| Objective:To investigate the effects of heavy water(2H2O)as a tracer on the kinetics of plasma albumin synthesis under lysine intake(70 mg/kg/d).The result of the required amount is 70mg/kg/d,and the lysine requirement of 74.2±13.1mg/kg/d is obtained from the topic "Effects of plasma protein metabolism on healthy young males in daily dietary lysine intake".To compare and explore the experimental methods and provide theoretical basis for the development of recommended intakes of dietary essential amino acids in different populations in China.Method:1.Dietary recipe design,-H2O labeling and food sample testingThe reference books for dietary design are food composition table,dietary guide for Chinese residents,2016 edition,and design recipes.Recruitment of 5 healthy young men with light physical activity,age 21±1.73 years old,body mass index(BMI):21.99±1.72kg/m2.The theoretical value of the subject’s intake of lysine was set at 70 mg/kg/d.Adaptation period:1-5 weeks.Five subjects ate according to a preset experimental dietary recipe and performed a body measurement every weekend.Marking period:6-10 weeks.On the first day of the 6th week,5 subjects took 70%2H2O after breakfast,60mL each time,6 times/day.During the next 5 weeks,subjects took 70%2H2O 60 mL twice everyday(after breakfast,after dinner);body measurements were taken every weekend.The blood samples of the subjects were collected one day before the labeling period as a baseline blood sample at the marking period of 6-10 weekends.Blood samples were collected,centrifuged,and stored in a-70 0C refrigerator.A three-day meal survey(chemical analysis)was performed every week.Collect the samples,break it up and mix well,and store the processed samples in a-20 0C refrigerator.Determine and calculate protein,fat,ash,moisture,carbohydrate,energy,and lysine content in food samples.Calculate the total energy,the energy supply ratio of the three major nutrients,and the ratio of the high quality protein.2.Plasma protein,lysine concentration detectionPlasma albumin and total protein were detected by enzyme-linked immunosorbent assay.Plasma lysine content was determined by high performance liquid chromatography.3.Determination of plasma albumin synthesis kinetics parameters in healthy young males by heavy water labeling40 uL of plasma sample,2μL of 10 mol/L NaOH,and 4μL of 5%(voL/voL)acetone in acetonitrile were mixed and allowed to stand for 24 hours to exchange 2H in water with acetone.0.6 mL of chloroform and 0.5 g of Na2SO4 were added to extract acetone.The chloroform extract was analyzed by gas chromatography-mass spectrometry(GC-MS)to detect the body water abundance marked by 2H.Plasma albumin was purified by trichloroacetic acid/ethanol method and identified by polyacrylamide gel electrophoresis.The purified albumin was hydrolyzed and derivatized by gas chromatography-mass spectrometry.The instrument detects the abundance of 2H-alanyl in plasma albumin and 2H in plasma free alanine.The fractional synthesis rate(FSR)and synthetic kinetic equation of albumin were calculated4.Comparison with the free diet phaseWhether the three major nutrient intakes,energy supply ratio,plasma albumin,total protein concentration and albumin fraction synthesis rate have significant differences under the condition of free diet and control of lysine diet,whether the results of the previous project meet the human body Physiological needs.Result:1.The nutrient and energy intake of the SubjectsThe energy intake of 5 subjects was 2628.50±123.01kcal;the protein energy supply ratio was 13.30±1.21%,the quality protein accounted for 47.43±3.74%;the carbohydrate energy supply ratio was 61.97±2.81%;the fat supply ratio was 24.43±2.87%.The lysine intake was 68.06±2.32mg/kg/d,which was lower than the daily dietary lysine intake state of 74.2±13.2mg/kg/d.2.Body measurement resultsThe average body weight(kg)before 2H2O labeling and 2H2O labeling were:68.00±4.60,68.74±4.12;average muscle rate(%):37.5±2.62,36.30±3.93;average fat percentage(%):22.5±3.26,22.56±3.51;mean basal metabolism(kcal):1524± 123.21,1539.721106.53,there was no significant difference between the above indicators before and after 2H2O labeling(p>0.05).There was no significant difference between the above indicators before and after the 2H2O labeling period(p>0.05).3.Detection of plasma protein and lysine concentrationThe mean plasma total protein concentration(mg/mL)of the 5 subjects before and during 2H2O labeling period was:68.84±9.89,73.13±8.09,there was no significant difference between the two periods(p>0.05),and the total protein concentration was in the normal range.The plasma albumin concentration(mg/mL)was 34.82±5.43 and 35.69±3.29 before and after 2H2O labeling.The plasma albumin concentration of the first subject was slightly lower than the normal value.The plasma albumin concentrations of other subjects were within the normal range,and there was no significant difference before and after 2H2O labeling(p>0.05).4.Detection of plasma albumin synthesis kinetics parameters in healthy young men by heavy water labelingThe plasma albumin fraction synthesis rate(%/week)of the five subjects was 27.3,25.4,29.7,29.9,28.9,respectively.Albumin synthesis kinetic equation"No.1 ft=0.1348 ×(1-e-0.426t),No.2ft=0.1306 ×(1-e-0.471t),No.3ft=0.1354 ×(1-e-0.473t),No.4ft=0.1302×(1-e-0.151t),No 5 ft=0.1330×(1-e-0.488t).5.Comparison with data from the free-meal phaseThere was a significant difference in the lysine intake of the subjects before and after the dietary intervention(p<0.05).There was no significant difference in protein intake and energy supply ratio(p>0.05),and there was a significant difference in fat,carbohydrate intake and energy supply ratio(p<0.05).There was no statistical difference in plasma total protein concentration and albumin concentration(p>0.05).Subjects had significant differences in albumin FSR before and after dietary intervention(p<0.05).Conclusion:Under the control of dietary lysine intake,the lysine intake of the subjects was significantly lower than that of the daily dietary lysine intake(p<0.05),and the FSR of plasma albumin was also significantly lower than the daily dietary status(p<0.05).This indicates that 70 mg/kg/d does not meet the physiological requirements of healthy young male subjects. |
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