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A simple scanning method for the quantitative estimation of serum lecithin
CLINICA
CCA
CHIMICA
535
ACTA
4806
A simple scanning method for the quantitative estimation of serum lecithin Serum phospholipids
are normally
determined
as total phospholipids
after they
have been extracted by organic solvents, or precipitated by acid addition, then subjected to vigorous oxidation. The liberated inorganic phosphorus moiety is then estimated using a calorimetric techniquel,z. Various workerP5 have separated the individual phospholipids after organic extraction, using paper or thin layer chromatography; the individual spots are then eluted or scraped off the plates and their phosphorus content measured. The proposed scanning method offers a procedure which enables a direct quantitative value of the individual phospholipids to be determined in normal or patlrological serum.
I. Chloroform
A.R.-Methanol
A.R.
(2 :I) mixture.
2. 0.1 RI aq. NaCl. 3. Silica gel paper. SG 81. Whatman. 4. 1% Sodium thiosulphate (stock), dilute I ml to IOO for use. 5. Dragendorff reagent - made up as follows : Soln. A, 0.85 g bismuth oxynitrate in IO ml glacial acetic acid plus 40 ml distilled water. Soln. B, 8 g potassium iodide in 20 ml distilled water. Add soln. A to soln. B = stock solution. For use: 2 ml Dragendorff stock+4 ml glacial acetic acid+zo ml distilled water-mix---make up fresh each day. 6. Stock standard. 50 mg lecithin in 50 ml chloroform Co., Colnbrook, England, U.K. 7. Chemistry Control Serum Special Q-PAK Laboratories,
Thetford,
Norfolk,
obtained
from L. Light CV
TM Hyland, obtained
from Baxters
England.
TECHNIQUE
One ml serum was added to 40 ml chloroform-methanol mixture (2: I) in a Kober tube and the tube shaken for I min. After standing for 4~h the mixture was filtered through Whatman No. I II cm filter paper to remove the precipitated protein. To the clear filtrate was added 8 ml of 0.1 M sodium chloride solution and the mixture again shaken for 30 sec. After shaking, the tubes were placed in a refrigerator at 4’ and allowed to separate overnight. Next morning the chloroform phase (lower layerj was separated using a roo-ml separating funnel and evaporated under reduced pressure to dryness. The lipid residue was redissolved in 0.5 ml chloroform; 5-,~1 and IO-$ amounts of the dissolved residue from each test were spotted on a sheet of silica gel paper 20 cm ><20 cm in a line z cm from the bottom, to produce narrow bands I cm in width. The paper was then stapled to form a cylinder and placed in a glass Museum type jar, which contained IOO ml solvent chloroform-methanol-water (65 : 25 : 4) and Clin.
ChiWL. Acta,
37 (1972) 53j-537
536
BRIEF
SOTES
the lid securely sealed. Development was allowed to proceed for I l-h before the chromatogram was removed and allowed to dry for i h at room temperature. The chromatogram when dry was dipped twice through the diluted Dragendorff solution and blotted free of excess liquid. The reaction was allowed to proceed for exactly 2 min and then the strip COTItaining the orange-coloured spots of lecithin was cut out and immersed completely in a bath of diluted sodium thiosulphate solution. The bath was rocked gently to “clear” the chromatogram of excess unbound iodine. After the background was clear (approximately I min) the strip was taken out and blotted dry. The individual spots were then cut out as square blocks and placed one behind the other on a piece of 3 1131 filter paper which had been cut to fit the Chromoscan slide-holder (Mark II, Joyce, Loebel
and Co. Ltd.). The row of spots were covered with a strip of Selotape and the slide holder was then mounted in the Chromoscan carriage. The spots were then scanned using a blue filter Ko. 465 at a I :I ratio. Duplicate
amounts equivalent to 5o iu,g of lecithin were run with each batch and read as tests. Any test which gave a higher reading than the standard peak was re-cllromatographed using a smaller inoculum. A graph of lecithin standards was prepared corresponding to 25, 50, 75, IOO, and zoo pg amounts of lecithin. These were chromatographed in duplicate, developed and scanned. The graph was found to bc linear up to the IOO ,ug value- above this the graph curved sharply. It was decided therefore to run duplicate 5o+g amounts of lecithin standard with each batch of tests. The mean value was taken and the results were calculated in mg/roo ml serum based on the following: 150
(4
Test value x 50 ~- ~-~ X 5 = x mg lecithin/I00 ml serum. Standard value If IO ~1 of the chloroform extract is chromatographed.
value x (b)Test ___.~~~
50 x IO = x mg lecithin/r00 ml serum. Standard value If 5 ~1 of the chloroform extract is chromatographed
The results TABLE
are shown in Table
I
ro-30 30-P w-50 50-60 60-70 70-80 80-90 Kangc
I5 1 .i 15 I6 15 15 Total
ro-90
ClLn. Chim.
.4cta,
9;
37 (1972)
535-,j37
I.
BRIEF
NOTES
537
To test the reproduciblity procedure. analysed
of the method two sera were taken through the whole
Each serum was assayed six times-Group
A and Group B and the results
to give:
Group A Mean 245.4 5 9.5 mg/roo ml Group B Mean 258.6 & 12.7 mg/roo ml To test the accuracy of the method, Hyland Chemistry Control Serum, Special Q-PAKTM was taken through the procedure with each batch of tests. Six estimations were done on each control batch and all the values obtained were found to lie within I 7%) of the stated value for each lot. Unlike the more conventional routine methods of measuring total phospholipi&b%“, the proposed method offers a technique which allows the direct measurement of serum lecithin as well as lysolecithin and sphingomyelin. After their rapid separation on silica gel paper, the phospholipids are located using the Dragendorff reagent and then visualised by removing the background stain with a very dilute solution of sodium thiosulphate. The individual phospholipids can then be quantitatively measured using a Chromoscan Mark II, Joyce, Loebel and Co. Ltd., equipped with the normal blue filter used routinely in protein scanning. The method has been found to be rapid, easy to perform and very reproducible and has been used to produce normal ranges for serum lecithin in 96 normal subjects, whose ages ranged from 20-90 years. The mean serum lecithin values obtained by this method (apart from the 8090 age group) appear to be in agreement with previous workers’ findingF9 using other methods, that in normal subjects, serum phospholipids as a group tend to increase with age.
_&iochcmistry Dqbartnzent, Scarborough Gerwral Hospital, Scarborough, Yorkslzire, En&ad
A. \II’.
STOTT
(U.K.)
1 c;.E. YOUXG~~RG AKD M. V. YOUNGB~RC, J. Lab. Clin. Med., 50 (1930) 158. L I-I. V. (‘ONNERTY, ‘1.R. BRICGS AND E. H. EATON, JR.,Clix.Chews.,7 (1961) 37. 589. 3 J. ETIESNF. AXD J. POLOXOVSKI, Bull. Sm. Chin%. Biol., 62 (1960) 857. _IW. C. \'OGEL, L. &EVE AND K. 0. CARLETON, J. Lab. Cli~z. Med., 59 (1966) 335. .j JOSEPHISE GLOSTER ASL) K. F. FLETCHER, Clin.Chinz. Acta, 13 (1966) 235. 6 I,. ;I. CARLSOS, Acta Med. Stand., 167 (1960) 377. 7 H. LINDHOLM, Stand. ,[,Clin. Lab. Invest.. 8 (1~56) Suppl. 23. N E. B. 1Im 4x1) J, P. PETERS, J. Lab. Clin. Med., 41 (1953) 738. c) I,. 1. SCHAEFFEK, D. ADLERSRERC ANI) 4. G. STEINRERG, Circulatzm, 18 (1958) 341.
Received
September
17,1971 Clin. C/ZZ’WZ. Ada,
37 (1972) .j35-.i37
CCA
CHIMICA
535
ACTA
4806
A simple scanning method for the quantitative estimation of serum lecithin Serum phospholipids
are normally
determined
as total phospholipids
after they
have been extracted by organic solvents, or precipitated by acid addition, then subjected to vigorous oxidation. The liberated inorganic phosphorus moiety is then estimated using a calorimetric techniquel,z. Various workerP5 have separated the individual phospholipids after organic extraction, using paper or thin layer chromatography; the individual spots are then eluted or scraped off the plates and their phosphorus content measured. The proposed scanning method offers a procedure which enables a direct quantitative value of the individual phospholipids to be determined in normal or patlrological serum.
I. Chloroform
A.R.-Methanol
A.R.
(2 :I) mixture.
2. 0.1 RI aq. NaCl. 3. Silica gel paper. SG 81. Whatman. 4. 1% Sodium thiosulphate (stock), dilute I ml to IOO for use. 5. Dragendorff reagent - made up as follows : Soln. A, 0.85 g bismuth oxynitrate in IO ml glacial acetic acid plus 40 ml distilled water. Soln. B, 8 g potassium iodide in 20 ml distilled water. Add soln. A to soln. B = stock solution. For use: 2 ml Dragendorff stock+4 ml glacial acetic acid+zo ml distilled water-mix---make up fresh each day. 6. Stock standard. 50 mg lecithin in 50 ml chloroform Co., Colnbrook, England, U.K. 7. Chemistry Control Serum Special Q-PAK Laboratories,
Thetford,
Norfolk,
obtained
from L. Light CV
TM Hyland, obtained
from Baxters
England.
TECHNIQUE
One ml serum was added to 40 ml chloroform-methanol mixture (2: I) in a Kober tube and the tube shaken for I min. After standing for 4~h the mixture was filtered through Whatman No. I II cm filter paper to remove the precipitated protein. To the clear filtrate was added 8 ml of 0.1 M sodium chloride solution and the mixture again shaken for 30 sec. After shaking, the tubes were placed in a refrigerator at 4’ and allowed to separate overnight. Next morning the chloroform phase (lower layerj was separated using a roo-ml separating funnel and evaporated under reduced pressure to dryness. The lipid residue was redissolved in 0.5 ml chloroform; 5-,~1 and IO-$ amounts of the dissolved residue from each test were spotted on a sheet of silica gel paper 20 cm ><20 cm in a line z cm from the bottom, to produce narrow bands I cm in width. The paper was then stapled to form a cylinder and placed in a glass Museum type jar, which contained IOO ml solvent chloroform-methanol-water (65 : 25 : 4) and Clin.
ChiWL. Acta,
37 (1972) 53j-537
536
BRIEF
SOTES
the lid securely sealed. Development was allowed to proceed for I l-h before the chromatogram was removed and allowed to dry for i h at room temperature. The chromatogram when dry was dipped twice through the diluted Dragendorff solution and blotted free of excess liquid. The reaction was allowed to proceed for exactly 2 min and then the strip COTItaining the orange-coloured spots of lecithin was cut out and immersed completely in a bath of diluted sodium thiosulphate solution. The bath was rocked gently to “clear” the chromatogram of excess unbound iodine. After the background was clear (approximately I min) the strip was taken out and blotted dry. The individual spots were then cut out as square blocks and placed one behind the other on a piece of 3 1131 filter paper which had been cut to fit the Chromoscan slide-holder (Mark II, Joyce, Loebel
and Co. Ltd.). The row of spots were covered with a strip of Selotape and the slide holder was then mounted in the Chromoscan carriage. The spots were then scanned using a blue filter Ko. 465 at a I :I ratio. Duplicate
amounts equivalent to 5o iu,g of lecithin were run with each batch and read as tests. Any test which gave a higher reading than the standard peak was re-cllromatographed using a smaller inoculum. A graph of lecithin standards was prepared corresponding to 25, 50, 75, IOO, and zoo pg amounts of lecithin. These were chromatographed in duplicate, developed and scanned. The graph was found to bc linear up to the IOO ,ug value- above this the graph curved sharply. It was decided therefore to run duplicate 5o+g amounts of lecithin standard with each batch of tests. The mean value was taken and the results were calculated in mg/roo ml serum based on the following: 150
(4
Test value x 50 ~- ~-~ X 5 = x mg lecithin/I00 ml serum. Standard value If IO ~1 of the chloroform extract is chromatographed.
value x (b)Test ___.~~~
50 x IO = x mg lecithin/r00 ml serum. Standard value If 5 ~1 of the chloroform extract is chromatographed
The results TABLE
are shown in Table
I
ro-30 30-P w-50 50-60 60-70 70-80 80-90 Kangc
I5 1 .i 15 I6 15 15 Total
ro-90
ClLn. Chim.
.4cta,
9;
37 (1972)
535-,j37
I.
BRIEF
NOTES
537
To test the reproduciblity procedure. analysed
of the method two sera were taken through the whole
Each serum was assayed six times-Group
A and Group B and the results
to give:
Group A Mean 245.4 5 9.5 mg/roo ml Group B Mean 258.6 & 12.7 mg/roo ml To test the accuracy of the method, Hyland Chemistry Control Serum, Special Q-PAKTM was taken through the procedure with each batch of tests. Six estimations were done on each control batch and all the values obtained were found to lie within I 7%) of the stated value for each lot. Unlike the more conventional routine methods of measuring total phospholipi&b%“, the proposed method offers a technique which allows the direct measurement of serum lecithin as well as lysolecithin and sphingomyelin. After their rapid separation on silica gel paper, the phospholipids are located using the Dragendorff reagent and then visualised by removing the background stain with a very dilute solution of sodium thiosulphate. The individual phospholipids can then be quantitatively measured using a Chromoscan Mark II, Joyce, Loebel and Co. Ltd., equipped with the normal blue filter used routinely in protein scanning. The method has been found to be rapid, easy to perform and very reproducible and has been used to produce normal ranges for serum lecithin in 96 normal subjects, whose ages ranged from 20-90 years. The mean serum lecithin values obtained by this method (apart from the 8090 age group) appear to be in agreement with previous workers’ findingF9 using other methods, that in normal subjects, serum phospholipids as a group tend to increase with age.
_&iochcmistry Dqbartnzent, Scarborough Gerwral Hospital, Scarborough, Yorkslzire, En&ad
A. \II’.
STOTT
(U.K.)
1 c;.E. YOUXG~~RG AKD M. V. YOUNGB~RC, J. Lab. Clin. Med., 50 (1930) 158. L I-I. V. (‘ONNERTY, ‘1.R. BRICGS AND E. H. EATON, JR.,Clix.Chews.,7 (1961) 37. 589. 3 J. ETIESNF. AXD J. POLOXOVSKI, Bull. Sm. Chin%. Biol., 62 (1960) 857. _IW. C. \'OGEL, L. &EVE AND K. 0. CARLETON, J. Lab. Cli~z. Med., 59 (1966) 335. .j JOSEPHISE GLOSTER ASL) K. F. FLETCHER, Clin.Chinz. Acta, 13 (1966) 235. 6 I,. ;I. CARLSOS, Acta Med. Stand., 167 (1960) 377. 7 H. LINDHOLM, Stand. ,[,Clin. Lab. Invest.. 8 (1~56) Suppl. 23. N E. B. 1Im 4x1) J, P. PETERS, J. Lab. Clin. Med., 41 (1953) 738. c) I,. 1. SCHAEFFEK, D. ADLERSRERC ANI) 4. G. STEINRERG, Circulatzm, 18 (1958) 341.
Received
September
17,1971 Clin. C/ZZ’WZ. Ada,
37 (1972) .j35-.i37