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Determination of disaturated lecithin in rhesus monkey amniotic fluid as an index of fetal lung maturity
Determination of disaturated lecithin in rhesus monkey amniotic fluid as an index of fetal lung maturity VIOLETA
CURBELO,
DOROTHY PHILIP
BERLIN M.
M.D.* GAIL,
FARRELL,
Bethesda, Maryland,
PH.D.
M.D.,
Washington,
PH.D.
D. C., and Madison,
Wisconsin
Although increased concentrations of total lecithin in amniotic fluid allow prenatal assessment of fetal lung maturation, it has become clear that routine use of the US index may lead to a substantial number of inaccurate predictions. Since disaturated lecithin (DL) is a more specific marker of pulmonary surfactant than total lecithin, we developed a convenient method for measuring this phospholipid in amniotic fluid, and then evaluated its level in pregnant rhesus monkeys of 120 to 163 days of gestation. The method involves osmic acid destruction of unsaturated lipids, chromatographic isolation of disaturated lecithin, and quantitatiin by phosphorus assay. It can be performed in approximately 5 hours on 4 ml. of amniotic fluid and yields 67 2 3 per cent average recovery of added 14C-dipalmitoyl lecithin. The results of analyzing 36 rhesus amniotic fluid specimens showed that disaturated lecithin and the disaturated lecithinlsphingomyelin ratio (DUS) increase sharply after 150 days of gestation, consistent with the pattern of lung maturation in this species. We conclude that dlsaturated lecithin can be readily quantitated in primate amniotic fluid and that its concentration, the DUS ratio, and percentage of disaturated lecithin are potentially useful indices of fetal lung maturity for the clinical laboratory. (AM. J. OBSTET. GYNECOL. 131: 764, 1976.)
SURFACE-ACTIVE components of the lung are primarily phospholipids. It has been shown that the most abundant of these are lecithins (3-sn-phosphatidylcholine species). In the lung, because of variations in fatty acid composition, there are several types of lecithin. Total lecithin, however, is not surface active, and only the disaturated fraction displays this property, particularly dipalmitoyl lecithin (DPL). Data gathered from several studies, as reviewed by Farrell and Avery,’ have shown that the surface-active material
THE
From the Neonatal and Pediatric Medicine Branch, National Institute of Child Health and Human Deuelofmumt, National Institutes of Health, the Deparknts of Medicine and Pediatrics, George Washiwton Uniuer&. and the Deeartment of Pediatrics, Unive&y of Wi5scim.s~~. Received for publication
August
Revised December
5, 1977.
Accepted January
5, 1978.
3, 1977.
Philip M. Farrell, Department oj Pediatrics, University of Wisconsin, 1300 University Reprint
Ave.,
requests: Dr.
Madison,
*Fogarty National
764
Wisconsin
53706.
International Fellow, 1976-l Institutes
of Health.
977, at the
of the fetal lung reaches amniotic fluid in late gestation, leading to an increase in the concentration of lecithin. Consequently, assessment of fetal lung maturation can be carried out by analyzing lecithin concentrations in amniotic fluid, a measurement which at present is usually performed in clinical laboratories by determining the lecithin/sphingomyelin (L/S) ratio described by Gluck, Kulovich, and Bore?; since the sphingomyelin concentration does not change during gestation, it is used in the L/S ratio method as an “internal standard” or reference base. Although the L/S procedure has proved reliable for identifying lung maturity in normal pregnancies, several investigators 3, 4 have reported that both falsepositive (“mature”) and false-negative (“immature”) L/S ratios may occur, particularly in complicated pregnancies. According to Morrison and associates,4 falsenegative L/S ratios can increase fetal mortality rates due to the fact that in distressed pregnancies, delivery tends to be delayed as the obstetrician waits for a higher L/S ratio on a repeat amniocentesis. Even though disaturated lecithin is a more specific marker of pulmonary surfactant than total lecithin,’ procedures for measuring this fraction in the past have 0002-9378/78/07131-0764$00.60/O
0
1978
The C. V. Mosby
Co.
Volume Number
13 I 7
been tedious, extremely time consuming, and not applicable to the clinical laboratory. These considerations led to the present study, in which we report a convenient approach for the measurement of disaturated lecithin in primate amniotic fluid, based on ada’ptation of the method described by Mason, Nellebogen, and Clements.” The goals of this study, therefore, were: (1) to devise a method which would permit relatively rapid and reproducible quantitation of disaturated lecithin in amniotic fluid, (2) to delineate the normal relationship of amniotic fluid disaturated lecithin concentradons to gestational age, and (3) to determine if disaturated lecithin in amniotic fluid could possibly serve as an index of fetal lung maturity in uncomplicated pregnancies. Because of similarities between the rhesus monkey (Macaca mulatta) and the human being with respect to pulmonary surfactant and the process of fetal lung development,6H and in view of our access to normal rhesus pregnancies, we focused on this animal model of primate lung development.
Materials and methods Thirty-six pregnant rhesus monkeys from the primate colony of the National Institutes of Health, with accurately known gestational ages (? 1 day), were used for these studies. Amniocenteses were performed on animals under ketamine anesthesia from 120 to 163 days of gestation (term = 164 + 3 days). The amniotic fluid was immediately centrifuged at 400 x g for 5 minutes and the supernatant fractions were stored at -20” C. until analyses could be performed (usually in less than 1 week). Under these conditions of storage, we did not detect any loss of phospholipids based on measurement of total lecithin and monitoring of the recovery of added ‘“C-dipalmitoyl Iecithin (14C-DPL). Samples contaminated with blood or meconium were discarded. Three samples of human amniotic fluid with known L/S ratios were also obtained from an affiliated hospital and were processed similarly. Lipids from 4 to 8 ml.* of amniotic fluid were extracted by means of the methods of Bligh and Dyer9 and Lindback, Frantz, and Graven.‘O Chloroform: methanol (1: 2) in a volume of 15 ml. per 4 ml. of *For pregnancies of >145 niotic fluid always sufficed
days of gestation, 4 ml. of amfor the measurement of total lecithin, disaturated lecithin, and sphingomyelin. In animals of 120 to 145 days of gestation, however, where lower amniotic fluid lipid levels reflecting pulmonary immaturity were found, 8 ml. were required. In practice, we found it convenient to extract two amniotic fluid samples of 4 ml. each when analyzing specimens from presumably immature pregnancies. After the initial processing steps described above, the two chloroform layers were pooled just prior to weighing for determination of the total lipids present in 8 ml. of amniotic fluid.
Disaturated
lecithin
as index
of lung maturity
765
amniotic fluid, and I%-dipalmitoyl lecithin (Applied Sciences, State College, Pennsylvania). a> an internal standard, were added to the samples. Thev were mixed vigorously for 5 minutes and then passed through a scintered glass filter with ultrapure nitrogen under positive pressure to expedite the filtration. Chloroform (5 ml.) was added through the filter and .5 ml. of distilled water were added to the filtrate to establish hydrophilic and hydrophobic phases. The samples were then centrifuged at 400 x g for 5 minutes, and the lower chloroform layer was removed and taken to dryness under nitrogen in a 40” C. water b,tth. The dry total lipid fraction was carefully weighed and dissolved in chloroform : methanol (2 : 1), and small aliquots were spotted on activated silica G HR thin-layer chromatography (TLC) plates (Brinkman Instruments, Westbury, New York). The plates were developed in chloroform : methanol : water (60 : 35 : 5). and ;I solution of bromthymol blue* was used to detect the lipid spots after chromatography. The lecithin and sphingomyelin spots were scraped from the plates and analyzed for phosphorus” or radioactivity. To determine the latter, the silica gel samples were placed in scintillation vials containing 10 ml. Aquasol; the vials were then counted in a Beckman LS-255 scintillation counter for 10 minutes. For the determination of disaturated lecithin, we used the method of Mason and associates” with minor modifications, after examining other methods described in the literature.‘*, I3 Aliquots of amniotic fluid lipid extract, obtained as described previously and representing approximately 1 mg. of rota1 lipid, were treated with 0.5 ml. of osmium tetroxide in carbon tetrachloride (100 mg. per milliliter) for 15 minutes. The lipids were then dried under nitrogen, dissolved in 0.5 ml. of chloroform : methanol (20: I), and added to columns containing 0.8 Gm. of neutral alumina (100 to 200 mesh, Bio-Rad Laboratories, Richmond, California). Neutral lipids were eluted first with 10 ml. of chloroform : methanol (20: I), and a disaturated lecithin fraction was then eluted with 5 ml. of chloroform : methanol : 7M ammonium hydroxide (70 : 32 : 2) (v/v/v). In order to determine if lipids other than disaturated 1 lecithin were present in the second alumina column eluates, we dried these 5 ml. fractions under nitrogen and routinely applied them to TLC plates for chromatography on silica gel as described previously. Gasliquid chromatography of fatty acid methyl esters was performed on a limited number of samples to determine the fatty acid composition of the disaturated lecithin fractions obtained both from the column eluate and from the TLC plates.
766
Curbelo, Gail, and Farrell
August I, 1978 Am. J. Obstet. C;ynerol
Cls Cl7 (internal standard)
Fig. 1. Gas chromatographic analysis of fatty acid methyl esters prepared from a lipid extract of amniotic fluid obtained from a pregnant rhesus monkey at 162 days of gestation. The sample was processed as described in the text, with osmic acid treatment, column chromatography, and thinlayer chromatography. Carbon numbers designate the fatty acids detected: C14 = myristic, Cl5 = pentadecanoic, Cls = palmitic, C,, = heptadecanoic (not present in amniotic fluid, but added as an internal standard), Cl8 = stearic, C,,:, = oleic, C18:*= linoleic. Calculations revealed that 98 per cent of the fatty acids present were saturated and that palmitate accounted for 91 per cent of the total fatty acids in this sample. The line represents an actual tracing from the gas chromatograph, with the time of injection (INJ) indicated by the arrow.
From data obtained by phosphorus analyses and measurement of r4C-DPL recovery, the following were calculated: total lecithin, sphingomyelin, the molar L/S ratio (the ratio of the absolute amounts of the two phospholipids), the percentage of the disaturated species in total lecithin, and the disaturated lecithin/ sphingomyelin (DLIS) ratio. The t test for unpaired data was used for statistical analysis.
Results The method of Mason and associates” was found to be applicable to amniotic fluid phospholipid analysis with minor modifications as described in this report. The procedure proved convenient and relativeiy rapid compared to other methods.r2* I3 Thus, it was possible to complete the analysis of total lecithin, sphingomyelin, disaturated lecithin, and the derived ratios in approxrmately 5 hours. The method also showed satisfactory reproducibility. The over-all recovery of added r4C-dipalmitoyl lecithin was 67 + 3 per cent (mean & S.E. for 34 analyses). Whatever losses of lecithin OCcured during the procedure seemed to be confined
primarily to the osmic acid treatment and column chromatographic steps. Analysis by thin-layer chromatography of the second eluate from the alumina column, i.e., the “disaturated lecithin fraction,” revealed only one lipid spot, and this migrated identically with standard dipalmitolyl lecithin. Furthermore, 99 per cent of the phosphorus and radioacnvity detected in each lane of the TLC plates was recovered with the disaturated lecithin spots. No radioactivity or phosphorus was detected in the neutral lipid fractions eiuted from the column. Gas-Jiquid chromatographic examination of selected column eluates and “disaturated lecithin spots” from TLC plates revealed these fractions to be highly saturated with respect to fatty acid composition. Representative results obtained upon analysis of an amniotic fluid extract from a rhesus monkey of 162 days of gestation are shown in Fig. 1. This experiment revealed 98 per cent of the fatty acids to be saturated, with palmitate (C,,) accounting for 91 per cent of the methyl esters. As shown in Fig. 2 and Table I, total and disaturated
Volume
Disaturated lecithin as index of lung maturity
13 I
767
Number 7
.
.
.
TERhn l Lf
t
,.
130
120
d 140’
l
lb0
160
1
GESTATlONALAGEfdays)
Fig. 2. Total lecithin and disaturated related to gestational age. Table
I. Phospholipids
Gestationd age
of rhesus amniotic
lecithin concentrations
fluid as related
Sphingomyelin (mg. I1 00 ml.)
130-139
0.274 k 0.041 (7) 0.670 +- 0.123
0.390 + 0.044 (7) 0.515 -r- 0.078
140-149
1.063 +- 0.354
(8)
(8)
150-159
2.912 -+ 0.862 (9) 5.139 k 0.853 (3)
0.651 + 0.060 (9) 0.431 k 0.093 (3)
120-129
(8)
2160
to gestational
age*
Disatwated Total lecithin (mg. I1 00 ml.)
(days)
in rhesus monkey amniotic fluid as
03) 0.494 2 0.037
Mgll
00 ml.
0.087 f 0.008 (7) 0.223 2 0.044
(6) 0.535 2 0.179 (8) 1.399 + 0.456 (9) 3.104 * 0.579 (3)
lecithin ‘r %
of total lecithin 33 + 3.5 (7) 30 t 2.5 (8) 53 c 5.0
03) 46 + 2.1 (9) 60 2 1.53 (3)
DlsatUR&d LIS ratio
0.23 r 0.003 (7) 0.38 2 0.04
(6) 1,06 + 0.31 (8) 1.95 rfr 0.49 (9) 8.13 t 2.26 (3)
*Means and standard errors are shown with the number of samples enclosed in parentheses. ICorrected for loss of disaturated lecithin, as determined with W-dipalmitoyl lecithin. lecithin concentrations were found to increase gradtially from 120 days until 150 days. After 150 days of gestation, a significant date in the development of the fetal rhesus monkey lung according to our previous work,s an especially sharp increase was noted in the concentratioin of the total lecithin and disaturated lecithin fractions. Disaturated lecithin reached a value 16-fold higher at 150 to 159 days as compared to the interval of 120 to 129 days of gestation. The mean disaturated lecithin concentration was 0.33 mg. per 100
ml. before 150 days and 1.8 mg. per 100 ml. after 150 days (p < 0.001). The concentration of sphingomyelin, in contrast, did not show significant changes from 120 to 160 days of gestation (Table I). Reflecting these changes in phospholipid concentrations, the ratio of lecithin to sphingomyeiin was found to increase in late gestation, especially after 150 days (Fig. 2). The Di/S ratio showed a similar surge at that point, after exhibiting a more gradual rise between 140 and 150 days of gestation. Thus, the somewhat
768
Curbelo, Gail, and Farrell
August Am. J. Obstet.
’
I
--------5-8
.
l
1. 1978 Gynecol
.
’
+--xi
:. .g**..
1 I
130
I
140
150
160 GESTATIONAL
Fig. 3. Phospholipid
AGE (days)
ratios in rhesus monkey amniotic fluid as related to gestational age.
abrupt increase in the DL/S ratio at 150 days led to a significantly higher (p < 0.01) mean of 3.69 after 150 days, as compared to a value of 0.67 prior to 150 days of gestation. As expected from our observation that disaturated lecithin accounts for approximately 50 per cent of the total lecithin (Table I), and our previous finding that fetal lung maturation is evident in rhesus monkeys after 150 days of gestation when the L/S ratio is greater than 2.0, 7* 8 it was found that the apparent “critical value” of the DL/S ratio is unity. When the individual values for analysis of amniotic fluid were examined as a function of gestational age, only one sample in 11 was found to have a DL/S ratio of less than 1 .O beyond 150 days. The percentage of total lecithin which is disaturated in amniotic fluid was found to increase slightly earlier than the disaturated lecithin concentration or the DL/S ratio. As shown in Table I, the percentage of disaturated lecithin was found to increase somewhat sharply during the interval of 140 to 149 days and remained high with little variation until term was reached. The average value for the percentage of disaturated lecithin was significantly higher between 140 to 149 days of gestation, as compared to the period before 140 days of gestation (p < 0.001). Based upon analysis of a limited number of human amniotic fluid samples, it appears that the procedure described in this report can be applied to such specimens without any additional technical difficulties being
encountered. The concentrations of disaturated lecithin in the three samples obtained from pregnant women of 34 to 37 weeks of gestation were similar to values found in rhesus monkeys of comparabIe gestation (0.844, 2.08, and 2.50 mg. per 100 ml.). The recoveries of ‘%-DPL for these samples were 64,66, and 79 per cent, respectively.
Comment Because of the complex and time-consuming nature of the previous methods used for measurement of disaturated lecithin,‘*’ I3 this important component of pulmonary surfactant has been only estimated in earlier work with amniotic fluid. Such measurements were made through the use of gas-liquid chromatography.‘* This approach does not provide direct information on the concentration of disaturated lecithin, but has revealed that palmitic acid is the predominant fatty acid in amniotic fluid lecithin during late gestation, after fetal pulmonary maturity is attained.” Thus, we felt that it was appropriate to pursue more convenient analytical techniques in an attempt to devise a method for routinely measuring this constituent as an index of fetal lung maturation. The method described in this paper allows measurement of disaturated lecithin directly, and the data reported herein are, to our knowledge, the first such values published for primate amniotic fluid. It was found that with adaptation of the method of Mason and associates,” disaturated lecithin
Volume Number
Disaturated lecithin as index of lung maturity
13 1 7
could be conveniently and reproducibly measured in amniotic fluid specimens from rhesus monkeys. The increased phospholipid levels observed in this study (Fig. 3) during late gestation agree with previous reports that in normal human and rhesus pregnancies, changes in lecithin concentration and the L/S ratio reflect gestational age and fetal lung development.‘* *, 6, ’ In this work, no amniotic fluid specimens were found with L/S values less than 2.0 after 150 days of gestation. The abrupt elevation of lecithin, disaturated lecithin, the L/S ratio, and the DL/S ratio at 150 days correlates with the fact that after this date, newborn monkeys do not develop symptoms of the respiratory distress syndrome. Our results also agree with those of Kotas and associates,* who evaluated physiologic indices of fetal lung development in rhesus monkeys and recently reported their findings on the ontogeny of pulmonary surfactant in this species. They found that functional alveolar surfactant, minimal surface tension, and total lecithin production in lung slices all increased significantly between 140 and 150 days of gestation. All rhesus fetuses examined after 150 days of gestation had mature lungs, whereas two of five animals studied between 140 and 149 days of gestation showed the presence of surfactant. Thus, the timing of physiologic changes in the fetal rhesus lung, as observed by Kotas and associates,’ and the pattern of increased disaturated lecithin con-
769
centrations in amniotic fluid, as reported herein, show good concordance. We therefore conclude that the rises in amniotic fluid disaturated lecithin occurs simultaneously with the attainment of fetal lung maturation. These correlations suggest that the method used in this study to measure disaturated lecithin yirida data that can serve as an index of fetal primate lung maturity. As we have shown that the pr0cedut.e of Mason and associates” with minor modifications is applicable to human amniotic fluid, we believe that it will be worthwhile to determine if the concentration of disaturated lecithin and the DLiS ratio are better predictors of neonatal respiratory outcome than the other methods now available. If so. measurement of disaturated lecithin and the DLiS ratio could prove to be a valuable means of predicting fetal lung maturity in the clinical setting. This approach may be especially helpful in assessing lung maturation in complicared gestations. We thank Dr. Ron Chanderbhan of (George Washington University for performing the gas-lipid chromatrography experiments, Dr. Ronald Chez of the Pregnancy Research Branch, NICHD, f‘cjr providing some of the rhesus amniotic fluid samples analyzed in this project, and Mr. Anthony J. i2dams of the Neonatal and Pediatric Medicine Branch, NICHD, for assisting us in the collection of specimc-*ns by percutaneous amniocentesis.
REFERENCES
1.
Farrell, P. M., and Avery,
M. E.: Hyaline membrane disease, Am. Rev. Resp. Dis. 111: 657, 1975. 2. Gluck, L., Kulovich, M., and Borer, R.: Estimates of fetal lung maturity, Clin. Perinatol. 1: 125, 1974. 3. Donald, 1. R., and Freeman, .R. K.: Clinical experience with the amniotic fluid L/S ratio, AM. J. OBSTET. GYNECOL. 115: 547, 1973. 4. Morrison, J. C., Whybrew, W. D., Bucovacz, E. T., Wiser, W. L., and Fisch, S. A.: The lecithin/sphingomyelin ratio in cases associated with fetomaternal disease, AM. J. OBSTET. GYNECOL. 127: 363, 1977. 5. Mason, R., Nellebogen, J., and Clements, J.: Isolation of disaturated phosphatidylcholine with osmium tetroxide. J. Lipid Res. 17: 281, 1976. 6. Gluck, L., Chez, R. A., Kulovich, M., Hutchinson, D. L., and Niemann, W. H.: Comparison of phospholipid indicators of fetal lung maturity in amniotic fluid of monkey (Mncaca mulatta) and baboon (Pupio papio), AM. J. OBSTET. GYNECOL. 120: 524. 1974. 7. Epstein, M. F., Fa&l, P. M., and Chez, R. A.: Fetal lung lecithin metabolism and the amniotic fluid L/S ratio in rhesus monkey gestations, AM. J. OBSTET. GYNECOL. 125: 545. 1976.
8.
Kotas, R. V., Farrell, P. M., Ulane, R. E.. and Chez.
R. A.: Fetal rhesus monkey lung development: Lobar differences and discordances between stability and distensibility. J. Appl. Physiol. 43: 1, 1977. 9. Bligh, E. G., and Dyer, W. J.: A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37: 911, 1959. 10. Lindback, T., Frantz, T.. and Graven, S. N.: Phospholipids in amniotic fluid, Acta Obstet. Gvnecol. &and. 53: 219. 1974. 11. Morrison, W. R.: A fast, simple, and reliable method for the microdetermination of phosphorus in biological materials, Anal. Biochem. 7: 218, 1964. 13 --. Shimojo, T., Abe, M., and Ohta, M.: A method for determinations of saturated phosphatidylchclline. J. lipid
Res. 15: 525, 1974. 13. Mangold, H. K.: Thin-layer chromatography of lipids, J. Am. Oil Chem. Sot. 38: 708. 1971. 14. Ogawa, Y., Okamoto. T.. &kuda, M.. Hiratani, Y., and Ogawa, J.: Estimation of surfactant lecirhin in amniotic Huid and prediction of respirator-y distress
CURBELO,
DOROTHY PHILIP
BERLIN M.
M.D.* GAIL,
FARRELL,
Bethesda, Maryland,
PH.D.
M.D.,
Washington,
PH.D.
D. C., and Madison,
Wisconsin
Although increased concentrations of total lecithin in amniotic fluid allow prenatal assessment of fetal lung maturation, it has become clear that routine use of the US index may lead to a substantial number of inaccurate predictions. Since disaturated lecithin (DL) is a more specific marker of pulmonary surfactant than total lecithin, we developed a convenient method for measuring this phospholipid in amniotic fluid, and then evaluated its level in pregnant rhesus monkeys of 120 to 163 days of gestation. The method involves osmic acid destruction of unsaturated lipids, chromatographic isolation of disaturated lecithin, and quantitatiin by phosphorus assay. It can be performed in approximately 5 hours on 4 ml. of amniotic fluid and yields 67 2 3 per cent average recovery of added 14C-dipalmitoyl lecithin. The results of analyzing 36 rhesus amniotic fluid specimens showed that disaturated lecithin and the disaturated lecithinlsphingomyelin ratio (DUS) increase sharply after 150 days of gestation, consistent with the pattern of lung maturation in this species. We conclude that dlsaturated lecithin can be readily quantitated in primate amniotic fluid and that its concentration, the DUS ratio, and percentage of disaturated lecithin are potentially useful indices of fetal lung maturity for the clinical laboratory. (AM. J. OBSTET. GYNECOL. 131: 764, 1976.)
SURFACE-ACTIVE components of the lung are primarily phospholipids. It has been shown that the most abundant of these are lecithins (3-sn-phosphatidylcholine species). In the lung, because of variations in fatty acid composition, there are several types of lecithin. Total lecithin, however, is not surface active, and only the disaturated fraction displays this property, particularly dipalmitoyl lecithin (DPL). Data gathered from several studies, as reviewed by Farrell and Avery,’ have shown that the surface-active material
THE
From the Neonatal and Pediatric Medicine Branch, National Institute of Child Health and Human Deuelofmumt, National Institutes of Health, the Deparknts of Medicine and Pediatrics, George Washiwton Uniuer&. and the Deeartment of Pediatrics, Unive&y of Wi5scim.s~~. Received for publication
August
Revised December
5, 1977.
Accepted January
5, 1978.
3, 1977.
Philip M. Farrell, Department oj Pediatrics, University of Wisconsin, 1300 University Reprint
Ave.,
requests: Dr.
Madison,
*Fogarty National
764
Wisconsin
53706.
International Fellow, 1976-l Institutes
of Health.
977, at the
of the fetal lung reaches amniotic fluid in late gestation, leading to an increase in the concentration of lecithin. Consequently, assessment of fetal lung maturation can be carried out by analyzing lecithin concentrations in amniotic fluid, a measurement which at present is usually performed in clinical laboratories by determining the lecithin/sphingomyelin (L/S) ratio described by Gluck, Kulovich, and Bore?; since the sphingomyelin concentration does not change during gestation, it is used in the L/S ratio method as an “internal standard” or reference base. Although the L/S procedure has proved reliable for identifying lung maturity in normal pregnancies, several investigators 3, 4 have reported that both falsepositive (“mature”) and false-negative (“immature”) L/S ratios may occur, particularly in complicated pregnancies. According to Morrison and associates,4 falsenegative L/S ratios can increase fetal mortality rates due to the fact that in distressed pregnancies, delivery tends to be delayed as the obstetrician waits for a higher L/S ratio on a repeat amniocentesis. Even though disaturated lecithin is a more specific marker of pulmonary surfactant than total lecithin,’ procedures for measuring this fraction in the past have 0002-9378/78/07131-0764$00.60/O
0
1978
The C. V. Mosby
Co.
Volume Number
13 I 7
been tedious, extremely time consuming, and not applicable to the clinical laboratory. These considerations led to the present study, in which we report a convenient approach for the measurement of disaturated lecithin in primate amniotic fluid, based on ada’ptation of the method described by Mason, Nellebogen, and Clements.” The goals of this study, therefore, were: (1) to devise a method which would permit relatively rapid and reproducible quantitation of disaturated lecithin in amniotic fluid, (2) to delineate the normal relationship of amniotic fluid disaturated lecithin concentradons to gestational age, and (3) to determine if disaturated lecithin in amniotic fluid could possibly serve as an index of fetal lung maturity in uncomplicated pregnancies. Because of similarities between the rhesus monkey (Macaca mulatta) and the human being with respect to pulmonary surfactant and the process of fetal lung development,6H and in view of our access to normal rhesus pregnancies, we focused on this animal model of primate lung development.
Materials and methods Thirty-six pregnant rhesus monkeys from the primate colony of the National Institutes of Health, with accurately known gestational ages (? 1 day), were used for these studies. Amniocenteses were performed on animals under ketamine anesthesia from 120 to 163 days of gestation (term = 164 + 3 days). The amniotic fluid was immediately centrifuged at 400 x g for 5 minutes and the supernatant fractions were stored at -20” C. until analyses could be performed (usually in less than 1 week). Under these conditions of storage, we did not detect any loss of phospholipids based on measurement of total lecithin and monitoring of the recovery of added ‘“C-dipalmitoyl Iecithin (14C-DPL). Samples contaminated with blood or meconium were discarded. Three samples of human amniotic fluid with known L/S ratios were also obtained from an affiliated hospital and were processed similarly. Lipids from 4 to 8 ml.* of amniotic fluid were extracted by means of the methods of Bligh and Dyer9 and Lindback, Frantz, and Graven.‘O Chloroform: methanol (1: 2) in a volume of 15 ml. per 4 ml. of *For pregnancies of >145 niotic fluid always sufficed
days of gestation, 4 ml. of amfor the measurement of total lecithin, disaturated lecithin, and sphingomyelin. In animals of 120 to 145 days of gestation, however, where lower amniotic fluid lipid levels reflecting pulmonary immaturity were found, 8 ml. were required. In practice, we found it convenient to extract two amniotic fluid samples of 4 ml. each when analyzing specimens from presumably immature pregnancies. After the initial processing steps described above, the two chloroform layers were pooled just prior to weighing for determination of the total lipids present in 8 ml. of amniotic fluid.
Disaturated
lecithin
as index
of lung maturity
765
amniotic fluid, and I%-dipalmitoyl lecithin (Applied Sciences, State College, Pennsylvania). a> an internal standard, were added to the samples. Thev were mixed vigorously for 5 minutes and then passed through a scintered glass filter with ultrapure nitrogen under positive pressure to expedite the filtration. Chloroform (5 ml.) was added through the filter and .5 ml. of distilled water were added to the filtrate to establish hydrophilic and hydrophobic phases. The samples were then centrifuged at 400 x g for 5 minutes, and the lower chloroform layer was removed and taken to dryness under nitrogen in a 40” C. water b,tth. The dry total lipid fraction was carefully weighed and dissolved in chloroform : methanol (2 : 1), and small aliquots were spotted on activated silica G HR thin-layer chromatography (TLC) plates (Brinkman Instruments, Westbury, New York). The plates were developed in chloroform : methanol : water (60 : 35 : 5). and ;I solution of bromthymol blue* was used to detect the lipid spots after chromatography. The lecithin and sphingomyelin spots were scraped from the plates and analyzed for phosphorus” or radioactivity. To determine the latter, the silica gel samples were placed in scintillation vials containing 10 ml. Aquasol; the vials were then counted in a Beckman LS-255 scintillation counter for 10 minutes. For the determination of disaturated lecithin, we used the method of Mason and associates” with minor modifications, after examining other methods described in the literature.‘*, I3 Aliquots of amniotic fluid lipid extract, obtained as described previously and representing approximately 1 mg. of rota1 lipid, were treated with 0.5 ml. of osmium tetroxide in carbon tetrachloride (100 mg. per milliliter) for 15 minutes. The lipids were then dried under nitrogen, dissolved in 0.5 ml. of chloroform : methanol (20: I), and added to columns containing 0.8 Gm. of neutral alumina (100 to 200 mesh, Bio-Rad Laboratories, Richmond, California). Neutral lipids were eluted first with 10 ml. of chloroform : methanol (20: I), and a disaturated lecithin fraction was then eluted with 5 ml. of chloroform : methanol : 7M ammonium hydroxide (70 : 32 : 2) (v/v/v). In order to determine if lipids other than disaturated 1 lecithin were present in the second alumina column eluates, we dried these 5 ml. fractions under nitrogen and routinely applied them to TLC plates for chromatography on silica gel as described previously. Gasliquid chromatography of fatty acid methyl esters was performed on a limited number of samples to determine the fatty acid composition of the disaturated lecithin fractions obtained both from the column eluate and from the TLC plates.
766
Curbelo, Gail, and Farrell
August I, 1978 Am. J. Obstet. C;ynerol
Cls Cl7 (internal standard)
Fig. 1. Gas chromatographic analysis of fatty acid methyl esters prepared from a lipid extract of amniotic fluid obtained from a pregnant rhesus monkey at 162 days of gestation. The sample was processed as described in the text, with osmic acid treatment, column chromatography, and thinlayer chromatography. Carbon numbers designate the fatty acids detected: C14 = myristic, Cl5 = pentadecanoic, Cls = palmitic, C,, = heptadecanoic (not present in amniotic fluid, but added as an internal standard), Cl8 = stearic, C,,:, = oleic, C18:*= linoleic. Calculations revealed that 98 per cent of the fatty acids present were saturated and that palmitate accounted for 91 per cent of the total fatty acids in this sample. The line represents an actual tracing from the gas chromatograph, with the time of injection (INJ) indicated by the arrow.
From data obtained by phosphorus analyses and measurement of r4C-DPL recovery, the following were calculated: total lecithin, sphingomyelin, the molar L/S ratio (the ratio of the absolute amounts of the two phospholipids), the percentage of the disaturated species in total lecithin, and the disaturated lecithin/ sphingomyelin (DLIS) ratio. The t test for unpaired data was used for statistical analysis.
Results The method of Mason and associates” was found to be applicable to amniotic fluid phospholipid analysis with minor modifications as described in this report. The procedure proved convenient and relativeiy rapid compared to other methods.r2* I3 Thus, it was possible to complete the analysis of total lecithin, sphingomyelin, disaturated lecithin, and the derived ratios in approxrmately 5 hours. The method also showed satisfactory reproducibility. The over-all recovery of added r4C-dipalmitoyl lecithin was 67 + 3 per cent (mean & S.E. for 34 analyses). Whatever losses of lecithin OCcured during the procedure seemed to be confined
primarily to the osmic acid treatment and column chromatographic steps. Analysis by thin-layer chromatography of the second eluate from the alumina column, i.e., the “disaturated lecithin fraction,” revealed only one lipid spot, and this migrated identically with standard dipalmitolyl lecithin. Furthermore, 99 per cent of the phosphorus and radioacnvity detected in each lane of the TLC plates was recovered with the disaturated lecithin spots. No radioactivity or phosphorus was detected in the neutral lipid fractions eiuted from the column. Gas-Jiquid chromatographic examination of selected column eluates and “disaturated lecithin spots” from TLC plates revealed these fractions to be highly saturated with respect to fatty acid composition. Representative results obtained upon analysis of an amniotic fluid extract from a rhesus monkey of 162 days of gestation are shown in Fig. 1. This experiment revealed 98 per cent of the fatty acids to be saturated, with palmitate (C,,) accounting for 91 per cent of the methyl esters. As shown in Fig. 2 and Table I, total and disaturated
Volume
Disaturated lecithin as index of lung maturity
13 I
767
Number 7
.
.
.
TERhn l Lf
t
,.
130
120
d 140’
l
lb0
160
1
GESTATlONALAGEfdays)
Fig. 2. Total lecithin and disaturated related to gestational age. Table
I. Phospholipids
Gestationd age
of rhesus amniotic
lecithin concentrations
fluid as related
Sphingomyelin (mg. I1 00 ml.)
130-139
0.274 k 0.041 (7) 0.670 +- 0.123
0.390 + 0.044 (7) 0.515 -r- 0.078
140-149
1.063 +- 0.354
(8)
(8)
150-159
2.912 -+ 0.862 (9) 5.139 k 0.853 (3)
0.651 + 0.060 (9) 0.431 k 0.093 (3)
120-129
(8)
2160
to gestational
age*
Disatwated Total lecithin (mg. I1 00 ml.)
(days)
in rhesus monkey amniotic fluid as
03) 0.494 2 0.037
Mgll
00 ml.
0.087 f 0.008 (7) 0.223 2 0.044
(6) 0.535 2 0.179 (8) 1.399 + 0.456 (9) 3.104 * 0.579 (3)
lecithin ‘r %
of total lecithin 33 + 3.5 (7) 30 t 2.5 (8) 53 c 5.0
03) 46 + 2.1 (9) 60 2 1.53 (3)
DlsatUR&d LIS ratio
0.23 r 0.003 (7) 0.38 2 0.04
(6) 1,06 + 0.31 (8) 1.95 rfr 0.49 (9) 8.13 t 2.26 (3)
*Means and standard errors are shown with the number of samples enclosed in parentheses. ICorrected for loss of disaturated lecithin, as determined with W-dipalmitoyl lecithin. lecithin concentrations were found to increase gradtially from 120 days until 150 days. After 150 days of gestation, a significant date in the development of the fetal rhesus monkey lung according to our previous work,s an especially sharp increase was noted in the concentratioin of the total lecithin and disaturated lecithin fractions. Disaturated lecithin reached a value 16-fold higher at 150 to 159 days as compared to the interval of 120 to 129 days of gestation. The mean disaturated lecithin concentration was 0.33 mg. per 100
ml. before 150 days and 1.8 mg. per 100 ml. after 150 days (p < 0.001). The concentration of sphingomyelin, in contrast, did not show significant changes from 120 to 160 days of gestation (Table I). Reflecting these changes in phospholipid concentrations, the ratio of lecithin to sphingomyeiin was found to increase in late gestation, especially after 150 days (Fig. 2). The Di/S ratio showed a similar surge at that point, after exhibiting a more gradual rise between 140 and 150 days of gestation. Thus, the somewhat
768
Curbelo, Gail, and Farrell
August Am. J. Obstet.
’
I
--------5-8
.
l
1. 1978 Gynecol
.
’
+--xi
:. .g**..
1 I
130
I
140
150
160 GESTATIONAL
Fig. 3. Phospholipid
AGE (days)
ratios in rhesus monkey amniotic fluid as related to gestational age.
abrupt increase in the DL/S ratio at 150 days led to a significantly higher (p < 0.01) mean of 3.69 after 150 days, as compared to a value of 0.67 prior to 150 days of gestation. As expected from our observation that disaturated lecithin accounts for approximately 50 per cent of the total lecithin (Table I), and our previous finding that fetal lung maturation is evident in rhesus monkeys after 150 days of gestation when the L/S ratio is greater than 2.0, 7* 8 it was found that the apparent “critical value” of the DL/S ratio is unity. When the individual values for analysis of amniotic fluid were examined as a function of gestational age, only one sample in 11 was found to have a DL/S ratio of less than 1 .O beyond 150 days. The percentage of total lecithin which is disaturated in amniotic fluid was found to increase slightly earlier than the disaturated lecithin concentration or the DL/S ratio. As shown in Table I, the percentage of disaturated lecithin was found to increase somewhat sharply during the interval of 140 to 149 days and remained high with little variation until term was reached. The average value for the percentage of disaturated lecithin was significantly higher between 140 to 149 days of gestation, as compared to the period before 140 days of gestation (p < 0.001). Based upon analysis of a limited number of human amniotic fluid samples, it appears that the procedure described in this report can be applied to such specimens without any additional technical difficulties being
encountered. The concentrations of disaturated lecithin in the three samples obtained from pregnant women of 34 to 37 weeks of gestation were similar to values found in rhesus monkeys of comparabIe gestation (0.844, 2.08, and 2.50 mg. per 100 ml.). The recoveries of ‘%-DPL for these samples were 64,66, and 79 per cent, respectively.
Comment Because of the complex and time-consuming nature of the previous methods used for measurement of disaturated lecithin,‘*’ I3 this important component of pulmonary surfactant has been only estimated in earlier work with amniotic fluid. Such measurements were made through the use of gas-liquid chromatography.‘* This approach does not provide direct information on the concentration of disaturated lecithin, but has revealed that palmitic acid is the predominant fatty acid in amniotic fluid lecithin during late gestation, after fetal pulmonary maturity is attained.” Thus, we felt that it was appropriate to pursue more convenient analytical techniques in an attempt to devise a method for routinely measuring this constituent as an index of fetal lung maturation. The method described in this paper allows measurement of disaturated lecithin directly, and the data reported herein are, to our knowledge, the first such values published for primate amniotic fluid. It was found that with adaptation of the method of Mason and associates,” disaturated lecithin
Volume Number
Disaturated lecithin as index of lung maturity
13 1 7
could be conveniently and reproducibly measured in amniotic fluid specimens from rhesus monkeys. The increased phospholipid levels observed in this study (Fig. 3) during late gestation agree with previous reports that in normal human and rhesus pregnancies, changes in lecithin concentration and the L/S ratio reflect gestational age and fetal lung development.‘* *, 6, ’ In this work, no amniotic fluid specimens were found with L/S values less than 2.0 after 150 days of gestation. The abrupt elevation of lecithin, disaturated lecithin, the L/S ratio, and the DL/S ratio at 150 days correlates with the fact that after this date, newborn monkeys do not develop symptoms of the respiratory distress syndrome. Our results also agree with those of Kotas and associates,* who evaluated physiologic indices of fetal lung development in rhesus monkeys and recently reported their findings on the ontogeny of pulmonary surfactant in this species. They found that functional alveolar surfactant, minimal surface tension, and total lecithin production in lung slices all increased significantly between 140 and 150 days of gestation. All rhesus fetuses examined after 150 days of gestation had mature lungs, whereas two of five animals studied between 140 and 149 days of gestation showed the presence of surfactant. Thus, the timing of physiologic changes in the fetal rhesus lung, as observed by Kotas and associates,’ and the pattern of increased disaturated lecithin con-
769
centrations in amniotic fluid, as reported herein, show good concordance. We therefore conclude that the rises in amniotic fluid disaturated lecithin occurs simultaneously with the attainment of fetal lung maturation. These correlations suggest that the method used in this study to measure disaturated lecithin yirida data that can serve as an index of fetal primate lung maturity. As we have shown that the pr0cedut.e of Mason and associates” with minor modifications is applicable to human amniotic fluid, we believe that it will be worthwhile to determine if the concentration of disaturated lecithin and the DLiS ratio are better predictors of neonatal respiratory outcome than the other methods now available. If so. measurement of disaturated lecithin and the DLiS ratio could prove to be a valuable means of predicting fetal lung maturity in the clinical setting. This approach may be especially helpful in assessing lung maturation in complicared gestations. We thank Dr. Ron Chanderbhan of (George Washington University for performing the gas-lipid chromatrography experiments, Dr. Ronald Chez of the Pregnancy Research Branch, NICHD, f‘cjr providing some of the rhesus amniotic fluid samples analyzed in this project, and Mr. Anthony J. i2dams of the Neonatal and Pediatric Medicine Branch, NICHD, for assisting us in the collection of specimc-*ns by percutaneous amniocentesis.
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1.
Farrell, P. M., and Avery,
M. E.: Hyaline membrane disease, Am. Rev. Resp. Dis. 111: 657, 1975. 2. Gluck, L., Kulovich, M., and Borer, R.: Estimates of fetal lung maturity, Clin. Perinatol. 1: 125, 1974. 3. Donald, 1. R., and Freeman, .R. K.: Clinical experience with the amniotic fluid L/S ratio, AM. J. OBSTET. GYNECOL. 115: 547, 1973. 4. Morrison, J. C., Whybrew, W. D., Bucovacz, E. T., Wiser, W. L., and Fisch, S. A.: The lecithin/sphingomyelin ratio in cases associated with fetomaternal disease, AM. J. OBSTET. GYNECOL. 127: 363, 1977. 5. Mason, R., Nellebogen, J., and Clements, J.: Isolation of disaturated phosphatidylcholine with osmium tetroxide. J. Lipid Res. 17: 281, 1976. 6. Gluck, L., Chez, R. A., Kulovich, M., Hutchinson, D. L., and Niemann, W. H.: Comparison of phospholipid indicators of fetal lung maturity in amniotic fluid of monkey (Mncaca mulatta) and baboon (Pupio papio), AM. J. OBSTET. GYNECOL. 120: 524. 1974. 7. Epstein, M. F., Fa&l, P. M., and Chez, R. A.: Fetal lung lecithin metabolism and the amniotic fluid L/S ratio in rhesus monkey gestations, AM. J. OBSTET. GYNECOL. 125: 545. 1976.
8.
Kotas, R. V., Farrell, P. M., Ulane, R. E.. and Chez.
R. A.: Fetal rhesus monkey lung development: Lobar differences and discordances between stability and distensibility. J. Appl. Physiol. 43: 1, 1977. 9. Bligh, E. G., and Dyer, W. J.: A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37: 911, 1959. 10. Lindback, T., Frantz, T.. and Graven, S. N.: Phospholipids in amniotic fluid, Acta Obstet. Gvnecol. &and. 53: 219. 1974. 11. Morrison, W. R.: A fast, simple, and reliable method for the microdetermination of phosphorus in biological materials, Anal. Biochem. 7: 218, 1964. 13 --. Shimojo, T., Abe, M., and Ohta, M.: A method for determinations of saturated phosphatidylchclline. J. lipid
Res. 15: 525, 1974. 13. Mangold, H. K.: Thin-layer chromatography of lipids, J. Am. Oil Chem. Sot. 38: 708. 1971. 14. Ogawa, Y., Okamoto. T.. &kuda, M.. Hiratani, Y., and Ogawa, J.: Estimation of surfactant lecirhin in amniotic Huid and prediction of respirator-y distress