344
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[39]
[39] Platelet-Activating Factor Acetylhydrolase from Human Plasma B y D I A N A M . STAFFORINI, THOMAS M . M C I N T Y R E , a n d STEPHEN M . PRESCOTT
Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) is a phospholipid that has been shown to possess potent biological activity. PAF induces hypotension, leukopenia, and thrombocytopenia, 1 and increases vascular permeability. 2 PAF also activates platelets, neutrophils, and macrophages. A variety of cells, e.g., neutrophils, macrophages, and endothelial cells, Synthesize PAF on appropriate stimulation. 3 Monocytes and macrophages secrete most of their PAF, and this phospholipid has been identified in human blood, saliva, urine, and amniotic fluid. 4-6 Thus, the rate of its removal could be an important means of regulating its bioactivity. The degradation of PAF occurs by removal of the acetoyl group esterifled at the sn-2 position of the glycerol backbone. The products lack the bioactive properties of PAF. Farr et al. 7 w e r e the first to demonstrate the occurrence, in mammalian plasma, of an enzyme that catalyzed the reaction shown in Eq. (I). --O ~
PAF
acetylhydrolase
II to--P--O---x] I
x-- N+--
o-
I PAF
i
OH 0
o
II
to__P__O__.X I (1) ] x'-- N ÷ - - + "~'x O -
olyso-PAF
} Acetate
I M. Halonen, J. D. Palmer, I. C. Lohman, L. M. McManus, and R. N. Pinckard, Annu. Rev. Respir. Dis. 122, 915 (1980). 2 j. Bjork and G. Smedegard, J. Allergy Clin. Immunol. 71, 145 (1983). a S. M. Prescott, G. A. Zimmerman, and T. M. McIntyre, Proc. Natl. Acad. Sci. U.S.A. 81, 3534 (1984). 4 K. E. Grandel, R. S. Farr, A. A. Wanderer, T. C. Eisenstadt, and S. I. Wasserman, N. Engl. J. Med. 313, 405 (1985). 5 C. P. Cox, M. L. Wardlow, R. Jorgensen, and R. S. Farr, J. Immunol. 127, 46 (1981). 6 M. M. Billah and J. M. Johnston, Biochem. Biophys. Res. Commun. 113, 51 (1983). 7 R. S. Farr, C. P. Cox, M. L. Wardlow, and R. Jorgensen, Clin. Immunol. Immunopathol. 15, 318 (1980).
METHODSIN ENZYMOLOGY,VOL. 187
Copyright @1990by Academic Press, Inc. All rights of reproductionin any form reserved.
[39]
P A F ACETYLHYDROLASE FROM HUMAN PLASMA
345
The PAF acetylhydrolase activity in human plasma is selective for PAF since a short acyl residue at the sn-2 position is required for hydrolysis to occur; long-chain phosphatidylcholines are not hydrolyzed by this enzyme. The cellular sources of the human plasma PAF acetylhydrolase are not known, but likely candidates include liver cells as well as macrophages. A PAF acetylhydrolase activity is present in the cytosolic fraction of a variety of mammalian tissues) This activity seems to belong to a family of intracellular PAF acetylhydrolases that differ from the plasma enzyme by a variety of criteria, although they all require a short-chain residue at the sn-2 position and are calcium independent. Assay for PAF Acetylhydrolase
Principle. The most convenient assay for thc determination of PAF acetylhydrolase activity utilizes 2-[acetyI-3H]PAF as substratc; thc [3H]acetate generated by hydrolysis is quickly and efficiently separated from labeled substrate since the product is soluble in water, while the substrate is a lipid. We separate them by reversed-phase column chromatography on disposable octadecylsilica gel cartridges; the substrate binds to the column, while the radioactive acetate passes through. Thus, thc amount of radioactivity released represents the amount of enzymatic activity in a given fraction, and can be conveniently quantified by liquid scintillation spectrometry. 9 Reagents [acetyl-3H]PAF: 4 ml of a 0.1 mM solution are prepared by first mixing 400 nmol of PAF (supplied in chloroform, Avanti Polar Lipids, Birmingham, AL), with 4.5/zCi of hexadecyl-2-acetyl-sn-glyceryl-3phosphorylcholine, 1-O-[acetyl-3H(N)] (supplied in ethanol, New England Nuclear, Boston, MA). After evaporation of the solvents (by a stream of nitrogen), 4 ml of HEPES buffer are added and the solution then sonicated for 5 min at 4° and 100 watts, using a 4-mm needle probe in a Braun Sonicator (model 1510). This solution should be stored frozen to avoid nonenzymatic hydrolysis; it can be reused for at least 1 week. The sonication step is repeated each time the substrate is thawed. Duplicate aliquots should be counted to determine the specific radioactivity of the substrate prepared each time; our working solutions are 100 IzM with 10,000 cpm/nmol. HEPES buffer: 0.1 M; adjust the pH to 7.2 with 1 M potassium hydroxide. Prepare 100 ml. g M. L. Blank, T-c. Lee, V. Fitzgerald, and F. Snyder, J. Biol. Chem. 256, 175 (1981). 9 D. M. Stafforini, S. M. Prescott, and T. M. Mclntyre, J. Biol. Chem. 261, 4223 (1987).
346
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[39]
Acetic acid: prepare 100 ml of a 10 M solution. Slowly add 57. l ml of glacial acetic acid to 42.9 ml of distilled water. Sodium acetate: 0.1 M; prepare 500 ml. Octadecylsilica gel cartridges; purchased from Baker Chemical Co. (Phillipsburg, NJ). Each assay requires an individual column which can be reused several times if they are properly regenerated. Wash each column with 3 ml of chloroform : methanol (I : 2), followed by 3 ml of 95% ethanol, and, finally, 3 ml of water. Standard Procedure. Aliquots of 10/zl of the samples to be assayed (diluted in H E P E S buffer, if necessary) are mixed with 40/zl of 0.1 mM [acetyl-3H]PAF in polypropylene tubes and then incubated for 30 min at 37°. Glass tubes should be avoided to minimize substrate binding to the glass surface. After incubation, 50/xl of acetic acid are added followed by 1.5 ml of sodium acetate solution. Each reaction mixture is then passed through a C ~8 gel cartridge and the filtrates collected in 15-ml scintillation vials. Each assay tube is then washed with an additional 1.5 ml of sodium acetate solution and the wash also passed through the cartridge and combined with the original effluent. Ten milliliters of Opti-Fluor (Packard Instruments, Downers Grove, IL) are added to the vials and the amount of radioactivity determined in a liquid scintillation counter. When there are many samples, it is convenient to use a multiplace vacuum manifold to allow several samples to be processed simultaneously. If only a few samples need to be assayed, one can obtain satisfactory results by manually pushing the product of the reaction through a syringe attached to a C~s cartridge. Enzyme Activity. The amount of enzymatic activity present is expressed in micromoles per milliliter per hour, after correction for quenching, incubation time, dilution factors, and the amount of enzyme present in the assay.
Comments We have also used another C~s silica gel cartridge, the Waters Sep-Pak (Milford, MA), but have found the Baker product to be more suitable for this assay as the Waters Sep-Pak gave more variable results. However, both types of cartridges can be reused at least ten times without loss of binding capacity. We have used this assay for the determination of hydrolase activity in serum and plasma samples, and found that both sources contain the same amount of activity. The collection of plasma in EDTA, citrate, or heparin resulted in equal activities. The amount of protein permissible in the solution to be applied to the cartridges should be determined, since large amounts of protein will
[39]
P A F ACETYLHYDROLASE FROM HUMAN PLASMA
347
prevent binding of PAF to the resin. This results in what appears to be high activity since the product becomes contaminated with substrate. This should be examined by carrying out mock assays terminated at zero time. The Baker cartridge has a higher capacity than the Waters cartridge, since only 3.4% of the labeled substrate leached from the column in the presence of 10/xl of plasma, in contrast to the Waters Sep-Pak, which allowed 10.1% of the label to leach. These considerations are particularly important when crude, protein-rich samples such as plasma, lipoproteins, and tissue extracts are assayed for PAF acetylhydrolase activity. Following any change in procedure, one should verify that the apparent product is not contaminated with substrate. The effluent should be extracted into CHCI3 and examined by TLC 1° or HPLC. 11
Other Assays for P A F Acetylhydrolase Other assays have been described for the determination of PAF acetylhydrolase activity. Farr et al. 7 used an assay that consisted of incubation of PAF with serum, a phase-partitioning step, and, finally, measurement of platelet aggregation. This assay is cumbersome, and lacks sensitivity since it detects the loss of substrate rather than the appearance of product. In addition, the PAF concentrations where platelet aggregation is linear are in the nanomolar range, while the PAF acetylhydrolase requires micromolar concentrations of substrate to achieve maximal initial rates. To ensure a linear response, one is forced to dilute the samples, and, consequently, increase the source of error. Blank et al. 8 employed 2-[acetyl-3H]PAF as a substrate and measured the release of radiolabeled acetate. The separation of released product from the lipid substrate was by phase-phase partitioning and measurement of radioactivity in the aqueous phase. This assay is slower than the one described above and care must be taken to remove all CHCI3 to avoid quenching of the liquid scintillation counting. Determination of Half-Life of P A F It is occasionally useful to determine the ability of a given biological sample to hydrolyze subsaturating concentrations of PAF, as discussed below.
Reagents [alkyl-3H]PAF: prepare 100 ~1 of a PAF solution with a final concentration of 10 -8 to 10-6 M by mixing unlabeled PAF (Avanti Polar ~o H. W. Mueller, J. T. O'Flaherty, and R. L. Wykle, J. Biol. Chem. 258, 6213 (1983). ~l A. R. Brash, C. D. Ingrain, and T. M. Harris, Biochemistry 26, 546 (1987).
348
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
Lipids)
with
[39]
hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine,
l-O-[hexadecyi-l', 2'-3H(N)] (New England Nuclear). Dry the solvents under a stream of nitrogen. Then add 100/zl of 50 mM Tris-HCl buffer (pH 7.5) and briefly sonicate this solution at 4 °. Procedure. Add 50/zl of the [alkyl-3H]PAF solution to 250/zl of the biological sample to be tested. Remove 50/xl-aliquots after 0, 2, 4, 6, 9, and 15 min at room temperature and place each one in a microcentrifuge tube containing 62.5 /~1 of chloroform and 125 /zl of methanol to stop the reaction. This results in the formation of a Bligh-Dyer monophase.12 Add approximately 40 nmol of PAF and lyso-PAF as carriers and then break the phases by adding 62.5/zl of water and 62.5/zl of chloroform. Remove the lower phase after a brief centrifugation step and dry it under nitrogen. Then, resuspend the extracted phospholipids in 50 /zl of chloroform: methanol (9: 1). Spot the samples on thin-layer chromatography plates that had been previously activated by heating at 100° for 1 hr. Develop the plates using chloroform : methanol : acetic acid : water (50 : 25 : 8 : 4).1° Scrape the spots corresponding to PAF, lyso-PAF, and the rest of the lane and determine the amount of radioactivity present in the scrapings after adding 5 ml of Opti-Fluor (Packard Instruments). Expression of Results. The amount of PAF hydrolyzed is determined by calculating the percentage of counts present in the PAF spot for all the time points tested. The percentage of PAF remaining is then plotted on a log scale versus time (linear scale). In human plasma, this usually gives pseudo-first-order kinetics and the half-life can then be calculated from this line. Properties of PAF Acetylhydrolase
Effect of Metals and Chelators The plasma acetylhydrolase does not require the presence of divalent cations for maximal activity. The addition of EDTA to whole blood as an anticoagulant does not alter enzymatic activity, and the activity of the nearly homogeneous enzyme is not altered by the addition of calcium. 9
Association with Lipoproteins The plasma PAF acetylhydrolase activity is found exclusively in the lipoprotein fraction. About two-thirds of the activity is associated with low-density lipoproteins (LDL) and one-third with high-density lipo12 E. G. Bligh and W. F. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959).
[39]
P A F ACETYLHYDROLASE FROM HUMAN PLASMA
349
0.04
A LDL
C"
0.03 HDL
O
E 0.02
5
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[39]
[39] Platelet-Activating Factor Acetylhydrolase from Human Plasma B y D I A N A M . STAFFORINI, THOMAS M . M C I N T Y R E , a n d STEPHEN M . PRESCOTT
Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) is a phospholipid that has been shown to possess potent biological activity. PAF induces hypotension, leukopenia, and thrombocytopenia, 1 and increases vascular permeability. 2 PAF also activates platelets, neutrophils, and macrophages. A variety of cells, e.g., neutrophils, macrophages, and endothelial cells, Synthesize PAF on appropriate stimulation. 3 Monocytes and macrophages secrete most of their PAF, and this phospholipid has been identified in human blood, saliva, urine, and amniotic fluid. 4-6 Thus, the rate of its removal could be an important means of regulating its bioactivity. The degradation of PAF occurs by removal of the acetoyl group esterifled at the sn-2 position of the glycerol backbone. The products lack the bioactive properties of PAF. Farr et al. 7 w e r e the first to demonstrate the occurrence, in mammalian plasma, of an enzyme that catalyzed the reaction shown in Eq. (I). --O ~
PAF
acetylhydrolase
II to--P--O---x] I
x-- N+--
o-
I PAF
i
OH 0
o
II
to__P__O__.X I (1) ] x'-- N ÷ - - + "~'x O -
olyso-PAF
} Acetate
I M. Halonen, J. D. Palmer, I. C. Lohman, L. M. McManus, and R. N. Pinckard, Annu. Rev. Respir. Dis. 122, 915 (1980). 2 j. Bjork and G. Smedegard, J. Allergy Clin. Immunol. 71, 145 (1983). a S. M. Prescott, G. A. Zimmerman, and T. M. McIntyre, Proc. Natl. Acad. Sci. U.S.A. 81, 3534 (1984). 4 K. E. Grandel, R. S. Farr, A. A. Wanderer, T. C. Eisenstadt, and S. I. Wasserman, N. Engl. J. Med. 313, 405 (1985). 5 C. P. Cox, M. L. Wardlow, R. Jorgensen, and R. S. Farr, J. Immunol. 127, 46 (1981). 6 M. M. Billah and J. M. Johnston, Biochem. Biophys. Res. Commun. 113, 51 (1983). 7 R. S. Farr, C. P. Cox, M. L. Wardlow, and R. Jorgensen, Clin. Immunol. Immunopathol. 15, 318 (1980).
METHODSIN ENZYMOLOGY,VOL. 187
Copyright @1990by Academic Press, Inc. All rights of reproductionin any form reserved.
[39]
P A F ACETYLHYDROLASE FROM HUMAN PLASMA
345
The PAF acetylhydrolase activity in human plasma is selective for PAF since a short acyl residue at the sn-2 position is required for hydrolysis to occur; long-chain phosphatidylcholines are not hydrolyzed by this enzyme. The cellular sources of the human plasma PAF acetylhydrolase are not known, but likely candidates include liver cells as well as macrophages. A PAF acetylhydrolase activity is present in the cytosolic fraction of a variety of mammalian tissues) This activity seems to belong to a family of intracellular PAF acetylhydrolases that differ from the plasma enzyme by a variety of criteria, although they all require a short-chain residue at the sn-2 position and are calcium independent. Assay for PAF Acetylhydrolase
Principle. The most convenient assay for thc determination of PAF acetylhydrolase activity utilizes 2-[acetyI-3H]PAF as substratc; thc [3H]acetate generated by hydrolysis is quickly and efficiently separated from labeled substrate since the product is soluble in water, while the substrate is a lipid. We separate them by reversed-phase column chromatography on disposable octadecylsilica gel cartridges; the substrate binds to the column, while the radioactive acetate passes through. Thus, thc amount of radioactivity released represents the amount of enzymatic activity in a given fraction, and can be conveniently quantified by liquid scintillation spectrometry. 9 Reagents [acetyl-3H]PAF: 4 ml of a 0.1 mM solution are prepared by first mixing 400 nmol of PAF (supplied in chloroform, Avanti Polar Lipids, Birmingham, AL), with 4.5/zCi of hexadecyl-2-acetyl-sn-glyceryl-3phosphorylcholine, 1-O-[acetyl-3H(N)] (supplied in ethanol, New England Nuclear, Boston, MA). After evaporation of the solvents (by a stream of nitrogen), 4 ml of HEPES buffer are added and the solution then sonicated for 5 min at 4° and 100 watts, using a 4-mm needle probe in a Braun Sonicator (model 1510). This solution should be stored frozen to avoid nonenzymatic hydrolysis; it can be reused for at least 1 week. The sonication step is repeated each time the substrate is thawed. Duplicate aliquots should be counted to determine the specific radioactivity of the substrate prepared each time; our working solutions are 100 IzM with 10,000 cpm/nmol. HEPES buffer: 0.1 M; adjust the pH to 7.2 with 1 M potassium hydroxide. Prepare 100 ml. g M. L. Blank, T-c. Lee, V. Fitzgerald, and F. Snyder, J. Biol. Chem. 256, 175 (1981). 9 D. M. Stafforini, S. M. Prescott, and T. M. Mclntyre, J. Biol. Chem. 261, 4223 (1987).
346
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[39]
Acetic acid: prepare 100 ml of a 10 M solution. Slowly add 57. l ml of glacial acetic acid to 42.9 ml of distilled water. Sodium acetate: 0.1 M; prepare 500 ml. Octadecylsilica gel cartridges; purchased from Baker Chemical Co. (Phillipsburg, NJ). Each assay requires an individual column which can be reused several times if they are properly regenerated. Wash each column with 3 ml of chloroform : methanol (I : 2), followed by 3 ml of 95% ethanol, and, finally, 3 ml of water. Standard Procedure. Aliquots of 10/zl of the samples to be assayed (diluted in H E P E S buffer, if necessary) are mixed with 40/zl of 0.1 mM [acetyl-3H]PAF in polypropylene tubes and then incubated for 30 min at 37°. Glass tubes should be avoided to minimize substrate binding to the glass surface. After incubation, 50/xl of acetic acid are added followed by 1.5 ml of sodium acetate solution. Each reaction mixture is then passed through a C ~8 gel cartridge and the filtrates collected in 15-ml scintillation vials. Each assay tube is then washed with an additional 1.5 ml of sodium acetate solution and the wash also passed through the cartridge and combined with the original effluent. Ten milliliters of Opti-Fluor (Packard Instruments, Downers Grove, IL) are added to the vials and the amount of radioactivity determined in a liquid scintillation counter. When there are many samples, it is convenient to use a multiplace vacuum manifold to allow several samples to be processed simultaneously. If only a few samples need to be assayed, one can obtain satisfactory results by manually pushing the product of the reaction through a syringe attached to a C~s cartridge. Enzyme Activity. The amount of enzymatic activity present is expressed in micromoles per milliliter per hour, after correction for quenching, incubation time, dilution factors, and the amount of enzyme present in the assay.
Comments We have also used another C~s silica gel cartridge, the Waters Sep-Pak (Milford, MA), but have found the Baker product to be more suitable for this assay as the Waters Sep-Pak gave more variable results. However, both types of cartridges can be reused at least ten times without loss of binding capacity. We have used this assay for the determination of hydrolase activity in serum and plasma samples, and found that both sources contain the same amount of activity. The collection of plasma in EDTA, citrate, or heparin resulted in equal activities. The amount of protein permissible in the solution to be applied to the cartridges should be determined, since large amounts of protein will
[39]
P A F ACETYLHYDROLASE FROM HUMAN PLASMA
347
prevent binding of PAF to the resin. This results in what appears to be high activity since the product becomes contaminated with substrate. This should be examined by carrying out mock assays terminated at zero time. The Baker cartridge has a higher capacity than the Waters cartridge, since only 3.4% of the labeled substrate leached from the column in the presence of 10/xl of plasma, in contrast to the Waters Sep-Pak, which allowed 10.1% of the label to leach. These considerations are particularly important when crude, protein-rich samples such as plasma, lipoproteins, and tissue extracts are assayed for PAF acetylhydrolase activity. Following any change in procedure, one should verify that the apparent product is not contaminated with substrate. The effluent should be extracted into CHCI3 and examined by TLC 1° or HPLC. 11
Other Assays for P A F Acetylhydrolase Other assays have been described for the determination of PAF acetylhydrolase activity. Farr et al. 7 used an assay that consisted of incubation of PAF with serum, a phase-partitioning step, and, finally, measurement of platelet aggregation. This assay is cumbersome, and lacks sensitivity since it detects the loss of substrate rather than the appearance of product. In addition, the PAF concentrations where platelet aggregation is linear are in the nanomolar range, while the PAF acetylhydrolase requires micromolar concentrations of substrate to achieve maximal initial rates. To ensure a linear response, one is forced to dilute the samples, and, consequently, increase the source of error. Blank et al. 8 employed 2-[acetyl-3H]PAF as a substrate and measured the release of radiolabeled acetate. The separation of released product from the lipid substrate was by phase-phase partitioning and measurement of radioactivity in the aqueous phase. This assay is slower than the one described above and care must be taken to remove all CHCI3 to avoid quenching of the liquid scintillation counting. Determination of Half-Life of P A F It is occasionally useful to determine the ability of a given biological sample to hydrolyze subsaturating concentrations of PAF, as discussed below.
Reagents [alkyl-3H]PAF: prepare 100 ~1 of a PAF solution with a final concentration of 10 -8 to 10-6 M by mixing unlabeled PAF (Avanti Polar ~o H. W. Mueller, J. T. O'Flaherty, and R. L. Wykle, J. Biol. Chem. 258, 6213 (1983). ~l A. R. Brash, C. D. Ingrain, and T. M. Harris, Biochemistry 26, 546 (1987).
348
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
Lipids)
with
[39]
hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine,
l-O-[hexadecyi-l', 2'-3H(N)] (New England Nuclear). Dry the solvents under a stream of nitrogen. Then add 100/zl of 50 mM Tris-HCl buffer (pH 7.5) and briefly sonicate this solution at 4 °. Procedure. Add 50/zl of the [alkyl-3H]PAF solution to 250/zl of the biological sample to be tested. Remove 50/xl-aliquots after 0, 2, 4, 6, 9, and 15 min at room temperature and place each one in a microcentrifuge tube containing 62.5 /~1 of chloroform and 125 /zl of methanol to stop the reaction. This results in the formation of a Bligh-Dyer monophase.12 Add approximately 40 nmol of PAF and lyso-PAF as carriers and then break the phases by adding 62.5/zl of water and 62.5/zl of chloroform. Remove the lower phase after a brief centrifugation step and dry it under nitrogen. Then, resuspend the extracted phospholipids in 50 /zl of chloroform: methanol (9: 1). Spot the samples on thin-layer chromatography plates that had been previously activated by heating at 100° for 1 hr. Develop the plates using chloroform : methanol : acetic acid : water (50 : 25 : 8 : 4).1° Scrape the spots corresponding to PAF, lyso-PAF, and the rest of the lane and determine the amount of radioactivity present in the scrapings after adding 5 ml of Opti-Fluor (Packard Instruments). Expression of Results. The amount of PAF hydrolyzed is determined by calculating the percentage of counts present in the PAF spot for all the time points tested. The percentage of PAF remaining is then plotted on a log scale versus time (linear scale). In human plasma, this usually gives pseudo-first-order kinetics and the half-life can then be calculated from this line. Properties of PAF Acetylhydrolase
Effect of Metals and Chelators The plasma acetylhydrolase does not require the presence of divalent cations for maximal activity. The addition of EDTA to whole blood as an anticoagulant does not alter enzymatic activity, and the activity of the nearly homogeneous enzyme is not altered by the addition of calcium. 9
Association with Lipoproteins The plasma PAF acetylhydrolase activity is found exclusively in the lipoprotein fraction. About two-thirds of the activity is associated with low-density lipoproteins (LDL) and one-third with high-density lipo12 E. G. Bligh and W. F. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959).
[39]
P A F ACETYLHYDROLASE FROM HUMAN PLASMA
349
0.04
A LDL
C"
0.03 HDL
O
E 0.02
5
