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Objectives: This study was designed to assess the stability and functional activity of fibrinogen concentrates subjected to the changes in temperature and duration observed in field conditions.Methods: Fibrinogen concentrate was stored at -20°C (12 vials), 22°C (12 vials), and 50°C with 80% humidity (12 vials), for up to 6 mo. At each temperature, three vials of fibrinogen concentrate were taken out at 0, 1, 3, and 6 mo and reconstituted. On analysis days, blood samples were taken from a single healthy donor to collect plasma samples. The donor plasma was mixed with commercial fibrinogen-deficient plasma to make fibrinogen-adjusted plasma (FAP). An aliquot of the reconstituted fibrinogen concentrate was used for quantification of stored fibrinogen content (using STA-R) and function (Rotem - Fibtem) in FAP.Results: At 22°C for 0, 1, 3, and 6 mo, there were no significant changes observed in fibrinogen content (1,223 ± 42 mg/vial, 1,286 ± 86 mg/vial, 1,234 ± 76 mg/vial, and 1,178 ± 64 mg/vial), prothrombin time (13.5 ± 0.1 s, 13.7 ± 0.6 s, 13.3 ± 0.4 s, and 13.7 ± 0.2 s), or activated partial thromboplastin time (31.1 ± 0.2 s, 32.0 ± 0.2 s, 31.5 ± 0.2 s, and 32.0 ± 0.8 s), respectively. There were also no significant changes observed in any of the Fibtem measurements. Similarly, no differences were observed in these variables over time at -20°C and 50°C with 80% humidity.Conclusions: Fibrinogen concentrate maintained its content and function when stored at -20°C to 50°C with up to 80% humidity for 6 mo. [ABSTRACT FROM AUTHOR]

Introduction: Platelet apheresis is a routine clinical practice, but the physiological impact on the donors has been incompletely characterized. This study measured the effects of platelet apheresis on hemodynamics, oxygen metabolism, and coagulation in pigs to assess its impact before employing the animals in experimental studies.Methods: Forty pigs (39.8 ± 0.6 kg) were anesthetized and catheterized with an apheresis catheter in the femoral vein. During the platelet apheresis process, blood was withdrawn from the pig to separate platelets, and the remaining red blood cells and plasma returned back to the pigs, using the Haemonetics MCS+9000 system. A total of 12 cycles of blood withdrawn and return were performed during the entire apheresis procedure to reduce platelet counts to a target of 50% of baseline. During the process, hemodynamics was recorded in each cycle. Blood samples were collected before and after apheresis to assess changes in oxygen metabolism and coagulation by prothrombin time, activated partial thromboplastin time (STA-R Evolution Stago), and using Rotem thrombelastometry, and platelet aggregation using a Chrono-Log 700 aggregometer.Results: During each cycle of the apheresis, mean arterial pressure (MAP) was decreased and heart rate was increased by blood withdrawal, but both recovered after blood return. On the completion of the apheresis, platelet count decreased from baseline 345 ± 15 109/L to 141 ± 14 109/L and fibrinogen levels were reduced from 124 ± 5 to 99 ± 4 mg/dL (both p < 0.05). Although oxygen delivery remained unchanged, oxygen consumption was decreased from 4.0 ± 0.2 to 3.2 ± 0.0 mL O2/kg/min (p < 0.05). Rotem alpha (clotting speed) decreased from 79 ± 0 to 69 ± 1° and maximum clot firmness (MCF or clot strength) decreased from 71 ± 1 to 57 ± 1 mm (both p < 0.05). No changes were observed in prothrombin time or activated partial thromboplastin time. Platelet aggregation induced by arachidonic acid or collagen was decreased to 28 ± 6% or 71 ± 3% of baseline values (p < 0.05), respectively.Conclusion: Platelet apheresis caused significant fluctuations in hemodynamics, reduced oxygen consumption, in addition to the compromised platelet aggregation and clotting function expected. The observations warrant consideration in humans undergoing apheresis over extended periods. [ABSTRACT FROM AUTHOR]

Introduction: Ibuprofen is commonly used by warfighters in the deployed environment. This study investigated its dose effects on in vitro coagulation in human and pig blood.Methods: Blood samples were collected from 6 normal volunteers and 6 healthy pigs and processed to make platelet-adjusted samples (100 × 10(3)/μL, common transfusion trigger in trauma). Ibuprofen was added to the samples at concentrations of 0 μg/mL (control), the concentration from the highest recommended oral dose (163 μg/mL, 1×), and 2×, 4×, 8×, 10×, 12×, 16×, and 20×. Platelet aggregation by Chrono-Log aggregometer and coagulation by rotational thrombelastogram (Rotem) were assessed at 15 minutes after the addition of ibuprofen.Results: A robust inhibition of ibuprofen on arachidonic acid-induced platelet aggregation was observed at all doses tested in human or pig blood. Collagen-stimulated platelet aggregation was inhibited starting at 1× in human blood and 4× in pig blood. Rotem measurements were similarly compromised in pig and human blood starting at 16×, except clot formation time was prolonged at 1× in human blood (all p < 0.05).Conclusion: Ibuprofen inhibited platelet aggregation at recommended doses, and compromised coagulation at higher doses. Human blood was more sensitive to ibuprofen inhibition. Further effort is needed to investigate ibuprofen dose responses on coagulation in vivo. [ABSTRACT FROM AUTHOR]

Introduction: Ibuprofen is commonly used by Soldiers in the deployed environment. This study investigated its dose-effects on in vitro coagulation.Methods: Blood samples were collected from 4 normal healthy pigs and were processed to make platelet-adjusted (100×10(3)/μL) blood samples. Ibuprofen was added to the samples at doses of 0 μg/mL (control), recommended oral dose (163 μg/mL, 1×), 2×, 4×, 8×, 10×, 12×, 16×, and 20×. Arachidonic acid or collagen-stimulated platelet aggregation was assessed at 15 minutes after the addition of ibuprofen. Coagulation was assessed with measurements of prothrombin time (PT) and activated partial thromboplastin time (aPTT), and thrombelastography by Rotem.Results: A robust inhibition of ibuprofen on arachidonic acid-induced platelet aggregation was observed at all doses tested. Collagen-stimulated platelet aggregation was inhibited to 71%±5% and 10%±5% of the control values at ibuprofen doses of 4× and 20×, respectively (both p<0.05). No changes were observed in PT at any dose, but aPTT was prolonged at dose of 16× and 20×. Rotem measurements of coagulation time, clot formation time, maximum clot firmness, and A10 were compromised at dose 16× and 20× (all p<0.05).Conclusion: Ibuprofen inhibited platelet aggregation at recommended doses, but did not compromise aPTT or coagulation profile until at 16 times the recommended doses and higher. Further effort is needed to clarify whether there are different dose-responses between human and pig blood samples in trauma situations. [ABSTRACT FROM AUTHOR]

Hemorrhagic coagulopathy (without neurological injuries) constitutes 40% of injury-related death in civilian hospitals and on the battlefield, and the underlying contributing mechanisms remain unclear. The purpose of this study is to investigate the effects of fibrinogen availability on coagulation function after hemorrhage in pigs. Sixteen crossbred commercial Yorkshire swine were randomized into the control group (group C) (n = 8) and hemorrhage group (group H) (n = 8). Hemorrhage was induced in group H by bleeding 35% of the estimated total blood volume, followed by resuscitation with lactated Ringer solution at three times the bled volume. Pigs in group C were not hemorrhaged or resuscitated. Blood samples were withdrawn at baseline, 15 min, 3 h, 6 h, and 24 h after hemorrhage and lactated Ringer (LR) resuscitation (H-LR). Coagulation was assessed by using thrombelastography. All baseline measurements were similar between groups C and H. Hemorrhage caused a decrease in mean arterial pressure and an increase in heart rate in group H, but LR resuscitation corrected these changes within 1 h. Compared to baseline values, fibrinogen concentrations in group H decreased at 15 min, 3 h and 6 h after H-LR, but increased to double that of the baseline value at 24 h; platelet counts decreased throughout the study; clot strength was decreased at 15 min, 3 h and 6 h, but returned to baseline value at 24 h after H-LR. Hemorrhage caused decreases in fibrinogen and platelets, and compromised clot strength. The rebound of fibrinogen at 24 h restored clot strength despite platelet deficit. These data suggest the potential compensatory role of fibrinogen in restoring coagulation function in vivo after hemorrhagic shock. [ABSTRACT FROM AUTHOR]

Hemorrhagic coagulopathy is involved in the morbidity and mortality of trauma patients. Nonetheless, many aspects of the mechanisms underlying this disorder are poorly understood. We have therefore investigated changes in fibrinogen metabolism and coagulation function after a moderate hemorrhagic shock, using a new stable isotope approach. Twelve pigs were randomly divided into the control (C) and hemorrhage (H) groups. Hemorrhage was induced by bleeding 35% total blood volume over a 30-min period. A primed constant infusion of [1-13C]phenylalanine (Phe), d5-phenylalanine, and α-[1-13C]-ketoisocaproate (KIC) was given to quantify fibrinogen synthesis and breakdown, together with measurements of circulating liver enzyme activities and coagulation function. Mean arterial pressure was decreased by hemorrhage from 89 ± 4 mmHg in C to 47 ± 4 mmHg in H (P « 0.05), followed by a rebound to 68 ± 5 mmHg afterward. Fibrinogen fractional synthesis rate increased from 2.7 ± 0.2%/h in C to 4.2 ± 0.4%/h in H by Phe (P « 0.05) and from 3.1 ± 0.4%/h in C to 4.4 ± 0.5%/h in H by KIC (P « 0.05). Fibrinogen fractional breakdown rate increased from 3.6 ± 1.0%/h in C to 12.9 ± 1.8%/h in H (P « 0.05). The absolute breakdown rate accelerated from 3.0 ± 0.4 mg·kg-1·h-1 in C to 5.4 ± 0.6 mg·kg-1·h-1 in H (P « 0.05), but the absolute synthesis rate remained unchanged. These metabolic changes were accompanied by a reduction in blood clotting time to 92.7 ± 1.6% of the baseline value by hemorrhage (P « 0.05). No changes were found in liver enzyme activities. We conclude that the observed changes in coagulation after hemorrhagic shock are mechanistically related to the acute acceleration of fibrinogen degradation. [ABSTRACT FROM AUTHOR]

We assessed the approach of using intracellular free amino acid enrichment as precursor enrichment for calculating the fractional synthetic rate of using the stable isotope tracer incorporation technique. We assumed that the true rate of protein synthesis was reflected by the rate of tracer incorporation over time divided by the plateau enrichment in protein. Isolated human fibroblasts and myocytes were cultured in medium supplemented with [(15)N]glycine, [(15)N]proline, and [d(5)]phenylalanine. Culture medium and cells were collected daily from d 1 to 5. A portion of cells harvested on d 5 was subcultured for an additional 3 passages to d 20. Protein enrichments in both cell types reached a plateau after 20 d of cell culture. In fibroblasts, the true protein synthesis rates were 0.74, 0.85, and 0.86%/h, using protein plateau enrichments of [(15)N]glycine, [(15)N]proline, and [d(5)]phenylalanine as precursor enrichments, respectively. When the corresponding intracellular free amino acid enrichments were used, protein synthesis rates were 0.76, 0.79, and 0.76%/h, respectively. Similarly, in myocytes, the true protein synthesis rates were 0.98 and 1.14%/h by protein plateau enrichments of [(15)N]glycine and [d(5)]phenylalanine, respectively. The synthesis rates were 0.94 and 1.01%/h by the corresponding intracellular enrichments, respectively. Extracellular amino acid enrichments resulted in underestimation of protein synthesis by a variable amount. We conclude that the intracellular free amino acid enrichment is an optimal surrogate for precursor enrichment to quantify protein synthesis. [ABSTRACT FROM AUTHOR]

Normal cardiac metabolism requires continuous replenishment (anaplerosis) of catalytic intermediates of the citric acid cycle. Little is known about the quantitative aspects of propionate as a substrate of in vivo anaplerosis; therefore, we measured the rate of propionate entry into the citric acid cycle in hearts of anesthetized pigs. [U-[SUP13]C[SUB3]]propionate (0.25 mM) was infused in a coronary artery branch for I h via an extracorporeal perfusion circuit, and cardiac biopsies were analyzed for the mass isotopomer distribution of citric acid cycle intermediates. Infusion of propionate did not affect myocardial oxygen consumption, heart rate, or contractile function. In the infused territory, propionate infusion did not affect uptake of glucose and lactate but decreased free fatty acid uptake by one-half (P < 0.05). Propionate extraction and uptake were 57.4 ± 3.3% and 0.078 ± 0.009 μmol-min[SUP-1]-g[SUP-1]. Anaplerosis from propionate, calculated from the mass isotopomer distribution of succinate, accounted for 8.9 ± 1.3% of the citric acid cycle flux. Propioylcarnitine release accounted for only 0.033 ± 0.002% of propionate uptake. Methylcitrate did not accumulate. Thus administration of a low concentration of propionate appears to be a convenient and safe way to boost anaplerosis in the heart. [ABSTRACT FROM AUTHOR]

OBJECTIVES:We investigated the effect of various dietary fats on pulmonary surfactant composition and lung function changes that occur before and after endotoxin infusion in pigs.METHODS:Eighteen pigs were assigned to three groups (n = 6 per group) to receive a diet of protein (20% of calories), carbohydrate (20% of calories), and fat (40% of calories). In one group the fat content consisted entirely of palmitic acid. In the second group, fat came from Intralipid, which provided predominantly linoleic acid. The third group was fed fish oil. Pigs were maintained on these diets for 21 d before the experiment. Cardiovascular and pulmonary functions were determined on day 22. Pigs then were infused with endotoxin (80 mg · kg−1 · min−1) until the pulmonary arterial pressure reached a pressure similar to that found in trauma victims (45 to 50 mmHg). Cardiovascular and pulmonary function tests were then repeated, the animals killed, and the lungs removed for study.RESULTS:Compliance was reduced in the linoleate and fish-oil groups compared with the palmitate group before and after endotoxin. Compliance changes in pigs fed the linoleate and fish-oil diets were consistent with significant increases in lung wet:dry weight ratios, increased CO2 retention, histologic evidence of vascular congestion, intra-alveolar edema, and alveolar septa thickening. Changes in surfactant phosphatidylcholine composition between groups were consistent with the notion that increased unsaturated fatty acids could affect surfactant function.CONCLUSIONS:We concluded that the common practice of providing calories in the form of polyunsaturated fatty acids to critically ill patients carries the risk of being detrimental to lung function. [ABSTRACT FROM AUTHOR]

We developed gas chromatography–mass spectrometry assays for the concentration and mass isotopomer distribution of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA in tissues. The assays involves perchloric acid extraction of the tissue, spiking the extract with [2H5]propionyl-CoA and [2H4]succinyl-CoA internal standards, and isolation of short-chain acyl-CoA fraction on an oligonucleotide purification cartridge. Propionyl-CoA is reacted with sarcosine and the formed N-propionylsarcosine is assayed as its pentafluorobenzyl derivative. Methylmalonyl-CoA and succinyl-CoA are hydrolyzed and the corresponding acids assayed as tert-butyl dimethylsilyl derivatives. The assay was applied to a study of [U-13C3]propionate metabolism in perfused rat livers. While propionyl-CoA is only M3 labeled, succinyl-CoA is M3, M2, and M1 labeled because of isotopic exchanges in the citric acid cycle. Methylmalonyl-CoA is M3 and M2 labeled, reflecting reversal of S-methylmalonyl-CoA mutase. Thus, our assays allow measuring the turnover of the coenzyme A derivatives involved in anaplerosis of the citric acid cycle via precursors of propionyl-CoA, i.e., propionate, odd-chain fatty acids, isoleucine, threonine, and valine. [Copyright &y& Elsevier]

Investigates the effects of glucose on lung surfactant kinetics in conscious pigs. Blood measurements; Pool sizes and fatty acid (FA) composition of surfactants; Rates of synthesis and loss; Contribution of De Novo synthesized FA of different origins to phosphatidylcholine synthesis.

Presents information on a study which investigated the effects of glucose infusion on surfactant phosphatidylcholine (PC) metabolic kinetics in the lungs. Fatty acid composition in surfactant PC; Methodology; Results and discussion.

Studies lung surfactant kinetics in conscious pigs. Isotropic enrichment of substrates; Secretion, synthetic, and recycling rate of lung surfactants; Endogenous surfactant turnover in preterm infants.

Explores the contributions of plasma free fatty acids (FFA) and de novo synthesized fatty acids (FA) to lung surfactant phosphatidylcholine (PC) synthesis. Isotopic enrichment of substrates; Synthetic rates of FFA and FA; Secretion rate and recycling rate of FFA and FA.

Recent human and animal studies have demonstrated that in severe end-stage heart failure (HF), the cardiac muscle switches to a more fetal metabolic phenotype, characterized by downregulation of free fatty acid (FFA) oxidation and an enhancement of glucose oxidation. The goal of this study was to examine myocardial substrate metabolism in a model of moderate coronary microembolization-induced HF. We hypothesized that during well-compensated HF, FFA oxidation would predominate as opposed to a more fetal metabolic phenotype of greater glucose oxidation. Cardiac substrate uptake and oxidation were measured in normal dogs (n = 8) and in dogs with microembolization-induced HF (n = 18, ejection fraction = 28%) by infusing three isotopic tracers ([9,10-3H]oleate, [U-14C]glucose, and [1-13C]lactate) in anesthetized open-chest animals. There were no differences in myocardial substrate metabolism between the two groups. The total activity of pyruvate dehydrogenase, the key enzyme regulating myocardial pyruvate oxidation (and hence glucose and lactate oxidation) was not affected by HF. We did not observe any difference in the activity of carnitine palmitoyl transferase I (CPT-I) and its sensitivity to inhibition by malonyl-CoA between groups; however, malonyl-CoA content was decreased by 22% with HF, suggesting less in vivo inhibition of CPT-I activity. The differences in malonyl-CoA content cannot be explained by changes in the Michaelis-Menten constant and maximal velocity for malonyl-CoA decarboxylase because neither were affected by HF. These results support the concept that there is no decrease in fatty acid oxidation during compensated HF and that the downregulation of fatty acid oxidation enzymes and the switch to carbohydrate oxidation observed in end-stage HF is only a late-stage phenomemon. [ABSTRACT FROM AUTHOR]

We developed a method for measurement of skin DNA synthesis, reflecting cell division, in conscious rabbits by infusing D-[U-13C6]glucose and L-[15N]glycine. Cutaneous protein synthesis was simultaneously measured by infusion of L-[ring-²H5]phenylalanine. Rabbits were fitted with jugular venous and carotid arterial catheters, and were studied during the infusion of an amino acid solution (10% Travasol). The fractional synthetic rate (FSR) of DNA from the de novo nucleotide synthesis pathway, a reflection of total cell division, was 3.26 ± 0.59%/d in whole skin and 3.08 ± 1.86%/d in dermis (P = 0.38). The de novo base synthesis pathway accounted for 76 and 60% of the total DNA FSR in whole skin and dermis, respectively; the contribution from the base salvage pathway was 24% in whole skin and 40% in dermis. The FSR of protein in whole skin was 5.35 ± 4.42%/d, which was greater (P < 0.05) than that in dermis (2.91 ± 2.52%/d). The FSRs of DNA and protein were not correlated (P = 0.33), indicating that cell division and protein synthesis are likely regulated by different mechanisms. This new approach enables investigations of metabolic disorders of skin diseases and regulation of skin wound healing by distinguishing the 2 principal components of skin metabolism, which are cell division and protein synthesis. [ABSTRACT FROM AUTHOR]