Oxidized plant sterols in human serum and lipid infusions as measured by combined gas-liquid chromatography-mass spectrometry
2001; Elsevier BV; Volume: 42; Issue: 12 Linguagem: Inglês
10.1016/s0022-2275(20)31532-7
ISSN1539-7262
AutoresJogchum Plat, H. Brzezinka, Dieter Lütjohann, Ronald P. Mensink, Klaus von Bergmann,
Tópico(s)Lipid metabolism and biosynthesis
ResumoSome oxidized forms of cholesterol (oxysterols) are thought to be atherogenic and cytotoxic. Because plant sterols are structurally related to cholesterol, we examined whether oxidized plant sterols (oxyphytosterols) could be identified in human serum and soy-based lipid emulsions. We first prepared both deuterated and nondeuterated reference compounds. We then analyzed by gas-liquid chromatography-mass spectrometry the oxyphytosterol concentrations in serum from patients with phytosterolemia or cerebrotendinous xanthomatosis, in a pool serum and in two lipid emulsions. 7-Ketositosterol, 7β-hydroxysitosterol, 5α, 6α-epoxysitosterol, 3β,5α,6β-sitostanetriol, and probably also 7α-hydroxysitosterol were present in markedly elevated concentrations in serum from phytosterolemic patients only. Also, campesterol oxidation products such as 7α-hydroxycampesterol and 7β-hydroxycampesterol were found. Interestingly, sitosterol was oxidized for approximately 1.4% in phytosterolemic serum, which is rather high compared with the approximate 0.01% oxidatively modified cholesterol normally seen in human serum. The same oxyphytosterols were also found in two lipid emulsions in which the ratio of oxidized sitosterol to sitosterol varied between 0.038 and 0.041. In conclusion, we have shown that oxidized forms of plant sterols are present in serum from phytosterolemic patients and two frequently used soy-based lipid emulsions. Currently, it is unknown whether oxyphytosterols affect health, as has been suggested for oxysterols. However, 7β-hydroxycholesterol may be one of the more harmful oxysterols, and both sitosterol and campesterol were oxidized into 7β-hydroxysitosterol and 7β-hydroxycampesterol. The relevance of these findings therefore deserves further exploration.—Plat, J., H. Brzezinka, D. Lütjohann, R. P. Mensink, and K. von Bergmann. Oxidized plant sterols in human serum and lipid infusions as measured by combined gas-liquid chromatography-mass spectrometry. J. Lipid Res. 2001. 42: 2030–2038. Some oxidized forms of cholesterol (oxysterols) are thought to be atherogenic and cytotoxic. Because plant sterols are structurally related to cholesterol, we examined whether oxidized plant sterols (oxyphytosterols) could be identified in human serum and soy-based lipid emulsions. We first prepared both deuterated and nondeuterated reference compounds. We then analyzed by gas-liquid chromatography-mass spectrometry the oxyphytosterol concentrations in serum from patients with phytosterolemia or cerebrotendinous xanthomatosis, in a pool serum and in two lipid emulsions. 7-Ketositosterol, 7β-hydroxysitosterol, 5α, 6α-epoxysitosterol, 3β,5α,6β-sitostanetriol, and probably also 7α-hydroxysitosterol were present in markedly elevated concentrations in serum from phytosterolemic patients only. Also, campesterol oxidation products such as 7α-hydroxycampesterol and 7β-hydroxycampesterol were found. Interestingly, sitosterol was oxidized for approximately 1.4% in phytosterolemic serum, which is rather high compared with the approximate 0.01% oxidatively modified cholesterol normally seen in human serum. The same oxyphytosterols were also found in two lipid emulsions in which the ratio of oxidized sitosterol to sitosterol varied between 0.038 and 0.041. In conclusion, we have shown that oxidized forms of plant sterols are present in serum from phytosterolemic patients and two frequently used soy-based lipid emulsions. Currently, it is unknown whether oxyphytosterols affect health, as has been suggested for oxysterols. However, 7β-hydroxycholesterol may be one of the more harmful oxysterols, and both sitosterol and campesterol were oxidized into 7β-hydroxysitosterol and 7β-hydroxycampesterol. The relevance of these findings therefore deserves further exploration. —Plat, J., H. Brzezinka, D. Lütjohann, R. P. Mensink, and K. von Bergmann. Oxidized plant sterols in human serum and lipid infusions as measured by combined gas-liquid chromatography-mass spectrometry. J. Lipid Res. 2001. 42: 2030–2038. Plant sterols are nonnutritive compounds that differ from cholesterol only by an additional ethyl (β-sitosterol) or methyl (campesterol) group at the 24-carbon atom of the sterol side chain. Western diets provide about 160–360 mg/day of plant sterols, which consist of approximately 80% β-sitosterol, some campesterol and stigmasterol, minor amounts of brassicasterol, and only traces of delta-5 saturated plant stanols (1Ling W.H. Jones P.J.H. Minireview dietary phytosterols: a review of metabolism, benefits and side effects.Life Sciences. 1995; 57: 195-206Google Scholar). Cholesterol is susceptible to oxidation, and cholesterol oxidation products, called oxysterols, can be formed by physical processes such as heating and radiation, by nonenzymatic processes involving reactive oxygen and free radical species (2Smith L.L. Review of progress in sterol oxidations: 1987–1995.Lipids. 1996; 31: 453-487Google Scholar), or enzymatically, by specific cytochrome P450 (CYP450) monooxygenases (3Russell D.W. Setchell K.D. Bile acid biosynthesis.Biochemistry. 1992; 31: 4737-4749Google Scholar). Consequently, cholesterol oxidation products, as present in the human body, may be derived from absorption of oxidized sterols present in the food, as well as from endogenous origin. The nuclear B-ring of the cholesterol molecule is mainly oxidized by nonenzymatic processes. In this way, 7β-hydroxycholesterol (7α-OH-Chol), 7β-hydroxycholesterol (7β-OH-Chol), 7-ketocholesterol (7=O-Chol), 5α,6α-epoxycholesterol (5α,6α-epoxy-Chol), 5β,6β-epoxycholesterol (5β,6β-epoxy-Chol), and 3β,5α,6β-tri-hydroxy-cholesterol (3β,5α, 6β-trihydroxy-Chol) can be formed. The side chain, on the other hand, is mainly oxidized by CYP450-specific enzymes, and various hydroxy derivatives of cholesterol are then formed (24S-OH-Chol and 27-OH-Chol) (4Brown A.J. Jessup W. Oxysterols and atherosclerosis.Atherosclerosis. 1999; 142: 1-28Google Scholar). The difference between the products formed by nonenzymatic and enzymatic oxidation is clearly illustrated by findings that during in vitro copper-catalyzed LDL oxidation, mainly oxysterols oxidized at C7 and C5-C6 are identified (5Chang Y.H. Abdalla D.S.P. Sevanian A. Characterization of cholesterol oxidation products formed by oxidative modification of low density lipoprotein.Free Rad. Biol. Med. 1997; 23: 202-214Google Scholar, 6Patel R.P. Diczfalusy U. Dzeletovic S. Wilson M.T. Darley-Usmar V.M. Formation of oxysterols during oxidation of low density lipoprotein by peroxynitrite, myoglobin, and copper.J. Lipid Res. 1996; 37: 2361-2371Google Scholar), whereas 24S-OH-Chol and 27-OH-Chol are not found (6Patel R.P. Diczfalusy U. Dzeletovic S. Wilson M.T. Darley-Usmar V.M. Formation of oxysterols during oxidation of low density lipoprotein by peroxynitrite, myoglobin, and copper.J. Lipid Res. 1996; 37: 2361-2371Google Scholar). Because plant sterols have a great structural similarity to cholesterol, analogous oxidation products can be formed from plant sterols (7Daly G.G. Finocchairo E.T. Richardson T. Characterization of some oxidation products of sitosterol.J. Agric. Food Chem. 1983; 31: 46-50Google Scholar). The terminology for oxidized plant sterols (oxyphytosterols) is similar to that described for oxysterols. After stereospecific peroxidation of sitosterol to 7α- and/or 7β-hydroperoxy sitosterol (7α-OOH-Sit and 7β-OOH-Sit, respectively), reduction of these compounds results in the formation of 7α- or 7β-hydroxysitosterol (7α- or 7β-OH-Sit; Fig. 1), while dehydration of the hydroperoxide leads to the formation of 7-ketositosterol (7=O-Sit; Fig. 1). Further, 5α,6α-epoxysitosterol (5α,6α-epoxy-Sit) and 5β,6β-epoxysitosterol (5β,6β-epoxy-Sit) are formed by epoxidation of the double bound between the C5 and C6 atoms of sitosterol. Epoxysterols at C5-C6 are rapidly converted into their triol end products such as 3β,5α,6β-sitostanetriol (3β,5α,6β-tri-hydroxy-Sit; Fig. 1). Comparable products can be formed from campesterol and other plant sterols. Plant sterols may be oxidized even more easily than cholesterol (8Li W. Przybylski R. Oxidation products formed from phytosterols.INFORM. 1995; 6: 499-500Google Scholar). However, only little information is available on the presence of oxyphytosterols in food, but small amounts have been found in coffee, fried potatoes, wheat flour, and vegetable oils (9Dutta P.C. Appelqvist L. Studies on phytosterol oxides I: effect of storage on the content in potato chips prepared in different vegetable oils.JAOCS. 1997; 74: 647-657Google Scholar, 10Dutta P.C. Studies on phytosterol oxides. II: content in some vegetable oils and in French fries prepared in these oils.JAOCS. 1997; 74: 659-666Google Scholar). Recently, it has been shown in rats that oxyphytosterols [7=O-Sit, 7-ketocampesterol (7=O-Camp), 5α,6α-epoxy-Sit, 5β,6β-epoxy-Sit, 5α,6α-epoxycampesterol (5α,6α-epoxy-Camp), and5β,6β-epoxycampesterol (5β,6β-epoxy-Camp)] are absorbed from the diet and incorporated into mesenteric lymph (11Grandgirard A. Sergiel J.P. Nour M. Demaison-Meloche J. Ginies C. Lymphatic absorption of phytosterol oxides in rats.Lipids. 1999; 34: 563-570Google Scholar). Information on endogenous formation of oxyphytosterols, either enzymatically or nonenzymatically, and information on the presence of oxyphytosterols in the human circulation is scanty (12Grandgirard A. Bretillon L. Martine L. Beaufrere B. Oxyphytosterols in human plasma.Chem. Phys. Lipids. 1999; 101: 198Google Scholar). Expanding our knowledge on the presence of oxyphytosterols in the circulation is of great importance, in particular because oxysterols, in general, may be atherogenic. Further, it was recently found that oxysterols and oxyphytosterols showed similar cytotoxic effects in cultured macrophages (13Adcox C. Boyd L. Oehrl L. Allen J. Fenner G. Comparative effects of phytosterol oxides and cholesterol oxides in cultures macrophage-derived cell lines.J. Agric. Food Chem. 2001; 49: 2090-2095Google Scholar). In this way, oxidation of plant sterols and the presence of these oxyphytosterols in the circulation might have implications for health. Phytosterolemia is a rare inherited sterol storage disease characterized by highly elevated serum plant sterol concentrations of up to 65 mg/dl (1.57 mM) (14Salen G. Horak I. Rothkopf M. Cohen J.L. Speck J. Tint G.S. Shore V. Dayal B. Chen T. Shefer S. Lethal atherosclerosis associated with abnormal plasma and tissue sterol composition in sitosterolemia with xanthomatosis.J. Lipid Res. 1985; 26: 1126-1133Google Scholar), tendon and tuberous xanthomas, and by a strong predisposition to premature coronary atherosclerosis (15Björkhem I. Boberg K.M. Inborn errors in bile acid biosynthesis and storage of sterols other than cholesterol.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Diseases. McGraw Hill, New York1994: 2073-2100Google Scholar). Therefore, the primary aim of this study was to examine whether and what kinds of oxyphytosterols are present at all in serum of phytosterolemic patients. In addition, we analyzed serum from patients suffering from cerebrotendinous xanthomatosis (CTX). Patients suffering from CTX, a rare autosomal recessive inherited disease, have decreased concentrations of 27-OH-Chol caused by a genetic absence or restriction of the CYP450-dependent 27-hydroxylase. As a consequence, these patients have a reduced formation of normal C24 bile acids. Moreover, there is a negative feedback from bile acids on cholesterol 7α-hydroxylase (CYP7A). Consequently, CTX patients have extremely high concentrations of 7α-hydroxylated bile acid precursors because they lack the feedback on cholesterol 7α-hydroxylase due to the absence of bile acids (15Björkhem I. Boberg K.M. Inborn errors in bile acid biosynthesis and storage of sterols other than cholesterol.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Diseases. McGraw Hill, New York1994: 2073-2100Google Scholar). It is therefore interesting to examine whether an increased activity of cholesterol 7α-hydroxylase will result in the formation of endogenous 7α-OH-Sit. We therefore hypothesized that oxyphytosterols, when formed in relevant concentrations and present in the circulation, should be found in serum of phytosterolemic and CTX patients. In addition, two different soybean oil-based lipid emulsions known to be rich in plant sterols and frequently used as parenteral infusion for short- and long-term nutritive therapy were analyzed for the presence of oxyphytosterols. To address these issues, we first developed a sensitive gas-liquid chromatography-mass spectrometry (GC-MS) method and synthesized all necessary reference compounds and deuterated internal standards. All reagents and solvents used were of analytical or HPLC grade. We analyzed by GC-MS a plant sterol mixture purchased from Sigma-Aldrich (Steinheim, Germany) that consisted of 60.19% sitosterol, 36.09% campesterol, and traces (2.72%) of stigmasterol. Chemical synthesis of oxyphytosterols was based on the methods as described for oxygenation of cholesterol by Li et al. (16Li S. Pang J. Wilson W.K. Schroepfer G.J. Sterol synthesis. Preparation and characterization of fluorinated and deuterated analogs of oxygenated derivatives of cholesterol.Chem. Phys. Lipids. 1999; 99: 33-71Google Scholar), with some minor modifications. Several oxygenated plant sterols were synthesized from the plant sterol mixture. In this way, a mixture of 7α- and 7β-OH-Sit, 7α- and 7β-hydroxycampesterol (7α- and 7β-OH-Camp, respectively), and 7α- and 7β-hydroxystigmasterol (7α- and 7β-OH-stigmasterol, respectively), a mixture of 7=O-Sit/-Camp/-stigmasterol, a mixture of 5α,6α-epoxy and 5β,6β-epoxy-Sit/-Camp/-stigmasterol, and a mixture of 3β,5α,6β-tri-hydroxy-Sit, 3β,5α,6β-tri-hydroxy-campesterol (3β, 5α,6β-tri-hydroxy-Camp), and 3β,5α,6β-tri-hydroxy-stigmasterol was prepared (Fig. 2). We did not separate sitosterol oxidation products from campesterol oxidation products, as they are always present as a mixture in the circulation and can be sufficiently separated by GC-MS. Tentative identification of the synthesized oxyphytosterols and interpretation of the mass spectra was performed by comparison of the spectra with a standard oxyphytosterol mixture, which was kindly provided by Dr. André Grandgirard (INRA, Unité de Nutrition Lipidique, Dijon, France). Acetylation of plant sterols. All plant sterols were acetylated prior to further use for synthesis of all oxyphytosterols, except for the triols. To this end, 2 g of the plant sterol mixture (Fig. 2, Ia-Ic) was acetylated in toluene after addition of 20 ml acetic anhydride (Fluka Chemika, Buchs, Switzerland), which finally resulted in the acetylated products (Fig. 2, IIa-IIc) as white crystals. 7-Hydroxyphytosterols. Two hundred milligram molecular sieve 0.4 nm (Merck, Darmstadt, Germany) and 3.2 g pyridinium chlorochromate (Sigma-Aldrich) were added to a solution of 340 mg acetylated plant sterols (Fig. 2, IIa -IIc) in 200 ml benzene and heated under reflux. The white crystals that formed (Fig. 2, IIIa-IIIc) were used for further synthesis of the two isomeric 7-hydroxyphytosterols. One gram of lithium aluminium hydride (Sigma-Aldrich) was added to a solution of 100 mg of IIIa-IIIc crystals (Fig. 2) dissolved in 100 ml di-ethyl ether, and was stirred overnight undernitrogen at room temperature. Next, the acetylated compound IVa-IVc (Fig. 2) was saponified to result in the formation of white to light-yellow crystals (Fig. 2, Va-Vc). The purity of the synthesized 7-hydroxy plant sterols was checked by TLC. TLC showed that in addition to hydroxy sterols, nonhydroxylated plant sterols were present. Therefore, the 7-hydroxylated plant sterols were purified by using a silica column (25 cm, diameter 2 cm). The fractions containing pure 7-hydroxy plant sterols (a mixture of alpha and beta isomers) were combined and crystallized in n-hexane. The GC-MS spectra showed a pure mixture of 7α-OH-Sit, 7α-OH-Camp, 7β-OH-Sit, and 7β-OH-Camp, and traces of 7-OH-stigmasterol. The ratio of 7α-OH-Sit to 7α-OH-Camp was 69:31, whereas the ratio of 7β-OH-Sit to 7β-OH-Camp was 70:30. This indicates that both sitosterol and campesterol from the initial plant sterol mixture were equally converted into 7-hydroxy plant sterols. Retention times and m/z used for analysis of 7-hydroxy sterols are shown in Table 1.TABLE 1.Retention times and m/z values for the detection of plant sterols, oxyphytosterols, cholesterol, and oxysterols as TMS derivativesPlant SterolsRTm/zM+B+7=O-Sit25.66500x7=O-Camp24.12486x7α-OH-Sit19.80484x7β-OH-Sit21.51484x7α-OH-Camp18.92470x7β-OH-Camp20.55470x5β,6β-epoxy-Sit22.63412x5α,6α-epoxy-Sit22.97412x5β,6β-epoxy-Camp21.47398x5α,6α-epoxy-Camp21.80398x3β,5α,6β-tri-hydroxy-Sit23.81484x3β,5α,6β-tri-hydroxy-Camp22.52470x2,2,4,4,6-d5-3β,5α,6β-tri-hydroxy-Sit23.72488x2,2,4,4,6-d5-3β,5α,6β-tri-hydroxy-Camp22.42474x2,2,4,4,6-d5-7=O-Sit25.55504x2,2,4,4,6-d5-7=O-Camp24.09490xSitosterol20.85486xCampesterol19.89472xBrassicasterol19.11470xStigmasterol20.23484xCholesterol18.74458x26,26,26,27,27,27-d6-Chol18.63464x26,26,26,27,27,27-d6-7=O-Chol22.18478x26,26,26,27,27,27-d6-7α-OH-Chol17.81462x26,26,26,27,27,27-d6-7β-OH-Chol19.29462x26,26,26,27,27,27-d6-3β,5α,6β-tri-hydroxy-Chol21.01462x26,26,26,27,27,27-d6-5β,6β-epoxy-Chol20.11390x26,26,26,27,27,27-d6-5α,6α-epoxy-Chol20.43390x7=O-Chol22.44472x7α-OH-Chol17.90456x7β-OH-Chol19.38456x3β,5α,6β-tri-hydroxy-Chol21.07456x5β,6β-epoxy-Chol20.15384x5α,6α-epoxy-Chol20.45384xRT, retention time; M+, molecular ion; B+, base peak. Open table in a new tab RT, retention time; M+, molecular ion; B+, base peak. 7-Keto plant sterols. 7-Keto plant sterols were formed from IIIa-IIIc (Fig. 2) via saponification, and resulted in the formation of white crystals (Fig. 2, VIa-VIc). The synthesized 7-keto plant sterols were also contaminated with free plant sterols, which were removed similarly as described for the hydroxy plant sterols. The purified 7-keto plant sterols were crystallized in n-hexane. GC-MS spectra showed a pure mixture of 7=O-Sit and 7=O-Camp. The ratio of 7=O-Sit to 7=O-Camp was 67:33. Retention times and m/z used for analysis of 7-keto sterols are shown in Table 1. 5,6-Epoxy plant sterols. Acetylated phytosterols (Fig. 2, IIa-IIc) were dissolved in chloroform, and by adding m-chloroperoxybenzoic acid (Sigma-Aldrich) and potassium bicarbonate, white crystals (Fig. 2, VIIa-VIIc) were formed, which were saponified as described for the hydroxy plant sterols (Fig. 2, VIIIa-VIIIc). GC-MS spectra showed a pure mixture of 5α,6α-epoxy-Sit, 5α,6α-epoxy-Camp, 5β,6β-epoxy-Sit, and 5β,6β-epoxy-Camp, and traces of 5β,6β-epoxy-stigmasterol. The ratio of 5β,6β-epoxy-Sit to 5β,6β-epoxy-Camp was 63: 37, whereas the ratio of 5β,6β-epoxy-Sit to 5β,6β-epoxy-Camp was 59:41. Mainly the 5α,6α-epoxy forms were formed during synthesis, as the ratio of 5α,6α-epoxy-Sit to 5β,6β-epoxy-Sit was 79:21 and the ratio of 5α,6α-epoxy-Camp to 5β,6β-epoxy-Camp was 76:24. Retention times and m/z used for analysis of epoxy sterols are shown in Table 1. 3α,5β,6α-triols. For plant sterols (Fig. 2; Ia-Ic) dissolved in formic acid (95%) and worked up to the triols (Fig. 2, IXa-IXc), GC-MS spectra showed a pure mixture of 3β,5α,6β-tri-hydroxy-Sit and 3β,5α,6β-tri-hydroxy-Camp. The ratio of 3β,5α,6β-trihydroxy-Sit to 3β,5α,6β-tri-hydroxy-Camp was 67:33. Retention times and m/z used for analysis of tri-hydroxy sterols are shown in Table 1. Internal standard preparation [2,2,4,4,6-2H5]-3β,5α,6β-tri-hydroxyphytosterols and [2,2,4,4,6-2H5]-7-keto-phytosterols. As internal standards, we synthesized deuterated tri-hydroxy-phytosterols and deuterated 7-keto-phytosterols from of mixture of d5-2,2,4,4,6-sitosterol/-campesterol/-stigmasterol (Medical Isotopes Inc., Pelham, USA) by similar procedures as described for the unlabeled plant sterols. Identification of the synthesized triols revealed that both deuterated tri-hydroxy-Sit (d-tri-hydroxy-Sit) and deuterated tri-hydroxy-Camp (d-tri-hydroxy-Camp) were formed in a ratio 66:34, as well as were traces of stigmasterol. Moreover, analyzing the isotope differentiation (corrected for the natural background of 13C) showed the deuterium distribution as d0-tri-hydroxy-Sit, 8.9%; d1-tri-hydroxy-Sit, 3.9%; d2-trihydroxy-Sit 9.2%; d3-tri-hydroxy-Sit, 21.2%; d4-tri-hydroxy-Sit, 29.8%; d5-tri-hydroxy-Sit, 22.4%; d6-tri-hydroxy-Sit, 2.7%; and d7-tri-hydroxy-Sit, 1.9%. Because d4-tri-hydroxy-Sit was the most abundant isotope, we measured on m/z 488 for d4-tri-hydroxy-Sit as internal standard. The deuterated 7-keto standards were formed in a 60:40 distribution, with some minor traces of stigmasterol as well. Analyzing the isotope differentiation (corrected for the natural background of 13C) showed the deuterium distribution as d0-7=O-Sit, 6.5%; d1-7=O-Sit, 4.2%; d2-7=O-Sit, 7.3%; d3-7=O-Sit, 20.3%; d4-7=O-Sit, 31.9%; d5-7=O-Sit, 22.4%; d6-7=O-Sit, 5.7%; and d7-7=O-Sit, 1.8%. Because d4-7=O-Sit was the most abundant isotope, we measured on m/z 504 for d4-7=O-Sit as internal standard. The deuterated 7-keto standards were used as internal standard for the calculation of 7=O, 7-OH, and 5,6-epoxy plant sterols, and the deuterated tri-hydroxy standards were used to calculate tri-hydroxy sterols. Retention times and m/z used for analysis of both deuterated sterols are shown in Table 1. Butylated hydroxy toluene (BHT) and EDTA were added to the blood collection tube prior to sampling and to the serum just before analysis. Final amounts added corresponded with 10 μl BHT (25 mg/ml in methanol) and 20 μl EDTA (10 mg/ml in methanol) for 1 ml serum. Before saponification and extraction, 6 μg each of each internal standard, [2,2,4,4,6-2H5]-3β,5α,6β-trihydroxy-phytosterol and [2,2,4,4,6-2H5]-7-keto-phytosterol, were added to the serum sample. Next, the sample was saponified in a closed tube under nitrogen for 2 h at room temperature by adding 10 ml of 0.35 M ethanolic KOH solution. In addition, prior to saponification, the samples were extensively saturated with nitrogen for removal of oxygen to minimize autoxidation. After saponification, 130 μl phosphoric acid 50% in water (v/v) was added to neutralize the solution, followed by addition of 6 ml NaCl solution in water (9 mg/ml), according procedures for oxysterol analysis as described previously (17Lutjohann D. Papassotiropoulos A. Bjorkhem I. Locatelli S. Bagli M. Oehring R.D. Schlegel U. Jessen F. Rao M.L. von Bergmann K. Heun R. Plasma 24S-hydroxycholesterol (cerebrosterol) is increased in alzheimer and vascular demented patients.J. Lipid Res. 2000; 41: 195-198Google Scholar). After extraction with 20 ml di-chloro-methane, the bottom layer was transferred into a round bottom flask and evaporated to dryness under vacuum at 50°C. The residue was dissolved in 5 ml ethanol and again evaporated under vacuum to remove all traces of water. The nonsaponifiable serum lipids were dissolved in 1 ml toluene. Silica cartridges (Bond Elut, bonded phase SI, 100 mg, 1 ml; Varian, Harbor City, CA) were equilibrated with cyclohexane before the toluene fractions were loaded. Neutral sterols (including cholesterol) were eluted from the column with 4 ml 0.5% ethylacetate–cyclohexane (v/v), whereas the absorbed oxysterols were eluted with 9 ml ethyl-acetate. The oxysterol fraction was dried under vacuum. Finally, the oxidized sterols were dissolved in 100 μl cyclohexane and transferred to an injection vial. Sterols were silylated by addition of 100 μl silylation reagent (dry pyridine–hexamethyl-disilazane–trimethylchlorosilane (TMS) 3:2:1 (v/v/v) and incubation for 1 h at 90°C for GC-MS analysis. TMS derivates of the oxyphytosterols were analyzed by GC-MS. For this, 2 μl of the TMS derivatives in cyclohexane were injected via an AS2000 autosampler (Thermoquest CE Instruments, Egelsbach, Germany) on a Trace GC2000 (Thermoquest CE Instruments) gas chromatograph equipped with a RTX5MS column (30 m × 0.25 mm internal diameter, 0.25 μm film thickness) coupled to a GCQ-plus ion trap (Thermoquest CE Instruments). Analysis was carried out in single ion monitoring (SIM) mode, making m/z the primary resolving parameter other than retention time. The injector temperature was set at 280°C. Helium was used as carrier gas (constant flow 1 ml/min). The oven temperature gradient was programmed for 150 s at 150°C, then increased by 10°C/min toward 290°C, and then increased by 7°C/min toward 320°C and kept there for 20 min. Thus, one analytical run lasted approximately 42 min. The ions and retention times of all individual compounds are given in Table 1. In addition to plant sterols and oxyphytosterols, we also indicated retention times and m/z of cholesterol oxidation products (and also of d6-26,26,26,27,27,27-cholesterol oxidation products), which illustrates that interference of oxysterols and oxyphytosterols in GC-MS identification is not a problem. Serum sampled at three different time points from a phytosterolemic patient was used for analysis of oxyphytosterols. The patient in 1998 was a 12-year-old girl being treated for her illness with cholestyramine (4 g/day). Two serum samples that were obtained at different time points in 1998 and stored at −20°C with BHT added as well as a recent fresh blood sample from the year 2000 obtained on the day of analysis were used. Furthermore, serum from two CTX patients not treated at the moment of blood sampling was used: The blood samples were taken in 1998 and were stored at −20°C with BHT since that time. In addition, serum from 15 patients not suffering from any sterol metabolism-related disease was pooled and analyzed. Two frequently used soybean oil-based lipid emulsions in total parenteral nutrition protocols were analyzed for oxyphytosterols by the same procedures as described for serum samples. 7α-OH-Chol (18Hahn C. Reichel C. von Bergmann K. Serum concentration of 7α-hydroxycholesterol as an indicator of bile acid synthesis in humans.J. Lipid Res. 1995; 36: 2059-2066Google Scholar) and plant sterol concentrations (19Lütjohann D. Björkhem I. Beil U.F. von Bergmann K Sterol absorption and sterol balance in phytosterolemia evaluated by deuterium-labeled sterols: effect of sitostanol treatment.J. Lipid Res. 1995; 36: 1763-1773Google Scholar) were analyzed as described before. Serum plant sterol concentrations from the phytosterolemic patient contained large amounts of plant sterols at all time points (Table 2). Serum plant sterol concentrations (sitosterol plus campesterol) from the phytosterolemic patient were 47.3, 44.5, and 40.3 mg/dl compared with 1.3 mg/dl in the pool serum of nonphytosterolemic patients (Table 3). Because esterified plant sterols are transported in the circulation in lipoproteins, concentrations are usually expressed relative to those of cholesterol. However, because serum cholesterol concentrations were not increased to the same extent as plant sterol concentrations, cholesterol-standardized sitosterol and campesterol concentrations were also still extremely high. Sitosterol concentrations were 19,714 × 102, 18,007 × 102, and 16,714 × 102 μmol/mmol cholesterol, whereas campesterol concentrations were 9,200 × 102, 8,289 × 102, and 8,909 × 102 μmol/mmol cholesterol. Compared with concentrations from the pool serum of nonphytosterolemic patients, cholesterol-standardized plant sterol concentrations were approximately 60 to70 times increased.TABLE 2.Serum plant sterol and oxidized sitosterol concentrations in a phytosterolemic patient treated with cholestyramine (4 g/day)199819982000Plant sterols (mg/dl)aPlant sterols are calculated as the sum of sitosterol and campesterol.47.344.540.3Oxidized sitosterol forms (μg/ml)7=O-Sit0.820.990.947β-OH-Sit0.820.93ND5α,6α-epoxy-Sit2.772.231.903β,5α,6β-tri-hydroxy-Sit0.080.151.38Total oxidized sitosterol4.494.304.21Serum from a phytosterolemic patient at three different time points was used for analysis of oxidized plant sterols. Two serum samples were obtained at different time points in 1998, stored at −20°C with BHT added, and analyzed together with a fresh blood sample from the year 2000, obtained on the day of analysis. ND = not detectable.a Plant sterols are calculated as the sum of sitosterol and campesterol. Open table in a new tab TABLE 3.Serum plant sterol and oxidized sitosterol concentrations in three samples of a phytosterolemic patient versus two CTX patients and pool serumControl SerumPhytosterolemiaCTXPlant sterols (mg/dl)aPlant sterols are calculated as the sum of sitosterol and campesterol.1.344.0 ± 3.51.2 and 1.4Oxidized sitosterol forms (μg/ml)7=O-SitND0.92 ± 0.090.04 and ND7β-OH-SitND0.88 ± 0.080.01 and ND5α,6α-epoxy-SitND2.33 ± 0.44ND and ND3β,5α,6β-tri-hydroxy-SitND0.54 ± 0.73ND and ND7α-OH-Chol (ng/ml)76.383.3 ± 36.91,221 and 671Values are mean ± SD. ND = not detectable.a Plant sterols are calculated as the sum of sitosterol and campesterol. Open table in a new tab Serum from a phytosterolemic patient at three different time points was used for analysis of oxidized plant sterols. Two serum samples were obtained at different time points in 1998, stored at −20°C with BHT added, and analyzed together with a fresh blood sample from the year 2000, obtained on the day of analysis. ND = not detectable. Values are mean ± SD. ND = not detectable. Serum concentrations of 7α-OH-Chol were approximately83.3 ng/ml in the phytosterolemic patient receiving 4 g/day of cholesteryramine. This concentration was comparable to the value of 76.3 ng/ml, as observed in pool serum (Table 3). As expected, CTX patients had very high concentrations of 7α-OH-Chol, about 10 to15 times higher than pool- and phytosterolemic serum. For the identification and quantification of oxyphytoste
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