The Effects Of Recombinant Human Growth Hormone On Albumin Synthesis Rate During Surgical Trauma And Intra-abdominal Infection

Part I The method establishment for albumin fractional synthesis rate measurement employing stable isotope tracerObjectives:Improve methods for the albumin purification and hydrolysis; explore conditions and methods for gas chromatography and mass spectrometry analysis and detection; establish methods for the measurement of albumin fractional synthesis rate by employing stable isotope tracer.Methods:1. The serum albumin was purified by using trichloroacetic acid / ethanol, identification of the purity deployed SDS-PAGE, the protein quantification employed Bradford method; amino acids quantification in hydrolysis fluid used HPLC method, and solid phase extraction method was applied for prior derivation treatment of phenylalanine.2. MSTFA was applied for the derivation of amino acids, sample solved in 40ul n-heptane and inlet into GC for 1ul.3. Compared with the spectral library, we found out the feature fragments of derivatives, then established the expression method for relative enrichment according to the analysis for areas under mass to charge M +5, M +2 and M +0.4. Determination of the fractional synthesis rate of albumin used standard curve method, according to the change to time of isotopic relative enrichment of TTR in albumin and precursor pool, FSR (Fractional Synthesis Rate) was calculated.Results:Extracted and purified albumin were identified by using SDS-PAGE, and it was found that there was a high quality strip at 66 KD; albumin concentration was measured by employing Bradford method, and the quantified outcome was about 0.9mg/ml; the amino acids concentration quantified by using HPLC was about 0.6umol/L; the derivation product N,O-Bis(trimethylsily)-L-phenylalanine and N,O-Bis(trimethylsily)-[2H5]L-phenylalanine were from natural L- phenylalanine and [2H5]-L-phenylalanine respectively, and 197/194 (m/z) was alternative for indicating relative enrichment; the tracer to tracee ratio of phenylalanine in precursor pool achieved the plat phase 1 hour after infusion, and linear increasing came out in the TTR incorporated into albumin; based on that, fractional synthesis rate were calculated in rabbit which had been given parenteral nutrition, and the value was 17.26±0.92 (n=8).Conclusions:1. The albumin purification, hydrolysis methods and amino acids derivation satisfied the requirement for albumin FSR determination2. The detecting conditions and analysis methods for the measurement of stable isotope tracer under GC / MS were reliable.3. The methods for albumin synthesis rate determination employing stable isotope tracer [2H5]-L-phenylalanine were feasible, and supported the following research which assess the effects on albumin FSR during the surgical and infection stress. Part II The effects of recombinant human growth hormone on albumin synthesis rate during surgical trauma and intra-abdominal infectionObjectives:Improve the New Zealand rabbit cecal ligation and puncture model, and establish the nutrition infusion and blood extraction methods; the effects on the albumin fractional synthesis rate during intra-abdominal infection and surgical stress would be evaluated by using the established isotope tracer methods; assess the role of recombinant growth hormone on albumin anabolism during the stress flow phase.Methods:1. Peritoneal infection model used the improved cecal ligation and puncture method, and intra-abdominal infection drainage operation was carried out for the infection removal; external jugular vein catheter and a spring fixed method were respectively applied for blood extraction and parenteral nutrition infusion.2. Sixteen New Zealand rabbits were randomly divided into four groups including normal group, sham-operated group, intra-abdominal infection and growth hormone intervention group, and intra-abdominal infection drainage operation was done in intra-abdominal infection and growth hormone intervention group after the isotope infusion experiment for the first time. Four groups were supported by total parenteral nutrition, and changes of body weight and serum albumin concentration were observed.3. Animals were given recombinant growth hormone by 0.3u/kg to intervention group after the first infusion experiment at 0:00, while others were given placebo.4. Stable isotope [2H5] L-phenylalanine solution was sterilized and got rid of pyrogen. According to the dose to weight, isotope was mixed with parenteral nutrition and pumped into the body by ear vein. From the beginning of the isotope infusion experiment, 3ml blood samples were reserved hourly until the experiment finished six hours later.5. The calculation of albumin fractional synthesis rate used standard curve method, and the change to time of relative enrichment TTR in albumin and precursor pool was obtained for calculating FSR, then compared the values intra-group and inter-group. Results:In our research, it was found that intra-abdominal adhesion was severe, mesenterium congestion and edema appeared, and abdominal cavity purulent effusion showed up; the changes of the animal body weight and serum albumin concentration were observed, and it was found that the 4th day body weight and serum albumin concentration were significantly declined in contrast to that preoperatively in intra-abdominal infection group (p values were 0.037 and 0.001 respectively); compared intra-group, it was found that 4th day albumin FSR was significantly higher than that of growth hormone group in the first day (p=0.006); compared inter-group, it was found that the first day albumin FSR of four groups had significant differences (F=33.687,p<0.001), and the FSR of the sham-operated group, intra-abdominal infection group and the growth hormone group was significantly higher than that of normal group(p<0.05); abdominal infection group and the growth hormone group was also higher than the sham-operated group (p<0.001); the fourth day FSR inter-group had significant differences (F=65.794, P<0.001), and intra-abdominal infection group was higher than the normal group (p=0.001) and sham operation group (p=0.002); growth hormone group was higher than that of other three groups (p<0.001).Conclusions:1. Intra-abdominal infection was effectively formed by using improved puncture cecal ligation method, the improvement of blood extraction and nutrition infusion equipment met needs of the experiment2. The albumin synthesis rate sped up in surgery and intra-abdominal infection stress early shock phase, the overlay effects increased albumin anabolic and catabolic rate to a higher level.3. Albumin synthesis rate maintained a high level in surgery and intra-abdominal infection stress flow phase, since the anabolic rate was even higher, the level of serum albumin concentration could not be kept at this time.4. The recombinant growth hormone applied for more than 3 days increased rabbit albumin synthesis rate during intra-abdominal infection stress flow phase, corrected albumin metabolic imbalance, and avoided concentration further declining.