrin saturation showed substantially less improvement in iron absorption when given ascorbic acid. Dose-response curves relating ascorbic acid dosage and iron absorption have been reported many times in the literature, but they usually were the results of studies in relatively small populations and did not include associations with the degree of transferrin saturation. For example, Hallberg et al. [23] concluded that a linear relationship existed when ascorbic acid dosage (between 12.5 and 1000 mg) was plotted against iron absorption ratio (i.e. % iron absorption before and after ascorbic acid administration). Stekel et al. [45] reported a flattening of the dose-response curve for iron absorption when infant milk formulas contained more than 200 mg/l ascorbic acid. These apparent differences reflect a need for additional research on the effect of dietary regimens on the ascorbic acid dose-response curve as well as a better understanding of the physiological and physicochemical factors operating at different k ratios of iron to ascorbic acid. The level of transferrin saturation had a marked effect on the amount of iron absorbed. Despite a moderate degree of intergroup variability, subjects with normal

(25% or more)

transferrin

saturation

consistently

showed

a significantly

lower ab-

sorption of iron than did subjects with deficient ( < 15%) transferrin levels. While the relationship between these two parameters does not necessarily indicate cause and effect, the level of transferrin saturation appears to be a ‘built-in safety factor’ that is a major contributor to the control of iron absorption in most individuals. Future studies status. become Only

should include transferrin saturation levels as well as other indicators of iron More subtle aspects of the relationship between iron and ascorbic acid may evident with these data. one study was found that attempted to relate iron dosage, ascorbic acid dos-

age and iron absorption [3 11. The data suggested that within the range of 2@ 60 mg, the higher dose of iron was absorbed to a similar or lesser extent than a lower dose of iron. Ascorbic acid at 600 mg appeared to significantly increase absorption of 20 mg ferric iron, but this effect did not occur with other doses of ferric iron or with ferrous iron. The inconsistency of this pattern may be a function of experimental design, since intergroup comparisons from other studies in this review provided somewhat equivocal results. While the relatively poor absorption of dietary iron is a wellknown phenomenon [3, 7, I I], the available evidence suggests that ascorbic acid intake above the U.S. RDA level does not significantly enhance the absorption of high iron doses. An attempt to estimate the risk of iron toxicity was made by determining the number of ‘high’ absorbers of iron in subjects with transferrin saturation of 25% or more. This subpopulation was chosen because individuals in this category appear to be at greatest risk. The incidence of ‘high’ iron absorbers was not increased in the group taking ascorbic acid at doses up to 100 mg. Limited data with ascorbic acid doses ranging from 250 to 1000 mg suggested that the vitamin had no dose-related effect on the degree of transferrin saturation that was associated with ‘high’ iron absorption and did not appear to change the incidence of ‘high’ iron absorption. Most iron overload disorders are characterized by iron being excreted at a much slower rate than it is absorbed or stored. When the iron-binding capacity of serum transferrin is saturated, an increase in circulating levels of unbound iron can result in toxicity [22]. The role that ascorbic acid may have in the dynamics of iron storage is not completely understood, and clinical research in this area has not clearly defined all the physiological pathways that may be involved. For example, Stekel et al. [45] reported that low plasma levels of ascorbic acid appeared to protect against toxic effects of high iron intake, but Cook et al. [24] reported that high ascorbic acid doses did not affect iron stores when the vitamin was administered over a 4-month period. Two possible mechanisms by which ascorbic acid might increase the risk of iron toxicity are by changes in iron absorption or mobilization of iron reserves. The doseresponse curves obtained for ascorbic acid enhancement of iron absorption in this review indicated that the effect of the highest ascorbic acid doses (501~1000 mg) on iron status was not significantly greater than doses in the range 101-500 mg. In subjects with marginal (I 5. I-24%) or normal (3 25%) transferrin saturation, iron absorp-

199

tion in the group with ascorbic

acid intake of > 100 mg was not greater than at doses

in the X%100 mg range. Thus, in these populations,

no evidence

corbic acid significantly affected iron absorption. Clinical studies on the effect of high-dose ascorbic

was found

acid on iron reserves

that asin iron

overload disorders have been largely limited to anecdotal reports [15-181. However, Cook et al. [24] studied the effects of high-dose ascorbic acid administration on iron reserves in healthy and iron-deficient subjects. Serum ferritin levels (an indicator of iron reserves) were measured before and after ingestion of ascorbic acid 2000 mg/d for 16 weeks; no change was found. An additional study lasting 20 months in 5 ironreplete and 4 iron-deficient subjects gave similar results. These limited data suggest that high ascorbic acid doses do not have a marked effect on iron reserves. Data from the studies evaluated in this review support the conclusion that ascorbic acid doses above the RDA level of 60 mg do not increase the susceptibility to iron overload. This appears to be true for the absorption phase of iron metabolism as well as the plasma transport. Additional epidemiological and ascorbic acid supplementation studies are needed to examine further the mechanism of this interaction in population groups at risk for iron toxicity. ACKNOWLEDGEMENTS

The assistance of Dr. Ruth Velez in evaluating the data, the critical review of the manuscript by Drs. Lawrence J. Machlin and Vishwa N. Singh, and the preparation of the manuscript by Mrs. Veronica Mushinskie and Ms. Shanda Reed are gratefully acknowledged.

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