Enhanced inflammatory responses in alpha-tocopherol transfer protein null mice

The liver preferentially secretes alpha-tocopherol into plasma under the control of the hepatic alpha-tocopherol transfer protein (alpha-TTP). alpha-TTP-null mice (Ttpa(-/-) mice) are vitamin E deficient, therefore were used for investigations of in vivo responses to sub-normal tissue alpha-tocopherol concentrations during inflammation. Increased basal oxidative stress in Ttpa(-/-) mice was documented by increased plasma lipid peroxidation, and superoxide production by bone marrow-derived neutrophils stimulated in vitro with phorbol 12-myristate 13-acetate. Lipopolysaccharide (LPS) injected intraperitoneally induced increases in lung and liver HO-1 and iNOS, as well as plasma NO(x) in Ttpa(+/+) mice. LPS induced more modest increases in these markers in Ttpa(-/-) mice, while more marked increases in plasma IL-10 and lung lavage TNF alpha were observed. Taken together, these results demonstrate that alpha-tocopherol is important for proper modulation of inflammatory responses and that sub-optimal alpha-tocopherol concentrations may derange inflammatory-immune responses.     

Reduced amino acid availability inhibits muscle protein synthesis and decreases activity of initiation factor eIF2B

We have examined the effect of a hemodialysis-induced 40% reduction in plasma amino acid concentrations on rates of muscle protein synthesis and breakdown in normal swine. Muscle protein kinetics were measured by tracer methodology using [(2)H(5)]phenylalanine and [1-(13)C]leucine and analysis of femoral arterial and venous samples and tissue biopsies. Net amino acid release by muscle was accelerated during dialysis. Phenylalanine utilization for muscle protein synthesis was reduced from the basal value of 45 +/- 8 to 25 +/- 6 nmol x min(-1) x 100 ml leg(-1) between 30 and 60 min after start of dialysis and was stimulated when amino acids were replaced while dialysis continued. Muscle protein breakdown was unchanged. The signal for changes in synthesis appeared to be changes in plasma amino acid concentrations, as intramuscular concentrations remained constant throughout. The changes in muscle protein synthesis were accompanied by a reduction or stimulation, respectively, in the guanine nucleotide exchange activity of eukaryotic initiation factor (eIF)2B following hypoaminoacidemia vs. amino acid replacement. We conclude that a reduction in plasma amino acid concentrations below the normal basal value signals an inhibition of muscle protein synthesis and that corresponding changes in eIF2B activity suggest a possible role in mediating the response.     

Carboxyhemoglobin formation following smoke inhalation injury in sheep is interrelated with pulmonary shunt fraction

Carboxyhemoglobin (COHb) formation is triggered by the inducible isoform of heme oxygenase (HO-1) catalyzing carbon monoxide (CO) production through breakdown of heme molecules, exposure to CO or both. In the setting of CO poisoning, COHb is regarded as a reliable marker characterizing both severity of injury and efficacy of treatment strategies. This study was designed as a prospective laboratory experiment to elucidate potential interdependencies between COHb generation, oxygenation, and pulmonary shunt fraction (Qs/Qt) in an ovine model of smoke inhalation injury. Chronically instrumented ewes (n=15) were repeatedly subjected to cotton smoke (4 x 12 breaths) according to an established protocol. This approach resulted in a progressive increase in COHb formation that was interrelated with the degree of Qs/Qt (P<0.001) and inversely correlated with both arterial and mixed venous HbO(2) saturation (r=-0.96 and -0.93). Although the arteriovenous COHb gradient successively decreased over time, COHb determined in venous blood underestimated the arterial content.     

Role of anti-L-selectin antibody in burn and smoke inhalation injury in sheep

We hypothesized that the antibody neutralization of L-selectin would decrease the pulmonary abnormalities characteristic of burn and smoke inhalation injury. Three groups of sheep (n = 18) were prepared and randomized: the LAM-(1-3) group (n = 6) was injected intravenously with 1 mg/kg of leukocyte adhesion molecule (LAM)-(1-3) (mouse monoclonal antibody against L-selectin) 1 h after the injury, the control group (n = 6) was not injured or treated, and the nontreatment group (n = 6) was injured but not treated. All animals were mechanically ventilated during the 48-h experimental period. The ratio of arterial PO2 to inspired O2 fraction decreased in the LAM-(1-3) and nontreatment groups. Lung lymph flow and pulmonary microvascular permeability were elevated after injury. This elevation was significantly reduced when LAM-(1-3) was administered 1 h after injury. Nitrate/nitrite (NO(x)) amounts in plasma and lung lymph increased significantly after the combined injury. These changes were attenuated by posttreatment with LAM-(1-3). These results suggest that the changes in pulmonary transvascular fluid flux result from injury of lung endothelium by polymorphonuclear leukocytes. In conclusion, posttreatment with the antibody for L-selectin improved lung lymph flow and permeability index. L-selectin appears to be principally involved in the increased pulmonary transvascular fluid flux observed with burn/smoke insult. L-selectin may be a useful target in the treatment of acute lung injury after burn and smoke inhalation.     

A rapid method for the extraction and determination of vitamin E metabolites in human urine

A method for the direct extraction and routine analysis of the vitamin E metabolites gamma- and alpha-carboxyethyl hydroxychroman (gamma- and alpha-CEHC) from human urine has been developed. A relatively small sample volume (5 ml) can be used and, after enzymatic hydrolysis of the conjugated forms and acidification, the metabolites are extracted with diethyl ether. Recovery of alpha- and gamma-CEHC was compared to that of trolox, used as an internal standard, added to 24-h urine collections from vitamin E-unsupplemented volunteers. Various solvent conditions were initially tested; acidification and ether extraction gave the highest recovery. It was found that after addition and extraction from urine, trolox, alpha- and gamma-CEHC are recovered to a similar extent, hence trolox is viable as an internal standard. The samples were analyzed by both GC and HPLC with electrochemical detection (ECD). HPLC-ECD was found to give higher selectivity and higher sensitivity compared to GC or HPLC with UV detection at 290 nm. The HPLC-ECD detection limit was 10 fmol, linearity (r(2) > 0.98) was achieved in the range of 40 to 200 fmol, which was found to be optimal for 24-h urines from unsupplemented subjects. Inter-sample variability was typically 2-5%. This greater sensitivity and selectivity means that vitamin E metabolites can be analyzed even in unsupplemented subjects. It is also possible to measure unconjugated forms of the metabolites. Typically these were found to represent approximately 10% of the total alpha- and gamma-CEHC. This method can be used routinely for the determination of vitamin E metabolites in urine. The new extraction and detection methods described are relatively quick, less laborious, and more cost-effective than previously available methods.     

Oxidative stress in athletes during extreme endurance exercise

Despite the many known health benefits of exercise, there is a body of evidence suggesting that endurance exercise is associated with oxidative stress. To determine whether extreme endurance exercise induces lipid peroxidation, 11 athletes (3 females, 8 males) were studied during a 50 km ultramarathon (trial 1) and during a sedentary protocol (trial 2) 1 month later. The evening before each trial, with dinner, subjects consumed 75 mg each d(3)-RRR and d(6)-all rac-alpha-tocopheryl acetates. Blood was obtained at baseline, 30 min pre-race, mid-race, post-race, 1 h post-race, 24 h post-race, and at corresponding times during trial 2. All 11 subjects completed the race; average run time was 391 +/- 23 min. Plasma F(2)-isoprostanes increased from 75 +/- 7 pg/ml at pre-race to 131 +/- 17 (p <.02) at post-race, then returned to baseline at 24 h post-race; F(2)-isoprostanes were unchanged during trial 2. Deuterated alpha-tocopherol disappearance rates were faster (2.8 x 10(-4) +/- 0.2 x 10(-4)) during the race compared to the sedentary trial (2.3 x 10(-4) +/- 0.2 x 10(-4); p <.03). These data suggest that extreme endurance exercise results in the generation of lipid peroxidation with a concomitant increase in vitamin E disappearance.