Normal- and reverse-phase HPLC separations of fluorescent (NBD) lipids

ANALYTICAL BIOCHEMISTRY159, 101-108 (1986)

Normal- and Reverse-Phase HPLC Separations Fluorescent (NBD) Lipids ONA C. MARTIN Department

of Embryology,

AND RICHARD

Carnegie Institution Baltimore, Maryland

of Washington,

of

E. PAGANO 115 West University

Parkway,

21210-3301

Received August 5, 1986 We have developed two high-performance liquid chromatography methods for separating a number of fluorescent 4-nitrobenzo-2-oxa-1,3diazole (NBD) analogs of glycerolipids and sphingolipids. Samples of fluorescent lipid analogs containing NBDaminocaproyl (C6-NBD) or NBDaminododecanoyl (&NBD) acyl chains were synthesized and analyzed by the following HPLC methods. (i) An isocratic normal-phase method permitted resolution of a mixture of the 1,2(palmitoyl, C6-NBD)-analogs of triacylglycerol, diacylglycerol, phosphatidic acid, phosphatidylethanolamine, and phosphatidylcholine in less than 10 min, while a mixture of the (Cs-NBD)labeled analogs of ceramide, glucocerebroside, and sphingomyelin was separated in approximately 15 min. This method also detected various (C6-NBD)-phosphatidylcholine and -phosphatidylethanolamine molecules which differed only in their nonfluorescent acyl (oleoyl or palmitoyi) chains, and readily separated nonfluorescent dipalmitoylphosphatidylcholine from both (C6-NBD)- and (C,,NBD)-phosphatidylcholine derivatives. (ii) An isocratic reverse-phase system permitted separation of isomers of fluorescent phosphatidylcholine, -ethanolamine, -glycerol, -inositol, -serine, and phosphatidic acid in which the NBD-fatty acid was present in either the sn-1 or sn-2 position of the glycerol backbone. 0 1986 Academic press,IIIC. KEY WORDS: HPLC, lipids; fluorescence; glycerolipids; sphingolipids; isomers.

Fluorescent NBD’-lipid derivatives have become increasingly useful in biophysical, biochemical, and cell biological research. They

have been used to study the lateral organization of membrane lipids (l), membrane phase transitions (2), and membrane fusion events (3). NBD-lipid derivatives have also been used ’ Abbreviations used (To indicate that the NBD-labeled as model compounds for studying lipid transgiycerolipids were mixed-chain isomers having fluorescent fer between membranes (4), and as substrates

and nonfluorescent acyl moieties present in both the sn1 and sn-2 positions of the glycerol backbone, we have abbreviated the compounds as 1,2-(X, Cg- or C,?-NBD) where X designates the nonfluorescent acyl chain moiety.): DOPC, dioleoylphosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine; HCMF, 10 mM Hepes-buffered, calcium- and magnesium-free Puck’s saline, pH 7.4; HMEM, 18 mM Hepes-buffered Eagle’s minimal essential medium, pH 7.4, without indicator; NBD, 4-nitrobenzo-2-oxa- 1,3diazole; (C6-NBD)-Cer, IV-NBD-aminocaproyl D-erythro sphingosine; (&NBD)-Cb, IV-NBD-aminocaproyl sphingosine monoglucoside; CDP-(oleoyl, C,-NBD>DG, cytidine diphosphate-I-oleoyl, 2-(NBD-aminocaproyl)-diacylglycerol; (acyl, C,-NBD)-DG, 1,2-(acyl, NBD-aminocaproyl) diacylglycerol; (&NBD)-FA, NBD fatty acid (NBD-aminocaproic acid); (palmitoyl, CcNBD)-PA, 1,2(palmitoyl, NBD-aminocaproyl) phosphatidic acid; (acyl, C6-NBD)-PC, 1,2-(acyl, NBD-aminocaproyl) phosphati-

dylcholine; (palmitoyl, &NBD)-PC, I ,2-(palmitoyl, NBDaminocaproyl) phosphatidylcholine; (oleoyl, &NBD)-PC, 1,2-(oleoyl, NBD-aminocaproyl) phosphatidylcholine; (palmitoyl, C12-NBD)-PC, 1,2-(palmitoyl, NBD-aminododecanoyl) phosphatidylcholine; (palmitoyl, &NBD)PE, l,t(palmitoyl, NBD-aminocaproyl) phosphatidylethanolamine; (oleoyl, C,NBD)-PE, 1,2-(oleoyl, NBDaminocaproyl) phosphatidylethanolamine; (palmitoyl, Ce-NBD)-PG, l,Z(palmitoyl, NBD-aminocaproyl) phosphatidylglycerol; (acyl, C,-NBD)-TG, 1,2,3-(acyl, NBDaminocaproyl) triacylglycerol; (oleoyl, C6-NBD)-PI, 1,2(oleoyl, NBD-aminocaproyl) phosphatidylinositol; (palmitoyl, C+-NBD)-PS, 1,2-(pahnitoyl, NBD-aminocaproyl) phosphatidylserine; (&NBD)-Sm; N-NBD-aminocaproylD-erythro sphingosine- 1-phosphocholine. 101

0003-2697186

$3.00

Copyright 0 1986 by Academic Fxss, Inc. All rigbh of reproduction in any form reserved.

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for assaying enzymatic activity in vitro (5,6). Our own laboratory has developed an NBDlipid system to study lipid metabolism and translocation in animal cells (7). With this methodology, it is possible to observe the intracellular (re)distribution of fluorescent lipid analogs in living cells by high-resolution fluorescence microscopy and correlate NBD-lipid location with biochemical investigations of the fluorescent metabolites found in the cell lipid extracts. We were interested in HPLC analysis of the NBD-lipids to augment our TLC analyses of these materials. Unfortunately, the polarity and solubility properties of the NBD-lipids did not permit use of published HPLC methods (8- 10) for analysis and purification. We report here two rapid and sensitive HPLC methods which should be extremely useful for in vitro and in vivo studies using these fluorescent compounds.

NBD)-PA and -PE. (Palmitoyl, Cs-NBD)-PC (Avanti No. 8 10130) was used in base exchange (14) to synthesize (palmitoyl, CgNBD)-PA, -PE, and -PS. (Oleoyl, C6-NBD)PI ( 13) and (C6-NBD)-Cer, -Cb, and -Sm were synthesized as described (15). (Cs-NBD)-lyso PC was obtained as a byproduct of the acylation of glycerophosphorylcholine with the imidazolide of palmitic and (Cs-NBD)-fatty acids essentially as previously described for the acylation of glycerol-3-phosphate (12). (C,NBD)-FA, DPPC, (C6-NBD)-PG (Avanti, special order), and (palmitoyl, Ciz-NBD)-PC (Avanti No. 8 10 13 1) were used without further purification. All lipids were stored at -70°C periodically monitored for purity by thin-layer chromatography, and repurified as necessary. Equipment. Two Waters Model 5 10 HPLC pumps, a McPherson Model 749 spectrofluorometric detector equipped with a xenonmercury lamp, and a Shimadzu C-R3A plotter/integrator were used. Fluorescence was detected using a 24-~1 (illuminated volume) MATERIALS flow cell. Maximum sensitivity was obtained Solvents and other chemicals. Glass-distilled by excitation at the 437-nm mercury line chloroform and methanol from Burdick and which is on the excitation peak of NBD, and Jackson Laboratories, glass-distilled hexane emission was detected using a 5 15-nm cut-off from EM Science, and phosphoric acid (re- filter. (The excitation and emission maxima agent grade) and triethylamine (Gold label) of NBD are 470 and 530 nm, respectively.) from Aldrich Chemical Company were ob- Samples were injected in 20 ~1 of the HPLC tained. Phospholipase D (cabbage) was solvent (see below) using a Rheodyne 7 125 injector. from Boehringer-Mannheim, phospholipase C (Clostridium perfringens) was from Calbiochem, and phospholipase A2 (Naja naja) was METHODS from Sigma. For normal-phase separations, two Waters Lipids. All lipids were purchased from Avanti Polar Lipids, Inc. or synthesized using Resolve (4.3 X 150 mm; 5 pm silica) columns the indicated Avanti lipid as the starting ma- connected in series were used. In later studies, terial. (Acyl, Ca-NBD)-PC (Avanti No. a single Alltech (4.6 X 250 mm; 5 pm silica) 8 10 12 1) was treated with phospholipase C to column was used and gave similar results. The solvent system consisted of hexane/chloroproduce (acyl, Cs-NBD)-DG (11) and further phosphoric acid acylated with palmitic acid imidazolide essen- farm/methanol/water/85% (281/650/300/30/4), and was used at a flow tially as previously described for the acylation of glycerol-3-phosphate (12) to synthesize rate of 1.0 ml/min. (acyl, C6-NBD)-TG. (Oleoyl, &NBD)-PC Thin-layer chromatography. When deter(Avanti No. 8 10132) was used for the synthesis mining retention volumes (Table I), each fluof CDP-(oleoyl, &NBD)-DG (13), and in orescent lipid was chromatographed separately base exhange (14) to synthesize (oleoyl, Cg- on the normal-phase column, extracted, and

HPLC SEPARATION

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LIPID ANALOGS

103

presence compared to the absence of the column was determined. For reverse-phase separations, a Regis HiChrom Reversible ODS column (4.6 X 250 TG 2.31 (acyl, C6-NBD)mm; 5 pm silica) was used. Similar separations DG 2.51 (acyl, &NBD)(C6-NBD)Cer 2.59 were achieved on a Beckman (Altex) ODS PA 2.64 column (4.6 X 250 mm; 5 pm silica). The sol(oleoyl, C6-NBD)PA 2.64 vent system, consisting of methanol/water (881 (palmitoyl, C,NBD)(palmitoyl, C6-NBD)PG 2.61 12, v/v), was buffered to pH 7.0 with triethFA 3.11 (&NBD)ylaminophosphate to a final concentration of (&NBD)cb 3.51 (palmitoyi, Ch-NBD)PS 3.82 45 mM. The flow rate was 1.5 ml/min. 3.83 (oleoyl, C,-NBD)PI IdentiJcation of isomers. (&NBD)-lipids (oleoyl, C,-NBD)PE 4.00 were digested to completion with phospholi(palmitoyl, Cs-NBD)PE 4.10 dipalmitoyl PC 5.50 pase A2 (17) and subsequently analyzed by CDP-(oleoyl, C6-NBD)DG 6.70 TLC to determine the position of the fluores6.78 cent acyl chain as follows. Approximately 1 (palmitoyl, C12-NBD)PC (oleoyl, &NBD)PC 9.21 nmol of (G,-NBD)-lipid was dried under ni(palmitoyl, C6-NBD)PC 9.43 trogen in a screw cap test tube. One milliliter (C,NBD)Sm 16.25 of ethyl ether/methanol (98/2, v/v) and 0.1 ml 0.1 M sodium borate, pH 7.5, containing 0.4 its identity confirmed by TLC on Silica gel 60 mg CaC12. 2Hz0 and 140 units phospholipase plates. The TLC developing solvent was chlo- A2 (N. naja) were added. The tube was capped roform/methanol/ammonium hydroxide (65/ and mixed thoroughly by frequent vortex 35/5), and the lipids were visualized by exci- mixing over a 2-h period at 22°C. The reaction tation with ultraviolet light. mixtures were dried first under nitrogen, then Quantitation of NBD-lipids. Standard so- under vacuum for several hours, and the reslutions of each NBD-lipid (see Table 1) con- idue was suspended in chloroform/methanol taining 0.01 I’nM NBD-fluorescence in the (2/l). Samples of the starting materials and HPLC solvent system were prepared by ref- the phospholipase A2 extracts were applied to erence to a stock solution of (palmitoyl, Cg- separate lanes of a silica gel 60 TLC plate NBD)-PA of a concentration determined by (Merck). The plate was developed in chlorolipid phosphorus analysis (16). Samples con- form/methanol/ammonium hydroxide (65/ taining 1O-200 pmol each NBD-lipid were in- 35/5), dried, and photographed under uv iljected in triplicate onto the normal-phase col- lumination. umn, eluted, and the peak areas were deterFluorescent lipid extracts from cultured cells. mined. The peak area of each NBD-lipid was Monolayer cultures of Chinese hamster V79 correlated in a linear manner to the quantity fibroblasts were grown as described ( 18). Small injected over the entire concentration range unilamellar vesicles (liposomes) containing 1 (data not shown). mM total lipid were prepared by ethanol in(Palmitoyl, Cr2-NBD)-PC was used as an jection ( 19) of either (palmitoyl, Cs-NBD)-PA/ internal standard to determine recovery of the DOPC (20/80 mol%) or (C&IBD)-Cer/DOPC (Cc-NBD)-lipids. To verify recovery of (pal- (20/80 mol%) into HCMF, dialyzed overnight mitoyl, Crz-NBD)-PC from the column, sam- against HMEM, and diluted to 200 PM total ples containing lo-200 pm01 were injected lipid in HMEM. Cells were trypsinized as deboth onto the column, and into the HPLC scribed (20), washed by centrifugation, and the system with the column replaced by stainless cell pellet was resuspended at a concentration steel tubing. The lipid was eluted, peak areas of 1 X lo7 cells/ml liposomes and incubated were determined, and the recovery in the for 90 min at 2°C. The cells were then washed TABLE 1 RETENTION VOLUMES (ml) OF LIPID STANDARDS INNORMAL-PHASE HPLC

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three times with HMEM and the lipids were extracted (21) immediately or the cells were warmed to 37°C for 90 min, washed, and then extracted. The lipid extracts were dissolved in chloroform/methanol (2/l), the fluorescence content was determined by reference to a (palmitoyl, C6-NBD)-PA fluorescence standard (see below), and the lipid extracts were then diluted to a concentration corresponding to 0.01 mM total NBD fluorescence prior to HPLC analysis. RESULTS AND DISCUSSION

Normal

Phase

NBD-glycerohpid separations. The isocratic normal-phase HPLC method described in this report was rapid, sensitive, and capable of resolving both neutral and polar NBD-lipids in the same chromatogram. The NBD-glycerolipid standards (palmitoyl, Cs-NBD)-TG, -DG, -PA, -FA, -PE, and -PC were separated using the isocratic HPLC solvent system in less than 10 min at a flow rate of 1.O ml/min (Fig. 1A). Addition of 2.5 pmol of nonfluorescent cell lipid extract to the injected sample containing 2.5 nmol of each of these NBD-lipids did not affect the retention volume or the amount of the NBD-lipids recovered from the column (data not shown). Although retention volume of each NBDlipid depended mainly on the structure of the polar head group, this HPLC system also sep arated (oleoyl, Cs-NBD)-PE from (palmitoyl, Cs-NBD)-PE, as well as (oleoyl, C6-NBD)-PC from (palmitoyl, &NBD)-PC (see Table 1). Complete HPLC separation of a standard mixture containing DPPC, (palmitoyl, ClzNBD)-PC, and (palmitoyl, C6-NBD)-PC was also achieved (see Table 1). Analysis of (palmitoyl, C6-NBD)-PA treated cells. Lipid extracts of cells treated with (palmitoyl, &NBD)-PA for 90 min at 2°C which were extracted immediately, or which were subsequently washed and warmed to 37°C for 90 min prior to extraction, were analyzed by HPLC (Figs. 1B and C). Similar to previous results obtained by TLC (22), the extract ob-

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MINUTES FIG. 1. Separation of NBD-glycerolipids. (A) A standard mixture containing 50 pmol each (palmitoyl, C6-NBD)TG, -DG, -FA, -PE, and -PC, as well as 50 pmol (pahnitoyl, Crr-NBD)-PC as an internal standard (IS.) was analyzed. (B) and (C) Cell lipid extracts containing fluorescence equivalent to 100 pmol NBD-lipid, as well as 50 pmol (palmitoyl, C,r-NBD)-PC as an internal standard (IS.) were analyzed. Trypsinized cells were treated with (palmitoyl, C6-NBD)-PA for 90 min at 2”C, washed, and extracted and analyzed after further incubation at 37°C for 0 min (B) or 90 min (C).

tained from the 2°C incubation contained 72.4 + 1.9% and 27.3 k 0.2 1% fluorescent diacylclycerol and phosphatidic acid, respectively. After the cells had been warmed, however, the extract contaiued 23.5 f 1.0% fluorescent triacylglycerol, 28.0 + 2.6% fluorescent diacylglycerol, and 48.3 k 3.6% fluorescent phosphatidylcholine. Values are the mean f SD of three determinations. In contrast to previous results (22), little fluorescent phosphatidic acid was detected by HPLC or TLC in the 37°C extract, and may reflect a difference between the trypsinized cells and the monolayer cultures previously used. NBD-sphingolipid separations. The sepa-

HPLC SEPARATION

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ration of the NBD-sphingolipids (Ch-NBD)Cer, -Cb, and -Sm using the isocratic HPLC solvent system on the normal phase column was achieved in approximately 15 min at a flow rate of 1.0 ml/min (Fig. 2A). Synthetic (C&NBD)-Sm prepared ( 15) from commercially available sphingosinephosphorylcholine exhibited two peaks in this system and in some TLC systems. The second isomer eluted by HPLC, which corn&rated with (&NBD)-Sm synthesized by cells provided with (Cs-NBD)-Cer, was purified by

LIPID ANALOGS

105

preparative TLC for use as the (Ch-NBD)-Sm standard (see Fig. 2A). Analysis of (C6-NBD)-Cer treated cells. Lipid extracts of cells treated with (C,-NBD)Cer for 90 min at 2°C and which were either extracted immediately, or which were subsequently washed and warmed to 37°C for 90 min prior to extraction, were analyzed by HPLC (Figs. 2B and C). Similar to previous results using TLC (15) the extract obtained from the 2°C incubation contained 97.2 + 1.9% (C,-NBD)-Cer and 2.8 + 1.2% (C,NBD)-Cb. After the cells were warmed to 37°C 39.7 -t 2.0% (C,-NBD)-Cer, 27.6 + 1.8% (C,-NBD)-Cb, and 32.5 + 2.1% (C6-NBD)-Sm were found in the extract. Values are the mean f SD of three determinations. Recovery of (C6-NBD)-lipids. Complete recovery of (palmitoyl, Ciz-NBD)-PC from the normal-phase column (see Methods) was achieved at each concentration over the entire lo-200 pmol range injected. Using (palmitoyl, C,,-NBD)-PC as an internal standard, recovery of all NBD-lipids except (Ch-NBD)-Sm was greater than 90% over the entire range of lo200 pmol injected. (C6-NBD)-Sm recovery increased from approximately 70 to 85% over the same concentration range. Reverse Phase

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FIG. 2. Separation of NBDsphingolipids. (A) A standard mixture containing 50 pmol each (C&BD)-Cer, -Cb, and -Sm, as well as 50 pmol (palmitoyl, C,,-NBD)-PC as an internal standard (LS.) was analyzed. (B) and (C)Cell lipid extracts containing fluorescence equivalent to 100 pm01 NBD-lipid, as well as 50 pmol (palmitoyl, C,*-NBD)-PC as an internal standard (I.S.) were analyzed. Trypsinized cells were treated with (&NBD)-Cer for 90 min at 2”C, washed, and extracted and analyzed after further incunation at 37°C for 0 min (B) or 90 min (C).

When samples of (acyl, CcNBD)-PC, (oleoyl, GNBD)-PC, (palmitoyl, C6-NBD)PC, and (palmitoyl, Ci2-NBD)-PC were digested to completion with phospholipase A2 ( 17), TLC analysis of the products indicated that approximately 20% of the NBD-FA was located at the sn- 1 position and 80% at the sn2 position. It was therefore of interest to learn if the mixed-chain isomers of the undigested lipids could be analyzed and purified by HPLC. Figure 3A shows that the elution profile of (palmitoyl, &NBD)-PC on reversephase HPLC consisted of two peaks. TLC analysis of the products of phospholipasc A2 digestion of each of these two peaks showed that only (Ch-NBD)-lyso PC was formed from the first peak, while only (Cb-NBD)-FA was

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fluorescent (NBD) fatty acid-labeled analogs of glycerolipids and sphingolipids. The use of these lipids is becoming increasingly popular, and we anticipate that the methods presented here will be particularly useful in purification and/or analysis of NBD-lipids in the following types of studies. (i) In vitro assays:Fluorescent fatty acid-labeled derivatives of various lipids, including an NBD-labeled glucocerebroside, have been used as substrates for several lipolytic enzymes (5,6). The use of these fluorescent substrates permits detection of extremely low enzyme activities without the use of radiolabeled lipids. In addition, the use of fluorescence methods such as resonance energy transfer (4) or self-quenching of fluorescence (24) may permit continuous kinetic measurements of enzyme activity. (ii) Cellular studies with NBD-glycerolipids of high isomericpurity:

As shown in this paper, commercially available glycerophospholipids with an NBD-label on the fatty acyl chain principally at the sn-2 poI 2 3 4 5 6 sition of the molecule, as well as some NBDFIG. 3. Reverse-phase separation and analysis of (pal- labeled lipids synthesized in this laboratory mitoyl, C,-NBD)-PC. (A) HPLC chromatogram of (palmitoyl, C6-NBDFPC showing resolution into two peaks, (25), contain significant amounts of the flu1 and 2. (B) Thin-layer chromatogram showing that peaks orescent fatty acid at the sn-1 position. Althis 1 and 2 are the purified isomers of tluorescent PC. Lanes though some synthetic routes minimize 1, 3, and 5 correspond to starting (palmitoyl, C6-NBD)contamination (26) the purification of each PC, and peaks 1 and 2 obtained by reverse-phase HPLC, of the molecular species by reverse-phase respectively. Lanes 2,4, and 6 show the same lipids after HPLC is simple, and yields both isomers for digestion with phospholipase AZ. Positions of lyso-(CsNBD)-PC (LPC), (palmitoyl, CsNBD)-PC (PC), and (C,- further study. In the future, it will be of interest to examine the metabolism and intracellular NBD)-FA (FA) standards are indicated. distribution of these isomers to learn whether fatty acid composition plays a role in directing obtained from the second (Fig. 3B). This result their intracellular translocation. This may be indicates that the two peaks correspond reparticularly important in studies with fluoresspectively to [ 1-C&JBD, 2palmitoyl]-PC and cent phosphatidylinositol, in which the fatty [ I-palmitoyl, 2-Ca-NBD]-PC. Similar results sn-2 position of the acid composition at the were obtained for each of the fluorescent molecule plays an important role in its meglycerolipids examined, and the retention voltabolism (27). In addition, the ability to readily umes of each of the isomers of these comseparate nonfluorescent lipids from the corpounds is listed in Table 2. Inclusion of triethresponding fluorescent ones (Table 1) will be ylaminophosphate (23) in the column solvent studies in was essential for resolution of the acidic gly- useful in deacylation/reacylation doubly labeled cells treated with fluorescent cerolipids in the reverse-phase system. NBD-lipids and radiolabeled lipid precursors. CONCLUSIONS (iii) Single-cell biochemistry: In the present study, the limit of detection of fluorescence We have developed two simple isocratic HPLC methods for analysis and separation of eluted as a single NBD peak was approxi-

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TABLE 2 RETENTION VOLUMES (ml) OF LIPIDS IN REVERSE-PHASE HPLC Lipid”

Volume (ml)

46 (C,NBD)-FA in sn-2 position’

I&-NBD), 2-palmitoyl-PC 1-palmitoyl, 2-(&NBD)-PC

15.8 17.5

79

I-(C,NBD), 2-oleoyl-PC I-oleoyl, 2-(C,-NBD)-PC

17.4 19.1

77

I-(C,-NBD), 2-palmitoyl-PE I-palmitoyl, 2-(C6-NBD)-PE

15.0 16.5

81

I-(C,NBD), 2-palmitoyl-PS 1-palmitoyl, 2-(CsNBD)-PS

10.7 12.1

73

I-(C6-NBD), 2-palmitoyl-PA I-palmitoyl, 2-(C6-NBD)-PA

13.3 14.3

76

I-(C,-NBD), 2-oleoyl-PI I-oleoyl, 2-(C,-NBD)-PI

11.3 12.5

72

I-(C,-NBD), 2-palmitoyl-PG I-palmitoyl, 2-(C6-NBD)PG

11.4 12.6

75

a All lipids were synthesized from commercially available (palmitoyl, &NBD)-PC Materials). * Determined by integration of peak areas of HPLC chromatogram.

mately 1 pmol. Similar separation characteristics but an additional 40-fold increase in sensitivity resulted when using a normal phase microbore column system (unpublished observations). Since approximately 1 fmol NBDlipid was present per cell in extracts of cells treated with (palmitoyl, C6-NBD)-PA at 2°C for 90 min, relatively minor improvements in detector sensitivity may make it possible to detect NBD-fluorescence in a small number of cells which have been microinjected (7) with NBD-labeled lipids. ACKNOWLEDGMENTS We thank Dr. M. Snider for helpful discussions and Dr. K. Longmuir for critically reading the manuscript. This work was supported by USPHS Grant GM-22942.

6. 7. 8. 9. IO. 11.

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12.

or (oleoyl, &NBD)-PC

(see

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13. Uster, P. S., and Pagano, R. E. (1985) in Enzymes of Lipid Metabolism, Vol. II (Freysz, L., Dreyfus, H., Massarelli, R., and Gatt, S., eds.), in press. 14. Comfurius, P., and Zwaal, R. F. A. (1977) Biochim. Biophys. Acta 488, 36-42. 15. Lipsky, N. G., and Pagano, R. E. (1983) Proc. Natl. Acad. Sci. USA g&2608-26 12. 16. Rouser, G., Siakatos, A. N., and F&her, S. (1966) Lipids 1, 85-86. 17. Kates, M. (1972) in Techniques of Lipidology (Work, T. S., and Work, E., eds.), p. 568, North-Holland/ American Elsevier, New York. 18. Pagano, R. E., Longmuir, K. J., Martin, 0. C., and Struck, D. K. (1981) J. Cell Biol. 91,872-877. 19. Kremer, J. M. H., v. d. Esker, M. W. J., Pathmamanoharan, C., and Wiersema, P. H. (1977) Biochemistry 16, 3932-3935.

20. Nichols, J. W., and Pagano, R. E. (1982) Biochemistry 21, 1720-1726. 21. Struck, D. K., and Pagano, R. E. ( 1980) J. Biol. Chem. 255,5404-5410. 22. Pagano, R. E., Longmuir, K. J., and Martin, 0. C. ( 1983) J. Biol. Chem. 258,2034-2040. 23. Rivier, J. E. (1978) J. Liquid Chromatogr. 1, 343366. 24. Schroit, A. J., and Pagano, R. E. (198 1) Cell 23, 105112. 25. Longmuir, K. J., Martin, 0. C., and Pagano, R. E. ( 1985) Chem. Phys. Lipids 36, 197-207. 26. Schmidt, N., and Gercken, G. (1985) Chem. Phys. Lipids 38,309-3 14. 27. Majerus, P. W., Neufeld, E. J., and Wilson, D. B. (1984) Cell 37,701-703.