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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Recent epidemiological studies have shown a positive association of a high-fat diet with the risk of colon cancer. Indeed, increments in the serum levels of triglycerides (TG) and cholesterols are positively related with colon carcinogenesis. We previously reported that an age-dependent hyperlipidemic state is characteristic of Min mice, an animal model for human familial adenomatous polyposis (FAP). However, qualitative and quantitative changes of lipid metabolism are poorly understood in this state. Here, we provide detailed analysis of serum lipids in Min mice using reverse-phased liquid chromatography/electrospray ionization mass spectrometry (RPLC/ESI-MS). We also demonstrate local analysis of lipid droplets in the villi of the small intestine using laser capture microdissection and a sensitive chip-based nanoESI-MS system. As a result, oxidized phosphatidylcholines (PC) such as aldehyde and carboxylic acid types were increased, even at an early stage of intestinal polyp formation in serum. In addition, hydroperoxidizable TG precursors containing linoleic acid (18:2n-6) were deposited at the tip of the villi with aging, and these hydroperoxidized TG were also increased in serum. Meanwhile, increments of the oxidizable TG precursors in serum and small intestinal mucosa were suppressed by treatment with pitavastatin, a novel third generation lipophilic statin. These results suggest that quantitative and qualitative lipid changes such as hydroperoxidizable TG precursors are important in the course of intestinal polyp formation and oxidative stress might lead to the development of intestinal polyp formation in Min mice. (Cancer Sci 2011; 102: 79–87)

The incidence and mortality of colon cancer, which is associated with obesity, a high-fat diet and hyperlipidemia according to several epidemiological studies, has increased in developed countries.(1–5) We previously reported an age-dependent hyperlipidemic state in adenomatous polyposis coli (Apc)-deficient Min and Apc1309 mice, animal models of familial adenomatous polyposis (FAP).(6–8) Min mice develop large numbers of intestinal polyps due to truncation mutation in both alleles of the Apc gene, leading to activation of Wnt signaling to promote cell growth, which is increased by consumption of a high-fat diet.(9)

Although the direct link between Apc-deficiency and hyperlipidemia is yet to be clarified, it is notable that serum triglyceride (TG) levels in Min mice are almost 10 times higher than those observed in wild-type littermates (C57BL/6J) at 20 weeks of age, even though both mice were fed a non-high-fat diet, AIN-76A, including corn oil (5% of total components).(6) Both groups of mice ate almost the same amount of diet, but the mean bodyweight was 16% lower in Min mice compared with that of their wild-type littermates at 20 weeks of age, which might be due to the development of intestinal polyps. Consumption of a high-fat diet is associated with hyperlipidemia and leading obesity.(10) However, Min mice featured hyperlipidemia without a high fat-diet intake or obesity. The reasons for the pathology is correlated with decreased mRNA expression levels of lipoprotein lipase (LPL), which hydrolyzes TG into free fatty acids and monoglyceride, in the liver and small intestine.(11,12) We demonstrated that induction of LPL mRNA by peroxisome proliferator-activated receptor (PPAR)-α and -γ agonists and selective LPL-inducing agent, NO-1886, which lacks potential for activating the PPAR pathways, suppressed the hyperlipidemic status, steatosis of the liver and intestinal polyp formation.(6–8) In addition, we indicated that a number of large lipid droplets were found in the surface epithelial cells of small intestinal polyps by Oil-red O staining and electron microscopy.(13) These results suggest that lipid metabolism changes might play important roles in intestinal polyp formation, but the quantitative and qualitative changes have not been well defined in the serum and small intestine.

In this study, we examined a detailed analysis of serum lipids in Min mice using electrospray ionization-mass spectrometry (ESI-MS) coupled with reverse-phased ultra-performance liquid chromatography (UPLC).(14,15) Moreover, lipid droplets in the villi of the small intestine were collected by laser microdissection (LMD) under direct microscopic visualization and analyzed using a sensitive chip-based nanoESI-MS system by neutral loss scanning of identical fatty acyl groups from individual TG molecular species and precursor ion scanning of phosphoryl choline from individual phosphatidylcholine (PC) molecular species.(16–22) The possible lipid markers for development of intestinal polyps in Min mice are discussed. Furthermore, the relationship between the markers and polyp formation in Min mice are demonstrated by administration of pitavastatin, a novel 3-hydroxy-3-methylglutaryl coenzyme-A (HMGCoA) reductase inhibitor, which possesses pleiotropic functions including anti-inflammation and anti-oxidant functions.(23–26)

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Animals.  Male C57BL/6-ApcMin/+ mice (Min mice) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) at 6 weeks of age and genotyped as previously reported.(27) Heterozygotes of Min strain and wild-type (C57BL/6J) mice were acclimated to laboratory conditions for 1 week. Less than five mice were housed per plastic cage with sterilized softwood chips as bedding in a barrier-sustained animal room at 24°C ± 2°C and 55% humidity on a 12 h light/dark cycle.

To investigate lipid metabolism changes, male Min mice (n = 9) and wild-type mice (n = 6) at 5 weeks of age were given AIN-76A powdered basal diet (CLEA Japan, Tokyo, Japan). Min mice (n = 3) and wild-type mice (n = 2) were killed at 10, 15 and 20 weeks of age. To examine the effects of pitavastatin ([+]-monocalcium bis [3R,5S,6E]-7-[2-cyclopropyl-4-(4-fluoro-phenyl)-3-quinolyl]-3,5-dihydroxy-6-heptenoate), which was kindly provided by Kowa Pharmaceutical Co., Ltd (Aichi, Japan), male Min mice at 6 weeks of age were given pitavastatin, which was mixed well at concentrations of 40 p.p.m. for 14 weeks in AIN-76A. Min mice with pitavastatin treatment (n = 3) and with pitavastatin untreatment (n = 3) were killed at 20 weeks of age. Food and water were available ad libitum. The mice were observed daily for clinical signs and mortality. Bodyweights and food consumption were measured weekly. The experimental protocol was in accordance with the guidelines for Animal Experiments in the National Cancer Center and was approved by the Institutional Ethics Review Committee for Animal Experimentation.

Materials.  All solvents for HPLC or MS grade were purchased from Wako Pure Chemicals (Osaka, Japan). Deionized water was obtained from a Milli-Q water system (Millipore, Milford, MA, USA).

Extraction and isolation of serum and small intestinal mucosa lipids.  Total lipids from serum and small intestinal mucosa were extracted using Bligh and Dyer’s method in the following procedure:(28) serum (75 μL) and small intestinal mucosa in the proximal segments (50 mg) removed by scraping were individually homogenized with 6 mL chloroform/methanol (1:2) for 10 strokes and left for 1 h at room temperature. In this process, 1 nmol of sphingomyelin (SM) (d18:1-12:0) was added as an internal standard. Each phase separation was achieved by adding 2 mL chloroform and 2 mL water. After vortexing, the mixture was centrifuged at 1000g. for 10 min. The bottom organic layer containing the total lipid extract was collected and dried under a gentle stream of nitrogen, and then dissolved by 200 μL chloroform-methanol (2:1) and normalized to the serum volume and intestinal mucosa weight.

Laser capture microdissection of the small intestine.  The intestinal tracts of male Min mice at 10 and 20 weeks old (n = 3) were removed and separated into the small intestine. Each small intestine was divided into the proximal segment (4 cm in length), and then opened longitudinally and fixed flat between sheets of filter paper in 10% buffered formalin for further Oil-red O staining and LMD.(6) Lipid droplets of villi in the small intestine were observed using modified Oil-red O staining method for confirmation of the locus.(29) In brief, each frozen section as Swiss rolls was treated with 60% isopropanol for 1 min, and stained with Oil-red solution for 15 min at room temperature. The dye was then removed, and the section was washed with PBS and 60% isopropanol each for 2 min. For local analysis of the villi, each frozen section was mounted on DIRECTOR LMD slide (AMR Inc., Tokyo, Japan), and lipid droplets from the tip and basis of the villi were collected by Leica LMD system, LMD 6000 (Leica Microsystems, Wetzlar, Germany) with a pulsed 355-nm diode laser and individually extracted by methanol.(22) The correction intensity in each graph was calculated by the ratio of TG to the endogenous standard (16:0-18:2 PC).

Reverse-phased liquid chromatography/ESI-MS (RPLC/ESI-MS) conditions.  The RPLC/ESI-MS analysis was performed using a quadrupole/time-of-flight hybrid mass spectrometer, Q-TOF micro (Waters Corporation, Milford, MA, USA) with an ACQUITY UPLC system (Waters Corporation).(14) The scan range of the instrument was set at m/z 200–1100, the scan duration of MS and MS/MS was at 0.5 s and the collision gas used for the MS/MS experiments was at 7.5 × 105 mbar (argon). The capillary voltage in positive ion mode was set at 3.5 kV, cone voltage at 30 V and collision energy of MS/MS at 30 V, whereas capillary voltage was at −2.5 kV, cone voltage at −30 V and collision energy of MS/MS at −30 V in negative ion mode.

The RPLC separation was achieved using an ACQUITY UPLC BEH C18 column (150 × 1.0 mm inner diameter [i.d.], Waters Corporation) at 45°C. Two microlitre of total lipids normalized to serum volume was individually injected. The mobile phase was acetonitrile/methanol/water : 19/19/2 (0.1% formic acid + 0.028% ammonia) (A) and isopropanol (0.1% formic acid + 0.028% ammonia) (B), and the composition was produced by mixing these solvents. The gradient consisted of holding solvent (A/B : 90/10) for 7.5 min, then linearly converting to solvent (A/B : 70/30) for 32.5 min and finally linearly converting solvent (A/B : 40/60) for 50 min. The mobile phase was pumped at a flow rate of 40–50 μL/min. The MS data processing was applied by Mass++ software (http://masspp.jp/) to detect each chromatogram peak with quantitative accuracy. The correction intensity in each graph was calculated by the ratio of TG or phosphatidylcholine (PC) to the internal standard.

Multiple reaction monitoring (MRM) conditions.  The MRM analysis was performed using a quadrupole-linear ion trap hybrid mass spectrometer, 4000Q TRAP (AB SCIEX, Foster City, CA, USA) with the same ACQUITY UPLC system as previously reported.(15) Ten microlitre of total lipids normalized to serum volume was individually injected. The mobile phase was acetonitrile/methanol/water : 2/2/1 (0.1% formic acid + 0.028% ammonia) (A) and isopropanol (0.1% formic acid + 0.028% ammonia) (B), and the composition was produced by mixing these solvents. The gradient consisted of holding solvent (A/B : 100/0) for 5 min, then linearly converting to solvent (A/B : 50/50) for 20 min and finally holding solvent (A/B : 50/50) for 34 min at a flow rate of 70 μL/min and column temperature of 30°C.

NanoESI-MS conditions.  Chip-based nanoESI-MS analysis was performed using a 4000Q TRAP with chip-based ionization source, TriVersa NanoMate (Advion BioSystems, Ithaca, NY, USA). The ion spray voltage was set at 1.25 kV, gas pressure at 0.3 pound per square inch (psi) and flow rates at 200 nL/min. The scan range was set at m/z 400–1100, declustering potential at 100 V, collision energies at 50–70 V and resolutions at Q1 and Q3, “unit”. The mobile phase composition was chloroform/methanol: 1/2 (0.1% ammonium formate). Total lipids were directly subjected by flow injection and selectivity analyzed by neutral loss scanning of identical fatty acyl groups from individual TG molecular species and precursor ion scanning of phosphorylcholine from individual PC molecular species.(16–21)

Statistical analysis.  The student’s t-test was used for statistical analysis. Values with P < 0.05 were considered to be statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Global analysis of serum lipids from Min mice by 2-D profiling.  Intestinal polyp counts in Min mice were obviously increased with age compared with wild-type mice due to downregulation of LPL expression. Indeed, the average number of intestinal polyps developed in Min mice was 82.4 ± 8.4 (mean ± SE). However, none of the wild-type mice developed intestinal polyps.

To further examine the effects on lipid metabolism at the molecular species level, we globally analyzed serum lipids at 10, 15 and 20 weeks of age by RPLC/ESI-MS and then created 2-D lipid maps of the individual precursor ion peaks for searching quantitative and qualitative changes. The 2-D lipid maps were constructed with X (retention time) and Y (m/z value) axes, and the intensity of these peaks was adjusted by color density spots.

As a result, TG increments in Min mice were detected on the map of positive ion mode (55–70 min) even at 10 weeks of age and were markedly found at 20 weeks of age (Fig. 1). In addition, several spots were abundantly observed on the map of negative ion mode (5–15 and 45–55 min) in Min mice compared with wild-type mice (Fig. 2). These distinctive spots were analyzed by MS/MS and respectively identified as oxidized PC and TG, which we first detected from mouse TG in white adipose tissue as previously reported.(14) At 20 weeks of age, these oxidized PC and TG were highly increased in Min mice, suggesting that enhancement of oxidative stress might be caused in the course of intestinal polyp formation.

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Figure 1.  2-D profiling analysis of serum lipids from Min mice by reverse-phased liquid chromatography/electrospray ionization mass spectrometry (RPLC/ESI-MS) in positive ion mode. Each of the lipid classes was detected in the following order: lysophosphatidylcholine (lysoPC) > phosphatidylcholine (PC) > triglyceride (TG), cholesteryl ester (CE). The TG of Min mice were increased at 10 weeks of age and highly elevated at 20 weeks of age. 2-D lipid maps were constructed with X (retention time) and Y (m/z value) axes, and the intensity of these peaks was adjusted by color density spots.

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image

Figure 2.  2-D profiling analysis of serum lipids from Min mice by reverse-phased liquid chromatography/electrospray ionization mass spectrometry (RPLC/ESI-MS) in negative ion mode. Each of the lipid classes was detected in the following order: oxidized phosphatidylcholine (PC), lysophosphatidylcholine (lysoPC), fatty acid (FA) > PC > sphingomyelin (SM), ceramide (Cer), oxidized triglyceride (TG) > TG. Oxidized PC and TG of Min mice were highly increased even at 10 weeks of age and highly elevated at 20 weeks of age.

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Quantitative and qualitative changes of serum TG molecular species of Min mice in the course of intestinal polyp formation.  These spots were more precisely quantified by Mass++, MS data processing software to detect individual chromatogram peaks with quantitative accuracy. In Min mice, serum TG molecular species such as hydroperoxidized (OOH) TG, which were derived from peroxidation of intramolecular linoleic acid (18:2n-6), and hydroperoxidizable TG precursors containing 18:2, which were oxidizable to the hydroperoxidized TG, were increased in a time-dependent manner (Fig. 3A). In particular, hydroperoxidizable TG precursors were highly elevated from 15 weeks of age. Meanwhile, non-oxidizable TG containing saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) were quantitatively unchanged until 15 weeks of age compared with wild-type mice and somewhat increased at 20 weeks of age. These results suggest that individual TG molecular species were not uniformly elevated, but oxidant-related TG were preferentially increased. As for the TG ratios of Min mice to wild-type mice, hydroperoxidized TG ratios were elevated in a time-dependent manner, and hydroperoxidized TG of Min mice at 20 weeks of age were 30–50 times larger than those of wild-type mice (Fig. 3B). Hydroperoxidizable TG precursor ratios were highly increased at 15 weeks of age and then somewhat decreased at 20 weeks of age, whereas non-oxidizable TG ratios were gradually elevated in a time-dependent manner. There were significant differences between hydroperoxidized TG ratios and non-oxidizable TG ratios at 20 weeks of age, and between hydroperoxidizable TG precursor ratios and non-oxidizable TG ratios at 15 weeks of age. It seems that these ratios might be effective indications for the pathology of intestinal polyp formation.

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Figure 3.  Quantitative and qualitative profiles of serum triglyceride (TG) molecular species of Min mice in the course of intestinal polyp formation. (A) Compared with wild-type mice, hydroperoxidized TG and hydroperoxidizable TG precursors of Min mice were highly increased from 15 weeks of age, whereas non-oxidizable TG of Min mice were somewhat elevated at 20 weeks of age. Values are mean ± SD (n = 3; *P < 0.05, **P < 0.01 versus correction intensity of Min mice at 10 weeks of age). (B) Regarding ratios of Min mice to wild-type mice in serum TG molecular species, hydroperoxidized TG ratios were elevated in a time-dependent manner, and hydroperoxidized TG of Min mice at 20 weeks of age were 30–50 times larger than those of wild-type mice. Hydroperoxidizable TG precursor ratios were highly increased at 15 weeks of age and then somewhat decreased at 20 weeks of age, whereas non-oxidizable TG ratios were gradually increased in a time-dependent manner. Values are mean ± SD (n = 3; *P < 0.05, **P < 0.01 versus intensity ratio at 10 weeks of age) and the individual mean values of these TG are indicated by crossbars.

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Quantitative and qualitative changes of serum PC molecular species of Min mice in the course of intestinal polyp formation.  Regarding serum PC molecular species, hydroperoxidizable PC precursors containing 18:2 and non-oxidizable PC containing SFA were quantitatively unchanged between Min and wild-type mice at 10 and 20 weeks of age (Fig. S1). Meanwhile, oxidized PC of Min mice were abundantly observed on the 2-D map, and then a large variety of these oxidized types such as OOH, aldehydes (CHO) and carboxylic acids (COOH) were analyzed in detail by MRM with theoretically expanded data sets as previously reported.(15) These oxidized types were derived from enzymatic and non-enzymatic reactions of peroxidation of polyunsaturated fatty acids (PUFA) such as 18:2, arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3) at the sn-2 position and important in inflammatory biomarkers for the physiological and pathological phenomena.(30,31) Interestingly, aldehyde and carboxylic acid types derived from peroxidation of 18:2, 20:4 and 22:6 were increased even at 10 weeks of age in Min mice (Fig. 4A), indicating that oxidative stress might occur at the early stage of polyp formation. Besides these aldehyde and carboxylic acid types, hydroperoxide types were highly elevated at 20 weeks in Min mice (Fig. 4B). In addition, PC ratios of Min mice to wild-type mice in these oxidized types were increased at 20 weeks of age compared with 10 weeks of age in the following order: carboxylic acid types > aldehyde types > hydroperoxide types (Fig. 4C). These data support the ideas that Min mice at 20 weeks of age were at a high oxidative stress level and these oxidants might also be effective indications of the exacerbation stage and inflammatory state.

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Figure 4.  Quantitative and qualitative profiles of serum oxidized phosphatidylcholine (PC) molecular species of Min mice in the course of intestinal polyp formation. (A) Oxidized PC derived from peroxidation of polyunsaturated fatty acids (PUFA) such as linoleic acid (18:2n-6), arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3) at the sn-2 position were analyzed by multiple reaction monitoring. Aldehyde (CHO) and carboxylic acid (COOH) types of Min mice (n = 3) were increased even at 10 weeks of age compared with wild-type mice (n = 2). Values are mean ± SD. (B) In addition to these types, hydroperoxide (OOH) types of Min mice (n = 3) were elevated at 20 weeks compared with wild-type mice (n = 2). Values are mean ± SD. (C) The PC ratios of Min mice to wild-type mice were highly increased at 20 weeks of age compared with 10 weeks of age in the following order: carboxylic acid types > aldehyde types > hydroperoxide types. Individual mean values of these types are indicated by crossbars.

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Local analysis of lipid droplets in the villi of Min mice.  Because serum lipid contents might be affected by dietary fat absorption in the small intestine, detailed analysis of the villi were investigated at 10 and 20 weeks of age in Min mice by LMD, which permits procurement of the locus from frozen sections of Swiss-rolled middle and distal parts of the small intestines. Lipid accumulation was observed at the tip of the villi in the small intestine with Oil-red O staining and the locus was collected. These lipid mixtures were individually extracted by methanol from the collected tissues and analyzed using a chip-based nanoESI-MS system. As a result, PC molecular species were quantitatively and qualitatively unchanged at 10 and 20 weeks of age in Min mice (Fig. S2). Meanwhile, hydroperoxidizable TG precursors were more abundant at 20 weeks of age than those at 10 weeks of age in Min mice (Fig. 5A). Moreover, the TG at the tip of the villi were 1.5–2.5 times larger than those at the basis at 20 weeks of age (Fig. 5B). These results suggest that hydroperoxidizable TG precursors were comparatively deposited at the tip of the villi with age in Min mice and might be subjected to oxidative stress and inflammation in the small intestine. As discussed previously, hydroperoxidized TG and hydroperoxidizable TG precursors in the serum of Min mice were increased at 20 weeks of age, which might be caused by the increments of hydroperoxidizable TG precursors at the tip of the villi.

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Figure 5.  Local analysis of lipid droplets in Min mice by laser microdissection and the nano-electrospray ionization-mass spectrometry (nanoESI-MS) system. (A) Hydroperoxidizable triglyceride (TG) precursors at villous tips and bases of Min mice were more increased at 20 weeks of age than 10 weeks of age. Values are mean ± SD (n = 3; *P < 0.05, **P < 0.01 versus correction intensity at 10 weeks of age). (B) From hydroperoxidizable TG precursor ratios of villous tips to villous bases, hydroperoxidizable TG precursors of villous tips were 1.5–2 times larger than those of villous bases at 20 weeks of age. Values are mean ± SD (n = 3).

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Suppression of hydroperoxidizable TG precursor production in the serum and small intestinal mucosa of Min mice by pitavastatin treatment.  To examine the relationship between oxidative stress and polyp formation in Min mice, detailed analysis of total lipid extract from the serum and small intestinal mucosa of Min mice treated with pitavastatin at doses of 40 p.p.m. for 14 weeks were investigated by a chip-based nanoESI-MS system. Intestinal polyp formation in the pitavastatin-treated groups was significantly suppressed (the data will be presented in a separate report). The PC molecular species were quantitatively and qualitatively unchanged between the pitavastatin-treated and untreated groups (data not shown). Meanwhile, the intensity ratios of hydroperoxidizable TG precursors to non-oxidizable TG (16:0-18:1-18:1) were significantly decreased in the serum and intestinal mucosa of the pitavastatin-treated groups compared with the untreated groups (Fig. 6). These results suggest that hydroperoxidizable TG precursors are important in developing intestinal polyp formation and hydroperoxidized TG derived from the oxidizable TG precursors might induce oxidative stress and inflammation.

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Figure 6.  Suppression of hydroperoxidizable triglyceride (TG) precursor production in serum and the small intestinal mucosa of Min mice by pitavastatin treatment. (A) Intensity ratios of hydroperoxidizable TG precursors to non-oxidizable TG (16:0-18:1-18:1) were significantly decreased in the serum of the pitavastatin-treated groups at doses of 40 p.p.m. for 14 weeks along with suppression of intestinal polyp formation compared with the untreated groups. (B) Similarly, the intensity ratios in the intestinal mucosa of the pitavastatin-treated groups were significantly reduced. Values are mean ± SD (n = 3; *P < 0.05, **P < 0.01 versus intensity ratios of the pitavastatin-untreated groups).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

In this study, oxidant-related TG and oxidized PC such as aldehyde and carboxylic acid types were increased even at the early stage of intestinal polyp formation in the serum of Min mice. The oxidized PC of Min mice were highly elevated even at 10 weeks of age. In particular, hydroperoxidized TG and hydroperoxidizable TG precursors were significantly increased from 15 weeks of age in a time-dependent manner. We previously reported that Min mice had higher levels of 18:2 in plasma compared with wild-type mice by gas–liquid chromatography method.(32) These results suggest that oxidant-related TG in the blood increased in the course of intestinal polyp formation, and these TG might be useful as biological markers for the formation and development of intestinal polyps.

The present study also showed that hydroperoxidizable TG precursors were comparatively deposited at the tip of villi with age in Min mice from our local analysis using LMD and sensitive chip-based nanoESI-MS system. Dietary fat mainly constituted of TG is absorbed at the tip of the villi and accumulates in the cytoplasm of intestinal epithelial cells, but not in stromal cells. The origin of the accumulated lipids seems to be derived from the contents in the digestive tract, not from blood vessels. Therefore, our detailed analysis of the villi in Min mice is thought to be effective for directly detecting qualitative and quantitative changes. To our knowledge, local lipid analysis of the villi has not yet been reported and accumulation of hydroperoxidizable TG precursors with age was first detected in Min mice. In addition, detailed analysis of serum and the intestinal mucosa in Min mice treated with pitavastatin revealed that hydroperoxidizable TG precursors were significantly decreased along with suppression of intestinal polyp formation. It is speculated that accumulation of hydroperoxidizable TG precursors in the small intestine mucosa leads to an increased serum level of hydroperoxidized TG, and oxidative stress and inflammation might be systemically induced by these oxidized TG. For one reason, the fact that malabsorption of the hydroperoxidizable TG precursors occurs at the tip of the villi, which might evoke a source for increments of hydroperoxidized TG in serum. For another reason, hydroperoxidizable TG precursor increments at the tip of the villi lead to an elevated influx into the general circulation, and hydroperoxidized TG might be abundantly generated by peroxidation of these precursors. Moreover, hydroperoxidized TG derived from these precursors could induce DNA damage, which might link to truncation mutation in both alleles of the Apc gene. However, further experiments are needed to sensitively detect oxidized PC and TG from the small regions of the villi by our local analysis, and to clarify the role of hydroperoxidized TG in intestinal polyp formation.

In conclusion, hydroperoxidizable TG precursors of Min mice in serum and the small intestine mucosa were increased in the course of intestinal polyp formation, and increase of these oxidizable TG precursors was suppressed by pitavastatin. These oxidant-related lipids might be useful as biological indications for the formation and development of intestinal polyps. Furthermore, hydroperoxidized TG, hydroperoxidizable TG precursors and oxidized PC such as aldehyde and carboxylic acid types were highly increased even at 10 weeks of age, and these lipids might be possible markers for early intestinal polyp formation. These results also suggest that qualitative changes of TG and PC are important in the course of intestinal polyp formation and might lead to their development in Min mice.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

The authors thank Reiko Yajima for technical assistance. This study was performed with the help of Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST).

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Fig. S1. Quantitative and qualitative profiles of serum phosphatidylcholine (PC) molecular species of Min mice in the course of intestinal polyp formation. The PC molecular species in the serum of Min mice (n = 3) were quantitatively and qualitatively similar to wild-type mice (n = 2). Values are mean ± SD. The PC containing docosahexaenoic acid (22:6n-3) was not included in the AIN-76A diet and the correction intensities were very small.

Fig. S2. Local analysis of phosphatidylcholine (PC) molecular species at the villous tips and bases of Min mice by laser microdissection and the nano-electrospray ionization-mass spectrometry (nanoESI-MS) system. The PC molecular species at the villous tips and bases of Min mice were quantitatively and qualitatively unchanged at 10 and 20 weeks of age. There was no significant difference between the villous and base values. Values are mean ± SD (n = 3). The PC containing 22:6 was not included in the AIN-76A diet and the intensities were very small.

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