Lipoprotein profiles in sheep plasma and lung lymph.
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Biomedical subjects
G C Kramer
Publications and source records attributed to G C Kramer.
Characterization of sheep lung lymph lipoproteins: chemical and physical properties.
We have determined the composition and distribution of plasma and lung lymph lipoproteins from unanesthetized ewes. Cholesterol, triglyceride, and phospholipid levels in lung lymph were 45%, 50%, and 50%, respectively, of those in plasma. Lipoproteins from both lymph and plasma were separated into two major fractions: d less than 1.063 g/ml or "LDL", and d 1.063-1.21 g/ml or HDL. HDL was the major lipoprotein species in the plasma and lymph. Gradient gel electrophoresis of HDL on 4-30% gels showed that, in lymph, HDL particles were shifted to larger sizes; in addition to a peak at 8.5 nm, which was similar to plasma HDL, there were two additional components of larger size, one at 9.2 nm and the other at 12 nm. Electron microscopy revealed that lymph HDL contained two new particles not seen in plasma: large, round particles, 13.6 nm diameter, and discoidal particles, 18.7 by 4.9 nm, long and short axis, respectively. Compositional analysis of lymph HDL revealed a relative enrichment in free cholesterol as well as an enrichment in apolipoprotein E. Lymph "LDL" on gradient gel electrophoresis was extremely heterogeneous. Several peaks were evident in the 23-30 nm size range (similar to plasma "LDL"), but a supplementary component at approximately 15-16 nm was also present. Whereas plasma "LDL" on electron microscopy contained only round particles 26 nm in diameter, lymph contained an additional, unusual particle which was close-packed, with square geometry, and was 15 nm in diameter. Lymph apolipoprotein composition differed from that of plasma by the appearance of apoE and A-I as well as apoB. Particles containing apoE and A-I were separated from apoB-containing particles in a fraction of d 1.047-1.063 g/ml by density gradient centrifugation. On electron microscopy, this fraction revealed square-packing particles; the density and apolipoprotein composition suggest that these unusual particles are a continuum of HDL. Changes in the physical and chemical properties of lung lymph lipoproteins suggest that these particles are metabolically modified.
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Lipoprotein distribution in sheep lung lymph.
The composition of lymph draining the sheep caudal mediastinal lymph node is believed to reflect the composition of lung interstitial fluid. Although long-term lymph fistulas have been extensively utilized in studies related to lung microvascular fluid and protein flux, attention has thus far not been focused on lipoproteins as they might appear in lung interstitial space. We therefore characterized the lipoprotein distributions and measured triglyceride and cholesterol concentrations of lung lymph and plasma in nine unanesthetized sheep with long-term embedded instrumentation. We also measured triglyceride and cholesterol concentrations in prefemoral and thoracic duct lymph. The plasma LDL concentration was 55.3 +/- 20.2 mg/dl and HDL concentration was 99.9 +/- 27.2 mg/dl. By comparison, the lung lymph LDL concentration was 27.7 +/- 3.0 mg/dl and the HDL concentration was 45.4 +/- 11 mg/dl. The major sheep lipoprotein classes (LDL, HDL), although low in concentration compared to humans, are present in lung lymph in relative proportions similar to those of plasma. Analytical ultracentrifugation revealed the presence of a fast-floating component in lung lymph HDL that was not present in plasma. Electron microscopy showed two sizes of particles in lung lymph HDL, 135A and 97A, which probably correspond to the fast-floating and slow-floating components, respectively; plasma HDL contained only 94 A particles. Changes in the physical properties of HDL present in lung lymph suggest modification of the HDL either in the endothelial barrier or interstitial fluid of the lung.
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Microvascular fluid and protein flux in pulmonary and systemic circulations after thermal injury.
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Mechanisms for redistribution of plasma protein following acute protein depletion.
Acute plasma protein depletion is followed by a rapid and substantial replenishment of the protein deficit. We studied the effects of plasmapheresis on flow and composition of peripheral lymph in 11 unanesthetized sheep. Whole blood was replaced with red blood cells and lactated Ringer solution to reduce plasma protein concentration ([P]) 26-54%. At 24 h after plasmapheresis, [P] had returned halfway to base line. Lymph flow (L) increased immediately after plasma protein reduction, was maximal 3 h later, and remained elevated for more than 3 days. The increase in L was coupled with a decrease more than 3 days. The increase in L was coupled with a decrease in lymph-to-plasma protein concentration ratio ([L/P]). The plasma-to-lymph oncotic gradient was reestablished by 24 h due to the reduction in lymph protein and the partial return of [P]. After 24 h, L remained elevated despite base-line levels for all measured vascular pressures and plasma-to-lymph oncotic gradients. Although lymph flow was increased, the permeability-surface area product for protein was decreased below base line. The data confirm that the partial return of [P] in the first day after plasmapheresis is due largely to a shift of extravascular protein mass into the vascular compartment and show that redistribution is initiated by increased lymphatic return and maintained by a sustained increase in L and a decrease in protein permeability of the plasma-lymph barrier.
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Effect of dextran-70 on increased microvascular fluid and protein flux after thermal injury.
The effect of a colloid (dextran-70) infusion on increased microvascular fluid and protein flux after thermal injury was compared with that seen with a crystalloid infusion. Lymph flow (QL) and lymph/phasma (L/P) protein content were used to monitor microvascular fluid flux and protein permeability in the lung and in burned and nonburned soft tissues, namely, skin and subcutaneous tissue, for 72 h in 13 sheep given a 30% TBS full-thickness burn. Vascular pressures were maintained constant with dextran in saline or lactated Ringer's. Mean fluid requirements for dextran were one-half that for crystalloid during the 24-h resuscitation period. However, plasma proteins decreased by 50% with dextran compared to 30% with crystalloid. An increase of 2-2.5 fold in QL was seen during resuscitation in the lung and nonburned soft tissue with crystalloid while the L/P protein ratio decreased, indicating no change in protein permeability. This QL response was prevented during dextran infusion. A rebound increase in soft tissue QL occurred after discontinuation of dextran, probably as a result of the severe residual hypoproteinemia. Burn QL and L/P were markedly increased during the entire 72-h period, indicating increased permeability. Dextran infusion accentuated the QL response in the burn microcirculation. We conclude that dextran decreases edema in nonburned tissue, but appears to increase the protein loss from burn tissue.
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Effect of hypoproteinemia on pulmonary and soft tissue edema formation.
The effect of acute hypoproteinemia on the rate of fluid flux across the pulmonary and soft tissue microcirculation was studied in the unanesthetized sheep. Lymph flow was used to monitor fluid flux, a protein depletion of 30-50% of baseline value was produced by plasmapheresis. Vascular hydrostatic pressures and cardiac output were maintained constant with crystalloid infusion. The measured oncotic pressure in plasma, pi rho rapidly decreased as did the oncotic gradient between plasma and lymph. Lung and soft tissue lymph flow increased 2- to 3-fold immediately after protein depletion. Lung interstitial oncotic pressure, pi L, as measured in lymph, decreased to return the oncotic gradient and lymph flow to baseline by 24 h. Soft tissue oncotic gradient also returned to baseline by 24 h, but lymph flow remained significantly elevated for the next 48 h, indicating an increase in fluid flux unrelated to changes in oncotic pressure. Lymph flow rapidly returned to baseline when protein was returned. Protein depletion may alter the soft tissue interstitial matrix, allowing for edema formation. More effective mechanisms prevent this from occurring in the lung.
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The effects of hypoproteinemia on blood-to-lymph fluid transport in sheep lung.
We studied the effects of reducing the plasma protein concentration on flow and composition of pulmonary lymph in 12 unanesthetized sheep. Whole blood was removed while red cells were returned and lactated Ringers was infused at a rate sufficient to maintain pulmonary vascular pressures at baseline values. A 44-54% reduction in plasma protein concentration resulted in a decrease in the plasma oncotic pressure from 18.6 +/- 1.1 to 7.8 +/- 0.9 mm Hg. Within an hour after plasmapheresis, lymph flows increased to a maximum of 4 times baseline. Subsequently, lymph flow gradually decreased and were close to baseline at 24 hours. The plasma-to-lymph oncotic gradient was reestablished in 5 hours due to decreased lymph protein. Maintained elevation of lymph flow with hydrostatic and oncotic gradients at baseline values suggest that the blood-to-lymph barrier offers less resistance to fluid transport. The calculated filtration coefficient increased 2- to 3-fold after plasmapheresis. Protein clearances remained normally coupled to lymph flows. Thus the enhanced fluid transport cannot be attributed to a permeability change in the large pore pathways. Hypoproteinemia may alter the interstitial gel so that there is less resistance to fluid movement. Such changes in fluid conductivity between blood capillaries and lymphatics may augment the lymphatic safety factor against pulmonary edema during hypoproteinemia.
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Tumor necrosis factor monoclonal antibody prevents alterations in leukocyte populations during cardiopulmonary bypass.
Tumor necrosis factor-alpha (TNF-alpha) has been implicated as causing the systemic inflammatory response to cardiopulmonary bypass (CPB) that contributes to the postoperative sequelae of coagulopathy, increased capillary permeability, leukocytosis, fever, and multiple organ dysfunction. To define the role of TNF-alpha on leukocyte populations during CPB, pigs (n = 6) were pretreated with 20 mg TNF-alpha monoclonal murine antibody before normothermic CPB (2 hr) in a blinded prospective randomized study with saline used as a control (n = 6). The leukocyte response to CPB was measured at 10, 30, 60, and 120 min during CPB and at 60 and 120 min after CPB. Repeated measures analysis of variance was performed and the null hypothesis was discarded at the 5% level. The control group displayed the typical leukocyte profile associated with CPB: and initial leukopenia (36% reduction) followed by leukocytosis (11% increase, P = 0.0001). The initial leukopenia was due to a fall in both polymorphonuclear neutrophils (33% reduced, P < 0.05) and monocytes (37% reduced, P < 0.05). In the TNF-alpha monoclonal murine antibody group the total leukocyte profile did not change significantly from baseline, (8.7% reduction to a 16% increase, P = 0.24) nor were there significant changes in populations including neutrophils and lymphocytes. In the treatment group the initial reduction in monocytes was prevented and total circulating monocytes increased during bypass. The experimental data suggest that TNF-alpha may play an important role in the early alterations in leukocyte populations associated with CPB, and TNF-alpha monoclonal murine antibody pretreatment ameliorates the leukocyte response.
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Effects of inhalation injury on airway blood flow and edema formation.
We measured airway blood flow in unanesthetized sheep under control conditions and after lung injury induced by inhalation of cotton smoke. Blood flows in trachea, carina, main stem bronchi, intraparenchymal bronchi, and whole lung were measured by injection of radioactive microspheres. In 10 control sheep mean blood flow (+/- SD) was trachea, 17.2 +/- 10.5; main stem bronchi, 17.5 +/- 7.6; and whole lung (parenchyma inclusive of all small intraparenchymal airways), 20.5 +/- 11.9 ml.min-1/100 gm tissue weight. After injury, measurements were made 8 to 30 hours after smoke inhalation when respiratory distress was evident by arterial oxygen tensions of less than 60 mm Hg. Inhalation injury had little effect on cardiac output or blood flow to peripheral tissue. However, after inhalation injury airway blood flow (n = 6) was increased nine times in trachea, eight times in main stem bronchi, twelve times in intraparenchymal bronchi, and two times in whole lung. The increased airway blood flow resulted from a selective vasodilation of the airway vasculature because arterial driving pressures were unchanged by inhalation injury. Other investigators have shown that the microvascular permeability of the bronchial circulation is remarkably sensitive to inflammation, and the present experiments suggest that a selective vasodilation of the airway vasculature is another aspect of the airway response to inflammation. Increased airway blood flow through a leaky microvasculature may increase capillary filtrate from the bronchial circulation and contribute to the pulmonary edema of inhalation injury.
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A comparison of two different 2400 mOsm solutions for resuscitation of major burns.
The reduction of burn edema is a common goal in the resuscitation of patients with thermal injury. Initial infusion of a 2400 mOsm hypertonic 7.5% NaCl 6% dextran (HSD) has been shown to reduce volume needs, but elevated serum sodium levels limit the dose that can be safely used. This study tested the hypothesis that a 2400 mOsm solution of NaCl, amino acids, glucose, and 6% dextran (Isosal-D) would reduce similar volume requirements while maintaining normal plasma sodium levels. Hemodynamics, plasma sodium, fluid balance, and tissue water content were measured after an initial baseline period and during resuscitation of a large scald injury in 21 anesthetized sheep. Resuscitation was begun 30 minutes after the scald with infusion of 10 ml/kg of either lactated Ringer's (LR), Isosal-D, or HSD and was continued with LR to restore and maintain baseline oxygen delivery throughout the 8-hour period. Oxygen delivery, cardiac output, and mean arterial pressure were rapidly reestablished by all three solutions, although a persistent tachycardia was noted with Isosal-D. Net fluid requirements of both HSD (35 +/- 13 ml/kg) and Isosal-D (72 +/- 13 ml/kg) were significantly lower than in the LR group (203 +/- 39 ml/kg). Mean serum sodium increased 11 mEq with HSD to a peak after 4 hours of 152 +/- 5 mEq, whereas with LR sodium fell 7 mEq to 132 +/- 4. Isosal-treated animals had minimal change in serum sodium. HSD significantly decreased tissue water content in colon, liver, pancreas, and nonburned skin compared with LR, whereas Isosal-D reduced edema only in the colon. It is concluded that in this protocol Isosal-D was not as effective as HSD at reducing volume needs and edema and had unexpected chronotropic effects.
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The role of interstitial starling forces in the pathogenesis of burn edema.
The formation and sustainability of burn edema require substantial change in net microvascular forces. We directly measured interstitial hydrostatic pressure (Pi) and total interstitial absorptive pressure (Pi + IIi), in dermis of anesthetized sheep, before and after a 70% to 85% total body surface area scald and during fluid resuscitation. The most rapid change occurred in Pi in the burn wound, which rapidly decreased from its baseline value of approximately -2 mm Hg to -11 mm Hg in the first 5 minutes, and thereafter increased but remained approximately -4 mm Hg through 4 hours of resuscitation. Pi in nonburned skin slowly increased from its preburn level -2 mm Hg, to become positive +1 mm Hg after 4 hours of resuscitation. The total interstitial absorptive pressure, Pi + IIi, slowly declined similarly from 15 to 16 mm Hg to approximately 10 to 11 mm Hg over 6 hours of resuscitation in both burned and nonburned dermis. Taken together, these data suggest that the rapid formation of burn edema is the result of development of a negative Pi in the burn wound, and its sustainability is the result of a large increase in interstitial compliance. Edema in nonburned skin did not start until after fluid resuscitation was initiated, and then developed as the plasma oncotic pressure declined from 21 to 10 mm Hg.
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Hypertonic acetate-alpha alpha hemoglobin for small volume resuscitation of hemorrhagic shock.
Hypertonic acetate solution in small volumes greatly improves cardiac output and corrects acid-base disturbances in hemorrhaged animals. We hypothesized that the combination of alpha alpha-crosslinked human hemoglobin (alpha alpha Hb), an oxygen carrier and vasoconstrictor, with hypertonic sodium acetate (HAHb), a vasodilator, may be effective for small volume resuscitation of hemorrhagic shock. Six pigs hemorrhaged to a mean arterial pressure of 40 mmHg for 60 min (bled volume: 23.6 +/- 2.5 ml.kg-1) received a single bolus of 4 ml.kg-1 of HAHb infused over two min. HAHb restored arterial pressure, increased systemic vascular resistance and caused a modest increase in cardiac output and SvO2, while pulmonary arterial pressure and vascular resistance were markedly increased. In two animals, transient severe hypotension and low cardiac output may have been due to acute pulmonary hypertension during injection. Compared to our previous study, in which animals received 4 ml-kg-1 of alpha alpha Hb alone, HAHb produced higher cardiac output and a smaller increase in systemic and pulmonary vascular resistance. However, slower, titrated infusions may be needed when hemoglobin solutions are combined with drugs or solutions that cause vasodilation in order to decrease the likelihood of acute hemodynamic instability.
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