27.2.1 Lifelong Supplementation of Coenzyme Q10
In order to identify differences in survival and longevity, we followed all 150 male Sprague-Dawley rats and 86 male c57/B17 strain mice throughout their life spans. Both rats and mice were randomly assigned to a study group receiving 10 mg/kg/day of coenzyme Q10 or control group receiving a standard diet.
Ubiquinone Q10 was mixed into the normal animal diet by using soybean oil as a vehicle. Soybean oil was also added to the control food. All the food was kindly provided by Pharma Nord (Vejle, Denmark). The feeding was adjusted so that the daily intake of Q10 was 10 mg/kg/day in the experimental group and less than 0.5 mg/kg/day in the control group. For rats weighing less than 150 g, the quantity of food made available was 20 g/rat/day; for rats weighing over 150 g, the amount was 25 g/rat/day. For the mice, the amount was 5 g/mouse/day.
Animals were regularly weighed and inspected to follow their growth and general well-being. Also, survival of animals was followed. An autopsy was performed on all rats that died naturally, whenever possible within 24 hours. A total of 31 treated and 29 control rats were autopsied. An autopsy included a macroscopic evaluation of skin, internal tumors, and pathology. After macroscopic evaluation, samples were taken of heart, liver, kidney, lung, hypophysis, adrenals, and tumors for later microscopic examination. This included a normal pathological examination of tissues as carried out by a pathologist.
27.2.2 Coenzyme Q Measurements in Plasma and Tissues
Coenzyme Q concentrations in plasma, CSF, and different tissues were measured by the high performance liquid chromatography (HPLC) method as described by Lang et al.26 The serum samples were extracted with w-propanol and coenzyme Q7 was added as an internal standard. The coenzymes were reduced with NaBH4 prior to HPLC employing a Gilson 232-401 automated sampler (Gilson Medical Electronics Inc., Villiers le Bel, France). The HPLC equipment consisted of two Wallac 2258 pumps (Pharmacia Biotechnology, Uppsala, Sweden), a Beckman Gold C18-ultrasphere column (Beckman Instruments Inc., CA, USA), a Gilson C18 precolumn, and an ESA electrochemical detector (ESA Inc., MA, USA). Coenzyme Q concentrations in the heart tissue were measured according to the method described by Lang et al.26 with some modifications. An identical piece of left ventricle from each heart was dry homogenized with a microdismembrator (Micro-Dismembrator, B. Braun, Melsungen, Gemany). An accurate amount of ventricle was dissolved in 300 ¡l of 1:2 ethanol-water solution. We added 100 ¡l of methyl substituted Q10 (0.5 g/l, dissolved in ethanol) as an internal standard. The tissue samples were stirred with an ultrason-icator, and coenzyme Q was extracted into 500 ¡l of hexane, which was dried under nitrogen and resuspended into 200 ¡l of methanol-ethanol (80:20). UV detection of reduced and oxidized coenzyme Q was performed by high performance liquid chromatography (HPLC) under the following conditions; pump: LKB 2249, column: Chromsphere C-18, mobile phase, methanol-ethanol 80:20, flow rate: 0.5 ml/min., Detector: LKB 2141, wavelength: 275 nm. The standard samples of Q9 and Q10 were extracted and analyzed accordingly as the heart tissue samples.
A fluorescence histochemistry of adrenal gland and superior cervical ganglia was carried out to estimate lipopigment accumulation. This was determined by a quantitative fluorescence microscopy as described in detail elsewhere.27 Briefly, the fixed tissues were embedded in paraffin, sectioned serially, and examined under a Nikon Mikrophot FXA fluorescence microscope. Quantitation of pigment autofluorescence was performed with an image analyzer (DPS-200 MTI image processor with Microscale software). Autofluorescence intensity was measured at random from 80 sympathetic neuronal pericarya or cells of adrenal cortex at four different levels. The data are expressed as mean arbitrary units.
After an autopsy, samples of heart, liver, kidney, lung, hypophysis, adrenals, and tumors were taken for later microscopic examination. Samples were immediately immersion fixed (4% parafor-madehyde for 2 to 6 h at room temperature) and stored at +4 °C. Later on samples were embedded in Tissue-Tek (Miles Inc., Elkhart, USA) and sectioned into 10 pm slices with a cryotomy (Micron, Heiselberg, Germany). After staining the slices with hematoxylin-eosin, a standard pathological examination was carried out by a pathologist.
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