Determination and Clinical Significance of Phospholipids 25 Papasani V. Subbaiah Phospholipids are integral and essential components of all lipoproteins and cell membranes. in quantitative terms,the amount of phospholipids in fact exceeds that of triglycerides (TG) as well as cholesterol (measured as unesterified) in normal human plasma, However, phospholipids are chemically more diverse than the neutral lipids, and are composed of several subclasses with distinct physicochemical properties. ‘There are two major classes of phospholipids in plasma lipoproteins: glycerophospho- lipids, which have a glycerol backbone, and sphingophospholipids, which have a sphingosine backbone. One sphingophospholipid—sphingomyelin (SPH)—and five subclasses of glycerophospholipids are found in plasma, Thesesubclasses include phosphatidyl choline (PC), lysophosphatidy! choline (LPC), phosphatidyl ethanolamine (PE), phosphatidyl serine (PS), and + phosphatidyl inositol PD) ood Each subclass of phospholipids is actually composed of several molecular species that differ from each other in their fatty acid composition. The structures of the major phospholipids in human plasme and their normal concentrations are shown in Figure 25-1. Despite their high concentration in plasma, phospholipid measurement is not routinely performed in the clinical chemistry laboratory. One reason is that most methods for the measurement of total phospholipids have been rather time consum- ing and tedious, and they are less amenable to automation, compared to the methods for the measurement of the neutral lipids. Another reason is that the phospholipid concentration in plasma is not altered as markedly as that of cholesterol and TG in various pathological conditions. However, the following considerations show that the estimation of lipoprotein phospholipids provides information that is not merely sup- plemental to the neutral lipid values, but at times more important than either choles- terol or TG concentration. 1. Only 10%-20% of the total weight of high-density lipoprotein (HDL) is choles- terol, but 25-30% of it is phospholipids.! Although HDL concentration is ex- set522 Chapter 25 Figure 25-1 ¢ Structures of Major Phospholipids of Normal Plasma. General Structure of Phosphoglyceride H,C-0-C-R, RCOCH H,C-O-P-0-X g Phosphogiyceride Phophatidyl choline (PC) Lysophosphatidy! choline (LPC) Phosphatidyl ethanolamine (PE) Phosphatidy! serine (PS) Phosphatidy! inositol (PI) Ry = Usually saturated fatty acid Re = Usually unsaturated fatty acid (absent in LPC) Structure of X H,C-H,C-N(CH,), H,C-H,C-N(CH,), H,C-H,C.NH, H,C-HC.NH, co0- on on SO,, Structure of Sphingomyelin (SPH) HC(CH,)2 HC=CH ° CH-CH-CH,0-P-O-CH,-CH, N(CH), OH NH-R, O- Cone. in normal plasma mmoi/t. (% of total) 1.795 (68.4) 0.178 (68) 0.115 (4.4) 0.021 (08) 0.060(2.3) 0.464 (17.3) R3 = Long-chain saturated fatty acid pressed routinely in terms of its cholesterol content, the phospholipid content reflects the concentration of HDL much more accurately. 2. Functionally, the phospholipids of HDL are more important than cholesterol or even apolipoprotein Ad in reverse cholesterol transport. Abnormalities in phospholipid composition affect the function of HDL more than abnormalities in TG or cholesterol concentration, Phospholipids are substrates for several important enzymes in plasma such as lecithin:cholesterol acyltransferase (LCAT), lipoprotein lipase, and hepatic lipase, and therefore changes in their composition may affect the activities of these enzymes.Determination and Clinical Significance of Phospholipids S23 4. Unlike TG or cholesteryl esters, phospholipids exchange more readily with cell membranes and therefore alterations in plasma phospholipid composition ikely reflect possible alterations in cell membrane composition and function in 5. Since the polyunsaturated fatty acids in plasma are mainly carried by the phospholipids, the longterm dietary status of essential fatty acids is more accu- rately assessed by the fatty acid composition of phospholipids rather than that in total lipids. The saturated fatty acid content of plasma phospholipids has been reported to bean independent risk factor for atherosclerosis.“ Table 25-1 lists some of the pathological conditions in which the plasma phospholipid composition has been shown to be altered. Table 25-1 + Pathological Significance of Plasma Phospholipids (PL) Disease Effects on plasma phospholipids Reference Coronary heart PC/FC ratio significantly lower in ischemic heart disease. | 36 disease (CHD) —_| HDL phospholipids correlate with atherogenic risk even in | 37 normolipidemics, PCISPH ratio decreased in HDI. 38,38 Decrease in HDL phospholipid: 40 decrease in HDL cholesterol. Free cholesteroVPL ratio increased in HDL in ischemic heart | 41 disease. yperipidemia ‘35% increase in cholesterol/L in LDL (Type lle a hyperlipidemia. Insulin-dependent | Decrease in choline PL in apo B-containing lipoproteins. a diabetes Increase in glycated aminophospholipids. " Free cholesteroV/PL ratio increased. a4 Liver diseases HDL-PL correlate better then HDL cholesterol with liver 6 disoase. Free cholesterol and PL increased in cholestatic liver disease. | 46 Concer Decreased PL in hematologic cancer. a7 Decrease in plasma PL in acute leukemia and malignant 23 lymphoma, LPC peak most sensitive indicator for monitoring treatment. Increase in lysophosphatidic acid in ovarian cancer, multiple | 48.49 myeloma. Cerebrovascular | HDL PL significantly decreased. 50 disease Cystic fibrosis: Polyunsaturated phospholipids correlate with pulmonary [51 function. ‘Schistosomiasis _| Phospholipid concentration elevated. 62 infection Trauma (burns) Elevated PC, and decreased SPH and lyso PC. 53Sze Chapter 25 Review of Existing Methods for Phospholipid Estimation Methods for phospholipid estimation can be divided into two groups: (1) estimation of total phospholipid concentration in plasma or lipoproteins, and (2) estimation of the composition of individual phospholipid subclasses. In addition, there are special methods (which will not be discussed here) to determine the molecular species com- position of individual phospholipid subclasses,’ and to estimate the concentration of minor phospholipids with specific biological functions, such as platelet-activating fac- tor,‘ disaturated PC,’ plasmalogens, phospholipid — hydroperoxides,? truncated (oxi- dized) PCs,10 glycated aminophospholipids,!! and lysophosphatidic acid.12 Total Phospholipids of Plasma ‘The only unique structural feature common to all phospholipids is the presence of lipid-bound phosphate. Therefore most of the methods originally designed to deter- mine plasma phospholipids depended upon the estimation of lipid phosphorus. In general. the total lipids are first extracted from the plasma, the lipid phosphorus is re- leased by acid-digestion, and then the released phosphorus is estimated by colori- metric methods. The various methods differ from each other in the reagents used to release the inorganic phosphate (P1): sulfuric acid and hydogen peroxide, perchloric acid, mixtures of perchloric and sulfuric acids, with vanadium pentoxide as catalyst. ‘The most common reagent used for color is ammonium molybdate, which forms a phosphomolybdate complex with the phosphate, which is then reduced by stannous chloride-hydrazine sulfate or aminonapihol sulfonate at high temperature. Phospholipids have also been estimated without acid digestion, by complexing the intact lipids with chromogenic reagents such as ammonium ferrothiocyanate!3 and prussian blue complex!* or fluorogenic reagents such as diphenylhexatriene.!5 Because about 95% of the total plasma phospholipids contain choline as the base (PC, LPC, SPH), many laboratories use the measurement of lipid-bound choline as a close approximation of total phospholipids. The method is based on the release of choline by phospholipase D from Sirepiomyces chromofuscus, which acts on all three choline-containing phospholipids. The released choline is oxidized with choline ‘oxidase to produce betaine and hydrogen peroxide. The hydrogen peroxide is then re- acted with peroxidase in the presence of 4amino antipyrene and phenol to produce a red quinone complex, which is measured at 505 mm, Pc —PhosoholinaseD_, phosphatidic acid + choline Lpc —Phospholipsse® , tysophosphatidic acid + choline SPH —fhosehelizese_, Ceremide phosphate + choline Choline —Shaline exidese_, Botaine +H,0, 2 Hy +Phenol + 4-aminoantipyrene "#88" _, Quinineimine (urmax 505 nm)+2H,0Determination and Clinical Significance of Phospholipids $25 This method has the advantage of not requiring lipid extraction and not involv- ing corrosive reagents such as perchloric acid and a special fume hood, The drawback is that the method does not measure PS, PI, and PE. High concentrations of bilirubin and ascorbic acid have been reported to interfere in color development, although the concentrations encountered in most clinical samples are without effect. Interference by high concentration of EDTA in plasma has been reported, |6 but including sufficient CaCl; in the buffer overcomes this. Choline can also be estimated with choline kinase by the following sequence of reactions. Choline kinase Choline + ATP Cholinephosphate + ATP ATP + phosphoenol pyruvate—Prewatekinase_, ATP + Pyruvate Pyruvate +NADH +Ht —Lsctate dehydrogenase. | actate +NAD ‘The NADH consumed is measured at 340 nm. The main advantage of this method is that it can be used on dilute solutions where the interference from bilirubin and ascorbate may be more problematic. However, the requirement for additional en- zymes and for a UV range spectrophotometer are disadvantages. Quantitation of Individual Phospholipid Classes As mentioned earlier, the total PC concentration of whole plasma or lipoproteins does not changes dramatically as either cholesterol or TG, and therefore, the quantitation of phospholipids as potential markers of disease has been neglected. However, the composition of phospholipids may be more important than the total amount of phospholipids in some cases. The importance of determining the SPH/PC ratio in amniotic fluid for the estimation of lung maturity of the fetus is well established." Ta- ble 25-1 lists other pathological conditions, several of which are characterized by ab- normalities in individual phospholipid subclasses. About 70% of plasma PE is in the plasmalogen form,!8 which is considered to have strong antioxidant properties. ‘Therefore the estimation of plasma PE provides an indirect measure of the anti-ox dant reserve of the plasma. Furthermore, since LPC is the product of lipolytic actions in the plasma, an increase in its concentration may indicate increased lypolysis. Separation of phospholipids into individual classes has been performed by clas- sical chromatographic procedures using silicic acid column, silicic acidimpregnated paper, and silica gel thin layer chromatography (TLC),}? as well as high-performance liquid chromatography (HPLC),20 and capillary gas-liquid chromatography (GLC)2! In addition, nuclear magnetic resonance (NMR) methods that do not require the phys+ cal separation of the phospholipids from each other for quantitation have been devel- ‘oped.22.22 At present, the TLC and HPLC methods are the most widely used. TLC separation is performed on silica gel-coated plates, with a variety of solvent systems, the most effective being those containing chloroform, methanol, and water. The quantitation of individual phospholipid spots after TLC separation is performed et ther by densitometry after color development on the plate? or by determining the526 Chapter 25 lipid phosphorus in individual spots after scraping from the plate.25 A combination of TLC and flame ionization detection is used in the Iatroscan method, employing sil- ica-coated chromarods.25 While this method has high sensitivity, in our experience it suffers from poor reproducibility. HPLC separation is usually carried out on normal phase silica columns with a variety of solvent systems, the choice of which is dictated by the detection method used. The various methods for quantitation following HPLC separation include UV absorbance,” post-column fluorescence derivatization,’ elec- trochemical detection 28 flame ionization,29 mass spectroscopy,” light-scattering de- tection,>! and refractive index. Although the UV detection method has been used by ‘several investigators, its drawback is that it is mainly dependent upon the number of double bonds in the acyl chains, and is, therefore, highly variable. In addition, the choice of solvents is limited because the most effective solvents interfere in UV mea- surements at the low wavelengths. The molar extinction coefficients are also very low with consequent low sensitivity. However, if the fatty acid composition is not signifi. cantly different among samples, one can use calibrated standards of similar composi- tion and quantitate the phospholipids quite accurately. The refractive index method is not dependent upon the structure of the phospholipid, but its sensitivity is very low and it is unsuitable for gradient separation of the phospholipids. The electrochemical method is also independent of the phospholipid structure, but suffers from poor reproducibility and interference from other solutes.28 While mass spectroscopy yields detailed information that is not obtainable with other methods, the instrumen- tation Is expensive and difficult to operate. The flame ionization technique is very sen- sitive, and allows the use of most solvents for HPLC separation. Although this method is promising, it has not yet gained wide acceptance for phospholipid estimations. In our experience the light-scattering detection method is more practical and reproduc- ible and the results correlate highly with the estimation of lipid phosphorus. Enzymatic methods to estimate the major phospholipids of plasma without prior chromatographic separation have also been reported. Blaton et al? used phospholipase C and sphingomyelinase from B. cereus, which release choline phos- phate specifically from PC and SPH respectively, to determine these phospholipids separately. LPC concentration can be determined as the difference between the total lipid-bound choline as estimated by the phospholipase D method, and the PC and SPH values as determined by this procedure. One drawback of this procedure is that since both phospholipase C and sphingomyelinase are from the same source, the possible cross-contamination of the enzymes can give rise to spurious results for elther PC or SPH, or both. Recommended Methods for Total Phospholipid Estimation ESTIMATION OF LIPID PHOSPHORUS BY CHEMICAL METHOD Apparatus Required 4 Heating block with thermostatic control > — Spectrophotometer525 Chapter 25 Lipid Digestion and Color Development Transfer aliquots of lipid extract (equivalent to 25 ul. whole plasma) into dispos- able glass tubes (13 x 100 mm), and evaporate the solvent completely under a stream of nitrogen. — Similarly, transfer KHaPO, standards containing 0.5 to 5.0 ug Pi, and a reagent blank containing 100 uL water. Using a glass pipette and Pipet-Aid dispenser, add 0.4 ml. of 70% perchloric acid to each tube, including the standards and blanks, 4 Place alll tubes in a block heater that has been preheated to 200° C in a perchloric acid hood and digest the samples for 20 min, or until all the yellow color disappears from the sample tubes. Cool the tubes to room temperature (in the fume hood) and add 3 mL de-ionized water, followed by 0.2 mL of ammonium molybdate solution and 80 uL of the ANSA reagent. Mix the tubes by vortexing and place them in a boiling water bath for 10 min. % Remove the tubes from the water and allow to cool to room temperature. & Take the readings in a spectrophotometer at 830 nm, adjusted for the reagent blank. The blue color Is stable for at least 24h and therefore, analysis of several samples in the same batch is possible without loss of sensitivity or accuracy. Calculations From the optical density (OD) values of the standards, calculate the absorbance units/ug phosphorus. Divide the OD of the unknown sample by the above value to obtain the jg of lipid phosphorus in the unknown sample (25 iL plasma). Be- cause there is one atom of phosphorus per mole of phospholipid, dividing the phos- phorus value by 31 (atomic weight of phosphorus) gives the mol of phospholipid in the sample. To convert yg phosphorus into mg weight percentage of phospholipids, multiply by 25 (taking the average mol weight of the phospholipid as 775) and the di- lution factor. .D of unknown (25 1 plasma), 40 1/L of pla 0.0 of standard perug Pi 31 ™™O!/L OF Plasma 0.0 of unknown (25 pt. pI 2540x100 _ 149 phoepholipid/ dL plasma O.Dofstandard pergPi 1000 ESTIMATION OF LIPID-SOUND CHOLINE BY ENZYMATIC METHOD Reagents All reagents described here are based on the “phospholipids B” kit supplied by Wako Chemicals, Inc. (Richmond, VA). These reagents can be purchased in kit formDetermination and Clinical Significance of Phospholipids 529 from this company, or prepared in the laboratory, using ingredients purchased from other manufacturers (eg., Sigma Chemical Co, St. Louis, MO; Roche Diagnostics, Indi- anapolis, IN). 1, Buffer (200 mL): 0.5 mmol/L Tris-Cl buffer, pH 8.0, containing 50 pg/mL CaCl,.H20 and 0.05% phenol. Store at 4'C. This solutions is stable for at least two months. 2 Color reagent (50 mL): Phospholipase D from Streptomyces chromofuscus (EC 3.1.4.4) 24 units Choline oxidase (EC 1.13.17) 100 units Horse radish Peroxidase (EC 1.11.7) 270 units 4amino antipyrene 750 mg Make up all the ingredients in 50 mL of the above buffer solution (1). This solu- tion is stable for at least two weeks at 4°C. 3. Standard solution (10 ml): Choline chloride: 3.87 mmol/L. (equivalent to 300 mg/dl. of phospholipid) Phenol: 0.1% (w/v) ‘This solution is stable for at least one month at 4°C. Procedure Transfer 20 ul aliquots of plasma, standard solution, and 0.15 mol/L NaCl (re- agent blank) into disposable glass tubes (13 x 100 mm). Add 3.0 mL of color reagent and mix the contents by vortexing. Place all the tubes in a water bath at 37°C for 10 min. Take the readings at 505 nm in a spectrophotometer adjusted for the reagent blank. The color is stable for more than two hours at room temperature. Calculation 0.0 of sample 0.D of standard x 300 = mg phospholipid /dt The above value may be converted to SI units by dividing by 775: phospholipids (mg/dL)/77.6 = phospholipids (mmol/L) When lipoprotein fractions are assayed, use samples equivalent to 50 ul of plasma. Interfering Substances Bilirubin up to 20 mg/dL (342 umol/L) does not affect the reaction. However, ascorbic acid above 5 mg/dL. (284 umol/L) inhibits the color development. The pres- ence of free choline in plasma gives spurtously high values, but less than 1% of total532 Chapter 25 The gradient system described by Becart et al¥$ is recommended with the flow rate set at 1.0 mL/min throughout. Solvent A: chloroform: methanol: 30% ammonium hydroxide (80: 19.5: 0.5 v/v) Solvent B: chloroform: methanol: water: 30% ammonium hydroxide (60: 34: 5.5: 0.5, viv) Set the gradient program as follows: Imeiminn %OfA B%OfB 0 100 0 14 ° 100 23 0 100 29 100 0 34 100 0 The last step returns the column to the initial setting, ready for the injection of the next sample. Figure 25-3 + HPLC Separation of Human Plasma Phospholipids, and Quantitation by a Light-Scattering Detector A; Neutral lipids, including free and esterified cholesterol, and triglycerides. B: Free fatty acids and unidentified neutral lipids. ‘The detector response is in arbitrary units. Detector response Time (min)Determination and Clinical Significance of Phospholipids S83 ELSD Detector Settings No gas flow, 1.6 L/min; drift tube temperature, 50° C. Inject total lipid extract corresponding to 50 il plasma into the column and re- cord the detector output using a computer-based data management system. ‘The response factor for each phospholipid should be determined by using stan- dards, and the concentrations of various phospholipids should then be calculated by entering the predetermined response factors into the program. Figure 25-3 shows a typical chromatogram for the separation of human plasma phospholipids (corre- sponding to 50 jl plasma). The peak for PS is barely visible at this concentration, be- tween PC and PI. Both SPH and LPC appear as double peaks, and the two peaks are combined for the calculation. ‘The concentrations determined by this method agree closely with those mea- sured by the TLC method, However, because the factors are non-linear and vary signif icantly among different phospholipids, it is important to determine these factors in the ranges expected in the samples and with the specific data management program employed. It should be pointed out that the response factors are lower for the phospholipids normally present in low concentrations in human plasma (PI, PS, LPC), than for PC, PE, and SPH. Acknowledgments: The originel research referred to in this chapter was supported by a grant from the National Institutes of Health, #L 52597. The technical assistance of Mr. Wilfred Buchanan is gratefully acknowledged. REFERENCES 1. Edelstein C. General properties of plasma lipoproteins and apolipoproteins. In: Scanu ‘AM, Spector AA, eds. Biochemistry and biology of plasma lipoproteins. 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A high-performance liquid chro- ‘matographic procedure for the determination of disaturated phosphatidyicholine in human plasma, Anal Biochem 1990;191:156-9. 8. Schulz R, Strynadka KD, Panas DL, Olley PM, Lopaschuk GD. Analysis of myocardial plasmalogen and arachidonic acid content using high-performance liquid chromatography. Anal Biochem 1993;213:140-6. iyazawa T, Fujimoto K, Oikawa S, Determination ot lipid hydroperoxides in low-den-