Validation of biomarkers of food intake-critical assessment of candidate biomarkers.

Biomarkers of food intake (BFIs) are a promising tool for limiting misclassification in nutrition research where more subjective dietary assessment instruments are used. They may also be used to assess compliance to dietary guidelines or to a dietary intervention. Biomarkers therefore hold promise for direct and objective measurement of food intake. However, the number of comprehensively validated biomarkers of food intake is limited to just a few. Many new candidate biomarkers emerge from metabolic profiling studies and from advances in food chemistry. Furthermore, candidate food intake biomarkers may also be identified based on extensive literature reviews such as described in the guidelines for Biomarker of Food Intake Reviews (BFIRev). To systematically and critically assess the validity of candidate biomarkers of food intake, it is necessary to outline and streamline an optimal and reproducible validation process. A consensus-based procedure was used to provide and evaluate a set of the most important criteria for systematic validation of BFIs. As a result, a validation procedure was developed including eight criteria, plausibility, dose-response, time-response, robustness, reliability, stability, analytical performance, and inter-laboratory reproducibility. The validation has a dual purpose: (1) to estimate the current level of validation of candidate biomarkers of food intake based on an objective and systematic approach and (2) to pinpoint which additional studies are needed to provide full validation of each candidate biomarker of food intake. This position paper on biomarker of food intake validation outlines the second step of the BFIRev procedure but may also be used as such for validation of new candidate biomarkers identified, e.g., in food metabolomic studies.

Dietary medium-chain saturated fatty acids induce gene expression of energy metabolism-related pathways in adipose tissue of abdominally obese subjects.

BACKGROUND: Dietary medium-chain saturated fatty acids (MC-SFAs) have been shown to reduce total body fat. Previously, we showed that MC-SFAs prevent body fat accumulation, despite weight gain. Here, we aim to explore potential molecular mechanisms underlying the protective effect of MC-SFAs on body fat gain. METHODS: The DairyHealth study examined the long-term effects of milk protein and milk fat with a low or high content of MC-SFA. In this 12 week, randomized, double-blind, diet intervention study, participants consumed 60 g milk protein (whey or casein) and 63 g milk fat (high MC-SFA or low MC-SFA) daily in a two by two factorial design. We used microarrays to measure whole genome gene expression changes in subcutaneous adipose tissue in a subpopulation of 12 participants, 6 in the low MC-SFA+casein group and 6 in the high MC-SFA+casein group. Gene expression of several genes that were found to be changed by MC-SFAs was confirmed in the full study population using qPCR. RESULTS: High MC-SFA resulted in an upregulation of gene expression related to citric acid cycle and oxidative phosphorylation, and a downregulation of gene expression related to complement system and inflammation. CONCLUSIONS: We hypothesize that the beneficial effects of MC-SFAs on prevention of fat accumulation are mediated via increased gene expression related to energy metabolism in the adipose tissue. Decreases in inflammation-related gene expression may have beneficial effects in relation to cardiometabolic diseases.

The impact of protein quantity during energy restriction on genome-wide gene expression in adipose tissue of obese humans.

BACKGROUND: Overweight and obesity is a growing health problem worldwide. The most effective strategy to reduce weight is energy restriction (ER). ER has been shown to be beneficial in disease prevention and it reduces chronic inflammation. Recent studies suggest that reducing the protein quantity of a diet contributes to the beneficial effects by ER. The organ most extensively affected during ER is white adipose tissue (WAT). OBJECTIVE: The first objective was to assess changes in gene expression between a high-protein diet and a normal protein diet during ER. Second, the total effect of ER on changes in gene expression in WAT was assessed. METHODS: In a parallel double-blinded controlled study, overweight older participants adhered to a 25% ER diet, either combined with high-protein intake (HP-ER, 1.7 g kg-1 per day), or with normal protein intake (NP-ER, 0.9 g kg-1 per day) for 12 weeks. From 10 HP-ER participants and 12 NP-ER participants subcutaneous WAT biopsies were collected before and after the diet intervention. Adipose tissue was used to isolate total RNA and to evaluate whole-genome gene expression changes upon a HP-ER and NP-ER diet. RESULTS: A different gene expression response between HP-ER and NP-ER was observed for 530 genes. After NP-ER, a downregulation in expression of genes linked to immune cell infiltration, adaptive immune response and inflammasome was found, whereas no such effect was found after HP-ER. HP-ER resulted in upregulation in expression of genes linked to cell cycle, GPCR signalling, olfactory signalling and nitrogen metabolism. Upon 25% ER, gene sets related to energy metabolism and immune response were decreased. CONCLUSIONS: Based on gene expression changes, we concluded that consumption of normal protein quantity compared with high-protein quantity during ER has a more beneficial effect on inflammation-related gene expression in WAT.

Differences in genome-wide gene expression response in peripheral blood mononuclear cells between young and old men upon caloric restriction.

BACKGROUND: Caloric restriction (CR) is considered to increase lifespan and to prevent various age-related diseases in different nonhuman organisms. Only a limited number of CR studies have been performed on humans, and results put CR as a beneficial tool to decrease risk factors in several age-related diseases. The question remains at what age CR should be implemented to be most effective with respect to healthy aging. The aim of our study was to elucidate the role of age in the transcriptional response to a completely controlled 30 % CR diet on immune cells, as immune response is affected during aging. Ten healthy young men, aged 20-28, and nine healthy old men, aged 64-85, were subjected to a 2-week weight maintenance diet, followed by 3 weeks of 30 % CR. Before and after 30 % CR, the whole genome gene expression in peripheral blood mononuclear cells (PBMCs) was assessed. RESULTS: Expression of 554 genes showed a different response between young and old men upon CR. Gene set enrichment analysis revealed a downregulation of gene sets involved in the immune response in young but not in old men. At baseline, immune response-related genes were higher expressed in old compared to young men. Upstream regulator analyses revealed that most potential regulators were controlling the immune response. CONCLUSIONS: Based on the gene expression data, we theorise that a short period of CR is not effective in old men regarding immune-related pathways while it is effective in young men. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00561145.

The effects of 30 days resveratrol supplementation on adipose tissue morphology and gene expression patterns in obese men.

Polyphenolic compounds, such as resveratrol, have recently received widespread interest because of their ability to mimic effects of calorie restriction. The objective of the present study was to gain more insight into the effects of 30 days resveratrol supplementation on adipose tissue morphology and underlying processes. Eleven healthy obese men were supplemented with placebo and resveratrol for 30 days (150 mg per day), separated by a 4-week washout period in a double-blind randomized crossover design. A postprandial abdominal subcutaneous adipose tissue biopsy was collected to assess adipose tissue morphology and gene expression using microarray analysis. Resveratrol significantly decreased adipocyte size, with a shift toward a reduction in the proportion of large and very-large adipocytes and an increase in small adipocytes. Microarray analysis revealed downregulation of Wnt and Notch signaling pathways and upregulation of pathways involved in cell cycle regulation after resveratrol supplementation, suggesting enhanced adipogenesis. Furthermore, lysosomal/phagosomal pathway and transcription factor EB were upregulated reflecting an alternative pathway of lipid breakdown by autophagy. Further research is necessary to investigate whether resveratrol improves adipose tissue function.

Homocysteine interference in neurulation: a chick embryo model.

BACKGROUND: Periconceptional folic acid supplementation reduces the occurrence and recurrence risk of neural tube defects (NTD). Mothers of children with NTD have elevated plasma homocysteine levels. Administering homocysteine to chick embryos is reported to cause 27% NTD. Therefore, elevated plasma homocysteine levels per se or a disturbed homocysteine metabolism may be teratogenic to the embryo and may interfere with neural tube closure. Our aim was to obtain a chick embryo model to explore the interference of homocysteine in neural tube closure. METHODS: Homocysteine or saline was administered to chick embryos in ovo at 3 hr, 30 hr, and 60 hr of incubation and harvested at 74 hr. Homocysteine was then applied to chick embryos in vitro at a defined time window of four to six somites and followed for 6 hr. RESULTS: Homocysteine administration to chick embryos in ovo resulted in several malformations but not in an increased number of NTDs. Homocysteine administration to chick embryos in vitro resulted in a transient, dose-dependent widening of the anterior neuropore and closure delay of the rhombencephalic neuropore. After 16 hr of incubation the neural tube was closed. CONCLUSIONS: The in vitro chick embryo model appears a good model to explore the interference of a disturbed homocysteine metabolism in neurulation.

Reduced vitamin B12 binding by transcobalamin II increases the risk of neural tube defects.

Periconceptional folic acid supplementation reduces the risk of neural tube defects (NTD). Homocysteine levels are elevated in mothers of NTD children, which may be due to decreased cellular vitamin B12 levels, as vitamin B12 is a cofactor for the methylation of homocysteine. Transcobalamin II (TC II) transports vitamin B12 to the tissues. To examine whether altered plasma transcobalamin levels are a risk factor for NTD, we determined the apo and holo form of TC II and haptocorrin (TCI+TCIII), vitamin B12 and homocysteine concentrations in the plasma of 46 mothers with NTD children, and in 73 female controls. Holo-tc II levels and holo-tc II percentages (holo-tc II/total tc II) in the first quartile of the control distribution were related to a three-fold (OR 2.9, 95%CI 0.9-9.2) and five-fold (OR 5.0, 95%CI 1.3-19.3) risk, respectively, for having a child with NTD, when compared with the last quartile. Homocysteine levels were significantly higher among individuals with low holo-tc II, low total vitamin B12 concentrations and low holo-tc II percentages. These low holo-tc II percentages are probably caused by reduced affinity of TC II for vitamin B12, which may be explained by genetic variation in the TC II gene. Vitamin B12 supplementation might therefore be warranted, in addition to folate, in the prevention of NTD.

Cafestol increases serum cholesterol levels in apolipoprotein E*3-Leiden transgenic mice by suppression of bile acid synthesis.

Cafestol, a diterpene present in unfiltered coffee, potently increases serum cholesterol levels in humans. So far, no suitable animal model has been found to study the biochemical background of this effect. We determined the effect of cafestol on serum cholesterol and triglycerides in different mouse strains and subsequently studied its mechanism of action in apolipoprotein (apo) E*3-Leiden transgenic mice. ApoE*3-Leiden, heterozygous low density lipoprotein-receptor (LDLR+/-) knockout, or wild-type (WT) C57BL/6 mice were fed a high- (0.05% wt/wt) or a low- (0.01% wt/wt) cafestol diet or a placebo diet for 8 weeks. Standardized to energy intake, these amounts are equal to 40, 8, or 0 cups of unfiltered coffee per 10 MJ per day in humans. In apoE*3-Leiden mice, serum cholesterol was statistically significantly increased by 33% on the low- and by 61% on the high-cafestol diet. In LDLR+/- and WT mice, the increases were 20% and 24%, respectively, on the low-cafestol diet and 55% and 46%, respectively, on the high-cafestol diet. These increases were mainly due to a rise in very low density lipoprotein (VLDL) and intermediate density lipoprotein cholesterol in all 3 mouse strains. To investigate the mechanism of this effect, apoE*3-Leiden mice were fed a high-cafestol or a placebo diet for 3 weeks. Cafestol suppressed enzyme activity and mRNA levels of cholesterol 7alpha-hydroxylase by 57% and 58%, respectively. mRNA levels of enzymes involved in the alternate pathway of bile acid synthesis, ie, sterol 27-hydroxylase and oxysterol 7alpha-hydroxylase, were reduced by 32% and 48%, respectively. The total fecal bile acid output was decreased by 41%. Cafestol did not affect hepatic free and esterified cholesterol, but it decreased LDLR mRNA levels by 37%. The VLDL apoB and triglyceride production rates, as measured after Triton injection, were 2-fold decreased by cafestol, indicating that the number of particles secreted had declined and that there was no change in the amount of triglycerides present in the VLDL particle during cafestol treatment. However, the VLDL particles contained a 4-times higher amount of cholesteryl esters, resulting in a net 2-fold increased secretion of cholesteryl esters. The decrease in triglyceride production was the result of a reduction in hepatic triglyceride content by 52%. In conclusion, cafestol increases serum cholesterol levels in apoE*3-Leiden mice by suppression of the major regulatory enzymes in the bile acid synthesis pathways, leading to decreased LDLR mRNA levels and increased secretion of hepatic cholesterol esters. We suggest that suppression of bile acid synthesis may provide an explanation for the cholesterol-raising effect of cafestol in humans.