A n n a l s o f C l i n i c a l L a b o r a t o r y S c i e n c e , Vol. 2 , No. 4C o p y r ig h t © 1 9 7 2 , I n s t i t u t e for C lin ic a l S c ie n c e

The Measurement o f Serum Total Phospholipids*

E U G E N E S. BAGINSKI, Ph .D ., E M A N U E L E PST E IN , Ph .D .,A N D B E N N IE ZAK, Ph .D .

From the Pathology Departments of Saint Joseph Mercy Hospital, Pontiac, MI 48053

William Beaumont Hospital, Royal Oak, MI 48072, and Wayne State University School of Medicine,

Detroit, MI 48201

IntroductionThe structural and functional roles of

phospholipids in the body are well recog­nized and the measurement of phospho­lipids in blood plasma and tissues is helpful in the diagnosis. The application of column and thin layer chromatography to the lipid analysis made it possible to separate phos­pholipids, thus placing the entire lipid re­search in a broader perspective. W ith bet­ter and more refined techniques, it is possi­ble now to study phospholipid composition of blood plasma, tissues, cerebrospinal fluid, etc., and to relate these findings to the condition of the patient. The literature covering the usefulness of phospholipid de­termination is very extensive. It is, however, beyond the scope of this manuscript to dis­cuss it here. This paper will be limited to the various phases of phospholipid analysis which includes extraction, release of inor­ganic phosphate, and the determination of phosphate. Our own version for the deter­mination of phospholipids will also be ofFered.

ExtractionBoth serum and plasma have been used

for phospholipid determination. However,

* Presented at the Applied Seminar on the Clin­ical Pathology of the Lipids, November, 1971.

255

it was suggested that plasma should be used rather than serum, since the latter may become contaminated with phospholipids released from platelets dining coagula­tion.67 When total fat was determined in heparinized and citrated plasma, the value for heparinized plasma was about 13 per­cent higher.67 Similar differences in values were observed when oxalated plasma was used for the determination of phospho­lipids,97 cholesterol,48’103’106 and practically all other lipid components.25 The discrep­ancy was attributed to the alteration of red cell-plasma volumes caused by the presence of citrate or oxalate.97 No significant differ­ence in total phospholipids was found, however, between heparinized and citrated plasma when correction was made for ci­trate dilution.50 A slight increase in ceph- alin content was observed in heparinized plasma having markedly elevated platelet count.50 To avoid errors due to the water shift between erythrocytes and plasma, the use of heparinized plasma50’123 or serum73 was recommended.

Bloor18’22 proposed an ethanol-ether mix­ture, 3:1 v/v, for plasma lipid extraction and showed that the extraction was com­plete when the mixture was brought just to a boil. To eliminate contamination with organic compounds, he evaporated the ex­

256 BAGINSKI, EPSTEIN AND ZAK

tract to dryness and redissolved the lipids in petroleum ether, whereas urea, glucose, amino acids, inorganic salts, and some phosphatides failed to solubilize.45 To obvi­ate the purification step, Folch and Van Slyke45 precipitated lipoproteins with col­loidal iron and extracted lipids from the precipitate with the Bloor’s reagent. Some claimed that the Bloor’s extraction mixture had to be boiled for one hour to completely extract the lipids.73 Others found heating unnecessary, since it did not increase the yield of total lipids25 or phospholipids,24’25’39 and eliminated the step entirely. It was re­ported that the ethanol-ether extract con­tained not only urea and chloride,87 but also inorganic phosphate.26 Using radioactive phosphate, Van Slyke and Sacks118 dem­onstrated the presence of less than 0.05 percent of inorganic phosphate in the ex­tract. Other authors found no contamina­tion of the extract with inorganic phos­phate39’74’107 or organophosphate compounds other than phospholipids.67 To purify the Bloor’s extract, some workers used chloro­form66’122 or ethyl ether24 rather than pe­troleum ether, since the latter was found to be a poor extractant for phospho­lipids. 3̂ 9 ,«,45

Zilversmit and Davis125 described a method for phospholipid determination in blood plasma which did not include ex­traction. These authors precipitated lipo­proteins with trichloroacetic acid and used the precipitate for phosphate determina­tion. WEFn a number of plasma specimens were analyzed for phospholipids, employ­ing the new extraction technique and the ethanol-ether mixture, the results for the two methods were in good agreement. A similar approach to that of Zilversmit and Davis was used earlier by Bessey et al.17 These authors precipitated proteins from leukocytes, destroyed the precipitate with sulfuric-perchloric acid mixture, and ana­lyzed phosphate in the residue. The obvious advantage of this technique seemed to lie

in its ability to separate lipids from organic contaminants other than proteins. However, phospholipids still had to be separated from proteins, before they could be fractionated by chromatography. To accomplish this, Tourtellotte et al114’115 extracted the lipids from the precipitate with alcoholic acetate, Redman92 employed chloroform-methanol- HC1 mixture, McArdle and Zilkha78 used chloroform-methanol, and Hirsch et al59 precipitated protein with the Somogyi rea­gents105 and extracted lipids with the eth­anol-ether mixture. Papadopoulos et al86 observed that some phospholipids escaped precipitation when the technique was ap­plied to cerebrospinal fluid.

Folch et al46 devised a method for ex­traction of lipids from brain tissues by em­ploying chloroform-methanol, 2:1 v/v, mix­ture. To eliminate non-lipid contaminants, they washed the extract with water. How­ever, since about 1 percent of phospho­lipids entered the water phase during wash­ing and were discarded, chlorides of Na+, K+, Ca++, Mg*+ were added to the water to prevent loss of lipids.47 Sperry and Brand107 applied a similar technique but in addition heated the serum-solvent mixture to assure complete extraction. When the latter au­thors compared results for serum phospho­lipids using their modified extraction tech­nique to that of Bloor’s, the results were in close agreement.

The Folch’s method of extraction has be­come popular. However, some found the procedure inadequate when applied to erythrocytes and used chloroform-isopro- panol, 7:11, instead.95 These authors agreed that washing the extract with KC1 was necessary to eliminate inorganic phosphate. Zollner and Warnock128 both heated the extract and washed it with CaCl2. Albrink,1 however, found the heating step unneces­sary. Amenta2 noticed that washing the chloroform-methanol extract with water containing NaCl led to a loss of phospho­lipids, but this was not the case when

THE MEASUREM ENT OF SERUM TOTAL PHOSPHOLIPIDS 257

CaCl2 was employed. Hanahan et al57 ex­tracted phospholipids from red cells with ethanol-ether, washed the extract with a small amount of water and found this pro­cedure as effective in removing non-lipid contaminants as using Folch’s method.

A number of papers have been published in which either of the three methods of extraction are used. However, some work­ers boil the Folch’s mixture, while others heat it to 50 °.93’122 Some may use chloro- form-ethanol, 1:1, at room temperature,35 others prefer ether-methanol,96 or methanol- chloroform, 1:2,104 mixtures. Some reflux the ethanol-ether extract for 1 hour,73 others prefer it cold.25 To redissolve lipids from the dry residue, Bloor22 suggested the use of petroleum ether. However, some authors used methylal-methanol mixture43 or eth­anol-ether.59 Holmes60 indicated a basic dif­ference between extraction from an aque­ous medium or from a dried residue. A particular mixture may extract lipids in the first case but fail in the second. Folch and Van Slyke45 pointed out that, although urea and other organic compounds may not be soluble in petroleum ether alone they will solubilize in the presence of lipids, whereas phosphatides may completely fail to redis­solve. Acidic phospholipids present in or­ganic solvents may form salts with some metals47’75’100 dissolved in the water used for washing. These salts exhibit different partition characteristics from the free phos­pholipids when they are separated by thin layer chromatography, and may also differ in solvent solubilities when extracted from the plates. Lysolecithin, being water-sol­uble on warming,88 may entirely escape the solvent extraction. In spite of the differ­ences in techniques employed for lipid ex­traction, the results obtained for phospho­lipid determination using ethanol-ether vs. chloroform-methanol, 1: l ,89 ethanol-ether vs. chloroform-methanol, 2: l ,66’95’107 or eth- anol-ether vs. precipitation125 seem to agree fairly well.

Phosphate ReleasePhospholipids extracted into organic sol­

vent mixtures, whether present in the orig­inal extract, eluted from a column, or scraped off thin layer chromatographic plates, are subjected to oxidation during which the organic portion is destroyed and inorganic phosphorus is released as phos­phate. Phospholipids precipitated with lipo­proteins, crude tissue homogenates, or any biological fluid, can be treated in the same fashion. The common approach has been the wet acid digestion in which sulfuric or perchloric acids are used in mixtures, in­cluding sulfuric-perchloric,17’71’115 sulfuric- nitric,19’29’67’108 sulfuric-selenious,86 sulfuric- nitric-H20 274; as well as chloric acid,53 nitric acid,5 and nitric-H2O2 .03 Magnesium nitrate94 or calcium acetate54 have been employed in a dry ash approach. By far the most common is the use of sulfuric acid with hydrogen peroxide, as originally sug­gested by Baumann12 and Briggs.27 How­ever, the presence of residual amounts of H20 2 which may remain in the acid residue after completion of digestion is detrimental to the color reaction and must be re­moved.12 It is generally assumed that H2O2

rapidly decomposes during heating. How­ever, Mortimer and Raine82 found even a 15 minute boiling period insufficient to completely decompose hydrogen peroxide and suggested that the excess of H20 2 be titrated with permanganate, while Shin102 added a 5 percent urea solution to the residue. The latter compound was also em­ployed by Dryer et al,38 who found the results more reproducible in the presence of urea. However, urea may inhibit the final color reaction when aminonaphthol- sulfonic acid is used as a reducing agent.91 To destroy H20 2 completely, Bartlett,10 and Shin and Lee101 heated the acid mixture for1.5 hours.

The use of other acid mixtures may also be troublesome. Sulfuric acid volatilizes on heating108 and nitric acid is not recom­

258 BAGINSKI, EPSTEIN AND ZAK

mended75 because it inhibits the heteropoly blue complex formation.44’108’124 Perchloric acid is also volatile on heating,71 and be­cause it is an explosive, its use requires special precautions.79 These acids, either used individually or as mixtures, leave var­iable acidities and volumes at the end of digestion, making it necessary to adjust the final pH, since the color reaction is acid dependent.15’44’71’108 The adjustment is sometimes based on the assumption that a certain amount of acid has evaporated,68 while in other cases alkali is added to neu­tralize the acid to a definite pH.19’53 These steps obviously contribute to dilutions and decrease the sensitivity of the final color. In addition, it is believed that during ash­ing, phosphate atoms combine to form poly-phosphates,30 meta- or pyro-phos- phates or both,4 and these must subse­quently be converted to orthophosphate.12 The conversion is usually accomplished by adding water to the residue and heating for several minutes.12’124 This step further dilutes the final color and limits the min­imum amount of phosphate which can be analyzed. Another important factor should not be overlooked, that is, volatilization of phosphoric acid during digestion.12’58’108 According to Baginski,9 at about 500° orthophosphate is converted to pyrophos­phate and above 600° phosphoric acid salts volatilize. According to Ashton,4 volatiliza­tion can be prevented between 600°-800° by addition of magnesium salts, although conversion to meta- and pyro-phosphates still takes place. However, above 800° phosphate volatilizes even in the presence of Mgw.4 It was reported that losses due to volatilization could occur during evap­oration of sulfuric acid even below 150°, unless the heating was stopped when fumes appeared.58 Goodwin53 believed that the addition of sodium chloride to the digestion mixture could prevent pyro-phosphate for­mation, whereas Baumann12 claimed that no loss of phosphate by volatilization took place when the sulfuric-H20 2 mixture was

used. Martland and Robison77 thought some phosphate could be taken up by glass dur­ing heating. To avoid possible loss of phos­phate through volatilization and or meta- or pyro-phosphate formation, Baginski et al7 added a calcium salt to the nitric acid used for digestion.

When phospholipids are scraped off thin layer chromatographic plates together with silicic acid and then subjected to digestion, a problem may arise. The presence of silicic acid may retard the final color forma­tion36’122 or contribute to the color by virtue of its ability to form silicomolybdic acid.91 To circumvent this difficulty, phospholipids are eluted from silicic acid with various solvent mixtures104 and the eluate digested. A good recovery is claimed with this ap­proach, although the elution may be diffi­cult because the salts formed between some phospholipids and cations100 exhibit extrac­tion characteristics which may be different from those of the free lipids. According to Hruska,62 there is no need to extract phos­pholipids from silica prior to ashing, when the citrate-arsenite system described in this manuscript is used, because silicic acid does not interfere in the reaction.

D eterm ination o f Inorganic PhosphateAfter completion of digestion, inorganic

phosphate is determined in the acid res­idue. The most popular approach has been to use molybdate to form a phosphomolyb- date complex which is then reduced to the blue phosphomolybdous acid. The intensity of the chromophore can be measured at either 700 nm or 840 nm,5 although differ­ent wavelengths have been used, since the peaks are rather broad. The ultraviolet por­tion of the spectrum has also been utilized for the studies of the blue complex.32’38’83

Numerous reducing agents have been employed for the reduction of phospho- molybdate. Reportedly Osmond85 in 1887 was first to use stannous chloride. The method was modified and improved by Deniges34 and Kuttner and Cohen.69 In

THE M EASUREM ENT OF SERUM TOTAL PHOSPHOLIPIDS 259

1914 Taylor and Miller113 used phenylhy- drazine to reduce phosphomolybdate pre­cipitated from ashed samples. Bell and Doisy13 introduced hydroquinone which was later applied by Baumann12 for the determination of phospholipids and by Briggs27 for total acid-soluble phosphate. Fiske and SubbaRow14 introduced 1-amino- 2-naphthol-4-sulfonic acid, Ammon and Hinsberg3 used ascorbic acid, and Gomori52

employed methyl-p-aminophenol sulfate (elon). Other reducing agents were sub­sequently described, including ferrous sul­fate, 1 1 1 stannous chloride-hydrazine, 68 N- phenyl-p-phenylenediamine, 38 monothiol glycerol, 55 metamizol, 56 etc. Most of the reducing agents were employed in various modifications developed through search for a better analytical tool to determine phos­phate concentration under different experi­mental conditions. The prevailing principle has been to find an agent capable of reduc­ing phosphomolybdate without reducing molybdate. This was accomplished by se­lecting reaction conditions most suitable for the agent used.

The reaction between molybdate, phos­phate, and a reducing agent is highly acid- dependent. Below a certain range of acidity molybdate is reduced, whereas above the range the color formation due to the reduc­tion of phosphomolybdate is retarded.10’15’ 27,52,70,108 Each reducing agent seems to operate best within a certain acidity range, although there is a substantial overlap in acidity requirements from one agent to another. Berenblum and Chain15 studied the effect of stannous chloride and amino- naphtholsulfonic acid on the reduction of phosphomolybdate and concluded that, although the former produced a more in­tense color, the latter agent could tolerate a wider pH range. Man and Peters74 carried out similar studies earlier comparing ami- nonaphtholsulfonic acid with hydroqui­none, using the method described by Bell and Doisy13 or its Briggs’ 27 modification. According to Sumner, 1 1 1 the chief disadvan­

tage of stannous chloride was its ability to reduce molybdate itself, unless a relatively high acidity was employed. The author sug­gested that stannous chloride be replaced by ferrous sulfate and acknowledged that the latter reagent was not stable. The rea­gent was later stabilized somewhat with thiourea . 51 Taussky and Shorr112 employed ferrous sulfate in their method for phos­phate determination, but found that the final reaction mixture had to be at least 1 N in sulfuric acid.

Stanford and Wheatley108 studied the effect of hydroquinone on phosphomolyb­date and observed that an increase in acid­ity caused the color to become more in­tense, but the system failed to stabilize; at a lower acidity the color was more stable, but the intensity was low. The rate of the reaction and the intensity could be in­creased when the final color was developed in a boiling water bath .14 Lowry et al71

found that ascorbate produced a highly sensitive reaction at a strongly acid pH when the mixture was incubated at 38° for two hours. Using the same reducing agent, McClare79 incubated the mixture at 50° for one hour. Heating was also applied to the aminonaphtholsulfonic acid reaction by Zinzadze127 who used 100° for 30 minutes. Bessey and Lowry16 heated at 60° for 1.5 hours, and others10’6 1’86’93’102 used 1 0 0 ° but limited the time of heating to several min­utes. The purpose of heating was to in­crease sensitivity and stability of the color, but the step contributed to a poor repro­ducibility, 84 especially at 100° . 16 The use of l-amino-2-naphthol-4-sulfonic acid, as introduced by Fiske and SubbaRow, 44 is by far the most frequently used method among biochemists, in spite of the fact that it has been criticized for lack of reproducibility , 38

narrow range of acid tolerance, 10 poor sen­sitivity,15 ’84 lack of linearity , 109 instability of the final color, 52 and turbidity formation. 52

Zinzadze126 described an interesting ap­proach to phosphate determination which does not require a reducing agent. A mix­

260 BAGINSKI, EPSTEIN AND ZAK

ture of molybdic acid, M0 O3 and MoOg produces a blue color in the presence of phosphate. The method was subsequently modified98 and automated . 6 Phosphomolyb- date was also found to form a very sensi­tive chromophore with the dye Malachite green28’64*110 and Methyl green 00.117

Since phosphomolybdate itself absorbs in the ultraviolet region of the spectrum, it is possible to eliminate the reduction step or the reaction of the complex with dyes and determine it directly. Usually the complex is extracted into an organic solvent first be­fore it is measured. Spectra of the complex were studied in various organic solvents119

and good sensitivity was obtained . 32’33 The measurement of phosphomolybdate was also applied to the determination of phos­phate in biological fluids. 37’83’116 A molyb- dovanadophosphoric acid complex forma­tion with absorption at 315 nm was described by Misson81 and Michelsen.80

The spectral characteristics of the complex were studied by Gee and Deitz,49 and the principle was applied to phosphate deter­mination in biological media .1 1 ’76

With the increased emphasis on automa­tion in recent years it appeared desirable to automate phospholipid determination. Two steps are usually involved in automa­tion: ( 1 ) destruction of the organic portion, and (2 ) inorganic phosphate determina­tion. The latter is not difficult since it in­cludes only the colorimetric step, the former, however, is rather involved. A number of workers automated only the color reaction, 68’72’12 1 whereas others, using the Technicon system, automated both steps. 90’120 The extraction is still done by hand.

ExperimentalR eagents

Phosphate Stock Standard, 1 mg P per1.0 ml. Desiccated potassium dihydrogen phosphate (438.1 mg) is dissolved in water and the volume made up to 1 dl.

Phosphate Working Standard, 5 mg P per dl. Five ml of the stock phosphate standard are diluted to 1 dl with water.

Two percent ascorbic acid in 10 percent trichloroacetic acid (A-TCA).

One percent ammonium molybdate tetra- hydrate (AM).

Arsenite-Citrate solution (AC). Sodium citrate dihydrate ( 2 0 . 0 g) and anhydrous sodium arsenite (2 0 . 0 g) are dissolved in water, 2 0 . 0 ml of glacial acetic acid are added and the volume made up to 1L.

Absolute ethanol-ethyl ether mixture, 3:1 v/v.

Nitric acid-calcium nitrate solution (NAC). Thirty mg of calcium carbonate are dissolved in 1L of concentrated nitric acid.

Antibumping granules. These granules (BDH anti-bumping granules, Gallard- Schlesinger Chemical Mfg. Corp., Carle Place, NY 11514), are prewashed by boil­ing them in concentrated nitric acid for several minutes. They are then rinsed with distilled water and dryed.

P rocedure

Fifty /j.I of serum are pipetted into 13 X 75 mm test tube containing 2.0 ml of ethanol-ether mixture. This is best done by vortexing the solvent while adding the serum. The tube is stoppered with Parafilm and centrifuged at about 3,000 rpm for three minutes.

One ml aliquot of the clear supernatant fluid is transferred into 25 X 150 mm boro- silicate glass tube and the contents evapo­rated to dryness. Two other 25 X 150 mm tubes are prepared, one for the reagent blank and the other for the standard. Fifty /¿I of the working phosphate standard are added to the latter tube.

Two ml of the NAC solution are pipetted into each tube and three antibumping gran­ules are then added. The tubes are heated over Bunsen burners and the acid boiled off continually until yellow fumes of nitrogen

THE M EASUREM ENT OF SERUM TOTAL PHOSPHOLIPIDS 261

oxides appear. At this time there is little or no liquid left in the tube. The heating step is continued uninterrupted until no more fumes are present. The entire process takes about three minutes. Overheating or ex­tending the heating period for a short time beyond that necessary for complete disap­pearance of fumes seems to have no detri­mental effect on recoveries.

One ml of A-TCA is added to the tube followed by 0.5 ml of the AM reagent; a thorough mixing at this point is essential. One ml of AC is added and the mixing repeated again.

Fifteen minutes are allowed for complete color development. The stable absorbances of the sample and the standard are deter­mined against the reagent blank using a spectrophotometer set at 700 nm or 840 nm.

Calculations

Absorbance of Sample „e _ mo.Absorbance of Standard

phospholipid phosphate per dl

DiscussionThere seems to be no uniform agreement

as to whether serum or plasma should be used for phospholipid determination. If plasma is used, volume changes due to water shift between red cells and plasma can be avoided by employing heparin as the anticoagulant. When using tartrate as anticoagulant it is doubtful whether one can apply a correction for the water shift between plasma and cells as suggested. 50

Shrinkage of cells is not directly propor­tional to the amount of anticoagulant pres­ent and the degree of cell shrinkage does not follow a regular pattern .40

When the ethanol-ether mixture is em­ployed for phospholipid extraction, 18 a proper technique must be used .2 1 ’22’23 Se­rum is slowly added to the continually agitated solvent. A well dispersed fine pre­cipitate should be obtained, or the extrac­tion will be incomplete. It has been stated

that the greatest source of error lies in the extraction process. 12 The tube is then imme­diately well stoppered with Parafilm and centrifuged any time after two minutes of standing at room temperature. The precip­itate forms a tightly packed button on the bottom of the tube and a portion of the clear supernatant fluid can be easily with­drawn. When the chloroform-methanol mixture, 2 : 1 v/v, is used instead of ethanol- ether, a fluffy precipitate results which seems more difficult to separate from the supernatant.

The ethanol-ether is an effective extract­ant for lipids. It was reported that when lipoproteins were precipitated with TCA and lipids extracted with ether-ethanol or chloroform-methanol mixture, the former solvent produced a higher yield. 114

The nitric acid-Ca++ ashing technique utilized here has some highly desirable features. There is no loss of phosphate or rearrangement to an unreactive form dur­ing ashing, even if the digestion is unduly prolonged. Nitric acid is completely volatil­ized during the short three-minute diges­tion period and no residual acidity remains which must be neutralized or diluted. De­struction of the organic material is also complete when serum or red cell extracts are ashed, and there is no need for hydro­gen peroxide, as suggested by Ichida and Hine . 63 However, a strict adherence to the described procedure is essential for success­ful analysis. The extract should be evapo­rated to dryness before nitric acid is added. This step may be performed by placing the tubes containing aliquots of the extract in a sand bath set at a temperature sufficiently low to prevent excessive boiling, otherwise losses may be encountered. Three acid- washed boiling stones (see Procedure) are placed in the tubes followed by addition of nitric acid-Ca++. The tubes are placed over open flames of multiple micro-bumers and the acid boiled continually until no more liquid remains and all yellow fumes of

262 BAGINSKI, EPSTEIN AND ZAK

nitrogen oxides disappear. When raw se­rum or tissue homogenates are digested one may occasionally find a few black particles left on the side of the tube. In such a case the side of the tube can be exposed to the flame for a few seconds until all black par­ticles disappear.

After the TCA-ascorbic acid and ammo­nium molybdate reagents have been added to the dry residue, it is essential to mix the content very well before the citrate-arsenite addition follows. During digestion nitric acid refluxes and creeps up the walls of the tube carrying phosphate with it. Therefore, after the addition of TCA-ascorbate and molybdate reagents, it is important to vor­tex the contents up and down to wash phosphate off the walls. The 1.5 ml liquid volume is sufficient for thorough washing. After the citrate-arsenite reagent has been added, phosphate, washed off the walls at this point, will not react. After the analysis is complete the tubes should be rinsed with distilled water, since phosphate tends to adhere to glass when the tubes are left to dry, and rinsing alone may not be helpful. Boiling in concentrated nitric acid followed by a thorough rinsing with distilled water is then necessary before the tubes can be used again. New tubes should be treated in the same fashion before they can be used. When the absorbance of the reagent blank is determined against water at 700 nm in a 1 cm cuvet, it is usually 0.03 to0.04. It may vary slightly according to im­purities present in reagents, but should be constant for each set of reagents. An in­crease in the blank absorbance may indi­cate that some contamination has occurred.

The described procedure has an addi­tional feature which makes the method more flexible and can expand its range of applicability. The reaction can tolerate a wide range of acidity in the final mixture. This indicates that when the use of a dif­ferent acid is more desirable for digestion, the residual acid should not interfere in the reaction. However, there seems no need to

employ any other acid since nitric acid is capable of destroying serum extracts or small amounts of raw serum or tissue ho­mogenates. Since the final color is sensitive, the procedure requires a very small sample which is easily destroyed. A high acidity leads to precipitation of citric acid and the color is not as stable as it is at a lower acid strength. Lowry and Lopez70 using ascorbic acid studied the effect of acidity on the reaction and concluded that the reaction could proceed between pH 0.4 to 0.9 and 2.8 to 4.6 but not at intermediate pH levels because a large blank resulted. They also found that an increase in ascorbate led to a continuous color development. The final pH in the citrate-arsenite reaction is about1.5 and the blank is negligible. In our pro­cedure, a 1 0 -fold increase in ascorbic acid concentration has no effect. Chen et al29

also analyzed the effect of acidity, ascorbic acid concentration, time necessary for color development, temperature, etc. They found the final acid concentration optimum to be0.5 to 1.0 N. Above 1.0 N no reduction took place and below 0.1 N the blank itself de­veloped color. These authors also stated that the color did not develop fully even after three hours of standing at room tem­perature, but at 37° it developed com­pletely within one hour.

Inorganic phosphate determination fol­lowing digestion does not require precau­tions concerning the presence of phosphate esters which may hydrolyze and release inorganic phosphate. For this reason rigid control of acidity seems unnecessary. How­ever, since the final reaction is highly acid- dependent in various methods used the acidity must generally be adjusted before the reaction is carried out. Furthermore, any pyro- or meta-phosphate must be con­verted to orthophosphate. Although the ash does not contain organic compounds which may interfere in the color reaction, inor­ganic contaminants such as arsenate or silicate may be present and they are known to interfere10 ’29’38,99 by forming arseno- and

THE M EASUREM ENT OF SEBUM TOTAL PHOSPHOLIPIDS 263

silico-molybdate complexes. However, these do not interfere in the citrate-arsenite sys­tem, nor is there a need to adjust acidity.

The final acid concentration employed in the citrate-arsenite system was chosen pur­posely to enable one to analyze inorganic phosphate in the presence of acid-labile phosphate-containing organic compounds known to be present in plasma.20’42’129 Fur­thermore, the method was adapted to the determination of Glucose-6-phosphatase in tissue homogenates8 which contain appreci­able amount of acid-labile phosphate esters. Under the relatively mild acid conditions employed here, one would expect molyb- date to undergo reduction in the absence of phosphate. Indeed, when a mixture of TCA, ascorbic acid, and molybdate is al­lowed to stand at room temperature, the solution becomes blue and turbidity de­velops,5 indicating some reduction of mo­lybdate itself. However, this phenomenon does not occur when arsenite-citrate is pres­ent. The purpose of the citrate addition is to complex molybdate and prevent the lat­ter from reduction by ascorbic acid. Oxalic acid was employed for the same purpose by James and Allen65 in their method for in­organic phosphate determination. Marsh76 also used citrate to bind molybdate after phosphomolybdate was extracted into buta­nol. The ability of citrate and other car- boxylic acids to bind molybdate was re­ported by Davies and Davies.31 In the citrate-arsenite system described by the present authors, the presence of citrate con­tributes to an increase in sensitivity, pre­vents reduction of molybdate, and stabil­izes the system. The role of arsenite is not clear, although it also contributes some­what to the overall increase in sensitivity.

Sum m aryThe various aspects of phospholipid de­

termination in blood plasma are discussed, including the extraction of phospholipids, release of phosphate from the organic por­tion, and the colorimetric determination of

inorganic phosphate. A method for phos­pholipid determination is proposed which is simple, rapid, and relatively free of the various shortcomings encountered in other methods.

References1. A l b r i n k , M. J.: The microtitration of total

fatty acids of serum, w ith notes on the esti­mation of triglycerides. J. L ipid Res. 2:53-59, 1959.

2. A m e n t a , J. S.: A rapid extraction and quan­tification of total lipids and lipid fractions in blood and feces. Clin. Chem. 26:339-346, 1970.

3. A m m o n , R. a n d H i n s b e r g , K.: Colorimetri- sche Phosphor- und Arsensaure-bestimmung m it Ascorbinsäure. Physiol. Chem. 239:207- 216, 1936.

4. A s h t o n , F. L.: Influence of the tem perature of ashing on the accuracy of the determina­tion of phosphorus in grass. J. Soc. Chem. Ind. 55:106-108, 1936.

5. B a g i n s k i , E. S. a n d Z a k , B . : Microdetermina­tion of serum phosphate and phospholipids. Clin. Chem. Acta 5:834—838, 1960.

6. B a g i n s k i , E . S., E p s t e i n , E . , a n d Z a k , B . : Automation of methods for the determination of serum phosphorus. Clin. Chim. Acta 20: 76-80, 1964.

7. B a g i n s k i , E. S., W e i n e r , L. M., a n d Z a k , B .: The simple determination of nucleotide phos­phorus. Clin. Chim. Acta 20:378—379, 1964.

8. B a g i n s k i , E. S., F o a , P. P., a n d Z a k , B . : Glucose-6-Phosphatase. Methoden der E n­zymatischen Analyse. Verlag Chemie W ein- heim/Bergstr. vol. 2:837-843, 1970.

9. B a g i n s k i , S.: Mikroveraschung. Einige prak­tische Hinweise. Z. Wiss. Mikr. 55:241-248, 1938.

10. B a r t l e t t , G. R.: Phosphorus assay in column chromatography. J. Biol. Chem. 234:466-468, 1959.

11. B a r t l e t t , E. M. a n d L e w i s , D. H.: Spectro- photometric determination of phosphate es­ters in the presence and absence of ortho­phosphate. Anal. Biochem. 36:159-167, 1970.

12. B a u m a n n , E. J.: On the estimation of or­ganic phosphorus. J. Biol. Chem. 59:667-674, 1924.

13. B e l l , R. D . a n d D o i s y , E. A.: Rapid colori­metric methods for the determination of phosphorus in urine and blood. J. Biol. Chem. 44:55-67, 1920.

14. B e n e d i c t , S. R. a n d T h e i s , R. C.: A modifi­cation of the molybdic method for the deter­mination of inorganic phosphorus in serum. J. Biol. Chem. 62:63-66, 1924.

15. B e r e n b l u m , I . a n d C h a i n , E .: Studies on the colorimetric determination of phosphate. Biochem. J. 32:286-294, 1938.

264 BAGINSKI, EPSTEIN AND ZAK

16. B e s s e y , O. A. a n d L o w r y , O. H.: Factors influencing the riboflavin content of the cornea. J. Biol. Chem. 155:535-643, 1944.

17. B e s s e y , O. A., L o w r y , O. H., a n d B r o c k , M. J.: The quantitative determination of as­corbic acid in small amounts of white blood cells and platelets. J . Biol. Chem. 264:197- 205, 1946.

18. B l o o r , W. R.: A method for the determina­tion of fat in small amounts of blood. J. Biol. Chem. 27:377-384, 1914.

19. B l o o r , W. R.: Studies on blood fat. II. Fat absorption and the blood lipoids. J. Biol. Chem. 23:317-326, 1915.

20. B l o o r , W. R.: The distribution of phosphoric acid in normal human blood. J. Biol. Chem. 36:49-57, 1918.

21. B l o o r , W. R.: Methode nephelometrique pour la determination de l’acide phospho- rique et de ses composes contenus dans de petites quantités de sang. Bull. Soc. Chim. Biol. 3:451-475, 1921.

22. B l o o r , W. R.: The determination of small amounts of lipid in blood plasma. J. Biol. Chem. 77:53-73, 1928.

23. B l o o r , W. R.: The oxidative determination of phospholipid (lecithin and cephalin) in blood and tissues. J. Biol. Chem. 82:273-286, 1929.

24. B o y d , E. M.: L o w phospholipid values in dog plasma. J. Biol. Chem. 91:1-12, 1931.

25. Boyd, E. M.: Extraction of blood lipids. J. Biol. Chem. 114:223-234, 1936.

26. B r a n t e , G.: Studies on lipids in the nervous system with special reference to quantitative chemical determination and typical distribu­tion. Acta Physiol. Scand. 28:Suppl. 63, 1 - 189, 1949.

27. B r ig g s , A. P.: Some applications of the col­orimetric phosphate method. J. Biol. Chem. 59:252-264, 1924.

28. C h a l v a r d j i a n , A. a n d R u d n ic k i , E.: Deter­mination of lipid phosphorus in the nano­molar range. Anal. Biochem. 36:225-227,1970.

29. C h e n , P. S., T o r ib a r a , T . Y., a n d W a r n e r ,H. : Microdetermination of phosphorus. Anal. Chem. 28:1756-1758, 1956.

30. C o h e n , L . E. a n d C h e c h , F. W.: The deter­mination of phosphorus in organic com­pounds via flash combustion. Chemist-Analyst 47:86-87, 1958.

31. D a v ie s , D . R. a n d D a v ie s , W. C.: The col­orimetric determination of phosphorus in the presence of interfering substances. Biochem. J. 26:2046-2055, 1932.

3 2 . D e l s a l , J . L . a n d M a n h o u r i , H.: Etude comparative des dosages colorimetriques du phosphore. II. Spectrophotometrie dans l’ul- tra-violet. Bull. Soc. Chim. Biol. 40:1169- 1177, 1958.

33. D e l s a l , J. L . a n d M a n h o u r i , H.: Etude comparative des dosages colorimetriques du

phosphore. III. Dosage de l’orthophosphate en presence d’esters phosphoriques. Bull. Soc. Chim. Biol. 40:1179-1187, 1958.

34. D e n i g e s , G.: Reaction de coloration extre- m ent sensible des phosphates et de arsenates. Compt. Rend. Acad, de Sciences. 171:802, 1920.

35. D o d g e , J. a n d P h i l l i p s , G. B.: Composition of phospholipid fatty acids and aldehydes in human red cells. J. Lipid Res. 8:667-675, 1967.

36. D o i z a k i , W. M. a n d Z i e v e , L . : Quantitative estimation of some phosphatides and their hydrolysis products by th in layer chromatog­raphy. Proc. Soc. Exp. Biol. Med. 223:91-94, 1963.

37. D r e i s b a c h , R. H.: Submicrogram determina­tion of inorganic phosphate in biological ma­terial. Anal. Biochem. 20:169—171, 1965.

38. D r y e r , R . L., T a m m e s , A. R ., a n d R o u t h ,I . : The determination of phosphorus and phosphatase w ith N-phenyl-p-phenylenedia- mine. J. Biol. Chem. 225:177-183, 1957.

39. E g s g a a r d , J.: On the determination of the phosphatide content of serum. Acta Physiol. Scand. 26:171-178, 1948.

40. E i s e n m a n , A. J.: The effect of potassium ox­alate on electrolytes of blood and plasma. J. Biol. Chem. 71:587-605, 1926-27.

41. E l l i s , G. a n d M a y n a r d , L. A.: The deter­mination of phospholipids in bovine blood. J. Biol. Chem. 228:701-709, 1937.

42. F e i g l , J.: U ber das Vorkommen von Phos- phaten im menschlichen Blutserum. II. Saur- eloslicher (G esam t-) Phosphor, vorgebildetes Orthophosphat und “Restphosphor” beim Gesunden. Biochem. Z. 83:82—95, 1917.

43. F i l l e r u p , D. L. a n d M e a d , J. F . : Chroma­tographic separation of the plasma lipids. Proc. Soc. Exp. Biol. Med. 83:574-577, 1953.

44. F i s k e , C. H. a n d S u b b a R o w , Y.: The colorimetric determination of phosphorus. 66:375-400, 1925.

45. F o l c h , J. a n d V a n S l y k e , D. D .: Prepara­tion of blood lipid extracts free from non­lipid extractives. Proc. Soc. Exp. Biol. Med. 41:514-515, 1939.

46. F o l c h , J., A s c o l i , I., M e a t h , J. A ., a n d L e B a r o n , F . N.: Preparation of lipid ex­tracts from brain tissue. J. Biol. Chem. 292:833-841, 1951.

47. F o l c h , J., L e e s , M . , a n d S l o a n e S t a n l e y ,G. H.: A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509, 1957.

48. G a r d n e r , J. A., G a i n s b o r o u g h , H., a n d M u r r a y , R .: CXCI. Studies on the cholesterol content of normal hum an plasma. VIII. A note on the effect of anticoagulants. Biochem. J. 32:1457-1459, 1938.

49. G e e , A. a n d D e i t z , V. R.: Determ ination of phosphate by differential spectrophotometry. Anal. Chem. 25:1320-1324, 1953.

THE M EASUREM ENT OF SERUM TOTAL PHOSPHOLIPIDS 265

5 0 . G j o n e , E. a n d O r n in g , O . M .: Plasma phospholipids in patients with liver disease. A quantitative thin layer chromatographic study. J. Clin. Lab. Invest. 2 8 :2 0 9 - 2 1 6 , 1 9 6 6 .

51. G o l d e n b e r g , H . a n d F e r n a n d e z , A.: Simplified method for the estimation of inorganic phosphorus in body fluids. Clin. Chem. 22:871-882, 1966.

52. G o m o r i , G . : A modification of the colori­metric phosphorus determination for use with the photelectric colorimeter. J. Lab. Clin. Med. 27:955-960, 1942.

53. G o o d w i n , J. F.: Total, phospholipid and labile phosphorus in serum and tissue em­ploying chloric acid and N-phenyl-p-phenyl- enediamine. Proc. Soc. Exp. Biol. Med. 100: 217-219, 1959.

54. G r e e n , H . H . : Studies in mineral metabolism. IV. Determination o f phosphorus compounds in blood by dry combustion. J. Agr. Sci. 28: 372-375, 1928.

55. G r i n d e y , G . B. a n d N i c h o l , C. A.: Micro procedure for determination of pyrophos­phate and orthophosphate. Anal. Biochem. 33:114-119, 1970.

56. G u i r g i s , F. K. a n d H a b i b , Y. A.: Use of a new reducing agent, Metamizol, in determin­ing inorganic phosphorus in blood and urine. Clin. Chem. 17:78-81, 1971.

5 7 . H a n a h a n , D. J., W a t t s , R. M ., a n d P a p p a - j o h n , D.: Some chemical characteristics of the lipids of human and bovine erythrocytes and plasma. J. Lipid Res. 2:421-432, 1960.

58. H il l e b r a n d , W. F. a n d L u n d e l l , G. E. F.: Volatilization losses of phosphorus during evaporations of phosphates with sulfuric acid or fusions with pyrosulfate. J. Amer. Chem. Soc. 42:2609-2615, 1920.

59. H i r s c h , J. a n d A h r e n s , H . : The separation of complex lipid mixture by the use of silicic acid chromatography. J. Biol. Chem. 233:311-320, 1958.

60. H o l m e s , F. E.: A turbulent drier-extractor and a wide-interface extractor. A method for determining total lipid in serum, feces, and urine. Clin. Chem. 10:1007-1024, 1964.

61. H o r e c k e r , B. L., M a , T. S., a n d H a s s , E.: Note on the determination of microquantities of organic phosphorus. J. Biol. Chem. 136: 775-776, 1940.

62. H r u s k a , K. J.: Interference of pyrophosphate in the determination of orthophosphate. Clin. Chim. Acta 22:454^155, 1968.

63. I c h id a , T. a n d H i n e , N.: Improvement of Baginski’s method for phosphorus determina­tion. Clin. Chim. Acta 23:378—379, 1969.

64. I t a y a , K. a n d Ui, M.: A new micromethod for the colorimetric determination of in­organic phosphate. Clin. Chim. Acta 24:361- 366, 1966.

65. J a m e s , R. a n d A l l e n , J . L.: The estimation

of phosphorus. Biochem. J. 34:858-865, 1940.

6 6 . J e s t i n g , E. a n d B a n g , H. O.: Methods of lipid extraction from blood plasma. Scand. J . Clin. Lab. Invest. 25:654-656, 1963.

67. K i r k , E. K ., P a g e , I. H., a n d V a n S l y k e ,D. D.: Gasometric microdetermination of lipids in plasma, blood cells and tissues. J . Biol. Chem. 206:203-234, 1934.

6 8 . K r a m l , M.: A semi-automated determina­tion of phospholipids. Clin. Chim. Acta 23: 442-448, 1966.

69. K u t t n e r , T. a n d C o h e n , H. R . : Micro colorimetric studies. I. A molybdic acid, stannous chloride reagent. The micro esti­mation of phosphate and calcium in pus, plasma, and spinal fluid. J . Biol. Chem. 75: 517-531, 1927.

70. L o w r y , O. H. a n d L o p e z , J. A.: The deter­mination of inorganic phosphate in the pres­ence of labile phosphate esters. J. Biol. Chem. 162:421-428, 1946.

71. L o w r y , O. H., R o b e r t s , N. R . , L e i n e r , K . Y., Wu, M„ a n d F a r r , A. L . : The quantitative histochemistry o f brain. I. Chemical Methods. J. Biol. Chem. 207:1-17, 1954.

72. M a l l e i n , R . , P i n a t e l , H., a n d R i v i e r e , J . F . : Determination semi-atomatique des phospholipides seriques. Ann. Biol. Clin. 28: 257-264, 1970.

73. M a n , E. B. a n d G i l d e a , E. F . : A modifica­tion of the Stoddard and Drury titrimetric method for the determination of the fatty acids in blood serum. J. Biol. Chem. 99:43- 60, 1932.

74. M a n , E. B. a n d P e t e r s , J . P . : Gravimetric determination of serum cholesterol adapted to the Man and Gildea fatty acid method, with a note on the estimation of lipoid phosphorus. J . Biol. Chem. 101:685-695, 1933.

75. M a r i n e t t i , B. V .: Chromatographic separa­tion, identification, and analysis of phos- phatides. J . Lipid Res. 3:1-20, 1962.

76. M a r s h , B. B.: The estimation of inorganic phosphate in the presence of adenosine triphosphate. Biochim. Biophys. Acta 32: 357-361, 1959.

77. M a r t l a n d , M . a n d R o b i s o n , R . : XCVII. Note on the estimation of phosphorus in blood. Biochem. J. 28:765-768, 1924.

78. M c A r d l e , B. a n d Z i l k h a , K . J . : The phos­pholipid composition of cerebrospinal fluid in neurologic disorders. Brain 85:389-402, 1962.

79. M c C l a r e , C . W. F . : An accurate and con­venient organic phosphorus assay. Anal. Biochem. 39:527-530, 1971.

80. M i c h e l s e n , O. B.: Photometric determina­tion of phosphorus as molybdovanadophos- phoric acid. Anal. Chem. 29:60-62, 1957.

266 BAGINSKI, EPSTEIN AND ZAK

81. M i s s o n , G.: Colorimetrische Phosphorbestim- mung in Stahl. Chem.-Ztg. 32:633-634, 1908.

82. M o r t i m e r , J. G. a n d R a i n e , D. N.: An improved estimation of phosphorus following digestion with hydrogen peroxide. Anal. Biochem. 9:492-495, 1964.

83. M o z e r s k y , S. M ., P e t t i n a t i , J. D., a n d

K o l m a n , S. D.: An improved method for the determination of orthophosphate suitable for assay of adenosine triphosphatase activity. Anal. Chem. 38:1182-1187, 1966.

84. N o r b e r g , B.: The working out of a method for histo- and cyto-chemical determination of phosphorus. Acta Physiol. Scand. 5:1-99, 1942.

85. O s m o n d , F.: Sur une reaction pouvantservir au dosage colorimetrique du phosphore dans les fontes, les aciers, etc. Bull. Soc.Chim. 47:745, 1887.

8 6 . P a p a d o p o u l o s , N. M., C e v a l l o s , W., a n d H e s s , W. C .: Determination of phospholipids in spinal fluid and brain. Arch. Neurol. 3: 677-682, 1960.

87. P e r n o k i s , E . W., F r e e l a n d , M. R., a n d

K r a u s , I.: The determination of blood lipids in blood dyscrasias. J. Lab. Clin. Med. 26: 1978-1980, 1941.

8 8 . P h i l l i p s , B. M. a n d R o b i n s o n , N.: Quanti­tative thin layer chromatography of cerebro­spinal fluid phospholipids. Clin. Chim. Acta 8:832-842, 1963.

89. P h i l l i p s , G. B.: The isolation and quantita­tion of the principle phospholipid compo­nents of human serum using chromatography on silicic acid. Biochim. Biophys. Acta 29: 594-602, 1958.

90. P o l l a r d , F. H., N ic k l e s s , G., R o g e r s , D. E., a n d R o t h w e l l , M. T.: Quantitative in­organic chromatography. Part XII. Automatic analysis of phosphorusanion mixtures by anion exchange chromatography. J. Chro- matog. 17:157-167, 1965.

91. R a e , J. J. a n d E a s t c o t t , E . V.: The effect of urea and sodium chloride on the colori­metric determination of organic phosphate by King’s method. J. Biol. Chem. 129:255- 262, 1939.

92. R e d m a n , C. M.: Phospholipid metabolism in intact and modified erythrocyte membranes. J. Cell Biol. 49:35-49, 1971.

93. R o b i n s o n , N . a n d P h i l l i p s , B. M.: Quantita­tive thin layer chromatography of serum phospholipids. Clin. Chim. Acta. 8:385-392, 1963.

94. Roepke, R. R.: Determination of phosphorus fractions in blood serum. Ind. & Eng. Chem. Anal. Ed. 7:78, 1935.

95. R o s e , H. G. a n d O k l a n d e r , M.: Improved procedure for the extraction of lipids from human erythrocytes. J. Lipid Res. 6:428-431,1965.

96. R o s e n t h a l , A. F. a n d C h i n g - H s i e n , S.: A simple nonchromatographic determination of

glycerophosphatide in serum. Anal. Chem. 15:197-203, 1969.

97. S c h m i d t , L . H.: The nature of the difference in phospholipid content of oxalated and heparinized plasma. J. Biol. Chem. 109:448- 453, 1935.

98. S c h r i c k e r , J. A. a n d D a w s o n , P. R.: Im­proved molybdenum blue reagent for deter­mination of phosphorus and arsenic. J. Assoc. Off. Agr. Chem. 22: 167-176, 1939.

99. S h e n , C. Y. a n d D y r o f f , D. R.: Determina­tion of phosphate in presence of silicates by molydenum bine method. Anal. Chem. 34: 1367-1370, 1962.

100. S h i m o j o , T., K a n o h , H., a n d O h n o , K .: The elution behaviors of acidic phospholipids on column chromatography. J. Biochem. 69: 255-263, 1971.

101. S h i n , Y. S . a n d L e e , J. C.: Microdetermina­tion of cholesterol and phospholipid in cerebrospinal fluid and serum by silicic acid column chromatography. Clin. Chem. 8:598- 605, 1962.

102. S h i n , Y. S .: Spectrophotometric ultramicro­determination of inorganic phosphorus and lipid phosphorus in serum. Anal. Chem. 34: 1164-1166, 1962.

103. S h o p e , R. E.: Differences in serum and plasma content of cholesterol ester. J. Biol. Chem. 80:125-126, 1928.

104. S k i p s k i , V. P . , P e t e r s o n , R. F., a n d B a r ­c l a y , M.: Quantitative analysis of phospho­lipids by thin layer chromatography. Bio­chem. J. 90:374-378, 1964.

105. S o m o g y i , M.: Method for preparation of blood filtrates for determination of sugar. J. Biol. Chem. 86:655-663, 1930.

106. S p e r r y , W . M . a n d S c h o e n h e i m e r , R.: A comparison of serum, heparinized plasma, and oxalated plasma in regard to cholesterol content. J. Biol. Chem. 110:655-648, 1935.

107. S p e r r y , W . M. a n d B r a n d , F. C.: The deter­mination of total lipids in blood serum. J. Biol. Chem. 213:69-76, 1955.

108. S t a n f o r d , R. V. a n d W h e a t l e y , A. H. M .: The estimation of phosphorus compounds in blood. Biochem. J. 19:697-705, 1925.

109. S t a n t o n , M. G .: Colorimetric determina­tion of inorganic phosphate in the presence of biological material and adenosine tri­phosphate. Anal. Biochem. 22:27-34, 1968.

110. S t e p a n o v a , I.: Bestimmung des anorgan- ischen Phosphors in einer Mikromenge von Serum. Clin. Chim. Acta. 16:330-332, 1967.

111. S u m m e r , J . B.: A method for the colori­metric determination of phosphorus. Science 100:413-414, 1944.

112. T a u s s k y , H. H. a n d S h o r r , E.: A micro- colorimetric method for the determination of inorganic phosphorus. J. Biol. Chem. 202: 675-685, 1953.

113. T a y l o r , A. E. and M i l l e r , C. W . : On the estimation of phosphorus in biological ma­terial. J. Biol. Chem. 18:215-224, 1914.

THE M EASUBEM ENT OF SERUM TOTAL PHOSPHOLIPIDS 267

114. T o u r t e l l o t t e , W. W., V a n d e r , A. J . , S k r e n t n y , B. A., a n d D e J o n g , R. N.: A study of lipids in the cerebrospinal fluid. II. The determination of total lipids. J . Lab. Clin. Med. 52:481-490, 1958.

115. T o u r t e l l o t t e , W. W., P a r k e r , F. M., a n d D e J o n g , R . N .: A study of lipids in the cerebrospinal fluid. III. The determina­tion of total phospholipids. J. Lab. Clin. Med. 52:491-495, 1958.

116. T u r a k u l o w , J. C., K u r g u l e v a , L. I., a n d G a g e l g a n c , A. I.: Determination of in­organic phosphate in biological materials. Biokhim. 32:106-110, 1967.

117. V a n B e l l e , N.: New and sensitive reaction for automatic determination of inorganic phosphate and its application to serum. Anal. Biochem. 33:132-142, 1970.

118. V a n S l y k e , D. D. a n d S a c k s , J.: Prepara­tion of serum lipid extracts free of inorganic phosphate. J. Biol. Chem. 200:525-528, 1953.

119. W a d e l i n , C. a n d M e l l o n , M . G .: Extraction of heteropoly acids. Application to determina­tion of phosphorus. Anal. Chem. 25:1668- 1673, 1953.

120. W e i n s t e i n , L. H., B o z a r t h , R. F., P o r t e r , C. A., M a n d l , R. H., a n d T w e e d y , B . G.: Automated analysis of phosphorus containing compounds in biological materials. Contrib. Boyce Thompson Inst. 22:389-397, 1964.

121. W h i t l e y , R. W . a n d A l b u r n , H. E.: A semi-automated method for the determination of serum phospholipids. Technicon Inter­national Symposium, New York, 1946.

122. W i l l i a m s , J. H., K u c h m a k , M., a n d W i t t e r , R. F.: Quantitative determination of phospholipid classes in human serum by combined thin-layer chromatography and phosphorus analysis. Clin. Chim. Acta 25: 447-452, 1969.

123. W i n t r o b e , M. W . : The size and hemoglobin content of the erythrocyte. J. Lab. Clin. Med. 17:899-912, 1932.

124. Y o u n g b u r g , G . E. a n d Y o u n g b u r g , M. V .: Phosphorus metabolism. I. A system for blood phosphorus analysis. J. Lab. Clin. Med. 16:158-166, 1930.

125. Z i l v e r s m i t , D . B. a n d D a v i s , A. K.: Microdetermination of plasma phospholipids by trichloroacetic acid precipitation. J. Lab. Clin. Med. 35:155-160, 1950.

126. Z i n z a d z e , S. R.: Neue Methoden zurkolorimetrischen Bestimmung der Phosphor- und Arsensaure. Z . Pflanzenernahr. 16:129- 180, 1930.

127. Z i n z a d z e , S. R.: Colorimetric methods for the determination of phosphorus. Ind. Eng. Chem., Anal. Ed. 7:227-230, 1935.

128. Z ö l l n e r , N. a n d W a r n o c k , S. A.: The enzymic determination of glycerophosphates and its application to the determination of phosphoglycerides, particularly those of serum phospholipids. Clin. Chim. Acta 7: 607-613, 1962.

129. Z u c k e r , T. F. a n d G u t m a n , M.: Distribu­tion of phosphorus in blood. Proc. Soc. Exp. Biol. Med. 20:133-136, 1922.