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INTRODUCTION
Glutathione S-transferases (GSTs), a family of phase II enzymes found in all eukaryotic species, play a critical role in detoxifying both naturally occurring and xenobiotic compounds, including carcinogens, environmental toxins, and reactive oxygen species, by catalyzing the transfer and conjugation of glutathione [1]. Eight classes of mammalian cytosolic GSTs are currently recognized, designated as alpha (A), mu (M), kappa (K), omega (O), pi (P), sigma (S), theta (T), and zeta (Z) [2]. Among them, both GSTM1 and GSTT1 are known to be polymorphic in humans and both of them have null alleles resulting from gene deletion [3]. The null genotypes (homozygous for the non-functional allele) of GSTM1 and GSTT1 have a decreased capability of detoxifying some carcinogens. Also, GST genetic polymorphisms imply variations in enzyme activities that can result in oxidative stress susceptibility through alterations in GSH metabolism [4]. Because of the role of GSTs in detoxifying xenobiotics and the products of oxidative stress, the effect of GST deletion has been investigated for numerous conditions. Meta-analyses have indicated that the deletion of either GSTM1 or GSTT1 is associated with a significant increased risk of coronary heart disease [5], and several forms of cancer [6,7,8]. Several epidemiological studies indicated that cancer and cardiovascular diseases share common risk factors [9].
Cruciferous vegetables are widely consumed in people's diets. These vegetables include kale, as well as broccoli, cauliflower, radish, Brussels sprouts, watercress, and cabbage and are consumed either fresh (salads), cooked, or in vegetable juices. Kale, classified into the Brassica oleracea species (Brassica oleracea acephala), is one of the most popular cruciferous vegetable consumed in South Korea. Besides nutritional components, these vegetables are also rich in health beneficial secondary metabolites, which include sulfur containing glucosinolates, flavonoids, anthocyanins, coumarins, carotenoids, antioxidant enzymes, terpenes, and other minor compounds [10]. Glucosinolates upon hydrolysis form biologically active compounds such as indoles and isothiocyanates (ITC) [11]. ITC are potentially anticarcinogenic phytochemicals formed from the metabolism of glucosinolates and are found in cruciferous vegetables as well as a number of other foods [12]. ITC are both substrates for and inducers of glutathione S-transferase (GST) phase II metabolizing enzymes involved in carcinogen detoxification as well as effectors of phase I pathways [13]. A few human intervention trials have evaluated the ability of the GST genotype to modulate the response to cruciferous vegetable intake on biomarkers [13]. It has been suggested that the capacity of a moderate intake of watercress [14] or cruciferous vegetable [15] to induce detoxification is dependent in part on the GSTM1 genotype. In the same manner, GSTM1 genotypes have a significant effect on the metabolism of sulforaphane derived from glucosinolate broccoli, and this difference in metabolism may explain the greater protection that GSTM1-positive persons gain from consuming broccoli [16].
Hypertensive patients who may be genetically impaired in their ability to handle oxidative stress, by virtue of deletion of the GSTM1 gene, are more susceptible to the impact of ROS exposure. Therefore, supplementation with antioxidants might compensate for this genetic susceptibility [2]. Recently, it has been reported that regular meals supplemented with kale juice could exhibit favorable influence on serum lipid profiles and antioxidant systems, and hence contribute to reduce the risks of coronary artery disease in male subjects with hyperlipidemia [17]. However, little is known about the potential effects of kale juice on human health, especially concerning the effect of GST polymorphisms on any health benefits that kale juice may provide. Therefore, the present study was undertaken to examine whether daily supplementation of kale juice modulates the blood pressure (BP), blood glucose, and levels of lipid profiles in subclinical hypertensive subjects, and whether this modulation could be affected by the GSTM1 and GSTT1 polymorphisms.
SUBJECTS AND METHODS
Participants and dietary intake assessment
This research was carried out for 6 weeks on male borderline isolated subclinical hypertensive patients [systolic BP (SBP) > 130 mmHg or diastolic BP (DBP) > 85 mmHg] who had never been treated for hypertension. Participants included members of the staff of Hannam University, government employees in Daejeon, and volunteers among the participants of a previous study [18]. The study was conducted according to a study protocol that passed the standards of the Institutional Review Board at Hannam University, South Korea (approval code: 2012-04k). Informed written consent including purpose, nature, and potential risks was obtained from all subjects. Information regarding individual characteristics, health status, and lifestyle factors including smoking, alcohol, and exercise were collected by questionnaires. Participants suffering from poor health or who were consuming prescribed medications were excluded from the study. The participants' weight, height, and waist and hip circumferences were measured using standard protocols. These measurements were then used for calculating body mass index (BMI) and waist-hip ratio (WHR). Body fat was measured by Bio-electrical Impedance Fatness Analyzer (Inbody 520, Biospace, South Korea). Blood pressure was the mean of three measurements in a seated position, using an automatic BP monitor (Watch BP Home, Microlife, Switzerland) on weeks 0, and 6. Dietary information provided by the participants was recorded using 24-hour recall and a food frequency questionnaire. Total nutrient intake was estimated using the CAN Pro 3.0 (Nutrition Information Committee, Korean Nutrition Society), and evaluated using the Korean Dietary Reference Intakes (2010) [19].
Kale juice supplementation
Two bottles (total 300 ml) of 100% pure kale juice (Pulmuone, South Korea) were freshly delivered and supplemented every day to the participants for 6 weeks. The participants were instructed to consume the kale juice daily and record their intake on a daily log. A depletion period restricting the consumption of kale products and fruits and vegetables with high antioxidant nutrients was established 2 weeks prior to the supplementation of kale juice. This period was intended to ensure antioxidant vitamin status within similar levels at baseline. Also, they were reminded and advised over the phone individually to refrain from consuming foods which may affect the antioxidant index during the experimental period.
Blood analysis
Blood was drawn from the participants at the beginning (0 week) and after 6 weeks of the supplementation of kale juice. Blood samples drawn from the survey participants after a minimum 12 hours overnight fasting were put in a 10 ml heparinated sterile tube (Vacutainer, Becton Dickinson, U.S.A.), and brought to the laboratory. Some of the blood (500 uL) was put in microcentrifuge tubes separately for the analysis of GSTM1 and GSTT1 polymorphisms. The remaining blood was centrifuged at 1,000 rpm for 15 minutes to collect PRP (platelet-rich plasma), and then it was centrifuged again at 3,000 rpm for 30 minutes to collect PDP (platelet-deficient plasma) following the separation of blood plasma. The blood plasma was divided for each analysis item and kept at -80℃ in a freezer until use. The blood glucose level was measured immediately after drawing the blood using a testing device (GLUCOTREND, Roche Diagnostics GmbH, Germany).
Analysis of GSTM1 and GSTT1 genetic polymorphisms
The GSTM1 and GSTT1 genotypes were determined as previously described without any modification [20,21]. Briefly, the β-globin primer pair (sense: 5'-CAACTTCATCCACGTTCACC-3' and antisense: 5'-GAAGAGCCAAGGACAGGTAC-3'), which had not been deleted, was used as an internal control. The primers for amplifying the GSTM1 gene were (sense) 5'-CGCCATCTTGTGCTACATTGGCCGTC-3' and (antisense) 5'-TTCTGGATTGTAGCAGATCA-3'. The primers for the GSTT1 gene were (sense) 5'-TTCCTTACTGGTCCTCACATCTC-3' and (antisense) 5'-TCACCGGATCATGGCCAGCA-3'. The polymerase chain reaction (PCR) was performed in a 50 µL reaction mix containing 0.1 µg DNA, 5 mM deoxyribonucleoside triphosphates, 30 pmol of each primer, 30 mM MgCl2, and 0.5 U thermos table Taq DNA polymerase. After 2 minutes of pretreatment at 95℃ the reaction was subjected to 30 cycles of amplification at 94℃ for 1 minute, annealing at 64℃ for 1 minute, and 1 minute of elongation at 72℃. A final extension step of 7 min at 72℃ terminated the process. The products of the PCR amplification were separated by electrophoresis in a 1.5% agarose gel and stained with ethidium bromide (0.1 ug/mL). The internal standard fragment amplified from β-globin gene was 268 bp. A 215 bp fragment was amplified for the GSTM1 gene, and a 480 bp fragment was obtained for the GSTT1 gene. The absence of amplified product was consistent with the null genotypes. All reagents and chemicals for the genetic polymorphism were purchased from Bioneer (South Korea).
Determination of plasma lipid profiles
Plasma lipid, total cholesterol, triglyceride, and HDL-Cholesterol contents were analyzed using a semi-auto biochemistry analyzer (Shining Sun A6, Beijing Shining Sun Technology, China) with 1 ml of enzyme solution from a kit reagent produced by STANBIO Laboratory (U.S.A.) and reactivated for 5 minutes in a water bath under 37℃. LDL-Cholesterol was calculated using the Friedewald equation [22].
LDL-Cholesterol
= Total cholesterol - HDL-Cholesterol - (Triglyceride/5) (mg/dl)
Statistical analysis
All the data were entered into a Microsoft Excel database and the statistical tasks were performed with the SPSS-PC+ statistics package (version 20.0). Mean and standard error of the mean (S.E) were obtained for each item, and the mean difference among the 4 types of polymorphism of the 2 groups (GSTM1 null and present types; GSTT1 null and present types) was verified by independent t-test, and all statistical significances evaluated at the level of α = 0.05. The significance of the mean comparison before and 6 weeks after supplementations of kale juice was tested by paired t-test. Also, a chi-square test was performed on the frequency of smoking habits, existence of alcohol intake, and exercise habits.
RESULTS
General characteristics of the participants
General characteristics of the participants are shown in Table 1. All of the participants in this study were aged 20-57 years, and the average age was 38 years. After 6 weeks of kale juice supplementation, the percentage body fat of the participants was decreased significantly, regardless of GSTM1 or GSTT1 genotype. The WHR of the participants was decreased only in GSTM1-null genotype after 6 weeks of supplementation. Participants' smoking habits indicated a daily average of 13.9 ± 1.4 cigarettes with 11.1 ± 2.1 pack years. The drinking habits of the participants showed that the percentage of drinkers was 81.0% among all participants and the total alcohol consumption was 56.8 ± 5.1 mL/day. The portion of participants who regularly exercised was 79.8% and the average exercise time was 30.3 ± 2.9 min/day.
Nutrient intake of the participants
Nutrient intake before kale juice supplementation (0 week) and after 6 weeks of supplementation was surveyed using the 24-hour recall method to find out changes in dietary intake during the 6 weeks of kale juice supplementation. The result is shown in Table 2. The nutrient intake showed that the participants maintained their usual intake in energy, protein, fat, carbohydrate, calcium, vitamin C, vitamin E, vitamin A, and retinol before and after kale juice supplementation (Table 2). The nutrient contents of 300 ml of kale juice are: 30 kcal of energy, 4 g of carbohydrate, 4 g of sugars, 4 g of protein, 1253.4 ug RE of vitamin A, 262 mg of vitamin C, 1 mg of iron, 334.6 mg of calcium, and 230 mg of total polyphenols.
GSTM1 and GSTT1 polymorphism frequency analysis
Among 84 participants, the GSTM1-null genotype was found in 49 (58.3%) and the GSTM1-present genotype in 35 (41.7%) (Table 3), The GSTT1-null genotype was found in 45 participants (53.6%) and the GSTT1-present genotype in 39 (46.4%). The number of participants who had both the GSTM1 and GSTT1 present genotypes was 13 (15.5%), and those who had either one of the present genotypes was 48 (57.1%), and who had neither of the present genotypes was 23 (27.4%).
Changes in blood pressure
The changes in blood pressure of the participants after kale juice supplementation showed that the systolic and the diastolic pressures were decreased in the participants when divided according to GST polymorphism (Table 4). Systolic pressure was decreased by 5.0%, and diastolic pressure by 3.7% in the GSTM1-null genotype, and also the systolic and diastolic pressures were decreased by 5.2% and 3.1%, respectively, in the GSTM1-present genotype. In the case of GSTT1, on the other hand, the systolic and diastolic pressures were significantly decreased by 4.4% and 3.6% in the GSTT1-null genotype, and 6.0% and 4.2% in the GSTT1-present genotype, respectively, after kale juice supplementation.
Comparison of blood glucose and plasma lipid levels
The results of blood glucose, plasma total cholesterol (TC), LDL-C, HDL-C, and triglyceride (TG), before and after 6 weeks of kale juice supplementation are shown in Fig. 1 and 2. The blood glucose levels of the participants were significantly decreased in the GSTM1-present and GSTT1-null genotypes (Fig. 1). The plasma TC and TG levels of the participants were not changed after kale juice supplementation regardless of GST polymorphisms. The plasma HDL-C level was significantly increased, and the plasma LDL-C level was significantly decreased after kale juice supplementation in the GSTT1-present genotype, but those levels in the GSTM1-null, GSTM1-present, and GSTT1-null genotypes were not significantly changed after kale juice supplementation (Fig. 2).
DISCUSSION
Lack of consistent GSTM1 and GSTT1 modulation of cruciferous vegetable intervention studies is probably due to multiple factors, including tissue-specific responses, differences in end points measured, and the type and amount of crucifers fed [13]. It is unknown whether these differences in glucosinolate profiles, and therefore ITC, lead to different biological effects in humans; however, several laboratories have shown differences in the potency and function of ITC in vitro [23,24,25]. Navarro et al. [13] reported that individuals with one or more null genotypes of GSTM1 or GSTT1 responded to a greater extent than individuals with both genotypes intact. These results also suggest that the intact GSTT1 allele may be compensating for the lack of active GSTM1 enzyme activity by playing a larger role in ITC metabolism among GSTM1-null individuals; when both alleles are absent, this compensation is no longer possible [13].
The aims of this study were to assess a GSTM1 and/or GSTT1 genotype-dependent effect of kale juice consumption on the blood pressure, glucose, and lipid profiles found in human plasma in subclinical hypertensive patients. In this study, there was a significant reduction of blood pressure after kale juice supplementation in both GSTM1-null and -present as well as GSTT1-null and -present individuals, however, the genetic polymorphisms of GSTM1 and GSTT1 are not associated with SBP and DBP reduction in subclinical hypertensive patients. From these results, it is suggested that this blood pressure reducing effect is more dependent on the composition of kale juice than on GSTM1 or GSTT1 polymorphisms. Furthermore, it is worth noticing that the reduction of blood pressure of GSTT1-present individuals was higher than those of GSTT1-null, as well as those of GSTM1-present or GSTM1-null individuals. Several studies have shown that blood pressure variation between 30% and 40% in a population is thought to have a genetic basis [26]. The results of our previous study of smokers indicated that the reduction of DBP was only observed in GSTM1-null, GSTT1-null, or GSTT1-present individuals after the 8-weeks of grape juice supplementation [27]. However, Saadat et al. [28] showed that alteration in SBP was only observed in subjects who possess the GSTM1-null, GSTT1-present combination genotype. Conversely, Delles et al. [29] did not find an association between GSTM1 gene variants and hypertension. A number of genome-wide linkage analyses concerned with blood pressure have been reported, and most studies have reported linkage with SBP rather than DBP, but there was no obvious explanation for that [30]. The conflicting results for the GST genes and blood pressure could be due not only to publication bias and sample size but also to extreme gene-environment interactions characterizing the hypertensive phenotypes [31]. Moreover, this discrepancy could be due to differences in the ethnic, genetic, and environmental background of the population studied [32].
A significant reduction of blood glucose was seen in the GSTM1-present and GSTT1-null genotypes after kale juice consumption. Several researchers have shown that consumption of vegetables in the Brassica family may improve insulin resistance and glycemic control in type 2 diabetes. Bahadoran et al. [33] observed that the consumption of 10g of broccoli sprout powder a day resulted in a significant decrease in serum insulin concentration and improved insulin resistance in type 2 diabetic patients. Supplementation of type 2 diabetics with high sulforaphane content broccoli sprouts resulted in increased plasma total antioxidant capacity and decreased oxidative stress index, serum insulin, and insulin resistance [34]. However, there has been no research which observes the modulation of blood glucose level after consumption of Brassica plants in hypertensive patients linked with GST polymorphisms. Several observational studies reported an association between GSTM1 or GSTT1 polymorphisms and the risk of diabetes mellitus. Amer et al. [32] demonstrated that the GSTT1- and GSTM1-null genotypes, alone or combined, are associated with increased risk of type-2 diabetes mellitus. The GSTM1-null genotype had an effect on glycemic control in type-2 diabetes patients, but they did not observe any significant effect of GSTT1-null on glycemic control. US-based epidemiologic study have correlated broccoli or crucifer consumption with the risk of cancer stratified by GSTM1 genotype, which suggests that GSTM1-present persons gain a greater protection than do GSTM1-null persons [16]. So, it is hypothesized that, due to the potential differences in ITC metabolism between GSTM1-present and GSTM1-null individuals [35], blood glucose differs by GSTM1 genotype. However, in this study's result, a decrease of blood glucose in the GSTT1-null genotype was also observed in addition to the decrease in the GSTM1-present genotype after kale juice supplementation. Reasons for these modulations are unclear. Further research is needed on the modulation of blood glucose by GSTM1 and/or GSTT1 polymorphisms and on the difference of the mechanism by which kale juice contributes to blood glucose.
In individuals from the general population, triglycerides, HDL-cholesterol, and the triglycerides/HDL ratio were significantly associated with a double-deleted genotype, suggesting that individuals without any copy of both the GSTM1 and GSTT1 genes are at increased risk for cardiovascular disease [36]. Amer et al. [32] attempted to evaluate the association of the GSTM1 (present, null) and GSTT1 (present, null) genotypes with different lipid profiles in diabetic subjects. Patients with the GSTT1-null genotype had higher levels of triglycerides and very low-density lipoprotein cholesterol compared to those with the GSTT1-present genotype. In the same manner, our previous observation study showed that TC and LDL-C levels were significantly higher in non-smokers with the GSTT1-null genotype than those with the GSTT1-present genotype [37]. Thus, it was hypothesized that the plasma lipid profiles of individuals with the GSTT1-null genotype who might be susceptible to coronary diseases responded to a greater extent after kale juice intervention Contrary to this study's hypothesis, an increase of plasma HDL-C and a decrease of LDL-C with kale juice supplementation were seen among GSTT1-present individuals only, and not among individuals with the GSTT1-null genotype. Reasons for these modulations are unclear. However, these GSTT1-genotype specific responses of plasma lipid profiles to kale juice intake suggest that a differential response to ITC exposure in GSTT1-present and GSTT1-null individuals may influence plasma lipid levels. Whether this genotype difference is due to a difference in ITC metabolism [16] or another factor remains to be determined. To date, studies have not shown consistent pharmacokinetic differences in ITC handling by the GSTT1 genotype. Brassica ITCs induce Phase II enzymes, and, in turn, Phase II enzymes conjugate ITCs leading to excretion. Seow et al. [38] observed a statistically significant difference in levels of urinary excretion of total ITC between GSTT1-positive versus GSTT1-null individuals with similar intakes of dietary ITC; the null genotype was associated with a lower excretion level. From these results, they suggested that GSTT1 may be a key enzyme in the metabolism of ITCs in humans, and suggest the presence of GSTT1 inducer(s) in cruciferous vegetables. At present, however, it is not known if cruciferous vegetables contain GSTT1 inducers. Conversely, Fowke et al. [39] found that urinary ITC excretion was marginally higher with the GSTT1-null genotype and trends between ITC levels and habitual Brassica intake was more consistent within subjects with the GSTT1-null genotypes. Reasons for these inconsistencies are unclear but may include differences in urine collection protocols, dietary assessment methods, types or amounts of Brassica consumed, or genetic profiles between populations [39]. On the other hand, it has been reported that GSTT1 and GSTM1 genotypes are not likely to be involved in the rate of excretion of ITCs in watercress juice [40]. The demonstrated differences in protection among subjects with the two genotypes are not likely due to differences in overall ITC excretion rates. Other yet to be identified mechanisms(s) may underlie the diet and gene interactions between dietary ITCs and GST genotypes in human populations [40]. Meanwhile, Gasper et al. [16] suggested that the differences from contrasting patterns of crucifer consumption and the most prevalent isothiocyanate (i.e. sulforaphane or 2-propenyl isothiocyanates) in the diet within different locations may be critical in interpreting the effect of the GSTM1 and GSTT1 genotypes. Hence the enzymology of sulforaphane and 2-propenyl isothiocyanates with GST isoenzymes is different [41], so it is expected that a GSTM1 and/or GSTT1 deletion will have contrasting effects on the metabolism of sulforaphane and alkenyl isopthiocyanates. This may explain the apparent paradoxical diet-gene interactions observed in different locations and in different studies. Further research is needed to evaluate the protective metabolism of the main metabolites of kale against hyperlipidemia with the GSTM1 or GSTT1 genotypes.
While the strengths of this study include the recruitment of participants based on GSTM1 and GSTT1 genotype, and the 6-week duration of kale juice supplementation, the limitation of the study is that there was no placebo group to compare with, although this study's purpose was to observe the effect of kale juice according to different GSTM1 and/or GSTT1 genotypes. The authors have already proven that green vegetable juice (Angelica keiskei) has an effect compared to the placebo [42]. Another potential limitation is modest sample sizes, which limited our power to further stratify the data by GST genotype and other possibly confounding factors, although significant changes were detected after supplementation. Thus, this study's results need to be confirmed by a larger-scaled, controlled study in the future.
In summary, our findings suggest that the supplementation of kale juice affected blood pressure, blood glucose, and lipid profiles in subclinical hypertensive patients depending on their GST genetic polymorphisms, and the decrease of blood glucose was greater in the GSTM1-present and GSTT1-null genotypes, and the improvement of lipid profiles were mainly greater in the GSTT1-present genotype. This finding suggests that kale juice intervention might be effective in plasma lipid profile control in the subgroup of hypertensive patients who are GSTT1-present, although the strength of this study's findings is limited by the sample size of the study. Much larger studies will be required to accurately measure the modest effects of genes, such as GSTT1, and identify the extent of gene-diet interactions. The relationship between genetic susceptibility biomarkers and the expression of a specific genotype needs to be assessed in vitro, in vivo, or in clinical trials of human volunteers [43]. Further investigation is necessary to establish the mechanism by which kale juice contributes to blood pressure, blood glucose, and lipid profile changes in relation to the GSTM1 and GSTT1 genotypes.
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