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Simultaneous determination of total cholesterol concentration and radioactivity in plasma
Journal of Biochemical and Biophysical Methods, 8 (1983) 1-7
1
Elsevier
Simultaneous determination of total cholesterol concentration and radioactivity in plasma B. Emmanuel, T.W. Fenton, B.V. Turner and L.P. Milligan Department of Animal Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5
(Received 4 January 1983) (Accepted 16 March 1983)
Summa~ Total plasma cholesterol concentration and radioactivity were measured simultaneously using a gas chromatograph equipped with a flame ionization detector and an effluent splitter. More than 99% of the recovered radioactivity was in the cholesterol peak. Specific activities were highly correlated with the a m o u n t s of labeled cholesterol present in plasma. The recovery of label was quantitative over a wide range of carrier cholesterol concentrations. The method is highly reproducible, accurate, rapid and specific. Key words: total cholesterol concentration; radioactivity; plasma.
Introduction Cholesterol has a significant role in lipid, hormone, vitamin and bile acid metabolism. Studies of in vitro, as well as whole body cholesterol kinetics require an accurate method to measure specific radioactivity in tissue samples. During preparations for studying the kinetics of incorporation of labeled acetate and propionate into cholesterol by the liver of several species in this laboratory it became apparent that the existing methods were not satisfactory. Methods used for labeled cholesterol isolation following saponification and extraction involve thin-layer chromatography [1,2] or precipitation with either totamine [3] or digitonin [4-7]. Direct counting of tissue containing labeled cholesterol by a two-phase liquid scintillation method was reported by Viikari [8]. The procedure of precipitation of labeled cholesterol with digitonin is tedious and time consuming, and the recovery is not complete. David [4] using the digitonin method [7] recovered only 81% of labeled standard cholesterol. Direct counting [8] is not specific: other labeled compounds present in the lipid fraction can markedly contaminate cholesterol, in particular, in studies in which labeled acetate is used which will incorporate simultaneously into fatty acids and cholesterol. The recovery of radioactive cholesterol in this procedure is 94%. Using thin-layer chromatography partial e|ution of radioactive compounds, and variation in the nature, quantity and orientation of the solid support in the scintillation liquid 0165-022X/83/$03.00 © 1983 Elsevier Science Publishers B.V.
can have a profound effect on counting efficiency [9]. Solubilization of the solid support with a proper solvent can improve the recovery [10]. Lillienberg and Svanborg [11] have reported that the gas-liquid chromatographic separation of cholesterol was more accurate than enzymatic and colorimetric procedures. This information gave impetus to the development of a method to measure the concentration and radioactivity of cholesterol simultaneously in tissues utilizing a gas chromatograph.
Material and Methods
Chemicals Cholesterol (AS-cholesten-3fl-ol) and a-cholestane were purchased from Sigma Chemical Co., St. Louis, Mo. [7(n)-3H]Cholesterol (8 Ci/mmol), [2-14C]propionic acid, sodium salt, and [l-14C]acetic acid, sodium salt were products of Amersham Radiochemical Centre, Arlington Heights, Ill. Aquasol-2 was purchased from New England Nuclear, Boston, Mass. The packing material (3% OV-I on 80-100 mesh Gas-Chrom Q) was obtained from Applied Science Laboratories Inc., State College, Pa. Tissue incubation Liver from freshly killed rats was removed and kept on ice. Liver slices (400-500 mg) were incubated in the presence of either 1 #Ci [l-lnC]acetate (200 ~Ci/mmol), or 1 ~Ci [2-J4C]propionate (200 ~Ci/mmol). Incubation conditions and extraction of cholesterol were according to the procedures of Danielsson [12]. Aliquots (3-5/~1) were injected into the gas chromatograph. Sample preparation Standard solutions were made of constant amounts of cholesterol (132 /~g) and cholestane (104 #g) and different quantities of [3H]cholesterol (0.27-4.32 nCi) in 120 /~1 toluene, or constant amounts of cholestane (104 /~g) and [3H]cholesterol (1.08 nCi), and varying amounts of cholesterol (33-528 ~g). [3H]cholesterol (0.27-4.32 nCi) was added to 0.2 ml ovine plasma, and the mixture was saponified and extracted [11]. Aliquots (2-5 /~1) of the standard and plasma extract were injected into the gas chromatograph. Gas chromatography Cholesterol was analyzed by gas chromatography using a Bendix 2500 (Ronceverte, W.Va.) equipped with 'off column' flame ionization detectors. A U-tube glass column (50 cm long × 6 mm o.d.) packed with 3% OV-1 on 80-100 mesh Gas-Chrom Q was used. The column was operated at 260°C with N 2 as the carrier gas at a flow rate of 60 ml/min. Peak areas were integrated using a Hewlett Packard 1000 System (Palo Alto, Calif.) To avoid changes in split ratios due to the variable back pressure introduced by the traps as was experienced using a commercially available effluent splitter (Hamil-
ton), the device described below was utilized. The effluent splitter (Fig. 1) which had a measured 1 : 1 flow rate ratio was made of type 304 stainless-steel capillary tubing (0.33 m m o.d. x 0.18 m m i.d.; 15 cm long) silver soldered into 3.2 m m o.d. stainless-steel tubing. A 3.2 m m brass swagelock tee and 3.2 m m silicone rubber O-rings were used to make the connections between the two arms of the splitter and the outlet end of the column. One arm of the splitter was attached to the flame ionization detector. The other arm with a 13 m m length of hypodermic needle (20 gauge) soldered over the outlet end of the capillary ended in the heated transfer zone of the gas chromatograph. A short length of thin wall copper tubing (5 m m o.d.) with a flared end to make a seat for the traps was attached to the end of the collector arm such that it protruded 13 m m through a hole in the cover of the transfer zone. Glass traps were made from modified ' b r e a k seal' vials which were inserted into the copper tubing through a silicone rubber O-ring to ensure a gas tight seal; the end of the trap was pressed against the septum. The trapping medium was made by soaking white absorbant cotton in 1% solution of light silicone oil in acetone. This solvent was allowed to evaporate before each glass trap was packed. The absorbant was necessary to avoid losses of material resulting from aerosol formation.
Counting After peak capture, the absorbant cotton was transferred to a scintillation vial and the trap was washed with 15 ml of a scintillation cocktail (Aquasol-2). The
Ili 1
10
-7 -6
i i i ii i i i
3 1
Fig. 1. Gas c h r o m a t o g r a p h splitter and trap. 1, column outlet; 2, swageiock tee; 3, O-ring; 4, gas c h r o m a t o g r a p h oven; 5, to detector; 6, stainless-steel tubing; 7, capillary tubing; 8, septum; 9, hypodermic needle; 10, transfer zone; 11, copper tube; 12, O-ring seal; 13, glass tubing; 14, wad of cotton.
samples were counted in a Mark III Liquid Scintillation System (Searle Analytic Inc., Middletones, Wisc.). The presence of the absorbant cotton had no effect on the counting efficiency. Correction for the quenching was made by the external standard procedure. Calculations
Cholesterol specific activity in plasma and in standard solutions containing constant amounts of cholesterol was expressed as dpm per cholesterol peak area. In standard solutions containing varying amounts of cholesterol and constant quantities of [3H]cholesterol, the total recovered radioactivity is shown. Correction was made for the recovered radioactivity taking into account the split ratio.
Results and Discussion
Incorporation rates of labeled acetate and propionate into cholesterol of rat liver (Emmanuel and Robblee, submitted) using the present gas-chromatographic procedure for the determination of cholesterol specific radioactivity were 41 and 7 nmols/2 h per g tissue at 37°C, respectively. The relationships between cholesterol specific activities and [3H]cholesterol added to the standard and plasma samples are shown in Figs. 2 and 3. Specific activities were highly correlated ( r = 0.996-0.998) with the amounts of labeled cholesterol present in the samples. The recovery of [3H]cholesterol added to plasma was complete; more than 99% of the radioactivity recovered was in the cholesterol peak. The data confirm other studies in which gas-liquid radiochromatographic procedures were used to measure radioactivities in steroid compounds [13]. When constant proportions of [3H]cholesterol and cholestane, and different quantities of
c'1o
I"
(J
[3H]CHOLESTEROL ADDED (102 `
DPM )
Fig. 2. Regression of cholesterol specific activity on [3Hlcholesterol in standard samples containing constant amounls of cholesterol and cholestane (internal standard). Mean values + S.E. of 5 observations are shown.
5
cholesterol were injected, the recovery of [3H]cholesterol was constant except at the lowest cholesterol level (Fig. 4) which is probably due to the adsorption of [3H]cholesterol on the column. However, this has no effect on the values of
3(
== t3
r=0.
I-
O
== 2'4
2.
~
[3H]CHOLESTEROLADDED (I02"
.'6 DPM )
Fig. 3. Regression of cholesterol specific activity on [3H]cholesterol in plasma samples. Mean values _+ S.E. of 4 observations are shown.
cholesterol specific activities since cholesterol peaks will be decreased concomitantly by the same fraction. Furthermore, the lowest level of carrier cholesterol used (0.55 /~g) is several times lower than the final concentration of cholesterol in the plasma samples. Under the conditions used in the present studies, the retention time for the cholesterol peak was 2.6 rain, therefore, it requires only a few minutes to measure both the radioactivity and concentration of cholesterol in samples.
w < w Z < Ill "r
r
,
,
e~
¢qo #..
CHOLESTEROL
INJECTED
(~Jg)
Fig. 4. The relationships between [3H]cholesterol recovered and amounts of carrier cholesterol added. Mean values + S.E. of 4 observations are shown.
With the split ratio (1 : 1) used in the present studies, the system can detect levels of radioactivity in standard labeled cholesterol as low as 100 d p m per injection. In biological samples containing markedly higher levels of unlabeled cholesterol than standard samples, injection must be at levels not exceeding the column capacity, thereby avoiding tailing in the cholesterol peak. Taking this into consideration the volume of plasma extract used for injection corresponded to [3H]cholesterol levels as low as 3000 d p m per ml plasma which are normal in kinetic studies. Nevertheless, the system can detect amounts of radioactivity several fold lower by applying one, or a combination of the following procedures: (1) an increase in column diameter or in liquid phase loading, to allow larger sample size injection, (2) a change in the split ratio, and (3) repeated injections of the same sample pooled in one trap. In measuring the concentration and activity of cholesterol separately by two different procedures, errors resulting from inefficiencies and lack of specificity of the systems would have a c o m p o u n d i n g effect on the calculated value of cholesterol specific radioactivity. Recovery efficiencies of gas-liquid r a d i o c h r o m a t o g r a p h y for cholesterol concentration [11], and radioactivity (present studies) are almost 100%. Furthermore, any experimental error during extraction and injection would affect equally cholesterol concentration and radioactivity, therefore, cholesterol-specific radioactivity would remain unchanged. To rule out any possibility of contamination of labeled cholesterol with other c o m p o u n d s it is not desirable to use a procedure where mass is determined by an aliquot of plasma extracts using gas-chromatography and radioactivity is measured by direct counting of another aliquot.
Simplified description of the method and its advantages A new chromatographic method has been developed to measure simultaneously the concentration and radioactivity of plasma cholesterol. The analysis involves the utilization of a gas chromatograph which has been equipped with an effluent splitter for diverting the flow at a 1 : 1 ratio to both an 'off' column flame ionization detector to quantitate cholesterol, and a cotton-filled glass trap to collect cholesterol for subsequent liquid scintillation counting. Since the method is sensitive in measuring small quantities of labeled cholesterol, and it is highly reproducible, accurate, inexpensive, rapid and specific, it can be successfully applied to kinetic and clinical studies of cholesterol metabolism.
Acknowledgement The authors wish to thank Mirjana F e n t o n for her technical assistance.
References I Mitropoulos, K.A., Myant, N.B., Gibbons, G.F., Balasurbramanium, S. and Reeves, B.E.A. (1974) J. Biol Chem. 249, 6052-6056 2 Teekell, R.A., Breidenstein, C.P. and Watts, A.B. (1975) Pouh. Sci. 54, 1036-1042 3 Gubler, C.J., Peterson, J.W., Turpin, K.K., Crane, L.W., Turner, L.G.W. and Bennion, M. (1974) J. Nutr. 104, 1690-1695
4 5 6 7 8 9 10 11 12 13
Davis, R.A. (1978) Steroids 31,593-600 Math~, D. and Chevallier, F. (1979) J. Nutr. 109, 2076-2084 Mathias, M.M., Sullivan, A.C. and Hamilton, J.G. (1981) Lipids 16, 739-743 Sperry, W.M. (1963) J. Lipid Res. 4, 221-225 Viikari, J. (1975) Anal. Biochem. 63, 566-571 Bransome, E.D., Jr. and Grower, M.F. (1970) Anal. Biochem. 38, 401-408 McKenzie, R.M. and Gholson, R.K. (1973) Anal. Biochem. 54, 17-31 Lillienberg, L. and Svanborg, A. (1976) Clin. Chim. Acta 68, 223-233 Danielsson, H. (1972) Steroids 20, 63-72 Nice, E. and Williams, K. (1974) Anal. Biochem. 59, 399-406
1
Elsevier
Simultaneous determination of total cholesterol concentration and radioactivity in plasma B. Emmanuel, T.W. Fenton, B.V. Turner and L.P. Milligan Department of Animal Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5
(Received 4 January 1983) (Accepted 16 March 1983)
Summa~ Total plasma cholesterol concentration and radioactivity were measured simultaneously using a gas chromatograph equipped with a flame ionization detector and an effluent splitter. More than 99% of the recovered radioactivity was in the cholesterol peak. Specific activities were highly correlated with the a m o u n t s of labeled cholesterol present in plasma. The recovery of label was quantitative over a wide range of carrier cholesterol concentrations. The method is highly reproducible, accurate, rapid and specific. Key words: total cholesterol concentration; radioactivity; plasma.
Introduction Cholesterol has a significant role in lipid, hormone, vitamin and bile acid metabolism. Studies of in vitro, as well as whole body cholesterol kinetics require an accurate method to measure specific radioactivity in tissue samples. During preparations for studying the kinetics of incorporation of labeled acetate and propionate into cholesterol by the liver of several species in this laboratory it became apparent that the existing methods were not satisfactory. Methods used for labeled cholesterol isolation following saponification and extraction involve thin-layer chromatography [1,2] or precipitation with either totamine [3] or digitonin [4-7]. Direct counting of tissue containing labeled cholesterol by a two-phase liquid scintillation method was reported by Viikari [8]. The procedure of precipitation of labeled cholesterol with digitonin is tedious and time consuming, and the recovery is not complete. David [4] using the digitonin method [7] recovered only 81% of labeled standard cholesterol. Direct counting [8] is not specific: other labeled compounds present in the lipid fraction can markedly contaminate cholesterol, in particular, in studies in which labeled acetate is used which will incorporate simultaneously into fatty acids and cholesterol. The recovery of radioactive cholesterol in this procedure is 94%. Using thin-layer chromatography partial e|ution of radioactive compounds, and variation in the nature, quantity and orientation of the solid support in the scintillation liquid 0165-022X/83/$03.00 © 1983 Elsevier Science Publishers B.V.
can have a profound effect on counting efficiency [9]. Solubilization of the solid support with a proper solvent can improve the recovery [10]. Lillienberg and Svanborg [11] have reported that the gas-liquid chromatographic separation of cholesterol was more accurate than enzymatic and colorimetric procedures. This information gave impetus to the development of a method to measure the concentration and radioactivity of cholesterol simultaneously in tissues utilizing a gas chromatograph.
Material and Methods
Chemicals Cholesterol (AS-cholesten-3fl-ol) and a-cholestane were purchased from Sigma Chemical Co., St. Louis, Mo. [7(n)-3H]Cholesterol (8 Ci/mmol), [2-14C]propionic acid, sodium salt, and [l-14C]acetic acid, sodium salt were products of Amersham Radiochemical Centre, Arlington Heights, Ill. Aquasol-2 was purchased from New England Nuclear, Boston, Mass. The packing material (3% OV-I on 80-100 mesh Gas-Chrom Q) was obtained from Applied Science Laboratories Inc., State College, Pa. Tissue incubation Liver from freshly killed rats was removed and kept on ice. Liver slices (400-500 mg) were incubated in the presence of either 1 #Ci [l-lnC]acetate (200 ~Ci/mmol), or 1 ~Ci [2-J4C]propionate (200 ~Ci/mmol). Incubation conditions and extraction of cholesterol were according to the procedures of Danielsson [12]. Aliquots (3-5/~1) were injected into the gas chromatograph. Sample preparation Standard solutions were made of constant amounts of cholesterol (132 /~g) and cholestane (104 #g) and different quantities of [3H]cholesterol (0.27-4.32 nCi) in 120 /~1 toluene, or constant amounts of cholestane (104 /~g) and [3H]cholesterol (1.08 nCi), and varying amounts of cholesterol (33-528 ~g). [3H]cholesterol (0.27-4.32 nCi) was added to 0.2 ml ovine plasma, and the mixture was saponified and extracted [11]. Aliquots (2-5 /~1) of the standard and plasma extract were injected into the gas chromatograph. Gas chromatography Cholesterol was analyzed by gas chromatography using a Bendix 2500 (Ronceverte, W.Va.) equipped with 'off column' flame ionization detectors. A U-tube glass column (50 cm long × 6 mm o.d.) packed with 3% OV-1 on 80-100 mesh Gas-Chrom Q was used. The column was operated at 260°C with N 2 as the carrier gas at a flow rate of 60 ml/min. Peak areas were integrated using a Hewlett Packard 1000 System (Palo Alto, Calif.) To avoid changes in split ratios due to the variable back pressure introduced by the traps as was experienced using a commercially available effluent splitter (Hamil-
ton), the device described below was utilized. The effluent splitter (Fig. 1) which had a measured 1 : 1 flow rate ratio was made of type 304 stainless-steel capillary tubing (0.33 m m o.d. x 0.18 m m i.d.; 15 cm long) silver soldered into 3.2 m m o.d. stainless-steel tubing. A 3.2 m m brass swagelock tee and 3.2 m m silicone rubber O-rings were used to make the connections between the two arms of the splitter and the outlet end of the column. One arm of the splitter was attached to the flame ionization detector. The other arm with a 13 m m length of hypodermic needle (20 gauge) soldered over the outlet end of the capillary ended in the heated transfer zone of the gas chromatograph. A short length of thin wall copper tubing (5 m m o.d.) with a flared end to make a seat for the traps was attached to the end of the collector arm such that it protruded 13 m m through a hole in the cover of the transfer zone. Glass traps were made from modified ' b r e a k seal' vials which were inserted into the copper tubing through a silicone rubber O-ring to ensure a gas tight seal; the end of the trap was pressed against the septum. The trapping medium was made by soaking white absorbant cotton in 1% solution of light silicone oil in acetone. This solvent was allowed to evaporate before each glass trap was packed. The absorbant was necessary to avoid losses of material resulting from aerosol formation.
Counting After peak capture, the absorbant cotton was transferred to a scintillation vial and the trap was washed with 15 ml of a scintillation cocktail (Aquasol-2). The
Ili 1
10
-7 -6
i i i ii i i i
3 1
Fig. 1. Gas c h r o m a t o g r a p h splitter and trap. 1, column outlet; 2, swageiock tee; 3, O-ring; 4, gas c h r o m a t o g r a p h oven; 5, to detector; 6, stainless-steel tubing; 7, capillary tubing; 8, septum; 9, hypodermic needle; 10, transfer zone; 11, copper tube; 12, O-ring seal; 13, glass tubing; 14, wad of cotton.
samples were counted in a Mark III Liquid Scintillation System (Searle Analytic Inc., Middletones, Wisc.). The presence of the absorbant cotton had no effect on the counting efficiency. Correction for the quenching was made by the external standard procedure. Calculations
Cholesterol specific activity in plasma and in standard solutions containing constant amounts of cholesterol was expressed as dpm per cholesterol peak area. In standard solutions containing varying amounts of cholesterol and constant quantities of [3H]cholesterol, the total recovered radioactivity is shown. Correction was made for the recovered radioactivity taking into account the split ratio.
Results and Discussion
Incorporation rates of labeled acetate and propionate into cholesterol of rat liver (Emmanuel and Robblee, submitted) using the present gas-chromatographic procedure for the determination of cholesterol specific radioactivity were 41 and 7 nmols/2 h per g tissue at 37°C, respectively. The relationships between cholesterol specific activities and [3H]cholesterol added to the standard and plasma samples are shown in Figs. 2 and 3. Specific activities were highly correlated ( r = 0.996-0.998) with the amounts of labeled cholesterol present in the samples. The recovery of [3H]cholesterol added to plasma was complete; more than 99% of the radioactivity recovered was in the cholesterol peak. The data confirm other studies in which gas-liquid radiochromatographic procedures were used to measure radioactivities in steroid compounds [13]. When constant proportions of [3H]cholesterol and cholestane, and different quantities of
c'1o
I"
(J
[3H]CHOLESTEROL ADDED (102 `
DPM )
Fig. 2. Regression of cholesterol specific activity on [3Hlcholesterol in standard samples containing constant amounls of cholesterol and cholestane (internal standard). Mean values + S.E. of 5 observations are shown.
5
cholesterol were injected, the recovery of [3H]cholesterol was constant except at the lowest cholesterol level (Fig. 4) which is probably due to the adsorption of [3H]cholesterol on the column. However, this has no effect on the values of
3(
== t3
r=0.
I-
O
== 2'4
2.
~
[3H]CHOLESTEROLADDED (I02"
.'6 DPM )
Fig. 3. Regression of cholesterol specific activity on [3H]cholesterol in plasma samples. Mean values _+ S.E. of 4 observations are shown.
cholesterol specific activities since cholesterol peaks will be decreased concomitantly by the same fraction. Furthermore, the lowest level of carrier cholesterol used (0.55 /~g) is several times lower than the final concentration of cholesterol in the plasma samples. Under the conditions used in the present studies, the retention time for the cholesterol peak was 2.6 rain, therefore, it requires only a few minutes to measure both the radioactivity and concentration of cholesterol in samples.
w < w Z < Ill "r
r
,
,
e~
¢qo #..
CHOLESTEROL
INJECTED
(~Jg)
Fig. 4. The relationships between [3H]cholesterol recovered and amounts of carrier cholesterol added. Mean values + S.E. of 4 observations are shown.
With the split ratio (1 : 1) used in the present studies, the system can detect levels of radioactivity in standard labeled cholesterol as low as 100 d p m per injection. In biological samples containing markedly higher levels of unlabeled cholesterol than standard samples, injection must be at levels not exceeding the column capacity, thereby avoiding tailing in the cholesterol peak. Taking this into consideration the volume of plasma extract used for injection corresponded to [3H]cholesterol levels as low as 3000 d p m per ml plasma which are normal in kinetic studies. Nevertheless, the system can detect amounts of radioactivity several fold lower by applying one, or a combination of the following procedures: (1) an increase in column diameter or in liquid phase loading, to allow larger sample size injection, (2) a change in the split ratio, and (3) repeated injections of the same sample pooled in one trap. In measuring the concentration and activity of cholesterol separately by two different procedures, errors resulting from inefficiencies and lack of specificity of the systems would have a c o m p o u n d i n g effect on the calculated value of cholesterol specific radioactivity. Recovery efficiencies of gas-liquid r a d i o c h r o m a t o g r a p h y for cholesterol concentration [11], and radioactivity (present studies) are almost 100%. Furthermore, any experimental error during extraction and injection would affect equally cholesterol concentration and radioactivity, therefore, cholesterol-specific radioactivity would remain unchanged. To rule out any possibility of contamination of labeled cholesterol with other c o m p o u n d s it is not desirable to use a procedure where mass is determined by an aliquot of plasma extracts using gas-chromatography and radioactivity is measured by direct counting of another aliquot.
Simplified description of the method and its advantages A new chromatographic method has been developed to measure simultaneously the concentration and radioactivity of plasma cholesterol. The analysis involves the utilization of a gas chromatograph which has been equipped with an effluent splitter for diverting the flow at a 1 : 1 ratio to both an 'off' column flame ionization detector to quantitate cholesterol, and a cotton-filled glass trap to collect cholesterol for subsequent liquid scintillation counting. Since the method is sensitive in measuring small quantities of labeled cholesterol, and it is highly reproducible, accurate, inexpensive, rapid and specific, it can be successfully applied to kinetic and clinical studies of cholesterol metabolism.
Acknowledgement The authors wish to thank Mirjana F e n t o n for her technical assistance.
References I Mitropoulos, K.A., Myant, N.B., Gibbons, G.F., Balasurbramanium, S. and Reeves, B.E.A. (1974) J. Biol Chem. 249, 6052-6056 2 Teekell, R.A., Breidenstein, C.P. and Watts, A.B. (1975) Pouh. Sci. 54, 1036-1042 3 Gubler, C.J., Peterson, J.W., Turpin, K.K., Crane, L.W., Turner, L.G.W. and Bennion, M. (1974) J. Nutr. 104, 1690-1695
4 5 6 7 8 9 10 11 12 13
Davis, R.A. (1978) Steroids 31,593-600 Math~, D. and Chevallier, F. (1979) J. Nutr. 109, 2076-2084 Mathias, M.M., Sullivan, A.C. and Hamilton, J.G. (1981) Lipids 16, 739-743 Sperry, W.M. (1963) J. Lipid Res. 4, 221-225 Viikari, J. (1975) Anal. Biochem. 63, 566-571 Bransome, E.D., Jr. and Grower, M.F. (1970) Anal. Biochem. 38, 401-408 McKenzie, R.M. and Gholson, R.K. (1973) Anal. Biochem. 54, 17-31 Lillienberg, L. and Svanborg, A. (1976) Clin. Chim. Acta 68, 223-233 Danielsson, H. (1972) Steroids 20, 63-72 Nice, E. and Williams, K. (1974) Anal. Biochem. 59, 399-406