Content Determination And Effects Of Compound Choline And Betaine Methyl Nutrient On Hyperhomocysteinemia In Rats

BackgroundHyperhomocysteins（HHcy）is an independent risk factor for cardiovascular and cerebrovascular diseases.Folic acid and/or vitamin B12 tablets are commonly used for clinical intervention in patients with homocysteine（Hcy）over 15μmol/L.The compound methyl nutrient preparation with choline or betaine as the main component developed in this study can be intervened from three pathways（see Figure 1）,so as to reduce Hcy level more effectively.Therefore,it can be added from the diet to play a preventive and auxiliary role in reducing Hcy.ObjectiveThe content of vitamin B6,vitamin B9（folic acid）and vitamin B12 in methyl nutrient tablets was determined by high performance liquid chromatography,and the content of betaine in compound betaine solid beverage was determined by perchloric acid non-aqueous method.Investigate the effect of methyl nutrients characterized by choline and betaine on hyperhomocysteinemia in rats,and provide data support for the methyl nutrient dietary formulations to prevent hyperhomocysteinemia.MethodsUsing Kromasil C18（250 mm×4.6 mm,5μm）chromatographic column to test vitamin B6and vitamin B9,the column temperature was 25℃,the mobile phase was acetonitrile:sodium heptanesulfonate solution（0.003 mol/L）（20:80）.The detection wavelength was 280 nm and the flow rate was 1.0 ml·min^-1.The chromatographic column is Kromasil C18（250 mm×4.6 mm,5μm）,the column temperature is 25℃,the mobile phase is methanol:sodium dihydrogen phosphate（0.01 mol/L）（25:75）,the detection wavelength is 361 nm,The content of vitamin B12was detected under the condition of a flow rate of 1.0 mL·min^-1.Perchloric acid solution（0.1mol/L）non-aqueous titration method was used to detect betaine content.Ninety-six Sprague Dawley rats were randomly divided into 12 groups,and they were divided into two groups,namely the prevention group 6 groups and the treatment group 6 groups.Modeling by feeding 2%methionine feed,folic acid was added to the feed in the prevention group 1,folic acid,vitamin B6 and vitamin B12 in the prevention group 2,folic acid,vitamin B6,vitamin B12 and choline in the prevention group 3,folic acid,vitamin,B6,vitamin B12 and betaine in the prevention group 4,fed for a total of 45 days.The treatment group was fed with2%methionine feed for the first 30 days,and then fed with the corresponding feed supplemented with nutrients for 45 days.The content was detected using the rat homocysteine Elisa kit,and the detection was performed step by step.The microplate reader was at 450 nm wavelength Determine the absorbance（OD value）.ResultsThe concentration of vitamin B₆ within 10～500μg/mL has a good linear relationship with the peak area.The standard curve of B6 is A=303.57C+27.64,and the average content of vitamin B6 in the test solution is 7.718 mg,and the relative deviation is 3.52%;The concentration of vitamin B9 has a good linear relationship with the peak area within 2-20μg/mL.The standard curve of vitamin B9 obtained by plotting is A=507.27C+30.51,and the content of vitamin B9 in the test solution is 0.4142 mg,with an relative deviation It is 1.38%.Vitamin B12 concentration in 2～10μg/mL showed a good linear relationship with the peak area.The standard curve of B₁₂was A=30.55C-60.585and the content of vitamin B12 in 513 mg solution was 3.97μg,equivalent to 0.8g/tablet containing vitamin B₁₂4.94μg,which was compared with the labeled amount of 5μg in prescription.The relative deviation is 1.2%.The betaine product was titrated with perchloric acid standard solution（C=0.0979 mol/L）.The average value of the five results was 5.214%,and the blank 0.2867%was deducted.The result was 4.927%,which was 5%compared with the indicated content of the prescription,and the relative deviation was 0.073%.The formula with choline,betaine and B vitamins as the main components can effectively reduce the hyperhomocysteine in the prevention group,but the treatment group has no significant difference compared with the corresponding model group,and it cannot effectively reduce the high homocysteine content of the treatment group.ConclusionThe method of determination of three B-vitamins by HPLC is rapid,accurate and simple,which provides a guarantee for the accuracy and reliability of the final experimental results,and can be used as the detection method of B-vitamins in dietary formula.Betaine is titrated by perchloric acid glacial acetic acid solution.The method is accurate,simple and maneuverable,and can be used as the detection method of betaine in dietary formula.The experiment on rats showed that methyl rich nutrients with choline,betaine and B vitamins as compound formula had certain preventive effect on hyperhomocysteinemia.Compared with the model group,the results of the prevention group are significantly different,which can effectively reduce the HHcy level of of the prevention group;while the treatment group has no significant difference compared with the corresponding model group,and it cannot effectively reduce the HHcy content of the treatment group.