A Mixture of Histidine-Dipeptides, Vitamin C, and Ferulic Acid Reduces Comet Assay Scores in Normal Middle-Aged Men.

Abstract

Specific antioxidants have previously been found to target particular reactive oxygen species. For example, histidine-dipeptides neutralize hypochlorite, while ascorbic acid targets peroxynitrite and ferulic acid scavenges hydroxyl radicals. In this study, we investigated the effects of a mixture of these antioxidants on oxidative stress in middle-aged men. Seventeen male volunteers ingested an antioxidant mixture containing histidine-dipeptides, vitamin C, and ferulic acid for 8 weeks. DNA damage in peripheral leukocytes was measured at 4-week intervals using comet assays. For comparison, oxidative status in twelve normal volunteers who did not ingest the test drink (control group) were examined. DNA damage was remarkably reduced at the 8- and 12- week follow-ups. Plasma LDL-cholesterol levels were also reduced at 8 weeks. In contrast, notable changes in DNA damage were not observed in the control group. In addition, the antioxidant mixture administered in this trial did not produce toxic effects on the liver, kidney, or pancreatic function. Thus, such antioxidant combinations may contribute to the maintenance of health in middle-aged men.

Introduction

Aging and chronic disease involve cellular apoptosis, which is promoted by reactive oxygen species (ROS) (Harman, 1972; Richter et al., 1995; Johnson et al., 1996). Since ROS are constitutively generated from energy metabolism and immune processes (Marnett, 2003), antioxidants and enzymes play important roles in preventing aging and in the progression of chronic diseases such as hypertension, diabetes, other cardiovascular diseases, and cancer. Endogenous antioxidants, ubiquinones such as Coenzyme Q10 (CoQ10), and antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase, and catalase, are all involved in mitochondrial and cytoplasmic defense systems and act to neutralize a variety of ROS (Chaudière et al., 1999; Powers et al., 1999; Curtin et al., 2002). Therefore, ingestion of sufficient quantities of naturally occurring antioxidants from the diet and supplements is also considered to be very important for suppressing the aging progress and ameliorating the risk of ROS-mediated chronic diseases. Accordingly, standard methods for measuring antioxidant activity in foods have been devised using artificial ROS, leading to informed recommendations for daily antioxidant intake (Aruoma et al., 2003; Prior et al., 2005).

However, only a few studies have directly indicated that antioxidants reduce the risk of chronic and degenerative diseases. For instance, although vitamin C and other hydrophobic polyphenolic compounds possess strong antioxidant and anti-genotoxic activities against specific ROS in vitro (Noroozi et al., 1998; Kontek et al., 2010), human trials have often failed to demonstrate preventive effects on DNA oxidation (Retana-Ugalde et al., 2008). Furthermore, methods for evaluating antioxidant activity in naturally occurring substances have not yet been established.

Ironically, studies of antioxidants administrated alone and in combination have failed to demonstrate the prevention of chronic diseases, and have exhibited subsequent increases in the incidence of lung cancer and cardiovascular diseases in smokers (Gaziano et al., 2009; Heinonen et al., 1994; Hennekens et al., 1996). Nonetheless, the ingestion of naturally occurring antioxidants may contribute to the maintenance of oxidative balance in vivo, suggesting that further consideration of antioxidant ingestion is necessary to reduce the risk of ROS-mediated chronic diseases.

In human tissues, at least 5 types of ROS are constitutively generated (Marnett et al., 2003); however, it is not known which antioxidants are the most effective for each ROS. Hydrogen peroxide (H₂O₂), a typical by-product of mitochondrial energy metabolism, is produced from superoxide-anions (O₂⁻) by SOD. Although H₂O₂ is rapidly degraded to oxygen gas and water by catalase, O₂⁻ and H₂O₂ can lead to the formation of hydroxyl (HO·), hypochlorite (ClO·), and peroxynitrite (ONOO·) radicals. In the presence of Fe or Cu, hydroxyl radicals are generated from H₂O₂ (Fenton's reaction) (Halliwell et al., 1987), both hypochlorite and peroxynitrite radicals are produced by leukocytes, neutrophils, monoytes and macrophages (Winterbourn et al., 1985; Hazen et al., 1999). It is widely accepted that hydroxyl, peroxynitrite, and hypochlorite radicals are the most pathologically relevant ROS and should therefore be the major targets of antioxidants that prevent DNA oxidation and associated cell death.

The histidine-dipeptides carnosine and anserine are distributed in active animal tissues and exhibit antioxidant activity (Boldyrev et al., 1988; Kohen et al., 1988; Hipkiss et al., 2000). However, they are not considered strong antioxidants because physicochemical measurements generally indicate relatively low activities (Aruoma et al., 1989; Hirayama et al., 1997). Consistent with the findings of Hipkiss et al. (1998), we previously observed that histidinedipeptides from chicken meat specifically inhibited protein degradation by the ClO· radical and that ascorbic acid strongly scavenged the ONOO· radical in vitro (Yanai et al., 2008). Furthermore, the polyphenolic antioxidant ferulic acid, which is slightly soluble in water, exhibits strong antioxidant activity against the HO· radical but weak activity against both the ClO· and ONOO· radicals. Based on these observations, it was hypothesized that an antioxidant combination including histidine-dipeptides, ascorbic acid, and a polyphenolic compound such as ferulic acid may effectively prevent oxidative stress caused by endogenous ROS in humans.

To test this hypothesis, we performed an open clinical trial of an antioxidant combination containing carnosine and anserine, ascorbic acid, and ferulic acid to determine effects on peripheral lymphocyte DNA damage in middle-aged men.

Materials and Methods

Study design. The primary endpoint of this study was to confirm whether the antioxidant combination resulted in an in vivo reduction of oxidative stress as reflected by the comet assay scores, which indicates ROS-mediated peripheral lymphocyte DNA damage. In addition, we surveyed how the reduction of oxidative stress influenced other clinical parameters, including glucose and fat metabolism. Referring to a previous study (Hróbjartsson and Gøtzsche, 2001), a placebo-controlled system was not used in this study because the vehicle (mango juice) had no physiological effect in a preliminary double blind placebo-controlled crossover trial (n = 10 per each group), and ascorbic acid and carotenoids contents were < 3 mg and < 0.1 mg, respectively. In addition, several volunteers refused the consumption of mango juice alone for 8 weeks.

To determine the effects of the antioxidant combination on peripheral lymphocyte DNA damage, comet assay scores and blood biochemical parameters were measured before and after ingestion of the antioxidant mixture in the same subjects. Except for the comet assay and blood tests, which were performed on blindly selected blood samples, this study was conducted as an open trial.

All volunteers were allowed to smoke and drink alcoholic and/or caffeinated beverages such as green tea, coffee, and fruit juice, but were not allowed to consume additional antioxidant supplements containing vitamins A, C, or E, CoQ10, or histidine-dipeptides. Subjects in the treatment group took a daily 50 mL dose of the test drink at appropriate times for 56 days (8 weeks). The test drink contained 400 mg of anserine-carnosine mixture from chicken extract (purity > 90%), 300 mg of ascorbic acid (V.C, reagent grade), 20 mg of ferulic acid (Tsuno Rice Fine Chemicals, Wakayama, Japan; purity > 95%), and 30 mL of mango juice. This formulation was prepared according to previous studies (Levine et al., 1996; Balasubashini et al., 2003), and its safety was confirmed in animal experiments. Subjects in the control group did not receive the antioxidant mixture and were monitored for the same 12-week period while continuing their usual lifestyle with no changes in diet, smoking, and drinking or exercise habits. Fasting blood samples of all volunteers were taken at the Health Care Center of Tokyo Metal Industry Association (Chiyoda-ku, Tokyo, Japan) every 4 weeks. Concomitantly, subjects submitted a monthly record of their drinking, smoking, and supplement intakes.

Volunteers. After obtaining approval from the Ethics Committee of Tokai Bussan Co., Ltd. and written informed consent from healthy volunteers, a total of 29 employees of Tokai Bussan Co., Ltd. were recruited in accordance with the Declaration of Helsinki. Healthy volunteers had not received any medical treatment and did not exhibit any abnormalities in clinical parameters. Exclusion criteria included systolic blood pressure over 150 mmHg, total serum cholesterol over 260 mg/dL, fasting blood glucose over 120 mg/dL, and aspartate amino transferase (AST), alanine aminotransferase (ALT), and γ-glutamyl transpeptidase (γ-GTP) levels over 10% of normal upper limits.

Prior to volunteer allocation, the number of volunteers necessary to enable measurements of significant reductions in DNA oxidation was calculated, and the necessary sample size was determined to be 11 or more subjects, using treatment effects producing an expected 30% reduction. After their selection and approval, volunteers were assigned to either the ingestion group (n = 17) or the control group (non-ingestion group, n = 12) for the monitoring of the influence of lifestyle and seasonal changes. The average age in the ingestion group was 50.6 years and 49.7 years in the non-ingestion group (Table 1).

Table 1. Characteristics of volunteers

	Ingestion group	Non-ingestion group	ANOVA
Number	17	12
Sex	Male	Male
Age (years)	50.6 ± 8.1	49.7 ± 9.8	N.S.
Height (cm)	168.9 ± 6.3	170.6 ± 7.1	N.S.
Body weight (kg)	65.9 ± 8.1	68.4 ± 7.7	N.S.
Body mass index (BMI)	23.1 ± 2.4	23.5 ± 1.7	N.S.
Non-smokers	7	5	N.S.
Smokers	10	7	N.S.
Drinkers	11	6	N.S.

Data are presented as mean ± S.D. N.S.: not significant. Significant differences between ingestion and non-ingestion groups were identified using analysis of variance (ANOVA).

Comet assay. Blood samples were blindly selected and evaluated at the National Food Research Institute, Tsukuba, Japan according to the procedures of Collins, et al. (Collins et al., 1995; Collins et al., 2008). Prior to evaluation, 5 mL of blood was added to the same volume of Dulbecco's buffered saline (Mediatech, USA) and layered onto 20 mL of Ficoll-Paque Plus solution (GE Health Care Japan, Tokyo, Japan) in a 50-mL centrifuge tube (Becton Dickinson Lab Wear, USA). Blood samples were then centrifuged at 400 × g for 30 min at room temperature and the lymphocyte fractions were collected. After washing twice with buffered saline, the lymphocyte fractions were re-suspended in RPMI-1640 medium containing 10% dimethyl sulfoxide and 10% fetal bovine serum (GIBCO, Invitrogen, USA) at a cell concentration of 10⁶/mL and were stored at −80°C in 0.5 mL aliquots until further use. After thawing at 37°C, lymphocyte cells were washed twice with buffered saline and centrifuged at 200 × g for 5 min. The average viability of lymphocytes subjected to the comet assay exceeded 90%. Duplicate smears from > 200 lymphocytes were measured using fluorescent microscopy (Nikon E600, Tokyo, Japan) and were given comet scores of 0 – 4 according to Collins et al. (2008). Comet scores were expressed as total counts per 100 lymphocytes.

Biochemical and hematological blood examinations. Height, body weight, body mass index (BMI), and blood pressure were measured at the Health Care Center. Biochemical and hematological assessment of blood samples were performed at the Mitsubishi BCL Laboratory, Tokyo, Japan. Biochemical parameters included total cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL) and very low density lipoprotein (VLDL) cholesterols, triglycerides, oxidized LDL (Oxi-LDL), glyco-albumin, lactate dehydrogenase (LDH), amylase, AST, ALT, γ-GTP, alkaline phosphatase (ALP), blood urea nitrogen (BUN), uric acid, creatinine, hemoglobin A1C (HbA1C), c-reactive protein (CRP), and fasting glucose levels. Red blood cells, white blood cells, platelets counts, hemoglobin content, and hematocrit were determined in the hematological examinations.

Statistical Analysis. Comet assay scores and other clinical parameters were compared between the initial examination (before ingestion, 0 weeks) and each follow-up time point (4-week intervals) using the paired Student's t-test (two-tailed). Differences between treatment groups identified using analysis of variance (ANOVA) and were considered significant when p < 0.05.

Results

Demographic data from volunteers is presented in Table 1. Mean age, body weight, height, BMI, and smoking habits were the same in test and control groups. No differences were observed in blood pressure or liver and kidney functions between the two groups. The volunteers did not consume antioxidant supplements or change their smoking or drinking habits during the trial. Subjects in the ingestion group received a test drink at breakfast (n = 11), lunch (n = 2) or dinner (n = 4), and consumed the full quantity within 60 days.

Initial comet assay scores were 15.1 in the test group and 17.4 in the control group and were slightly higher at 4 weeks. After 8 weeks, comet scores were significantly decreased in the ingestion group (p = 0.045) and were slightly higher in the control group. At 12 weeks (4 weeks after discontinuing the test drink), comet scores were further decreased from baseline scores in the treatment group (p = 0.0018) but remained higher in the control group (Fig. 1). These results, using paired Student's t-tests, were in good agreement with the results obtained using the ANOVA test. Baseline comet scores were higher among smokers (n = 17, 17.5 ± 7.6) than non-smokers (n = 12, 13.6 ± 7.1) in both groups, but the differences were not significant (p = 0.1669).

Fig. 1.

Changes in comet assay scores in ingestion (A) and non-ingestion (B) groups. Closed circles indicate individual comet assay scores. Open circles represent mean values. The paired Student's t-test was used for comparisons between 0 weeks and each follow-up time point. *: p = 0.045; **: p = 0.0018.

Although initial blood concentrations of total and LDL cholesterol levels were higher in the ingestion group than among control subjects, these decreased after consumption of the test drink (Tables 2 and 3). At 8 weeks, LDL cholesterol levels were significantly decreased (p = 0.029), whereas total cholesterol levels also decreased, but this was not significant (p = 0.072). In contrast, in the control group, total and LDL cholesterol levels were lower than in the ingestion group at base line, but this did not change during the study. Moreover, fasting glucose concentrations did not change significantly in either group.

Table 2. Changes in lipid and fasting glucose concentrations in blood from subjects of the ingestion group

Parameters	0 weeks	4 weeks	8 weeks	12 weeks
	(mg/dL)
Total Cholesterol	209.8 ± 30.1	205.6 ± 29.4	201.2 ± 28.0a	202.7 ± 30.5
HDL Cholesterol	58.8 ± 14.3	58.4 ± 15.0	57.9 ± 13.7	56.8 ± 12.2
Triglycerides	106.1 ± 57.9	100.9 ± 54.9	111.4 ± 6 2.4	114.8 ± 74.4
LDL Cholesterol	133.2 ± 29.1	127.0 ± 26.2	121.0 ± 32.1b	123.2 ± 32.1
Fasting Glucose	96.1 ± 11.6	95.8 ± 11.8	93.1 ± 10.6	94.6 ± 9.5
LDL/HDL	2.38 ± 0.80	2.31 ± 0.73	2.27 ± 0.87	2.27 ± 0.78
Body weight (kg)	65.9 ± 8.1	67.0 ± 8.3	67.3 ± 8.3	66.8 ± 8.3
BMI	23.1 ± 2.4	23.2 ± 2.7	23.3 ± 2.8	23.2 ± 2.7

Data are presented as mean ± S.D. The paired Student's t-test was used for comparisons between 0 weeks and each follow-up time point. ^a: p = 0.072, ^b: p = 0.029

Table 3. Changes in lipid and fasting glucose concentrations in blood from subjects of the non-ingestion group

Parameters	0 weeks	4 weeks	8 weeks	12 weeks
	(mg/dL)
Total Cholesterol	184.3 ± 25.6a	182.2 ± 28.2a	186.3 ± 29.2	191.4 ± 27.1
HDL Cholesterol	58.6 ± 15.1	60.3 ± 15.0	61.1 ± 19.3	59.8 ± 14.5
Triglycerides	123.8 ± 98.3	105.6 ± 59.5	116.3 ± 96.3	117.9 ± 72.0
LDL Cholesterol	100.9 ± 30.0a	99.6 ± 28.7a	99.7 ± 38.0	106.9 ± 26.8
Fasting Glucose	93.8 ± 8.0	93.8 ± 8.2	91.1 ± 7.3	95.6 ± 9.0
LDL/HDL	1.85 ± 0.72	1.84 ± 0.93	1.90 ± 0.80	1.89 ± 0.73
Body weight (kg)	68.4 ± 7.7	69.8 ± 7.8	69.5 ± 7.6	69.3 ± 7.2
BMI	23.5 ± 1.7	23.5 ± 1.9	23.4 ± 1.9	23.4 ± 1.8

Data are presented as mean ± S.D. Statistical analysis between ingestion and non-ingestion groups was performed using analysis of valiance (ANOVA). ^a: p < 0.05.

The test drink did not affect kidney or liver function, and had no side effects during the trial (Table 4). At base line, HbA1C and glyco-albumin concentrations were 4.9 ± 0.3% and 14.2 ± 2.0%, respectively. These indicators of diabetes did not change with ingestion of the test drink. Moreover, Oxi-LDL cholesterol levels, which indicate oxidative stress in vivo, were not influenced by the ingestion of antioxidants. In addition, neither CRP levels, which indicate inflammatory oxidative stress, nor hematological parameters changed throughout the study.

Table 4. Blood biochemical parameters

Parameters	Ingestion group		Non-ingestion group
	0 weeks	12 weeks	0 weeks	12 weeks
AST (IU/L)	20.8 ± 5.1	22.2 ± 5.6	22.1 ± 6.9	20.8 ± 7.7
ALT (IU/L)	20.2 ± 6.6	22.5 ± 8.5	24.9 ± 11.0	21.9 ± 5.8
ALP (IU/L)	191.4 ± 58.3	202.7 ± 72.7	190.6 ± 46.8	193.7 ± 51.2
γ-GTP (IU/L)	39.5 ± 31.8	36.7 ± 26.6	51.2 ± 33.7	48.3 ± 31.9
Amylase (IU/L)	94.6 ± 33.7	82.7 ± 21.5	82.8 ± 33.1	64.5 ± 18.4
BUN (mg/dL)	13.4 ± 2.5	13.9 ± 3.2	12.8 ± 3.1	12.8 ± 2.6
Creatinine (mg/dL)	0.8 ± 0.1	0.8 ± 0.1	0.8 ± 0.2	0.8 ± 0.2
Uric acid (mg/dL)	5.9 ± 0.8	6.5 ± 0.8	5.7 ± 1.5	5.7 ± 1.7
CRP (mg/dL)	0.09 ± 0.10	0.16 ± 0.32	0.11 ± 0.08	0.19 ± 0.35
HbA1C (%)	4.9 ± 0.3	5.0 ± 0.2	4.7 ± 0.5	5.0 ± 0.3
Glyco-Alb (%)	14.2 ± 2.0	13.9 ± 1.0	14.2 ± 1.1	13.8 ± 0.9
Oxi-LDL (U/mL)	6.6 ± 2.1	7.6 ± 2.3	6.7 ± 2.4	7.3 ± 1.9

Data are presented as mean ± S.D.

Discussion

Comet assay scores in peripheral lymphocytes are considered an in vivo index of DNA damaging oxidative stress. However, only few studies show reduced comet assay scores with single or combined antioxidant treatments. In this study, comet assay scores were significantly reduced after 8 weeks of ingesting a test drink containing 3 different antioxidants. Furthermore, this reduction was sustained for 4 weeks after discontinuation (12 weeks follow-up). In contrast, comet scores tended to increase in the control group over the course of the trial (Fig. 1).

Lifestyle and antioxidant intake monitoring did not reveal any violations of the protocol, and no marked changes in drinking or smoking habits were found in either group during the trial period. Although the delay in reduction of comet assay scores in the ingestion group is unexplained, we speculated that because the average life span of circulating lymphocytes is approximately several weeks to a month, this reduction may be a result of the replacement time of circulating lymphocytes.

In agreement with a previous study (Yanai et al., 2008), we recently showed specificity of antioxidant activities against various types of DNA-degrading ROS. Specifically, histidine-dipeptides suppressed 4 kinds of ROS, including H₂O₂, in vitro (Takahashi et al., 2011). However, no reports indicate that histidine-dipeptides can reduce DNA damage, as evaluated using comet assay scores in vivo. Some reports indicate failure of single antioxidants to reduce comet assay scores in human trials (Møller et al., 2002). Hence, the present data indicate that DNA oxidation is mediated by complex networks of multiple ROS and that single naturally occurring antioxidants or combinations of similar antioxidants cannot completely suppress all ROS. Thus, although this study was conducted as an open trial, the results in Fig. 1 strongly suggest that combined treatment with diverse antioxidant types remarkably reduces DNA oxidation in healthy humans. In addition, a placebo-controlled double-blind trial of this combination recently performed in middle-aged females also resulted in a significant reduction in comet assay scores.

Furthermore, the present trial indicated an additional advantageous effect on lipid metabolism. Due to higher concentrations of total and LDL cholesterol levels in the ingestion group at baseline (0 weeks), these were reduced by ingesting the antioxidant combination (Table 2). When the ingestion group was divided into two groups (> 200 mg/dL total cholesterol, and > 120 mg/dL of LDL cholesterol, Table 5), differential analysis showed statistically significant reductions in both levels at 8 weeks. On the other hand, groups with < 200 mg/dL of total and < 120 mg/dL of LDL cholesterol levels were unchanged, as well as the non-ingestion group, throughout the trial. These results indicated that the antioxidant mixture reduces total and LDL cholesterol levels in subjects with elevated levels, but produces no effect on subjects with normal or lower levels.

Table 5. Differential analysis of total and LDL cholesterol levels in the ingestion group

Weeks	Total cholesterol		LDL cholesterol
	>200 mg/dL (n = 12)	<200 mg/dL (n = 5)	>120 mg/dL (n = 12)	<120 mg/dL (n = 5)
0	225.6 ± 14.9	172.0 ± 22.0	145.5 ± 12.4	93.8 ± 12.1
4	214.3 ± 23.9	180.2 ± 27.0	136.0 ± 19.2	100.0 ± 23.6
8	209.5 ± 21.4a	181.2 ± 34.2	134.7 ± 17.1b	88.0 ± 37.4
12	211.4 ± 2 5.7	172.6 ± 26.7	133.8 ± 22.2	90.1 ± 33.4

Data are presented as mean ± S.D. The paired Student's t-test was used for comparisons between 0 weeks and each follow-up time point. ^a: p = 0.0029; ^b: p = 0.0039.

As previously reported (Arad et al., 2005; Costabile et al., 2008), ascorbic acid and ferulic acid alone or in combination with other compounds can reduce total cholesterol, LDL cholesterol, and triglyceride levels in serum. However, it is not clear whether these antioxidants or combinations specifically improved lipid metabolism by suppressing oxidative stress. In contrast, this study confirms that antioxidant combinations can normalize serum cholesterol levels accompanied by a reduction in oxidative DNA damage. However, the mechanisms and interactions of each antioxidant in lipid metabolism and in reducing comet assay scores remain unclear, thereby necessitating further studies that make direct comparisons of single antioxidants with antioxidant combinations.

The antioxidant mixture administered in this study had no adverse effects on liver, kidney, or pancreatic function (Table 4). Together with demonstrating its safety, the results of this trial indicate that combinations of different types of naturally occurring antioxidants can protect DNA from oxidative ROS damage and may contribute to the maintenance of good health in middle-aged men. In next stage of human trials, we will confirm the benefit of antioxidant combinations by performing a randomized, double blind study that compares single and combination antioxidants in elderly subjects.

Acknowledgements    The authors are grateful to all the volunteers of Tokai Bussan Co., Ltd. for their participation and cooperation in this study. This study was supported in part by a grant-in-aid (development of evaluation and management methods for supply of safe, reliable, and functional food and farm produce) from the Ministry of Agriculture, Forestry, and Fisheries of Japan.

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