- Email: [email protected]
Influence of proteasome inhibitors (MG132, ZL3VS, ADAAHX3L3VS) on protein metabolism in incubated rat skeletal muscle
S29
PROTEIN AND AMINO ACID METABOLISM
t&ions of dimethylarginines, as both ADMA and SDMA were eliminated from the systemic circulation in substantial amounts. Fmthemore, evidence for the role of endotoxemia on the metabolism of dimethylarginines was obtained as plasma levels of ADMA wele significantly lower in endotoxemic rats.
107-P. GUT AND LIVER HANDLING OF ASYMMETRICAL (ADMA) AND SYMMETRICAL (SDMA) DIMETHYLARGININE IN THE RAT UNDER BASAL CONDITIONS AND DURING ENDOTOXEMIA R.J. Niiveldt’, T. Teerlink’, A.A. Van Lambalgen3, M.P.C. Siroen’, J.A. Rauwerda’, P.A.M. Van Leeuwen’ ‘Departments of Surgery, 2Clinical Chemistry, 3Physiology, VU University Medical Center, Amsterdam, Netherlands Rationale: Asymmetrical dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase enzymes, whereas symmetrical dimethylxginine (SDMA) competes with arginine transport. Although both dimethylarginines may be important regulators of the arginine-NO pathway, their metabolism is lxgely unknown. We aimed to investigate dimethylarginine handling of the gut and the liver in detail under basal conditions and during endotoxemia. Method: Twenty-one male Wistx rats were used for this study. Endotoxemia was induced by lipopolysaccharide (LPS) infusion (Smg/kg). Blood flow was measured using radiolabeled microspheres according to the reference sample method. Concentration of dimethylarginines were measured by HPLC. The combination of arteriovenous concentration difference and organ blood flow allowed calculation of net organ fluxes and fractional extraction rates. Results: Arterial plasma concentration of ADMA was lower in LPS rats, in contrast to a higher SDMA concentration. For the gut, net release of ADMA was found, which was higher in LPS rats. In contrast, for the gut, net uptake of SDMA was found, which was lower in LPS rats. For the liver, a high net uptake of ADMA was found in both groups, while fractional extraction was significantly increased in LPS rats. Hepatic handling of SDMA was negligible. Conclusions: The liver plays an important role in eliminating ADMA from the circulation, and endotoxemia stimulates this capacity. In contrast to the liver, the gut releases ADMA. Endotoxemia results in a reduced systemic ADMA concentration.
108-P. INFLUENCE OF PROTEASOME INHIBITORS (MG132, ZL3VS, ADAAHX3L3VS) ON PROTEIN METABOLISM IN INCUBATED RAT SKELETAL MUSCLE .I. Kadlcikova ‘, M. Holecek’, R. Safranek’, I. Tilser ’ ‘Pharmacology, Faculty of Pharmacy, Charles university, ‘Physiology, Faculty of Medicine, Hradec Kralove, Czech Republic Rationale: Ubiquitin-proteasomal system is believed to degrade most of the cytosolic proteins and is activated under many catabolic conditions. We investigated direct effects of three different proteasome inhibitors: MG132 (peptide aldehyde), ZL3VS and AdaAhxxLxVS (vinyl sulfones) on muscle protein metabolism during sepsis. Method: Sepsis was induced in rats by caecal ligation and puncture. Parameters of protein metabolism were measured in incubated rat skeletal muscles - m. soleus (SOL) and m. extensor digitorum longus (EDL). Muscles were incubated in medium containing 30 umol/I MG132, ZL3VS and AdaAhxxLxVS, respectively. Total proteolysis was detelminated according to the rates of tyrosine release into the medium during incubation. The rate Rate of proteolysis (nmol of tyrosinelg wet w/hour)
SOL EDL
control IId3
MG132 IId3
control n=9
ZL3VS n=9
control IId3
AdaAhx3L3VS IId3
x3 * 13 76 zk 9
36 zk 2”,#,t 30 * 3s
x1 *4 x0 * 5
75 * 4 66 * 4s
6X&5 70 * 3
55 * 3s 47 * 2s
Paired t-test “~50.05; ANOVA#p50.05
vs ZL3VS, jP50.05 vs AdaAhx3L3VS
of leucine oxidation was evaluated by incubating muscles in medium containing L-[l-t4C]leucine. Statistical compz&ons were performed by paired t-tests and ANOVA followed by Bonferroni test. Results: The main changes were found in the rates of proteolysis as indicated in the table. Data xe presented as mean f SE. All proteasome inhibitors tested had no significant effect on leucine oxidation. Conclusions: We conclude that in our model of sepsis MG132 was more potent inhibitor of muscle proteolysis then ZLxVS and AdaAhxxLxVS both in SOL and EDL muscles.
109-P. THE DUMAS METHOD NITROGEN EVALUATION
FOR THE TOTAL URINARY
F. Rancini, R. De Toni, L. Caregaro, L. Di Pascoli, G. Bucciante Department of Clinical and Experimental Medicine, Azienda Ospedaliera di Padova, Padua, Italy Rationale: The Kjeldhal method (Km) is the standxd technique for nitrogen evaluation, but it is time-consuming, labour intensive and uses hazardous chemicals. The minxy urea determination is widely used in clinical practice, but urea nitrogen is not a constant shanz of total nitrogen excretion. The Dumas method (Dm) is a common technique for the protein detelmination in food; it is rapid and do not require hazardous chemicals. In Dm the sample is combusted at high temperature; nitrogen oxides are reduced to gaseous nitrogen and measured by thermal conductivity. The aim of the study was to compxe the Dm and Km in total mine nitrogen evaluation. Method: The total nitrogen content was measured in single urine sample of 31 healthy adults with Dm (FP-528, LECO Nitrogen Analyses) and with an automatized macro-Km; the repeatability of Dm was estimated by two trials in the same urine samples. The agreement of the two methods and the repeatability of Dm were evaluated by Bland-Altman test. Results: Mean urinary nitrogen was 10.7f3.4 g/L with Dm and 9.9f3.lg/L with Km (r=0.99; piO,OS). Mean difference between urinary nitrogen data with Dm and Km was O.Sf0.5 g/L (relative difference Sf5.3%). The limits of agreement showed by the Bland-Altman test were 1.85 and -0.27 g/L. The two trials performed with Dm showed: bias 0.13f0.15 g/L (1.2f1.2%), CV 0.9% and agreement limits 0.43 and -0.17 g/L. Conclusions: The Dm agrees with Km in total urinary nitrogen measurement and shows a high repeatability. The positive bias, evidence heady known in food analysis, may be due to the determination of some nitrogen that the Km cannot measure. The speedy, simplicity and safety make Dm a better approach for routine urinary nitrogen evaluation in clinical practice.
110-P. EFFECTS OF FREE L-GLUTAMINE VERSUS L-ALANYL-L-GLUTAMINE ON PLASMA ARGININE CONCENTRATIONS AND ORGAN FLUXES IN MICE UNDERGOING SURGERY P.G. Bcelens’, P.A.M. Van Leeuwen’, P.B. Soeters’, P. F rst3, C.H.C. Dejong’, N.E.P. De& ‘Surgery, VU University medical center; Amsterdam, ‘Surgery, Maastricht University, Maastricht, Netherlands, 31nst Biol Chem Nutrition, University of Hohenheim, Stuttgart, Germany Rationale: An increasing number of studies show clinical benefits of glutamine (GLN)-enriched nutrition in surgical patients. Since free-GLN is unstable in aqueous solutions, the dipeptide L-Alanyl-GLN (ALA-GLN) might be good alternative. Previous studies suggest a pathway of GLN as substrate for xginine (ARG) synthesis in the kidney. We studied the effect of GLN and ALA-GLN infusions on plasma ARG concentrations and fluxes across the gut and kidney. Method: In the post absorptive state, mice (n=40) underwent a lapxotomy and catheters were inserted for infusion and sampling across the gut and kidney. A primed continuous infusion of either free L-GLN or dipeptide ALA-GLN, was administered during 1 horn in order to reach a steadystate, via the jugular vein (IV) or via the duodenum (EN). Plasma flow was measured with infusion of 14C-para-aminohippuric acid. Amino acid concentrations were determined using HPLC. Significance was tested using a two-way ANOVA.
PROTEIN AND AMINO ACID METABOLISM
t&ions of dimethylarginines, as both ADMA and SDMA were eliminated from the systemic circulation in substantial amounts. Fmthemore, evidence for the role of endotoxemia on the metabolism of dimethylarginines was obtained as plasma levels of ADMA wele significantly lower in endotoxemic rats.
107-P. GUT AND LIVER HANDLING OF ASYMMETRICAL (ADMA) AND SYMMETRICAL (SDMA) DIMETHYLARGININE IN THE RAT UNDER BASAL CONDITIONS AND DURING ENDOTOXEMIA R.J. Niiveldt’, T. Teerlink’, A.A. Van Lambalgen3, M.P.C. Siroen’, J.A. Rauwerda’, P.A.M. Van Leeuwen’ ‘Departments of Surgery, 2Clinical Chemistry, 3Physiology, VU University Medical Center, Amsterdam, Netherlands Rationale: Asymmetrical dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase enzymes, whereas symmetrical dimethylxginine (SDMA) competes with arginine transport. Although both dimethylarginines may be important regulators of the arginine-NO pathway, their metabolism is lxgely unknown. We aimed to investigate dimethylarginine handling of the gut and the liver in detail under basal conditions and during endotoxemia. Method: Twenty-one male Wistx rats were used for this study. Endotoxemia was induced by lipopolysaccharide (LPS) infusion (Smg/kg). Blood flow was measured using radiolabeled microspheres according to the reference sample method. Concentration of dimethylarginines were measured by HPLC. The combination of arteriovenous concentration difference and organ blood flow allowed calculation of net organ fluxes and fractional extraction rates. Results: Arterial plasma concentration of ADMA was lower in LPS rats, in contrast to a higher SDMA concentration. For the gut, net release of ADMA was found, which was higher in LPS rats. In contrast, for the gut, net uptake of SDMA was found, which was lower in LPS rats. For the liver, a high net uptake of ADMA was found in both groups, while fractional extraction was significantly increased in LPS rats. Hepatic handling of SDMA was negligible. Conclusions: The liver plays an important role in eliminating ADMA from the circulation, and endotoxemia stimulates this capacity. In contrast to the liver, the gut releases ADMA. Endotoxemia results in a reduced systemic ADMA concentration.
108-P. INFLUENCE OF PROTEASOME INHIBITORS (MG132, ZL3VS, ADAAHX3L3VS) ON PROTEIN METABOLISM IN INCUBATED RAT SKELETAL MUSCLE .I. Kadlcikova ‘, M. Holecek’, R. Safranek’, I. Tilser ’ ‘Pharmacology, Faculty of Pharmacy, Charles university, ‘Physiology, Faculty of Medicine, Hradec Kralove, Czech Republic Rationale: Ubiquitin-proteasomal system is believed to degrade most of the cytosolic proteins and is activated under many catabolic conditions. We investigated direct effects of three different proteasome inhibitors: MG132 (peptide aldehyde), ZL3VS and AdaAhxxLxVS (vinyl sulfones) on muscle protein metabolism during sepsis. Method: Sepsis was induced in rats by caecal ligation and puncture. Parameters of protein metabolism were measured in incubated rat skeletal muscles - m. soleus (SOL) and m. extensor digitorum longus (EDL). Muscles were incubated in medium containing 30 umol/I MG132, ZL3VS and AdaAhxxLxVS, respectively. Total proteolysis was detelminated according to the rates of tyrosine release into the medium during incubation. The rate Rate of proteolysis (nmol of tyrosinelg wet w/hour)
SOL EDL
control IId3
MG132 IId3
control n=9
ZL3VS n=9
control IId3
AdaAhx3L3VS IId3
x3 * 13 76 zk 9
36 zk 2”,#,t 30 * 3s
x1 *4 x0 * 5
75 * 4 66 * 4s
6X&5 70 * 3
55 * 3s 47 * 2s
Paired t-test “~50.05; ANOVA#p50.05
vs ZL3VS, jP50.05 vs AdaAhx3L3VS
of leucine oxidation was evaluated by incubating muscles in medium containing L-[l-t4C]leucine. Statistical compz&ons were performed by paired t-tests and ANOVA followed by Bonferroni test. Results: The main changes were found in the rates of proteolysis as indicated in the table. Data xe presented as mean f SE. All proteasome inhibitors tested had no significant effect on leucine oxidation. Conclusions: We conclude that in our model of sepsis MG132 was more potent inhibitor of muscle proteolysis then ZLxVS and AdaAhxxLxVS both in SOL and EDL muscles.
109-P. THE DUMAS METHOD NITROGEN EVALUATION
FOR THE TOTAL URINARY
F. Rancini, R. De Toni, L. Caregaro, L. Di Pascoli, G. Bucciante Department of Clinical and Experimental Medicine, Azienda Ospedaliera di Padova, Padua, Italy Rationale: The Kjeldhal method (Km) is the standxd technique for nitrogen evaluation, but it is time-consuming, labour intensive and uses hazardous chemicals. The minxy urea determination is widely used in clinical practice, but urea nitrogen is not a constant shanz of total nitrogen excretion. The Dumas method (Dm) is a common technique for the protein detelmination in food; it is rapid and do not require hazardous chemicals. In Dm the sample is combusted at high temperature; nitrogen oxides are reduced to gaseous nitrogen and measured by thermal conductivity. The aim of the study was to compxe the Dm and Km in total mine nitrogen evaluation. Method: The total nitrogen content was measured in single urine sample of 31 healthy adults with Dm (FP-528, LECO Nitrogen Analyses) and with an automatized macro-Km; the repeatability of Dm was estimated by two trials in the same urine samples. The agreement of the two methods and the repeatability of Dm were evaluated by Bland-Altman test. Results: Mean urinary nitrogen was 10.7f3.4 g/L with Dm and 9.9f3.lg/L with Km (r=0.99; piO,OS). Mean difference between urinary nitrogen data with Dm and Km was O.Sf0.5 g/L (relative difference Sf5.3%). The limits of agreement showed by the Bland-Altman test were 1.85 and -0.27 g/L. The two trials performed with Dm showed: bias 0.13f0.15 g/L (1.2f1.2%), CV 0.9% and agreement limits 0.43 and -0.17 g/L. Conclusions: The Dm agrees with Km in total urinary nitrogen measurement and shows a high repeatability. The positive bias, evidence heady known in food analysis, may be due to the determination of some nitrogen that the Km cannot measure. The speedy, simplicity and safety make Dm a better approach for routine urinary nitrogen evaluation in clinical practice.
110-P. EFFECTS OF FREE L-GLUTAMINE VERSUS L-ALANYL-L-GLUTAMINE ON PLASMA ARGININE CONCENTRATIONS AND ORGAN FLUXES IN MICE UNDERGOING SURGERY P.G. Bcelens’, P.A.M. Van Leeuwen’, P.B. Soeters’, P. F rst3, C.H.C. Dejong’, N.E.P. De& ‘Surgery, VU University medical center; Amsterdam, ‘Surgery, Maastricht University, Maastricht, Netherlands, 31nst Biol Chem Nutrition, University of Hohenheim, Stuttgart, Germany Rationale: An increasing number of studies show clinical benefits of glutamine (GLN)-enriched nutrition in surgical patients. Since free-GLN is unstable in aqueous solutions, the dipeptide L-Alanyl-GLN (ALA-GLN) might be good alternative. Previous studies suggest a pathway of GLN as substrate for xginine (ARG) synthesis in the kidney. We studied the effect of GLN and ALA-GLN infusions on plasma ARG concentrations and fluxes across the gut and kidney. Method: In the post absorptive state, mice (n=40) underwent a lapxotomy and catheters were inserted for infusion and sampling across the gut and kidney. A primed continuous infusion of either free L-GLN or dipeptide ALA-GLN, was administered during 1 horn in order to reach a steadystate, via the jugular vein (IV) or via the duodenum (EN). Plasma flow was measured with infusion of 14C-para-aminohippuric acid. Amino acid concentrations were determined using HPLC. Significance was tested using a two-way ANOVA.