The Journal of Nutrition Vol. 128 No. 12 December 1998, pp. 2383-2390

Serum Antioxidant Capacity Is Increased by Consumption of Strawberries, Spinach, Red Wine or Vitamin C in Elderly Women^1,2

Guohua Cao^*, , Robert M. Russell^*, Neal Lischner^*, and Ronald L. Prior^{*, 3}

^* USDA-ARS, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111 and  Nutritional Science Department, University of Connecticut, Storrs, CT 06269.

	ABSTRACT

Abstract Introduction Methods Results Discussion References

It is often assumed that antioxidant nutrients contribute to the protection afforded by fruits, vegetables, and red wine against diseases of aging. However, the effect of fruit, vegetable and red wine consumption on the overall antioxidant status in human is unclear. In this study we investigated the responses in serum total antioxidant capacity following comsumption of strawberries (240 g), spinach (294 g), red wine (300 ml) or vitamin C (1250 mg) in eight elderly women. Total antioxidant capacity was determined using different methods: oxygen radical absorbance capacity (ORAC) assay, Trolox equivalent antioxidant capacity (TEAC) assay and ferric reducing ability (FRAP) assay. The results showed that the total antioxidant capacity of serum determined as ORAC, TEAC and FRAP, using the area under the curve, increased significantly by 7-25% during the 4-h period following consumption of red wine, strawberries, vitamin C or spinach. The total antioxidant capacity of urine determined as ORAC increased (P < 0.05) by 9.6, 27.5, and 44.9% for strawberries, spinach, and vitamin C, respectively, during the 24-h period following these treatments. The plasma vitamin C level after the strawberry drink, and the serum urate level after the strawberry and spinach treatments, also increased significantly. However, the increased vitamin C and urate levels could not fully account for the increased total antioxidant capacity in serum following the consumption of strawberries, spinach or red wine. We conclude that the consumption of strawberries, spinach or red wine, which are rich in antioxidant phenolic compounds, can increase the serum antioxidant capacity in humans. J. Nutr. 2383-2390, 1998

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Considerable epidemiological evidence suggests an association between consumption of diets rich in fruits and vegetables and a decreased risk of cardiovascular disease, hypertension, and certain forms of cancer (Ames et al 1993, Ascherio et al 1992; Doll 1990, Gillman et al 1995, Kohlmeier et al 1995, Rimm et al 1996, Steinmetz et al 1996, Willet 1994). It is not known for certain which active dietary constituents contribute to these protective effects, but it is often assumed that antioxidant nutrients contribute to this defense. However, results from intervention trials on the protective effect of the supplementation with antioxidants such as -carotene and vitamin E are not conclusive (Hennekens et al 1996, Omenn et al 1996, Rapola et al 1996, Stephens et al 1996). It was recently reported that two months of supplementation with -tocopherol acetate (100 mg, twice daily), ascorbic acid (250 mg, twice daily), -tocopherol acetate (100 mg, twice daily) plus ascorbic acid (250 mg, twice daily) or coenzyme Q10 (30 mg, three times daily) in human subjects did not result in significant changes in the urinary excretion rate of 8-oxo-7,8-dihydro-2'-deoxyguanosine, a repair product of oxidative DNA damage (Priemé et al 1997); whereas intervention with Brussels sprouts (300 g daily) for 3 wk resulted in a significant 28% decrease in the 8-oxo-7,8-dihydro-2'-deoxyguanosine excretion rate (Verhagen et al 1995). Therefore, the beneficial effect of a high intake of fruits and vegetables on the risk of cardiovascular disease and cancer may rely not on the effect of the well characterized antioxidants, such as vitamins E and C and -carotene, but rather on some other antioxidants or nonantioxidant phytochemicals or a concerted action of different compounds present in these foods.

View larger version (27K): [in this window] [in a new window]   Fig 1. Change in serum total antioxidant capacity during the initial 4 h following consumption of a control breakfast meal or a control breakfast meal with strawberries, spinach, red wine, or vitamin C. Data are presented as mean ± SEM (n-8). SB: strawberry, SP: spinach, RW: red wine, VC: vitamin C, Ctrl: control. a) ORACPCA, oxygen radical absorbance capacity analyzed using serum deproteinized with perchloric acid. b) FRAP, ferric reducing ability.

View larger version (21K): [in this window] [in a new window]   Fig 2. Change in plasma vitamin C during the 24 h following consumption of breakfast meal. The changes of vitamin C concentration following consumption of vitamin C was much more dramatic than those folowing other treatments, and thus indicated by the right y axis. Ctrl: control, SP: spinach, SB: strawberry, RW: red wine, VC: vitamin C.

View larger version (17K): [in this window] [in a new window]   Fig 3. Change in serum glucose during the 24-h period following consumption of breakfast meal. Ctrl: control, SP: spinach, SB: strawberry, RW: red wine, VC: vitamin C.

We (Cao et al. 1996, Wang et al. 1996) have found that, in general, more than 80% of the total antioxidant capacity in fruits and vegetables comes from ingredients other than vitamin C, indicating the presence of other potentially important antioxidants in these foods. In those studies, total antioxidant capacity was quantitated by using the oxygen radical absorbance capacity (ORAC⁴) assay (Cao et al. 1993 and 1995). ORAC varies considerably (20-30-fold) from one kind of fruit or vegetable to another. Brussel sprouts are one of the vegetables that show high ORAC activity. Garlic, kale and spinach are particularly high, as are strawberries and plums (Cao et al. 1996, Wang et al. 1996).

Flavonoids and other phenolic compounds appear to be antioxidants that contribute to the high antioxidant capacity observed in certain fruits and vegetables (Guo et al 1997). There are several thousand different flavonoids present in plants, and many of them have antioxidant activities (Cotelle et al. 1996, Hanasaki et al. 1994, Salah et al. 1995, van Acker et al. 1996). The antioxidant capacities, measured as ORAC, of some flavonoids were found to be several times stronger on the basis of molar concentration than vitamins E and C (Cao et al. 1997, Wang et al. 1997). Such phenolic compounds have already been implicated as playing a role in the protection that fruits and vegetables have against chronic diseases (Hertog et al. 1993 and 1995, Keli et al 1996, Knekt et al 1996). However, the extent to which these potentially important antioxidants can be absorbed is not clear, although early evidence indicates that substantial quantities of the flavonoids are absorbed (Hollman et al 1995 and 1996). For example, absorption of quercetin (a common flavonoid) defined as oral intake minus ileostomy excretion and corrected for the degradation within the ileostomy bag was 52 ± 15% for quercetin glucosides from onions (Hollman et al, 1995). It is critical to provide evidence that a diet containing high antioxidant activity from fruits and vegetables can either increase the overall antioxidant capacity or change the relative balances between individual antioxidant components in the human body; otherwise, any antioxidant hypotheses related to the protection from fruits and vegetables against diseases would not be sustained. The objectives of this study were to determine if antioxidant compounds other than vitamin C are absorbed from spinach, strawberries and red wine in sufficient amounts to change the total antioxidant capacity of human serum. Vitamin C was used to demonstrate the effect of a single antioxidant on the total antioxidant capacity.

	SUBJECTS AND METHODS

Abstract Introduction Methods Results Discussion References

Subjects.  Eight healthy female subjects (age: 66.9 ± 0.6; body mass index: 26.1 ± 0.7 kg/m²; mean ± SEM) were recruited to participate in this study (one subject did not finish all experiments). All study participants were in good health as determined by a medical history questionnaire, physical examination and normal results of clinical laboratory tests. All of the subjects fulfilled the following eligibility criteria: 1) no history of cardiovascular, hepatic, gastrointestinal, or renal disease; 2) no alcoholism; 3) no antibiotic or supplemental vitamin and/or mineral use for at least 4 wk before the start of the study and 4) no smoking. The study protocol was approved by the Human Investigation Review Committee of Tufts University and the New England Medical Center, and written informed consent was obtained from each study participant.

Study Design.  Each subject was admitted to the Metabolic Research Unit at the Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University in the evening and fasted overnight. In the morning, an intravenous catheter was inserted into one forearm. A 10 mL blood sample (zero baseline sample) was obtained from fasting subjects, following which they were given a glass of breakfast beverage (coconut drink, Table 1) containing one of the following: 1) strawberries (240 g); 2) ascorbic acid (1250 mg); 3) spinach (raw, 294 g); 4) red wine (300 mL) (the red wine was lyophilized to remove alcohol); or 5) control (coconut drink only). The strawberry, ascorbic acid, spinach and red wine treatments were all formulated to provide an antioxidant capacity equal to 3.7 mmol Trolox (a water soluble vitamin E analogue) equivalent in the ORAC assay. Blood samples (10 mL) were collected again at 0.25, 0.5, 1, 2 and 4 h after the consumption of the beverage. Following the blood sampling at 4 h, a lunch meal was given (Table 1). Additional blood samples were obtained at 7, 9 and 11 h following the breakfast drink. A snack was given after the 7 h blood sampling and a dinner was given immediately after the 11 h blood sampling (Table 1) . The breakfast, lunch and dinner were designed to be low in foods containing antioxidant activity (no substantial amounts of vitamin C, -tocopherol, carotenoids, and flavonoids), but provide the Recommended Dietary Allowance for protein and energy (Table 1). Each subject received each of the five treatments (drinks) on a separate day 2 wk apart during a period of 10 wk. Treatments were assigned to each subject in a random sequence. Four subjects completed a treatment each time with four of the five treatments represented at each time.

After the breakfast beverage, over a 24-h test period, urine was collected into two separate pooled collections. Collection 1 was from baseline to 4 h; Collection 2 was from 4 to 24 h. Pooled specimens were collected into urine jugs containing HCl as an additive. Volumes were recorded and four separate aliquots were stored at -80°C.

Antioxidant Capacity Analyses.  Three different methods were used for the antioxidant capacity analyses: 1) ORAC assay, 2) Trolox equivalent antioxidant capacity (TEAC) assay and 3) ferric reducing ability (FRAP) assay.

The automated ORAC assay was carried out on a COBAS FARA II spectrofluorometric analyzer (Roche Diagnostic System Inc., Branchburg, NJ) with fluorescent filters (Ex: 540 nm; Em: 565 nm) as previously described (Cao et al, 1995). Briefly, in the final assay mixture (0.4 mL total volume), R-phycoerythrin (16.7 nmol/L) (Sigma, St. Louis, MO) was used as a target of free radical attack, with 2,2'-azobis(2-amidinopropane) dihydrochloride (4 mmol/L) (Wako Chemicals USA Inc., Richmond, VA) as a peroxyl radical generator. Trolox (Aldrich, Milwaukee, WI) was used as a control standard. Final results were calculated using the differences of areas under the R-phycoerythrin decay curves between the blank and a sample. The ORAC assay has been used by different laboratories and provided important information regarding the antioxidant capacity of various biological samples from pure compounds such as melatonin, dopamine and various flavonoids, to complex matrices such as tea, fruits, vegetables, herbs and animal tissues (Cao and Prior 1998). Both serum and serum nonprotein fractions extracted with perchloric acid (PCA) and acetone were used in the ORAC assay. For preparation of the serum nonprotein fraction, serum was diluted with 0.5 mol/L PCA (1:1, v/v) or pure acetone (1:8, v/v). The samples were then centrifuged at 100,000 x g for 10 min at 4°C, and the supernatants were removed as the serum nonprotein fractions and appropriately diluted for the ORAC assay.

TEAC was measured in serum using the method of Miller et al. (1993) with commercially available kits (Total Antioxidant status; Lot 21440, Randox Laboratories, Lakewood, CA). The TEAC assay is based on the inhibition by antioxidants of the absorbance of the radical cation of 2,2'-azinobis(3-ethylbenzothiazoline 6-sulfonate).

FRAP was determined in serum by the method of Benzie and Strain (1996), which measures the ferric to ferrous iron reduction in the presence of antioxidants. The 2,4,6-Tripyridyl-s-triazine used in this assay was from Sigma (St. Louis, MO).

The final results of ORAC, TEAC and FRAP assays were all expressed using µmol Trolox equivalents per L. The relative activity of Trolox in the FRAP assay was 2.0 (Benzie and Strain 1996).

Determination of Glucose, Protein, Urate, Bilirubin and Vitamin C.  Glucose, protein, urate and bilirubin were measured in serum using a COBAS MIRA spectrophotometric analyzer with reagent kits purchased from Roche Diagnostic Systems, Inc. (Branchburg, NJ). Vitamin C was determined by HPLC analysis of plasma deproteinized immediately following separation with 0.5 mol/L PCA (Behrens and Madere 1987).

Statistical Analyses.  Serum responses to dietary treatment were evaluated by calculating the area under the curve following the consumption of the treatment beverage (Matthews et al. 1990). The effects of the treatments were determined by ANOVA with repeated measures (SYSTAT for Windows, version 5, Systat, Inc., Evanston, IL). The differences between two groups were also analysed by paired t-test.

	RESULTS

Abstract Introduction Methods Results Discussion References

Serum antioxidant capacity, which was measured as ORAC_TOTAL, ORAC_PCA, ORAC_ACETONE, FRAP and TEAC, plasma vitamin C, and serum urate, bilirubin and protein concentrations at time 0 following a 12-h fast are presented in Table 2. No significant differences were observed among different treatments at the zero sampling time. A detailed comparison of the relative merits of the different methods for determining total antioxidant capacity is presented elsewhere (Cao and Prior 1998). ORAC_PCA was found to be the preferred method for evaluating changes in water soluble antioxidants.

The area-under-curve (AUC) technique was used to assess the serum response of antioxidants following consumption of the 5 treatment drinks. The AUC for the different measures of antioxidant capacity and specific antioxidants is presented in Table 3. Data are presented for the time period of 0-4 h. The AUC for the time period of 0-11 h was also calculated but not presented in Table 3. Generally, the responses were similar for the two time periods, although for some responses the changes were diminished for the 0-11 h period, because all treatment groups received the same meals after the 4-h sampling time point. AUC for ORAC_ACETONE (data not shown) and bilirubin did not change with any of the meal treatments as determined by ANOVA using repeated measures. Increases in the AUC for the 0-4 h period for ORAC_PCA and FRAP were observed for all treatments (compared to the control, P < 0.05). The largest increase in these two parameters of ~25% was observed following the spinach meal. The increases in the AUC for ORAC_PCA and FRAP relative to the control drink were 13 and 10%, respectively, following the strawberry treatment. The relative increases were 11 and 7%, respectively, following the red wine treatment, and were 21 and 20%, respectively, following the vitamin C treatment. The increase in the AUC for the 0-4 h period for ORAC_TOTAL (data not shown) and TEAC was only seen for spinach treatment (P < 0.05).

The percentage of change in ORAC_PCA concentration following consumption of the different treatment beverages is presented in Figure 1a. ORAC_PCA concentration changed very little in the control group during the first 4 h. Other treatments reached a maximum at 1 or 2 h following consumption of the drink. Levels returned toward baseline at 4 h after the beverage. ORAC_PCA concentration increased by 9% for spinach at 2 h, 14% for strawberry at 1 and 2 h, 16.9% for red wine at 1 h, and 17.3% for vitamin C at 2 h.

Changes in serum FRAP concentration are presented in Figure 1b. Spinach consumption produced a 12.2% increase in FRAP at 1 h, which declined toward baseline by 4 h. Maximal responses due to strawberry and red wine were 4.8 and 5.5%, respectively. Vitamin C treatment produced an increase in FRAP of 12.1% at 4 h, but a maximum of 18% was not reached until 7 h after time zero. However, plasma vitamin C concentrations reached a maximum of 118 µmol/L by 4 h following the vitamin C treatment (Figure 2), which was almost a two-fold increase in plasma vitamin C relative to the baseline concentration of 41.6 µmol/L (Table 2).

Besides the vitamin C drink, the strawberry drink was the only treatment that produced a significant response in serum vitamin C. For strawberry treatment, the increase in the vitamin C AUC during the 4 h was 25%, and the vitamin C concentration reached a maximum of 58 µmol/L (40% increase) at 4 h. Serum urate AUC increased by 30% during the 4 h after the spinach drink, and 14% after the drink containing strawberries.

The percentage contribution of vitamin C, urate and other antioxidants to the serum ORAC_PCA is presented in Table 4. Vitamin C accounted for about 7% of the activity measured by ORAC_PCA. The percentage contribution from vitamin C approximately doubled with the vitamin C treatment (P < 0.001). Urate accounted for approximately 38% of the antioxidant activity measured by ORAC_PCA and did not change with dietary treatment. The percentage contribution by other antioxidants was ~52% for ORAC_PCA and was not significantly altered by dietary treatment. These calculations also included contributions to the antioxidant capacity measures from protein, bilirubin, vitamin E and carotenoids. These contributions were not altered by dietary treatment (P > 0.05).

A measure of total antioxidants excreted in the urine as reflected in the ORAC measure is presented in Table 5. Vitamin C treatment produced a 44.9% increase in urine ORAC compared to the control, whereas the ORAC increase with spinach was 27.5%. Strawberry and red wine produced much smaller urinary ORAC increases of 9.6 and 8.2%, respectively (Table 5). Because 3,700 µmol Trolox equivalents were given with each of the diet treatments, the percentage of this dose that could contribute to the increased ORAC activity in the urine collected during the 24 h period was 69, 42.6, 14.8 and 12.7% for vitamin C, spinach, strawberry and red wine treatments, respectively.

Responses in serum glucose concentrations are presented in Figure 3. Dietary treatments did not alter responses in glucose. A 20-40% increase in serum glucose within 1 h of the breakfast drink was observed, which declined to fasting levels by 4 h. Another peak was observed at 9 h, which followed the afternoon snack.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The results of this study demonstrated that the consumption of strawberries, spinach, red wine or vitamin C could increase the antioxidant capacity of serum in elderly women. Increases in ORAC_PCA and FRAP were observed for all these treatments compared to the control. Because serum proteins were removed in the ORAC_PCA assays and protein produced negligible activity in the FRAP assay, the significant increase of serum antioxidant capacity following these drinks was mainly due to the nonprotein antioxidants in the serum. The increase of total antioxidant capacity of serum after consumption of strawberries, spinach, or vitamin C was further supported by the significant increase of urine ORAC following these treatments.

The increased antioxidant capacity in serum (ORAC_PCA and FRAP) and urine (ORAC) following the consumption of strawberries or spinach indicated a direct absorption and/or an enhanced production of antioxidants. The absorbed antioxidants included vitamin C, and the produced antioxidants may include uric acid from purine metabolism; plasma vitamin C level was significantly increased after the strawberry drink, and serum urate levels were increased by both the strawberry and spinach treatments. Based on the data from USDA handbooks (USDA 1986), we calculated the strawberry drink contained about 120 mg vitamin C. However, the increased blood vitamin C and urate could be a result of the sparing effect on them by the other antioxidants absorbed from strawberries or spinach. Also, other antioxidants, rather than vitamin C and urate, contributed to half of the increased serum antioxidant capacity. The increased serum ORAC_PCA AUC and FRAP AUC (1-4 h) after the strawberry drink were 275 and 315 µmol · h/L Trolox equivalents, respectively; whereas the increased vitamin C AUC following the strawberry drink during the same time period was only 44 µmol · h/L, or 23-44 µmol · h/L Trolox equivalents in the ORAC_PCA assay and 44 µmol · h/L Trolox equivalents in the FRAP assay. The antioxidant capacity (Trolox equivalents) of vitamin C was 0.52-1.0 in the ORAC and FRAP assays. Therefore, the contribution of vitamin C to the increased serum ORAC_PCA and FRAP after the strawberry drink was only 8-14%. Similarly, it was calculated that the contribution of urate to the increased serum ORAC_PCA and FRAP following the strawberry treatment was 39-42%, and to the increased serum ORAC_PCA and FRAP following the spinach treatment (1-4 h) was 36-44%.

Significant increases in serum antioxidant capacity were observed in the strawberry and red wine treatments even though total carotenoids decreased (Paiva et al. 1998). Because of the low plasma carotenoid concentration and the relatively lower antioxidant activity of carotenoids as assessed by the ORAC assay (Cao et al. 1993), carotenoids do not make a major contribution to the ORAC in serum.

The other antioxidants responsible for the increased serum antioxidant capacity following the consumption of strawberries or spinach are likely phenolic compounds including flavonoids, although it was not clear which specific phenolic compounds were absorbed. Some phenolic compounds, such as ellagic acid, catechin, rutin, naringin and anthocyanins have been identified in strawberries (Daniel et al. 1989, Guo et al. 1997, Wang et al 1997). It was suggested that phenolic compounds were responsible for the high antioxidant capacity found in strawberries and spinach and also for the differences in the antioxidant capacities between fruits and vegetables (Guo et al. 1997).

The absorption of some individual flavonoids in humans was reported by several laboratories. Xu et al. (1994) reported that in adult women the average plasma concentration of total isoflavones reached 4.4 µmol/L at 6.5 h after a dose of 2 mg soybean isoflavones/kg body weight. Two studies by Hollman et al. (1995 and 1996) indicate that quercetin glucosides from fried onions were absorbed in humans and that quercetin absorption was enhanced by conjugation with glucose. This was supported by a recent study that suggested quercetin glucosides are capable of interacting with the sodium dependent glucose transport receptors in the mucosal epithelium (Gee et al. 1998). The aglycones naringenin and hesperitin were detected by positive chemical ionization-collisionally activated dissociation tandem mass spectrometry (PCI-CAD MS/MS) in human plasma and/or urine after oral administration of naringin and hesperidin (Ameer et al. 1996). Naringenin was also detected by HPLC/UV either on or in saline-washed erythrocytes 2 and 4 h after the oral administration of naringin (Ameer et al. 1996).

Red wine contains phenolic compounds, such as quercetin, rutin, catechin and epicatechin; the concentration is about 1 g/L, 20-fold the level found in the average white wine (Singleton 1982). Red wine also has a relatively high ORAC activity of 12.3 mmol/L, which is 5.3-fold the activity measured in white wine (Cao et al. 1995). Red wine, or the phenolic compounds in the red wine, were shown to reduce the susceptibility of human plasma and low-density lipoprotein to lipid peroxidation both in vitro (Frankel et al. 1993) and in vivo (Fuhrman et al. 1995), although the in vivo study has not yet been confirmed (Rijke et al. 1996). The results of the present study support the antioxidant hypothesis regarding the role of red wine in the `French Paradox' (apparent compatibility of a high fat diet with a low incidence of coronary atherosclerosis); the red wine drink significantly increased the overall antioxidant capacity as reflected in the serum ORAC_PCA and FRAP.

A single antioxidant, vitamin C, also affected the overall antioxidant status. It appears that strawberries and spinach are as effective in enhancing the overall antioxidant status in serum as a large dose of vitamin C.

In conclusion, we found that the overall antioxidant capacity in serum or urine of elderly women was significantly increased following the consumption of strawberries, spinach, red wine or vitamin C. The increased serum antioxidant capacity after the treatments of strawberries, spinach, and red wine indicated the possible absorption of phenolic compounds in these diets.

	FOOTNOTES

Manuscript received 13 May 1998. Initial reviews completed 23 July 1998. Revision accepted 20 August 1998.

	ACKNOWLEDGMENTS

We thank John McEwen and Christine O`Brien for their technical assistance; Gayle Perrone and the staff of the Nutrition Evaluation Laboratory for their expertise in performing many of the laboratory analyses; Helen Rasmussen for her assistance in the formulation of the diets; and the nursing staff of the Metabolic Research Unit at the HNRC for their assistance in the care of the subjects.

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