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J Appl Physiol 82: 2020-2027, 1997;
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Journal of Applied Physiology
Vol. 82, No. 6, pp. 2020-2027, June 1997
GAS EXCHANGE, MECHANICS, AND AIRWAYS

Effect of triiodothyronine augmentation on rat lung surfactant phospholipids during sepsis

Sergey M. Ksenzenko, Scott B. Davidson, Amer A. Saba, Alexander P. Franko, Aml M. Raafat, Lawrence N. Diebel, and Scott A. Dulchavsky

Department of Surgery, Wayne State University School of Medicine, Detroit, Michigan 48201

ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES

ABSTRACT

Ksenzenko, Sergey M., Scott B. Davidson, Amer A. Saba, Alexander P. Franko, Aml M. Raafat, Lawrence N. Diebel, and Scott A. Dulchavsky. Effect of triiodothyronine augmentation on rat lung surfactant phospholipids during sepsis. J. Appl. Physiol. 82(6): 2020-2027, 1997.---Surfactant functional effectiveness is dependent on phospholipid compositional integrity; sepsis decreases this through an undefined mechanism. Sepsis-induced hypothyroidism is commensurate and may be related. This study examines the effect of 3,3',5-triiodo-L-thyronine (T3) supplementation on surfactant composition and function during sepsis. Male Sprague-Dawley rats underwent sham laparotomy (Sham) or cecal ligation and puncture (CLP) with or without T3 supplementation [CLP/T3 (3 ng/h)]. After 6, 12, or 24 h, surfactant was obtained by lavage. Function was assessed by a pulsating bubble surfactometer and in vivo compliance studies. Sepsis produced a decrease in surfactant phosphatidylglycerol and phosphatidic acid, with an increase in lesser surface-active lipids phosphatidylserine and phosphatidylinositol. Phosphatidylcholine content was not significantly changed. Sepsis caused an alteration in the fatty acid composition and an increase in saturation in most phospholipids. Hormonal replacement attenuated these changes. Lung compliance and surfactant adsorption were reduced by sepsis and maintained by T3 treatment. Thyroid hormone may have an active role in lung functional preservation through maintenance of surfactant homeostasis during sepsis.

fatty acids; respiratory distress

INTRODUCTION

THE PULMONARY SURFACTANT SYSTEM is of primary importance in normal lung function and homeostasis. Surfactant consists of a complex mixture of phospholids and lipoproteins which act in concert to reduce lung surface tension, maintain fluid balance, and possibly reduce infection. Acute alterations in the availability and functional integrity of lung surfactant have been demonstrated in animal models of respiratory distress and during the acute respiratory distress syndrome (ARDS) in humans (8). Similar changes in lung surfactant are noted during sepsis, which is often remote from the lung (17). Although it is estimated that 200,000 people die of ARDS annually in the US, the acute biochemical changes in lung surfactant during infection remain poorly characterized.

3,3',5-Triiodo-L-thyronine (T3) is a ubiquitous growth hormone and is necessary for the maintenance of lung morphology, type II alveolar cell function (surfactant synthesis), and lung repair after injury (2, 22). The "Sick Euthyroid" or "Low T3" syndrome is frequently coexistent with ARDS and consists of a normal or low serum thyroxine, with a profound reduction in circulating levels of metabolically active T3 (1, 3, 11, 12). The physiological ramifications of these acute changes in thyroid economy are not clear. An acute decrease in thyroid hormone-dependent metabolism may provide a beneficial reduction in energy requirements during periods of stress. In contrast, thyroid hormone is necessary for optimal cellular repair after injury and for normal homeostasis. Furthermore, recent studies have suggested that an intact thyroid axis is essential for survival during hemorrhagic and septic shock (10, 14). The purpose of this study was to determine the time course and effect of sepsis with or without T3 augmentation on surfactant compositional and functional integrity.

METHODS

Animals and surfactant preparation. The experiments described herein conform to the NIH Guidelines for the Care of Laboratory Animals and were approved by the Wayne State University Animal Care Committee. Adult male Sprague-Dawley rats (250-350 g) were acclimated to the animal care facility for 1 wk and fed standard rat chow. Animals were anesthetized and underwent sham laparotomy (Sham; n = 60), cecal ligation and puncture (CLP; n = 60), or CLP with T3 replacement (CLP/T3; n = 60). T3 replacement was administered by a subcutaneous Alzet osmotic pump at 3 ng/h, which has previously been shown to correct the sepsis-induced decrease in serum free T3 levels (10). In each group, animals were randomly killed at 6, 12, or 24 h after CLP. Alveolar surfactant was obtained by repeated lavage through a tracheostomy with 0.15 M sodium chloride to total lung capacity × 3. The surfactant pellet was isolated, following the procedures of Curstedt and co-workers (7), with a low-speed centrifugation to remove the cellular pellet followed by high-speed centrifugation to isolate the surfactant fraction.

Measurements of lung compliance and surface tension. In situ dynamic pulmonary compliance was determined in a subset of animals from the slope of the pressure curve during serial lung inflation. The animals were anesthetized, and a tracheostomy was performed. The lungs were inflated with humidified room air at 4 ml/min, and the change in pressure was monitored with a pressure transducer. Dynamic lung compliance was calculated from the midslope region of the pressure-volume curve. Stock solutions of surfactant extract were prepared from pelleted surfactant from each animal group and dissolved in 0.15 M sodium chloride to a concentration of 4 mg phospholipid/ml and dispersed by mechanical vortexing. Surfactant adsorption and surface tension were measured on a pulsating bubble surfactometer (Electronetics, Amherst, NY), as described by Enhorning (15). Surfactant adsorption was calculated from the pressure-volume loop during initial bubble inflation over a 10-s interval. Surface tensions are calculated from the Young-Laplace equation for a sphere (P = 2 × T/r), where P is the pressure drop across the bubble interface, T is the surface tension, and r is the bubble radius. Temperature was set at 37°C, and the pulsation rate was 20 cycles/min. Oscillation was continued for 15 min or until the minimum tension (Tmin) was <3 mN/m; surface Tmin and maximum tension were continuously recorded.

Results are expressed as the means ± SD for each animal group. Statistics were done on the IBM INSTAT program via analysis of variance (ANOVA) with Bonferroni correction. Significance was inferred when P < 0.05.

Lipid extraction. Surfactant phospholipids were extracted according to the method of Bligh and Dyer (5). After centrifugation of the extract at 4000 g for 20 min, the extract was passed through a glass filter (10-20 µm), mixed with water (3:1 vol/vol), and separated by centrifugation. The chloroform layer was washed with methanol-water (1:2 vol/vol), concentrated in a vacuum, and subjected to column chromatography on a silica gel (130-270 mesh, Aldrich Chemical, Milwaukee, WI). The lipids were eluted sequentially with ether, chloroform, and alternating mixtures of chloroform and methanol with the amount of methanol increasing stepwise from 10 to 100% (vol/vol). The fractions were assayed by two-dimensional thin-layer chromatography (TLC) on silica gel HP-K plates (Fisher Scientific, Pittsburgh, PA) with chloroform-methanol-water (65:25:4 vol/vol/vol) in the first dimension, and chloroform-methanol-25% ammonia (14:6:1 vol/vol/vol) in the second dimension. Supplementary in situ visualization of phospholipid spots was done with modified Jungnickel's reagent; aminophospholipids were visualized with ninhydrin (26). Lipid standards for TLC were obtained from Sigma Chemical (St. Louis, MO). Fractions containing phospholipids were collected, concentrated in a vacuum, and separated by TLC to recover individual phospholipid classes on silica gel plates as described above. Lipid phosphorous was determined by the method of Vaskovsky et al. (25).

Gas chromatography. Fatty acids of individual phospholipids, purified by TLC as described above, were determined as methyl esters, according to Morrison and Smith (21). Fatty acid methyl esters were purified by TLC in hexane-ether-acetic acid (85:15:1 vol/vol/vol) and analyzed by chromatography on a model 5890 gas chromatograph (Hewlett-Packard, Wilmington, DE) with a HP capillary Ultra 2 column (25 m × 0.2 mm × 0.33 mm), using a flame-ionized detector, with He (12 ml/min) as a gas carrier with a ramped 190 to 250°C temperature regimen. Data were collected, integrated, and analyzed with HP Peak-96 software (Hewlett-Packard). Commercially available saturated and unsaturated fatty acids (C8-C28) were used as standards (Sigma Chemical). Peak positions of the unsaturated fatty acids were doubly checked after hydrogenation of the prime fatty acid methyl esters in the presence of platinum oxide according to Kates (18). Nonadecanoic acid methyl ester (Sigma Chemical) was used as the external standard in quantitative analysis of constituent fatty acids. All reagents and materials were analyzed as blank samples to eliminate any background peaks that could interfere with the fatty acid determinations.

RESULTS

Lung compliance and surface tension. In vivo dynamic lung compliance was significantly decreased in septic animals by 24 h compared with Sham, nonseptic animals (Sham = 0.66 ± 0.02, CLP = 0.47 ± 0.06 ml/cmH2O; P < 0.01 compared with Sham by ANOVA). In contrast, thyroid hormonal augmentation modulated the sepsis-induced changes in lung compliance toward control nonseptic animals (CLP/T3 = 0.56 ± 0.02 ml/cmH2O; P < 0.05 compared with CLP by ANOVA).

All pressures of the inflation and deflation surface tension-hysteresis loop of CLP animals were significantly higher than for control animals at 12 or 24 h after CLP (Figure 1; P < 0.05 by ANOVA for all points). Surfactant from animals treated with T3 produced intermediate hysteresis plots that were significantly less than those for CLP animals at all points (P < 0.05 by ANOVA). Surfactant adsorption rate, an index of interface stability, was adversely affected by sepsis (45 ± 3.2 vs. 32 ± 4.6 mN/m, Sham vs. CLP, respectively; P < 0.05 by ANOVA). T3 treatment produced intermediate surfactant adsorption values (CLP/T3 = 38 ± 3.5 mN/m).
Fig. 1. Surface tension was determined on a pulsating bubble surfactometer from the Young-Laplace equation for a sphere at 37°C, a pulsation rate of 20 cycles/min, and phospholipid concentration of 4 mg/ml. Representative surface tension isotherms are shown for 50-100% of maximal bubble size from each animal group at 24 h after cecal ligation and puncture (CLP; bullet ), CLP and 3,3,5-triiodo-L-thyronine [(T3); X], and Sham (square ).
[View Larger Version of this Image (20K GIF file)]

Phospholipid content. Total content of lung phospholipids recovered by bronchoalveolar lavage decreased in septic animals and was significantly less than in control animals at 12 or 24 h after CLP (Fig. 2; P < 0.01 by ANOVA). In contrast, T3 replacement maintained phospholipid availability compared with nonseptic animals at all time periods, although a diminution of lavagable surfactant was seen by 24 h (CLP/T3 vs. CLP at 12 or 24 h, P < 0.05; CLP/T3 vs. Sham at 12 h, P < 0.05; and CLP/T3 vs. Sham at 24 h, P < 0.08).
Fig. 2. Total lung surfactant phospholipids were measured, following the method of Vaskovsky et al. (25), from each animal group and represent the average of 3 measurements ± SD. CLP caused acute decrease in total surfactant phospholipids at 12 and 24 h compared with normal (N) rats; P < 0.01 by analysis of variance (ANOVA). In contrast, T3 treatment (CLP/T3) modulated early decrease in surfactant phospholipid content, although to a lesser extent by 24 h after septic insult (12 h CLP/T3 vs. CLP; P < 0.05 by ANOVA).
[View Larger Version of this Image (17K GIF file)]

Phosphatidylcholine was the primary phospholipid constituent of surfactant in all of the animal groups (Table 1). Sepsis did not significantly alter the relative content of surfactant phosphatidylcholine compared with control animals. Similarly, the relative percentage of phosphatidylethanolamine in surfactant was not altered by CLP or T3 treatment at any time period. In contrast, sepsis caused an early (12 h) increase in the relative amount of surfactant phosphatidylinositol, cardiolipin, and lysophospholipids that was abrogated by concurrent replacement of thyroid hormone except for a late (24 h) decrease in phosphatidylinositol in the hormonally supplemented animals. Septic animals had a significant decrease in the amount of phosphatidylglycerol and phosphatidic acid compared with control animals; treatment with thyroid hormone maintained normal concentration of these phospholipids at all time periods.

Table  1.   Composition of total phospholipids in rat lung surfactant
Phospholipid N CLP
CLP/T3
6 h 12 h 24 h 6 h 12 h 24 h
Phosphatidylcholine 74.6 ± 0.6  74.0 ± 0.6  75.0 ± 0.9  75.2 ± 0.8  75.0 ± 0.7  74.2 ± 0.8  74.1 ± 0.6 
Phosphatidylethanolamine 4.9 ± 0.3  5.0 ± 0.4  5.2 ± 0.6  4.9 ± 0.5  4.8 ± 0.5  4.4 ± 0.4  4.9 ± 0.8 
Phosphatidylserine 2.8 ± 0.6  3.0 ± 0.8  3.2 ± 0.9  3.3 ± 1.0  3.0 ± 0.5  3.3 ± 0.7  2.9 ± 0.9 
Phosphatidylinositol 1.3 ± 0.4  1.6 ± 0.4  3.8 ± 0.8  3.4 ± 0.6  1.7 ± 0.5  0.9 ± 0.6  0.3 ± 0.5 
Cardiolipin 3.9 ± 0.5  3.8 ± 0.3  5.3 ± 0.5  5.1 ± 0.7  3.5 ± 0.6  4.3 ± 0.8  4.5 ± 1.0 
Phosphatidylglycerol 10.9 ± 0.4  9.3 ± 0.6  4.3 ± 0.6  4.5 ± 0.8  9.0 ± 0.3  9.2 ± 0.8  9.2 ± 0.8 
Phosphatidic acid 2.2 ± 0.3  2.3 ± 0.5  0.8 ± 0.4  1.0 ± 0.6  2.0 ± 0.7  2.7 ± 0.7  3.0 ± 0.8 
Lysophospholipids and other lipids 0.3 ± 0.3  1.0 ± 0.5  2.4 ± 0.8  2.6 ± 0.9  1.0 ± 0.6  1.0 ± 0.3  1.1 ± 0.9
Values are means ± SD from 4 experiments. Phospholipid composition of lung surfactant, determined from pooled surfactant from 60 animals in each treatment group, shown as %total phospholipids (organic phosphorus) at various time periods in each fraction. N, untreated animals; CLP, cecal ligation and puncture; T3, 3,3',5-triiodo-L-thyronine.

Composition of phospholipid fatty acids. CLP caused alterations in the relative fatty acid saturation of the individual phospholipid moieties. The fatty acid saturation of phosphatidylcholine, the most abundant phospholipid in surfactant, was decreased during sepsis. T3 replacement modulated this effect. In contrast, there was a significant increase in the relative fatty acid saturation of phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol, and cardiolipin at 24 (not shown) and 12 h after CLP (Tables 2, 3, 4). Hormonal treatment prevented these early alterations in the saturation index in the phospholipid moieties. However, by 24 h, the group was indistinguishable from nontreated septic animals.

Table  2.   Fatty acid composition of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine from total lung surfactant lipids
Fatty Acids Phosphatidylcholine
Phosphatidylethanolamine
Phosphatidylserine
N CLP CLP/T3
N CLP CLP/T3
N CLP CLP/T3
6 h 12 h 24 h 6 h 12 h 24 h 6 h 12 h 24 h
Lauric tr. 4 1 1 3 2 1 1 1 3
Myristic 3 3 3 3 4 2 4 2 2 3 2 3 2 2 4
Pentadecyclic 1 1 1 1 1 5 2 4 4 1 tr. 1 1 1
Palmitic 76 68 79 70 69 33 25 28 22 21 14 22 17 22 25
Hexadecenoic 5 5 4 3 5 3 2 3
Hydroxypalmitic 1 1 2 5 1 1 2 3
Methylpalmitic 1 1 4 1 tr.
Margaric 4 2 4 3 1 2 1 2 1 1
Heptadecenoic 1 tr. 1 1
Stearic 3 7 3 3 8 12 40 12 11 34 14 41 19 17 47
Oleic 1 1 1 1 1 3 1 3 3 1 2
Linoleic 1 2 tr. tr. 2 1 1 1
Linolenic 3 3 1 1 4 5 1 3 2
Methylstearic 2 1 1
Nonadecylic 2 1 2
Nonadecenoic 1 1 1 1 5 tr. 1 tr.
Arachidic 1 1 1 1 1 tr. 1 1
Eicosenoic 1 1 1 1 2 1 1 tr.
Arachidonic 1 1 3 4 1 8 3 9 14 3 5 tr. 6 7 1
Methylarachidic 1 1 1 1 1 1 1 1 8 2 tr. tr. tr.
Heneicosanoic 1 3 tr. 1
Hydroxyheneicosanoic 1 tr. 1 tr. 1 5 4 1
Behenic 6 1 2 tr. 25 tr. 5 3 1
Docosatetraenoic 4 3 1
Methylbehenic 1 tr. tr. 1 1 2 4 2 2
Tricosanoic tr. 1 2
Lignoceric 1 tr. 1 1 8 1 6 6 1
Nervonic 2 4 1 1
Pentacosanoic tr. 3 1 1 4 1
Cerotic 1 1 1 1 1 4 2 1
Hexacosatetraenoic 4 1 2
Unidentified 4 8 2 7 5 4 5 14 15 27 2 7 19 21 13
Unsaturated/saturated 0.13 0.15 0.10 0.12 0.16 0.32 0.08 0.30 0.44 0.06 0.23 0.11 0.21 0.22 0.01
Data represent an average of 4 measurements, with extreme range not exceeding 5% of data shown. Fatty acid composition of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine was measured as methyl esters by gas chromatography, as described in METHODS. Fatty acids are given as %total methyl esters in each fraction. Measurements were taken from combined samples of 60 animals for each group. CLP data are shown for animals 12 h after sepsis. Unsaturated/saturated fatty acid ratio of individual phospholipids was calculated for each treatment group from pooled surfactant data and is shown as the arithmetic ratio. tr, Trace.

Table  3.   Fatty acid composition of phosphatidic acid and phosphatidylglycerol from total lung surfactant lipids
Fatty Acids Phosphatidic Acid
Phosphatidylglycerol
N CLP CLP/T3
N CLP CLP/T3
6 h 12 h 24 h 6 h 12 h 24 h
Lauric 1 3 1 1 2
Myristic 1 4 2 3 3 1 3 1 1 1
Pentadecyclic 1 1 4 4 1 1 1 1 1 1
Palmitic 13 26 18 19 22 58 45 60 54 39
Palmitoleic 1 tr. 3 4 1 2 2 3 2 2
Hydroxypalmitic 1 2 4 tr. 1 tr. 2 2
Methylpalmitic 1 1 tr. 1
Margaric 1 1 1 2 1 1 1 1 1 1
Heptadecenoic 1 1 2
Stearic 16 47 12 13 33 7 23 7 8 28
Oleic 1 tr. 1 2 tr. 3 1 3 3 1
Linoleic 1 tr. tr. 1 2 2 1 3
Linolenic 1 tr. 1 1 1 4 5 3 2 6
Methylstearic 4 1 1
Nonadecylic tr. 1 tr. tr. 1 1 1 1 1 tr.
Nonadecenoic 2 1 1 1 tr. 1 1 tr.
Arachidic 2 1 1 1 1 tr. 1 1 1 1
Eicosenoic 5 1 tr. 1 1
Arachidonic 10 2 9 8 1 5 2 6 11 2
Methylarachidic 3 tr. 1 2 1 1 tr. 1 1 tr.
Hydroxyheneicosanoic 6 9 7 1
Behenic 2 1 1 1 1 1 tr. tr.
Erucic 4 2 2 1
Hydroxybehenic 3 2
Methylbehenic 1 1 1 1 2 tr. 1 3
Tricosanoic 1 1 1 1 tr.
Lignoceric 9 8 3 1 tr. tr.
Nervonic tr. 1 1
Pentacosanoic 2 tr. tr. 1
Cerotic 2 5 2 1 tr. 2 tr.
Unidentified 7 10 12 7 23 8 8 4 8 9
Unsaturated/saturated 0.39 0.02 0.28 0.31 0.05 0.23 0.15 0.23 0.29 0.18
Data represent an average of 4 measurements, with the extreme range not exceeding 5% of data shown. Measurements were taken from combined samples of 60 animals for each group. Fatty acid composition of phosphatic acid and phosphatidylglycerol was measured as methyl esters by gas chromatography and is shown as %total methyl esters in each fraction. CLP data are shown for animals 12 h after sepsis. Unsaturated/saturated fatty acid ratio of individual phospholipids was calculated for each treatment group and is shown as the arithmetic ratio.

Table  4.   Fatty acid composition of cardiolipin and phosphatidylinositol from total lung surfactant lipids
Fatty Acids Cardiolipin
Phosphatidylinositol
N CLP CLP/T3
N CLP CLP/T3
6 h 12 h 24 h 6 h 12 h 24 h
Lauric 1 1 1 1 1 2 1 1 3
Hydroxylauric 1 1 tr. tr. 3
Myristic 1 3 2 2 3 2 3 2 2 3
Pentadecyclic 1 tr. 2 1 1 3 1 2 2 1
Palmitic 29 23 34 30 20 40 28 38 29 21
Hexadecenoic 2 2 2 1 tr. 1 1
Hydroxypalmitic 1 1 2 1 tr. 1 1
Methylpalmitic 1 tr. 1 1 1 tr. 1
Margaric 1 1 1 1 1 4 1 1 1 1
Heptadecenoic 1 tr. 1 2 1
Stearic 15 41 15 14 37 9 45 11 10 40
Oleic 3 2 2
Linoleic 1 1 tr.
Linolenic 5 1 4 2
Methylstearic 3 2 tr. 3 tr. 1 tr.
Nonadecylic 3 2 tr. 1 tr. tr. 1
Nonadecenoic tr. 1 1
Arachidic tr. 1 1 1 1 1 tr. tr. 1
Eicosenoic 1 1 2 1 tr. tr. tr.
Arachidonic 9 8 9 4 2 5 5 1
Methylarachidic 1 1 1 3 1
Heneicosanoic 1 tr. 1 1 2 tr. 1 1 1
Hydroxyheneicosanoic 7 1 5 3 1 7 1 10 11 1
Behenic 1 2 tr. 1 tr. 1 1 1 1
Erucic 1 1 1 2 2 1 1 1
Hydroxybehenic tr. 1 4 1 tr. 3 1 2
Methylbehenic 1 2 2 7 tr. tr. 2
Lignoceric 2 1 2 1 1 1 1 6 4 tr.
Nervonic 1 tr. 1 1 tr. 3 1 1
Cerotic 2 1 2 4 tr. 1 3 5 tr.
Hexacosatetraeonic 2 1
Unidentified 4 22 5 10 26 4 6 10 12 21
Unsaturated/saturated 0.35 0.03 0.28 0.32 0.01 0.13 0.04 0.13 0.13 0.04
Data represent an average of 4 measurements, with extreme range not exceeding 5% of data shown. Measurements were taken from combined samples of 60 animals for each group. Fatty acid composition of cardiolipin and phosphatidylinositol was measured as methyl esters by gas chromatography and are shown as %total methyl esters in each fraction. CLP data are shown for animals 12 h after sepsis. Unsaturated/saturated fatty acid ratio of individual phospholipids was calculated for each treatment group and is shown as the arithmetic ratio.

The major fatty acid constituent of phosphatidylcholine in normal animals was palmitic acid (Table 2). Sepsis reduced this, with a commensurate twofold increase in the concentration of stearic acid. Hormonal augmentation of septic animals maintained early stearic acid concentrations. However, by 24 h, the fatty acid makeup of phosphatidylcholine was indistinguishable from that of nontreated septic animals.

The major components of phosphatidylethanolamine were palmitic acid and stearic acid, with moderate contributions by arachidonic and behenic acids. Septic animals had decreased concentrations of palmitate and arachidonic acids, with a large increase in stearic acid. Thyroid hormonal treatment maintained early septic stearic and arachidonic acid concentrations; however, these differences were diminished by 24 h.

Lung surfactant phosphatidylserine consisted primarily of palmitate, stearate, and behenic acids. In addition, there was a moderate amount of arachidonic, methylarachidic, and lignoceric acids. CLP increased the amount of saturated palmitic and stearic fatty acids with decreases in arachidonic, behenic, and lignoceric acids. T3 treatment decreased the early changes in these fatty acids, although to a lesser extent with behenic acid. However, these changes were manifest by 24 h.

The most abundant fatty acids in phosphatidic acid (Table 3) were palmitic, stearic, and arachidonic acids. Sepsis produced a significant increase in palmitic and stearic acids and decreases in arachidonic acid. The addition of T3 partially modulated these changes.

Normal rat phosphatidylglycerol was composed primarily of palmitic acid, with lesser contribution of stearic and arachidonic acids. Sepsis caused a significant increase in stearic acid, with small changes in palmitic acid. Hormone treatment transiently attenuated this change.

Cardiolipin was primarily composed of palmitic, stearic, and arachidonic acids (Table 3). Sepsis increased stearic acid and significantly reduced arachidonic acid. T3 supplementation produced a short-lasting modulation of these findings.

Phosphatidylinositol consisted primarily of palmitic acid with a variety of other fatty acids. CLP caused a dramatic increase in stearic acid, with a decrease in palmitic acid. Thyroid hormone reduced, but did not prevent, these alterations in phosphatidylinositol fatty acids.

DISCUSSION

Surfactant is a complex mixture of phospholipids and lipoproteins that coats the alveolar surface of the lung. It is manufactured by pulmonary alveolar type II cells and maintains normal lung biophysical function by lowering alveolar surface tension. Conditions that alter surfactant availability or integrity have been implicated in a number of pulmonary disease processes. Sepsis produces acute alterations in the pulmonary surfactant system, including a decrease in total phospholipid availability, an altered biochemical composition, and possibly a decreased functional effectiveness of the remaining surfactant. Septic patients demonstrate decreased pulmonary compliance, increased lung water, and decreased functional residual capacity (decreased alveolar volume).

Pison and co-workers (23) characterized the lung phospholipid alterations seen in septic patients and suggested that damage to the surfactant system is of primary importance in ARDS. In their study, they noted a decrease in the total amount of surfactant phospholipids, as well as an increase in lesser surface active components. Unfortunately, there is little data regarding the pathophysiology of the acute sepsis-induced changes in lung surfactant. Nonetheless, the altered surfactant integrity appears deleterious. Recent studies have suggested that surfactant augmentation, pharmacologically or via heterologous replacement, improves respiratory function during neonatal and adult ARDS (4).

The Low T3 or Sick Euthyroid syndrome is often coexistent with sepsis and respiratory dysfunction; however, the physiological ramifications of the altered thyroid hormone economy, specifically on lung surfactant integrity, are not clear. Although the regulation of surfactant synthesis is complex, thyroid hormone appears to be intimately involved in surfactant metabolic regulation (2). A stimulatory effect of T3 on phosphatidylcholine synthesis has been documented in studies utilizing rat, human, and rabbit lung in vitro (20). Also, a synergistic effect on fetal rat lung phospholipid synthesis has been shown between glucocorticoid and thyroid hormone (2).

In our experiments, sepsis produced acute alterations in surfactant availability after CLP. Septic animals had an acute decrease in total lavagable lung phospholipids at 12-24 h compared with control nonseptic animals. Thyroid hormonal treatment maintained lung phospholipid content in the normal range. This finding is in agreement with earlier experimental work and corroborates recent clinical trials utilizing thyroid hormone in respiratory distress in the neonate; these studies found an improved surfactant function and synthesis (1, 10).

Sepsis also caused dramatic alterations in surfactant phospholipid amount and biochemical composition. Although the major surfactant phospholipid, phosphatidylcholine, did not change appreciably after CLP with or without T3 treatment, the acidic phospholipids were profoundly affected. There was a coordinate increase in the relative concentration of phosphatidylinositol-cardiolipin and a corresponding decrease in phosphatidic acid-phosphatidylglycerol during sepsis. Thyroid hormonal augmentation attenuated these changes in surfactant phospholipids and maintained compositional integrity with the exception of a late decrease (at 24 h) in the content of phosphatidylinositol.

Sepsis induced acute changes in the fatty acid composition and saturation index of the major surfactant phospholipids. The most representative of the surfactant fatty acids are palmitate and stearate. Occasionally, behenic acid is a predominant species, such as in phosphatidylserine. The relative concentrations of arachidonic and lignoceric fatty acids approach 10% in phosphatidic acid and contribute substantially to the composition of cardiolipin, phosphatidylserine, and phosphatidylethanolamine. It is noteworthy that the surfactant phospholipids contain a significant amount of long-chain fatty acids and may comprise up to 50% of total acylated fatty acids.

Sepsis did not cause a significant alteration in phosphatidylcholine fatty acid composition, which has less fatty acid diversity compared with the other phospholipids. Phosphatidylcholine is composed of >75% palmitic acid and up to 90% saturated fatty acids. In addition, it occasionally consists of arachidonic and methylarachidic fatty acids. Although sepsis produced few compositional alterations in phosphatidylcholine, the fatty acid saturation was acutely decreased by CLP. This is in direct contrast to an increased fatty acid saturation seen in all of the other surfactant phospholipid moieties. Phosphatidylcholine fatty acid saturation was maintained in a more normal ratio when the septic animals were treated with T3. At least two mechanisms have been proposed for formation of unsaturated phosphatidylcholine (16, 19). We may assume that CLP inhibits either deacylation-reacylation or deacylation-transacylation, both of which transform unsaturated phosphatidylcholine molecules into primarily saturated species of the surfactant complex.

CLP generally caused a decrease in the amount of phospholipid palmitate, whereas the amount of stearic acid increased. An exception was seen in phosphatidylserine and phosphatidic acid, in which an increased amount of stearate and palmitate were seen during CLP. Also, there was a domination of stearate rather than palmitate in surfactant phospholipids of CLP animals, with counterchanges of fatty acids in T3-treated animals, with the exception of phosphatidylcholine and phosphatidylglycerol.

Phosphatidylserine and phosphatidylinositol in rat lung surfactant do not have unsaturated 18-carbon fatty acids. This is an unusual finding and may reflect phylogenesis of the phospholipids, such as phosphatidylethanolamine and phosphatidylcholine, that include cytidine 5'-diphosphate (CDP)-diacylglycerol rather than diacylglycerol (Fig. 3). In addition, the similarity of fatty acid composition between phosphatidylserine and phosphatidic acid, with domination of long-chain fatty acids, suggests a shortcut biosynthetic pathway of phosphatidylserine from phosphatidic acid through CDP-diacylglycerol, rather than through phosphatidylcholine or phosphatidylethanolamine (9).

Fig. 3. Biosynthetic pathways of rat lung surfactant are shown. Precursor lipid moieties are depicted in circles; major surfactant phospholipids are shown enclosed in boxes. CTP, cytidine 5'-triphosphate; CMP, cytidine 5'-monophospate; CDP, cytidine 5'-diphosphate; PPi, inorganic pyrophosphate.
[View Larger Version of this Image (20K GIF file)]

A relationship between phosphatidylserine and phosphatidylethanolamine is suggested by the similarity in unique fatty acid composition of both phospholipids, including the relatively high content of behenic acid. The differing fatty acid compositions of phosphatidylglycerol and cardiolipin may be secondary to participation of CDP-diacylglycerol in the biosynthesis of cardiolipin as described previously (24). There was no fatty acid compositional evidence in the lung to support the conversion of phosphatidylethanolamine to phosphatidylcholine previously reported in rat liver (6).

Interestingly, the fatty acid composition of phosphatidylethanolamine and phosphatidylserine became more similar during the CLP insult. Palmitate significantly decreased in phosphatidylethanolamine and was coordinately increased in phosphatidylserine (Table 2). These unusual observations suggest that the phosphatidylserine decarboxylase pathway of phospholipid biosynthesis and transformation is important in lung surfactant maintenance. This pathway may serve to stabilize diacylglycerol and the CDP-diacylglycerol biosynthetic subpools during stress (Fig. 3). This may also explain the relative resistance of surfactant zwitterionic phospholipids to change during infection. These phospholipids play an important role in the stabilization of the surfactant lipid matrix and may significantly contribute to surfactant homeostasis.

Thus we can distinguish three groups of surfactant phospholipids according to their response to CLP. The most abundant zwitterionic phospholipids (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine) had the least alterations in their composition during sepsis. Together, these phospholipids make up >80% of the total surfactant pool. The second group includes phosphatidylinositol and cardiolipin, which underwent an increase in their relative amounts by 12 h after CLP. The third group is composed of phosphatidylglycerol and phosphatidic acid; these phospholipids dramatically decreased during a septic insult.

The sepsis-induced changes in phospholipid amount and composition appear to be functionally significant. A difference in surfactant hysteresis isotherms was noted in the septic animals compared with control animals. Despite correction to total phospholipid content, surface tension isotherms from septic animals treated with T3 were improved over untreated CLP controls, suggesting an intrinsic difference in surfactant functional integrity. Surfactant adsorption rate correlates with the stability or fluidity of the phospholipid interface. Unsaturation of phospholipid fatty acids reduces interface mobility and increases the "rigidity" of the pulmonary alveolus at low volumes, preserving alveolar structure. In this study, surfactant adsorption rate was significantly decreased in septic compared with control animals; this may in part reflect the relative changes in saturation seen in the fatty acids of surfactant phospholipid during CLP.

The mechanism for the sepsis-induced and, specifically, hormone-sensitive alterations in surfactant composition is not known. Choline phosphate cytidyltransferase regulates the formation of CDP-choline and has been suggested to be the major rate-limiting enzyme involved in surfactant synthesis. Although the enzyme has been shown to be thyroid hormone responsive in rat liver, the effects of T3 on lung enzyme activity have not been extensively examined. In a previous report, we demonstrated a significantly increased cytidyltransferase activity in septic rat lung fractions compared with nonseptic controls (13). The addition of thyroid hormone did not significantly change the supranormal cytidyltransferase activity seen in the septic animals. Therefore, although cytidyltransferase activity may be important in the regulation of phosphatidylcholine synthesis during unstressed metabolism, it does not appear to be a key regulator of surfactant metabolism during sepsis.

The findings reported herein attest to the complex interactions that the pulmonary surfactant system undergoes during a septic challenge. Deleterious changes in lung compliance and alveolar pressure relationships can occur secondary to the changes in lung phospholipid environment. The role of surfactant-associated proteins was not addressed in these experiments. Alterations in the availability or function of the phospholipid-associated proteins may provide an alternative or additional explanation for the surfactant biophysical changes seen during sepsis. The surfactant functional and biochemical findings reported herein may also partially reflect contamination of lung surfactant with intra-alveolar proteins and phospholipids secondary to cellular injury. Although this factor is an important consideration when attempting to explain relative changes in phospholipid species, previous investigations have not demonstrated extensive cellular damage or necrosis at the time points selected in these experiments (12). Finally, a thyroid-pulmonary axis may exist, because surfactant homeostasis is in part hormonally responsive. The role of surfactant compositional alterations and thyroid hormonal augmentation during sepsis requires further study.

ACKNOWLEDGEMENTS

This work was supported by National Institute of General Medical Sciences Grant GM-49272-01A1.

FOOTNOTES

Address for reprint requests: S. A. Dulchavsky, Dept. of Surgery, Detroit Receiving Hospital, 4201 St. Antoine, Detroit, MI 48201.

Received 25 July 1996; accepted in final form 12 February 1997.

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