Shriners Burns Hospital, and Departments of Surgery and Anesthesiology, The University of Texas Medical Branch, Galveston, Texas 77555
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ABSTRACT |
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The primary goal of
this study was to determine the contributions of plasma free fatty
acids (FFA) and de novo synthesized fatty acids (FA) to lung surfactant
phosphatidylcholine (PC) synthesis. A new stable isotope tracer model
was developed in which
[1,2-13C2]acetate
and uniformly labeled
[U-13C16]palmitate
were infused in nine normal overnight fasted pigs to quantify
surfactant kinetics in the basal state and during low-dose glucose
infusion (2 mg · kg1 · min
1). There was no
effect of glucose; therefore, all data were pooled. The
surfactant PC-bound palmitate incorporation rate from plasma palmitate
was 20.9 ± 1.9 nmol
palmitate · h
1 · g
wet lung
1, compared with
the rate of 2.1 ± 0.3 nmol
palmitate · h
1 · g
wet lung
1 from de novo
synthesized palmitate. The PC-bound palmitate secretion rate from the
lamellar body pool to the alveolar surface pool was 239 ± 26 nmol
palmitate · h
1 · g
wet lung
1. Approximately
90% of the secreted PC recycled back to the lamellar bodies for
reutilization. We conclude that plasma is the primary contributor of FA
for surfactant PC synthesis under the conditions of this experiment.
stable isotopes; fatty acids; pulmonary surfactant; phosphatidylcholine; synthesis
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INTRODUCTION |
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THE IMPORTANCE of surface forces in the lungs was first demonstrated in 1929 (27). It was not until 1955 that a surfactant complex that markedly lowered surface tension was identified in the alveolar lining layer (28). In mammals, the surfactant complex reduces lung surface tension, maintains fluid balance, keeps the lungs dry, provides alveolar stability, and reduces lung infection (4, 34). The alveolar type II cell is the primary site of synthesis, storage, and release of the pulmonary surfactant complex. This complex is a lipid-protein mixture that is highly enriched with the most abundant zwitterionic phospholipids, dipalmitoyl phosphatidylcholine (DPPC), and lesser amounts of other phospholipids. The fatty acids (FA) for net surfactant phosphatidylcholine (PC) synthesis may be derived from circulating free fatty acids (FFA) and de novo synthesis (5, 9, 25). The relative contribution of each of these potential sources of FA for incorporation into the lung surfactant PC has not previously been quantified in vivo. The difficulty in this quantitation lies in the fact that the precursor enrichment for de novo FA synthesis cannot be directly measured, and thus it is difficult to quantify the corresponding synthesis rate as well as the total PC synthetic rate.
The novel aspect of our approach for quantifying de novo FA synthesis comes from the application of mass isotopomer distribution analysis, which was first presented by Hellerstein et al. (16). This approach enables calculation of the precursor enrichment for de novo FA synthesis from the labeling patterns of synthesized FA. By using mass isotopomer distribution analysis coupled with traditional tracer dilution methodology, we have quantified the relative contributions of the various sources of FA (i.e., de novo synthesized vs. preformed FA) to surfactant PC synthesis. We have also quantified the total PC synthetic and secretion rates and the rate of recycling of secreted PC. We used stable isotope tracer methodology involving the infusion of [1,2-13C2]acetate as a precursor for de novo FA synthesis and uniformly labeled [U-13C16]palmitate as a tracer of preformed FFA. We found that in the postabsorptive state de novo synthesized FA play a minor role as precursors for pulmonary surfactant synthesis. In contrast, preformed (plasma) FFA are the primary precursor for surfactant PC synthesis.
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METHODS |
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Tracer Infusion
Nine Yorkshire swine (from K-bar live stock, Sabinal, TX), weighing 16.4 ± 2.7 kg, were used. The animals were fasted overnight before the start of the infusion study and kept fasted during the isotope tracer infusion. All animals were given ketamine (20 mg/kg im, Ketaset, Fort Dodge Laboratories, Fort Dodge, IA) for the surgery of catheter placement and tracheotomy. Tracheotomy was used without ventilation in this study because these animals served as a control group for a concurrent study in which a tracheotomy was required. A venous catheter was inserted via the jugular vein for isotope tracer infusion, and a second catheter was inserted via the right common carotid artery into the abdominal aorta for blood sampling. After blood was taken to determine background enrichment, a constant infusion of [1,2-13C2]acetate {99% enriched, 95.5 molar percent excess (MPE) [1,2-13C2]acetate; Isotec, Miamisburg, OH; 24 µmol · kgBronchoaveolar Lavage
The right lung was isolated, weighed, vacuum degassed, and suspended in a Plexiglas vacuum chamber via the trachea cannula, which passed through the top of the chamber to two 150-ml syringes connected by a three-way valve. A subatmospheric chamber pressure was externally applied to the lungs, and the lungs were filled with fluid consisting of normal saline plus 0.01 M Tris buffer. This ensured that the lungs were fully filled with fluid, and any residual CO2 was absorbed without the introduction of air bubbles. This fluid was withdrawn from the lungs into a syringe by application of a positive chamber pressure to the lungs along with gentle suction. This procedure was repeated three times, and as much bronchoalveolar lavage (BAL) was collected as possible with positive external pressure, while care was taken not to introduce any air bubbles. The entire procedure was repeated six times, each starting with saline and the Tris buffer. Each volume of collected BAL was measured for later calculation of total PC content in BAL. Any fluid that leaked from the lungs was collected and added to the appropriate BAL fraction. The total alveolar PC pool size was calculated as the sum of these six washes.Sample Analysis
Alveolar surfactant isolation.
Lung surfactant from BAL was isolated with a previously described
technique (2). The BAL was filtered through gauze, and the volume was
measured. An aliquot of BAL was centrifuged at 160 g for 20 min to remove intact cells
and cellular debris. The supernatant was collected and centrifuged at
100,000 g for 1 h at 4°C in an
ultracentrifuge (model L7-55 Beckman Ultracentrifuge, SW 40 TI
rotor, Beckman Instruments, Palo Alto, CA). The precipitate pellet was
stored at 70°C for lipid extraction.
Lamellar body isolation.
After BAL collection, 0.5 g of lung tissue was minced and homogenized
in 1 M sucrose for 3 min (Brinkman Homogenizer, Brinkman Instrument,
Westbury, NY). The homogenate was filtered, and the volume was adjusted
to 5 ml with 1 M sucrose. A discontinuous sucrose density gradient from
0.9 to 0.2 M was loaded on top of the homogenates, as described by
Duck-Chong (7). The gradients were centrifuged at 200 g for 15 min and then at 116,000 g for 3 h at 4°C. The lamellar
bodies that sedimented in the 0.45 M sucrose layer were identified as a
white opaque band, similar to that described by Duck-Chong and Young et
al. (35). This band was aspirated, quantified by volume, and stored at
70°C for lipid extraction.
PC isolation from surfactant. The lipids from the lamellar bodies and from BAL were extracted with a mixture of hydrochloric acid, heptane, and 2-propanol (1:10:40 by vol). The upper layer was aspirated and dried under nitrogen. The lipid extract was separated by thin-layer chromatography on a silica gel plate with a two solvent system of 1) chloroform-methanol-water equalling 65:30:5 (by vol) and 2) heptane-ethyl ether-acetic glacial acid equaling 80:20:2 (by vol). The PC location was identified by iodine dye, scraped from the gel plate, and dissolved in 1 ml chloroform. A 250-µl aliquot was used for PC-bound palmitate enrichment measurements, a 250-µl aliquot for PC-bound FA composition determinations, and a 500-µl aliquot for phosphorus analysis with the method described by Bartlett (3). [14C]DPPC was used to monitor the recovery (54 ± 2%) of this isolation procedure. The total PC contents in the lamellar bodies and BAL were used for calculating surfactant PC synthetic rates and secretion rates, respectively.
Lung tissue PC content. The nonlavaged frozen lung tissue (0.5 g) was minced and homogenized, and PC was extracted with the same procedures used in isolating surfactant PC. The tissue PC content was determined by phosphorus analysis (3).
FA isolation from plasma and from PC of BAL and lamellar bodies.
Plasma (500 µl each) samples were extracted with a mixture of
hydrochloric acid, heptane, and propanol (1:10:40 by vol). The upper
layer was collected and dried under nitrogen. The FFA extract was
isolated by thin-layer chromatography with a mixture of heptane, glacial acetic acid, and ethyl ether (80:2:20 by vol). The FFA location
was identified by iodine staining, scraped, and stored at
70°C for the determination of isotope enrichment and composition.
FA enrichment measurement. The FA isolated from PC in BAL and lamellar bodies and from plasma were methylated with baron trifluoride (BF3) in methanol, and the isotopic enrichment of palmitate methyl ester was determined by gas chromatography-mass spectrometry (GC-MS, model 5992, Hewlett-Packard) in the electron impact ionization mode. Ions of mass-to-charge ratio (m/z) 270(m + 0), 271(m + 1), 272(m + 2), 273(m + 3), 274(m + 4), and 286(m + 16) were selectively measured.
Concentration and composition. Plasma FFA composition and concentration, FA compositions in lamellar body PC, alveolar surface PC, and lung tissue were determined by HPLC (Waters 2690, Milford, MA) following the procedure described by Uji et al. (30). Heptadecanoic acid was used as an internal standard. Plasma triglyceride concentration was determined enzymatically (Sigma Diagnostics, Triglyceride GPO-Trinder, St. Louis, MO). Plasma PC was isolated by TLC, and the content was measured by phosphorus analysis (3).
Plasma very low density lipoprotein triglyceride isolation. Plasma very low density lipoprotein (VLDL) was isolated from 3 ml of plasma by overlaying the plasma with a 0.9% normal saline with a density of 1.006 and subsequent ultracentrifugation at 50,000 rpm (171,500 g) for 20 h at 15°C. After ultracentrifugation, the VLDL was carefully removed along with the density solution by slicing the tube into sections (11, 15). VLDL-triglyceride (VLDL-TG)-bound FA were isolated and measured following the same procedures as in PC-bound FA.
Calculations
We calculated the rates of surfactant PC synthesis from plasma FFA and from de novo synthesized FA. In this model, we focused on the quantification of surfactant palmitate-bound PC kinetics. Because palmitate is the primary FA incorporated in surfactant PC, this was a reasonable simplification to investigate the surfactant PC kinetics. We also calculated the rates of secretion, recycling, and irreversible loss of surfactant PC. A schematic of the parameters calculated by our model is shown in Fig 1. Briefly, the FA component of newly synthesized surfactant PC can be derived from two sources: de novo synthesized FA and plasma FFA. Once synthesized, the PC is stored in the lamellar bodies of the alveolar type II cells. The PC in the lamellar bodies is then secreted onto the alveolar surface and can be either recycled back to lamellar bodies or irreversibly lost.
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The measurements required to quantify the parameters shown in Fig. 1 are plasma palmitate m + 2/m + 0, m + 4/m + 0, and m + 16/m + 0 enrichment; lamellar body PC-bound palmitate m + 2/m + 0, m + 4/m + 0, and m + 16/m + 0 enrichments; alveolar surface PC-bound palmitate m + 16/m + 0 enrichment; and the pool sizes of lamellar body PC and alveolar surface PC.
Incorporation of plasma FFA into surfactant PC is determined with the traditional precursor-product tracer approach. After the infusion of [U-13C16]palmitate, the enrichment of plasma palmitate [tracer to tracee ratio (TTR)(m + 16), the precursor, denoted EP(t)] is measured over the infusion duration (t), and the enrichment of lamellar body PC-bound palmitate [TTR(m + 16), the product, denoted EL(t)] is measured at the end of the infusion. The rate at which labeled palmitate appears in lamellar body PC for a given enrichment of the plasma palmitate pool will quantitatively reflect the rate that plasma FFA is incorporated into lamellar body PC. Thus the fractional synthetic rate (FSRsyn) of lamellar body PC from plasma palmitate is calculated with the traditional formula
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The measurement of the incorporation of de novo synthesized palmitate into lamellar body PC uses the same precursor-product principle as described above. The difficulty in applying the traditional precursor-product tracer approach to the measurement of incorporation of de novo synthesized palmitate into PC is that the precursor enrichment cannot be directly measured. The precursor pool, in this case, is the pool of two carbon acetyl-CoA molecules in the lung. However, by use of the general principles of mass isotopomer distribution analysis (16), the precursor pool enrichment can be deduced from the product enrichment. This method hinges on the fact that FA are polymers of acetyl-CoA. If some of the acetyl-CoA molecules contain two labels (achieved in this case by infusing [1,2-13C2]acetate), then the palmitate molecules, formed from the acetyl-CoA molecules, could have two, four, or more labels. The relative distribution of unlabeled, doubly labeled, and quadruply labeled palmitate molecules will be determined by the enrichment of the acetyl-CoA molecules. For example, if one-half the acetyl-CoA molecules are doubly labeled and one-half the acetyl-CoA molecules are unlabeled, then one would expect that on an average 8 out of the 16 carbons of palmitate formed from the acetyl-CoA molecules would be labeled and the other 8 carbons would be unlabeled. By this logic, every possible precursor enrichment of acetyl-CoA would result in a unique labeling pattern in the palmitate molecules. Therefore, by examining the distribution of labels in the palmitate molecules, we can deduce the precursor enrichment. We have previously described how this can be achieved in quantitative terms (1). The actual formula to determine the precursor enrichment (i.e., the enrichment of acetyl-CoA molecules in the lung, denoted p) is
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The fractional secretion rate (FSRsec) of lamellar body PC to alveolar surface PC is calculated with the traditional precursor-product tracer method, with the addition of a term t EA(t)/2 to account for the loss of tracer from alveolar surface PC pool. After the infusion of [U-13C16]palmitate, the lamellar body PC pool will become labeled. These labeled lamellar body PC molecules will be secreted into the alveolar PC pool. Thus, by use of the measurement the lamellar body PC-bound palmitate enrichment [TTR(m + 16), denoted EL(t)] and the alveolar surface PC-bound palmitate enrichment [TTR(m + 16), denoted EA(t)] at the end of the infusion, the secretion of lamellar body PC into alveolar surface PC can be calculated with the formula
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We assume that the precursor enrichment for surfactant PC synthesis reaches a plateau rapidly (<2 h) after the start of the infusion, and the enrichment of PC product increases linearly during the infusion. Therefore, only one lung tissue sample after the infusion starts is required for the calculation of the rates of surfactant PC synthesis. We also assume that a physiological steady state exists during the time of our measurements. In this case, the rate of irreversible loss of alveolar surface PC must be equal to the synthetic rates of PC from plasma FFA and de novo synthesized FA. Once these two synthetic rates are calculated (see above), the rate of irreversible loss can be calculated by adding the two synthetic rates together. The recycling rate is calculated by subtracting the irreversible loss rate from the lamellar body PC secretion rate. Thus all quantities pictured in Fig. 1 can be quantified.
Statistical Analysis
All results are expressed as means ± SE. We compared all parameters of the low-dose glucose infusion group and the nonglucose infusion group with the unpaired t-test. We found that none of the parameters were significantly different between the two groups. In addition, all the parameters from the 4- and 6-h infusion studies were in the range of those from 8-h infusion studies. Consequently, all the data from these nine pigs were pooled. ![]() |
RESULTS |
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We initially used a low-dose glucose infusion (2 mg · kg1 · min
1)
in three pigs because the method requires that a certain rate of de
novo FA synthesis occur in the lung for sufficient labeled acetate to
be incorporated to allow accurate detection in these FA. We found,
however, that accurate measurement of the enrichment of de novo
synthesized FA was possible even when glucose was not infused, so six
pigs were studied without glucose infusion. We found that the low rate
of glucose infusion did not cause a significant difference in the
various parameters.
The concentrations of individual plasma FFA, plasma PC, plasma
triglyceride, and the rate appearance of plasma palmitate were constant
throughout the infusion (data not shown), indicating a physiological
steady state. The palmitate composition in plasma FFA was 29.7 ± 0.4%, whereas the palmitate composition in lamellar body PC and
alveolar surface PC was 61.7 ± 2.1 and 71.9 ± 1.4%, respectively. The individual FA composition in plasma FFA, lamellar body PC, alveolar PC, and lung tissue PC is listed in Table
1.
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The surfactant PC pool sizes in lamellar bodies, alveolar surface, and lung tissue are 1.14 ± 0.02, 1.46 ± 0.12, and 12.6 ± 1.4 µmol PC/g wet lung, respectively. The PC content in lung tissue was 10.5 times as high as that in lamellar bodies. Our isotopic measurements were made on lamellar body PC and alveolar surface PC, which correspond to the most abundant pulmonary surfactant.
Precursor Enrichment
Plasma palmitate enrichment TTR(m + 16) reached an isotopic steady state (0.032 ± 0.005, EP(t)) within 1 h after [U-13C16]palmitate infusion. The precursor enrichments for de novo FA synthesis were 0.247 (in the 4-h infusion), 0.230 and 0.255 (in the 6-h infusions), and 0.255 ± 0.010 (in the 8-h infusions). These data demonstrated that the precursor for de novo FA synthesis in the lungs reached an isotopic steady state in <4 h after infusion started. In addition, we assessed FA synthesis in the liver by measuring the palmitate enrichment in circulating VLDL-TG. On the basis of the plasma VLDL-TG palmitate enrichments TTR(m + 2) and TTR(m + 4), the precursor enrichment for palmitate in VLDL-TG incorporation reached a steady state (0.265 ± 0.042) within 2 h of infusion.The isotope enrichments required for the calculations are summarized in
Table 2.
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Synthetic Rates
The calculated FSRsyn for surfactant PC from plasma palmitate and de novo synthesized palmitate in 8-h infusions was 1.44 ± 0.13 and 0.14 ± 0.02%/h, respectively. The FSRsyn of the two 6-h infusion studies was 0.13 and 0.15%/h from de novo synthesized palmitate and 1.52 and 1.45%/h from plasma palmitate. The FSRsyn of the 4-h infusion study was 0.141%/h (from de novo synthesized palmitate) and 1.35%/h (from plasma palmitate). Because the FSRsyn from 4- and 6-h infusions was in the range of 8-h infusion FSRsyn, all of these FSRsyn data were pooled to calculate synthetic rates. The rate of synthesis of surfactant PC from plasma palmitate was 20.9 ± 1.9 nmol palmitate · hSecretion Rate and Recycling Rate
The FSRsec from the 4- and 6-h infusion studies was also in the range of the 8-h infusion studies. The pooled value of FSRsec for surfactant PC was 12.31 ± 2.01%/h. The absolute palmitate-bound PC secretion rate from the lamellar body pool to the alveolar surface pool, calculated as the product of FSRsec and alveolar surface PC-bound palmitate pool size, was 239 ± 26 nmol palmitate · h ![]() |
DISCUSSION |
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We have developed a new stable isotope tracer model (Fig. 1) that allows, for the first time, the quantitation of the rates of surfactant synthesis from all pathways, including incorporation of FA synthesized de novo and circulating FFA. This method also enables us to quantify the irreversible loss of lung surfactant PC. Our central observations are that either in the postabsorptive state or during a low-dose glucose infusion, plasma is the major source of FA for newly synthesized PC and that ~90% of the secreted PC is recycled from the alveolar surface PC pool back into the lamellar body PC pool.
Assumptions
We have assumed that both plasma FFA enrichment and de novo FA synthesis precursor enrichment reached steady state during the infusion. We observed a steady state in plasma FFA enrichment within 1 h after [U-13C16]palmitate infusion started. We found no differences in the precursor enrichments for de novo FA synthesis in the lungs in 4-, 6-, and 8-h infusion studies. We also found in this study that the de novo FA synthesis precursor enrichment for VLDL-TG incorporation in liver was at a steady state within 2 h of infusion. Consequently, it is reasonable to assume that both plasma FFA enrichment and the precursor enrichment for de novo FA synthesis were at isotopic steady state during the infusion.We assumed the enrichments of synthesized PC increased linearly with no delay during the infusion. If there was a delay in product PC enrichment or the PC enrichment reached plateau before the end of infusion, the FSRsyn (calculated as the PC-bound palmitate enrichment change divided by the infusion duration and the precursor enrichment) would be different during different periods of the infusion. However, when we tested shorter infusion intervals, such as 4 and 6 h, the calculated values for FSRsyn were in the range of 8-h infusion, which were 0.14 ± 0.02%/h (from de novo synthesized palmitate) and 1.44 ± 0.13%/h (from plasma palmitate). These data indicated that although a delay in appearance of label in PC might be expected on a theoretical basis, particularly from the de novo FA synthesis pathway, any such delay was insignificant in comparison to the infusion intervals we used. A similar observation has been reported by Ikegami et al. (17) in which the labeled saturated FA PC accumulated in a linear fashion for 27 h in adult and 44 h in newborn sheep with no apparent initial delay. In addition, other literature values (19, 21) report that far more than 8 h was required for surfactant PC enrichment to achieve an isotopic plateau. Therefore, our calculation of the rates of surfactant PC synthesis based on one tissue sample was reasonably accurate.
We isolated the lamellar bodies from whole lung homogenate following
the same method as Duck-Chong (7) and Young et al. (35) used. Young et
al. have reported that the nonlamellar body contamination from other
organelles in this isolation procedure was insignificant, employing
morphological criteria and the enzyme markers of microsome,
mitochondria, and plasma membranes. They suggested a very high yield of
lamellar body isolation from this procedure based on the close
agreement of their lamellar body disaturated phosphatidylcholine (DSPC)
measurement value to the calculated values by Mason et al. (26) and
Kikkawa et al. (23). Because precise estimate of this recovery
procedure was difficult due to substantial technical problems, we
assumed 80% as lamellar body isolation recovery in our kinetics
calculation. We used 60 and 100% recoveries as lower and upper bounds
to estimate the calculated kinetics error range for comparison. From 60 to 100%, synthetic rates (nmol
palmitate · h1 · g
wet lung
1) changed from
2.7 to 1.6 (from de novo synthesized palmitate) and from 27.8 to 16.7 (from plasma palmitate), but the relative contribution of the two FA
sources to PC synthesis was the same. There were no changes in
secretion rate and only small changes in the recycling rate (from 208 to 220 nmol
palmitate · h
1 · g
wet lung
1). The loss
rates changed from 30.5 to 18.3 nmol
palmitate · h
1 · g
wet lung
1. Consequently,
although our assumed recovery rate of 80% was only an estimate, any
reasonable error in this assumption did not affect our conclusions.
We assumed that the secreted surfactant PC recycles back to the lamellar body pool intact. Hallman et al. (14) provided some evidence for this assumption. By intratracheal instillation of surfactant PC labeled with both [32P]palmitate and [3H]palmitate to rabbits, Hallman et al. were able to measured the ratio of specific activity 3H to 32P from PC and found that the ratio remained the same in both lavage fluid PC and the lamellar body PC, as that in the instilled fluid. This supported the notion that the secreted surfactant PC is recycled back to the lamellar bodies intact. This concept was confirmed by Glatz et al. (10) and Jacobs et al. (18) on the basis of similar experiments. They found that as much as 95% of alveoli PC was recycled intact in both newborn lambs and 3-day-old rabbits, respectively.
The formula we used to calculate the fractional synthesis of lamellar body PC assumes that there is no recycling of label from the alveolar surface PC pool. Our results show that recycling of unlabeled alveolar PC to the lamellar body pool is 10 times greater than the rate of synthesis of lamellar body PC, which raises a concern for this assumption. However, the enrichment of the precursor for synthesis is much greater than the enrichment of the alveolar surface PC pool over the 8 h of the study. Thus the amount of label in the lamellar body PC pool comes predominately from synthesis. We can express this effect of recycling on FSR in quantitative terms. In the APPENDIX, we demonstrate that if it is assumed that recycling does occur, then the formula to calculate fractional synthesis rate is
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Comparison to Other Results
Our approach to experimentally validating our model was to compare our results where possible with those from comparable studies. The rate of incorporation of de novo synthesized FA into PC, however, has not previously been quantified due to the lack of available methodology. Therefore, total PC synthesis has not previously been correctly measured. Consequently, there is no "gold standard" with which to compare the most novel values shown in Fig. 1. However, ballpark values expected for certain components of the model help us to evaluate if we have obtained reasonable values for other calculations for which there are no relevant data currently published. For example, two different reports indicate ~85% recycling of surfactant (12, 24), which is close to our value (90%). Wright (33) has reported the FSRsec (secretion rate/alveoli pool size) in 3-day-old and adult rabbits to be 12.8 and 17.5%/h, respectively, which compares well with our value of 12.31 ± 2.01%/h. Thus, where it is possible to compare our values with the literature, we are close to expected numbers. This, coupled with the theoretical basis for the methodology, gives us good reason to believe the numbers cited in Fig. 1 are reasonably accurate.Our measurement of alveolar pool size (11 ± 1 mg PC/kg body wt) in pigs is similar to measurements reported by Jacobs et al. (19) and Ennema (8) in adult rabbits (16 and 10 mg PC/kg, respectively). The total PC content in lung tissue in our study (12.6 ± 1.4 µmol PC/g wet lung) is also close to the reported value (9.3 ± 2.6 µmol PC/g wet lung) from Wichert and Wilke (31) in normal pigs. We further found that the pool sizes of lamellar body PC and alveolar surface PC were similar, which were 9 and 12% of the lung tissue PC pool size, respectively. This is comparable to the report by Young et al. (35) that lamellar body DSPC and alveolar DSPC were 13 and 14% of lung tissue DSPC, respectively.
Our conclusions regarding the proportional contributions of plasma FFA and de novo synthesized FA are at odds with some previous papers, but methodological problems of those previous experiments can explain the discrepancies. Thus [3H]acetate and [14C]palmitate have been used by many researchers in the past to investigate the incorporation of FA from plasma and de novo synthesis into surfactant PC (13, 20, 22). However, because previous investigators were unable to quantify the extent that de novo synthesized FA are incorporated into PC, it is difficult to draw valid conclusions regarding even the relative contributions of plasma FFA and de novo synthesized FA to the production of surfactant PC. For example, when [3H]acetate and [14C]palmitic acid were simultaneously injected into 3-day-old rabbits, the specific activities of 3H and 14C in PC and DPPC from lamellar body and BAL surfactant were measured to evaluate the relative contribution to surfactant PC incorporation of de novo FA and plasma FFA (20). Because the specific activity ratios of 3H to 14C in PC and DPPC in BAL were 2.02 and 1.86, respectively, it was concluded that the de novo synthesized FA were the preferred FA source for surfactant PC incorporation (20). The specific activities of 3H and 14C from product PC, however, are affected not only by the incorporation rates but also by the precursor enrichments. Because the precursor enrichments of acetate and palmitate were not measured, it is impossible to interpret the specific activities of product PC. Given the results of our study, it is likely that the higher 3H specific activity of PC in Ref. 20 resulted from the higher precursor enrichment of acetate, rather than a higher incorporation rate from de novo synthesized FA. This point is clarified with data from our study. After infusion with [1,2-13C2]acetate and [U-13C16]palmitic acid, the TTR(m + 2) (representing the incorporation from de novo synthesized palmitate) and TTR(m + 16) (representing the incorporation from plasma palmitate) in the PC from BAL are 0.003 and 0.002 (Table 2), respectively, which gives the ratio of TTR(m + 2) to TTR(m + 16) as 1.5. Because TTR from stable isotopes is equivalent to specific activity from radioactive isotopes (32), we would have concluded from the product enrichments [TTR(m + 2) and TTR(m + 16)] alone that the de novo synthesized FA are the preferred FA source for surfactant PC synthesis, if we had neglected the precursor enrichments. However, the measured synthesis rate from plasma FFA is 10 times that from de novo synthesized FA when appropriate account is taken of the precursor enrichments. Thus the comparison of FA contribution to surfactant PC incorporation based only on product enrichment is not valid. This comparison becomes even more problematic when different dosages of radioactive precursors are used, such as in Ref. 20 where the radioactive dosage of [3H]acetate was 20 times as high as that of [14C]palmitic acid.
Recently, Bunt et al. (6) used a new methodology to investigate endogenous surfactant turnover in preterm infants by infusing [U-13C6]glucose for 24 h. This is a step forward from previous attempts to quantify surfactant synthesis, because precursor enrichment was taken into consideration. However, there are some aspects that need to be addressed. First, they measured surfactant production from plasma glucose-derived de novo synthesized FA, not endogenous surfactant production as claimed. Our data show that in our experimental setting de novo synthesized FA is a minor contributor to total surfactant incorporation, compared with plasma FFA. Furthermore, plasma glucose may not be the only precursor source used for de novo FA synthesis. Therefore, Bunt et al. actually calculated a minimal value for the rate of surfactant incorporation from de novo synthesized FA, and even if that value was reasonably accurate, they likely significantly underestimated total surfactant synthesis. Second, there was a long delay (19 h) in the appearance of label in the alveolar wash enrichments in the paper by Bunt et al., whereas we found alveolar enrichments in our 4-, 6-, and 8-h infusion studies. The much longer delay reported by Bunt et al. may have resulted from the concurrent administration of a high dose of exogenous surfactant (17 times the endogenous pool size) in their study, which may have caused the inhibition of endogenous surfactant secretion as well as the dilution of alveolar wash enrichments. Third, they actually calculated neither synthesis nor secretion, because secretion surfactant enrichments were used together with synthesis precursor enrichments in the calculation. This problem is difficult to overcome in human subjects because it is unrealistic to obtain the necessary lung tissue samples for the correct calculation.
Our conclusion that the plasma is the primary source of FA for lung surfactant synthesis may seem at odds with the fact that palmitate constitutes only ~30% of plasma FFA, whereas surfactant PC contains ~70% palmitate. The most likely explanation for this discrepancy is that there is a deacylation-reacylation pathway that concentrates palmitate in the surfactant PC (29). Whereas we have not specifically quantified the contribution of the deacylation-reacylation pathway, that pathway and all other pathways are included in our calculation of the total rates of incorporation into lamellar body surfactant PC.
To obtain accurate enrichments in surfactant PC-bound palmitate made
from de novo synthesis,
[1,2-13C2]acetate
was infused at 24 µmol · kg1 · min
1
for 8 h in this study. This not only enabled adequate labeling of
surfactant-bound palmitate but also ensured relatively constant labeling of the acetyl-CoA pool throughout the lungs. This is reflected
by the high precursor enrichment (25%) in the palmitate. This high
infusion rate of acetate necessitated a significant concurrent infusion
of sodium (11.5 mmol/kg over 8 h) and water (76.6 ml/kg over 8 h).
Nonetheless, there were no clinical signs of electrolyte or water
imbalance. In one pig, arterial
Na+ concentration was increased
from 134 to 139 mM, and arterial pH was increased from 7.3 to 7.4. These changes are unlikely to have a physiological effect. On the other
hand, the high rate of acetate infusion may have stimulated the rate of
de novo FA synthesis. Nonetheless, even with this possible source of
overestimation, we found that the contribution to PC incorporation from
de novo synthesized FA was minor in comparison to that from plasma FFA. The same conclusion applied even when low-dose glucose was infused. Therefore, the high
[1,2-13C2]acetate
infusion rate used in our study did not affect our conclusion.
Because the contribution of de novo synthesized FA was small, we did not differentiate whether the de novo synthesized FA originated in the lungs or the liver. Thus we focused our investigation on preformed FFA vs. de novo synthesized FA as the sources of FA for net surfactant PC synthesis. The de novo synthesized FA could have been produced either in the lungs or in another tissue (e.g., liver). FA produced in the liver and transported to the lungs via VLDL-TG would not be distinguished by our method from FA synthesized in the lungs. Hydrolysis of intracellular triglyceride in type II pneumocyte pool could also be a source of FA for surfactant PC synthesis. However, the sources of FA (i.e., from de novo synthesized FA vs. preformed FFA) in that pool should be proportionally the same as in the surfactant PC, so our conclusion regarding the relative roles of these two FA sources should not be affected by ignoring the intracellular triglyceride pool. Further, because that pool is relatively constant, it could not contribute significantly to the net synthesis of surfactant.
In summary, we developed a new stable isotope model to quantify surfactant PC kinetics in conscious pigs. We found that plasma FFA was the primary source of FA used for new surfactant PC synthesis in postabsorptive pigs. Because plasma FFA and de novo synthesized FA can be potentially affected by diet, this study leads credence to the possibility of altering the quantity and composition of surfactant through dietary manipulation.
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APPENDIX. Fractional Synthetic Rate Calculation |
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We will calculate the fractional rate of synthesis (FSR) of lamellar body PC assuming that there is recycling of PC between the lamellar body and alveolar surface PC pools. We define FSR as the rate of synthesis of lamellar body PC (not including recycling, denoted as Ra) divided by the lamellar body PC pool size (denoted as QL). We will denote the rate that lamellar body PC is transported to the alveolar surface pool by FA,L and the rate that the PC returns to the lamellar body pool by FL,A. We will assume that alveolar pool PC is lost at rate F0,A. We will assume that the unlabeled PC pools are in a physiological steady state, so that the appearance of PC into the lamellar body pool is equal to the disappearance, i.e.
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(A1) |
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(A2) |
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(A3) |
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(A4) |
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(A5) |
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(A6) |
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(A7) |
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(A8) |
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(A9) |
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(A10) |
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(A11) |
Solving Eq. A11 for FSR gives
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ACKNOWLEDGEMENTS |
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We wish to thank Lillian Traber and colleagues for guidance in performing this study. We would also like to thank Zhanpin Wu for HPLC technical assistance.
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FOOTNOTES |
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This investigation was supported by Shriners Hospital Grants 8050, 8490, and 8550.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. R. Wolfe, Chief of Metabolism, Shriners Burns Hospital, 815 Market St., Galveston, TX 77550 (E-mail: rwolfe{at}sbi.utmb.edu).
Received 23 September 1998; accepted in final form 31 March 1999.
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