Abstract
A simple and precise micro-method for measurement of daily fecal excretion of neutral and acidic sterols has been developed which utilizes sitostanol (24-ethyl-5 alpha-cholestane-3 beta-ol) as fecal flow and recovery marker. Extractions of sterols were performed from 50 microliters of fecal homogenate (feces-water 1:1), and analyses of neutral and acidic sterols were carried out by gas-liquid chromatography. The method is sensitive, precise, and easy to perform; the intra-assay variability yielded coefficients of variations of 1.9% and 3.5% (n = 6) for neutral and acidic sterols, respectively. The results from this method were compared with those obtained with the standard fecal flow marker chromic oxide. The correlation coefficients between the two markers were compared in 16 subjects and were 0.938 and 0.998 for excretion of neutral sterols and acidic sterols, respectively. Comparison of the fecal excretion of neutral and acidic sterols in 12 subjects determined from frozen samples and aliquots (approximately 1 g) sent by ordinary mail to the laboratory (transport time 1 to 5 days) gave identical results using sitostanol as fecal flow marker (818 +/- (SEM) 85 mg/day vs. 838 +/- 89 mg/day for neutral sterols and 417 +/- 59 mg/day vs. 414 +/- 60 mg/day for acidic sterols). The new micro-method is ideally suited for research laboratories in need of a simple, accurate, inexpensive, and high through-put method for measuring daily fecal excretion of neutral and acidic sterols, as well as total cholesterol synthesis, and can be performed on an outpatient basis.
Highlights
A simple and precise micro-method for measurement of daily fecal excretion of neutral and acidic sterols has been developed which utilizes sitostanol(24-ethyl-5a-cholestane38-01) as fecal flow and recovery marker
Extractions of sterols were performed from 50 pl of fecal homogenate, and analyses of neutral and acidic sterols were carried out by gas-liquid chromatography
Comparison of the fecal excretion of neutral and acidic sterols in 12 subjects determined from frozen samplesand aliquots (=1 g) sent by ordinary mail to the laboratory gave identical results using sitostanol as fecal flow marker
Summary
In 16 healthy subjects (6 females and 10 males; age range 22 to 30 years), the fecal excretion of neutral and acidic sterols was determined by the fecal balance method [1, 2]. In the second experiment 6 of the 16 subjects, plus 6 additional volunteers who received only sitostanol (30 mg t.i.d.) as fecal marker, collected total fecal samples as described above. Sitostanol and neutral sterols were measured and day-today variation in baseline fecal excretion of sitostanol was calculated. Determination of neutral and acidic sterols in aliquots 1 ( 3 g) of fecal samples sent by mail (micro-method). Determination of neutral and acidic sterols in total fecal samples (macro-method). One mg 5a-cholestane (Serva Feinbiochemica, Heidelberg, Germany) and 1 mg hyodeoxycholic acid (Sigma Chemical Co., St. Louis, MO) were added to exactly 1.0 g of homogenate as internal standards for neutral and acidic sterols, respectively. After cooling to room temperature the samples were transferred quantitatively to 100-ml volurimetric flasks and distilled water was added to exactly 100 ml. Calculation of neutral and acidic sterol excretion using sitostanol as fecal flow marker. Linear regressions were calculated by the method of least squares
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