Associate Professor Mark Lucock

The biological role of two key vitamins, folic acid and vitamin D is so fundamental to life processes, it follows that their UV sensitivity, dietary abundance (both key exposomal factors) and variability in dependent genes will modify their functional efficacy, particularly in the context of maintaining the integrity and function of genome and epigenome. This article therefore examines folate and vitamin D-related phenotypic adaptation to environmental factors which vary across the human life cycle as well as over an evolutionary time-scale. Molecular mechanisms, key nutrigenomic factors, phenotypic maladaptation and evolutionary models are discussed.

Objective: To test the ¿vitamin D-folate hypothesis for the evolution of human skin pigmentation.¿. Methods: Total ozone mapping spectrometer (TOMS) satellite data were used to examine surface UV-irradiance in a large (n¿= 649) Australian cross-sectional study population. Genetic analysis was used to score vitamin D- and folate-related gene polymorphisms (n¿= 22), along with two pigmentation gene variants (IRF4-rs12203592/HERC2-rs12913832). Red cell folate and vitamin D3 were measured by immunoassay and HPLC, respectively. Results: Ultraviolet radiation (UVR) and pigmentation genes interact to modify blood vitamin levels; Light skin IRF4-TT genotype has greatest folate loss while light skin HERC2-GG genotype has greatest vitamin D3 synthesis (reflected in both TOMS and seasonal data). UV-wavelength exhibits a dose¿response relationship in folate loss within light skin IRF4-TT genotype (305 > 310 > 324 > 380 nm). Significant vitamin D3 photosynthesis only occurs within light skin HERC2-GG genotype, and is maximal at 305 nm. Three dietary antioxidants (vitamins C, E, and ß-carotene) interact with UVR and pigmentation genes preventing oxidative loss of labile reduced folate vitamers, with greatest benefit in light skin IRF4-TT subjects. The putative photosensitiser, riboflavin, did not sensitize red cell folate to UVR and actually afforded protection. Four genes (5xSNPs) influenced blood vitamin levels when stratified by pigmentation genotype; MTHFR-rs1801133/rs1801131, TS-rs34489327, CYP24A-rs17216707, and VDR-ApaI-rs7975232. Lightest IRF4-TT/darkest HERC2-AA genotype combination (greatest folate loss/lowest vitamin D3 synthesis) has 0% occurrence. The opposing, commonest (39%) compound genotype (darkest IRF4-CC/lightest HERC2-GG) permits least folate loss and greatest synthesis of vitamin D3. Conclusion: New biophysical evidence supports the vitamin D-folate hypothesis for evolution of skin pigmentation.

Purpose: The initiation of mandatory folic acid fortification using pteroylmonoglutamic acid (PteGlu) has reduced the rate of congenital malformations. However, it also appears to be responsible for several adverse effects, including increased cancer incidence. This may be related to physicho-chemical characteristics of PteGlu. This study examines the potential effect of high concentrations of PteGlu on a population subjected to mandatory folic acid fortification using an in vitro model. Methods: Caco-2 (colorectal cancer) and MCF7 (breast cancer) cell lines were cultured at 6 different PteGlu concentrations (0, 0.1, 1, 50, 250, and 500µg/ml) for 6 days. Cell growth was determined using thiazolyl blue tetrazolium bromide assay. The genotype of dihydrofolate reductase 19bp deletion/insertion (DHFR 19-del) was also scored in cell lines using a restriction fragment length polymorphism technique to examine whether genetic variations may factor in cell proliferation. Results: PteGlu exhibited differential growth promoting properties between cell lines. Caco-2 cells did not show a significant growth difference at low concentrations compared to control, however, at higher concentrations, the growth showed a contrasting trend in the early experimental period, while MCF7 showed enhanced cell growth at all concentrations. The DHFR 19-del genotype differed in the two cell lines. Conclusions: Altered response to PteGlu by Caco-2 and MCF7 may reflect a tissue specific disease aetiology or genotype specific differential enzyme activity, for example by DHFR, to critical levels of PteGlu. As folic acid fortification is a blanket intervention, and DHFR and other enzyme activities vary between individuals, PteGlu intake may have an as yet undefined effect on health. These findings may be relevant when considering mandatory folic acid fortification for disease prevention.

Vitamin D receptor (VDR) gene polymorphisms may influence risk for adenomatous polyps (AP), a benign precursor to colon cancer, via modulation of vitamin D sensitive pathways, including cell proliferation and differentiation. However, results have been mixed and any association remains contentious. Failure to clinically exclude the presence of (AP in control cohorts may contribute to the lack of consensus. Therefore, we assessed the role of the FokI, BsmI, ApaI, and TaqI VDR polymorphisms in modifying risk for AP, adjusting for a range of dietary and lifestyle variables. Blood was collected from colonoscopy patients (n = 258) and VDR polymorphisms assessed by restriction fragment length polymorphism. Dietary habits were estimated from food frequency questionnaires. Odds ratios for AP were calculated by genotype, stratified by sex, and adjusted for age, lifestyle, and dietary factors. FokI was associated with modified risk for AP in males, whereas the BsmI/ApaI/TaqI haplotype was associated with modified risk in females. No interaction was found between VDR variants and vitamin D intake. This study offers novel insight into the potential for VDR genetics to contribute to risk for AP and is the first to demonstrate a sex-specific relationship between these polymorphisms and risk for AP.

Vitamin D is known for its role in the regulation of gene expression via the Vitamin D receptor, a nuclear transcription factor. More recently, a role for Vitamin D in regulating DNA methylation has been identified as an additional mechanism of modulation of gene expression. How methylation status influences Vitamin D metabolism and response pathways is not yet clear. Therefore, we aimed to assess the relationship between plasma 25-hydroxycholecalciferol (25(OH)D) and the methylation status of Vitamin D metabolism enzyme genes (CYP2R1, CYP27B1 and CYP24A1) and the Vitamin D receptor gene (VDR). This analysis was conducted in the context of dietary Vitamin D, and background methyl donor related biochemistry, with adjustment for several dietary and lifestyle variables. Percentage methylation at CpG sites was assessed in peripheral blood cells using methylation sensitive and dependent enzymes and qPCR. Standard analytical techniques were used to determine plasma 25(OH)D and homocysteine, and serum folate and B12, with the relationship to methylation status assessed using multi-variable regression analysis. CYP2R1 and VDR methylation were found to be independent predictors of plasma 25(OH)D, when adjusted for Vitamin D intake and other lifestyle variables. CYP24A1 was related to plasma 25(OH)D directly, but not in the context of Vitamin D intake. Methyl-group donor biochemistry was associated with the methylation status of some genes, but did not alter the relationship between methylation and plasma 25(OH)D. Modulation of methylation status of CYP2R1, CYP24A1 and VDR in response to plasma 25(OH)D may be part of feedback loops involved in maintaining Vitamin D homeostasis, and may explain a portion of the variance in plasma 25(OH)D levels in response to intake and sun exposure. Methyl-group donor biochemistry, while a potential independent modulator, did not alter this effect.

Background/objectives Restricting energy intake for weight management in older adults has potential to adversely affect nutritional status and result in impairment of an already compromised immune system. Investigation of alternative strategies to combat adiposity and sustain lean muscle mass in older adults are warranted to minimise the risk of developing chronic diseases. Long chain omega-3 polyunsaturated fatty acids (LCn-3PUFA), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), may play an important role through their impact on increased fat oxidation and reduced inflammation. This study aimed to examine the association between erythrocyte membrane LCn-3PUFA and anthropometric measures in an older population. Subjects/methods A cross-sectional sample of older adults (n¿=¿620; age 65¿95 years; 56.3% females) from the Retirement Health and Lifestyle Study (RHLS) was analysed. Anthropometric measurements, including height, weight, body mass index (BMI), waist (WC) and hip circumference (HC) were taken. The fatty acid composition of erythrocyte membranes was analysed via gas chromatography (GC) to determine the omega-3 index (%EPA plus %DHA). Results An inverse association was detected between the omega-3 index and anthropometric measures, BMI (r¿=¿-0.076, p=0.06), WC (r¿=¿-0.118, p¿<¿0.01) and waist-to-hip ratio (WHR; r¿=¿-0.149, p¿<¿0.001). Stratification of data by sex (females, n¿=¿349; males, n¿=¿271) indicated that these associations were sex-specific. Females displayed an inverse association between the omega-3 index and BMI (r¿=¿-0.146, p¿<¿0.01) and WC (r¿=¿-0.125, p¿<¿0.05). In contrast, no significant association between the omega-3 index and anthropometric measures was detected in males. After correcting for the potentially confounding effects of age, household income, fish oil supplement status, daily dietary energy intake and total physical activity times, the omega-3 index was inversely associated with BMI and WC in females but not males. Conclusions Omega-3 status was associated with weight status, particularly in older women but not in men. These results suggest the need for sex-based intervention trials to examine the role of dietary intake and/or supplementation of LCn-3PUFA in weight management of older adults.

Background/Objectives Non-alcoholic fatty liver disease (NAFLD) is highly prevalent in older people but currently no specific drugs are available for its treatment. Omega-3 polyunsaturated fatty acids (n-3PUFA), known for their lipid-lowering, anti-inflammatory and anti-hypertensive properties, may have therapeutic potential for the management of NAFLD. The aim of this study was to determine whether n-3PUFA levels are associated with the prevalence of NAFLD in older adults. Methods A cross-sectional sample of older adults aged 65¿95 years (n¿=¿620) from the Retirement Health and Lifestyle Study (RHLS) was analysed. Fatty Liver Index (FLI) scores, used as an indicator of NAFLD risk, were calculated using a validated algorithm that incorporates body mass index, waist circumference, plasma triglycerides and ¿-glutamyl transferase. Omega-3 index scores (O3I, %eicosapentaenoic acid plus %docosahexaenoic acid) were determined by analysing the fatty acid composition of erythrocyte membranes by gas chromatography. Results Following application of exclusion criteria, 475 participants were included in the analysis (age 77.9¿±¿7.0 years; 60.4% females). Of these, 216 participants had FLI scores (=60) suggestive of NAFLD (age 77.0¿±¿6.6 years; 49.1% females). O3I was significantly lower in participants with NAFLD compared to those without NAFLD (p¿<¿0.01). A significant inverse relationship was found between O3I and FLI (r¿=¿-0.165; p¿<¿0.001). This relationship was gender specific with women, but not men, showing a significant association (r¿=¿-0.206; p¿<¿0.001). Conclusions The current study demonstrated a sex-dependent inverse relationship between erythrocyte n-3PUFA concentrations and NAFLD in older adults. The finding supports the proposal for sex-stratified n-3PUFA intervention trials in this high-risk age group.

Background Elevated levels of pro-inflammatory mediators heighten the risk of developing or aggravating a spectrum of chronic diseases and are a strong predictor of mortality in elderly cohorts. Omega-3 polyunsaturated fatty acids (n-3PUFA), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are known to possess anti-inflammatory properties. However, the relationship between erythrocyte membrane n-3PUFA and inflammation biomarkers has not been well established. Objective This study aimed to determine if n-3PUFA status, together with the omega-3 index (O3I, erythrocyte membrane % EPA plus DHA), is associated with pro-inflammatory mediators in older Australians. Methods The study was a cross-sectional analysis of randomly selected older men and women aged =65 years (n¿=¿620) recruited from the Central Coast of NSW, Australia. Fasted blood samples were analysed for C-reactive protein (CRP), fibrinogen and full blood count using standardised laboratory methods. The fatty acid composition of erythrocyte membranes was analysed via gas chromatography to determine n-3PUFA levels. The relationships between n-3PUFA and inflammatory mediators were evaluated in multivariate regression models after adjusting for known inflammatory confounders. Results After excluding participants who had an inflammatory disease, CRP levels >10¿mg/L, or who were taking anti-inflammatory medications or n-3PUFA supplements, 126 participants (age 77.6¿±¿7.3 years; females, 46%) were included in the analysis. After multivariate adjustments, O3I was inversely associated with CRP (ß¿=¿-0.209, p¿<¿0.05) and monocyte cell counts (ß¿=¿-0.205, p¿<¿0.05), and total n-3PUFA was inversely related to WBC (ß¿=¿-0.238, p¿<¿0.05), neutrophils (ß¿=¿-0.212, p¿<¿0.05) and monocytes (ß¿=¿-0.246, p¿<¿0.05). However no association between fibrinogen and O3I or total n-3PUFA was detected. Conclusions This study demonstrated a negative association between O3I and biomarkers of inflammation in an older population. The findings support a potential role for n-3PUFA supplementation in the management of inflammatory diseases.

Management of hyperlipidaemia remains a cornerstone therapy for the prevention of cardiovascular disease (CVD). Dietary supplementation with n-3 polyunsaturated fatty acid (PUFA) has been shown to modulate blood lipid profiles and reduce the risk of developing CVD. However, studies relating objective measures of long-term dietary n-3 PUFA intake and circulating lipid levels in older adults are limited. Thus, we aimed to determine whether there is an association between erythrocyte n-3 PUFA status (omega-3 index, O3I) and blood lipid profiles in older adults. A sample of adults aged 65-95 years who participated in the Retirement Health and Lifestyle Study was evaluated. Outcome measures included O3I (% eicosapentaenoic acid+% docosahexaenoic acid) and fasting blood lipid profiles [total cholesterol (TC), low-density lipoprotein (LDL)-cholesterol, high-density lipoprotein (HDL)-cholesterol and triglyceride (TG)]. Two hundred and seventy-six subjects were included in the analyses. The mean±SD age was 77.6±7.4 years, and 40.9% were males. O3I was significantly higher in females compared to males. O3I was inversely associated with plasma TG (P<.001) and TC/HDL-cholesterol ratio (P<.05), and positively associated with HDL-cholesterol (P<.05), in all subjects. Associations between O3I and TG were evident in both females (r=-0.250, P<.01) and males (r=-0.225, P<.05). In females only, the odds of being hypertriglyceridaemic were highest in those with lowest O3I (P=006). Trends for hypercholesterolaemia and elevated LDL risk were converse between males and females. Long-term n-3 PUFA status is associated with blood lipid profiles in older Australians. Our findings support the development and implementation of age-specific dietary strategies to reduce the risk of CVD via improving the O3I.

Interest in vitamin D and the VDR gene is increasing as putative roles in human health and evolutionary processes are explored. This review looks beyond the classic biochemistry that links vitamin D to calcium homeostasis; it explores how vitamin D interacts with light in a broader perspective than simple skin photosynthesis. It examines how the vitamin influences circadian rhythm, and how it may have helped drive the evolution of skin pigmentation. To this end, the nutrient¿nutrient relationship with folate is also explored. The VDR gene is additionally examined as a factor in the evolutionary selection of skin depigmentation at higher latitudes to allow vitamin D synthesis. Evidence is given to show that VDR polymorphisms exhibit a latitudinal gradient in allele prevalence consistent with such a paradigm. Overall, the review examines new evo-devo ideas that link light-sensitive vitamins to human health/phenotype, both within and across the lifecycle.

Purpose: The aim of this study is to explore whether a methylation diet influences risk for adenomatous polyps (AP) either independently, or interactively with one-carbon metabolism-dependent gene variants, and whether such a diet modifies blood homocysteine, a biochemical phenotype closely related to the phenomenon of methylation. Methods: 249 subjects were examined using selective fluorescence, PCR and food frequency questionnaire to determine homocysteine, nine methylation-related gene polymorphisms, dietary methionine, 5-methyltetrahydrofolate, vitamins B6 and B12. Results: 1). Both dietary methionine and 5-methyltetrahydrofolate intake are significantly associated with plasma homocysteine. 2). Dietary methionine is related to AP risk in 2R3R-TS wildtype subjects, while dietary B12 is similarly related to this phenotype in individuals heterozygous for C1420T-SHMT, A2756G-MS and 844ins68-CBS, and in those recessive for 2R3R-TS. 3). Dietary methionine has a marginal influence on plasma homocysteine level in C1420T-SHMT heterozygotes, while B6 exhibits the same effect on homocysteine in C776G-TCN2 homozygote recessive subjects. Natural 5-methyltetrahydrofolate intake is interesting: Wildtype A1298C-MTHFR, heterozygote C677T-MTHFR, wildtype A2756G-MS and recessive A66G-MSR individuals all show a significant reciprocal association with homocysteine. 4). Stepwise regression of all genotypes to predict risk for AP indicated A2756G-MS and A66G-MSR to be most relevant (p= 0.0176 and 0.0408 respectively). Results were corrected for age and gender. Conclusion: A methylation diet influences methyl group synthesis in the regulation of blood homocysteine level, and is modulated by genetic interactions. Methylation-related nutrients also interact with key genes to modify risk of AP, a precursor of colorectal cancer. Independent of diet, two methylation-related genes (A2756G-MS and A66G-MSR) were directly associated with AP occurrence.

Background: The C1561T variant of the glutamate carboxypeptidase II (GCPII) gene is critical for natural methylfolylpolyglutamte (methylfolate) absorption, and has been associated with perturbations in folate metabolism and disease susceptibility. However, little is known on C1561T-GCPII as a risk factor for colorectal cancer. Therefore, this study examined whether C1561T-GCPII influences folate metabolism and adenomatous polyp occurrence. Materials and Methods: 164 controls and 38 adenomatous polyp cases were analysed to determine blood folate and plasma homocysteine (Hcy) level, dietary intake of natural methylfolate, synthetic pteroylglutamic acid (PteGlu), vitamin C and C1561T-GCPII genotype. Results: In controls and cases, 7.3 and 18.4 percent of subjects respectively, were found to have the CT genotype, increasing the risk for adenomatous polyp occurrence 2.86 times (95% CI:1.37-8.0, p=0.035). Total dietary folate, methylfolate and PteGlu intake and the level of erythrocyte folate and plasma Hcy did not predict the occurrence of an adenomatous polyp. However, dietary natural vitamin C intake was associated with adenomatous polyp risk within C1561T-GCPII CT genotype subjects (p=0.037). Conclusions: The findings suggest that C1561T-GCPII variation may be associated with risk for adenomatous polyp, and vitamin C may modify risk by interacting with the variant gene, its expression product and/or folate substrates.

Background: Free circulating microRNA (miRNA) in serum may be valuable biomarkers for disease diagnosis and prognosis. miR-21, the archetypal oncogenic miRNA, has been proposed as a biomarker for colorectal cancer and its benign precursor, adenomatous polyps. However, it is now becoming clear that circulating miRNA profiles may be sensitive to lifestyle and environmental influences. Dietary components involved in one-carbon metabolism are particularly well placed to modulate miRNA expression through an influence on DNA methylation pathways. Methods: We investigated the role of methyl group donors (folate, B12, cysteine, homocysteine), polymorphisms of the enzymes of one-carbon metabolism, and serum miR-21 expression in a primary case-control cohort (colonoscopy confirmed adenomatous colon polyps vs controls; n. =. 253) and a secondary cross-sectional cohort (over 65s; n. =. 649). The relationships between these parameters and serum miR-21 levels were assessed, stratified by gender. Conclusions: Serum miR-21 expression was related to occurrence of adenomatous polyps in females, but not males. Folate levels and MTHFR-C677T genotype was associated with miR-21 expression in both genders. Additionally, DHFR-19 del and MSR-A66G were associated with miR-21 expression in females and males, respectively. Stimulation with excess folate increased expression of miR-21 in colon cancer cell lines. General significance: This study demonstrates that serum miR-21 expression correlates with folate status and related genetic status. This may have consequences for the proposed use of miR-21 as a colorectal cancer biomarker.

The impact of folate on health and disease, particularly pregnancy complications and congenital malformations, has been extensively studied. Mandatory folic acid fortification therefore has been implemented in multiple countries, resulting in a reduction in the occurrence of neural tube defects. However, emerging evidence suggests increased folate intake may also be associated with unexpected adverse effects. This literature review focuses on contemporary issues of concern, and possible underlying mechanisms as well as giving consideration the future direction of mandatory folic acid fortification. Folate fortification has been associated with the presence of unmetabolized folic acid (PteGlu) in blood, masking of vitamin B12 deficiency, increased dosage for anti-cancer medication, photo-catalysis of PteGlu leading to potential genotoxicity, and a role in the pathoaetiology of colorectal cancer. Increased folate intake has also been associated with twin birth and insulin resistance in offspring, and altered epigenetic mechanisms of inheritance. Although limited data exists to elucidate potential mechanisms underlying these issues, elevated blood folate level due to the excess use of PteGlu without consideration of an individual's specific phenotypic traits (e.g. genetic background and undiagnosed disease) may be relevant. Additionally, the accumulation of unmetabolized PteGlu may lead to inhibition of dihydrofolate reductase and other enzymes. Concerns notwithstanding, folic acid fortification has achieved enormous advances in public health. It therefore seems prudent to target and carefully monitor high risk groups, and to conduct well focused further research to better understand and to minimize any risk of mandatory folic acid fortification.

Nutrient-gene research tends to focus on human disease, although such interactions are often a by-product of our evolutionary heritage. This review explores health in this context, reframing genetic variation/epigenetic phenomena linked to diet in the framework of our recent evolutionary past. This "Darwinian/evolutionary medicine" approach examines how diet helped us evolve among primates and to adapt (or fail to adapt) our metabolome to specific environmental conditions leading to major diseases of civilization. This review presents updated evidence from a diet-gene perspective, portraying discord that exists with respect to health and our overall nutritional, cultural, and activity patterns. While Darwinian theory goes beyond nutritional considerations, a significant component within this concept does relate to nutrition and the mismatch between genes, modern diet, obesogenic lifestyle, and health outcomes. The review argues that nutritional sciences should expand knowledge on the evolutionary connection between food and disease, assimilating it into clinical training with greater prominence. © The Author(s) 2013.