We have demonstrated a GSTM1 genotype-dependent effect of a cruciferous diet on the proteins found in human serum using two independent feeding studies, EAT and 2EAT. We identified two peaks (6700 m/z and 9565 m/z) that were affected by the combination of diet and GSTM1 genotype and showed consistent trends across diets in both studies as a doubly charged isoform of TTR (6700 m/z) and a fragment of ZAG (9565 m/z). Using both MALDI-TOF and immunoassays, we demonstrated that both TTR and ZAG decrease with cruciferous vegetable intake among GSTM1+ individuals, but not among individuals with the GSTM1-null genotype.
TTR is a negative acute-phase protein that decreases in serum with early signs of inflammation. It is a known marker of nutritional status and with a half-life of ~2 days, its concentration closely reflects recent changes in diet [29
]. TTR has several known biological roles: In cerebrospinal fluid, it is a carrier of the thyroid hormone thyroxine (T4) and retinol. In serum, TTR transports 15% of T4 and retinol through the complex formation with retinol binding protein (RBP). It also binds many aromatic compounds, leading to speculation of its role in removal of toxic compounds [24
]. Constituents of cruciferous vegetables at high doses have been shown to suppress the function of the thyroid gland [25
]. Thus, although the types and doses of cruciferous vegetables fed in this study were unlikely to influence thyroid function appreciably, one possible explanation of our results is that changes in T4 due to cruciferous vegetable feeding result in less TTR being needed in serum to aid in transport. Another possible explanation is that cruciferous vegetable constituents more directly influence TTR gene expression or TTR protein half-life through other pathways [31
ZAG is an adipokine and apparently plays multiple roles in the human body, most concerning lipid mobilization from fat stores. Levels of ZAG expression are regulated by glucocorticoids [32
]. ZAG is synthesized in the liver and increased levels correlate with lipolysis [33
], implying that it may be regulated by fat intake. Decreased levels of ZAG associated with increased cruciferous vegetable intake could relate to adipokine signaling. Another possible explanation is that cruciferous vegetable constituents directly influence gene expression as it has been suggested that ZAG expression is likely mediated by the interaction of several synergistic transcription factors [34
]. Investigation into the levels of other adipokines and further analysis of ZAG processing will be necessary to shed light on the biology behind these cruciferous vegetable mediated changes.
-genotype-specific responses of TTR and ZAG to cruciferous vegetable intake suggest that differential response to ITC exposure in GSTM1+
-null individuals may influence the serum peptidome. Whether this genotypic difference is due to a difference in ITC metabolism [12
] or another factor remains to be determined. To date, studies have not shown consistent pharmacokinetic differences in ITC handling by GSTM1
genotype, but given the rather broad and overlapping substrate specificities of this superfamily of enzymes, other GST isozymes may compensate in part for the lack of GSTM1
] or alternative metabolic pathways may substitute for GSTM1
. By whatever mechanism, several interventions and observational studies suggest a modifying effect of GSTM1
genotype on serum GST-α in response to cruciferous vegetable exposure [8
] and as biomarkers of oxidative damage [36
]. Based on this previous work and our findings here, it appears that this effect may extend to other serum proteins.
We have also demonstrated that, in the context of a controlled study and systematic data analysis, MALDI-TOF MS is a suitable tool for identifying reproducible biomarkers that respond to diet. In an earlier MALDI-TOF MS analysis of the EAT study, a peptide identified as the B-chain of α2-HS glycoprotein (AHSG) decreased with cruciferous vegetable intake (p = 0.002) [15
]. In the current 2EAT single-dose cruciferous diet data this peptide showed a similar trend but without statistical significance (p = 0.197).
The strengths of our study include the controlled feeding study crossover design, the use of two separate datasets to examine the consistency of peak changes across both studies, and the availability of samples from individuals given two doses of cruciferous vegetables in 2EAT for the assessment of dose-response. Limitations of this study include the modest sample sizes, which limited our power to further stratify the data by sex, race, genotype and other possibly confounding factors. Nonetheless, with 78 (36 + 42) participants, we had 80% power (with Type I error probability of 5%) to detect a biomarker difference between the cruciferous and basal diets as small as one-third of its standard deviation. The two feeding studies were slightly different; the feeding period length was 6 days for EAT and 14 days for 2EAT. Further, participants were fed amounts of cruciferous vegetables based on their total body weight in 2EAT, but the same, absolute amount in EAT. In 2EAT, the 2X cruciferous diet also provided doses of cruciferous vegetables that were substantially higher than usual intake reducing generalizability of the results. To reduce inherent variability due to sample preparation and matrix crystallization inconsistencies, samples were run in quintuplicate with multiple preparations for each sample. All samples from each individual in the study were run on the same plate to decrease variability between plates. All plates were run over the course of two consecutive days to limit day-to-day and instrument variability. The peak identification process can be time-consuming and validation can be costly, hence identifying a large number of significant peaks is challenging. Despite these issues, we were able to find peaks with consistent changes in response to cruciferous diet. Further investigation is necessary to establish the mechanism by which crucifers contribute to these protein changes in relation to GSTM1 genotype.