1. Dietary phosphate restriction induces hepatic lipid accumulation through dysregulation of cholesterol metabolism in mice Nutrition Research Journal May 2013 Sarasa Tanaka, Hironori Yamaoto, Otoki Nakashi, Tomohiro Kagawa Presented by: Caitlyn Parry

2. Phosphorus  Dietary phosphorus occurs as inorganic phosphates (Pi) as well as organic forms.  Most phosphorus absorbed in gastrointestinal tract as inorganic phosphate ions.  Organic phosphates are bound to proteins, sugars and lipids.

3. Introduction  The relationship between inorganic phosphate and cholesterol metabolism has not been thoroughly investigated.  A recent study indicated that triiodothyronine (t3) can induce both hyperphosphatemia and hypocholesterolemia in mice, therefore hypothesizing a linkage between Pi and cholesterol metabolism.  It is suggested that dietary inorganic phosphate plays an important role in the development of fatty liver disease and hyperlipidemia induced by a high-cholesterol diet through lipid metabolism-related gene expression in the liver.

4. Methods  Animals: Eight week old C57BL/6J male mice were maintained on 12-hour light 12-hour dark cycles (0800-2000 hours) with free access to water and food.  Diets: Egg white-based protein  1.2% Pi (Pi-sufficient), 1.2% Pi + Chol (Pi sufficient + Cholesterol), 0.1% Pi (Pi- restricted), 0.1% Pi + Chol (Pi-restricted + Chol)  Mice were randomly divided into these 4 experimental groups of 7 mice each.  Fed 1 of the 4 diets for a period of 12 days.  Mice were sacrificed and blood and liver samples were collected.

6. Methods  One gram of frozen liver homogenized in a mixture of chloroform and methanol.  Used for lipid extraction.  Quantification of hepatic total cholesterol and TG determined by T- cholesterol E-Test and Triglyceride E-Test Wako kits. (Spectrophotometric assay)

7. Methods  Extraction of total RNA, complementary DNA synthesis and real-time polymerase chain reaction (PCR) were completed.  The PCR products were quantified by fit-point analysis.  Results normalized to those for b-actin.

8. Methods: PCR

9. Methods  Plasma levels of Pi, calcium, total cholesterol and total TG were determined using the Phospho C-test, Calcium test, T-cholesterol E-test, and Triglyceride E-test Wako kits.  Blood lipoprotein analysis to determine HDL,LDL and VLDL were performed using high performance liquid chromatography with gel permeation columns using LipoSEARCH.

10. Results  Plasma Pi levels in the Pi-restricted groups were significantly lower than those in the Pi-sufficient groups, and a high cholesterol diet did not effect plasma Pi levels.  Liver weight in Pi-restricted mice was significantly higher than in Pi-Sufficient groups.  High cholesterol diet significantly increased hepatic TG levels in the Pi- restricted + cholesterol, but not in the Pi-sufficient + cholesterol.  No change in plasma TG, chylomicron or HDL levels- increase I plasma VLDL

11. Results  Hepatic CYP7A1 and LDL-R mRNA expression decreased by Pi restriction  High Cholesterol and Pi restriction showed additive inhibitory effect on hepatic mRNA expression of HMGC-R  Hepatic mRNA levels of CPY7A1 were decreased in both Pi restricted and Pi-restricted + cholesterol group.

12. Discussion  This study indicated that dietary Pi plays an important role in cholesterol homeostasis.  Pi does this through regulation of lipid metabolism-related gene expression in the liver and the development of fatty liver induced by a high-cholesterol diet.  This study is the first known to demonstrate the relationship between dietary Pi intake and fatty liver disease.

13. Conclusion  Data suggests that controlling Pi and cholesterol levels in the diet and plasma may prevent the development of fatty liver disease.  Findings may indicate that dietary Pi restriction induces fatty liver disease and hypercholestemia through hepatic CYP7AI expression.  Further research needs to be concluded to determine the validity of this information.

14. References Gropper, S. S. (2012). Advanced nutrition and human metabolism. (6th ed.). Wadsworth Cengage Learning Linus Pauling Institute Higdon, J. (n.d.). Retrieved from http://lpi.oregonstate.edu/infocenter/minerals/phosphorus/http://lpi.oregonstate.edu/infocenter/minerals/phosphorus/ http://www.waters.com/waters/en_US/GPC---Gel-Permeation-Chromatography/nav.htm?cid=10167568&locale=en_US