ANALYTICAL RIOCHEMISTRY 77, ?.(o-36: c 1977)

Quantification of Dipalmitoyl Lecithin in Lung Wash and Extracts1

SHREE MULAY, FIROUZ KHAMSI, AND SAMUEL SOLOMON

Departments of Biochemistry and Experimental Medicine, McGill University and the University Clinic, Royal Victoria Hospital, Montreal, Canada

Received July 15, 1976; accepted September 24, 1976

A quantitative method for the measurement of dipalmitoyl lecithin in lung wash and extracts has been described. A known amount of tritiated dipalmitoyl lecithin is added to lung wash or tissues prior to homogenization and extraction. The lipid extract is subjected to a sequence of chromatographic separations, and after each separation the amount of radioactivity and phosphorus is determined. Specific activity is calculated in terms of disintegrations per minute per micromole of phosphorus. Constant specific activity is achieved in the iast two thin-layer separations. The losses incurred in the procedure are determined by the recovery of radioactivity in the final purification step; the total amount of dipalmitoyl lecithin present in the pulmonary extract is calculated after correcting for losses. The accuracy, sensitivity, and precision of the method have been evaluated by using adult and fetal lung extracts as well as fetal lung wash. The sensitivity was found to be 0.24 mg and the coefficient of variance ranged between 3.0 and 6.9% with an average of 5%. Changes in dipalmitoyl lecithin levels as well as wet lung weight, dry lung weight, protein, and DNA have been reported.

In the last decade the development and maturation of the fetal lung has been studied extensively in relation to the synthesis of surfactant by alveolar lining (1). Dipalmitoyl lecithin (DPL) has been considered to be the principal surface-active phospholipid in mammalian lungs (2). Until now, separation of lecithin with specific fatty acids has not been feasible and evidence for the presence of DPL comes primarily from the high percentage of palmitic acid found in the lecithin fraction isolated from the lung (3,4). Gluck et al. (5) studied the composition of the fatty acids on the first and second carbons of the glycerol molecule of surface-active and non-surface-active lecithins from the alveolar wash and in the extracts from residual lungs after wash in the late-gestation fetal rabbit and the newborn, the young, and the adult rabbit. They showed that in the acetone- precipitable fraction of the alveolar wash lecithins, DPL was the most abundant surface-active component. An intermediate in the methylation pathway, phosphatidyl dimethylethanolamine, another surface-active lecithin with a l- 16:0/2- 14:0 configuration was also identified (5). How-

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350 CopyrIght 0 1977 by Academic Resr. Inc. All rights of reproduction in any form reserved. ISSN OGil3-2697

QUANTIFICATION OF DIPALMITOYL LECITHIN 351

TABLE 1

SYSTEMS USED FOR THE SEPARATION OF PHOSPHOLIPIDS BY THIN-LAYER

CHROMATOGRAPHY

sys-

tern Type of plate used and conditions

of development Composition of the system References

0.5mm-thick silica gel G plate, Hexane:ethyl ether: activated for 1 hr at 110°C and glacial acetic acid, developed at room temperature 70:30: I

OS-mm-thick silica gel HR plate prepared with a 1 mM Na2C03 solution, and activated for 4 hr at 140°C; the plates chro- matographed at room temperature

Chlorofomnmethanol: Skipski ef al. glacial acetic acid:water, (11) 100:60:16:8

OS-mm-thick silica gel H plate prepared with a 10% AgNO, solution, dried at room tempera- ture for 24 hr, and then activated for 24 hr at 180°C; plates chromatographed at room temperature

Chloroformmethanol: water, 65:25:4

Same as those used in System 1; plates chromatographed at 0-W; system used for separation of mercury adducts

Chloroform:methanol: water, 70:30:4

-

Arvidson (12)

Blank et al.

(13)

ever, in all the earlier studies, direct measurement of DPL was not made. In order to study hormonal control of the synthesis and release of surfactant in the developing lung, it is necessary to measure the amount of DPL present in the alveolar wash and the lung parenchyma after wash in control and hormone-treated animals. In this publication, we report a method for the quantitative measurement of DPL in lung.

MATERIALS

Chemicals. 1-Palmitoyl-2-lysolecithin (LL), 1,2-dipalmitoyl lecithin (DPL), and other phospholipid standards as well as fatty acids were purchased from Sedary Research Laboratories, London, Ontario. The purity of DPL and LL was assessed on thin-layer chromatography (tic) in System 2 (Table l), and each compound was found to migrate as a single spot. Phospholipase AZ (Vipera russelli), ATP, and coenzyme A were purchased from Sigma Chemical Co. Silica gel G, silica gel H, and silica gel HR were purchased from Brinkmann Instruments, Rexdale, Ontario. DEAE-cellulose (Cellex D) and Dowex AG 5OW-X8 (200-400 mesh, hydrogen form) were purchased from Bio-Rad Laboratories,

352 MULAY, KHAMSI AND SOLOMON

Richmond, California. All other chemicals were purchased from Fisher Scientific Co., Montreal. All organic solvents were purchased redistilled from A & C Chemical Co., Montreal, and used without further purification.

[9,10-3H]Palmitic acid (220 mCi/mmol) was purchased from New England Nuclear Corp. Its radiochemical purity was established by mixing an aliquot with authentic standard, followed by chromatography of the mixture on tic in System 1 (Table 1). A single radioactive peak corresponding to the mobility of the authentic standard was observed.

Experimental animals. Pregnant New Zealand does, timed for gesta- tion, were purchased from Canadian Breeding Farm and Laboratories, St. Constant, Quebec.

METHODS

Synthesis of t3HJDPL. Tritiated DPL was prepared by the incubation of rat liver microsomes with LL and [9,10-3H]palmitic acid using the method described by Lands and Merkl (6). The reaction was terminated by the addition of a methanol:chloroform mixture (2:l). The incubation medium was extracted as described by Bligh a&Dyer (7). The residue of the chloroform-rich phase containing the phospholipids was chromato- graphed twice on tic in System 2 (Table l), The peak corresponding to authentic DPL was eluted and rechromatographed in System 2, and a single radioactive peak was observed. The specific activity of the tritiated DPL was determined and was found to be 46-67 mCi/mmol. The purity of the tritiated DPL was assessed in the following ways: (a) Standard DPL, 3.12 mg, was mixed with 43,000 cpm of the labeled sub- stance; the mixture was subjected to argentation chromatography in System 3 (Table 1) and then, after mercuric acetate adduct formation, in System 4 (Table 1). In both systems a single radioactive peak was observed, the mobility of which coincided with the uv-positive band obtained after spraying the plates with rhodamine 6G. No significant change in specific activity was observed. It was evident from these findings that the synthesized [3H]DPL fraction contained a single compound which was homogenous with the authentic DPL standard. (b) Two aliquots of tritiated DPL were mixed with authentic carrier and evaporated to dryness under nitrogen in glass-stoppered tubes. The mixture was hydro- lyzed with phospholipase A, as described by Lands and Merkl (6) in one aliquot; the enzyme was omitted in the second which served as the control. The phospholipids extracted from the two tubes were chromato- graphed on tic in System 1. It was evident from the radioactive scans that tritiated DPL was hydrolyzed by phospholipase A, to give a non- radioactive LL band and a radioactive palmitic acid band, whereas, in the absence of phospholipase A*, the peak of radioactivity corresponded to DPL in mobility. It can therefore be concluded that the tritiated

QUANTIFICATION OF DIPALMITOYL LECITHIN 353

Lung wash, residual lungs after lovage or intact lungs + known

1 amount of 3H-DPL

Extraction of lipids done as described by Bligh and Dyer (7).

1 Aliquots removed for counting and phosphorus determination.

Lipids chromatogrophed on o DEAE cellulose acetate column (10)

Fraction 3aAliquots removed for counting and phosphorus

c determination.

Chromatographed on TLC in system 2.

DPL fractionqAliquots removed for counting and phosphorus

1 determination.

Chromatographed on TLC in system 3.

DPL fraction -W Aliquots removed for counting and phosphorus

i determination.

Mercury adduct formed and extracted os described by King and

Clements (15) and chromatographed in system 4.

DPL fraction+Aliquot removed for counting and phosphorus

determination.

FIG. 1. Flow chart summarizing the procedure used for the purification of pulmonary DPL.

palmitic acid was present in the carbon-2 position of the DPL molecule. (c) Carrier DPL, 0.675 mg, was mixed with 79,800 cpm of tritiated DPL, and the mixture was subjected to mild alkaline deacylation as described by Kates (8). Aliquots of the fraction containing the methyl esters were subjected to gas chromatography and simultaneous recording of radio- activity. A single peak was observed by gas chromatography; this corresponded to a single radioactive peak which has the same retention time as authentic methyl palmitate.

We were therefore able to demonstrate that the tritiated DPL syn- thesized from 1-palmitoyl-2-lysolecithin and [9,10-3H]palmitic acid was pure and had labeled palmitic acid in the carbon-2 position.

Extraction of phospholipids from alveolar wash and lungs. Does were locally anesthetized with xylocaine, and fetuses were removed by laparotomy. Fetuses were decapitated before they could breathe. Intact lungs were removed and the lungs were lavaged with saline as described by Gluck et al. (9). The lung washings from each litter were pooled and then centrifuged at 3000 rpm for 10 min to remove red blood cells. Adult lungs were washed in a similar manner. Intact and residual lungs were weighed, then homogenized, and extracted as described by Bligh and Dyer (7). The alveolar wash was also extracted with chloroform: methanol (2: I), and the proportion of chloroform:methanol:water was adjusted to give a final ratio of 2:2: 1.8. The phases were allowed to separate, and the chloroform-rich phase was evaporated to dryness. The recovery of phospholipids in the extraction step was determined by the addition of a known amount of tritiated DPL prior to homogenization of intact and residual lungs and extraction of lung washes. In each

354 MULAY, KHAMSI AND SOLOMON

instance the recovery of [3H]DPL was greater than 95%. The lavage of lungs and homogenization and extraction of tissues were carried out in a cold room or by placing samples on ice in order to minimize degrada- tion of phospholipids. The lipid extracts were stored at - 15°C in an atmosphere of nitrogen pending further analysis.

DEAE-cellulose (acetate) column chromatography. Lipid extracts were chromatographed on DEAE-cellulose (acetate) columns as an initial purification step as described by Gluck et al. (10). We substituted small columns with Teflon stoppers for Pasteur pipets to facilitate the running of 12 columns at one time. The [3H]DPL was eluted in the third solvent mixture (chloroform:methanol, 7:3). Aliquots of this fraction were removed for determination of radioactivity and phosphorus content. The residue from this fraction was subjected to thin-layer chromatography. The sequence of these chromatographic steps is outlined in Fig. 1.

Thin-layer chromatography. Silica gel G and silica gel H were used for preparation of the plates. Plates, 5 x 20 cm, were coated with silica gel as described in Table 1. These plates could be scanned for radioactivity on Packard 7200 scanners. The composition of the solvent systems used is also listed in Table 1.

The phospholipids were detected by spraying the plates with 0.2% 4’,5’-dibromofluorecein in ethanol and observing the bands under uv light. Rhodamine 6G was found to be a better reagent for detection of phospholipids after argentation chromatography. In some instances the plates were sprayed with 50% H,SO, in ethanol, and then charred at 250°C for IO-15 min.

Elution of phospholipids from silica gel was done with a mixture of chloroform:methanol (2: 1). Silica gel bands corresponding to the radio- active peak were scraped in small sintered-glass funnels of medium porosity. Elution of phospholipids after argentation chromatography was done on Dowex AG SOW-X8 columns as described by Kyriakides and Balint (14).

Measurement of radioactivity. Tritium was counted with a Packard Model 3003, and the scintillation fluid mixture was that recommended by King and Clements (15).

Phosphorus determination. The amount of phosphorus present was determined by the method described by Bartlett (16). Standard curves were prepared with DPL, and standards were included in each assay to control variations. The ultramicromethod for measurement of phos- phorus as described by Bartlett (16) was used for extracts of fetal lung washes. The specific activity of the DPL fraction throughout the procedure (Fig. 1) was calculated as disintegrations per minute per micromole of phosphorus. The losses incurred were estimated from the disintegrations per minute in the final purification step, and the total amount of DPL was calculated from these data.

Protein and DNA determination. The protein content of lungs was

QUANTIFICATION OF DIPALMITOYL LECITHIN 355

4

7

a

J

es

2000 ISO

FIG. 2. Gas chromatographic scan of methyl esters obtained from the purified DPL fraction from lung extracts.

measured by the method of Lowry et al. (17), and DNA content was measured by the diphenylamine method (18).

RESULTS AND DISCUSSION

Tritiated DPL was used as an internal marker for assessment of procedural losses as well as for the determination of specific activity used as a criterion for the purity of the DPL at various stages of chromatog- raphy. The suitability of the method was assessed by considering the following factors.

Specijcity

The sequence of chromatographic steps described in Methods (Fig. 1) initially separates the acidic and nonacidic phospholipids on DEAE- cellulose acetate columns. The basic tic plates used next separate the nonacidic phospholipids on the basis of the phosphatidic group as well

1 Methyl myristate 2 Penta decanoic acid-

methyl ester 3 Unknown 4 Methyl palmitate 5 Ethyl palmitate 6 Hepta decanoic rcid-

methyl ether 7 Methyl oleate 6 Methyl stearate

I

I; 2

5. 0

356 MULAY, KHAMSI AND SOLOMON

TABLE 2

CHANGES IN SPECIFIC ACTIVITY WHEN FETAL LUNG HOMOGENATES CONTAINING KNOWN AMOUNTS OF CARRIER DPL ARE SUBJECTED TO THE

PURIFICATION PROCEDURE

Specific activity (dpmlpmol)” of sample containing

Chromatographic step 1 pmol of DPL 2 pmol of DPL 3 pmol of DPL

Before DEAE-cellulose (acetate) column 137,300 71,920 49,360

After DEAE-cellulose (acetate) column 146,960 77,320 50,930

a- 1 132,660 70,270 45,920 tic-2 138,550 76,260 51,770 k-3 139,960b 72,540 50,610 Calculated specific activity 151,030 75,5 10 50,340

a Specific activity is expressed as disintegrations per minute per micromole of phosphorus. b Amounts of DPL were calculated from the specific activities obtained after k-3. These

were found to be 1.079, 2.082, and 2.984 pmol, respectively.

as the presence of mono- and diglycerides. Argentation chromatography separates phospholipids on the basis of the number of ethylenic bonds in the fatty acid, although separation of the lecithin with monenoic and saturated fatty acids is incomplete. Chromatography of the mercury- adduct product of phospholipids brings about a separation of lecithin containing saturated fatty acids from that containing monoenoic fatty acids. Very small changes in the specific activity (<lo%) of the DPL fraction obtained from adult rabbit lungs in the last two chromatographic steps suggested that it was essentially pure. Its purity was confirmed by subjecting the DPL-containing fraction to mild alkaline deacylation (8). An aliquot containing the methyl ester of the fatty acid was subjected to gas-mass analysis (LKB-9000), and a separate aliquot was taken for counting. The scan obtained after gas chromatography is shown in Fig. 2. Eight different methyl esters were identified on the basis of their mass fragmentation patterns. However, quantitatively, 95% of the methyl esters was contained in the methyl palmitate peak. The specific activity of the methyl palmitate differed only by 4% from the calculated value. This finding demonstrated that the sequence of chromatographic steps outlined in the method (Fig. 1) successfully separated DPL from other closely related phospholipids in adult lung.

Reproducibility and Accuracy’

(1) To equal aliquots of fetal lung homogenates previously freed from lipids were added approximately 150,000 dpm of t3H]DPL as well as 1, 2, and 3 pmol of carrier DPL. The changes in specific activity

QUANTIFICATION OF DIPALMITOYL LECITHIN 357

6.0-

~=I.89 +0.92x

5.0 -

1.0 0

1 I I I 1.0 2.0 3.0 4.0

+mg OPL cldded

FIG. 3. A plot of the amount of the DPL added to aliquots of lung extract versus the amount of DPL assayed.

at each stage of purification are shown in Table 2. It is evident from these data that the changes in specific activity are within 5-10% of the calculated value, with the exception of the specific activity obtained after tic-1 for the 1-pmol sample which is 12%. The regression equation is y = 0.17 + 0.95x, and the correlation coefficient is 1.018.

(2) Two adult lungs were excised, freed of tracheal tissue, homogenized, and extracted. The extract was divided into 40 equal aliquots, and the samples were frozen at -15°C until processed. The amount of DPL present in each aliquot was determined by assaying eight of the 40 samples. To five replicate samples were added 0.25, 0.50, 1.0, 2.0, and 4.0 mg of DPL, and the amount present was assayed. The plot of amount of DPL added against amount of DPL assayed is shown in Fig. 3. The coefficient of variance of the mean values ranges from 6.9% for samples to which 0.25 mg of DPL was added to 3.5% for samples to which 4.0 mg of DPL were added, with the average value being 5%. This was found to be consistent over a large range of DPL assayed. The regression equation of the straight line obtained is y = 1.89 + 0.92~ and the cor- relation coefficient is r = 1.001. The fiducial limits at P = 0.01 are shown

358 MULAY, KHAMSI AND SOLOMON

TABLE 3

PURIFICATION OF FETAL LUNG WASHY

Sample DEAE column

Specific activity (dprn/pmol)

tic-l k-2 t1c-3 Corrected

(pm01 of DPL)b

1 139,350 165,360 230,260 225,340 0.46 2 I53,500 160,750 211,940 219,230 0.47 3 145,520 153.460 235,660 228,330 0.45 4 130,750 120,040 231,150 236,460 0.44 5 133,120 141,320 161,930 194,550 -

D Fetal lung wash from 29 fetuses of gestational ages between 27 and 29 days, pooled, extracted, and then divided into six parts. Each fraction contains 0.79 pmol of phosphorus and 103,510 dpm of [3H]DPL.

b DPL = 0.46 0.01 k (SD) pmol; DPL = 0.33 0.01 -C (SD) mg.

as dotted lines in Fig. 3. The precision was also calculated and found to be 0.04 mg when no DPL was added to the samples.

(3) Two adult lungs were excised intact, then lavaged with isotonic saline, The residual lungs were freed of tracheal tissue, homogenized, and extracted as described before. The extract was divided into 12 aliquots. In a set of six samples, an additional step of acetone precipitation (5) was introduced into the procedure after the DEAE-cellulose (acetate) columns. It was evident from the changes in specific activities that there was approximately a twofold purification after the acetone- precipitation step, suggesting that this precipitate contains phospholipids other than DPL and probably cannot be used as a measure of DPL in the rabbit adult lung. The amount of DPL in the two sets of extracts was found to be 13.32 k 0.32 and 12.94 + 0.38 mg. Statistically, the difference between the two means was not significant, and the coefficients of variance were 2.4 and 3.0%, respectively, which were lower than values obtained for smaller DPL samples. This is not surprising since the accuracy of measurement increases with larger sample sizes. The reproducibility and accuracy of the method outlined was assessed to be satisfactory for routine measurements of DPL.

Sensitivity

A Student’s t test was performed to compare the values obtained when no carrier DPL was added to aliquots of lung extract and when 0.25 mg of DPL was added. The difference between the two means was statistically significant at the 5% level. The sensitivity was calculated using the value obtained for precision when no carrier DPL was added to aliquots of lung extract; this was found to be 0.24 mg. The sensitivity of the method

QUANTIFICATION OF DIPALMITOYL LECITHIN 359

TABLE 4

CHANGES IN DPL LEVELS IN FETAL LUNGS DURING 27-30 DAYS OF GESTATION

Gestation (days)

DPUwet DPWdry weight weight (wk) hh)

DPL/protein (mgk)

DPL/DNA Owk)

DPL/fetus (mg/fetus)

27 (6) 2.18 s 0.12b 27.5 2 1.3 40.2 + 4.6 276.9 2 30.3 1.7 4 0.12 28 (61 2.78 -t_ 0.25 33.6 5 2.6 39.0 +- 3.1 334.5 2 23.5 2.86 2 0.16 29 (6) 3.80 -c 0.23 44.0 2 1.2 50.5 2 0.9 503.3 2 24.2 4.66 k 0.24 30 (5) 4.15 h 0.21 44.0 t 2.2 52.9 2 3.8 476.0 rt 37.9 5.19 5 0.47

n Numbers in parentheses indicate number of assays included. b Standard error of mean.

is a function of the procedural losses and the reliability of the phosphorus assay. The procedural losses when multiple chromatographic separations are used in quantitative measurements are quite high. In all the determina- tions described in the preceding sections the overall recoveries ranged from 20 to 40% with an average of 25%. The problem of the relative insensitivity of the method can be overcome by using a large sample size.

Measurement of DPL in Fetal Lung Wash

Intact lungs from 29 fetuses of gestational ages ranging from 27 to 29 days were removed and gently lavaged with isotonic saline. The lung washes were pooled and extracted as described earlier. The extract was divided into six aliquots; one sample was lost during processing. The specific activity data for the remaining five samples are shown in Table 3. The value for Sample 5 was omitted from the calculation because the amount of phosphorus obtained after tic-3 was higher than that after tic-2, and contamination of this sample was suspected. The mean for the remaining four values was found to be 0.33 k 0.01 mg. The coefficient of variance was 3.0% which was similar to the values obtained earlier.

Determination of DPL in Fetal Lung Extracts

The method described was found to be suitable for measuring DPL in adult lung tissues and fetal lung washes, which predictably contain a high proportion of the phospholipids in the DPL fraction (54.9% in adult residual lung and 58.2% in fetal lung washes). It was equally essential to evaluate the suitability of the method for fetal lung extracts as well as to establish the levels of DPL in the period crucial from the point of view of lung maturation, namely, Days 27-30. DPL measure- ments were made with lung extracts from a large number of litters obtained from pregnant does timed for gestation. Only those values where changes

360 MLJLAY, KHAMSI AND SOLOMON

- mg DPL x IO/fetus - mg DPL x IO/fetus o---o mg wet wt x lo/fetus o---o mg wet wt x lo/fetus e-a mg dry wt/fetus e-a mg dry wt/fetus 0 . . . . . . . . 0 0 . . . . . . . . 0 mg mg protein /fetus protein /fetus l . . . . . . . . . a l . . . . . . . . . a mg mg DNA/fetus DNA/fetus

28 29 30 Days

FIG. 4. Changes in DPL, wet weight, dry weight, protein, and DNA in each pair of fetal lungs between 27 and 30 days of gesfation.

in specific activity between k-2 and k-3 were less than 10% and when the methyl ester gas-mass pattern was similar to Fig. 2 were included in the results shown in Table 4. We were unable to achieve constant specific activity with lung extracts from fetuses of gestational ages less than 27 days. These results showed that there was an increase in DPL levels between Day 27 and 29 no matter which parameter was used for expressing these results. The changes in DPL, wet lung weight, dry lung weight, protein, and DNA in each pair of fetal lungs through Days 27-30 is shown in Fig. 4. A slight increase in DNA is observed throughout the 4 days. There is a significant increase in wet lung weight, dry lung weight, protein, and DPL between Day 27 and 29, but these changes are not significant between Day 27 and 30.

The method described for measurement of DPL is found to be suitable

QUANTIFICATION OF DIPALMITOYL LECITHIN 361

on the basis of accuracy, reproducibility, and specificity. The sensitivity of the method is found to be relatively unsatisfactory with small sample size, and this can be overcome by using larger aliquots of tissue extracts. The forgoing data demonstrate that it is feasible to measure DPL quan- titatively in fetal lung washes and lung extracts; they could be used as an index of maturity of fetal lungs. Furthermore, we have established the precise changes in the levels of DPL in the last 4 days of gestation in the rabbit. Currently, we are studying the effect of different hormones such as glucocorticoids, thyroxines, and prostaglandins on DPL levels in fetal lungs.

ACKNOWLEDGMENTS

The authors would like to express their appreciation to Dr. Orval Mamer for performing the gas-mass analyses and to Ms. C. Schwenter for technical assistance.

This work was supported by grants from the Medical Research Council of Canada (MT-1658) and the U. S. Public Health Service (HDO-4365).

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