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Loyola University Medical Center Stritch School of Medicine, Maywood, IL 60153;
*
University of Catania, Institute of Geriatrics and Gerontology, Catania, Italy;
Cornelli Consulting, Milano, Italy; and
**
CNR I.Co.C.E.A., Area della Ricerca di Bologna, I-40129 Bologna, Italy
1To whom correspondence should be addressed. E-mail: corcon{at}katamail.com.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-tocopheryl acetate) and 10 mg L-cysteine. Formula 2 was composed of 30 mg bioflavonoids from citrus, 30 mg vitamin C (as L-ascorbic acid), 10 mg coenzyme Q10 and 1 mg vitamin B-6 (as pyridoxine hydrochloride). Formula 3 was composed of Formula 1 plus Formula 2. Each formula was prepared in dry capsules (formulation D1, D2, D3) or in a fluid form (formulation P1, P2, P3). Each formulation was administered for 1 wk in a crossover design. A 15% deviation of U.CARR. levels was chosen as the cut-off value for a significant change in oxidative stress. Formulas F1 and F3 reduced mean U.CARR. levels in most of the treated subjects (t test, P < 0.05), whereas F2 was not active. Fluid formulations were more active than dry formulations (
2 test, P < 0.05). In some cases, a slight increase in oxidative stress was detected. These minimal increases were not related to any particular antioxidant formula. In one subject only, the administration of the dry formulation (D1), increased oxidative stress to a level that reached the cut-off value. In conclusion, when antioxidants are taken in combination at low dosages they reduce oxidative stress, and little relevant prooxidant activity is detectable.
KEY WORDS: • antioxidants • prooxidants • D-Roms test • bioavailability • crossover study • humans
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) that may be clinically useful. There are studies that show some clinical benefits after the intake of antioxidants (2
–4
), and others with opposite results (5
–7
). In these studies, it was impossible to evaluate the degree of oxidative stress in patients. As a consequence, two problems arise.
The first problem is related to the possibility that antioxidants could act as prooxidants. This may happen in cases of high dosages or because of unpredictable clinical/biochemical conditions, which do not require that particular kind of supplementation (8
,9
).
The second problem concerns the bioavailability of antioxidants taken as supplements, which may not be sufficiently absorbed to produce any biological effects. Therefore, the effect of their intake on the oxidative stress is not predictable, especially if there is no reliable measure of their activity.
Recently, a new test for the evaluation in serum of the derivatives of reactive oxygen metabolites, the (D-Roms)2
test, has become available (10
,11
). This test is a measure of the equilibrium between free radical production and antioxidant defense. In apparently healthy individuals, serum levels of the D-Roms test range between 250 and 300 U.CARR. (Carratelli Units).
The present study compared the activity and the bioavailability of some antioxidants, which were combined at dosages very close to those taken with an average daily meal. Three different formulas were tested. Each formula was prepared in both a fluid and a dry formulation and was given to the same group of subjects for 1 wk. The activity and the bioavailability of the above-mentioned antioxidants were determined using the D-Roms test.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Three different formulas F1, F2, F3 were prepared (Table 1
). The formulas were administered to subjects once each day. The quantities of most ingredients were lower than one recommended daily allowance (RDA) (12
), except for vitamin E which was 150% of the RDA. Those compounds with no established RDA, such as coenzyme Q10 and L-cysteine, were present in the formulas in arbitrarily chosen low quantities. The amount of L-cysteine (10 mg) was chosen because it is approximately the quantity excreted in urine in 24 h (13
); the amount of coenzyme Q10 (10 mg) was chosen because this daily dosage is allowed as a supplement in most European countries.
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Fluid formulations were prepared according to a two-phase technology (2-Phase) in which the powders are kept in a reservoir located in the cap of the vial until the moment of administration. At the moment of ingestion, the plunger cap is pushed down and the powders drop into the 10 mL liquid of the vial; the mixture is then carefully shaken and ingested. The liquid part of the 2-Phase vial contained distilled water in a base of fructose, citrus aroma, mannitol and potassium sorbate (as preservative). With this system, all of the ingredients remain very stable. Dry forms were prepared in capsules (1 capsule for F1 and 2 capsules for F2), mixing together the same amount of ingredients as for the 2-Phase vial. For each formula, F1, F2 and F3 (Table 1)
the 2-Phase formulation was identified as P (P1, P2, P3, respectively), and the dry formulation was identified as D (D1, D2, D3, respectively).
Subjects and study design.
All formulations were tested in 14 apparently healthy volunteers (11 men and 3 women) aged 25–58 y, who gave their written consent, and were ethically informed concerning the goals of the study. Subjects were treated for a period of 7 consecutive days with each formulation, according to a crossover design (Table 2
). Each formulation was administered before breakfast. No concomitant therapies were allowed. Six subjects were smokers and no women took oral contraceptives. Volunteers were selected among a group of 40 people during a run-in period of 2 wk in which they were instructed about the life style to maintain throughout the experiment. The request was to avoid excess food, alcohol, fatigue and sex. A weekly questionnaire was given to the subjects to report details about food intake and general life style (compliance). The selection of 14 candidates was based on compliance and the stability of the D-Roms test. Subjects who showed <10% variation of the D-Roms test in three determinations (at baseline and after 1 and 2 wk) were admitted to the trial (see also statistical analysis section below).
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) in which seven subjects were treated with the sequence P-D (first period of treatment with fluid formulations and second period of treatment with dry formulations) and seven subjects with the sequence D-P (first period of treatment with dry formulations and second period of treatment with fluid formulations). After a washout period of 1 wk, the treatments were reversed. In each study, oxidative stress was tested in serum four times (see D-Roms test section below), one time before and one time after the treatment with the given formulations. The entire study was completed in 23 wk. When the weekly questionnaire indicated that a subject was not compliant, the relative D-Roms test values were not included in the statistical evaluation (see Table 2
). The D-Roms test.
The test (10
,11
) is based on the concept that the amount of organic hydroperoxides present in serum is related to the free radicals from which they are formed. When the serum sample is dissolved in an acidic buffer, the hydroperoxides react with the transition metal ions liberated from the proteins in the acidic medium and are converted to alkoxy and peroxy radicals. These newly formed radicals are able to oxidize an additive (N,N-diethyl-para-phenylendiamine) to the corresponding radical cation. The concentration of this persistent species can be easily determined through spectrophotometric procedures (absorption at 505 nm). The normal values of the test are between 250 and 300 U.CARR. (Carratelli Units), where 1 U.CARR. corresponds to 0.8 mg/L H2O2. Values outside this range are considered indicative of an alteration in the equilibrium between prooxidant and antioxidant capability of subjects. Values >300 U.CARR. indicate a condition of oxidative stress. U.CARR levels were determined in serum samples drawn in the morning, after an overnight fast and before breakfast. Blood samples (2 mL) were drawn from the brachial vein before the administration and 24 h after the last administration of each formulation. Serum was isolated at room temperature, and after centrifugation (1 x g, 1 min) it was kept frozen at -80 C°. All the samples of one sequence of treatment were analyzed together.
Statistical analysis.
The aims of the study were to determine whether a combination of antioxidants at low dosages could be antioxidant or prooxidant and to compare the bioavailability of fluid and dry forms of the same antioxidants. The stability of the oxidative status of the subjects was fundamental to determine any significant modification due to the treatment with antioxidants. The selection of subjects was carried out over a period of 2 wk. Only those candidates who were compliant with the questionnaire were admitted to the D-Roms retesting. Among these subjects, only those who showed variation of U.CARR. values <10% when comparing the maximum and the minimum of three consecutive determinations (baseline, and after 1 and 2 wk of the run-in period) were admitted to the trial. Variations > 10% of the morning values of U.CARR. were never found in any of the candidates who were compliant with the questionnaire. However, only one candidate of three was compliant.
A change of at least 15% from the baseline value was taken as the cut-off value for a significant variation in oxidative stress, provided that the subject was compliant with the recommended behavior during the study periods. Because antioxidants may behave as prooxidants, after a given treatment, the average value of a group of subjects can be composed of some subjects showing a significant increase of the oxidative stress and also some subjects showing a significant decrease. As a consequence, the average value may be inappropriate to describe the differences between two different treatments. For this reason, the parametric statistics (mean, SD and t test for interdependent data) were not considered sufficient to determine significant differences among formulas and formulations. Therefore, the numbers of subjects who reached the cut-off value of ± 15% of the baseline value before the treatment with antioxidants were also compared using the 2 test (according to Fisher or Yates), when the differences between formulas and/or formulations were tested. All statistical analysis were performed using SPSS (Chicago, IL) software.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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reports the maxima (Mx), and minima (Mn) of the six baseline determinations of U.CARR. levels. The Mx/Mn ratio never exceeded 10% in any of the subjects, indicating that the cut-off value of 15% is a reliable index of modifications induced by the antioxidants administered, provided that lifestyle was controlled to avoid excesses of food, fatigue and sex. Seven determinations of U.CARR. were not considered reliable due to fatigue and lack of overnight fasting. The eligible subjects are reported in Table 2
.
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), and for F1 and F3, the two formulations (fluid vs. dry) differed (Table 5
). Both formulations of F1 decreased oxidative stress. However, the number of subjects who reached the cut-off value (see Table 5
) was more consistent after P1 than after D1 (Fisher 2 test, P < 0.05).
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The reduction of U.CARR. levels was more pronounced due to F3 than with the other formulas and both formulations were effective (t test, P < 0.05). The number of subjects reaching the cut-off value was higher after P3 administration than after D3 administration, indicating that the bioavailability of P3 was higher than that of D3 (Fisher test, P < 0.05).
Overall, the 2-Phase formulations (P1 + P2 + P3) were significantly more effective than the dry formulations (D1 + D2 + D3) (Table 4)
. The total cut-off reduction of 15% of U.CARR. levels (Table 5)
was shown in 63% of the subjects when treated with the fluid formulas and in 23% of the subjects when treated with dry formulas (Fisher test, P < 0.01).
The combination in F1 (vitamin A, ß-carotene, vitamin E, selenium, zinc and L-cysteine) was more active than the combination present in F2 (coenzyme Q10, citrus flavonoids, vitamin C, and vitamin B-6) (Fisher test P < 0.05). No synergism was shown in F3 (F1 plus F2)because the reduction of U. CARR. was very close to the addition of the effects of F1 and F2.
In some subjects, a slight increase in U.CARR. levels was detected after the administration of the formulas. In one subject, the value reached the cut-off value after the administration of D1. Furthermore, the remaining minimal increases in oxidative stress were not related to the bioavalability or to the potency of formulas because they were found similarly distributed among the two formulations and in different subjects.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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2 test, P < 0.05), and F3 seems to improve the activity of F1 without any real synergism. However, we cannot exclude the existence of a "roof " effect of antioxidants, particularly in healthy subjects. The activity of antioxidants may be much more evident in subjects with consistent oxidative stress. A formulation very similar to F3, in a fluid form, was tested in patients (11
,14
) to treat oxidative stress, and was found to be active.
We also considered the concept of the "prooxidant activity of antioxidants" (15
). In one subject, we found an increase of U.CARR. levels higher than 15% of the baseline value (subject 8 after D1). This means that the event is at least uncommon, even though we cannot exclude that in some subjects, antioxidants may act as prooxidants.
The D-Roms test, which was evaluated in 
5000 apparently healthy subjects (14
), measures the difference between the antioxidant capability of subjects and the production of free radicals derived from reactive oxygen metabolites. When U.CARR. values in serum are >300 U, the subject can be considered to be under oxidative stress. Some of the subjects enrolled in this study had stable baseline values > 300 U.CARR. These people were apparently healthy, and were admitted to the trial because the U.CARR. values during the run-in period were stable (<10% variation).
Some interfering factors must be considered when the D-Roms test is used because several vascular diseases, the use of oral contraceptives, as well as a state of fatigue after physical exercise (11
,14
), have been shown to increase U.CARR. levels. Oxidative stress is common in several physiologic and pathologic conditions, and a high value in the D-Roms test may indicate both an increase in the production of damaging free radicals and/or a decrease in antioxidant defenses.
In our study, the D-Roms test was confirmed to be a reliable tool for the determination of oxidative stress. It is sensitive to the administration of antioxidants and may represent an indirect measurement of their bioavailabilities.
The activity of antioxidants is much more important than the blood levels because they may behave as prooxidants; consequently, when the blood levels are above the normal values, there may be no curative effects. In many cases, antioxidants are administered to prevent illnesses such as cancer and cardiovascular diseases, which are supposed to be associated with oxidative stress. The tendency is to use very high dosages of a single or few antioxidants without monitoring the activity. Furthermore, in most patients, antioxidants are taken in conjunction with other therapies, which may interfere. Under such conditions, the final result in terms of oxidation is unknown. Moreover, apparently healthy people take antioxidants at high dosages in the hope of preventing illnesses. This is another example of misuse.
In conclusion, a pool of antioxidants combined at low dosages, particularly when formulas are administered as a fluid formulation, reduced oxidative stress as determined by U.CARR. values. If oxidative stress is considered important, either for the prevention or treatment of a disease, the D-Roms test seems to be an appropriate tool with which to determine the type and the dosage of antioxidants.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received July 31, 2001. Revision accepted September 4, 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Herbert, V. (1994) The antioxidant supplement myth. Am. J. Clin. Nutr. 60:157-158.
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