Xiao-Ming Lu

Cysteine metabolism and whole blood glutathione synthesis in septic pediatric patients.

ObjectiveTo investigate whole body in vivo cysteine kinetics and its relationship to whole blood glutathione (GSH) synthesis rates in septic, critically ill pediatric patients and controls. DesignProspective cohort study. SettingMultidisciplinary intensive care unit and pediatric inpatient units at a children’s hospital. PatientsTen septic pediatric patients and ten controls (children admitted to the hospital for elective surgery). InterventionsSeptic patients (age, 31 months to 17 yrs) and controls (age, 24 months to 21 yrs) received a 6-hr primed, constant, intravenous tracer infusion of l-[1-13C]cysteine. Blood samples were obtained to determine isotopic enrichment of plasma cysteine and whole blood [1-13C]cysteinyl-glutathione by gas-chromatography mass spectrometric techniques. The plasma flux and oxidation rate of cysteine and the fractional and absolute synthesis rates of GSH were determined. Septic patients received variable protein and energy intake, as per routine clinical management, and controls were studied in the early postabsorptive state. Measurements and Main Results Plasma cysteine fluxes were increased in the septic patients when compared with the controls (68.2 ± 17.5 [sd] vs. 48.7 ± 8.8 &mgr;mol·kg−1·hr−1;p < .01), and the fraction of plasma cysteine flux associated with oxidative disposal was similar among the groups. The absolute rates of GSH synthesis in whole blood were decreased (p < .01) in the septic patients (368 ± 156 vs. 909 ± 272 &mgr;mol·L−1·day−1). The concentration of whole blood GSH also was decreased in the septic group (665.4 ± 194 vs. 1059 ± 334 &mgr;M;p < .01) ConclusionsWhole blood glutathione synthesis rates are decreased, by about 60%, in critically ill septic children receiving limited nutritional support. Plasma cysteine fluxes and concentration of cysteine were increased in the septic patients, suggesting a hypermetabolic state with increased protein breakdown. The mechanisms whereby GSH synthesis rates are decreased in these patients are probably multifactorial, presumably involving an inflammatory response in the presence of limited nutritional support. The role of nutritional modulation and the use of cysteine prodrugs in maintaining GSH concentration and synthesis remain to be established.

Arginine and nitric oxide metabolism in critically ill septic pediatric patients.

ObjectiveTo investigate whole body, in vivo arginine metabolism and nitric oxide synthesis rates in septic, critically ill pediatric patients. DesignProspective study. SettingPediatric intensive care unit at a general hospital. PatientsTen consecutive septic patients age 6–16 yrs. InterventionsSeptic patients received an 8-hr primed, constant intravenous tracer infusion of l-[guanidino-15N2]arginine, l-[1-13C]leucine, and [13C]urea. A 24-hr urine collection was obtained for determination of [15N]nitrate enrichment (15NO3−) and urinary nitrogen. The next day they received an infusion of l-[5-13C]arginine and l-[5-13C-ureido, 5,5,2H2]citrulline. Blood samples were obtained for determination of plasma isotopic enrichment of the tracers given and of derived [15N]citrulline (nitric oxide synthesis), l-[13C-guanidino 5,5, 2H2]arginine (M+3 arg) (arginine synthesis), and [15N2]urea (urea formation). Data are compared with historic controls from studies in healthy young adults. Measurements and Main ResultsPlasma arginine fluxes were 67 ± 21 and 72 ± 17 &mgr;mol·kg−1·hr−1, respectively, for the [15N2 guanidino] and the [13C] arginine labels, which were not different from reported adult values. The rates of arginine oxidation were 22.9 ± 10.8 &mgr;mol·kg−1·hr−1 and were higher than de novo arginine synthesis rates of 9.6 ± 4.2 &mgr;mol·kg−1·hr−1 (p < .01); therefore, these patients were in a negative arginine balance. The rates of nitric oxide synthesis as estimated by the [15N]citrulline method were 1.58 ± 0.69 &mgr;mol·kg−1·hr−1 for septic patients and higher (p < .05) than values of 0.96 ± 0.1 &mgr;mol·kg−1·hr−1 in healthy adults. Septic patients were in a negative protein (leucine) balance of about −1.00 ± 0.40 g·kg−1·day−1. ConclusionsHomeostasis of plasma arginine in septic patients was impaired compared with reported adult values. The rates of arginine oxidation were increased whereas de novo net arginine synthesis was unchanged, leading to a negative arginine balance. The rates of nitric oxide synthesis and the fraction of plasma arginine used for nitric oxide and urea formation were increased. These findings suggest that under condition of sepsis, arginine becomes essential in critically ill children.

Effects of glucagon-like peptide 1 on glycemia control and its metabolic consequence after severe thermal injury--studies in an animal model.

BACKGROUND Hyperglycemia with insulin resistance is commonly seen in severely burned patients and tight glycemia control with insulin may be beneficial in this condition. The most potent insulinotropic hormone, glucagon-like peptide 1 (GLP-1), stimulates insulin secretion in a glucose-dependent manner. Because infusion of GLP-1 never reduces glucose levels to below ∼70 mg/dL, the risk of hypoglycemia by using insulin is reduced. In this study we investigated the metabolic effects of GLP-1 infusion after burn injury in an animal model. METHODS Male CD rats were divided in 3 groups: burn injury with saline, burn injury with GLP-1 treatment, and sham burn (SB). Burn injury was full thickness 40% total body surface area. The burn injury with GLP-1 treatment group received GLP-1 infusion via osmotic pump. Fasting blood glucose, plasma insulin, and plasma GLP-1 levels were measured during intraperitoneal glucose tolerance tests. Expressions of caspase 3 and bcl-2 were evaluated in pancreatic islets. In a subset of animals, protein metabolism and total energy expenditure were measured. RESULTS Fasting GLP-1 was reduced in burn injury with saline compared to SB or burn injury with GLP-1 treatment. Burn injury with GLP-1 treatment showed reduced fasting blood glucose, improved intraperitoneal glucose tolerance test results, with increased plasma insulin and GLP-1 responses to glucose. GLP-1 reduced protein breakdown and total energy expenditure in burn injury with GLP-1 treatment versus burn injury with saline, with improved protein balance. Increased expression of caspase 3 and decreased expression of bcl-2 in islet cells by burn injury were ameliorated by GLP-1. CONCLUSION Burn injury reduced plasma GLP-1 in association with insulin resistance. GLP-1 infusion improved glucose tolerance and showed anabolic effects on protein metabolism and reduced total energy expenditure after burn injury, possibly via insulinotropic and non insulinotropic mechanisms.

Nitric oxide activates intradomain disulfide bond formation in the kinase loop of Akt1/PKBα after burn injury

Severe burn injury is an acute inflammatory state with massive alterations in gene expression and levels of growth factors, cytokines and free radicals. During the catabolic processes, changes in insulin sensitivity and skeletal muscle wasting (unintended loss of 5–15% of lean body mass) are observed clinically. Here, we reveal a novel molecular mechanism of Akt1/protein kinase Bα (Akt1/PKBα) regulated via cross-talking between dephosphorylation of Thr308 and S-nitrosylation of Cys296 post severe burn injury, which were characterized using nano-LC interfaced with tandem quadrupole time-of-fight mass spectrometry (Q-TOF)micro tandem mass spectrometry in both in vitro and in vivo studies. For the in vitro studies, Akt1/PKBα was S-nitrosylated with S-nitrosoglutathione and derivatized by three methods. The derivatives were isolated by SDS-PAGE, trypsinized and analyzed by the tandem MS. For the in vivo studies, Akt1/PKBα in muscle lysates from burned rats was immuno-precipitated, derivatized with HPDP-Biotin and analyzed as above. The studies demonstrated that the NO free radical reacts with the free thiol of Cys296 to produce a Cys296-SNO intermediate which accelerates interaction with Cys310 to form Cys296-Cys310 in the kinase loop. MS/MS sequence analysis indicated that the dipeptide, linked via Cys296-Cys310, underwent dephosphorylation at Thr308. These effects were not observed in lysates from sham animals. As a result of this dual effect of burn injury, the loose conformation that is slightly stabilized by the Lys297-Thr308 salt bridge may be replaced by a more rigid structure which may block substrate access. Together with the findings of our previous report concerning mild IRS-1 integrity changes post burn, it is reasonable to conclude that the impaired Akt1/PKBα has a major impact on FOXO3 subcellular distribution and activities.

Targeted antisense modulation of inflammatory cytokine receptors.

Antisense technology is potentially a powerful means by which to selectively control gene expression. We have used antisense oligonucleotides to modulate the response of the hepatoma cell line, HepG2, to the inflammatory cytokine, IL-6, by inhibiting the expression of its multifunctional signal transducer, gp130. HepG2 cells respond to IL-6 by upregulating acute phase proteins, such as haptoglobin, by five- to tenfold. Gp130 is central to this response, as the upregulation of haptoglobin is almost completely blocked by the addition of high concentrations ( approximately 100 microg/ml) of a monoclonal antibody to gp 130. Antisense oligodeoxynucleotides complementary to the mRNA encoding gp 130 inhibited the upregulation of haptoglobin by IL-6-stimulated HepG2 cells by about 50%. However, a nonsense sequence also inhibited haptoglobin secretion by about 20%. To improve the specificity and efficiency of action, we targeted the antisense oligonucleotides to HepG2 cells using a conjugate of asialoglycoprotein-poly-L-lysine. The targeted antisense reduced the binding of IL-6 to HepG2 cells, virtually eliminating high affinity binding. In addition, it inhibited haptoglobin upregulation by over 70%. Furthermore, the dose of targeted antisense required for biological effect was reduced by about an order of magnitude as compared with unconjugated antisense. These results demonstrate the potential of antisense oligonucleotides as a means to control the acute phase response as well as the need for a greater understanding of the mechanism and dynamics of antisense molecules as they are developed toward therapeutic application.

SILAM for quantitative proteomics of liver Akt1/PKBα after burn injury

Akt1/protein kinase Bα (Akt1/PKBα) is a downstream mediator of the insulin signaling system. In this study we explored mechanism(s) for its role in burn injury. Akt1/PKBα in liver extracts from mice with burn injury fed with (2H7)-L-Leu was immunoprecipitated and isolated with SDS-PAGE. Two tryptic peptides, one in the kinase loop and a control peptide just outside of the loop were sequenced via nano-LC interfaced with quadruple time-of-flight tandem mass spectrometry (Q-TOF tandem MS). Their relative isotopologue abundances were determined by stable isotope labeling by amino acids in mammalians (SILAM). Relative quantifications based on paired heavy/light peptides were obtained in 3 steps. The first step included homogenization of mixtures of equal amounts of tissue from burned and sham-treated animals (i.e., isotope dilution) and acquisition of uncorrected data based on parent monoisotopic MS ion ratios. The second step included determination of isotopic enrichment of the kinase from burned mice on Day 7 and the third step enrichment correction of partially labeled heavy and light monoisotopic MS ion ratios for relative quantification of bioactivity (loop peptide) and expression level (control peptide). Protein synthesis and enrichment after injury were found to be dependent on tissue and turnover of individual proteins. Three heavy and light monoisotopic ion ratios for albumin peptides from burned mice indicated ~55% enrichment and ~16.7-fold downregulation. In contract, serum amyloid P had ~66% enrichment and was significantly upregulated. Akt1/PKBα had ~56% enrichment and kinase level in response to the burn injury was upregulated compared with the control peptide. However, kinase bioactivity, represented by the Cys296 peptide, was significantly reduced. Overall, we demonstrated that i) quantitative proteomics can be performed without completely labeled mice; ii) measurement of enrichment of acyl-tRNAs is unnecessary and iii) Cys296 plays an important role in kinase activity after burn injury.

Bacterial cell killing by antibody targeted photolysis : enhanced effect by OH radical generation

Two structurally distinct immunoconjugates were used for photolysis of bacterial cells. One contained a dextran carbazate (DC) polymer as a linker between the photosensitizer molecules and the Fc oligosaccharide moiety of the monoclonal antibody, while the other linked the photosensitizer molecules and the Fc oligosaccharide by a short ethylenediamine (ED) spacer. The two immunoconjugates exhibited remarkably different photophysical properties and cell killing potential with respect to their abilities to generate singlet oxygen. The DC conjugate exhibited poor singlet oxygen (lδg) yields, yet was shown to produce more efficient cell killing on the basis of 1δg dose than did the ED conjugate. In light of the enhanced cell killing capacity of the DC conjugate, a search for other toxic photoproducts was initiated. It was found that the DC conjugate was capable of generating hydroxyl radicals (OH.) upon light illumination. Quantum yields for OH. generation were evaluated. The DC-photosensitizer polymer appeared to initiate a cascade addition reaction presumably by adding peroxides and hydroperoxides to the glucose residues of the dextran carbazate linker. These results suggest that this radical formation could propagate down the DC polymer and account for the superior cell killing exhibited by the DC conjugate.

Synthesis of [1,2‐13C2] gly and [2,2‐2H2] gly glutathione

[1,2-13C2] Gly and [2,2-2H2] Gly isotopomers of the intracellular tripeptide glutathione were prepared by standard methods of solution phase peptide synthesis. The synthetic products were characterized by gas chromatography/mass spectroscopy (GC/MS) and 1H NMR spectroscopy. Optical purity was determined by hydrolysis, derivatization of the free amino acids with isopropanol-acetyl chloride and pentafluoropropionic anhydride and NCI/MS with a Chirasil-Val Heliflex column. These compounds should serve as useful tracers for the non-invasive study of glutathione synthesis and turnover rates in humans by GC/MS.

Post-Translational Modification Profiling of Burn-Induced — Insulin Resistance and Muscle Wasting

The maintenance of glucose levels represents one of the most tightly regulated systems in the body. All cells require glucose, however, it is only available from exogenous sources or hepatic production. Since glucose cannot be stored in significant amounts except as glycogen in the liver and muscle, glucose transport into the cell by specific transport proteins is critical for cell function. Insulin plays a major role in the maintenance of normal glucose levels. When blood glucose levels rise, insulin secretion is stimulated, resulting in increase in uptake of glucose by skeletal muscle via glucose transporter protein 4 (GLUT4), and decreased production by the liver. Binding of insulin to its receptor leads to activation of insulin receptor tyrosine kinase, which phosphorylates IRS proteins that function as docking platforms for the two main signalingpathways are responsible downstream regulation [1]; the phosphatidyl inositol 3kinase (PI3K)-Akt/protein kinase B (PKB) pathway and the Ras-mitogen-activated protein kinase (MAPK) pathway. There appear to be several key proteins in the insulin/glucose and protein turnover regulatory cascade, including IRS-1 the predominent form of IRS in muscle. When levels of insulin and glucose are abnormally high in the fasting state, a condition called insulin resistance exists. Insulin resistance and muscle wasting during the persistent high grade inflammation induced by severe burn injury increases the patients risk for infection, sepsis, and death. Clinical data indicate that a resting metabolic rate switch occurs after burn injuries that involve ~40-60% total body surface area (TBSA). Based on studies of 189 pediatric burn patients [2,3,4], it was determined that burn size determines the inflammatory and hypermetabolic response to injury; serum glucose and insulin levels increased to 144 mg/dl and 32 ng/ml on day 8 after injury. Metabolic alterations such as abnormal cytokine release [5, 6], altered gene expression [7,8] and increased protein catabolism are common in patients with

Burn injury differentially alters whole-blood and organ glutathione synthesis rates: An experimental model

Previous studies from our laboratories revealed a reduced rate of whole-blood (WB) glutathione (GSH) synthesis in severely burned patients. To determine whether WB GSH metabolism is an indicator of the status of GSH metabolism in one or more of the major organs, we used a burn rabbit model to determine GSH concentrations and rates of synthesis in WB, liver, lungs, kidney, and skeletal muscle. L-[1-13C]-cysteine was infused intravenously for 6 h in rabbits at 3 days post-burn and in sham burn controls. WB and organ 13C-enrichment of cysteine and GSH was determined by gas chromatography/mass spectrometry. Plasma cysteine metabolic flux was increased significantly (P < 0.01) following burn injury. WB, liver, and lung GSH concentrations (P = 0.054, P < 0.05, and P < 0.05, respectively) and fractional rates of GSH synthesis (P < 0.05, P < 0.01, and P < 0.05, respectively) were reduced at 3 days post-burn. Kidney was unaffected. There also appears to be an increased rate of GSH transport out of the liver after burn injury. Hence, there is a differential impact of burn injury on tissue and organ GSH status, with WB qualitatively reflecting the changes in lung and liver. It will be important to determine whether these changes are due to alterations in the intrinsic capacity for GSH synthesis and/or availability of amino acid precursors of GSH.