BioFactors 22 (2004) 271275 IOS Press

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Phase 1 study of multiple biomarkers for metabolism and oxidative stress after one-week intake of broccoli sprouts
Megumi Murashimaa, Shaw Watanabea, , Xing-Gang Zhuoa , Mariko Ueharaa and Atsushi Kurashigeb
Department of Applied Bioscience, Tokyo University of Agriculture, 1-1-1, Sakuragaoka Setagaya, Tokyo, 156-8502, Japan Tel.: +81 3 5477 2453; Fax: +81 3 5477 7222; E-mail: swatanab@nodai.ac.jp b The Japan Institute for the Control of Aging, Shizuoka, Japan
Abstract. Little is known about the direct effect of broccoli sprouts on human health. So we investigated the effect of broccoli sprouts on the induction of various biochemical oxidative stress markers. Twelve healthy subjects (6 males and 6 females) consumed fresh broccoli sprouts (100 g/day) for 1 week for a phase 1 study. Before and after the treatment, biochemical examination was conducted and natural killer cell activity, plasma amino acids, plasma PCOOH (phosphatidylcholine hydroperoxide), the serum coenzyme Q10 , urinary 8-isoprostane, and urinary 8-OHdG (8-hydroxydeoxyguanosine) were measured. With treatment, total cholesterol and LDL cholesterol decreased, and HDL cholesterol increased signicantly. Plasma cystine decreased signicantly. All subjects showed reduced PCOOH, 8-isoprostane and 8-OHdG, and increased CoQ10 H2 /CoQ10 ratio. Only one week intake of broccoli sprouts improved cholesterol metabolism and decreased oxidative stress markers. Keywords: Broccoli sprouts, phosphatidylcholine hydroperoxide, 8-isoprostane, coenzyme Q10, 8-hydroxydeoxyguanosine, phase 1 study
a The

1. Introduction Although many epidemiological studies have indicated that vegetables reduce the risk of chronic diseases, it remains unclear whether single extracted chemicals or whole vegetables are effective [1]. Cruciferous vegetables are rich in isothiocyanates which protect the human body against carcinogenesis [2]. In 1992, sulforaphane, an isothiocyanate, was isolated from broccoli and showed phase 2 enzyme inducer activity [3]. Extracts of broccoli sprouts have 10100 fold inducer activity of phase 2 enzymes compared to mature broccoli [4]. Mature broccoli also contains indole compounds that induce not only phase 2 enzymes but also phase 1 enzymes. Recently, a combination of biomarkers has become available for estimating the role and effects of antioxidants [5,6]. In this study, we estimated the effects of broccoli sprouts based on various biomarkers as a phase 1 study.

Corresponding author.

0951-6433/04/$17.00 2004 IOS Press and the authors. All rights reserved

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M. Murashima et al. / Phase 1 study of multiple biomarkers for metabolism and oxidative stress

2. Material and methods 2.1. Study design Six males and 6 females, aged 2036 y, were recruited. All subjects gave written informed consent. At baseline, subjects received a physical checkup, and answered a health questionnaire [7]. They consumed 100 g fresh broccoli sprouts everyday for one week keeping their lifestyle otherwise unchanged. After the treatment, they answered a questionnaire about their dietary behavior and health condition during the study. 2.2. Biochemical analysis Before and after the treatment, fasting blood and the second urine after wake-up were collected and urination time were recorded. They were immediately transported to the SRL (Serum Research Lab; Tokyo, Japan) and the JaICA (Japan Institute for the Control of Aging; Shizuoka, Japan) to measure blood lymphocyte markers, natural killer cell activity, total cholesterol, HDL cholesterol and triacylglycerol, urea nitrogen, uric acid, AST (asparate aminotransferase), ALT (alanine aminotransferase), -GPT ( glutamyltranspeptidase), plasma amino acids, serum CoQ 10 , urinary 8-isoprostane and 8-OHdG. Serum CoQ10 H2 (reduced form of CoQ10 ) was analyzed before and after the reduction by HPLC (CLASSUP; SHIMADZU CORPORATION, Kyoto, Japan) and the CoQ 10 H2 /CoQ10 ratio was calculated. The excretion rates (ng/kg/h) of 8-isoprostane and 8-OHdG were calculated by multiplying the concentrations (ng/mL) by sample size (mL), and dividing the result by the product of body weight (kg) and hours from last urination. For measurement of plasma PCOOH, total lipids were extracted from plasma using chloroformmethanol mixture (2:1, by vol. containing 0.002% butyl hydroxytoluene). After evaporation, the total lipids fraction was dissolved in chloroform-methanol and analyzed by chemiluminescence detection high performance liquid chromatography (CL-HPLC). The column is a Finepak SIL NH2-5 (250 4.6 mm, Japan Spectroscopic Co. Tokyo, Japan). The ow rate is maintained at 1.0 mL/min by a DS-4 pump (SHOWA DENKO Tokyo, Japan) and the column oven temperature was 35 C. The luminescent reagent was a mixture of cytochrome C (typeVI, Sigma-Aldrich. St. Louis, US) and luminol (Wako Pure Chemical Co. Tokyo, Japan) in borate buffer (pH 10.0). The chemiluminescence was detected by a CL-2 instrument (SHOWA DENKO Tokyo, Japan) [8]. 2.3. Statistical analysis All analyses were performed with SPSS Ver. 11.5 (SPSS Japan Inc.). Differences between sexes or smoking habits were assessed by the one-sample t test. Changes from baseline to post treatment were assessed by the paired-samples t test or Wilcoxon signed-rank test. 3. Results Male and female smokers smoked 35 22 and 22 8 cigarettes/day, respectively. Two of 12 subjects were daily drinkers, while only one was a non-drinker. Five had both drinking and smoking habits. All smokers did not eat breakfast regularly. Three non-smokers ate breakfast almost everyday. Females ate vegetables more than males and got the animal fat off. Four males lived alone while all females lived with their families.

M. Murashima et al. / Phase 1 study of multiple biomarkers for metabolism and oxidative stress Table 1 Biochemical markers by sex at baseline and post treatment unit Total cholesterol LDL cholesterol HDL cholesterol Triacylglycerol Urea nitrogen Uric acid AST ALT -GTP Natural killer cell activit
1

273

mg/dL mg/dL mg/dL mg/dL mg/dL mg/dL IU/L IU/L IU/L %

Males (n = 6) Baseline Post treatment 178 25 (163) 160 284 (145)6 94 22 (88) 75 402 (62)5 66 10 (67) 66 11 (66) 89 67 (64) 97 64 (77) 13.5 3.6 (13) 12.2 1.1 (12.7) 6.3 1.2 (6) 5.8 1.2 (5.5) 19.2 5.0 (20) 19.5 5.3 (19) 19.7 10.6 (15) 18.2 8.3 (15) 21.2 10.5 (22) 19.3 7.3 (21) 2.5 2.3 (2) 1.8 0.8 (2)

Females (n = 6) Baseline Post treatment 180 19 (186) 175 13 (173) 99 20 (103) 91 17 (91) 66 11 (65) 71 123 (73)6 70 28 (64) 64 19 (55) 8.5 2.5 (8.3) 13.4 3.82 (13.3) 4.1 0.6 (4.3) 4.3 0.53 (4.4)6 17.2 2 (17) 17 2.3 (18) 10.7 4.7 (12) 10.5 4.6 (11) 16.7 4.9 (15) 16.5 2.8 (15) 6.3 5.0 (6) 16.5 2.8 (3)

Mean SD (median). LDL cholesterol = Total cholesterolHDL cholesterolTriacylglycerol/5; AST, asparate aminotransferase; ALT, alanine aminotransferase; -GPT, -glutamyltranspeptidase. Different from baseline by Paired-samples t test: 2 p < 0.1, 3 p < 0.05, 4 p < 0.01. Different from baseline by Wilcoxon signed-rank test: 5 p < 0.1, 6 p < 0.05.

At baseline, average biochemical values were all within the normal range (Table 1). Throughout the treatment, no biomarkers except urea nitrogen and uric acid showed signicant sex differences. Oneweek intake of broccoli sprouts signicantly reduced total cholesterol and LDL cholesterol. Females had no signicant reduction in total cholesterol but showed increased HDL cholesterol. Triacylglycerol levels were abnormal in six subjects at baseline. After treatment, three of them moved into normal range. Urea nitrogen in one female was low at baseline, but it returned to normal after the treatment. Uric acid in two males was higher than 7.0 mg/dL at baseline. After the treatment, one returned to normal, but the other slightly increased. AST, ALT and -GTP were within the normal range. Natural killer cell activity was low in all subjects (reference value: 1040%). It did not change signicantly with treatment, but one female showed a remarkable increase. Results for protein-constituting amino acids in the plasma are shown in Table 2. They remained within the normal range throughout the treatment. With treatment, cystine decreased signicantly (p < 0.01) and lysine decreased slightly (p < 0.1). Glycine and glutamine also showed a decreasing trend. The BCAA/TAA ratio (branch-chain amino acids/total amino acids) tended to increase (p < 0.1). Oxidative stress markers are showed for smokers and non-smokers (Fig. 1). Both smokers and non-smokers showed decreased PCOOH. Smokers showed higher value than non-smokers. The CoQ10 H2 /CoQ10 ratio increased signicantly independent of smoking status. 8-isoprostane decreased signicantly, although the excretion rate of 8-isoprostane varied greatly in individuals. 8-OHdG decreased except for two subjects, while 8-OHdG excretion rate increased slightly. 4. Discussion In vitro and animal experiments reported that broccoli sprouts suppress carcinogenesis [3,4,9]. We evaluated the DNA damage by 8-OHdG, which is produced when deoxyguanosine (dG) is oxidized [6]. Reduction of the urinary 8-OHdG and increase in the excretion rate mean recovery from oxidative stress and our results support this [1,5]. Isoprostanes, among which 8-isoprostane is the major compound, are prostaglandin-like compounds formed in vivo from free radical-initiated peroxidation of arachidonic acid without involvement of cyclooxygenase, and are excreted in urine [10]. Non-smokers had signicant

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M. Murashima et al. / Phase 1 study of multiple biomarkers for metabolism and oxidative stress Table 2 Change of plasma amino acids of all subjects by the treatment1 Baseline (nmol/mL) TAA EAA BCAA BCAA/TAA Glycine Cystine Glutamicacid 2725 334 865 124 353.7 77.0 0.13 0.02 239.5 65.7 35.5 4.3 28.1 8.9 (2753) (895) (339) (0.13) (225) (35) (24) Post treatment 2696 345 873 126 373.5 70.5 0.14 0.02 230.4 43.5 33.2 3.5 29.6 15.3 (2546) (890) (381) (0.14)a (225) (33)a (24) Reference value2 21003500 6601200 270600 0.110.20 150350 2949 1263

1 Mean SD (median); n = 12. TAA, total amino acid; EAA, essential amino acid; BCAA, branched chain amino acids (valine, leucine and isoleoucine); Fisher ratio=BCAA/AAA. 2 Reference values are obtained from SRL.(Tokyo, Japan). Different from baseline by paired-samples t test: p < 0.01, p < 0.1. Different from baseline by Wilcoxon signed-rank test: a p < 0.05.

100

35

25

90

30 6

20

8-isoprostane (ng/mg creatinine)

8-OHdG (ng/mg creatinine)

PCOOH (pmol/mL)

CoQ 10 H2 /CoQ

80

25

15

70

20

10

2 60 15 5

50 Baseline Post treatment

10 Baseline Post treatment

0 Baseline Post treatment

0 Baseline Post treatment

Smoker Non-smoker

84.3 10.1 75.1 9.9

69.2 11.3* 63.1 6.3*

17.5 2.5 16.5 4.5

29.6 4.3** 21.5 5.9**

4.4 4.8

0.5 2.0

3.8 2.9

0.8 1.3*

10.5 2.3 15.4 5.8

9.4 11.4

3.4 2.1

Fig. 1. Plasma concentration of PCOOH (phosphatidylcholine hydroperoxide), the serum CoQ10 H2 /CoQ10 ratio, urinary 8-isoprostane and urinary 8-OHdG (8-hydroxydeoxyguanosine) adjusted to creatinine and the excretion rate at baseline and after the treatment for all subjects. Open circles show smokers and closed circles show non-smokers. Values are shown mean SD.

decreases in 8-isoprostane but there were great differences between individuals. PCOOH is associated with core lipid oxidation and reects in vivo oxidative damage to organs [11]. CoQ 10 H2 is the rst antioxidant when lipoproteins are exposed to free radicals [12]. In all subjects PCOOH decreased and CoQ10 H2 /CoQ10 ratio increased regardless of smoking habit. This means CoQ 10 H2 preve phospholipid peroxidation. Antioxidative effects of sulforaphane have not been reported in humans. Recently, induction of GSH by sulforaphane via stimulation of anantioxidant response element (ARE)related gene was reported [13]. So, glutathione could be attributable to antioxidant effects of broccoli sprouts. Cystine, glycine and glutamine in the amino-acid pool may be consumed to synthesize GSH [14]. After 1 week treatment, improvement of lipid metabolism was observed. Interaction of many compounds in broccoli sprouts may contribute to these effects [15]. HDL cholesterol increased signicantly only among females. Gender differences in lifestyle, dietary habits and hormonal state inuenced the results. Lymphocyte markers did not change remarkably, except for the low CD56 positive cells (data not

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shown). Natural killer cell activity is effective for primary prevention of cancer and virus infection. Subjects lifestyle would explain its low activity. A few human studies reported the activation of natural killer cell by vegetables after two weeks [1]. Longer administration may be necessary. This study showed that multiple oxidative biomarkers could be used to evaluate resistance to oxidative stress. The results suggest that further studies on the effectiveness of broccoli sprouts are warranted. Acknowledgements A part of this study was supported by the research fund from the Ministry of Education, Culture, Sports, Science and Technology. The authors thank Ms. Melissa Melby for her English revision. References
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