Effects Of Continuous Veno-venous Hemofiltration On Metabonomics And Protein Dynamics In Critical Ill Patients

Acute kidney injury (AKI) is one of surgical common complications experienced by critical ill patients. About 10%-30% of patients in the intensive care unit develop AKI, and 5%-10% of these patients are treated with renal replacement therapy. As one of most efficient RRT treatment, continuous renal replacement therapy (CRRT) has experienced a huge technological improvement since was first introduced in 1970s. It could rapidly and continuously remove metabolic wastes and inflammatory factors without significant interference of haemodynamics. And continuous veno-venous hemofiltration has become a widely used mode for renal replacement therapy.In this paper, we employed enzyme linked immunosorbent assay, high throughput GC/QMS detection and stable isotopic infusion test to compare metabolic differences between serum and filtrate, which was changed along with time. We also evaluated the difference of nutrient level, clearance of mediators, and fractional synthesis and breakdown rates of large molecular protein between two different CWH dosage treatments. We also employed scanning electronic microscope and loading plot to observe the filter microstructure and to assess the filter efficiency. By all above means, we tried to discuss the specific mechanism of metabolic changes in critically ill patients treated by CWH, and to provide more evidences for therapeutic regime.PART I Effects of continuous veno-venous hemofiltration on cytokines in serum and filtrateObjectives:enzyme linked immunosorbent assay was employed for concentration assessment of TNF-α、IL-10、leptin, adiponectin and TWEAK in serum and filtrate between two dose groups, and for concentration comparison among different time points. The direct effect of filter clearance on these cytokines was also observed.Methods:28 cases of critically ill patients were enrolled from Dec.2010 to Dec.2011 in our general surgery ICU. prospective observation was carried out for demography and laboratory indicators between two groups of patients; ELISA method was used for concentration detecting of TNF-α、IL-10、leptin, adiponectin and TWEAK in serum and filtrate.Results:demography, disease severity score and laboratory indicators showed a balance between different dose groups, except for CRP and PLT. Besides, CRP, PLT, Hb, TP, PT and APTT declined after CVVH treated for 3 days, but albumin level increased, there existed no significant difference in these changes. However, serum urea and creatinine concentration turn out to be significant declined. In cytokines, TNF-α、IL-10、leptin and TWEAK significantly decreased after CWH treated for 3 days, and high dose group declined much more rapidly than low dose group. However, there was no significant change in three days of adiponectin serum concentration.Conclusion:through convection and absorption, CWH could remove most substances which molecular weight below 100KD. CWH caused thrombopenia could be corrected by increase substitution dosage and accelerate blood flow.PART II Effects of continuous veno-venous hemofiltration on cytokines in serum and filtrateObjectives:By comparing two dose groups of serum and filtrate metabolic differences and changes over time, we assessed the CVVH overall loss of nutritional substrate; through the substrate level comparing before and after filter, we assessed the direct impact of semi-permeable membrane on the serum metabolism.Methods:28 cases of critically ill patients were enrolled from Dec.2010 to Dec.2011 in our general surgery ICU. Subjects were randomly assigned into low-dose group (2000ml/ h) and high dose group (4000ml/h); metabolomics analysis methods were used to compare metabolites level between two different dose group in serum and filtrate and the changes over time; loading plot and scanning electron microscopy (SEM) were used for filter microstructure observation and efficiency evaluation.Results:demography, disease severity score and laboratory indicators showed a balance between different dosage groups, except for CRP and PLT. The overall metabolites levels between serum samples and filtrate samples existed a significant different (P<0.05); serum concentrations of glucose, galactose, and maltose within 24h showing significant decreased (P<0.05); serum valine level within 24h was significantly decreased (P<0.05), whereas citrulline level was significantly increased (P>0.05); changes of lipids within 24h was no significant difference (P>0.05); most metabolites could pass off the filter whose serum levels declined; in 72h-used filter, micro-structure of the entry, export and mesh structure has been blocked; substrates in serum and filtrate were significant different between groups (P<0.05), but serum levels become more and more close, and the difference was getting smaller and smaller.Conclusion:Small molecule metabolites in serum could be cleared by convection, but serum abundance is much higher than the filtrate. Glucose, lactose and semilactose could be easily cross over the filtrate and lost; the lean body mass decomposition makes the serum levels of amino acids changed insignificantly, and serum lipids could not be removed. Two dose groups show difference only in the clearance rate, the final level of metabolites level would be similar.PART Ⅲ Effects of continuous veno-venous hemofiltration on protein dynamicsObjectives:by using the prime-constant tracer theory, isotope infusion trials for critical ill surgical patients have been carried out, and the changes of albumin synthesis rate and breakdown rate have also been evaluated; combined with the first two parts, we attempt to discuss albumin metabolic mechanism during CVVH treatment, and to provide experiment evidence for clinical nutrition regime.Methods:By using the isotopic theory, we simultaneously infused two stable isotopes of 1-13C-phenylalanine and d5--phenylalanine; by using gas chromatography mass spectrometry in the selected ion mode, we detected mass to charge ratios of 192,194,197, 218 and 219 and calculate the isotopic enrichment; by using mathematical model, we calculated the expected value of the enrichment, the albumin fractionalsynthesis rate and breakdown rate in both groups.Results:demography, disease severity score and laboratory indicators showed a balance between different dosage groups, except for CRP, urea and creatinine; external standard ion peak and PHE derivatives showed up at the retention time of 7.3 min and 7.6 min respectively; plasma and isotopic enrichment of the d5—phenylalanine and 1-13C-phenylalanine reached a plateau within an hour; when infusion ceased at 4h, d5--phenylalanine isotopic enrichment decreased slowly; albumin hydrolysis isotopic enrichment of 1-13C-phenylalanine showed a linear increase; albumin FSR in CVVH treatment group were significantly higher than control group(P<0.05); there existed no significant difference of albumin FBR among these groups (P> 0.05).Conclusion:Mixed infusion of multi-isotope for simultaneous determination of plasma albumin synthesis and breakdown rate, is an effective and feasible method for albumin dynamic assessment; CVVH could remove metabolic wastes, regulating body fluids and acid-base balance and improve the immune homeostasis, so as to increase albumin synthesis rate; After CVVH treatment, the albumin breakdown rate remains at a high level, so as to give priority to ensuring protein synthesis of vital organs; protein dynamic indicators could be directly and effectively reflecting body protein synthesis and breakdown state.