Effect of serum albumin on dynamic force-area curve of dipalmitoyl lecithin

Respiration Physiology (1978) 32,265279 @ Elsevier/North-Holland Biomedical Press

EFFECT OF SERUM ALBUMIN ON DYNAMIC FORCEAREA DIPALMITOYL LECITHIN ’

GIUSEPPE

COLACICCO

and MUKUL

CURVE OF

K. BASU

Departments of Pathology and Pediatrics, Albert Einstein College of Medicine, Bronx, New York 10461, N. Y.. U.S.A.

Abstract. In line with previous findings at 25 “C, solutions of serum albumin in the subphase stabilized the surface activity of DPL spread films at 25 “C as well as 37 “C. In contrast, films adsorbed from mixtures of DPL and albumin exhibited a marked inhibitory action of the albumin on DPL activity. The inhibitory effect increased with the relative protein concentration but, with albumin/DPL ratios smaller than 2, the DPL activity was regained gradually with cycling. With larger albumin/DPL ratios the adsorbed films retained the albumin character permanently. The negative effect of albumin was counteracted by higher temperatures (37°C us 25°C) and modest cholesterol concentrations; with greater cholesterol concentrations the known inhibitory effect of cholesterol prevailed. The inhibitory effect of albumin was potentiated by humidity; saturation of the atmosphere with water vapor at 37 “C abolished the DPL character of DPL-RSA mixtures and prevented its return (zero surface tension) upon reversal of the atmosphere from saturated water vapor to dry air. The data are important in the interpretation of the surface activity of pulmonary washings and other pulmonary extracts.

Alveolar surface tension Dynamic force-area curves Dipalmitoyl lecithin

Pulmonary surfactant Zero surface tension

A property ascribed to pulmonary surfactant is to lower surface tension at the air/ water interface of the alveolar lining layer and so prevent alveolar collapse during the compression or exhalation phase of respiration (Pattle, 1958; Clements, 1962). An essential component of pulmonary surfactant is dipalmitoyl lecithin (Brown, 1964), whose films in the surface balance (Colacicco and Scarpelli, 1970, 1973) and in the bubble model (Reifenrath and Zimmermann, 1973) can be compressed to produce a value of zero surface tension. Acceptedfor publication I October 1977.

’ This work was supported by NIH Grant HL 16137. 265

266

G. COLACICCO AND M. K. BASU

It is assumed that pulmonary washing is the most convenient source of pulmonary surfactant (Clements, 1970; Colacicco and Scarpelli, 1970, 1973) and is ordinarily collected after irrigation of the lung via trachea with 0.15 M NaCl. Since surface activity is a function of the molecular structure of a given species, an approach to the understanding of the correlations between chemistry and surface activity is the study of the interactions between the major components of the various surfactant fractions. Since DPL and albumin are the major constituents of rabbit pulmonary washings (Colacicco et al., 1973, 1976b), the influence of albumin on the dynamic surface tension in DPL films is an important question. Previous studies (Colacicco and Scarpelli, 1970, 1973; Hurst et al., 1973) were either incomplete or dealt with complex lipid and lipid-protein mixtures, from which it would be difficult to extricate the function of DPL. The present study considers the influence of various concentrations of rabbit serum albumin on the dynamic force-area curves of DPL. Two types of experiments were performed both at 25 “C and 37 “C. In one, DPL films were spread on albumin solutions; in the other, films were adsorbed from aqueous DPL-albumin dispersions. Finally, since cholesterol is also present in pulmonary washings (Colacicco and Scarpelli, 1973; Colacicco et al., 1973), inhibits the surface activity of DPL (Colacicco and Scarpelli, 1973; Reifenrath and Zimmermann, 1976; Colacicco and Basu, 1977) and interacts with proteins in bulk (Marinetti and Pettit, 1968), and since humidity also inhibits the dynamic surface activity of DPL (Colacicco et al., 1976a), the effects of humidity and cholesterol on the surface activity of aqueous DPL-RSA systems were studied at 37°C.

Materials and Methods LIPIDS AND PROTEINS

Cholesterol and synthetic L-a-dipalmitoyl lecithin were obtained from Sigma Chemical Co., St. Louis, MO. On precoated silica gel G plates, after resolution in chloroform-methanol-ammonium hydroxide-water (70 : 30 : 1:4) and I, stain, the lipids appeared as single spots of probably homogeneous material. The lipids were not purified further, particularly because the thin layer chromatography (TLC), surface tension and surface potential patterns did not change with respect to the starting material and no new spots appeared on TLC plates after our attempts to improve the lipid preparation by chromatography on silicic acid column (Colacicco, 1973). Rabbit serum albumin, ‘crystallized’, was obtained from Miles Laboratories Kankakee, Ill. Upon disc gel electrophoresis in 0.2% SDS and 0.2% fi-mercaptoethanol, RSA in quantity of 25 pg appeared as a typical single band having the migration of other serum albumin standards.

DYNAMIC SURFACE ACTIVITY OF

PREPARATION OF SOLVENTS AND LIPI~PRO~IN

DPL

267

SYSTf&fS

Water was distilled twice, the second time over alkaline permanganate; in order to avoid permanganate spraying or creeping over into the clean water, the distillation flask had a long neck (30 cm) on which was mounted upright a Vigreux fractionating column, 40 cm long. The electrolyte solutions were prepared with reagent grade salt and were foamed in order to remove surface-active impurities that are ordinarily present in commercial salts (Colacicco and Rapport, 1966). Organic solvents were spectral grade and were not purified further. The lipid, to be used in making spread films, was dissolved in chloroformmethanol (85 : 15). These solutions were stored at - 20 “C between experiments, and kept on ice during the surface balance experiments; they were discarded after 2 weeks. Using the criteria specified above, we observed that the lipids in organic solvent at - 20 “C maintained their integrity for a longer time (2 weeks) than at 2 “C (2 days: Colacicco, 1973). Dispersions of lipid-protein mixtures were prepared as follows. After evaporation of the organic solvent from the lipid solution, the aqueous protein solution in 0.15 M NaCl was added to the lipid residue, and the system was sonicated in ice for 15 min at 90 W in a sonifier cell disruptor (Model 185D, Branson Sonic Power Co., Plainview, Long Island, N.Y.). Lipid dispersions were prepared by sonication of the dry lipid residue in 0.15 M NaCl under the same conditions.

SURFACE TENSION MEASUREMENTS

A known modification of the combined Langmuir and Wilhelmy methods (Clements and Tierney, 1965) was used. The trough, with internal dimensions 14.6 x 5.7 x 1.l cm3, was carved out of a heavy teflon block. The film, contained by a flexible teflon strip and a light teflon piston, was compressed between a maximal (I~%) area of 65 cm2 and a minimal (20%) area of 13 cm2. The surface tension was measured by a platinum plate suspended from a Cahn electrabalance. The outputs from the amplified signal of the electromechanical transducer of the Cahn balance and from the compression-decompression mechanism fed the values of surface tension y and % area to the y and x axes respectively of an x-y pen chart recorder (Esterline Angus, Indianapolis, Ind.). The curves resulted in a r--A loop. The cycle’s period was 8 min.

RATIONALE OF PROCEDURES

Understanding of the molecular correlates of dynamic surface tension in the lung could derive from a knowledge of the exact molecular topography of the air/water interfaces. Since such knowledge is not accessible, useful information can be attained by probing the relevant lipid and lipid-protein systems with certain variables that modify the surface tension at such interfaces.

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G. COLACICCO AND M. K. BASU

Spread and adsorbedfilms Previous studies have considered indifferently various methods for the assay of surfactant quality: (a) films of lipid extracts or isolated lipids spread from organic solvent; (b) films spread from intact pulmonary washings or other aqueous dispersions of surfactant fractions; and (c) films adsorbed from aqueous dispersions of lipid or lipid-protein systems. Although methods (a) and (b) probably have little biological significance, they have been used the most and are valid in comparing the behavior of various surface-active molecules. Ultimately, in the third assay method, the adsorption of surfactant from its aqueous dispersion is probably more relevant to in uioo models, in which the surfactant system of the lung is the pertinent constituent of the alveolar lining layer. In previous studies we discussed the significance and evaluated the differences of the several assay procedures (Colacicco, 1969,1972 ; Colacicco and Scarpelli, 1970,1973 ; Colacicco et al., 1976a ; Colacicco and Basu, 1977). For obvious reasons the present study considers mainly films adsorbed from aqueous dispersions. Temperature Most of the experiments in the past were carried out at 25 “C. Although we prefer 37 “C, we shall also report a few experiments at 25 “C, for two reasons: (a) to make a comparison with 37 “C, and (b) to explain the results that were obtained in previous studies with pulmonary washings at 25 “C. Since molecular species are required to make a monomolecular film able to reduce surface tension, the higher temperatures would decrease viscosity and increase fluidity of molecules and particles. This would favor transport of molecules from bulk particles to film and promote surface activity. Humidity Inasmuch as the air/water interface in the lung is saturated with humidity at 37 “C, most of the previous studies were carried out with films exposed to ambient, relatively dry, air. A comparison in the behavior of dry and wet films should provide information about molecular mechanisms relevant to the functioning of ,alveolar interfaces. The surface tension response of dry and wet DPL films at 37 “C is known to be different (Colacicco et al., 1976a). Lipid-lipid and lipid-protein interactions Another variable that is capable of affecting the c,omposition, organization and surface tension response of films is the composition of the starting material and the molecular interactions that are bound to control the transport of lipid or protein from the aqueous to the film phase. Such interactions must be anticipated in lipidprotein mixtures as complex as pulmonary washing. Therefore we consider two binary systems, DPL-albumin and DPL-cholesterol (Colacicco and Basu, 1977), and a ternary system, DPL-albumin-cholesterol, since these three substances are major constituents of pulmonary washing.

DYNAMIC

SURFACE ACTIVITY

OF

DPL

269

Rt?SUItS PRESENTATION

AND INTERPRETATION

OF DATA

Unless otherwise specified, the y-A curves in cycles 1 and/or 2 are representative, since the curves of subsequent cycles were virtually identical. Each curve is the exact r--A tracing from one single ex~riment ; the y--A curves of triphcate experiments were practically superimposable, with an average deviation smaller than 1 dyne/cm at any point of the curve. A condition for the reproducibility of the data was a constant time of ultrasonic irradiation of the aqueous lipid or lipid-protein dispersion (15 min at 2 ‘C). DPL

FILMS SPREAD ON

RSA SOLUTIONS

It was reported that (a) films of DPL spread from organic solvent on 0.15 M NaCl tend to lose activity with the time of cycling (Hurst et al., 1973; Shahriari and Hurst, 1976; Cola&co and Scarpelli, 1973, fig. 15C), and (b) the protein stabilizes the DPL films spread on to albumin solutions (Hurst et ai., 1973).

Influence of temperature: 25 “C and 37 “C

Our data confirm and extend the conclusions that Hurst et al. (1973) reached in their work at 25 “C. We studied films of DPL spread on numerous concentrations of rabbit serum albumin, between 0.001 mgjml and lOm~m1, at 25 “C as well as 37 “C. For simplicity, we present the PA curves of DPL spread on only one albumin concentration, 10 mg/ml (fig. 1). Note the 7-A curve of the adsorbed albumin films:

0 100

f COMPRESSiON X AREA

20

Fig. I. Force-area curves of DPL films spread on hypophase containing 10 mgjml RSA in 0.15 M NaCl at 25 “C (panel A) and 37 ‘C (panel B). The results were virtually identical on all protein concentrations between 1 m&ml and 10 m&ml.

270

G. COLACICCOAND M. K. 3ASU

in the absence of DPL, the y,,, and yminvalues are not far apart, thus causing a long and shallow hysteresis at pressures above 30 dyne/cm. At a given temperature, 25 “C or 37”C, the 7-A curves were virtually superimposable at all protein concentrations (between 0.001 mg/ml and 10 mg/ml) and preserved all the characteristics of the DPL films spread on 0.15 M NaCl in the absence of protein. The only difference was in that, on the protein solutions, the zero surface tension of DPL persisted indefinitely, as opposed to the gradual loss of activity of DPL films spread on NaCl solutions (see tig. 15C in Colacicco and Scarpelli, 1973). The mechanism of this effect requires further study. Similar effects were reported by Tabak and Notter (1977) in experiments in which DPL films were spread from organic solvent on albumin films that had been spread from aqueous protein solutions. The typical y-A curves of DPL on 0.15 M NaCl, both at 25 “C and 37 “C, were not altered when serum albumin was applied from aqueous solution on to DPL films adsorbed from aqueous dispersion in 0.15 M NaCl. These results are consistent with similar findings of Tabak and Notter (1977). Influence of humidity Panel A, fig. 2, shows the force-area curve of a spread DPL film at 37 “C. A film of albumin adsorbed from a concentration of 200 pg/mI in 0.15 M NaCl produced a typical force-area curve through the 5th cycle. Then, upon application of DPL, the DPL character of the force-area loop at 37 “C immediately ensued at the beginning of the 6th compression (panel A). Cycling was continued through the end of the 8th cycle, at which time the hysteresis loop of DPL was unchanged. The compression mechanism was then stopped at maximal area, the trough was covered under a leucite chamber (Colacicco et al., 1976a) and, after 5 min, cycling was resumed under 80

A

DRY

60 -

X COMPRESSION % AREA

sb 20

Fig. 2. Effect of humidity at 37 “C on y-A curve of DPL spread on RSA solution in 0.15 M NaCl. (A) DPL on 200 pg/rnl RSA after completion of 5th cycle with film of RSA alone (upper loop); (B) the same film in (A) after closing the chamber and producing saturation of the atmosphere with water vapor.

DYNAMIC SURFACE ACTIVITY OF

271

DPL

humidity saturated atmosphere. Immediately ymin rose from zero to 13 dyne/cm and continued to rise to become 35 dyne/cm by the 16th cycle. Clearly, the effect of humidity was to restore the albumin character (ymin> 30 dyne/cm) and not just to cause a rise of yminfrom zero to 20 dyne/cm as was the case with DPL alone in water vapor saturated atmosphere (Colacicco et al., 1976a). Unlike with DPL, the phenomenon was not reversible: by returning the DPL film from the humidity chamber to open air, yminfell immediately from 20 dyne/cm to zero (Colacicco et al., 1976a); in contrast, in the presence of albumin yminremained at 35 dyne/cm.

FILMS ADSORBED FROM

DPL-RSA

CODISPERSIONS

Injluence of temperature: 25 “C and 37°C

At 25 “C, with a bulk concentration of 200 pg/ml, the adsorbed DPL film lowered surface tension to zero, whereas the yminvalue of RSA was 41 dyne/cm. Such films were stable over numberless cycles (fig. 3A, B). With a dispersion of 200 pg/ml each (DPL and RSA) the ymin value and the force-area loop in the first cycle (fig. 3C) were similar to those of RSA alone. However, upon repeated compressions and decompressions a full DPL characfer appeared after nearly 38 cycles (fig. 3D); the change was gradual. At 37°C (fig. 4) DPL and RSA films maintained ymin values of zero and 36 dyne/cm respectively (panels A, B) for numberless cycles; the film adsorbed from DPL-RSA dispersions, however, showed a gradual change from an albumin 8o

25’

DPL(200pghnl)

25’

A i

DPL (200pg~ml)+RSA(2OO~g/ml)

C

I I

40-

a

-42

20 -

/ 80

RSA (200pghl)

60;;

B

2 2

40 -

1

-41

200 0 100

Y, COMPRESSION X AREA

I 80 20

0 100

X COMPRESSION X AREA

80 20

Effect of cycling time at 25 “C. Force-area cuives of film adsorbed from a codispersion of 200 pg/ml each DPL and RSA in 0.15 M NaCI. Panels: (A) DPL alone; (B) RSA alone; (C) DPL-RSA mixture in 1st cycle; (D) DPL-RSA mixture through 38 cycles.

272

G. COLACICCO AND M. K. BASU

a0

a0

37’ DPL (200pghnl)

A

DPL(2OOpghnl) + RSA(200~ghnl)

C

37” RSA (200pghl)

602

40 = 2

I

w

-36

20 0 0 100

% COMPRESSION % AREA

I

a0

0

20

100

% COMPRESSION % AREA

I

1

80 20

Fig. 4. Effect of cycling time at 37 C. Force-area curves of film adsorbed from a codispersion of 200 pg/ml each DPL and RSA in 0.15 M NaCI. Panels: (A) DPL alone; (B) RSA alone; (C) DPL-RSA mixture in 1st and 6th cycles; (D) DPL-RSA mixture in 7th and 12th cycles.

character (30 dyne/cm) in the 1st cycle to a slightly modified DPL character (5 dyne/cm) in 6th cycle. The change from albumin to DPL character at 37 “C (panels C, D) was much faster than at 25 “C, as it took only 12 cycles for the ymin value to reach 2 dyne/cm as compared to nearly 38 cycles at 25 “C. This time-dependent lowering of surface tension with the films adsorbed from DPL-albumin mixtures explains the data and the inveterate practice of the physiologists who, working at 25 “C, left the film on pulmonary washings or other surfactant fractions in the surface balance to cycle for a very long time until a constant lowest value of ymin. Whether this was attained after 10 or 100 cycles could now be accounted for by different concentrations of those factors (albumin or other protein, and cholesterol) and lower temperature, which ordinarily prevent DPL from readily reaching the interface. Influence of albumin concentration Indeed, the greater the albumin concentration, the greater were the time and number of cycles required for the y-A curve to lose the albumin character and assume the DPL character, until, with a concentration of 10 mg/ml protein, the albumin character persisted indefinitely also at 37 “C (fig. 5). The yminvalue was 37 dyne/cm as opposed to 42 dyne/cm at 25 “C; similar effects were obtained with concentrations of 2, 3 and 5 mg/ml RSA. When the albumin concentration was small, 50 pg/ml (fig. 5A), the albumin character was imperceptible, meaning that such small albumin concentrations do not interfere with the surface activity of aqueous DPL dispersions, 200 pg/ml.

DYNAMIC SURFACE ACTIVITY

80

6

c

4 -

2 w

OF

DPL

273

37’C RSA(50pg/ml)*DPL(200pg/ml)

a0

RSA(500pg/ml)+DPL(200pg/ml)

B

60

01

80

54

RSA(10,000~g/ml)*DPL

0

(2OOpghnl)

C

I

0

100

X’COMPRESSION XAREA

a0 20

Fig. 5. Effect of protein concentration on the dynamic force-area curves of film adsorbed from codispersion of RSA with 200 pg/ml DPL in 0.15 M NaCl at 37 C.

Znjluence of cycling time The question arose if the appearance of the DPL character from the aqueous DPL-RSA mixture was a consequence of either the continuous compressions and decompressions of the film or the time of standing of the lipid-protein mixture. The experiment was performed at 37 “C. When a DPL-RSA mixture containing 200 pg/ml each was left to stand in the trough for 80 min, the time equivalent of 10 cycles, during the first compression the film had already lost the albumin character typical of the fresh DPL-RSA mixture of fig. 5 and acquired part of the DPL character, which was preserved in the subsequent cycles (fig. 6); further cycling did not bring out any additional DPL character. Indeed, after 10 cycles (80 min) yminwas about 2 dyne/cm (fig. 5), whereas after 80 min standing yminwas 18 dyne/cm and remained such through the subsequent 5 cycles observed. It is apparent that effects of standing and effects of cycling have some similarities but also marked differences. At present, an explanation for such differences is not possible, primarily because the molecular parameters of the interfacial structures of such simple mixtures are not accessible.

274

G. COLACICCO AND M. K. BASU

37’C RSA(200pg/ml)+DPL(2UOpp/ml)

100

X AREA

20

Fig. 6. Influence of standing. Dynamic y-area curve of film adsorbed from a codispersion of 200pg/ml each DPL and RSA in 0.15 M NaCl after 80 min standing at 37 C. Two cycles are reported, I and 5.

Injluence ofhumidity

With concentrations of 200 pg/ml each in the mixture of DPL and RSA in 0.15 M NaCl in the subphase at 37 “C, in open air, the adsorbed film attained a yminvalue of 30 dyne/cm in the 1st cycle and 5 dyne/cm in the 6th cycle. In humidity saturated atmosphere (fig. 7B) ymin was 48 dyne/cm in the 1st cycle and 20 dyne/cm at the 7th cycle, as opposed to 30 dyne/cm and 5 dyne/cm respectively in open air. Little change in ymi, and y-A loop was observed when, at the end of the 7th cycle (fig. 7B), the cover was removed and the film was returned to the open air through 20 cycles (fig. 7C). This contrasts with the behavior of adsorbed tilms of DPL alone, where the effect of the humidity was reversible (Colacicco et al., 1967a), and with the behavior of DPL films spread on albumin solutions, where continuous cycling in humidity saturated atmosphere (fig. 2B) promoted the albumin character instead of the DPL character of the film. Injluence of cholesterol

It is known that rabbit pulmonary washings contain cholesterol in quantities ranging between 5 % and 20 % of either DPL or albumin (Colacicco et al., 1973, 1976b). Since cholesterol is a potent inhibitor of DPL activity (Colacicco and Scarpelli, 1973; Colacicco and Basu,. 1977) and interacts with proteins in bulk (Marinetti and Pettit, 1968) and in films (Colacicco, 1972), the influence of albumin on the surface activity of DPL must be examined in the light of possible DPLcholesterol and albumin-cholesterol interactions. Although, for obvious reasons, a detailed presentation of such a multifaceted problem must be postponed, we offer only a summary of trends (fig. 8), with the view to pointing out certain major effects of cholesterol in surfactant fractions consisting of DPL-albumin-cholesterol mixtures. Whereas the presence of 2 mol ‘A cholesterol in the aqueous dispersion of DPL at 37 “C caused a rise in yminfrom zero to 17 dyne/cm (fig. 8A) (Colacicco

DYNAMIC SURFACE ACTIVITY OF DPL(200pgcg/ml)+RSA(200~g/ml)

80

2 c z

80

z

40

5

c -c

20t

k

80

37-C

(DRY)

A

37’C

(WET)

B

II. 60 x 0 si

275

DPL

40

3 i

1

7

20

37’C

2

0 100

(DRY)

% COMPRESSION % AREA

00 20

Fig. 7. Effect of humidity saturated atmosphere on the force-area curves of films adsorbed from codisp-ersion of 200 &ml each DPL and RSA in 0.15 M NaCl at 37 C (see text).

and Basu, 1977) serum albumin removed the effect of such small cholesterol concentrations. Concentrations of 50-100 pg/ml RSA were required to bring ymin to 6 dyne/cm at the 6 th cycle (fig. 8C) and down to zero at the 10th cycle; at 10 pg/ml albumin already had some effect (fig. 8B). Two features are worthy of note. The small protein concentration (10 pg/ml) removed a great part of the cholesterol character and restored the DPL character (lower y values) at relatively large areas, whereas at minimal area the ymin value was still high (13 dyne/cm). The greater protein concentrations, 100 and 200 pg/ml, removed the cholesterol effect and restored the DPL character of the film at small areas, where ymintended to approach zero (fig. 8C), but also removed the DPL character and introduced the protein character (high surface tension) at large areas, where the hysteresis was nearly identical with that of albumin (fig. 8D). Although larger amounts of RSA were needed to counteract the effect of greater concentrations of cholesterol, too high a protein concentration brought out the albumin character (ymin greater than 30 dyne/cm), which finally prevailed over both the cholesterol and humidity

276

G. COLACICCO AND M. K. BASU

DPL(200~g/ml)+Cholesterol + RSA(lOOpg/ml)

1

$0,

DPL (200pQlml)

+Choiesterol (Zmole %,) + RSA(lOpg/mf)

DP1(200/~g/ml)

B

+Cholesterol(Zmole%) lRSA (20Opglml)

D 1

O0 joo

I % COMPREWON % AREA

8’0 20

8. Influence of serum albumin concentration on the force-area curves of films adsorbed from codispersions of DPL (200 pg/ml), cholesterol (2 mol %) and RSA in 0.15 M NaCl at 37 T (see text).

characters of DPL films (20 dyne/cm) and the DPL character in open air (zero surface tension).

Discussion

The foregoing data and arguments warrant the following conclusions: (1) Both at 25 “C and 37 “C serum albumin stabilized the activity (y-A curves) of DPL when the latter was spread from organic solvent on to albumin solutions, irrespective of the very small or very large albumin concentration in the aqueous phase. (2) At both temperatures albumin inhibited the surface activity of DPL when the film was adsorbed from aqueous dispersions of DPL-albumin mixtures having albumin/DPL weight ratios 2 1; the inhibitory effect at 37 “C was less than at 25 “C. (3) Humidity at 37 “C tended to restore the albumin character (high surface tension) of the film adsorbed from DPL-RSA mixtures as opposed to the DPL character (zero surface tension) of the homologous dry $II?z; in brief, albumin potentiates and prevails over the DPL-humidity effect. (4) With films adsorbed from DPL-RSA+holesterol mixtures, modest albumin concentrations in the aqueous phase mitigated the effect of cholesterol, which otherwise is a potent inhibitor of DPL activity.

DYNAMIC SURFACE ACTIVITY BIOLOGICAL

SIGNIFICANCE

OF

DPL

277

OF THE SURFACE BALANCE DATA

Inasmuch as ultrastructural studies showed that the upper region of alveolar lining layer consisted of a protein-free thick film of lamellae of dipalmitoyl lecithin (Kaibara and Kikkawa, 1971) as well as myelinic figures containing serum albumin (Bignon et al., 1975), in view of such conflicting results and recent findings (Colacicco, 1978), the question of the composition of the surfactant system of the lung must be postponed. However, since the most abundant components of pulmonary lavage in 0.15 M NaCl are DPL and serum albumin (Colacicco et al., 1973, 1976b), and since the yminvalues of DPL and albumin on 0.15 M NaCl are zero and about 40 dyne/cm respectively, the influence of albumin on the dynamic surface activity of DPL is relevant to the study of lung mechanics under conditions in which either the alveolar spaces contain albumin or, more specifically, the structures of the alveolar wall release serum albumin or other protein into the lavage (Colacicco, 1978). The foregoing data, therefore, may be used only to set up empirical criteria for the diagnosis of the physiological, pathological and therapeutic states of the lung. Indeed, the composition of pulmonary washing reflects (a) the state of the air-blood barrier, whether normal or abnormal, as it was shown in the case of alveolar proteinosis (Passer0 et al., 1973), and (b) the insult the lavage medium inflicts on to the alveolar wall, as shown by micropuncture (Reifenrath, 1973) and as intimated by immunofluorescence studies (Colacicco et al., 1978; Colacicco, 1978). Irrespective of whether aibumin is a normal constituent of the alveolar lining layer and of the surfactant system of the lung (Bignon et al., 1975) or is an artifact of pulmonary lavage (Reifenrath, 1973; Colacicco et al., 1978), the surface activity of the latter could be interpreted only in relation to the lipid and protein compositions of the surfactant fractions, For instance, failure of the latter to lower surface tension to zero may be ascribed to either low temperature in the assay (25”C), low DPL concentration, high protein/DPL ratio and high cholesterol/DPL ratio. Such criteria are valid at 25 “C as well as 37 C, except that the higher temperature facilitates the appearance of the DPL character from a DPL-albumin mixture, which at 37 “C attains zero surface tension faster than at 25°C. Furthermore, in spite of normal or abundant concentrations of DPL in pulmonary washing or other lung preparation, the activity of DPL could be masked by the large quantities of cholesterol (and other lipids) or protein that may reflect specific pathologic or experimental states of the lung. Finally, if zero or very low surface tension were to be a requirement for normal alveolar mechanics, it may be important that albumin and cholesterol (as well as other lipids and proteins) be excluded from the alveolar lining layer.

278

G. COLACICCO AND M. K. BASU

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Clement% J. A. (1962). Surface phenomena in relation to pulmonary function. Physiologisr 5: I l-28. Clements, J. A. (1970). Pulmonary surfactants. Am. Rev. Respir. Dis. IO1: 984-990. Clement% J. A. and D. F. Tierney (1965). Handbook of Physiology. Respiration. Sect. 3, Vol. II. Washington, D.C., Am. Physiol. Sot., pp. 156551583. Colacicco, Cl. (1969). Application of monolayer techniques to biological systems: symptoms of specific lipid-protein interactions. J. Colloid Inferface Sri. 29: 345-364. Colacicco, Cl. (1972). Surface behavior of membrane proteins. Ann. N. Y. Acad. Sci. 195: 224261. Colacicco, G. (1973): I ipid monolayers: ionic impurities and their influence on the surface potentials of neutral phospholipids?Chem. Phys. Lipids 10: 66-72. Colacicco, G. (197Q’Pulmonary surfactant: Alveolar lining layer and surfactant system of the lung do not exist. Fed. Proc. 37 (3): 720. Colacicco, G. and M. M. Rapport (1966). Lipid monolayers: action of phospholipase A of Crorulus atrox and Nqja nqja venoms on phosphatidyl choline and phosphatidal choline. J. Lipid Rev. 7: 258-263.

Colacicco, G. and E. M. Scarpelli (1970). Pulmonary surfactants: phospholipid

or lipoprotein?

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