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Quantitative determination of phospholipid classes in human serum by combined thin-layer chromatography and phosphorus analysis
CLINICA
CHIMICA
ACTA
QUANTITATIVE
447
DETERMINATION
OF
PHOSPHOLIPID
HUMAN SERUM BY COMBINED THIN-LAYER PHOSPHORUS
ANALYSIS
J. H. WILLIAMS,
M. KUCHMAK
AND
R.
CLASSES
CHROMATOGRAPHY
IN AND
F. WITTER
Lipid Standardization Laboratory, Laboratory Division, National Communicable Disease Center, Health Services and Mental Health Administration, U.S. Department of Health, Education, and Welfare, Atlanta, Georgia 30333 (U.S.A.) (Received
April
I,
1969)
SUMMARY An improved procedure for the quantitative determination of phospholipids in human serum is decribed. The four main phospholipid classes, lecithin, sphingomyelin, phosphatidyl ethanolamine and lysolecithin, are separated on thin-layers of silica gel G with chloroform-methanol-water-acetic acid, 65/45/8/x, v/v/v/v. The silica gel with the cleanly separated phospholipids is transferred to small chromatotubes, phospholipids are eluted with methanol-chloroform-water, 80/10/10, v/v/v, and phosphorus is determined in the effluent. The average recovery of phosphorus for a reference phospholipid mixture was 96.8% and for serum extracts g5.20,$.
INTRODUCTION In recent years the determination of phospholipid classes by separation on thin-layer chromatographic plates followed by lipid phosphorus analysis has been applied to extracts from human serum1-3, plasma415,erythrocytesa*7, brains, and cerebrospinal fluids or rat liverrO-12,brainlo and thymus*s or rabbit liver and plasma14. In these procedures the phospholipid classes separated on the thin-layer either were extracted from the adsorbent before digestion and color developmenta~6~10~12-14 or were digested in the presence of the adsorbent and the color was developed without1~4-8~11 or after3 separation of the silica gel. When attempts were made to adopt a procedure for the determination of the phospholipids of serum, the intensity of color produced was found to be depressed as much as 17% when digestion and color development were carried out in the presence of silica gel. Similar results have been reported by Doizaki and Zievel. Also, much larger amounts of hydrogen peroxide were required for the completion of digestion with subsequent increase in magnitude and variation of the blank phosphorus levels. Concomitantly, the time required for digestion was greatly increased. Furthermore, extraction of the separated phospholipid classes from silica gel with various combinations of chloroform-methanol-water with added formic acid13, ammonia6, or acetic Cl&. Chim. Acta,
25
(1969)
447-452
448
WILLIAMS
et al.
acid1e114did not give quantitative recoveries of phospholipids. In addition, the solvent system had to be modified in order to assure adequate separation of the phospholipid classes under conditions of varying humidity. Hence, conditions for the chromatographic separation and quantitative recoveries of the phospholipid classes of serum were studied. Then the precision of the completed method was investigated. In this report a relatively simple procedure will be presented which in our hands give reliable results in the determination of the four major phospholipids of serum. These classes include lecithin, lysolecithin, sphingomyelin, and phosphatidyl ethanolamine and account for about 98 per cent of the phospholipid of this tissue. EXPERIMENTAL
Materials
All solvents are reagent grade and freshly redistilled. BHT (2, 6-di-tert-butylp-cresol) is added at o.o05% level to all solvents used for extraction and chromatography16. Lipid extractiolz
The serum samples are analyzed in duplicate starting with the extraction of duplicate samples. Lipids are extracted according to the method of Folch et al.10. 1.0 (or 2.0) ml of serum in duplicate are added dropwise with swirling to a flask containing 8 ml of methanol. Eight ml of chloroform are added, and the flask is placed in a 50” water bath for 15 min with occasional swirling. The cooled extract is centrifuged at IOOOG for 15 min. Then the extract is decanted into a separatory funnel. The insoluble residue in the centrifuge tube is reextracted at room temperature with 4 ml of chloroform. After centrifugation for 15 minutes the extract is decanted into the separatory funnel. The process of extraction of the insoluble residue is repeated with another 4 ml portion of chloroform. Water (5.1 ml for a r.o-ml or 4.2 ml for a z.o-mlserum sample) is added to the combined extract. The mixture is shaken and the phases allowed to separate. The lower chloroform layer is withdrawn, evaporated in vacua and the moist residue transferred with chloroform to a I- or z-ml volumetric flask. Thin-layer
chromatography
Chromatography is carried out on layers of silica gel 250 ,LLthick. The plates (20x20 cm) are air dried and just before use activated at IIO' for one hour. Then they are allowed to cool in a desiccating cabinet and spotted as soon as cool. From each extract a 250~1 aliquot (approximately 15-25 ,ugof lipid phosphorus) is applied as a streak with 250~~1pipette on the starting line 1.5 cm from the bottom of the plate. Each band is 7 cm long and starts 1.5 cm from the vertical edge of the plate. There is a clear zone of three cm between the two streaked areas. With the same pipette another aliquot of the same extract is pipetted into 5o-ml digestion flasks for determination of total lipid phosphorus. Chromatoplates are developed in a lined conventional chromatographic chamber at 22’C using a freshly prepared solvent system of chloroform-methanol-waterglacial acetic acid, 65145181 I, v 1v /v /v, and the solvent allowed to travel 15 cm. UsualClin. Chim.
Acta,
25 (1969)
447-452
QUANTITATIVE
DETERMINATION
OF PHOSPHOLIPIDS
, TLC of SmUM PH~~~~IOL~PIDS _L’‘
449
.’
CHCL~-CH5OH-H~0-CH~COOH 65-45-8-I Fig. I. Serum phospholipids separated on thin-layer chromatoplate. LL, Lysolecithin; SPH, Sphingomyelin; L, Lecithin; PE, Phosphatidyl ethanolamine. The areas near the solvent front represent neutral lipids, pigments and z, 6-di-tert-butyl-p-cresol.
ly this process requires about 40 minutes. The developed chromatogram is allowed to dry at room temperature. The bands are visualized with iodine vapor in a closed chamber and quickly outlined (Fig. I). Removal
of zones
Most of the iodine is allowed to evaporate before scraping off the outlined areas, The plate is placed with the starting line in the horizontal position on a glass rack at about a 75” angle to the vertical. The silica gel at the vertical edges of the plate outside of the phospholipid zones is removed first with a razor blade held by a paint scraper handle. This adsorbent is discarded. The plate is turned 90’ and each of the phospholipid zones is scraped onto a piece of glassine paper. The silica gel is transferred to a small chromatotube (I x 15 cm) with a fine porosity fritted disc. A small pledget of cotton, previously moistened with the elution mixture is wiped over the scraped area and added to the tube. After the zones are removed, the plate is rotated 90’ and an area of silica gel about equal to that of a phospholipid zone is removed from the center lane between the two aliquots of the sample for use as a blank for that plate. El&on
of phospholi+is
The phospholipids are eluted from the silica gel into 25 ml test tubes in the following manner. Three ml of eluting solvent, methanol-chloroform-water, S/I/I/, v/v/v, are added to each chromatotube to wet thoroughly the silica gel and cotton. After the mixture is allowed to stand for 30 minutes, the remaining solvent is forced through with nitrogen. The process is repeated twice with 2 ml of solvent. Each time the mixture is allowed to stand 15 min before nitrogen pressure is applied. Phosphorus
determination
Only one half of the lecithin eluted is used for the rest of the procedure. The Cl&. Chim.
Acta,
25
(1969)
447-452
WILLIAMS et al.
450
effluents are evaporated to dryness and digested with I ml of 7 N sulfuric acid at ISO” in an oven for z h. Then 2 drops of 30% hydrogen peroxide are added and digestion is completed in the oven after an additional 30 min. Orthophosphate is then determined by a micro modification of the method of Beveridge and Johnson17 in a final volume of 10.2 ml with optical densities being read at 830 m/A with a path length of I cm. Appropriate standards of orthophosphate are carried through the steps of digestion and color development. Total lipid phosphorus is determined in a similar manner on the lipid extracts, previously placed in digestion flasks, using the method of Beveridge and Johnson17 in a final volume of 50 ml. RESULTS AND DISCUSSION
Separation and recoveries
As noted in Fig. I, the solvent system employed yielded excellent separations of the phospholipids into four major classes. There is no overlap of these constituents even under conditions of varying humidity. Using the procedure described in the experimental section, (Table I) each constituent of a known mixture of phosphatidyl ethanolamine, lecithin, sphingomyelin, and lysolecithin which was similar in composition to serum phospholipids was recovered in 97% yield with the recovery of the sum of the 4 components being 96.8 & 1.7% of the lipid applied. Since the recoveries of each individual phospholipid were in a similar range regardless of their affinities for silica gel, the 3% loss of phospholipids cannot be attributed to the ineffectiveness of the solvent system used for elution. Under similar conditions g5.z% of the total serum lipid phosphorus applied was recovered from the representative group of samples for which analyses are presented in Table II. Previous studies have shown that non-lipid phosphorus is not present in lipid extracts of serum prepared according to the method outlined in this communication. The average recovery of serum phospholipid phosphorus in this table is lower by TABLE
I
RECOVERY
OF PHOSPHORUS
IN
REFERENCE
COW@OU%dS
PHOSPHOLIPIDS
Recovery of P* (%)
tLg Plml of Solution Mixture
Phosphatidyl ethanolamine Lecithin Sphingomyelin Lysolecithin Total
SEPARATED
4.22 78.61 18.68 9.67 111.18
96.9 96.8 96.4 96.8
f & zt *
5.7 1.7 3.5 4.1
96.8
*
1.7
TLC
BY
-
* Means and standard deviations of 12 replicates. Two replicates were made on a thin-layer plate. Standard deviation fS.D.l was calculated from the eauation: L’
S.D. = &
I
zjl” J N-I where x is a difference from the average and N = 12. The reference phospholipids were isolated from beef liver and purified by silicic acid column chromatography in this laboratory until they were chromatographically homogenous Lysolecithin was prepared by enzymatic hydrolysis of isolated lecithinlo. Clin. Chinz. Acta, 25 (1969) 447-452
QUANTITATIVE TABLE
DETERMINATION
4.51
OF PHOSPHOLIPIDS
II
PRECISION
OF HUMAN
Constituents
SERUM
PHOSPHOLIPIDS
pg P/ml
DETERMINATION
of serum
Range
Average
BY
TLC
S.D. of Duplicates*
P Factor’8
Average mg% of Phospholi$ds
0.2
24.0
6.0
Phosphatidyl ethanolamine Lecithin Sphingomyelin Lysolecithin
44.1-94.9 8.3-20.8 4.7-13.3
57.1 r4.r 8.4
I.4 0.7 0.4
26.2 23.4 17.5
149.6 33.0 14.7
Total
6r.4-129.0
82.1
2.0
25.0
205.2
1.0-4.5
* The standard deviation the formula:
2.5
was obtained from I I samples run in duplicate.
It was calulated from
S.D. = f ‘; Jwhere x is a difference of duplicate and N = I I. The average recovery of phosphorus from the I I samples was 95.2% of the determined total lipid phosphorus. All blood donors were adult males.
1.6% as compared with the average total recovery of reference phospholipids (Table I). This difference in recoveries is probably due to the fact that the minor phospholipid components constituting about 2% in normal human serum’8 were not included with the four major classes studied. Precision The precision of the analyses of a representative group of samples is given in Table II. The standard deviation of duplicates ranged from 0.2 to 1.4yg of phosphorus per ml of serum with the four major phospholipid classes. The latter ranged in concentration from 44.1 to g4.gltg P/ml (115.5 to 248.6 rng%) with lecithin to 1.0 to 4.5 pg P/ml (2.4 to 10.8 mg%) with phosphatidyl ethanolamine. Variables in Method During the course of development of this procedure several variables which influence the precision and accuracy of the method were investigated. The method of extraction is widely used and is generally accepted as yielding with tissue such as serum a quantitative extraction of lipids free of non-lipid phosphate compounds. However, the manipulations with the small volumes of extracts employed can lead to appreciable variation. Thus when filtration through Whatman No. I filter paper in a small Buchner funnel was substituted for centrifugation as a means of removing the insoluble portion of the serum, the precision of the extraction step was decreased. For example, in an experiment with 28 replicates from the same serum, the standard deviation for total lipid phosphorus in the 14 extracts clarified by centrifugation was 0.5 /Lg P/ml of lipid phosphorus per one ml of serum whereas in the same number of extracts clarified by filtration the standard deviation was raised to 1.3 c/g P/ml. Furthermore, the average lipid phosphorus for the filtered extracts (73.9 kbg/ml) was 1.7% lower than the mean for the centrifuged extracts (75.2 tlg/ml). Using a t-test for paired observations, this difference was significant at the 5’$/0level. Therefore, cenC&n. Chim. Acta,
25
(1969) 447-452
WILLIAMS
452
et at.
trifugation was selected as a means of clarifying the extracts and replication of the analysis was begun at the extract stage. Attempts to increase the recovery of each of the phospholipid classes, after chromatographic separation, to values greater than 970/b were unsuccessful. No measurable amounts of phosphorus other than the blank level was found in the clear areas between the various zones containing the four phospholipid classes of the model mixture. The same was true for the areas at the origin and at the solvent front. The blank taken from the center of the plate was equivalent to about 0.08 ,~g of phosphorus for an area equal to a phospholipid zone and was quite consistent from plate to plate. There was a fine film of silica gel left on the plate after careful scraping with a razor blade. By clearing off the film with a cotton pledget moistened with eluting mixture, approximately 2% more of the total amount could be recovered. During elution from the silica gel about 92”/” of the phospholipid was removed in the first elution and after the third elution no measurable amount could be eluted. Also, identical results were obtained when digestion of the phospholipids for the determination of lipid P was carried out at ISO' in an oven or at the relatively higher temperature of an electric hot plate. The former method was more convenient for the digestion of the phospholipids extracted from thin-layer plates. ACKNOWLEDGMENT
This work was performed as a cooperative effort with the Heart Disease & Stroke Control Program, Regional Medical Programs, Health Services and Mental Health Administration, U.S. Department of Health, Education, and Welfare, Arlington, Virginia 22203. REFERENCES I 2 3 d ;
6 7 8
g IO
W. M. DOIZAKI AND L. ZIEVE, Proc.
Sot. Exptl.
Biol.
Med.,
113 (1963)
gr-gq.
JOSEPHINE GLOSTER, AND R. F. FLETCHER,C&. Chim. Acta., 13(1g66) 235-240. N. ROBINSON AND B.M.PHILIPs,CZ~~. Chim. Acta., 8(rg63) 385-392. E. GTONE AND 0. M. ORNING. &and. l.Clin.Lab. Invest., 18 (1966) 209-216. E. G;ONE, Stand. J. Clin. Lab. Invest.: 18 (1966) 263-267. B. A. BRADLOW, R. RUBENSTEIN, AND J. LEE, S. Afr.J. Med. Sci., 2g(Ig64) 41-52. B. A. BRADLOW, J. LEE, AND R. RUBENSTEIN, Brit. J. Haematol., II (1965) 315-321. G. ROUSER. A. N. SIAKOTOS, AND S. FLEISCHER, Lipids, I (1966) 85-86. B. M. PHILLIPS AND N. ROBINSON, Clin.Chim. A&., 8 (1963) 832-842. LILIA S. E. DE BOHNER, F. SOTO, AND TAD~ARA DE COHAN, J. Chromatog., 17, (1965) 513519.
II F. PARKER AND N. F. PETERSON, J. LipidRes.. 6(rg65) 455-460. 12 V. P. SKIPSKI, R. F. PETERSON, ANDM. BARCLAY, Biochem.J., go(Ig64) 374-378. 13 D. ABRAMSON AND M. BLECHER, J. Lipid Res., 5 (1964) 628-631. 14 J. J. BIEZENSKI, J. Lipid Res., 8 (67) 409-410. 15 J. J. WREN,AND ANNAD. SZCZEPANOWSKA, J.Chromatog., 14(1964)405-410. 16 J. FOLCH,M.LEES, AND G.H.SLOANE-STANLEY, J.Biol.Chem., 226(1957)497-509 17 J. M. R. BEVERIDGE AND S. E. JOHNSON, Can. J. Res. Sect., E 27 (1949) 159-163. 18 J.H. WILLIAMS, M. KUCHMAK AND R. F.WITTER, Lipids, I (1966)89-97. Ig N. H. TATRIE, J. Lipid Res., I (1959) 60-65. Clin. Chim.
Acta,
25 (1969)
447-452
CHIMICA
ACTA
QUANTITATIVE
447
DETERMINATION
OF
PHOSPHOLIPID
HUMAN SERUM BY COMBINED THIN-LAYER PHOSPHORUS
ANALYSIS
J. H. WILLIAMS,
M. KUCHMAK
AND
R.
CLASSES
CHROMATOGRAPHY
IN AND
F. WITTER
Lipid Standardization Laboratory, Laboratory Division, National Communicable Disease Center, Health Services and Mental Health Administration, U.S. Department of Health, Education, and Welfare, Atlanta, Georgia 30333 (U.S.A.) (Received
April
I,
1969)
SUMMARY An improved procedure for the quantitative determination of phospholipids in human serum is decribed. The four main phospholipid classes, lecithin, sphingomyelin, phosphatidyl ethanolamine and lysolecithin, are separated on thin-layers of silica gel G with chloroform-methanol-water-acetic acid, 65/45/8/x, v/v/v/v. The silica gel with the cleanly separated phospholipids is transferred to small chromatotubes, phospholipids are eluted with methanol-chloroform-water, 80/10/10, v/v/v, and phosphorus is determined in the effluent. The average recovery of phosphorus for a reference phospholipid mixture was 96.8% and for serum extracts g5.20,$.
INTRODUCTION In recent years the determination of phospholipid classes by separation on thin-layer chromatographic plates followed by lipid phosphorus analysis has been applied to extracts from human serum1-3, plasma415,erythrocytesa*7, brains, and cerebrospinal fluids or rat liverrO-12,brainlo and thymus*s or rabbit liver and plasma14. In these procedures the phospholipid classes separated on the thin-layer either were extracted from the adsorbent before digestion and color developmenta~6~10~12-14 or were digested in the presence of the adsorbent and the color was developed without1~4-8~11 or after3 separation of the silica gel. When attempts were made to adopt a procedure for the determination of the phospholipids of serum, the intensity of color produced was found to be depressed as much as 17% when digestion and color development were carried out in the presence of silica gel. Similar results have been reported by Doizaki and Zievel. Also, much larger amounts of hydrogen peroxide were required for the completion of digestion with subsequent increase in magnitude and variation of the blank phosphorus levels. Concomitantly, the time required for digestion was greatly increased. Furthermore, extraction of the separated phospholipid classes from silica gel with various combinations of chloroform-methanol-water with added formic acid13, ammonia6, or acetic Cl&. Chim. Acta,
25
(1969)
447-452
448
WILLIAMS
et al.
acid1e114did not give quantitative recoveries of phospholipids. In addition, the solvent system had to be modified in order to assure adequate separation of the phospholipid classes under conditions of varying humidity. Hence, conditions for the chromatographic separation and quantitative recoveries of the phospholipid classes of serum were studied. Then the precision of the completed method was investigated. In this report a relatively simple procedure will be presented which in our hands give reliable results in the determination of the four major phospholipids of serum. These classes include lecithin, lysolecithin, sphingomyelin, and phosphatidyl ethanolamine and account for about 98 per cent of the phospholipid of this tissue. EXPERIMENTAL
Materials
All solvents are reagent grade and freshly redistilled. BHT (2, 6-di-tert-butylp-cresol) is added at o.o05% level to all solvents used for extraction and chromatography16. Lipid extractiolz
The serum samples are analyzed in duplicate starting with the extraction of duplicate samples. Lipids are extracted according to the method of Folch et al.10. 1.0 (or 2.0) ml of serum in duplicate are added dropwise with swirling to a flask containing 8 ml of methanol. Eight ml of chloroform are added, and the flask is placed in a 50” water bath for 15 min with occasional swirling. The cooled extract is centrifuged at IOOOG for 15 min. Then the extract is decanted into a separatory funnel. The insoluble residue in the centrifuge tube is reextracted at room temperature with 4 ml of chloroform. After centrifugation for 15 minutes the extract is decanted into the separatory funnel. The process of extraction of the insoluble residue is repeated with another 4 ml portion of chloroform. Water (5.1 ml for a r.o-ml or 4.2 ml for a z.o-mlserum sample) is added to the combined extract. The mixture is shaken and the phases allowed to separate. The lower chloroform layer is withdrawn, evaporated in vacua and the moist residue transferred with chloroform to a I- or z-ml volumetric flask. Thin-layer
chromatography
Chromatography is carried out on layers of silica gel 250 ,LLthick. The plates (20x20 cm) are air dried and just before use activated at IIO' for one hour. Then they are allowed to cool in a desiccating cabinet and spotted as soon as cool. From each extract a 250~1 aliquot (approximately 15-25 ,ugof lipid phosphorus) is applied as a streak with 250~~1pipette on the starting line 1.5 cm from the bottom of the plate. Each band is 7 cm long and starts 1.5 cm from the vertical edge of the plate. There is a clear zone of three cm between the two streaked areas. With the same pipette another aliquot of the same extract is pipetted into 5o-ml digestion flasks for determination of total lipid phosphorus. Chromatoplates are developed in a lined conventional chromatographic chamber at 22’C using a freshly prepared solvent system of chloroform-methanol-waterglacial acetic acid, 65145181 I, v 1v /v /v, and the solvent allowed to travel 15 cm. UsualClin. Chim.
Acta,
25 (1969)
447-452
QUANTITATIVE
DETERMINATION
OF PHOSPHOLIPIDS
, TLC of SmUM PH~~~~IOL~PIDS _L’‘
449
.’
CHCL~-CH5OH-H~0-CH~COOH 65-45-8-I Fig. I. Serum phospholipids separated on thin-layer chromatoplate. LL, Lysolecithin; SPH, Sphingomyelin; L, Lecithin; PE, Phosphatidyl ethanolamine. The areas near the solvent front represent neutral lipids, pigments and z, 6-di-tert-butyl-p-cresol.
ly this process requires about 40 minutes. The developed chromatogram is allowed to dry at room temperature. The bands are visualized with iodine vapor in a closed chamber and quickly outlined (Fig. I). Removal
of zones
Most of the iodine is allowed to evaporate before scraping off the outlined areas, The plate is placed with the starting line in the horizontal position on a glass rack at about a 75” angle to the vertical. The silica gel at the vertical edges of the plate outside of the phospholipid zones is removed first with a razor blade held by a paint scraper handle. This adsorbent is discarded. The plate is turned 90’ and each of the phospholipid zones is scraped onto a piece of glassine paper. The silica gel is transferred to a small chromatotube (I x 15 cm) with a fine porosity fritted disc. A small pledget of cotton, previously moistened with the elution mixture is wiped over the scraped area and added to the tube. After the zones are removed, the plate is rotated 90’ and an area of silica gel about equal to that of a phospholipid zone is removed from the center lane between the two aliquots of the sample for use as a blank for that plate. El&on
of phospholi+is
The phospholipids are eluted from the silica gel into 25 ml test tubes in the following manner. Three ml of eluting solvent, methanol-chloroform-water, S/I/I/, v/v/v, are added to each chromatotube to wet thoroughly the silica gel and cotton. After the mixture is allowed to stand for 30 minutes, the remaining solvent is forced through with nitrogen. The process is repeated twice with 2 ml of solvent. Each time the mixture is allowed to stand 15 min before nitrogen pressure is applied. Phosphorus
determination
Only one half of the lecithin eluted is used for the rest of the procedure. The Cl&. Chim.
Acta,
25
(1969)
447-452
WILLIAMS et al.
450
effluents are evaporated to dryness and digested with I ml of 7 N sulfuric acid at ISO” in an oven for z h. Then 2 drops of 30% hydrogen peroxide are added and digestion is completed in the oven after an additional 30 min. Orthophosphate is then determined by a micro modification of the method of Beveridge and Johnson17 in a final volume of 10.2 ml with optical densities being read at 830 m/A with a path length of I cm. Appropriate standards of orthophosphate are carried through the steps of digestion and color development. Total lipid phosphorus is determined in a similar manner on the lipid extracts, previously placed in digestion flasks, using the method of Beveridge and Johnson17 in a final volume of 50 ml. RESULTS AND DISCUSSION
Separation and recoveries
As noted in Fig. I, the solvent system employed yielded excellent separations of the phospholipids into four major classes. There is no overlap of these constituents even under conditions of varying humidity. Using the procedure described in the experimental section, (Table I) each constituent of a known mixture of phosphatidyl ethanolamine, lecithin, sphingomyelin, and lysolecithin which was similar in composition to serum phospholipids was recovered in 97% yield with the recovery of the sum of the 4 components being 96.8 & 1.7% of the lipid applied. Since the recoveries of each individual phospholipid were in a similar range regardless of their affinities for silica gel, the 3% loss of phospholipids cannot be attributed to the ineffectiveness of the solvent system used for elution. Under similar conditions g5.z% of the total serum lipid phosphorus applied was recovered from the representative group of samples for which analyses are presented in Table II. Previous studies have shown that non-lipid phosphorus is not present in lipid extracts of serum prepared according to the method outlined in this communication. The average recovery of serum phospholipid phosphorus in this table is lower by TABLE
I
RECOVERY
OF PHOSPHORUS
IN
REFERENCE
COW@OU%dS
PHOSPHOLIPIDS
Recovery of P* (%)
tLg Plml of Solution Mixture
Phosphatidyl ethanolamine Lecithin Sphingomyelin Lysolecithin Total
SEPARATED
4.22 78.61 18.68 9.67 111.18
96.9 96.8 96.4 96.8
f & zt *
5.7 1.7 3.5 4.1
96.8
*
1.7
TLC
BY
-
* Means and standard deviations of 12 replicates. Two replicates were made on a thin-layer plate. Standard deviation fS.D.l was calculated from the eauation: L’
S.D. = &
I
zjl” J N-I where x is a difference from the average and N = 12. The reference phospholipids were isolated from beef liver and purified by silicic acid column chromatography in this laboratory until they were chromatographically homogenous Lysolecithin was prepared by enzymatic hydrolysis of isolated lecithinlo. Clin. Chinz. Acta, 25 (1969) 447-452
QUANTITATIVE TABLE
DETERMINATION
4.51
OF PHOSPHOLIPIDS
II
PRECISION
OF HUMAN
Constituents
SERUM
PHOSPHOLIPIDS
pg P/ml
DETERMINATION
of serum
Range
Average
BY
TLC
S.D. of Duplicates*
P Factor’8
Average mg% of Phospholi$ds
0.2
24.0
6.0
Phosphatidyl ethanolamine Lecithin Sphingomyelin Lysolecithin
44.1-94.9 8.3-20.8 4.7-13.3
57.1 r4.r 8.4
I.4 0.7 0.4
26.2 23.4 17.5
149.6 33.0 14.7
Total
6r.4-129.0
82.1
2.0
25.0
205.2
1.0-4.5
* The standard deviation the formula:
2.5
was obtained from I I samples run in duplicate.
It was calulated from
S.D. = f ‘; Jwhere x is a difference of duplicate and N = I I. The average recovery of phosphorus from the I I samples was 95.2% of the determined total lipid phosphorus. All blood donors were adult males.
1.6% as compared with the average total recovery of reference phospholipids (Table I). This difference in recoveries is probably due to the fact that the minor phospholipid components constituting about 2% in normal human serum’8 were not included with the four major classes studied. Precision The precision of the analyses of a representative group of samples is given in Table II. The standard deviation of duplicates ranged from 0.2 to 1.4yg of phosphorus per ml of serum with the four major phospholipid classes. The latter ranged in concentration from 44.1 to g4.gltg P/ml (115.5 to 248.6 rng%) with lecithin to 1.0 to 4.5 pg P/ml (2.4 to 10.8 mg%) with phosphatidyl ethanolamine. Variables in Method During the course of development of this procedure several variables which influence the precision and accuracy of the method were investigated. The method of extraction is widely used and is generally accepted as yielding with tissue such as serum a quantitative extraction of lipids free of non-lipid phosphate compounds. However, the manipulations with the small volumes of extracts employed can lead to appreciable variation. Thus when filtration through Whatman No. I filter paper in a small Buchner funnel was substituted for centrifugation as a means of removing the insoluble portion of the serum, the precision of the extraction step was decreased. For example, in an experiment with 28 replicates from the same serum, the standard deviation for total lipid phosphorus in the 14 extracts clarified by centrifugation was 0.5 /Lg P/ml of lipid phosphorus per one ml of serum whereas in the same number of extracts clarified by filtration the standard deviation was raised to 1.3 c/g P/ml. Furthermore, the average lipid phosphorus for the filtered extracts (73.9 kbg/ml) was 1.7% lower than the mean for the centrifuged extracts (75.2 tlg/ml). Using a t-test for paired observations, this difference was significant at the 5’$/0level. Therefore, cenC&n. Chim. Acta,
25
(1969) 447-452
WILLIAMS
452
et at.
trifugation was selected as a means of clarifying the extracts and replication of the analysis was begun at the extract stage. Attempts to increase the recovery of each of the phospholipid classes, after chromatographic separation, to values greater than 970/b were unsuccessful. No measurable amounts of phosphorus other than the blank level was found in the clear areas between the various zones containing the four phospholipid classes of the model mixture. The same was true for the areas at the origin and at the solvent front. The blank taken from the center of the plate was equivalent to about 0.08 ,~g of phosphorus for an area equal to a phospholipid zone and was quite consistent from plate to plate. There was a fine film of silica gel left on the plate after careful scraping with a razor blade. By clearing off the film with a cotton pledget moistened with eluting mixture, approximately 2% more of the total amount could be recovered. During elution from the silica gel about 92”/” of the phospholipid was removed in the first elution and after the third elution no measurable amount could be eluted. Also, identical results were obtained when digestion of the phospholipids for the determination of lipid P was carried out at ISO' in an oven or at the relatively higher temperature of an electric hot plate. The former method was more convenient for the digestion of the phospholipids extracted from thin-layer plates. ACKNOWLEDGMENT
This work was performed as a cooperative effort with the Heart Disease & Stroke Control Program, Regional Medical Programs, Health Services and Mental Health Administration, U.S. Department of Health, Education, and Welfare, Arlington, Virginia 22203. REFERENCES I 2 3 d ;
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