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Microdetermination of lipids and serum total fatty acids
AN.4LTTICAL
BIOCHEMISTRT
Microdetermination
6,
415-423
11963)
of lipids
and
Serum
S. V. PANDE, R. PARVIN KHAN, T. A. VENKITASUBRAIMANIAN From
the
Vallabhbhai
Pate1
Chest
Rcccirrd
Institute, November
I’niversity
Total
Fatty
Acids
AND
of Delhi,
Delhi,
India
10, 1962
INTRODUCTION
Since the introduction of acid-dichromate reagent for lipid estimation (1, 2)) many methods making use of this principle have been evolved for the estimation of cholesterol (3, 5), phospholipids (2, 5, 6), fatty acids (3, 5, 7, 8)) and total lipids (9, 10). However different workers have employed different factors (3, 11) for the calculation of the quantity of lipid, as the extent of oxidation in these procedures differed depending upon the time and temperature employed for oxidation. Nevertheless, Bloor (7) was able to overcome this st,umbling block in the application of the acid-dichromate method for lipid estimation by defining the conditions for almost complete oxidation. Most of the earlier methods of lipid estimation were based on the titrimetric determination of excess dichromate by the starch-thiosulfate method. Because the procedure of Backlin (12) involving manometric determination of carbon dioxide evolved on chromic acid combustion of lipid was inconvenient, it did not find much application. However, a simple calorimetric method was developed for the first time by Bloor (7) ; the procedure was not entirely satisfactory because a linear relationship over a wide range was not obtained and a specially constructed calorimeter was required. This method was subsequently improved by Bragdon (13’). Considering the method of Bragdon (13) to be of a hazardous nature, Kibrick and Skupp (8) attempted to simplify it, but their use of a pressure cooker for oxidation is too cumbersome. Therefore simplified procedures of varyin g dcgrec of sensitivity for the determination of lipid are presented here. The availability of rapid and sensitive methods for the determination of serum lipid is of importance. For the determination of serum total fatty acids, the methods presently in use (14-16) are essentially based on the microtitration method of Dole (17) for unesterified fatty acids. A colorimetric method many times more sensitive than microtitration method5 is described here for the determination of total fat’ty acids in serum. 415
416
PANDE,
KHAN,
AND
VENKITASUBRAMANIAN
METHODS
Chemicals All chemicals used were of A.R. grade. Cholesterol, palmitic acid, stearic acid, soluble starch, ethanol, potassium dichromate, and sodium sulfite used were from E. Merck; solvent ether and petroleum ether from B. D. H., England. Cadmium iodide used in the earlier experiments was of unknown origin. In later work the Chemapol Prague product was used. Before use, solvents were distilled in an all-glass apparatus and the lipids recrystallized. The standard lipid solutions were prepared in petroleum ether (b.p. 40-60°C). Reagents (1) Ultramicro method for 2-12 pg lipid: (a) 0.034% (w/v) potassium dichromate in 97% (w/v) sulfuric acid is prepared by diluting aqueous 3.4% (w/v) potassium dichromate with 98% sulfuric acid before use as described by Halliwell (18). (b) Cadmium iodide-starch reagent. 1.4 gm cadmium iodide is dissolved in 60-70 ml water and the solution boiled for 15-20 min. To this is added a clear solution of starch obtained by boiling 0.33 gm soluble starch in about 75 ml water for 5 min, and the mixed solution boiled for 5 more min. The final solution is then filtered, made up to 500 ml at room temperature with distilled water, and stored protected from light. This is stable for about six months without precipitation. (6) Micro method for 20-140 pg lipid: (a) 0.15% potassium dichromate in 96% sulfuric acid. 1.5 gm potassium dichromate is dissolved with heating in 10 ml water and the solution cooled and made up to 1 liter with 98% (w/v) sulfuric acid. (b) Aqueous 20% (wiv) sodium sulfite solution, prepared fresh when required. (3) Xemimicro method for 170 pg to 1.33 mg lipid: 2.0y0 potassium dichromate (w/v) in 98% (w/v) sulfuric acid. 20 gm powdered potassium dichromate is dissolved in 1 liter 98% (w/v) sulfuric acid at a temperature below 100°C. The solution is stored in an all-glass bottle with an inverted tube over the mouth to protect from dust. It is stable for at least one year. Analytical
Procedure
An aliquot of the lipid solution to be analyzed was transferred, the solvent was removed by rapid evaporation under reduced pressure, and the lipid was oxidized with acid dichromate. The reaction was followed by direct calorimetry (micro method), by an iodometric calorimetry
MICRODETERMINATIOiV
OF
LIPIDS
417
(ultramicro method), and by the appearance of reduced chrome alum (semimicro method). The lipid solutions were delivered into the bottom of scrupulously clean glass-stoppered tubes (6J’ X a/4”‘) with standard joints, the volumes of the solution never exceeding that of the acid-dichromate reagent to be added subsequently. The tubes were placed vertically in a vacuum dessicator and the pressure was very gradually reduced by adjusting the stopcock to avoid any spurting during evaporation of the solvent.. These precautions were necessary to restrict the dried film to the bottom of the tube so that no lipid escaped contact with the acid-dichromate reagent during the process of oxidation. After complete removal of the solvent, the vacuum was released carefully by putting a piece of white paper at the mouth of the stopcock to keep the ingoing air free from dust. This procedure was carried out in a place with sufficient air circulation to avoid the entry of solvent vapors. A number of samples could thus be freed of the solvent simultaneously and in a short time. Acid-dichromate reagent was added almost immediat’ely, and tubes were stoppered and then heated for oxidation. The subsequent procedures for different ranges of lipid estimation are described below separately. Ultramicro
Method
(2-12 pg)
To 2 to 12 pg dried lipid sample was added exactly 1.0 ml 0.034% (w/v) potassium dichromate in 97% sulfuric acid (w/v). A control tube was also included which did not contain any lipid. The tubes were placed in a boiling water bath for 15 min and then cooled in running water; 9.0 ml water was added to all the tubes, contents were mixed well, and 0.5 ml of these solutions was pipetted separately into the bottom of widebore test tubes, followed by addition of 4.5 ml CdI,-starch reagent. For optimal color development, the solutions were t’horoughly mixed and allowed to stand at room temperature for exactly 20 min, and then diluted with water (1: 1). The reagent blank was prepared by taking 0.5 ml 3.6 N sulfuric acid, adding 4.5 ml CdI,-starch reagent, and proceeding as for samples. The color intensities were read against the reagent blank at 575 rnp (peak absorption). Micro
Method
(20-140
pg)
2.0 ml 0.15% potassium dichromate in 96% (w/v) sulfuric acid was added to tubes containing zero (two blank t,ubes required) to 140 pg solvent-free lipid. After heating and cooling as described above, 4.5 ml water was added and the solut,ions were retooled after mixing; 0.10 ml freshly prepared aqueous 20% Na,SO,.7H,O (w/v) was added to reduce the diehromate in one of the blanks. All tubes were read against the
418
PANDE,
KHAN,
AND
VENKITASUBRAMANIAN
reduced blank at 440 mp. The unreduced blank tube serves as a control, showing the amount of dichromate present initially. Semimicro
Method
(170 pg to l.SS mg)
The procedure of Bragdon (13) has been modified so that the colorimetry can be performed in test tubes with relative ease. The reduction in the volume of the solutions used obviously increases the sensitivity. After removing the solvent completely, 3.0 ml 2$% potassium dichromate (w/v) in 98% (w/v) sulfuric acid was added to 170 pg to 1.33 mg lipid. After heating and cooling 2s described in the ultramicro method, 4.5 ml water was added, the contents were mixed and the solutions were retooled. The color intensities were measured at 590 mp against a reagent blank prepared by taking 3.0 ml acid-dichromate reagent and treating like the rest. All calorimetric measurements were made in a Bausch 8.1Lomb colorimeter. Procedure for Determination
of Serum Total Fatty Acids
To 7 ml alcohol-ether mixture (3: 1 v/v) of Bloor (19) in a graduated centrifuge tube w2s added 40 ~1 serum, with constant and vigorous stirring during the addition. The contents were brought to boil and cooled and the volume was made up to 10 ml by addition of more alcohol-ether mixture; 3.0 ml of the clear supernatant obtained after centrifugation ~2s transferred to a 15-ml glass-stoppered tube with standard joint and the volume w2s reduced to about, half by dipping the tube in warm water. After adding 0.10 ml 4N NaOH, an air condenser w2s attached to the tube and the contents saponified in 2 water bath at 80°C for 30 min. After cooling, about 1.5 ml water was added and the nonsaponifiable lipids were removed by two extractions with about 8 ml petroleum ether, by giving end to end shakes for 2 few minutes and separating the two phases by centrifugation. The petroleum ether layer was withdrawn 2s much as possible without touching the aqueous phase by means of 2 capillary pipet. The small volume of ether left behind w2s evaporated off by putting the tubes in warm water. The contents were then acidified by the addition of 0.2 ml 4 N H,SO, solution cooled to room temperature (ZO-25’C) and the liberated fatty acids extracted into exactly 4.0 ml petroleum ether (b.p. 40-60°C). After centrifugation suitable aliquots were withdrawn for fatty acid estimation by the ultramicro method as already described. RESULTS
The calibration curves obtained for cholesterol, palmitic acid, and stearic acid by the ultramicro, micro, and semimicro method are presented in Figs. 1-3, respectively. As seen from the figures, the calibration curves
MICRODETERMINATION
OF
419
LIPIDS
for palmitic and stearic acid almost overlap each other, as is to bc cxpetted from the close dichromate equivalents of long-chain fatty acids (13). The theoretical calibration curves included in Figs. 1 and 2 are based on the presumption that the oxidation of lipids was complete and t,hat only carbon dioxide and no carbon monoxide xvas evolved during 0.6
6 0.2 d 0.1
,ug of
lipid
FIG. 1. Calibration curves for standard lipids by x represent values obtained with cholesterol, stearic tively. Solid and dashed lines represent theoretical acid and cholesterol, respectively.
ultramicro acid, and calibration
method. palmitic curves
A, 0, and acid, respecfor palmitic
0.6 0.5 20.4 $ * ‘0 6 6
0.3 0.2
d o.ll I , , T‘\
,
0.1
25
50
75 pg
100
125
150
175
of lipid
FE. 2. Calibration curves for standard lipids by micro method. A, 0, and X represent values obtained with cholesterol, stearic acid, and palmitic acid, respectively. Solid aud dashed lines represent theoretical calibration curves for palmitic acid and cholesterol, respectively.
420
PANDE,
KHAN,
AND
VENKITASUBRAMANIAN
0.7 r 0.6 t
mg FIG. 3. Calibration curves for represent values obtained with tively.
of
lipid
standard lipids by semimicro method. cholesterol, stearic acid, and palmitic
A, 0, acid,
and X respec-
oxidation. As seen from these figures, utilization of dichromate occurred corresponding to over 95% of the lipid, in agreement with the results of Bragdon (13). A number of human serum samples were analyzed for the estimation of total fatty acids by the procedure described herein. The results were independently compared with those obtained using the Bragdon method (13)) where fatty acids were isolated by a similar procedure involving saponification of solvent extracts of larger serum samples and removal of nonsaponifiable lipids prior to the acidification of saponified digests for the extraction of fatty acids. The data obtained are presented in Table 1. A good agreement between the two methods was observed.
COMPARISON
1 2 3 4 5
TABLE 1 OF SERUM TOTAL FATTY ACID ESTIMATIONS ULTRAMICRO METHOD AND THE BRAGDON (13) For details see text
355 210 371 286 240
357 207 367 285 237
BY THE PRESENT METHOD
-0.56 +1.45 +1.09 t-o.35 +1.27
MICRODETERMINATION
OF
LIPIDS
421
DISCUSSION
The basis of the calorimetric method of Bragdon (13) and the semimicro method described in this investigation is the appearance of chrome alum (maximum absorption 590 mp) due to the reducing action of lipids on acid dichromate. It was found that the simultaneous disappearance of dichromate (maximum absorption 440 rnp) results in a greater optical density change. This principle has been utilized in the micro method described here and is based on a modification of the Johnson procedure (20) for the determination of total nonvolatile organic material. The suggested calorimetric determination of iodine liberated by the action of acid-dichromate on an iodide after extraction into an organic solvent offers little advantage over the other methods described above. However its very sensitive determination in the starch-iodine complex as used by Halliwell (18) for the microdetermination of carbohydrates and proteins is utilized here in the ultramicro method for lipids. The color development process has been simplified here by employing a suitably diluted CdI,-starch reagent to obtain desired acid strength for optimal color development while dispcnsin,g with the use of 0.21 h: acid required by the Halliwell proccdurc ( 18‘). As the destruction of dichromate in concentrated sulfuric acid at higher temperature increases with decreasing concentration of dichromate below 0.034% (18)) it is essential that the heating should be done for a minimum period of time. Bragdon (13) found that the oxidation of lipids was complete in 30 min. A reinvestigation showed that most of the oxidation occurred in the first 8 min. A heating period of 15 nun has been found adequate, although subsequent heating in the semimicro and micro methods was not critical. The colors in the semimicro and micro methods remain stable for a few hours. In the ultramicro method, however, it is necessary that the colors be measured 20 nun after the addition of Ccl&starch reagent (18). These oxidative procedures of lipid estimation require complete removal of the solvent prior to oxidation. Since the removal of solvent by heating on a sand bath as recommended by Bang (1) and BIix (21) was not satisfactory (22), Bloor (3) was led to use a bath of Wood’s metal heated to 145°C. Attention has been drawn by Van Slyke (23) and Bloor (7) for exercising caution in the removal of solvent to avoid lipid loss clue to volatilization and air oxidation, especially when deposited as thin films. The removal of solvent as described here minimized the chances of lipid loss due to a considerable lowering of the temperature, and gave identical results with those obtained by passing nitrogen for solvent removal (13). This procedure was therefore preferred for it.s
422
PANDE,
KHAN,
AND
VENKITASUBRAMANIAN
convenience and quickness and because it restricted the dried lipid to the same area as that occupied by its solution. Although organic materials of all kind respond to acid dichromate oxidation (23), the wide applicability for lipid estimation (l-10) is possible because of the availability of selective extraction procedures. However, extreme precautions are required to avoid possible interference due to reducing substances by contact with acid-dichromate at all steps. The recently published methods for the determination of serum total fatty acids (14-16) require a microtitration assembly and not less than 0.2 ml serum for a single titration. The procedure described here allows replicate calorimetric determinations on lipid extracts of 15 ~1 serum. The present method makes use of solvent extracts of serum, as is often required for phospholipid and cholesterol determinations. Further, it avoids the contamination of fatty acid extract by tiny particles of nonlipid nature encountered during the direct saponification of serum. If cholesterol estimation is also carried out, the removal of nonsaponifiable lipids prior to the extraction of fatty acids is unnecessary. Hence, cholesterol and fatty acids are extracted t,ogether after acidification of the saponified digest, an aliquot is directly taken for oxidation, and the amout of fatty acids is evaluated by applying a suitable correction for the oxidation of cholesterol (see ref. 8). SUMMARY
Calorimetric methods for the estimation of lipids, covering a range of 2 pg to 1.33 mg are described. The principle involved is the oxidation of lipids in acid-dichromate. Using ioclometric calorimetry for the determination of microgram amounts of lipid, a procedure for the determination of serum total fatty acids requiring only 15 ~1 of serum is described. SCKNOWLEDGMENTS This investigation was supported in part by grants from the International Atomic Energy Agency, Vienna, and the Indian Council of Medical Research, New Delhi. Our thanks are due Dr. R. Viswanathan for his interest in this work. REFERENCES 1. 2. 3. 4. 5. 6. 7.
BANG,
I., &o&em. 2. 91, 36 (1918). BANG, I., Biochena. Z. 91, 235 (1918). BLOOR, W. R., J. Biol. Ckem. 77, 53 (1928). OKEY, R., J. Biol. Chem. 88, 387 (1939). BOYD, E. M., An&. J. Cl&. Path. 8, 77 (1938). BLOOR, W. R., J. Biol. Chem. 82, 273 (i929). BLOOR, W. R., J. Biol. Chem. 170, 671 (1947). 8. KIBRICK, A. C., AKD SKUPP, S. J., Arch. Biochem. Biophys. 9. BALAKHOUSBII, S. D., AND BAL.4KHOUSKI1, I. S., in “Methods of Blood” (in R.ussian). Moscow, 1953.
44, 34 (1953). of Chemical Analysis
MICRODETERMINATION
OF
LIPIDS
10. SPERRY, W. M., Methods Biochem. Anal. 2, 83 (1955). 11. MAN, E. B., AND GILDEA, E. B., J. Bid. Chem. 99, 43 (1933). 12. BACKLIN, E., &o&em. Z. 217,493 (1930). 13. BRAGDON, J. M., J. Biol. Chem. 190, 513 (1951). 14. ALBRINK, M. J., J. Lipid Res. 1, 53 (1959). 15. DRYSDALE, J., AND BILLIMORIA, J. D., Clin. Chim. Acta 5, 828 (1960). 16. DOLE, V. P., AND MEINERTZ, H., J. Biol. Chem. 235, 2595 (1960). 17. DOLE, V. P., J. Clin. Iwest. 35, 150 (1956). 18. HALLIWELL, G., Biochem. J. 74, 487 (19601. 19. BLOOR, W. R., J. Biol. Chem. 24, 227 (1916). 20. JOHNSON, M. J., J. Biol. Chem. 181, 707 (1949). 21. BLIX, G.. Skand. Arch. Ph?~siol. 48, 297 (1926). 22. FLEISCH, A., Biochem. Z., 177, 453 (1926). 23. VAN SLTKE, D. D., ASD FOLCH, J., /. Biol. Chem. 136, 509 (1940).
423
BIOCHEMISTRT
Microdetermination
6,
415-423
11963)
of lipids
and
Serum
S. V. PANDE, R. PARVIN KHAN, T. A. VENKITASUBRAIMANIAN From
the
Vallabhbhai
Pate1
Chest
Rcccirrd
Institute, November
I’niversity
Total
Fatty
Acids
AND
of Delhi,
Delhi,
India
10, 1962
INTRODUCTION
Since the introduction of acid-dichromate reagent for lipid estimation (1, 2)) many methods making use of this principle have been evolved for the estimation of cholesterol (3, 5), phospholipids (2, 5, 6), fatty acids (3, 5, 7, 8)) and total lipids (9, 10). However different workers have employed different factors (3, 11) for the calculation of the quantity of lipid, as the extent of oxidation in these procedures differed depending upon the time and temperature employed for oxidation. Nevertheless, Bloor (7) was able to overcome this st,umbling block in the application of the acid-dichromate method for lipid estimation by defining the conditions for almost complete oxidation. Most of the earlier methods of lipid estimation were based on the titrimetric determination of excess dichromate by the starch-thiosulfate method. Because the procedure of Backlin (12) involving manometric determination of carbon dioxide evolved on chromic acid combustion of lipid was inconvenient, it did not find much application. However, a simple calorimetric method was developed for the first time by Bloor (7) ; the procedure was not entirely satisfactory because a linear relationship over a wide range was not obtained and a specially constructed calorimeter was required. This method was subsequently improved by Bragdon (13’). Considering the method of Bragdon (13) to be of a hazardous nature, Kibrick and Skupp (8) attempted to simplify it, but their use of a pressure cooker for oxidation is too cumbersome. Therefore simplified procedures of varyin g dcgrec of sensitivity for the determination of lipid are presented here. The availability of rapid and sensitive methods for the determination of serum lipid is of importance. For the determination of serum total fatty acids, the methods presently in use (14-16) are essentially based on the microtitration method of Dole (17) for unesterified fatty acids. A colorimetric method many times more sensitive than microtitration method5 is described here for the determination of total fat’ty acids in serum. 415
416
PANDE,
KHAN,
AND
VENKITASUBRAMANIAN
METHODS
Chemicals All chemicals used were of A.R. grade. Cholesterol, palmitic acid, stearic acid, soluble starch, ethanol, potassium dichromate, and sodium sulfite used were from E. Merck; solvent ether and petroleum ether from B. D. H., England. Cadmium iodide used in the earlier experiments was of unknown origin. In later work the Chemapol Prague product was used. Before use, solvents were distilled in an all-glass apparatus and the lipids recrystallized. The standard lipid solutions were prepared in petroleum ether (b.p. 40-60°C). Reagents (1) Ultramicro method for 2-12 pg lipid: (a) 0.034% (w/v) potassium dichromate in 97% (w/v) sulfuric acid is prepared by diluting aqueous 3.4% (w/v) potassium dichromate with 98% sulfuric acid before use as described by Halliwell (18). (b) Cadmium iodide-starch reagent. 1.4 gm cadmium iodide is dissolved in 60-70 ml water and the solution boiled for 15-20 min. To this is added a clear solution of starch obtained by boiling 0.33 gm soluble starch in about 75 ml water for 5 min, and the mixed solution boiled for 5 more min. The final solution is then filtered, made up to 500 ml at room temperature with distilled water, and stored protected from light. This is stable for about six months without precipitation. (6) Micro method for 20-140 pg lipid: (a) 0.15% potassium dichromate in 96% sulfuric acid. 1.5 gm potassium dichromate is dissolved with heating in 10 ml water and the solution cooled and made up to 1 liter with 98% (w/v) sulfuric acid. (b) Aqueous 20% (wiv) sodium sulfite solution, prepared fresh when required. (3) Xemimicro method for 170 pg to 1.33 mg lipid: 2.0y0 potassium dichromate (w/v) in 98% (w/v) sulfuric acid. 20 gm powdered potassium dichromate is dissolved in 1 liter 98% (w/v) sulfuric acid at a temperature below 100°C. The solution is stored in an all-glass bottle with an inverted tube over the mouth to protect from dust. It is stable for at least one year. Analytical
Procedure
An aliquot of the lipid solution to be analyzed was transferred, the solvent was removed by rapid evaporation under reduced pressure, and the lipid was oxidized with acid dichromate. The reaction was followed by direct calorimetry (micro method), by an iodometric calorimetry
MICRODETERMINATIOiV
OF
LIPIDS
417
(ultramicro method), and by the appearance of reduced chrome alum (semimicro method). The lipid solutions were delivered into the bottom of scrupulously clean glass-stoppered tubes (6J’ X a/4”‘) with standard joints, the volumes of the solution never exceeding that of the acid-dichromate reagent to be added subsequently. The tubes were placed vertically in a vacuum dessicator and the pressure was very gradually reduced by adjusting the stopcock to avoid any spurting during evaporation of the solvent.. These precautions were necessary to restrict the dried film to the bottom of the tube so that no lipid escaped contact with the acid-dichromate reagent during the process of oxidation. After complete removal of the solvent, the vacuum was released carefully by putting a piece of white paper at the mouth of the stopcock to keep the ingoing air free from dust. This procedure was carried out in a place with sufficient air circulation to avoid the entry of solvent vapors. A number of samples could thus be freed of the solvent simultaneously and in a short time. Acid-dichromate reagent was added almost immediat’ely, and tubes were stoppered and then heated for oxidation. The subsequent procedures for different ranges of lipid estimation are described below separately. Ultramicro
Method
(2-12 pg)
To 2 to 12 pg dried lipid sample was added exactly 1.0 ml 0.034% (w/v) potassium dichromate in 97% sulfuric acid (w/v). A control tube was also included which did not contain any lipid. The tubes were placed in a boiling water bath for 15 min and then cooled in running water; 9.0 ml water was added to all the tubes, contents were mixed well, and 0.5 ml of these solutions was pipetted separately into the bottom of widebore test tubes, followed by addition of 4.5 ml CdI,-starch reagent. For optimal color development, the solutions were t’horoughly mixed and allowed to stand at room temperature for exactly 20 min, and then diluted with water (1: 1). The reagent blank was prepared by taking 0.5 ml 3.6 N sulfuric acid, adding 4.5 ml CdI,-starch reagent, and proceeding as for samples. The color intensities were read against the reagent blank at 575 rnp (peak absorption). Micro
Method
(20-140
pg)
2.0 ml 0.15% potassium dichromate in 96% (w/v) sulfuric acid was added to tubes containing zero (two blank t,ubes required) to 140 pg solvent-free lipid. After heating and cooling as described above, 4.5 ml water was added and the solut,ions were retooled after mixing; 0.10 ml freshly prepared aqueous 20% Na,SO,.7H,O (w/v) was added to reduce the diehromate in one of the blanks. All tubes were read against the
418
PANDE,
KHAN,
AND
VENKITASUBRAMANIAN
reduced blank at 440 mp. The unreduced blank tube serves as a control, showing the amount of dichromate present initially. Semimicro
Method
(170 pg to l.SS mg)
The procedure of Bragdon (13) has been modified so that the colorimetry can be performed in test tubes with relative ease. The reduction in the volume of the solutions used obviously increases the sensitivity. After removing the solvent completely, 3.0 ml 2$% potassium dichromate (w/v) in 98% (w/v) sulfuric acid was added to 170 pg to 1.33 mg lipid. After heating and cooling 2s described in the ultramicro method, 4.5 ml water was added, the contents were mixed and the solutions were retooled. The color intensities were measured at 590 mp against a reagent blank prepared by taking 3.0 ml acid-dichromate reagent and treating like the rest. All calorimetric measurements were made in a Bausch 8.1Lomb colorimeter. Procedure for Determination
of Serum Total Fatty Acids
To 7 ml alcohol-ether mixture (3: 1 v/v) of Bloor (19) in a graduated centrifuge tube w2s added 40 ~1 serum, with constant and vigorous stirring during the addition. The contents were brought to boil and cooled and the volume was made up to 10 ml by addition of more alcohol-ether mixture; 3.0 ml of the clear supernatant obtained after centrifugation ~2s transferred to a 15-ml glass-stoppered tube with standard joint and the volume w2s reduced to about, half by dipping the tube in warm water. After adding 0.10 ml 4N NaOH, an air condenser w2s attached to the tube and the contents saponified in 2 water bath at 80°C for 30 min. After cooling, about 1.5 ml water was added and the nonsaponifiable lipids were removed by two extractions with about 8 ml petroleum ether, by giving end to end shakes for 2 few minutes and separating the two phases by centrifugation. The petroleum ether layer was withdrawn 2s much as possible without touching the aqueous phase by means of 2 capillary pipet. The small volume of ether left behind w2s evaporated off by putting the tubes in warm water. The contents were then acidified by the addition of 0.2 ml 4 N H,SO, solution cooled to room temperature (ZO-25’C) and the liberated fatty acids extracted into exactly 4.0 ml petroleum ether (b.p. 40-60°C). After centrifugation suitable aliquots were withdrawn for fatty acid estimation by the ultramicro method as already described. RESULTS
The calibration curves obtained for cholesterol, palmitic acid, and stearic acid by the ultramicro, micro, and semimicro method are presented in Figs. 1-3, respectively. As seen from the figures, the calibration curves
MICRODETERMINATION
OF
419
LIPIDS
for palmitic and stearic acid almost overlap each other, as is to bc cxpetted from the close dichromate equivalents of long-chain fatty acids (13). The theoretical calibration curves included in Figs. 1 and 2 are based on the presumption that the oxidation of lipids was complete and t,hat only carbon dioxide and no carbon monoxide xvas evolved during 0.6
6 0.2 d 0.1
,ug of
lipid
FIG. 1. Calibration curves for standard lipids by x represent values obtained with cholesterol, stearic tively. Solid and dashed lines represent theoretical acid and cholesterol, respectively.
ultramicro acid, and calibration
method. palmitic curves
A, 0, and acid, respecfor palmitic
0.6 0.5 20.4 $ * ‘0 6 6
0.3 0.2
d o.ll I , , T‘\
,
0.1
25
50
75 pg
100
125
150
175
of lipid
FE. 2. Calibration curves for standard lipids by micro method. A, 0, and X represent values obtained with cholesterol, stearic acid, and palmitic acid, respectively. Solid aud dashed lines represent theoretical calibration curves for palmitic acid and cholesterol, respectively.
420
PANDE,
KHAN,
AND
VENKITASUBRAMANIAN
0.7 r 0.6 t
mg FIG. 3. Calibration curves for represent values obtained with tively.
of
lipid
standard lipids by semimicro method. cholesterol, stearic acid, and palmitic
A, 0, acid,
and X respec-
oxidation. As seen from these figures, utilization of dichromate occurred corresponding to over 95% of the lipid, in agreement with the results of Bragdon (13). A number of human serum samples were analyzed for the estimation of total fatty acids by the procedure described herein. The results were independently compared with those obtained using the Bragdon method (13)) where fatty acids were isolated by a similar procedure involving saponification of solvent extracts of larger serum samples and removal of nonsaponifiable lipids prior to the acidification of saponified digests for the extraction of fatty acids. The data obtained are presented in Table 1. A good agreement between the two methods was observed.
COMPARISON
1 2 3 4 5
TABLE 1 OF SERUM TOTAL FATTY ACID ESTIMATIONS ULTRAMICRO METHOD AND THE BRAGDON (13) For details see text
355 210 371 286 240
357 207 367 285 237
BY THE PRESENT METHOD
-0.56 +1.45 +1.09 t-o.35 +1.27
MICRODETERMINATION
OF
LIPIDS
421
DISCUSSION
The basis of the calorimetric method of Bragdon (13) and the semimicro method described in this investigation is the appearance of chrome alum (maximum absorption 590 mp) due to the reducing action of lipids on acid dichromate. It was found that the simultaneous disappearance of dichromate (maximum absorption 440 rnp) results in a greater optical density change. This principle has been utilized in the micro method described here and is based on a modification of the Johnson procedure (20) for the determination of total nonvolatile organic material. The suggested calorimetric determination of iodine liberated by the action of acid-dichromate on an iodide after extraction into an organic solvent offers little advantage over the other methods described above. However its very sensitive determination in the starch-iodine complex as used by Halliwell (18) for the microdetermination of carbohydrates and proteins is utilized here in the ultramicro method for lipids. The color development process has been simplified here by employing a suitably diluted CdI,-starch reagent to obtain desired acid strength for optimal color development while dispcnsin,g with the use of 0.21 h: acid required by the Halliwell proccdurc ( 18‘). As the destruction of dichromate in concentrated sulfuric acid at higher temperature increases with decreasing concentration of dichromate below 0.034% (18)) it is essential that the heating should be done for a minimum period of time. Bragdon (13) found that the oxidation of lipids was complete in 30 min. A reinvestigation showed that most of the oxidation occurred in the first 8 min. A heating period of 15 nun has been found adequate, although subsequent heating in the semimicro and micro methods was not critical. The colors in the semimicro and micro methods remain stable for a few hours. In the ultramicro method, however, it is necessary that the colors be measured 20 nun after the addition of Ccl&starch reagent (18). These oxidative procedures of lipid estimation require complete removal of the solvent prior to oxidation. Since the removal of solvent by heating on a sand bath as recommended by Bang (1) and BIix (21) was not satisfactory (22), Bloor (3) was led to use a bath of Wood’s metal heated to 145°C. Attention has been drawn by Van Slyke (23) and Bloor (7) for exercising caution in the removal of solvent to avoid lipid loss clue to volatilization and air oxidation, especially when deposited as thin films. The removal of solvent as described here minimized the chances of lipid loss due to a considerable lowering of the temperature, and gave identical results with those obtained by passing nitrogen for solvent removal (13). This procedure was therefore preferred for it.s
422
PANDE,
KHAN,
AND
VENKITASUBRAMANIAN
convenience and quickness and because it restricted the dried lipid to the same area as that occupied by its solution. Although organic materials of all kind respond to acid dichromate oxidation (23), the wide applicability for lipid estimation (l-10) is possible because of the availability of selective extraction procedures. However, extreme precautions are required to avoid possible interference due to reducing substances by contact with acid-dichromate at all steps. The recently published methods for the determination of serum total fatty acids (14-16) require a microtitration assembly and not less than 0.2 ml serum for a single titration. The procedure described here allows replicate calorimetric determinations on lipid extracts of 15 ~1 serum. The present method makes use of solvent extracts of serum, as is often required for phospholipid and cholesterol determinations. Further, it avoids the contamination of fatty acid extract by tiny particles of nonlipid nature encountered during the direct saponification of serum. If cholesterol estimation is also carried out, the removal of nonsaponifiable lipids prior to the extraction of fatty acids is unnecessary. Hence, cholesterol and fatty acids are extracted t,ogether after acidification of the saponified digest, an aliquot is directly taken for oxidation, and the amout of fatty acids is evaluated by applying a suitable correction for the oxidation of cholesterol (see ref. 8). SUMMARY
Calorimetric methods for the estimation of lipids, covering a range of 2 pg to 1.33 mg are described. The principle involved is the oxidation of lipids in acid-dichromate. Using ioclometric calorimetry for the determination of microgram amounts of lipid, a procedure for the determination of serum total fatty acids requiring only 15 ~1 of serum is described. SCKNOWLEDGMENTS This investigation was supported in part by grants from the International Atomic Energy Agency, Vienna, and the Indian Council of Medical Research, New Delhi. Our thanks are due Dr. R. Viswanathan for his interest in this work. REFERENCES 1. 2. 3. 4. 5. 6. 7.
BANG,
I., &o&em. 2. 91, 36 (1918). BANG, I., Biochena. Z. 91, 235 (1918). BLOOR, W. R., J. Biol. Ckem. 77, 53 (1928). OKEY, R., J. Biol. Chem. 88, 387 (1939). BOYD, E. M., An&. J. Cl&. Path. 8, 77 (1938). BLOOR, W. R., J. Biol. Chem. 82, 273 (i929). BLOOR, W. R., J. Biol. Chem. 170, 671 (1947). 8. KIBRICK, A. C., AKD SKUPP, S. J., Arch. Biochem. Biophys. 9. BALAKHOUSBII, S. D., AND BAL.4KHOUSKI1, I. S., in “Methods of Blood” (in R.ussian). Moscow, 1953.
44, 34 (1953). of Chemical Analysis
MICRODETERMINATION
OF
LIPIDS
10. SPERRY, W. M., Methods Biochem. Anal. 2, 83 (1955). 11. MAN, E. B., AND GILDEA, E. B., J. Bid. Chem. 99, 43 (1933). 12. BACKLIN, E., &o&em. Z. 217,493 (1930). 13. BRAGDON, J. M., J. Biol. Chem. 190, 513 (1951). 14. ALBRINK, M. J., J. Lipid Res. 1, 53 (1959). 15. DRYSDALE, J., AND BILLIMORIA, J. D., Clin. Chim. Acta 5, 828 (1960). 16. DOLE, V. P., AND MEINERTZ, H., J. Biol. Chem. 235, 2595 (1960). 17. DOLE, V. P., J. Clin. Iwest. 35, 150 (1956). 18. HALLIWELL, G., Biochem. J. 74, 487 (19601. 19. BLOOR, W. R., J. Biol. Chem. 24, 227 (1916). 20. JOHNSON, M. J., J. Biol. Chem. 181, 707 (1949). 21. BLIX, G.. Skand. Arch. Ph?~siol. 48, 297 (1926). 22. FLEISCH, A., Biochem. Z., 177, 453 (1926). 23. VAN SLTKE, D. D., ASD FOLCH, J., /. Biol. Chem. 136, 509 (1940).
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