Studies on liver phosphatidyl cholines: I. Effects of fatty liver induction on phosphatidyl cholines from liver mitochondria and microsomes

Protein, total phospholipid, phosphatidyl cholines and phosphatidyl choline fractions from liver mitochondria and microsomes of female rats were analyzed after treatment with CCl₄ (0.3 ml of CCl₄ suspended in corn oil) or ethionine (50 mg in 0.9% saline) or after feeding a choline deficient, low protein diet for seven days. Phosphatidyl cholines were separated into four fractions differing in the degree of fatty acid unsaturation. Over 50% of total phosphatidyl choline phosphorus was present in fraction 3 of liver mitochondria and microsomes. The major fatty acid in fraction 1 was docosahexaenoic acid. Fraction 4 contains oleic and linoleic acids. Arachidonic acid occurs in fraction 2 and 3. Ethionine decreased the amount of microsomal protein and phosphatidyl choline fraction 1 of mitochondria. Microsomal protein was decreased by CCl₄. The choline deficient, low protein diet caused a decrease in mitochondrial and microsomal phospholipids. The amount of the mitochondrial phosphatidyl choline decreased. Corn oil increased the level of phosphatidyl choline fraction 3. Choline deficiency decreased the amount of phosphatidyl choline fraction 3, increased fraction 4 of mitochondria and microsomes and increased fraction 1 of microsomes.     

On the phospholipids ofCulex pipiens fatigans

The lipids of different developmental stages ofCulex pipiens fatigans, vector of bancroftian filariasis, have been investigated. The phospholipid composition of the developmental stages and of the subcellular fractions of fourth instar larvae of the insects were analyzed. The composition of fatty acids and their positional distribution have also been examined in the major phospholipids of the larvae. The insect eggs contained higher amounts of lipids than larvae suggesting that they were utilized during embryogenesis. Phosphatidyl ethanolamine (PE) and phosphatidyl choline (PC) comprised over 75% of the insect phospholipids. Of these, PE was present in the greatest amounts during all stages of growth and in the subcellular fractions of larvae. An ethanolamine containing sphingolipid was found as a component of the phospholipids of the insects. About 50% of the lipids of the larvae were localized in the cell debris and nuclei fraction which also contained most of the lysolipids of the insects. As in other Diptera 16∶0, 16∶1 and 18∶1 were the major fatty acids present in the insect lipids of which the fatty acid found in greatest amounts was 16∶1. Similar to the phospholipids of animal species, saturated fatty acids were predominantly linked to the 1 position of the major phospholipids of the insects while the unsaturated fatty acids were in higher amounts at the 2 position.     

Chemical composition of subcellular particles from cultured cells of human tissue

Chemical composition of subcellular components of HeLa, KB, human heart and liver tissue-culture cell lines have been studied. The concentration of RNA, protein and phospholipid (μg/μg of DNA) of total subcellular particles was similar for all four cell lines studied. The greatest RNA concentration and lowest protein concentration is found in the microsomes as compared to the other subcellular fractions of HeLa and KB cells. The lipid P/Protein N ratio of mitochondria was greater than the other subcellular fractions from tissue-culture cell lines studied. Phosphatidyl choline and phosphatidyl ethanolamine are the major phospholipids with the former more predominant in all of the subcellular fractions of tissue-culture cells studied. Phosphatidyl inositol, phosphatidyl serine, sphingomyelin and polyglycerol phosphatide were shown to be present. Phosphatidyl choline composition (per cent of total lipid-P) is greatest in the microsomes when compared with the other subcellular fractions obtained from all of the cell lines studied except the nuclear fraction of human liver cells. Correspondingly, the mitochondrial fraction for all of the tissue culture cell lines contains the greatest composition of phosphatidyl ethanolamine except for the human liver and heart cells. The mitochondrial fraction contains the lowest amount of phosphatidyl inositol. Polyglycerol phosphatide is mainly present in the mitochondrial fraction of the tissue-culture cells. Part of a thesis submitted to the Graduate School of the University of North Dakota in partial fulfillment for the degree of Doctor of Philosophy.     

Distribution of cholesteryl esters and other lipid in subcellular fractions of the adrenal gland of the pig

Total lipids from whole pig adrenal glands as well as from their mitochondria, microsomes, liposomes, and cell sap were extracted and fractionated first into neutral lipids and phospholipids. The highest percentage of neutral lipids was found in the cell sap, and the lowest in the microsomal fraction. Neutral lipids were subfractionated into cholesteryl esters, free cholesterol, triglycerides, and free fatty acids. Cholesteryl esters were distributed throughout the liposomes. Free fatty acids represented a substantial part of cell sap lipids, but were present also in the mitochondria, microsomes, and liposomes. Fatty acids of all fractions were analyzed by gas liquid chromatography. Free fatty acids and cholesteryl ester fatty acids from all cellular fractions were similar in composition and were characterized by considerable quantities of linoleic and arachidonic acid. Triglycerides were characterized by an increased percentage of palmitic and a low content of arachidonic acid. Phosphatidyl choline, phosphatidyl ethanolamine, diphosphatidyl glycerol, and sphingomyelin plus phosphatidyl inositol were isolated from the lipids by preparative thin layer chromatography, and their fatty acids analyzed by gas liquid chromatography. Phosphatidyl choline and phosphatidyl ethanolamine from mitochondria, microsomes, and cell sap were very similar in respect of their fatty acid composition. Sphingomyelin plus phosphatidyl inositol was characterized by a high content of C22:2omega6. Diphosphatidyl glycerol was present in mitochondria and in the cell sap.     

Androgen regulation of lung lipids in the male rat

The aim of this study was to determine whether or not testosterone regulates the lipid concentration in rat lung tissue. Rats were either sham-operated controls, castrated, or castrated and injected with testosterone. Twenty-one days after castration, we observed in relation to the control: (i) Total lipids, phospholipids, and total cholesterol increased, while triglycerides decreased in whole lung. (ii) Phospholipid concentration increased in microsomes, lamellar bodies, and alveolar macrophages, but it decreased in extracellular surfactant. (iii) On a percentage basis, the concentration of phosphatidylcholine increased in microsomes, lamellar bodies, and alveolar macrophages, and it decreased in extracellular surfactant. (iv) Protein concentration decreased in extracellular surfactant and increased in microsomes, lamellar bodies, and alveolar macrophages. (v) The incorporation of [¹⁴C]glycerol into phospholipids of lung slices increased. (vi) The activity of CTP:phosphocholine cytidylyltransferase bound to the microsomal fraction increased without any change in the activity of the soluble form of the enzyme in the lung. The results obtained when testosterone was administered to castrated rats were similar to those obtained in the control in all cases. These results suggest that the lipid concentration in the lung is regulated at least partly directly or indirectly by androgens.     

Subcellular distribution of disaturated phosphatidylcholine in developing rabbit lung

To determine the subcellular distribution of disaturated phosphatidylcholine (DSPC) in lung tissue during perinatal development, fetal rabbits at 24, 26, 28 and 31 (term) days gestation and newborns were studied. Following alveolar lavage, fractions enriched in nuclei-cellular debris, mitochondria, microsomes, surfactant (lamellar bodies) and cytosol were prepared from the residual tissue homogenate, and their DSPC content was determined. The DSPC content of the unfractionated residual lung tissue homogenate progressively and significantly increased during fetal development, rising from 9.09±0.91 to 17.45±2.88 mg/g dry lung between 24 days gestation, and term. Between 24 and 26 days gestation the overall increase in tissue DSPC was due to a two-fold increase in the mitochondrial, microsomal and cytosolic pools. Lamellar bodies were first isolable at 26 days gestation. The DSPC content of this fraction increased six-fold (from 0.10±0.02 to 0.67±0.15 mg/g dry lung) between 26 and 28 days gestation and a further seven-fold (to 4.63±1.06 mg/g dry lung) by term, accounting for the overall increase in the tissue homogenate value during this time period. By the first postnatal day, microsomal and cytosolic DSPC increased another two-fold, but no significant change occurred in the other subcellular fractions. Alveolar lavage DSPC progressively increased over the time period studied. While there was no change in the lamellar body DSPC/total PC ratio during fetal development, each of the mitochondrial, microsomal and cytosolic ratios decreased between days 26 and 28 of gestation and then increased at term. Our results indicate that in addition to the pulmonary surfactant, for which DSPC is often used as a marker, other subcellular organelles contain significant DSPC pools that undergo dynamic changes in size during perinatal development.     

Phospholipids of the pulmonate land snailCepaea nemoralis (L.)

The phospholipids of the snailCepaea nemoralis, comprising the major lipid fraction (65%) in this terrestrial pulmonate, were investigated by thin-layer and column chromatography. Detailed gas chromatographic analyses of liberated fatty acid fractions and amino acid analyses of the water soluble moieties of isolated phospholipid classes were carried out. Phosphatidyl choline (47%) and phosphatidyl ethanolamine (21%) were found to be the predominant phospholipid classes, while phosphatidyl serine (8%), phosphatidyl inositol (6%), diphosphatidyl glycerol (3%), ceramide amino-ethylphosphonate (7%), lysophosphatidyl choline (1%), and phosphatidic acid (1%) were present in lesser amounts. In the phosphatidyl ethanolamine and phosphatidyl serine fractions, minor quantities of plasmalogen analogues were detected. Fatty acid profiles of the various phospholipid classes appeared to be strikingly diverse, e.g. a characteristic component, such as linoleic (18∶2ω6) acid, ranging from 3–54%. In vivo radioisotope studies using 1-¹⁴C-acetate demonstrated the high biosynthetic rate of all phospholipid classes and their respective fatty acid fractions. Results are discussed in relation to data on the phospholipids from other invertebrate species.     

Phospholipid Composition of <Emphasis Type="Italic">Jatropha curcus</Emphasis> Seed Lipids

Jatropha curcus L. oil has emerged as one of the most important raw materials for biodiesel production. However, no detailed study has been reported on characterizing the lipid constituents of jatropha oil. The present study revealed that the total oil content of jatropha seeds was 32% with a composition of 97.6% neutral lipids, 0.95% glycolipids and 1.45% phospholipids. The fatty acid composition of total lipids, neutral lipids, phospholipids and glycolipids was also determined and found to contain oleic acid (18:1) and linoleic acids (18:2) as major fatty acids. The phospholipids fraction was further characterized and quantified and found to contain phosphatidyl choline (PC) 60.5%, phosphatidyl inositol (PI) 24% and phosphatidyl ethanolamine (PE) 15.5%. The fatty acid composition and the positional distribution of the fatty acids of individual phospholipids were also reported.     

Coordinate packaging of newly synthesized phosphatidylcholine and phosphatidylglycerol in lamellar bodies in alveolar type II cells

Methylamine, a weak base, inhibits packaging of newly synthesized phosphatidylcholine (PC) in lamellar bodies in 20–22 h cultured alveolar type II cells, suggesting a role for acidic pH of lamellar bodies. In this study, we tested if (i) the packaging of PC is similarly regulated in freshly isolated type II cells and (ii) methylamine also inhibits the packaging of other surfactant phospholipids, particularly, phosphatidylglycerol (PG). The latter would suggest coordinated packaging so as to maintain the phospholipid composition of lung surfactant. During the short-term metabolic labeling experiments in freshly isolated type II cells, methylamine treatment decreased the incorporation of radioactive precursors into PC, disaturated PC (DSPC), and PG of lamellar bodies but not of the microsomes, when compared with controls. The calculated packaging (the percentage of microsomal lipid packaged in lamellar bodies) of each phospholipid was similarly decreased (∼50%) in methylamine-treated cells, suggesting coordinated packaging of surfactant phospholipids in lamellar bodies. Equilibrium-labeling studies with freshly isolated type II cells (as is routinely done for studies on surfactant secretion) ± methylamine showed that in methylamine-treated cells, the secretion of PC and PG was decreased (possibly due to decreased packaging), but the phospholipid composition of released surfactant (measured by radioactivity distribution) was unchanged; and the PC content (measured by mass or radioactivity) of lamellar bodies was lower, but the PC composition (as percentage of total phospholipids) was unchanged when compared with control cells. We speculate that the newly synthesized surfactant phospholipids, PC, DSPC, and PG, are coordinately transported into lamellar bodies by a mechanism requiring the acidic pH, presumably, of lamellar bodies.     

Studies on tamarind kernel oil II1L : analysis of phospholipids

Phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, phospha-tidic acid, and lyso derivatives of phosphotidyl choline and phosphatidyl ethanolamine were identi-fied from phospholipids of Tamarind kernel oil. Phosphatidyl choline and phosphatidyl ethanolamine were isolated and their fatty acid composition was determined by GLC. Reference 2 is considered as paper I of the series. Presented at Annual Convention of Chemists, Dec. 1977, at Jaipur, India.     

Phospholipids of human serum

Phospholipids extracted from normal human serum were fractionated into lecithin, lysolecithin, sphingomyelin, phosphatidyl ethanolamine, lysophosphatidyl ethanolamine, phosphatidyl serine, and phosphatidyl inositol. Identification of each was established by thin-layer chromatography and infrared spectrophotometry. The content of plasmalogen was determined in both lecithin and phosphatidyl ethanolamine fractions. The composition of fatty acids and fatty aldehydes in isolated phospholipids is presented. The degree of unsaturation as reflected in the average content of double bonds per molecule of the fatty acids in phospholipids was: lecithin 1.2, choline plasmalogen 2.1, lysolecithin 0.6, sphingomyelin 0.2, phosphatidyl ethanolamine 2.8, lysophosphatidyl ethanolamine 1.0, phosphatidyl serine 1.0, and phosphatidyl inositol 1.8. Both chlline and ethanolamine plasmalogen aldehydes were predominantly saturated. Molecular weight of each phospholipid was calculated from determined fatty acid and fatty aldehyde compositions; the phosphorus factor for each phospholipid was computed. On a weight percent basis, lecithin, sphingomyelin, and lysolecithin accounted for 95% of the total phospholipids. The ethanolamine-containing phospholipids accounted for 2.5%, and the remainder was divided among phosphatidyl inositol, choline plasmalogen and phosphatidyl serine. Presented in part at the AOCS Meeting, Houston, April, 1965. Dept. of Health, Education and Welfare, USPHS.     

Lung lipid composition in zinc-deficient rats

There have been a limited number of studies investigating surfactant lipid changes in lung with trace elements. The present investigation was designed to examine the effect of moderate zinc deficiency on the lipid metabolism in rat lung. We also evaluated whether zinc deficiency, which is a wide-spread problem, could play a role in adult respiratory distress syndrome (ARDS). For that purpose, adult male Wistar rats were fed two diets differing in zinc concentration. The rats were divided into two groups. One group was fed a zinc-deficient diet containing 3 mg Zn/kg, and the other group received a zincadequate control diet with 30 mg Zn/kg according to AIN 93-M. After 2 mon of treatment, we observed that in the zinc-deficient group (i) total lipids, phospholipids, and cholesterol increased whereas TG decreased in whole lung; (ii) phospholipid (PC) concentration increased in lamellar bodies and alveolar macrophages and decreased in extracellular surfactant but did not change in microsomes; (iii) protein concentration decreased in whole lung, extracellular surfactant, lamellar bodies, and macrophages; (iv) the incorporation of [Me-¹⁴C]choline into PC (phospholipids) of lung slices increased; and (v) the activity of CTP/phosphocholine cytidylyltransferase bound to the microsomes increased in the lung. These results suggest that the lipid concentration in the lung (especially the phospholipids) is modified directly or indirectly by a zinc-deficient diet. In a zincdeficient diet, the lung changes the pattern of PC for an adaptive or recovery stage. Therefore, zinc deficiency implications are important for the design of therapies and public health interventions involving targeted zinc supplementation for high-risk groups or groups with certain diseases, such as ARDS.     

Lipids of some thermophilic fungi

Total lipid content in the thermophilic fungi—Thermoascus aurantiacus, Humicola lanuginosa, Malbranchea pulchella var.sulfurea, andAbsidia ramosa—varied from 5.3 to 19.1% of mycelial dry weight. The neutral and polar lipid fractions accounted for 56.4 to 80.2% and 19.8 to 43.6%, respectively. All the fungi contained monoglycerides, diglycerides, triglycerides, free fatty acids, and sterols in variable amounts. Sterol ester was detected only inA. ramosa. Phosphatide composition was: phosphatidyl choline (15.9–47%), phosphatidyl ethanolamine (23.4–67%), phosphatidyl serine (9.3–17.6%), and phosphatidyl inositol (1.9–11.9%). Diphosphatidyl glycerol occurred in considerable quantity only inH. lanuginosa andM. pulchella var.sulfurea. Phosphatidic acid, detected as a minor component only inM. pulchella var.sulfurea andA. ramosa, does not appear to be a characteristic phosphatide of thermophilic fungi as suggested earlier. The 16∶0, 16∶1, 18∶0, 18∶1, and 18∶2 acids were the main fatty acid components. In addition,A. ramosa contained 18∶3 acid. Total lipids contained an average of 0.93 double bonds per mole of fatty acids, and neutral lipids tend to be more unsaturated than phospholipids.     

Liver phospholipids of rats fed a choline-deficient diet supplemented with choline or methionine

The low amount of arachidonic acid in the total phospholipids in the liver of rats fed a standard type of choline-deficient diet was corrected by either choline or methionine, which also increased food intake. Choline increased the content of this fatty acid in the phosphatidyl ethanolamine but not in the phosphatidyl choline. Methionine increased both the amount of phosphatidyl choline and its content of arachidonic acid.     

Lipid composition of beef and human pituitary glands

The lipid composition of beef and human pituitary was determined by chromatographic and spectrophotometric methods. Beef pituitary lipid contained about 25% nonpolar lipids and 75% phospholipids whereas nonpolar lipids made up approximately 60% of the total in human pituitaries. The main nonpolar (i.e., low polarity) lipids in human pituitary were triglycerides, cholesterol, free fatty acids and an unidentified component in the triglyceride fraction. Cholesterol was the major nonpolar lipid component in freshly collected beef anterior and posterior pituitary, but the amount of free fatty acids appeared to increase during storage. Preliminary investigation of the unknown nonpolar lipid in human pituitaries suggested that it was an unsaturated hydroxy compound with no carbonyl functions. Thin layer chromatography indicated that it was also present in smaller amounts in freshly collected beef pituitaries. The main phospholipids of beef anterior, posterior and human pituitary were phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl inositol, phosphatidyl serine and sphingomyelin. The fatty acid composition of total nonpolar lipids, free fatty acids, total phospholipids, phosphatidyl ethanolamine and phosphatidyl choline of beef anterior and posterior pituitary was determined by gas liquid chromatography. Mixtures of saturated and unsaturated fatty acids ranging from C₁₂ to C₂₂ were present; the main fatty acids were palmitic, stearic, oleic, linoleic and arachidonic.     

Lipid composition of further purified bovine liver nuclear membranes

The lipid content, distribution and fatty acid composition of highly purified bovine liver nuclear membranes was determined and compared to those of microsomes prepared in parallel. Contrasted with microsomes, nuclear membranes while containing nearly the same levels of lipid had more cholesterol and total neutral lipid and less phospholipid. Phospholipid and neutral lipid patterns generally were similar for the two types of membranes. The same fatty acids, in similar proportions, were observed in respective total lipid, total polar lipid, phosphatidyl choline and phosphatidyl ethanolamine fractions of the two membrane types. The microsomal lipid fractions contained slightly greater percentages of unsaturated fatty acids. With respect to previous results from preparations contaminated with nonmenbranous nuclear material, purified fractions contained more total lipid on a protein basis and more total unsaturated fatty acids. Only minor differences in levels and distribution of phospholipids and neutral lipids were observed between the crude and highly purified fractions. Purdue University AES Journal Paper No. 4482.     

Diet-induced alterations in the discoid shape and phospholipid fatty acid compositions of rat erythrocytes

For eight weeks young male rats were fed diets rich in 18∶2 (stock diet, or 10% corn oil, CO) or those devoid of 18∶2 (fat free, FF, or 10% hydrogenated coconut oil, HCNO). The CO and HCNO diets were fed in the absence or presence of eicosa-5,8,11,14-tetraynoic acid (TYA). When 18∶2 was excluded, an increase in the level of 16∶1, 18∶1 and 20∶3 and a decrease in 18∶2 was observed in the fatty acids of red cells. On feeding TYA, an increase in 18∶2 and in the case of the HCNO+TYA diet, a decrease of 12∶0 and 14∶0 was also observed. In all cases the levels of 20∶4 in erythrocyte fatty acids were similar. Saturated fatty acids were predominant in phosphatidyl choline (PC), lysophosphatidylcholine, (LPC) and sphingomyelin whereas unsaturated acids were predominant in phosphatidyl ethanolamine (PE), (PS), and phosphatidyl inositol (PI). Acids containing three or more double bonds comprised about 90% of the total acids in PI. In all the phospholipids, the characteristic changes in the composition of fatty acids were observed due to the exclusion of 18∶2 from the diet. However, changes due to the feeding of TYA were found only in PC and LPC. In rats fed the 18∶2-rich diet, about 60% of the red cells were discocytes. In those fed the 18∶2-free diet, the level of discocytes decreased to about 23%, and the levels of echinocytes II and III increased. The exclusion of 18∶2 for even a few days decreased the proportion of discocytes. The loss of discoid shape was reversed in a few days by feeding an 18∶2-rich diet. Fatty acid analysis of erythrocytes of rats of the various dietary manipulations showed that the change in the proportion of discocytes followed the change in the level of 18∶2.     

Studies on the lipid composition of developing soybeans

Studies are reported on changes in fatty acid and lipid class composition in developing soybeans picked at intervals from ca. nine days after flowering to maturity. In the early stages of development of the bean, the lipid was virtually devoid of triglyceride and the major constituents consisted of glycolipids and phospholipids. As the bean developed, there was a rapid synthesis of triglyceride that paralleled the deposition of lipid. Simultaneously, unknown substances which occurred in relatively large amounts in the neutral, as well as the glycolipid and phospholipid, fractions of the immature bean diminished to less than 2% of the total lipid in the mature bean. The glycolipids and phospholipids also increased as the bean developed but at a much slower rate than the triglycerides and became minor components in the mature bean. The major component of the phospholipids in the immature bean was phosphatidic acid. It decreased as the phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidyl inositol, as well as triglyceride, increased. The major component of the glycolipid fraction in the early stages of the development of the bean had the same migration pattern on two-dimensional thin layer chromatography as phosphatidic acid and gave a positive test for phosphorus; it also gave a positive test for glycolipids and was separated completely from phosphatidic acid and other phospholipids by column chromatography. It also decreased as the bean developed. Changes also occurred in the fatty acid composition of the developing bean. The percentage of saturated fatty acids decreased rapidly in the early stages of the development of the bean; oleic and linoleic increased rapidly as the bean developed. Linolenic acid increased rapidly to a maximum concentration in the early stages of the development of the bean and then gradually decreased as the bean matured.     

Lipid metabolism of the yellow clam,Mesodesma mactroides: I. Composition of the lipids

The lipid composition of the yellow clam,Mesodesma mactroides, that lives in the northern beaches of the Buenos Aires province of Argentina was studied. The main nonpolar lipids are triglycerides and alkoxyglycerides. Phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidyl serine are the main phospholipids. The predominant fatty acids are 16∶0, 16∶1ω7, 18∶0, 18∶1ω9, 20∶5ω3, and 22∶6ω3. The are mainly provided by the clam's food and stored in the hepatopancreas. The content of polyunsaturated acids increases in summer together with an increase in nonpolar lipids and is correlative with an increase in phytoplankton in the sea water. Sexual maturity modifies the lipid composition of gametes.     

Lung surfactant phospholipids in different animal species

A comparative study of adult mammalian lung surfactants was undertaken to determine which animal species might serve as appropriate models for surfactant alterations in human lung diseases. Phosphatidylcholine (PC) comprised 80–87% of the phospholipid and contained more than 65% palmitic acid in all species studied. Phosphatidylglycerol (PG) was found to vary significantly in fatty acid composition among the species. Rabbit, dog and rat surfactant PG contained 50–60% palmitic acid, while human and cat surfactant contained much lower levels of saturated fatty acids. Both the PC and PG of all species contained 2 positional isomers of fatty acids with 16 carbons and one double bond, but the relative amounts of the unusual isomer, 16∶1Δ7, and palmitoleic, acid, 16∶1Δ9, varied among the different animal species. Only cat and dog surfactant phospholipids contained 18∶1Δ5. Cat surfactant phospholipids also differed by the absence of 20∶4 and the presence of small amounts of several 20- and 22-carbon fatty acids. These results explain some discrepancies found in the literature concerning surfactant composition and delineate limiting factors in extrapolating results from animal studies for the evaluation of maturation and pathological alterations in human surfactant.