Identification of Plant Sterols in Plasma and Red Blood Cells of Man and Experimental Animals A. KUKSIS, L. MARAI, J.J. MYHER, and K. GEHER, Banting and Best Department of Medical Research, University of Toronto, Toronto, Canada, M5G 1 L6 ABSTRACT
storage disease characterized by 13-sitosterolemia and xanthomatosis in which the total plasma plant sterol levels reached 40 mg%. Furthermore, Mellies et al. (7) have shown that the plasma of infants on formula diets rich in plant sterols had plant sterol levels of 6 mg% compared to 1.5 mg% or less for babies on milk or ad lib diets. Since plant sterols are common components of most normal diets and are known to be absorbed by adults (8,9), we have monitored the GLC peaks for C28 and C29 sterols in the plasma of over 3,000 subjects participating in the hyperlipemia prevalence study. To distinguish the plant sterol peaks from the peaks of t h e companion sterols of cholesterol also reported to occur in plasma or serum in variable amounts (10-14), we have performed detailed mass spectrometric analyses on the free and esterified sterol fractions on a significant number of the plasma samples. As a result, we have shown that under normal conditions sterols other than cholesterol do not reach significant levels in the plasma of adult subjects, although molecular ions characteristic of both plant sterols and cholesterol companions can be obtained from the appropriate regions of the c h r o m a t o g r a m even when discernible mass peaks are not seen in the total lipid profile.
D i r e c t gas liquid chromatography (GLC) of total plasma lipids showed small peaks (0.5-1.5% of total free sterol area) corresponding to free C2s and C29 sterols in ca. 50% of some 3,000 normal subjects and patients with hyperlipemia. Comparable proportions of similar peaks were present in the sterol fraction isolated from the red blood cells of many of these subjects. The maximum levels of these components in the plasma and red blood cells of domestic and laboratory animals were up to I0 times higher than t h o s e seen in man. Detailed gas chromatography/mass spectrometry analyses of the plasma lipids from a much more limited number of subjects and animals s h o w e d that the GLC peaks corresponding to the free C2s and C29 sterols were largely due to the plant sterols campesterol, stigmasterol, and 13s i t o s t e r o l . I n all instances, variable amounts (0.05-0.2% of the total free s t e r o l a r e a ) of 7-dehydrocholesterol, desmosterol, lanosterol, and cholesterol t~-oxide were also detected. While the total content and composition of the plasma plant sterols appeared to vary greatly among the subjects, it never exceeded 2% of total sterol in the normal subjects and patients examined. There was no evidence for a significant increase in the plant sterol content of the plasma of patients with hypercholesterolemia or hypert rigiyceride mia.
MATERIALS AND METHODS Standards
I NTRODUCTION
Routine determinations of plasma lipid profiles of normal subjects and patients with hyperlipemia by gas liquid chromatography (GLC) have revealed the presence of small peaks with retention times corresponding to those of plant sterols (1-3). Other reports have claimed the occurrence of plant sterols in plasma and tissues o f normal subjects (4) and cancer patients (4,5). In most of these instances, the estimated plant sterol levels have averaged 0.5 mg% or less. Recently, liowever, Bhattacharyya and Connor (6) have described a new lipid
Purified desmosterol, campesterol, stigmasterol, and 13-sitosterol were obtained from Applied Science Laboratories, State College, PA. Cholestanol, coprostanol, and cholesterol oroxide were obtained from Mann Research Laboratories, New York, NY, while the Aldrich Chemical Co., Milwaukee, WI, supplied 7-dehydrocholesterol, l a n o s t e r o l , and dihydrolanosterol, ct-Tocopherol was obtained from Distillation Products Industries, Rochester, NY.
581
Sources of Plasma and Serum Samples
The bulk of the plasma samples (over 3,000) were obtained from a study of the prevalence of hyperlipemia in a free-living population in the Toronto area conducted by the TorontoMcMaster Lipid Research Clinic. The blood samples were collected in ethylenediamine
582
A. KUKSIS, L. MARAI, J.J. MYHER, AND K. GEHER
tetraacetic acid (EDTA) tubes according to a procedure published by the Lipid Research Clinics Program (15). Plasma of patients with various types of hyperlipemia (2 Type I, 22 Type II, 2 Type III, 9 Type IV, and 4 Type V) were obained through the courtesy of J.A. Little of St. Michael's Hospital and G. Steiner of Toronto General Hospital, Toronto, Canada. Isolation of Plasma or Serum Lipids
Total lipid extracts of whole plasma or neutral lipid extracts of Dlasrna following digestion with phospholipase C were prepared by extraction with chloroform:methanol, 2:1, as previously described (16). F o r a detailed examination of the free sterol and steryl ester fractions, the plasma lipid extracts were resolved into the lipid classes by thin layer chromat o g r a p h y ( T L C ) according to previously published methods (16,17). The steryl ester fraction was saponified with 0.5 N KOH in 70% ethanol and the unsaponifiable matter recovered by extraction with diethyl ether (18). NOTE: Care should be taken not to contaminate the plasma lipid samples with plant sterols present on laboratory equipment. Thus, the latex rubber bulbs c o m m o n l y employed to control Pasteur pipettes upon contact with organic solvents release readily detectable amounts of ~-sitosterol and other terpenoid impurities of comparable tool wt (unpublished results). Trimethylsilylation
The dried lipid extracts were transferred to sealed conical vials of 0.5 ml capacity, and 150-250/,d of TRISIL/BSA or a solution o f p y r i d i n e : h e x a m e t h y l d i s i l a z a n e : trimethylchlorosilane, 12:5:2, were added (19). After 2 hr at room temperature, the silylation was complete and the reaction mixture ready for injection into the gas chromatograph. Gas Liquid Chromatography
Routine determination of the plasma or serum lipid profiles was made by the manual (16) or automated ( 2 0 ) G L C techniques. High resolution GLC of plasma sterols was perf o r m e d on 6 f t x I / 8 i n . ID glass columns packed with 1% SE-30 or 3% SILAR 5CP on 100-120 mesh Gas Chrom Q (Applied Science Laboratories). The columns were installed in a V a r i a n m o d e l 2 7 0 0 Gas Chromatograph equipped with a flame ionization detector. The carrier gas was nitrogen at 40 ml[min. Isothermal runs were made at 200 C with the injector and detector heaters at 250 C. Temperature programmed runs were made in the range 180-250 C at a heating rate of 4 C/min. LIPIDS, VOL 11, NO. 8
Gas Chromatography/Mass Spectrometry (GC/MS)
Combined GC/MS analyses o f the trimethylsilyl (TMS) ethers of sterois and of the fatty acid esters of sterols were made with a Varian Mat CH-5 single focusing mass spectrometer coupled to Varian Data 620/i Computer and a peak matcher (21). The TLC separations of the TMS ethers of sterols were made on a Varian M o d e l 2700 Moduline gas chromatograph equipped with a 6 ft x 1/8 in. ID glass column packed with 1% SE-30 on Gas Chrom Q. The carrier gas was helium at 10 rnl/min. The gas chromatograph which did not have a separate detector was operated at 180 C isothermally with an injector heater at 225 C. The transfer line was maintained at 275 C. The mass spectrometer was operated at an ionization voltage of 3,000 V, electron emission energy of 100 pA, an d an ion source temperature o f 270 C. Scanning was done at 4 see/decade at-a resolution of 800-1,000. Mass acquisitions were made exponentially in a cyclic manner. All total spectra taken over the GLC peaks were corrected for total ion current variation. Also, spectra were taken of the column bleed and were subtracted from the total spectra of any minor components b y the c o m p u t e r using Varian Module Sub. GC/MS of t o t a l plasma l i p i d extracts was obtained by means o f 3 ft x 1]8 in. ID glass columns packed with 3% OV-1 on Gas Chrom Q (100-120 mesh). These columns were temperature programmed from 175-310 C, using helium as the carrier gas at a flow rate of 10 ml[min. F o r this purpose, the temperature of the injector heater was raised to 300 C and that of the transfer line to 325 C, while the helium separator and the ion source temperatures were adjusted to 310 C. Under these conditions, the presence of free plant sterols could be monitored by searching for the molecular ions of campesterol, stigmasterol, and fl-sitosterol, and that of the plant sterol esters could be monitored by searching for the M-fatty acid ions of these steryl esters. For a more sensitive detection of the plant sterols, the steryl ester fraction was saponified and free sterols isolated by TLC. The sterols were then resolved as the TMS ethrs on the 6 ft GLC columns described above and the appropriate molecular ions monitored b y the peak marcher. F o r this purpose, the low mass channel of the peak matcher was focused on m/e 458 and this channel used to monitor the elution o f the TMS ether of cholesterol. After the appearance of cholesterol, the second channel of the peak matcher, preset to focus on m/e 472, was used to monitor the TMS ether o f campesterol. Finally, after elution of campesterol, the dial settings of the second channel were switched to
PLANT STEROLS
allow the monitoring of m/e 486, which is the molecular ion for the TMS ether of/3-sitosterol. Peak height values were corrected for retention time variation and differences in response by using a standard mixture made up by weighing out predetermined amounts of cholesterol, campesterol, and /~-sitosterol. The presence of a-tocopherol in the cholesterol peak was detected by monitoring m/e 502, which represents the molecular ion of the TMS ether of a-tocopherol and m]e 237, which is a major fragment. The molecular ions and other appropriate fragments (M-15, M-90, and M-90+15) w e r e u s e d t o m o n i t o r the presence of AS-methostenol, AS-lanosterol, methostenol, lanosterol, and the a-oxide of cholesterol in the GLC elution pattern. RESULTS AND DISCUSSION GLC Identification of Plant Sterols-in Total Lipid Extracts of Plasma and Red Blood Cells of Man
An examination of the GLC elution profiles of total plasma lipids from normal subjects and patients with hyperlipemia showed that many samples possessed small peaks in the regions occupied by plant sterols. Figure 1 shows the plasma lipid profiles obtained for a Type I and for a Type IIb hyperlipemia patients. By decreasing the attenuation, it was possible to increase the size and to improve the shape of the peaks, which then allowed the recognition of
583
OF BLOOD
rI
FIG. 1. GLC profiles of plasma lipids of patients with hyperlipemia. A) Total lipids of a patient with Type l hyperlipemia; B) neutral lipids of a patient with Type lib hyperlipemia. Peaks 16 and 18, TMS esters of CI 6 and C18 fatty adds; Peaks 27, 28, and 29, TMS ethers of cholesterol, campesterol, and #-sitosterol; Peak 30, tridecanoylglycerol internal standard; Peak 34, TMS ether of palmitoylsphingosine; Peaks 36-40, TMS ethers of diacylglycerols of 36 to 40 acyl carbons; Peaks 43-47, cholesterol esters of C16 to C20 fatty acids; Peaks 50-54, triaeylgiyeerols with 50-54 acyl carbons. GLC conditions and samples as given in text. the characteristic plant sterol profile of various vegetable oils and fats. The minor amounts of plant sterols could be recovered along with free cholesterol by TLC of the plasma lipids in a nautral lipid system. However, TLC occasionally removed some of the nonsterol material emerging in the free sterol region of the total lipid profile. In all instances where plant sterols were found in the free sterol fraction, peaks corresponding to the C 28 and C 29 sterols were also recovered in the unsaponifiable matter of the steryl esters isolated from the total lipid
TABLE I
Plant Sterol Levels in Plasma and Red Blood Cells of Normal Subjects and Patients with Hyperlipemia as Estimated by Gas Liquid Chromatography Subjects a
Free sterols Ester• Campesterol 13-sitosterol Campesterol
sterols /~-sitosterol
Total plant sterols b
(mg/100 ml plasma or cells) Normal P~sma[35] c Cel~[20]
0.2• 0.3•
0.2• 0.3•
0.4•
0.4•
1.2• 0.6•
Hypercholesterolemi~ P~sma[22] Cells[lO]
0.5• 0.7•
0.3• 0.5•
0.8•
0.8•
2.4• 1.2•
Hypertfiglyceridemics P~sma[15] Cells[5]
0.3• 0.4•
0.3• 0.5•
0.6•
0.6•
1.8• 0.9•
aThe normal subjects had plasma-free cholesterol level of 45.9 +11.0 mg % and a total cholesterol level of 171.5 • 38.5 mg %. Total plasma triglyceride was 100 + 20 mg %. The hypercholesterolemics had plasma-free cholesterol levels of 100-200 mg % and total cholesterol levels of 300-600 mg %. The total plasma triglyceride ranged from 100-200 mg %. The hypertriglyceridemics had plasma-free cholesterol levels of 70-100 mg % and total cholesterol levels ranging from 200 to 300 mg %. The total plasma triglyceride levels ranged from 400 to 1,000 mg %. bThe presence of plant sterols was confirmed by GC/MS in all normolipemic [35] and hyperlipemie [12] plasma samples examined in this manner. CThe numbers in square brackets represent the number of individuals in each test group. LIPIDS, VOL. 11, NO. 8
A. KUKSIS, L. MARAI, J.J. MYHER, AND K. GEHER
584
TABLE II Plant Sterol Levels in Plasma or Serum and Red Blood Cells of Domestic and Laboratory Animals as Estimated by Gas Liquid Chromatography Animal speciesa Rat [10] c Mouse [2] Guinea pig [3] Rabbit 131 Dog" [ 2 ] Cow [ 1 ] Pig [21 Horse [2] Sheep [2] Chicken [5] Goose [ 1 ] Duck [ 1 ] Pigeon [2] Frog [2] Turkey [1 ]
Plasma or serum b Red blood cells Campesterol /~-sitosterol Campesterol /3-sitosterol (percent of total sterol in plasma, serum, or cells) 3.4• 1 2.3. 5.9 0.6 1.0 0.4 o.s 1.5 0.9 1.0 • 0.5 0.8 2.0 3.0 3.0 0.4
5.7• 1 6.4 3.0 3.7 3.0 5.3 3.0 2.8 6.2 4.3 +- 1 5.4 4.0 7.0 10.0 1.8
2.0•
5.0•
2.0• 0.6 1.0 i.0
0.5 2.5• 2.0 1.0
1.5•
3.5•
aThe total plasma cholesterol levels ranged from 70 mg % in the rat to 250 mg % in the pigeon, while the corresponding total plasma or serum triglycerides ranged from 25 to 50 rag%. The total red blood cell cholesterol averaged 100 mg % per 100 ml of cells. bThe presence of plant sterols in the plasma of the rat, chicken, and rabbit was confirmed by GC/MS. The plasma sterol samples from other animal species were not e x a m i n e d by GC/MS. CTotal number of animals in each test group. extracts o f the plasma of n o r m a l subjects and patients. Similarly, small a m o u n t s of the plant sterols were f o u n d in the total lipid extracts of the red b l o o d cells where such e x a m i n a t i o n s were made. On the basis of the GLC profiles o f the t o t a l plasma lipids, we have been able to show that > 50% of the 3,000 normal subjects e x a m i n e d exhibited small peaks with r e t e n t i o n times corresponding to C28 and C29 sterols. In a m u c h smaller n u m b e r o f patients with hyperlipemia, the incidence of occurrence of detectable plant sterol peaks appeared to be s o m e w h a t higher and the absolute c o n t e n t o f these sterols somewhat greater, although the relative p r o p o r t i o n s of cholesterol and plant sterols remained a b o u t the same as those in normal subjects. Table I summarizes t h e findings on the plasma and red blood cells o f man. The values for total free plant sterol in the plasma o f normal subjects averaged ca. 0.4 mg %, of which campesterol and stigmasterol made up ca. 50% and /3-sitosterol 50%. It was estimated that the steryl ester fraction contained a b o u t twice as m u c h plant sterol, which raised the total plasma plant sterol level in normal subjects to ca. 1.2 mg %. This estimate is in the u p p e r range o f the values given by Gray et al. (12) for plasma fl-sitosterol. The average values for the hyperlipemic subjects were nearly twice as high as those in the normals and averaged 1.8-2.4 LIPIDS, VOL 11, NO. 8
mg %. The latter estimates are of the order rep o r t e d by Salen et al. (22) for patients ingesting a m o u n t s of/~-sitosterol n o r m a l l y f o u n d in the diet. There was n o t significant difference in the plant sterol c o n t e n t of plasma o f patients with h y p e r c h o l e s t e r o l e m i a and hypertriglyceridemia, but the differences b e t w e e n n o r m a l subjects and patients with the hyperlipemias were statistically significant ( P < 0 . 0 1 ) . The present values for plasma plant sterols in adults are s o m e w h a t lower than those r e p o r t e d by Mellies et al. (7) for children on cholesterol-poor, plant sterol rich diets, which averaged 2-6 mg %. In no case did the patients with h y p e r l i p e m i a and x a n t h o m a t o s i s reach the plant sterol levels (up to 40 mg %) r e p o r t e d by B h a t t a c h a r y y a and C o n n e r (6) for their x a n t h o m a t o s i s subjects. In most instances, the red b l o o d cells showed a plant sterol p r o p o r t i o n comparable to that seen for the plasma sterols. In several cases, however, the peak for the C28 sterol was s o m e w h a t greater than that for the C29 sterol. This was probably due to a preferential uptake of campesterol by the red b l o o d eel m e m b r a n e , as already d e m o n s t r a t e d for the m e m b r a n e s of the intestinal mucosa of the dog (23), chicken (24), and pigeon (25). GLC Identification of Plant Sterols in Total Lipid Extracts of Plasma and Red Blood Cells of Domestic and Laboratory Animals
An e x a m i n a t i o n of the total lipid profiles of
PLANT STEROLS OF BLOOD
585
the plasma and serum samples collected from a variety of laboratory and domestic animals also revealed minor peaks for plant sterols. Table II summarizes the results obtained for the total plant sterols of both plasma or serum and red blood cells. Although only a few individuals of each species were assayed, their plant sterol l e v e l s appeared to be significantly higher (P
storage disease characterized by 13-sitosterolemia and xanthomatosis in which the total plasma plant sterol levels reached 40 mg%. Furthermore, Mellies et al. (7) have shown that the plasma of infants on formula diets rich in plant sterols had plant sterol levels of 6 mg% compared to 1.5 mg% or less for babies on milk or ad lib diets. Since plant sterols are common components of most normal diets and are known to be absorbed by adults (8,9), we have monitored the GLC peaks for C28 and C29 sterols in the plasma of over 3,000 subjects participating in the hyperlipemia prevalence study. To distinguish the plant sterol peaks from the peaks of t h e companion sterols of cholesterol also reported to occur in plasma or serum in variable amounts (10-14), we have performed detailed mass spectrometric analyses on the free and esterified sterol fractions on a significant number of the plasma samples. As a result, we have shown that under normal conditions sterols other than cholesterol do not reach significant levels in the plasma of adult subjects, although molecular ions characteristic of both plant sterols and cholesterol companions can be obtained from the appropriate regions of the c h r o m a t o g r a m even when discernible mass peaks are not seen in the total lipid profile.
D i r e c t gas liquid chromatography (GLC) of total plasma lipids showed small peaks (0.5-1.5% of total free sterol area) corresponding to free C2s and C29 sterols in ca. 50% of some 3,000 normal subjects and patients with hyperlipemia. Comparable proportions of similar peaks were present in the sterol fraction isolated from the red blood cells of many of these subjects. The maximum levels of these components in the plasma and red blood cells of domestic and laboratory animals were up to I0 times higher than t h o s e seen in man. Detailed gas chromatography/mass spectrometry analyses of the plasma lipids from a much more limited number of subjects and animals s h o w e d that the GLC peaks corresponding to the free C2s and C29 sterols were largely due to the plant sterols campesterol, stigmasterol, and 13s i t o s t e r o l . I n all instances, variable amounts (0.05-0.2% of the total free s t e r o l a r e a ) of 7-dehydrocholesterol, desmosterol, lanosterol, and cholesterol t~-oxide were also detected. While the total content and composition of the plasma plant sterols appeared to vary greatly among the subjects, it never exceeded 2% of total sterol in the normal subjects and patients examined. There was no evidence for a significant increase in the plant sterol content of the plasma of patients with hypercholesterolemia or hypert rigiyceride mia.
MATERIALS AND METHODS Standards
I NTRODUCTION
Routine determinations of plasma lipid profiles of normal subjects and patients with hyperlipemia by gas liquid chromatography (GLC) have revealed the presence of small peaks with retention times corresponding to those of plant sterols (1-3). Other reports have claimed the occurrence of plant sterols in plasma and tissues o f normal subjects (4) and cancer patients (4,5). In most of these instances, the estimated plant sterol levels have averaged 0.5 mg% or less. Recently, liowever, Bhattacharyya and Connor (6) have described a new lipid
Purified desmosterol, campesterol, stigmasterol, and 13-sitosterol were obtained from Applied Science Laboratories, State College, PA. Cholestanol, coprostanol, and cholesterol oroxide were obtained from Mann Research Laboratories, New York, NY, while the Aldrich Chemical Co., Milwaukee, WI, supplied 7-dehydrocholesterol, l a n o s t e r o l , and dihydrolanosterol, ct-Tocopherol was obtained from Distillation Products Industries, Rochester, NY.
581
Sources of Plasma and Serum Samples
The bulk of the plasma samples (over 3,000) were obtained from a study of the prevalence of hyperlipemia in a free-living population in the Toronto area conducted by the TorontoMcMaster Lipid Research Clinic. The blood samples were collected in ethylenediamine
582
A. KUKSIS, L. MARAI, J.J. MYHER, AND K. GEHER
tetraacetic acid (EDTA) tubes according to a procedure published by the Lipid Research Clinics Program (15). Plasma of patients with various types of hyperlipemia (2 Type I, 22 Type II, 2 Type III, 9 Type IV, and 4 Type V) were obained through the courtesy of J.A. Little of St. Michael's Hospital and G. Steiner of Toronto General Hospital, Toronto, Canada. Isolation of Plasma or Serum Lipids
Total lipid extracts of whole plasma or neutral lipid extracts of Dlasrna following digestion with phospholipase C were prepared by extraction with chloroform:methanol, 2:1, as previously described (16). F o r a detailed examination of the free sterol and steryl ester fractions, the plasma lipid extracts were resolved into the lipid classes by thin layer chromat o g r a p h y ( T L C ) according to previously published methods (16,17). The steryl ester fraction was saponified with 0.5 N KOH in 70% ethanol and the unsaponifiable matter recovered by extraction with diethyl ether (18). NOTE: Care should be taken not to contaminate the plasma lipid samples with plant sterols present on laboratory equipment. Thus, the latex rubber bulbs c o m m o n l y employed to control Pasteur pipettes upon contact with organic solvents release readily detectable amounts of ~-sitosterol and other terpenoid impurities of comparable tool wt (unpublished results). Trimethylsilylation
The dried lipid extracts were transferred to sealed conical vials of 0.5 ml capacity, and 150-250/,d of TRISIL/BSA or a solution o f p y r i d i n e : h e x a m e t h y l d i s i l a z a n e : trimethylchlorosilane, 12:5:2, were added (19). After 2 hr at room temperature, the silylation was complete and the reaction mixture ready for injection into the gas chromatograph. Gas Liquid Chromatography
Routine determination of the plasma or serum lipid profiles was made by the manual (16) or automated ( 2 0 ) G L C techniques. High resolution GLC of plasma sterols was perf o r m e d on 6 f t x I / 8 i n . ID glass columns packed with 1% SE-30 or 3% SILAR 5CP on 100-120 mesh Gas Chrom Q (Applied Science Laboratories). The columns were installed in a V a r i a n m o d e l 2 7 0 0 Gas Chromatograph equipped with a flame ionization detector. The carrier gas was nitrogen at 40 ml[min. Isothermal runs were made at 200 C with the injector and detector heaters at 250 C. Temperature programmed runs were made in the range 180-250 C at a heating rate of 4 C/min. LIPIDS, VOL 11, NO. 8
Gas Chromatography/Mass Spectrometry (GC/MS)
Combined GC/MS analyses o f the trimethylsilyl (TMS) ethers of sterois and of the fatty acid esters of sterols were made with a Varian Mat CH-5 single focusing mass spectrometer coupled to Varian Data 620/i Computer and a peak matcher (21). The TLC separations of the TMS ethers of sterols were made on a Varian M o d e l 2700 Moduline gas chromatograph equipped with a 6 ft x 1/8 in. ID glass column packed with 1% SE-30 on Gas Chrom Q. The carrier gas was helium at 10 rnl/min. The gas chromatograph which did not have a separate detector was operated at 180 C isothermally with an injector heater at 225 C. The transfer line was maintained at 275 C. The mass spectrometer was operated at an ionization voltage of 3,000 V, electron emission energy of 100 pA, an d an ion source temperature o f 270 C. Scanning was done at 4 see/decade at-a resolution of 800-1,000. Mass acquisitions were made exponentially in a cyclic manner. All total spectra taken over the GLC peaks were corrected for total ion current variation. Also, spectra were taken of the column bleed and were subtracted from the total spectra of any minor components b y the c o m p u t e r using Varian Module Sub. GC/MS of t o t a l plasma l i p i d extracts was obtained by means o f 3 ft x 1]8 in. ID glass columns packed with 3% OV-1 on Gas Chrom Q (100-120 mesh). These columns were temperature programmed from 175-310 C, using helium as the carrier gas at a flow rate of 10 ml[min. F o r this purpose, the temperature of the injector heater was raised to 300 C and that of the transfer line to 325 C, while the helium separator and the ion source temperatures were adjusted to 310 C. Under these conditions, the presence of free plant sterols could be monitored by searching for the molecular ions of campesterol, stigmasterol, and fl-sitosterol, and that of the plant sterol esters could be monitored by searching for the M-fatty acid ions of these steryl esters. For a more sensitive detection of the plant sterols, the steryl ester fraction was saponified and free sterols isolated by TLC. The sterols were then resolved as the TMS ethrs on the 6 ft GLC columns described above and the appropriate molecular ions monitored b y the peak marcher. F o r this purpose, the low mass channel of the peak matcher was focused on m/e 458 and this channel used to monitor the elution o f the TMS ether of cholesterol. After the appearance of cholesterol, the second channel of the peak matcher, preset to focus on m/e 472, was used to monitor the TMS ether o f campesterol. Finally, after elution of campesterol, the dial settings of the second channel were switched to
PLANT STEROLS
allow the monitoring of m/e 486, which is the molecular ion for the TMS ether of/3-sitosterol. Peak height values were corrected for retention time variation and differences in response by using a standard mixture made up by weighing out predetermined amounts of cholesterol, campesterol, and /~-sitosterol. The presence of a-tocopherol in the cholesterol peak was detected by monitoring m/e 502, which represents the molecular ion of the TMS ether of a-tocopherol and m]e 237, which is a major fragment. The molecular ions and other appropriate fragments (M-15, M-90, and M-90+15) w e r e u s e d t o m o n i t o r the presence of AS-methostenol, AS-lanosterol, methostenol, lanosterol, and the a-oxide of cholesterol in the GLC elution pattern. RESULTS AND DISCUSSION GLC Identification of Plant Sterols-in Total Lipid Extracts of Plasma and Red Blood Cells of Man
An examination of the GLC elution profiles of total plasma lipids from normal subjects and patients with hyperlipemia showed that many samples possessed small peaks in the regions occupied by plant sterols. Figure 1 shows the plasma lipid profiles obtained for a Type I and for a Type IIb hyperlipemia patients. By decreasing the attenuation, it was possible to increase the size and to improve the shape of the peaks, which then allowed the recognition of
583
OF BLOOD
rI
FIG. 1. GLC profiles of plasma lipids of patients with hyperlipemia. A) Total lipids of a patient with Type l hyperlipemia; B) neutral lipids of a patient with Type lib hyperlipemia. Peaks 16 and 18, TMS esters of CI 6 and C18 fatty adds; Peaks 27, 28, and 29, TMS ethers of cholesterol, campesterol, and #-sitosterol; Peak 30, tridecanoylglycerol internal standard; Peak 34, TMS ether of palmitoylsphingosine; Peaks 36-40, TMS ethers of diacylglycerols of 36 to 40 acyl carbons; Peaks 43-47, cholesterol esters of C16 to C20 fatty acids; Peaks 50-54, triaeylgiyeerols with 50-54 acyl carbons. GLC conditions and samples as given in text. the characteristic plant sterol profile of various vegetable oils and fats. The minor amounts of plant sterols could be recovered along with free cholesterol by TLC of the plasma lipids in a nautral lipid system. However, TLC occasionally removed some of the nonsterol material emerging in the free sterol region of the total lipid profile. In all instances where plant sterols were found in the free sterol fraction, peaks corresponding to the C 28 and C 29 sterols were also recovered in the unsaponifiable matter of the steryl esters isolated from the total lipid
TABLE I
Plant Sterol Levels in Plasma and Red Blood Cells of Normal Subjects and Patients with Hyperlipemia as Estimated by Gas Liquid Chromatography Subjects a
Free sterols Ester• Campesterol 13-sitosterol Campesterol
sterols /~-sitosterol
Total plant sterols b
(mg/100 ml plasma or cells) Normal P~sma[35] c Cel~[20]
0.2• 0.3•
0.2• 0.3•
0.4•
0.4•
1.2• 0.6•
Hypercholesterolemi~ P~sma[22] Cells[lO]
0.5• 0.7•
0.3• 0.5•
0.8•
0.8•
2.4• 1.2•
Hypertfiglyceridemics P~sma[15] Cells[5]
0.3• 0.4•
0.3• 0.5•
0.6•
0.6•
1.8• 0.9•
aThe normal subjects had plasma-free cholesterol level of 45.9 +11.0 mg % and a total cholesterol level of 171.5 • 38.5 mg %. Total plasma triglyceride was 100 + 20 mg %. The hypercholesterolemics had plasma-free cholesterol levels of 100-200 mg % and total cholesterol levels of 300-600 mg %. The total plasma triglyceride ranged from 100-200 mg %. The hypertriglyceridemics had plasma-free cholesterol levels of 70-100 mg % and total cholesterol levels ranging from 200 to 300 mg %. The total plasma triglyceride levels ranged from 400 to 1,000 mg %. bThe presence of plant sterols was confirmed by GC/MS in all normolipemic [35] and hyperlipemie [12] plasma samples examined in this manner. CThe numbers in square brackets represent the number of individuals in each test group. LIPIDS, VOL. 11, NO. 8
A. KUKSIS, L. MARAI, J.J. MYHER, AND K. GEHER
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TABLE II Plant Sterol Levels in Plasma or Serum and Red Blood Cells of Domestic and Laboratory Animals as Estimated by Gas Liquid Chromatography Animal speciesa Rat [10] c Mouse [2] Guinea pig [3] Rabbit 131 Dog" [ 2 ] Cow [ 1 ] Pig [21 Horse [2] Sheep [2] Chicken [5] Goose [ 1 ] Duck [ 1 ] Pigeon [2] Frog [2] Turkey [1 ]
Plasma or serum b Red blood cells Campesterol /~-sitosterol Campesterol /3-sitosterol (percent of total sterol in plasma, serum, or cells) 3.4• 1 2.3. 5.9 0.6 1.0 0.4 o.s 1.5 0.9 1.0 • 0.5 0.8 2.0 3.0 3.0 0.4
5.7• 1 6.4 3.0 3.7 3.0 5.3 3.0 2.8 6.2 4.3 +- 1 5.4 4.0 7.0 10.0 1.8
2.0•
5.0•
2.0• 0.6 1.0 i.0
0.5 2.5• 2.0 1.0
1.5•
3.5•
aThe total plasma cholesterol levels ranged from 70 mg % in the rat to 250 mg % in the pigeon, while the corresponding total plasma or serum triglycerides ranged from 25 to 50 rag%. The total red blood cell cholesterol averaged 100 mg % per 100 ml of cells. bThe presence of plant sterols in the plasma of the rat, chicken, and rabbit was confirmed by GC/MS. The plasma sterol samples from other animal species were not e x a m i n e d by GC/MS. CTotal number of animals in each test group. extracts o f the plasma of n o r m a l subjects and patients. Similarly, small a m o u n t s of the plant sterols were f o u n d in the total lipid extracts of the red b l o o d cells where such e x a m i n a t i o n s were made. On the basis of the GLC profiles o f the t o t a l plasma lipids, we have been able to show that > 50% of the 3,000 normal subjects e x a m i n e d exhibited small peaks with r e t e n t i o n times corresponding to C28 and C29 sterols. In a m u c h smaller n u m b e r o f patients with hyperlipemia, the incidence of occurrence of detectable plant sterol peaks appeared to be s o m e w h a t higher and the absolute c o n t e n t o f these sterols somewhat greater, although the relative p r o p o r t i o n s of cholesterol and plant sterols remained a b o u t the same as those in normal subjects. Table I summarizes t h e findings on the plasma and red blood cells o f man. The values for total free plant sterol in the plasma o f normal subjects averaged ca. 0.4 mg %, of which campesterol and stigmasterol made up ca. 50% and /3-sitosterol 50%. It was estimated that the steryl ester fraction contained a b o u t twice as m u c h plant sterol, which raised the total plasma plant sterol level in normal subjects to ca. 1.2 mg %. This estimate is in the u p p e r range o f the values given by Gray et al. (12) for plasma fl-sitosterol. The average values for the hyperlipemic subjects were nearly twice as high as those in the normals and averaged 1.8-2.4 LIPIDS, VOL 11, NO. 8
mg %. The latter estimates are of the order rep o r t e d by Salen et al. (22) for patients ingesting a m o u n t s of/~-sitosterol n o r m a l l y f o u n d in the diet. There was n o t significant difference in the plant sterol c o n t e n t of plasma o f patients with h y p e r c h o l e s t e r o l e m i a and hypertriglyceridemia, but the differences b e t w e e n n o r m a l subjects and patients with the hyperlipemias were statistically significant ( P < 0 . 0 1 ) . The present values for plasma plant sterols in adults are s o m e w h a t lower than those r e p o r t e d by Mellies et al. (7) for children on cholesterol-poor, plant sterol rich diets, which averaged 2-6 mg %. In no case did the patients with h y p e r l i p e m i a and x a n t h o m a t o s i s reach the plant sterol levels (up to 40 mg %) r e p o r t e d by B h a t t a c h a r y y a and C o n n e r (6) for their x a n t h o m a t o s i s subjects. In most instances, the red b l o o d cells showed a plant sterol p r o p o r t i o n comparable to that seen for the plasma sterols. In several cases, however, the peak for the C28 sterol was s o m e w h a t greater than that for the C29 sterol. This was probably due to a preferential uptake of campesterol by the red b l o o d eel m e m b r a n e , as already d e m o n s t r a t e d for the m e m b r a n e s of the intestinal mucosa of the dog (23), chicken (24), and pigeon (25). GLC Identification of Plant Sterols in Total Lipid Extracts of Plasma and Red Blood Cells of Domestic and Laboratory Animals
An e x a m i n a t i o n of the total lipid profiles of
PLANT STEROLS OF BLOOD
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the plasma and serum samples collected from a variety of laboratory and domestic animals also revealed minor peaks for plant sterols. Table II summarizes the results obtained for the total plant sterols of both plasma or serum and red blood cells. Although only a few individuals of each species were assayed, their plant sterol l e v e l s appeared to be significantly higher (P
