Gl was from Fermenta SA, Payerne, Switzerland. Mushrooms were cultivated on a defined formula of sawdust, wheat straw and millet grain. Substrate was sterilized at 90°C for 48 h, then incubated with Gl seed material from Mycotec Sàrl (Cernier, Neuchâtel). Cultivation was with controlled temperature, light, humidity and carbon dioxide concentration. Human hepatic T9A4 cells  were grown in LCM serum-free media under 3.5% CO2 at 37°C. Lovastatin was purchased as 20 mg Mevacor tablets (MSD Chibropharm GmbH, Haar, Germany). HMG-CoA reductase, DL-3-Glutaryl-3- [14C]-HMG-CoA (2216 MBq/mmol), R-[5-3H] mevalonic acid ammonium salt (1443 MBq/mmol), and [1-14C] acetic acid sodium salt (2070 MBq/mmol) were from Amersham (Upsala, Sweden). α-3-HMG-CoA (cold) and liquid scintillation cocktail were from Sigma (Buchs, Switzerland). LCM cell medium was from Biofluids (Rockville, MD). 5β-cholesteane-3α-ol, 5-α-cholestane and 2,3-nor-5β-cholanicacid-3α,7α,12α-triol were from Steraloids, Inc. (Newport, Rhode Island); other steroid standards were from Sigma, and Calbiochem (La Jolla, California). Methanolic HCl and Sylon HTP were from Supelco (Buchs, Switzerland). The Cobas Bio autosampler was from Hoffmann-La Roche (Basel, Switzerland) and reagents were from Roche Diagnostics (Rotkreuz, Switzerland). Total Cholesterol Kit 352 and Triacylglycerol Kit 336 were from Sigma. Deuterium was from Cambridge Isotope Laboratories (Andover, MA). Zn catalyst was from Biochemical laboratories (University Bloomington, IN). Silica gel thin layer chromatography (TLC) plates were from Merck Eurolab (Dietikon, Switzerland). Coomassie Plus-200 protein assay reagents and bovine serum albumin fraction V were from Pierce (Rockford, Illinois). All other chemicals were from Sigma.
Preparation of Gl for in vitro testing
Fruiting bodies from Gl (20 g) were dried, milled and macerated in 0.4 L MeOH/H2O (4:1, v/v) at room temperature for 3d. The mixture was then filtered, evaporated, re-dissolved in H2O, acidified to pH 3 with 3 M HCl, extracted 3 × with 150 mL ethyl acetate, and the organic phase evaporated under vacuum at 30°C, re-dissolved in 10 mL MeOH, and dried with Na2SO4, for HPLC analyses and in vitro testing.
Chemical analysis of Gl
The presence of lovastatin in Gl was determined by HPLC with a Nucleosil 100-5 C18 column (250 × 4 mm; Macherey-Nagel, Oensingen, Switzerland) and a Lichrospher 100 RP-18 post column (Merck, Glattbrugg, Switzerland). Solvent A was H3PO4/H2O (1:2000, by vol); solvent B was acetonitrile. Separation was initiated with a linear gradient of 95% A, 5% B, reaching 50% A, 50% B in 45 min, 30% A, 70% B in 46 min, 10% A, 90% B in 48 min, and 0% A, 100% B in 50 min; the run was continued isocratically 4 min. Initial conditions were maintained 6 min for re-equilibration; the flow rate was 1 mL/min. The detector was a G1315 A, series 1100 detector (Hewlett Packard, Meyrin, Switzerland); absorbance was measured at 254 nm. After selective extraction and purification with different adsorbents and solvents, ganoderols and ganoderic acids were detected by mass spectroscopy and NMR (details to be published separately).
In vitro activity of Gl extracts
Human hepatic T9A4 cells were grown in LCM serum-free media under 3.5% CO2 at 37°C. Cells were seeded in 24-well plates and at confluence, incubated with 1 mM 14C-acetate (1 mCi/mmol) for 20 h ± mushroom extracts. Lipids were extracted from cells by incubating 2 × with 1.5 mL hexane/isopropanol (3:2, by vol) for 30 min at room temperature. Combined organic extracts were dried under N2, re-dissolved in hexane, and separated by TLC with hexane/diethyl ether/acetic acid (75:25:1, by vol). Cholesterol synthesis was determined by measuring incorporation of 14C from acetate to cholesterol. Radioactivity was assessed with an instant imager and expressed as percent of control.
Administration of Gl and lovastatin to hamsters
Male Golden Syrian hamsters (Harlan, UK), 3–4 wks, 40–60 g, were housed individually in Macrolon Type 3 cages with 12 h alternating periods of light and darkness. During 3 wks preceding treatment, hamsters were fed Nafag 924 hamster complete diet (# 3132/20, Eberle Nafag AG, Gossau, Switzerland; Table 1). Following body weight randomization, groups consisted of 6 hamsters/group receiving either: Nafag diet (control), Nafag mixed with 2 mg lovastatin /100 g diet (powdered in liquid N2); or Nafag mixed with 2.5 or 5.0% dried Gl. Hamsters were fed experimental diets for 17 d. Lovastatin is an inhibitor of HMG-CoA reductase , and was used as a positive control. Dietary intake was recorded daily, body weights weekly. Feces were collected on D15-18. Hamsters were injected subcutaneously with 250 μL D2O on D17 and killed under anesthesia with isoflurane on D18. Following a 16 h fast, D1 (0.5 mL) and D18 blood (>3 mL) were obtained from the retro-orbital cavity and cardiac vein, respectively, and transferred to EDTA tubes. Plasma was prepared by centrifugation at 1500 g, 15 min, at 4°C. Plasma, and hepatic and cecum tissues were stored at -80°C. Animal procedures were authorized by Service Vétérinaire du Canton de Vaud, Switzerland, protocol 1247.
Administration of Gl and lovastatin to minipigs
Nine female and one male Göttingen minipig(s) (Jörg Farm in Bern Switzerland; Minipig-Primärzucht, Auswill, Switzerland) aged 6–12 mo (18–20 kg), with white (7) and black (3 minipigs) colorations, were housed in a 30 m2 box with normal light/dark cycle, and kept at room temperature. Females were chosen because they have fewer age-related lipid modifications and higher lipid concentrations than males . One male was accidentally provided in the delivery, however its total cholesterol (TC), lipoproteins, bile acids and neutral sterols were similar to that of other minipigs. Minipigs were randomly distributed by weight into two separately housed groups, marked with a plastic label in the ear, and fed twice daily for 11 d with powdered commercial pig chow (Diet 574, Minipig-Primärzucht). During a subsequent 4 d adaptation period, minipigs were fed an acclimatization mixture of chow and increasing amounts of powdered hypercholesterolemic control diet (custom diet 2604, Kliba, Kaiseraugst, Switzerland; Table 2) from 0% to 100%, in steps of 25%, designed after Burnett et al. [64, 74], that was consistent with Göttingen minipig nutritional needs . During the following 2 wks (D15-29), groups were fed control hypercholesterolemic diet pre-mixed with 2.5% Gl extract; or hypercholesterolemic diet plus 80 mg lovastatin/pig/d (in four 20 mg tablets) , hand fed to each minipig, mornings, in half an apple. For acclimatization, on D12-14, minipigs received a half apple without lovastatin. The study was blinded in that the diets were coded, and the mushroom extract was referred to as "Nestlé Special Fiber." Food intake was 3.5% of body wt/d (based on group average wt), readjusted weekly, to provide sufficient, but not excessive, calories [64, 65, 75]. Diets were distributed at 0700 and 15h00, and spread linearly on a clean cement surface to facilitate individual consummation. Distilled water was provided ad libitum. Toys and human contact were provided to avoid boredom. Fasting 16 h blood samples (10 mL; 20 mL on D29) were collected in EDTA tubes on D1, 15, 22, 28, and 29 from anterior vena cava. Plasma was prepared by centrifugation as described for hamsters, and stored at -80°C. Blood collection began at 0800 following injection of the intra-muscular relaxant Dormicum® (Hoffmann La Roche, Basel, Switzerland), then the tranqulizer Stresnil® (Janssen Pharmaceuticals, Beerse, Belgium). After blood sampling on D1, 15 and 29, minipigs were isolated for 2 h maximum for individual fecal collections. Some minipigs did not defecate during this period, whereas others defecated again following return to their groups. Hence, the morning fecal collection was qualitative. Feces were stored at -40°C under N2. Body weight was recorded weekly, and food intake recorded each morning. Minipigs were donated to the University of Geneva at the study's conclusion. Animal procedures were authorized by Service Vétérinaire du Canton de Geneve, Switzerland, protocol 1315, authorization 31.1.1014/1719/1.
Cholesterol and triacylglycerol measurements in hamsters and minipigs
Plasma total cholesterol and triacylglycerol were measured using commercial kits and a Roche Cobas Bio autosampler. Plasma lipoproteins were separated by size-exclusion HPLC as previously described .
Fractional cholesterol synthesis rate measurements in hamsters and minipigs
Measurements of water- and cholesterol deuterium enrichment were performed with a Finnigan Thermoquest Delta XL plus Isotopic Ratio Mass Spectrometer (Bremen, Germany) as previously described [76, 77]. Fractional synthesis rate (FSR) of free cholesterol was calculated from a plasma sample collected 24 h after deuterium oxide subcutaneous injection as follows: FSR (in % pool/d) = 100 × (cholesterol enrichment/(water enrichment × 0.478)). Due to technical reasons, there were insufficient values in the minipig experiments to reach interpretable conclusions.
Fecal bile acids and neutral sterol measurements in hamsters and minipigs
Fecal neutral sterols and bile acids were extracted from lyopholized feces, deconjugated, derivatized with Sylon HTP and analyzed by gas chromatography as previously described with internal standards: 5-α-cholestane for neutral sterols; 2,3-nor-5β-cholanic acid-3α,7α,12α-triol for bile acids .
Hepatic ex-vivo HMG-CoA reductase measurements in hamsters
Freshly excised liver (300 mg) was collected after 16 h fast of hamsters, minced with scissors, and homogenized with 0.4 mL buffer (50 mM KH2PO4, 0.1 M sucrose, 50 mM KCl, 50 mM NaCl, 30 mM EDTA, and 2 mM dithiothreitol, ± 50 mM NaF) with a Potter-Elvehjem S homogenizer with 400 rpm/5 strokes, on ice, after Conde et al. . NaF inhibits dephosphorylation of HMG-CoA reductase by inactivating phosphoprotein phosphatases, yielding total phosphorylated HMG-CoA reductase activity. After washing homogenizer with 0.2 mL buffer, homogenate was centrifuged at 10000 g, 15 min, at 4°C. Supernatant was decanted, 0.4 mL cold buffer added, and the tube vortexed and re-centrifuged. Pooled post-mitochondrial supernatants were spun in 1.5 mL ultracentrifuge tubes at 150000 rpm, 10 min, at 4°C in a Sorvall Discovery M 150 micro ultracentrifuge (Kendro Laboratory Products SA, Carouge-Geneva, Switzerland), and microsomal fractions stored at -80°C. Microsomal protein (200 μg, 10–18 μL) was pre-incubated 10 min at 37°C in an agitating bath, then incubated 15 min with 50 μL substrate solution (buffer plus 90 mM glucose-6-phosphate, 72 mM EDTA, 9 mM NADP, 6.2 nmol cold HMG-CoA (0.12 mM), 1.3 nmol [14C]HMG-CoA (0.0025 MBq), 0.3 IU glucose-6-phosphate dehydrogenase, and 0.024 MBq [3H]mevalonic acid as recovery standard). After 15 min, reaction was terminated with 25 μL 10 M HCL, then incubated 30 min at 37°C for mevalonate-mevalonolactone conversion. Following centrifugation at 1000 g, 1 min, at 4°C to remove denatured protein, supernatant was applied to activated (1 h, 105°C) TLC plates, developed in fresh benzene-acetone (1:1, by vol), the mevalonolactone region scraped (based on migration of cold standards and X-ray film visualization; Rf 0.42–0.5), and radioactivity measured in 10 mL scintillation cocktail.
Differences between groups were tested by unpaired/paired, one-tailed/two-tailed, student t-tests, equal variances, as appropriate for different measurements. Statistical significance was evaluated at P < 0.05 unless stated otherwise.