A n n a l s o f C l i n i c a l L a b o r a t o r y S c i e n c e , Vol. 3 , N o. 4 Copyright © 1973, Institute for Clinical Science

Biochemical Basis o f Hyaline Membrane Disease

DONALD T. FORMAN, Ph.D.

Division of Clinical Biochemisry, Evanston Hospital,Evanston, 1L 60201

andDepartment of Biochemistry and Pathology,

Northwestern University Medical School, Chicago, IL 60611

ABSTRACT

Pulmonary function in a normal fetus is dependent upon maturation and maintenance of the biochemical synthesis of a distinct surface-active lipid. Two major pathways for the biosynthesis of this major surface-active lipid, dipalmitoyl lecithin, are described and relative activities of each pathway in human lung are discussed. In normal human lung, the CDP-choline path­way is quantitatively most important for total lecithin synthesis. Infants with idiopathic respiratory distress syndrome (RD S) are usually prematurely bom. Their lungs lack adequate surface activity and are deficient in the principle surface-active component lecithin. Stress (hypoxia, acidosis, and hyper­thermia) may induce respiratory distress in the fetus. Biochemical methods are available to detect and characterize pulmonary surfactant in amniotic fluid. Studies show that developmental changes in phospholipids in lungs are reflected by the phospholipids in amniotic fluid, and it is possible to predict the fetus whose lung is too prematurely developed for delivery and who may develop RDS.

In trod u ction

The survival of a newly born infant de­pends upon the development of a respira­tory system capable of both pulmonary and cellular oxygenation and carbon dioxide excretion. During the development of this system, there must be both anatomical and biochemical maturation to allow for ade­quate lung function at birth. Fetal lung maturation serves as an excellent model for showing these important facets of pulmon­ary function.

In 1959, Avery and Mead1 reported that lung surface tension lowering material was

absent or markedly depleted in infants who died with hyaline membrane disease. Histo­logical studies13 of human lung have shown that the presence of osmiophilic granules correlated well with the presence of sur­factant. Both are usually found in fetuses and newborn infants of more than 29 weeks gestation. However, granules and surface tension lowering material are not found in infants dying from hyaline membrane disease (HMD) in the first few days of life. The role of surfactant deficiency in HMD remains controversial although such deficiency is probably responsible for the

2 4 2

B IO C H E M IC A L BA SIS O F H Y A L IN E M E M B R A N E D ISEA SE 2 4 3

progressive atelectasis and lack of alveolar stability found in HMD (figure 1).

B iosynthesis o f Pulm onary Surface- A ctive Lipid

Stability of lung depends upon a surface- active phospholipid rich complex which lines the pulmonary alveoli. This surfactant decreases the alveolar surface tension re­sulting in a decrease of their radii on ex­piration and stabilizing the fine air spaces of the lung.10 The unusual chemical, physical and physiologic properties of the principal pulmonary surface-active lipid, dipalmitoyl lecithin (D PL), have stimu­lated much research since the first descrip­tion of its occurrence by Clements & co­workers in 1961.17

The basic component of the surfactant is a lipoprotein composed largely of surface active phospholipids and smaller amounts of neutral lipids and proteins. Dipalmitoyl- phosphatidylcholine is a major surface active component of surfactant and com­prises about 50 percent of the total surfact­ant lipids.2 This dipalmitoyl lecithin (D PL) contains a phosphatidic acid structure in which the primary hydroxyl group of gly­cerol is esterified with phosphoric acid. This tertiary structure has a nitrogen base (choline) esterified to the hydroxyl group of phosphoric acid. Therefore, DPL con­tains an acidic hydroxyl group of phos­phoric acid and a basic hydroxyl of choline and behaves as a zwitterion (internal salt) and has an isoelectric pH at 6.7. When different fatty acid components are sub­stituted on the primary and secondary position of glycerol, lecithins of varying properties are derived. Lecithin containing stearic and oleic acids on the primary and secondary position of glycerol is different from lecithin containing palmitic and oleic acid substitutes. DPL is highly surface active owing to its bipolar chemical con­figuration, - a hydrophilic choline group and two hydrophobic saturated fatty acid

AERATION (LUNG)

*L IN IN G LAVER

FORMED AT'GAS/LIQUID INTERFACE

*SURFACTANT CONSUMPTION

*[s u r f a c t a n t d e f ic ie n c y ]

/ *ALVEOLAR COLLAPSE 1

iINTERSTITIAL EDEMA -*■ CELL INJURY

*INTERSTITIAL FLUID LEAKS INTO ALVEOLAR SPACE

[HYALINE MEMBRANE; FORMATION!

F ig u r e 1. Postulated chain of events resulting in surfactant deficiency and hyaline membrane formation.

side chains. It is remarkably effective in lowering the surface tension of aqueous solutions when chemically combined or adsorbed to proteins.

Dipalmitoyl lecithin (in contrast to total lung lecithin with acyl groups of variable length and unsaturation) may be syn­thesized in lung by random nonspecific acylation of a, B-diglyceride and subse­quent condensation with cytidine dipho- sphocholine (CDP-choline)18 (figure 2 ). An alternate biosynthetic route has been proposed by Bremer and Greenberg8(figure 3). In both phosphatidyl dimethyl- ethanolamine and phosphatidyl choline (PC ), palmitate accounts for 65 to 80 per­cent of fatty acyl groups, and both lipids possess a remarkable ability to lower sur-

O ' Oc h 2 - o - c - r , CH2- 0 - C - R |I O I O1 n PHOSPHOCHOLiNE 1 h

C H - O - C - R 2 *C 0 P -C H 0 U N £ ---------------------- ► C H - 0 - C - R 2I TRANSFERASE j 0

CHsjOH CH20 -P - 0 -0 H 2 - C H 2N*(CHi)3D - a , R DIGLYCERIDE '

PHOSPHATIDYL CHOLINE (LECITHIN) ♦ CMP

Ri ond Rg » Dipolmiroyl' F ig u r e 2 . Enzymatic pathway f o r de novo

biosynthesis o f lecithin.

244 FORMAN

CHg-0 " C‘ R,

I 01 nCH -O-C-R2

1 °CH2-0-P-0-0H2-CH2N+{CH3)3

IOH

CH2- 0 - C - R | PHOSPHATIDYL CHOLINE (LECITHIN)I OCH - O -C-Rz I oII

c h 2- o -p - o - c h 2 - c h 2- n h 2

PHOSPHATIDYL ETHANO LAM INE

C H 2 -O C -R 0 11

CH - O - C - R 2 0 11

C H 2 - 0 - P - O - CH 2CH 2N H c h 3IOH

PH O SPH ATID YL M ETH YLETH A N O LAMINE

/ FRO M \I m e t h i o n i n e ) m e t h y l

Qfu ________3 TRANSFERASE

F i g u r e 3. Other major pathway for de novo lecithin biosynthesis.

face tension. These findings further support the premise that the highly surface-active phospholipids may be synthesized in the lung by N-methylation of saturated phos­phatidyl ethanolamine and that this process in the lung might be selective leading to the concentration of surface-active lipids in the lung. Lipid N-methyltransferases cata­lyzing S-adenosyl methionine méthylation have been identified in liver, muscle, kid­ney, and lung of several species.6 Com­parative studies by Bjomstad and Bremer4 suggest that N-methyltransferase makes a relatively minor contribution to the total lecithin synthesis in lung.

Gluck15 has recently proposed two enzy­matic pathways for the biosynthesis of lecithin and suggests that these are principally responsible for the surfactant production in the human fetus.

Phosphocholine transferase reaction Cytidine Diphosphate Choline (CDP-Choline)+ D — a, B-Diglyceride —> Lecithin (Dipalmitoyl)

This pathway matures at 35 weeks and synthesizes primarily dipalmitoyl lecithin.

The other major pathway is catalyzed by the methyl transferase enzyme, where

Phosphatidyl Ethanolamine+ 3CHS (from Methionine)—*

Lecithin (Palmitoylmyristoyl)

This pathway is identifiable as early as 20 to 22 weeks. It synthesizes a second surface-active lecithin, palmitoylmyristoyl lecithin. The activity of this enzyme in­creases during gestation and is not active at term and beyond. This pathway permits the premature child to be bom successfully but is easily inhibited by acidosis, hypo­thermia, and hypoxia, resulting in respira­tory distress syndrome (RDS). It is in­teresting to note that in the course of evolu­tion, the major pathway in the lung for the biosynthesis of surfactant and the one which indicates a mature lung is the phosphocholine transferase pathway.15 This pathway is shared with lower mammals (rabbits, mice, and sheep). The methyla- tion pathway in the fetus shows little or no activity in most of these species but be­comes active after delivery. Thus, only the

BIOCHEMICAL BASIS OF HYALINE MEMBRANE DISEASE 245

human and other primates can be bom prematurely. Maturation of the phospho- choline transferase system takes place in all humans and animals at about 90 percent of their gestation period.

E ffect o f Oxygen an d C arbon D ioxide T en sion on Sy n th esis o f S u r fa c ta n t

Exposure of some species of animals (rabbits, dogs and man) to increased partial pressures of oxygen leads to a syn­drome of progressive respiratory distress, pulmonary edema, increased pulmonary surface tension, decreased pulmonary sur­factant and progressive irreversible histo­logic change with decreasing arterial oxygen saturation.8 These studies showed that variations in P 02 had little effect on the rate of incorporation of methyl-14C into phosphatidyl N-methethanolamine, but in­creased partial pressure (720 mm Hg) sig­nificantly depressed incorporation of radio­activity into phosphatidyl choline (table I). At low P 02, methyl transferase catalyzed synthesis of disaturated lecithin over CDP- choline lecithin synthesis (figure 4).

L u n g P h osph olip ids

Palmitic acid accounts for the largest fraction of the fatty acids of mature lung lecithins, constituting from 44 to 79 percent of the total fatty acids. The range of values of the other fatty acids are: oleic, 6 to 37 percent; stearic, 5 to 20 percent; palmito-

Po2 (mmHg)

MORGAN, T.E.' ARCH. INTERN. MED. 127-405, 1971

F i g u r e 4. Effect of P 02 on “Lecithin” synthesis.

leic, from a trace to 8 percent; myristic and linoleic, present in traces only.® Dipalmitoyl lecithin (DPL) is known to form the major component of lung phospholipids.7 DPL also has the property of forming a film, when spread on saline, with surface tension properties quite similar to those of lung ex­tracts. Lung specimens from perinatal deaths have demonstrated a distinct palmi- tic-lecithin relationship (figure 5). There is a highly significant linear relationship. In­fants bom with and without hyaline mem­brane disease show high lung surface

TABLE IE f f e c t o f O x y g e n o n L e c i t h i n S y n t h e s i s *

Subject (Dogs)fTime Ex-posed to

Oxygen, Hr.Lecithin

%

Lecithin Fatty Acids

Satu- % rated Palmitate

Normal (10) — 56 72 63Respiratory distress (5) 44.5-52.0 52 64 49Respiratory distress & pulmonary edema 47-52 53 20 45

* Morgan, T. E .: Arch. Inter. Med. 127:404, 1971. t Animals were exposed to P 0 2 greater than 550 mm Hg.

24<j FO RM A N

TOTAL LECITHIN (g/100g)

F i g u r e 5 . Relation of palmitic-lecithin to total lecithin in 29 perinatal deaths.

tension values even when lung lecithin is low.5 No such relationship is present in a comparable number of stillbirths. There­fore, the lecithin content of lung must provide some measure of reserve from which surfactant is formed. An infant born with relatively small amounts of lecithins may be at risk for developing surfactant deficiency but would only do so if addi­tional factors were present (acidosis, mas­sive lung hemorrhage).

Results of studies using radioactive palmitic acid to label DPL, has demon­strated that the surfactant has a rapid turnover with a half-life of about 14 hours.20 Recent studies, however, have shown that the rate of turnover of surfactant is con­siderably slower.19 The biological half-life for both choline and phosphate incorpo­rated into lecithin of surfactant is 43 hours

F i g u r e 6 . A . Expanded normal alveolus sur­rounded by small interstitial space and drained by lymphatic. B. Collapsed alveolus lacking sur­factant with increased interstitial fluid and de­creased lymphatic drainage.

and the biological half-life for leucine in­corporated into the protein of surfactant is 45 hours. This similarity in the biological half-life for choline, phosphate and leucine suggests that surfactant is a lipo-protein which turns over as a single unit.2 These latter findings intimate that a transient or relatively acute decrease of surfactant bio­synthesis would not result in measurable surfactant deficiency. Numerous biochemi­cal and ultrastructural observations9 postu­late that the pulmonary surfactant is syn­thesized by Type II alveolar epithelial cells, stored in the inclusion bodies of these cells and then are secreted into the alveolar space where it forms a layer on the surface of the alveolar wall. The previous concept that the surfactant layer is formed from a single phospholipid film interspaced be­tween air and liquid phase has been chal­lenged recently by a concept12 which views the surfactant layer as a multimolecular gel structure which protects the air spaces against transudation of fluid since gels and certain liquid-crystaline states can tolerate wide variations in water content ( figure 6).

B ioch em ical E va lu ation o f A m nio tic F lu id P h osph olip ids

Survival of the prematurely delivered human fetus depends upon its ability to synthesize surface-active alveolar lecithin after breathing begins.14 Previous reports by Biezenski3 attempting to correlate lipids in amniotic fluid with abnormal pregnancies showed no clearly defined patterns. How­ever, concentrations of total lipids rise as gestation progresses and relationships be­tween lecithin and sphingomyelin concen­trations permit evaluation of pulmonary maturity. Prior to 35 weeks gestation, sphingomyelin concentrations are either greater than or almost identical to those of lecithin (figure 7). Statistically, week 35 of gestation is the turning point when a sud­den increase in lecithin/sphingomyelin ratio occurs. By week 36, this ratio is 3: 1 or

Blood A

Surfactant

BIOCHEMICAL BASIS OF HYALINE MEMBBANE DISEASE 247

greater and thereafter the lung function becomes mature. An infant bom spon­taneously or by Cesarean section or in­duction at this time should have no RDS. Where the ratio of lecithin/sphingomyelin in amniotic fluid is one or less than one, RDS is probable, should birth take place at that time. Findings on thin-layer chro­matography11 (TLC) lend themselves to helpful interpretation by visual inspection. The TLC examination of amniotic fluid in­dicates whether or not biochemical develop­ment of the lung has progressed to the point of alveolar stability. When the lung has ma­tured sufficiently, RDS will not occur, should the fetus be delivered at that time or later.

The changes in both concentrations of lecithin in amniotic fluid and fatty acids esterified on a- and B-carbons of lecithin parallel changes observed in developing fetal lung and in lungs of infants after birth.16 The fatty acids from surface active lecithin are predominantly a-palmitic/B- palmitic. The alveolar stability seen much earlier in the human fetus is due to produc­tion from about week 20 to 22 of less stable surface active palmitoyl-myrisoyl lecithin.16 This is synthesized by the second (methyla- tion) pathway for lecithin biosynthesis in human fetal lung. This pathway is ex­tremely sensitive to metabolic insults, such as acidosis, hypoxia and hypothermia, which accounts for failure of the premature infant to survive.

A major advantage for examining amnio­tic fluid for lecithin/sphingomyelin is that it is reliable even when the fetus is dysma- ture. The timetable for lung maturity as reflected by a rise in lecithin is apparently unaffected when there is a deviation of growth in utero. This is exemplified by the baby who has a low birth weight and characteristically does not have RDS.16 When decisions to interrupt a pregnancy have to be made, other procedures for assessing fetal maturity (creatinine, lactic

* PO SS IB LE PULMONARY MATURITY

F i g u r e 7. Gestational changes in amniotic fluid phospholipids indicative of pulmonary maturity.

dehydrogenase) may be of little help when the fetus is dysmature.

This procedure for detecting adequate pulmonary surfactant promises to be help­ful in timing elective interruption of pregnancy, in deciding on inhibition of uncomplicated premature labor, and in predicting the clinical course of the infant whose birth is expected within hours. It can also help guide pharmacologic manipu­lation of the fetus.

Acknowledgment

Sincere thanks are extended to Dr. John U. Balis of the Department of Pathology, Loyola University Stritch School of Medicine, Chicago, II for scientific materials which were generously made available.

R eferences1. A v e r y , M . E. a n d M e a d , J.: Surface prop­

erties in relation to atelectasis and hyaline membrane disease. Amer. J. Dis Child 97: 517-523, 1959.

2. B a l i s , J. U. a n d S h e l l e y , S . A.: Quantitative evaluation of the surfactant system of the lung. Ann. Clin. Lab. Sci. 2:410—419, 1972.

3 . B i e z e n s k i , J. J., P o m e r a n c e , W., a n d G o o d ­m a n , J.: Studies on the origin of amniotic

248 FORMAN

fluid lipids—I. Normal Composition. Amer. J. Obstet. Gynec. 105:853-861, 1968.

4. B j o r n s t a d , P. a n d B r e m e r , J.: In vivo studies on pathways for the biosynthesis of lecithin in the rat. J. Lipid Res. 7:38-45, 1966.

5. B o u g h t o n , K., G a n d y , G ., a n d G a i r d n e r , D.: Hyaline membrane disease. II. Lung lecithin. Arch. Dis. Child. 45:311-20, 1970.

6. B r e m e r , J. a n d G r e e n b e r g , D. M.: Methyl transferring enzyme system of microsomes in the biosynthesis of lecithin (phosphatidyl choline). Biochim. Biophys. Acta 46:205-216, 1961.

7. B b o w n , E. S.: Chemical identification of a pulmonary surface active agent. Fed. Proc. 22:438, 1972.

8. B r u m l e y , G . W., C h e r n i c k , V., a n d H o d s o n , W. A.: Correlation of mechanical stability, moiphology, pulmonary surfactant, and phos­pholipid content in the developing lamb lung. J. Clin. Invest. 46:863-873, 1967.

9. B u c k in g h a m , S ., H e i n e m a n n , H . O., S o m e r s , S . C., a n d M c N a r y , W. F.: Phospholipid synthesis in the pulmonary alveolar cell. Amer. J. Path. 48:1027-1041, 1966.

10. C l e m e n t s , J. A.: Surface tension of lung ex­tracts. Proc. Soc. Exp. Biol. Med. 95:170-172, 1957.

11. F o r m a n , D. T. a n d B a l i s , J. U.: Measure­ment of phospholipids in amniotic fluid for the assessment of pulmonary maturity. Manual of Procedures of the Seminar on Clinical Pathology of Lipids, Institute for Clinical Science, Philadelphia, pp. 1-8, 1971.

12. F r o s o l o n o , M. F . , C h a r m s , B . L., P a w l o w - s k i , R., a n d S l i n k a , S . : Isolation, characteriza­

tion & surface chemistry of a surface active fraction from dog lung. J . Lipid Res. 11:439- 457, 1970.

13. G a n d y , G ., J a c o b s o n , W., a n d G a i r d n e r , D.: Hyaline membrane disease. I. Cellular changes. Arch. Dis. Child. 45:289-294, 1970.

14. G l u c k , L., K u l o v ic h , M. V., B o r e r , R. C., Jr., B r e n n e r , P. H., A n d e r s o n , G. G., a n d S p e l l a c y , W. N.: The diagnosis of the res­piratory distress syndrome (RD S) by amnio­centesis. Amer. J. Obstet. Gynec. 109:440- 445, 1971.

15. G l u c k , L.: Surfactant: 1972 Symposium on recent clinical advances. Pediat. Clin. N. Amer. 19:325-330, 1972.

16. G l u c k , L., K u l o v ic h , M. V., E i d e l m a n , A. I., C o r d e r o , L., a n d K h a z i n , A. F .: Biochemical development of surface activity in mammalian lung. IV. Pulmonary lecithin synthesis in the human fetus and newborn and etiology of the respiratory distress syndrome. Pediat. Res. 6:81-99, 1972.

17. K l a u s , M. H., C l e m e n t s , J. A., a n d H a v e l , R. J .: Composition of surface-active material isolated from beef lung. Proc. Nat. Acad. Sci. 47:1858-1859, 1961.

18. M o r g a n , T. E . : Biosynthesis of pulmonary surface-active lipid. Arch. Intern. Med. 127: 401-407, 1971.

19. S p i t z e r , H. L. a n d N o r m a n , J . J . : The bio­synthesis and turnover of surfactant lecithin and protein. Arch. Intern. Med. 127:429-435, 1971.

20. T i e r n e y , D. F., C l e m e n t s , J . A., a n d T r a h a n , H. J . : Rates of replacement of leci­thins and alveolar instability in rat lungs. Amer. J . Physiol. 213:671-676, 1967.