This study showed that chronic intake of quercetin in mice lowered serum lipid levels which are risk factors for CVD. Microarray analysis indicated that hepatic genes involved in lipid metabolism, in particular in ω-oxidation of fatty acids, could be responsible for these quercetin-induced effects.
Other studies have also observed that supplementation of quercetin to a high-fat diet decreases serum FFA and/or TG levels in mice 
. However, these circulating FFA and TG levels were measured with commercial enzymatic assays, which have recently been found to be sensitive to interference of quercetin and its major metabolite quercetin-3-O-glucuronide, resulting in apparently incorrect lower detected levels 
. Here, besides these enzymatic assays, we also used two independent analytical methods for quantification of serum lipid profiles; GC and 1
H NMR techniques. The observed effect of quercetin on lipid levels measured with the enzymatic FFA and TG assays (FFA -13% and TG -27%) was higher than measured with the two analytical techniques (GC: total fatty acids -7% and 1
H NMR: FFA -2%, TG -14%). This confirms interference of quercetin in the enzyme based assays 
in the physiological range of quercetin exposure and as a consequence overestimate the lipid lowering effect of quercetin. Nevertheless, with GC and 1
H NMR a significant reduction in serum lipid levels was found, proving that lipid lowering is a real biological effect of quercetin. The GC data revealed that the specific serum fatty acids palmitic acid (16
0), oleic acid (18
1(n-9)) and linoleic acid (18
2(n-6)), originating from total lipids, were all significantly decreased in the quercetin-fed mice. Moreover, with 1
H NMR, serum lipids were measured separately, which revealed that serum TG levels of the quercetin group were significantly decreased, while total FFA, cholesterol and phospholipid levels remained unchanged. This indicates that the decreased levels of palmitic acid (16
0), oleic acid (18
1(n-9)) and linoleic acid (18
2(n-6)) found by GC originated from TG. Moreover, the 1
H NMR data showed unchanged levels of total FFA and increased levels of PUFA in the serum of the mice on the quercetin diet, which indicate a shift from saturated fatty acids to PUFA, which are known as the more healthy fatty acids. Together, these data proved that quercetin significantly reduced serum lipid levels and resulted in a more beneficial lipid profile.
The increased levels of PUFA and the decreased levels of saturated fatty acids cannot be fully explained by the microarray data. Genes involved in beta oxidation or specific desaturases were not differentially regulated by the quercetin diet.
There were no significant differences found in the serum phospholipid levels, while pathway analysis revealed phospholipid metabolism as a regulated pathway. However, based on gene expression it was not clear how phospholipid metabolism would be affected, since up as well down regulated genes were observed in different parts of this pathway, and a relative small number of genes of the total pathway was regulated. Therefore, it was concluded that this was not a crucial pathway in this study. Quercetin induced a decrease in relative liver weight in our study. This decrease cannot be explained by a decrease in hepatic lipid accumulation, because hepatic lipid levels were not affected by quercetin. Other studies 
have shown a decrease in lipid accumulation in liver upon dietary administration of quercetin and thus seem to be in contrast with our study. While a study with mulberry leaves, high in quercetin, did report unmodified lipid accumulation in the liver 
and is thus in line with our data. The differences may be explained by the diets used in the different studies. We have used a mild-high-fat diet rich in unsaturated fatty acids, which did not result in extensive lipid accumulation in the liver, since the found hepatic lipid levels were in the same range as found for mice fed a normal-fat diet. The other studies that show a quercetin induced decrease in lipid accumulation used a high saturated fatty acid rich diet which induced lipid accumulation in the liver 
. This suggests that quercetin may prevent lipid accumulation in the liver under adverse dietary conditions, but not with relatively healthy diets. In general, quercetin induced altered lipid metabolism on a mild-high-fat diet (our study), a normal-fat diet 
, and different high-fat diets 
. Suggesting, that quercetin can affect lipid metabolism independent of the diet, although the impact of this effect can be different.
Using whole genome microarrays and confirmation by RT-qPCR, we showed that quercetin up-regulates Cyp4a10, Cyp4a14, Cyp4a31, Acot3, Por, and, possibly Car. An integration of these genes into a single ‘hepatic pathway’ differentially expressed by quercetin treatment is proposed in . Normally, fatty acids are mainly metabolized by β-oxidation first in peroxisomes (very long chain FFA) and subsequently in mitochondria (long, medium, and short chain FFA). Another type of fatty acid oxidation is ω-oxidation, which occurs in the endoplasmatic reticulum by members of the cytochrome P450 4A family 
. Omega-oxidation becomes more important during periods of increased influx of fatty acids into the liver, for example in our high-fat diet mice study, in obesity, and when the mitochondrial oxidation system is insufficient to metabolize fatty acids 
. In these situations ω-oxidation can prevent lipid toxicity 
. Fatty acids oxidized by ω-oxidation result in ω-hydroxy fatty acids which are then dehydrogenated to a dicarboxylic acid in the cytosol. These dicarboxylic acids are further degraded by peroxisomal β-oxidation to shorter chain dicarboxylic fatty acids, which can be excreted in the urine, metabolized by the peroxisomal oxidation system to succinate and acetyl CoA, or completely oxidized after transport into the mitochondrial β-oxidation system 
. A small increase of ketone bodies was found in the quercetin-fed mice suggesting an increase of β-oxidation (292.5±199.2 versus 185.6±118.1 µM, p
Schematic representation of the quercetin-regulated genes involved in ω-oxidation.
Acot3 was also up-regulated in our study, and the enzyme ACOT3 hydrolyses long-medium chain fatty acyl-CoA esters to FFA, and thus facilitate transport into peroxisomes. The FFA can subsequently be transported out of peroxisomes to mitochondria for further β-oxidation 
It has been described that, among others, palmitic acid (16
0) and oleic acid (18
1(n-9)) can be hydroxylated by CYP4A11, the human variant of murine Cyp4a10 
. This is especially consistent with the serum fatty acid profile obtained in the present study (), where levels of palmitic acid (16
0) and oleic acid (18
1(n-9)) were significantly lower in the quercetin-fed mice. The significant up regulation of Cyp4a10, Cyp4a14, Cyp4a31 and Acot3 therefore explains the observed reduced serum levels for these specific fatty acids.
In humans, various polymorphisms are described in the genes of cytochromes P450s and they can be considered as one of the major determinants of individual susceptibility to CVDs 
. Allelic variations in CYP4A11 are suggested to result in an increased risk for hypertension 
. Hypertension can be caused by increased serum lipid levels 
, which were decreased by quercetin in our study with concomitant up regulation of Cyp4a genes.
The up regulation of the Cyp4a genes is consistent with the significant, 1.97 fold up regulation of Por by quercetin. POR is an enzyme that is required for electron transfer to cytochrome P450 enzymes and is therefore rate limiting for P450 enzymes. Deletion of the Por gene in a mouse model reduced hepatic P450 activity by more than 95%. Moreover, hepatic Por knockout (Por-KO) mice showed decreased CYP4A protein levels, and an enlarged and fatty liver. Based on these observations, it was concluded that the P450 system plays a major role in regulating lipid homeostasis and hepatic lipid levels 
. Two to three-fold more genes were significantly regulated when WT mice were exposed to quercetin compared to Por-KO mice. These genes were, among others, involved in fatty acid metabolism pathways. This suggests that hepatic POR mediates many of the biological effects of quercetin, including fatty acid metabolism 
. These results underscores our data, which showed an up regulation of Por.
It is also suggested that P450 expression can be mediated via a CAR-dependent signaling pathway 
. CAR is a transcription factor that is highly expressed in the liver. It is shown that ligand dependent activation of CAR increased lipid metabolism in rodents 
and it is also shown that this can lead to specifically increased expression of genes involved in ω-oxidation 
. Furthermore, exposure of quercetin to HepG2 cells transfected with CAR showed that CAR can be activated by quercetin 
. Our data showed significant up regulation of Car (FC
1.37, FDR adjusted p-value
0.005), which suggests that Car has an important role in quercetin mediated regulation of lipid metabolism.
This study used male mice, therefore caution is needed in translating these data to female mice. It is known that there are sex differences in the sensitivity to CAR activators and also Cyp4a genes can be under sex-dependent control 
. In conclusion, quercetin can affect hepatic lipid metabolism, especially ω-oxidation. This is shown by the up regulation of Cyp4a10, Cyp4a14, Cyp4a31, Acot3, Por and the transcription factor Car. These effects are associated with decreased corresponding circulating lipid levels, which may contribute to potential beneficial effects on CVD.