Multilevel data analysis of a crossover designed human nutritional intervention study.

A new method is introduced for the analysis of 'omics' data derived from crossover designed drug or nutritional intervention studies. The method aims at finding systematic variations in metabolic profiles after a drug or nutritional challenge and takes advantage of the crossover design in the data. The method, which can be considered as a multivariate extension of a paired t test, generates different multivariate submodels for the between- and the within-subject variation in the data. A major advantage of this variation splitting is that each submodel can be analyzed separately without being confounded with the other variation sources. The power of the multilevel approach is demonstrated in a human nutritional intervention study which used NMR-based metabolomics to assess the metabolic impact of grape/wine extract consumption. The variations in the urine metabolic profiles are studied between and within the human subjects using the multilevel analysis. After variation splitting, multilevel PCA is used to investigate the experimental and biological differences between the subjects, whereas a multilevel PLS-DA model is used to reveal the net treatment effect within the subjects. The observed treatment effect is validated with cross model validation and permutations. It is shown that the statistical significance of the multilevel classification model ( p << 0.0002) is a major improvement compared to a ordinary PLS-DA model ( p = 0.058) without variation splitting. Finally, rank products are used to determine which NMR signals are most important in the multilevel classification model.

Anthocyanins and colonic metabolites of dietary polyphenols inhibit platelet function.

Maintenance and achievement of an optimal platelet function by dietary solutions might be considered an interesting target to influence cardiovascular health. Polyphenol-rich foods such as vegetables and fruits have been shown to significantly improve platelet function in ex vivo studies in humans. However, until yet, it still remains unclear if polyphenols itself, their metabolites or a mixture of both are responsible for the beneficial effects observed so far. Our study aims to evaluate the effect of anthocyanins, in vivo metabolites of different polyphenols from colonic origin and a representative mixture of both at physiological concentrations on platelet aggregation and activation function. Some anthocyanins [1 microM], colonic metabolites [10 microM] and a mixture (4 phenolic acid [1 microM], 4 anthocyanin [0.1 microM] showed significant platelet sedating and desensitizing effects. Activation of the platelets (P-selectin expression) was significantly reduced by 10-40% in resting platelets, TRAP-activated and hydrogen peroxide-stressed platelets and epinephrine pre-activated platelets relative to controls. The dose-response curve of the weak agonist TRAP was also significantly altered resulting in a >0.8 microM increase of threshold concentration to induce aggregation. The representative mixture was active despite ten times lower concentrations of the individual components, which showed no activity when tested individually at that concentration, indicating synergism of the different components. Platelet reactivity to strong agonists such as collagen and ADP was not influenced. These results show that anthocyanins and in vivo metabolites of polyphenols have anti-thrombotic properties, suggesting themselves as well as their dietary sources and precursors as potential cardiovascular health promoters.

IRS-1 mediates inhibition of Ca2+ mobilization by insulin via the inhibitory G-protein Gi.

Platelet agonists initiate aggregation and secretion by activating receptors coupled to the G-protein G(q), thereby raising cytosolic Ca(2+), [Ca(2+)](i). The rise in [Ca(2+)](i) is facilitated via inhibition of cAMP formation by the inhibitory G-protein of adenylyl cyclase, G(i). Since insulin attenuates platelet activation, we investigated whether insulin interferes with cAMP regulation. Here we report that insulin (0.5-200 nmol/liter) interferes with agonist-induced increases in [Ca(2+)](i) (ADP, thrombin), cAMP suppression (thrombin), and aggregation (ADP). The effects of insulin are as follows: (i) independent of the P2Y(12) receptor, which mediates ADP-induced cAMP lowering; (ii) not observed during G(s)-mediated cAMP formation; (iii) unaffected by treatments that affect phosphodiesterases (3-isobutyl-1-methylxanthine); and (iv) not changed by interfering with NO-mediated regulation of cAMP degradation (N(G)-monomethyl-l-arginine). Hence, insulin might interfere with G(i). Indeed, insulin induces the following: (i) tyrosine phosphorylation of the insulin receptor, the insulin receptor substrate-1 (IRS-1) and G(i)alpha(2); (ii) co-precipitation of IRS-1 with G(i)alpha(2) but not with other G alpha subunits. Despite persistent receptor activation, the association of IRS-1 with G(i)alpha(2) is transient, being optimal at 5 min and 1 nmol/liter insulin, which is sufficient to suppress Ca(2+) signaling by ADP, and at 10 min and 100 nmol/liter insulin, which is required to suppress Ca(2+) signaling by thrombin. Epinephrine, a known platelet sensitizer and antagonist of insulin, abolishes the effect of insulin on [Ca(2+)](i), tyrosine phosphorylation of G(i)alpha(2), and aggregation by interfering with the phosphorylation of the insulin receptor beta subunit. We conclude that insulin attenuates platelet functions by interfering with cAMP suppression through IRS-1 and G(i).

Fish oil inhibits photochemically induced thrombosis in the guinea pig in a dose dependent manner.

The dose-response effect of dietary fish oil was investigated in the photochemically induced thrombosis model in guinea pigs. In this arterial thrombosis model thrombus formation was evaluated by determination of different occlusion parameters (percentage of occlusion, area under the blood flow curve, time to first occlusion, spontaneous reflow). Sixty guinea pigs (7 weeks old) were randomly assigned to and fed a 40 energy % diet containing increasing amounts (0, 5.5, 17 and 36 energy %) of fish oil for four weeks. Arterial thrombosis was induced in the femoral artery by free radical damage and subsequent thrombus formation. Increasing fish oil concentrations in the diet were associated with a linear decrease (p<0.001) in the percentage of occlusion (calculated as a decrease in blood flow) and a linear increase in area under the blood flow curve/begin flow (p<0.001). The time to thrombus formation was not significantly prolonged in any group. However the frequency of animals in which complete occlusion of the femoral artery was not obtained during the thrombosis induction and subsequent observation period was higher in the groups receiving the two highest doses of fish oil. Spontaneous reflow correlated positively (p<0.013) with increasing dietary fish oil content. In conclusion, our data indicates that dietary fish oil inhibits photochemically induced thrombosis in this animal model of arterial thrombosis in a dose dependent manner.

Cyclic AMP raises intracellular Ca(2+) in human megakaryocytes independent of protein kinase A.

The immature megakaryoblastic cell line MEG-01 responds to iloprost with an increase in cytosolic Ca(2+) and cAMP. The Ca(2+) response is almost absent in CHRF-288-11 cells, but cAMP formation is preserved in this more mature megakaryoblastic cell line. Also, in human hematopoietic stem cells, iloprost induces a Ca(2+) response and cAMP formation. The Ca(2+) response is downregulated during megakaryocytopoiesis, but cAMP formation remains unchanged. The Ca(2+) increase may be caused by cAMP-mediated inhibition of Ca(2+) sequestration, because it is (1) independent of Ca(2+) entry; (2) mimicked by forskolin, an activator of adenylyl cyclase, and isobutylmethylxanthine, an inhibitor of phosphodiesterases; and (3) preserved in the presence of inhibitors of protein kinase A and inositol-1,4,5-triphosphate receptors. The small GTPase Rap1 has been implicated in the control of Ca(2+) sequestration. Indeed, Rap1 activation parallels the iloprost- and forskolin-induced Ca(2+) increase and is unaffected by the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N',-tetraacetic acid-AM. These findings reveal a novel mechanism for elevating cytosolic Ca(2+) by cAMP, possibly via GTP-Rap1.