Salka E. Rasmussen

Self-Reported Dietary Supplement Use Is Confirmed by Biological Markers in the Norwegian Mother and Child Cohort Study (MoBa)

Background/Aims: A food frequency questionnaire (FFQ) and a database for dietary supplements were developed for use in the Norwegian Mother and Child Cohort Study (MoBa). The aim of the present study was to investigate the relation between reported use and biomarkers in supplement and nonsupplement users and to validate self-reported intake of dietary supplements in mid pregnancy. Method: 120 women were recruited from MoBa, and 119 subjects completed the MoBa FFQ and a 4-day weighed food diary. Information on supplement use was collected by both methods. Venous blood specimens and 24-hour urine samples were obtained for measurement of dietary biomarkers. Results: Biomarker concentration/excretion and intake differed significantly between supplement and nonsupplement users for vitamin D, carotenoids, folate, the n–6/n–3 fatty acid ratio and iodine (p < 0.05 for all variables). Flavonoid excretion was higher in flavonoid-supplement users (p < 0.05). Significant correlations between total dietary intake (food and supplements) and biomarker concentration/excretion were found for vitamin D (r = 0.45, p < 0.001), folate (r = 0.26, p = 0.005), the n–6/n–3 fatty acid ratio (r = 0.36, p < 0.001) and iodine (r = 0.42, p < 0.001). Conclusion: The biochemical indicators examined in this study confirmed differences in self-reported micronutrient intake between supplement and nonsupplement users for vitamin D, beta-carotene, folate, n–3 fatty acids, flavonoids and iodine.

Urine flavonoids and plasma carotenoids in the validation of fruit, vegetable and tea intake during pregnancy in the Norwegian Mother and Child Cohort Study (MoBa)

OBJECTIVE To validate a new food-frequency questionnaire (FFQ) for measuring the intake of fruit, vegetables and tea reported by women participating in the Norwegian Mother and Child Cohort Study (MoBa). DESIGN Intake of fruits, vegetables and tea estimated by the FFQ was compared with urinary flavonoid excretion, plasma carotenoid concentration and intake measured by a 4-day weighed food diary (FD). The triangular method was applied to calculate FFQ validity coefficients using two independent biomarkers. SETTING AND SUBJECTS One hundred and nineteen women participating in MoBa. RESULTS The FFQ estimate of fruit intake was significantly correlated with urine phloretin (r = 0.33), citrus fruit/juice with urine hesperetin (r = 0.44), cooked vegetables with plasma alpha-carotene (r = 0.37), and tea with urine kaempferol (r = 0.41) (P < 0.01 for all). On average, 60% of the participants fell into the same or adjacent quintiles when classified by FFQ and biomarkers. Significant correlations between the FFQ and FD were found for fruit (r = 0.39), vegetables (r = 0.34), juices (r = 0.50) and tea (r = 0.53). The FFQ validity coefficient was 0.65 for citrus fruit/juice and 0.59 for cooked vegetables as calculated by the triangular method. CONCLUSIONS The validation study shows that the MoBa FFQ can be used to estimate fruit, juice, vegetable and tea intake in pregnant Norwegian women, and to rank individuals within the distribution.

Biological effects of fruit and vegetables

A strong and persistent effect of plant-derived foods on the prevention of lifestyle diseases has emerged from observational studies. Several groups of constituents in plants have been identified as potentially health promoting in animal studies, including cholesterol-lowering factors, antioxidants, enzyme inducers, apoptosis inducers etc. In human intervention studies the dose levels achieved tend to be lower than the levels found to be effective in animals and sampling from target organs is often not possible. A controlled dietary human intervention study was performed with forty-three volunteers, providing 600 g fruit and vegetables/d or in the controls a carbohydrate-rich drink to balance energy intake. Surrogate markers of oxidative damage to DNA, protein and lipids, enzymic defence and lipid metabolism were determined in blood and urine. It was found that a high intake of fruit and vegetables tends to increase the stability of lipids towards oxidative damage. Markers of oxidative enzymes indicate a steady increase in glutathione peroxidase (GPX1) activity in erythrocytes during intervention with fruit and vegetables but there is no effect on GPX1 transcription levels in leucocytes. No change occurs in glutathione-conjugating or -reducing enzyme activities in erythrocytes or plasma, and there are no effects on the transcription of genes involved in phase 2 enzyme induction or DNA repair in leucocytes. Fruit and vegetable intake decreases the level of total cholesterol and LDL-cholesterol, but does not affect sex hormones. In conclusion, it has been shown that total cholesterol and LDL-cholesterol, markers of peripheral lipid oxidation, and erythrocyte GPX1 activity are affected by high intakes of fruit and vegetables. This finding provides support for a protective role of dietary fruit and vegetables against CVD.

Evaluation of flavonoids and enterolactone in overnight urine as intake biomarkers of fruits, vegetables and beverages in the Inter99 cohort study using the method of triads.

Since collection of 24 h urine samples is very time consuming and difficult to obtain, epidemiological studies typically only obtain spot urine samples. The aim of the present study was to evaluate whether flavonoids and enterolactone in overnight urine could substitute flavonoids and enterolactone in 24 h urine as an alternative and more feasible biomarker of fruit, vegetable and beverage intake. A total of 191 individuals in the Inter99 cohort in Denmark completed the validation study. Concentrations of nine urinary flavonoid aglycones (quercetin, isorhamnetin, tamarixetin, kaempferol, hesperetin, naringenin, eriodictyol, phloretin and apigenin) and enterolactone were determined in overnight and 24 h urine samples, and their validity as biomarkers of fruit, vegetable and beverage intake was evaluated in relation to two independent reference methods (Inter99 FFQ data and plasma carotenoids) by using the method of triads. The intakes of fruit, juice, vegetables and tea reported in the FFQ were reflected by the flavonoid biomarker both in overnight and 24 h urine samples. Validity coefficients for the flavonoid biomarker in overnight urine ranged from 0·39 to 0·49, while the corresponding validity coefficients for the biomarker in 24 h urine ranged from 0·43 to 0·66. Although the validity coefficients were lower for overnight urine than for the 24 h urine flavonoid biomarker, they were still of acceptable magnitude. In conclusion, the results indicate that flavonoids and enterolactone in overnight urine samples may be used as a more feasible biomarker than 24 h urine for the assessment and validation of fruit, juice, vegetable and tea intakes in epidemiological studies.

Dietary supplementation with an extract of lycopene-rich tomatoes does not reduce atherosclerosis in Watanabe Heritable Hyperlipidemic rabbits

Tomatoes are rich in lycopene and other carotenoids which have shown beneficial effects on CVD in epidemiological and intervention studies. In the present study the effect of an extract of lycopene-rich tomatoes, Lyc-O-Mato on atherosclerosis was studies in Watanabe Heritable Hyperlipidemic rabbits. The rabbits were fed a control diet, a control diet supplemented with the tomato extract or a control diet supplemented with a mixture of plant oils for 16 weeks. Lycopene was detected only in plasma of rabbits receiving tomato extract. The tomato extract had no effect on cholesterol and triacylglycerol levels measured in total plasma, lipoprotein fractions and on aortic atherosclerosis evaluated biochemically and by microscopy. Oxidation of lipids in unfractionated plasma also was unaffected by the intake of tomato extract. In conclusion, the tomato extract increased plasma levels of lycopene in rabbits, but had no effect on hypercholesterolaemia, oxidation of plasma lipids or aortic atherosclerosis.

Relative bioavailability of the flavonoids quercetin, hesperetin and naringenin given simultaneously through diet

The bioavailability and urinary excretion of three dietary flavonoids, quercetin, hesperetin and naringenin, were investigated. Ten healthy men were asked to consume a ‘juice mix’ containing equal amounts of the three flavonoids, and their urine and plasma samples were collected. The resulting mean plasma area under the curve (AUC)0−48h and Cmax values for quercetin and hesperetin were similar, whereas the AUC0−48h of naringenin and, thus, the relative bioavailability were higher after consumption of the same dose. The study consolidates a significantly lower urinary excretion of quercetin (1.5±1%) compared with hesperetin (14.2±9.1%) and naringenin (22.6±11.5%) and shows that this is not due to a lower bioavailability of quercetin, but rather reflects different clearance mechanisms.

Bone marrow-derived and peritoneal macrophages have different inflammatory response to oxLDL and M1/M2 marker expression - implications for atherosclerosis research.

Macrophages are heterogeneous and can polarize into specific subsets, e.g. pro-inflammatory M1-like and re-modelling M2-like macrophages. To determine if peritoneal macrophages (PEMs) or bone marrow derived macrophages (BMDMs) resembled aortic macrophages from ApoE−/− mice, their M1/M2 phenotype, inflammatory status, and lipid metabolism signatures were compared. oxLDL accumulation was similar in PEMs and BMDMs. On protein expression level, BMDMs showed an M2-like CD206highCD11clow profile, while cholesterol loading led to enhanced CD11c expression and reduced MCP-1 secretion. In contrast, PEMs expressed low levels of CD206 and CD11c, and responded to cholesterol loading by increasing CD11c expression and MCP-1 secretion. mRNA expression of M1/M2 markers was higher in PEMS than BMDMs, while lipid metabolism genes were similarly expressed. Whole aorta flow cytometry showed an accumulation of M2-like CD206highCD11clow macrophages in advanced versus early atherosclerotic disease in ApoE−/− mice. In isolated lesions, mRNA levels of the M2 markers Socs2, CD206, Retnla, and IL4 were downregulated with increasing disease severity. Likewise, mRNA expression of lipid metabolism genes (SREBP2, ACSL1, SRB1, DGAT1, and cpt1a) was decreased in advanced versus early lesions. In conclusion, PEMs and BMDMs are phenotypically distinct and differ from macrophages in lesions with respect to expression of M1/M2 markers and lipid metabolism genes.

Liraglutide Reduces Both Atherosclerosis and Kidney Inflammation in Moderately Uremic LDLr-/- Mice

Chronic kidney disease (CKD) leads to uremia. CKD is characterized by a gradual increase in kidney fibrosis and loss of kidney function, which is associated with a progressive increase in risk of atherosclerosis and cardiovascular death. To prevent progression of both kidney fibrosis and atherosclerosis in uremic settings, insight into new treatment options with effects on both parameters is warranted. The GLP-1 analogue liraglutide improves glucose homeostasis, and is approved for treatment of type 2 diabetes. Animal studies suggest that GLP-1 also dampens inflammation and atherosclerosis. Our aim was to examine effects of liraglutide on kidney fibrosis and atherosclerosis in a mouse model of moderate uremia (5/6 nephrectomy (NX)). Uremic (n = 29) and sham-operated (n = 14) atherosclerosis-prone low density lipoprotein receptor knockout mice were treated with liraglutide (1000 μg/kg, s.c. once daily) or vehicle for 13 weeks. As expected, uremia increased aortic atherosclerosis. In the remnant kidneys from NX mice, flow cytometry revealed an increase in the number of monocyte-like cells (CD68+F4/80-), CD4+, and CD8+ T-cells, suggesting that moderate uremia induced kidney inflammation. Furthermore, markers of fibrosis (i.e. Col1a1 and Col3a1) were upregulated, and histological examinations showed increased glomerular diameter in NX mice. Importantly, liraglutide treatment attenuated atherosclerosis (~40%, p < 0.05) and reduced kidney inflammation in NX mice. There was no effect of liraglutide on expression of fibrosis markers and/or kidney histology. This study suggests that liraglutide has beneficial effects in a mouse model of moderate uremia by reducing atherosclerosis and attenuating kidney inflammation.

Urinary flavonoid excretion and risk of acute coronary syndrome in a nested case-control study

BACKGROUND Epidemiologic studies have suggested that a higher intake of flavonoids may be associated with lower risk of ischemic heart disease. However, the traditional estimation of flavonoid intake by using dietary assessment methods is affected by subjective measures. OBJECTIVE We examined whether the objective measurement of dietary flavonoids excreted in urine is associated with lower risk of acute coronary syndrome (ACS). DESIGN A case-control study was nested in the Danish Diet, Cancer and Health cohort study. Cases were identified in participants who had received a first-time ACS diagnosis in the Danish National Patient Registry after the time of enrollment into the Diet, Cancer and Health study. The excretion of 10 flavonoids, which represent 5 subclasses, was measured in spot urine samples by using liquid chromatography-mass spectrometry. RESULTS A total of 393 eligible cases with ACS were identified and matched to 393 noncases by using incidence density sampling. For kaempferol, most of the individual ORs were statistically significant and from 42% to 61% lower when the higher 4 quintiles were compared with the lowest quintile. The P-trend was not significant. For daidzein, individual ORs were 5-38% lower. None of the individual ORs were significant, but the P-trend was 0.041. For the remaining flavonoids, there were no significant relations between urinary excretion and risk of ACS. CONCLUSIONS Except for kaempferol and daidzein, there were no significant associations between the urinary excretion of flavonoids and risk of ACS. A lack of relations may be a result of the use of short-term exposure measures.