Kang-Jey Ho

A new instrument for the rapid preparation of tissue slices

Abstract A manually operated microtome for the rapid preparation of live tissue slices is described. The instrument operates submerged in an isotonic solution and incorporates an automatic mechanism for the removal of the cut slices which are gently carried by a stream of fluid to a strainer reservoir. An inexperienced operator can obtain circular slices of nearly identical thickness at a rate of one every 3 or 4 s. Mechanical trauma to the tissues is minimized and the temperature and oxygenation of the medium are easily controlled.

Effect of Cholesterol Feeding on Circadian Rhythm of Hepatic and Intestinal Cholesterol Biosynthesis in Hamsters

Summary Forty-eight adult hamsters were divided equally into two groups fed a control diet and a 2% cholesterol diet, respectively, under a rigid lighting (6 pm-6 am) and feeding (6 pm-8 am) schedule for three weeks. The cholesterol synthetic activity of the liver, stomach, small intestine, cecum, colon and kidney was measured by in vivo conversion of acetate-1-14C to cholesterol in four animals each time at 4 hour intervals. A remarkable circadian rhythm with the peak at midnight and the nadir at noon was found in the liver of the control hamsters, but was completely abolished in the cholesterol-fed animals since the activity was nearly totally suppressed at all times. The small intestine exhibited a similar rhythm with the peak at midnight but maintained a high baseline activity from 8 am to 6 pm. Cholesterol feeding did not alter the baseline activity but reduced 17% of the peak activity. Other organs failed to show such a circadian rhythm.

Cholesterol accumulation in various rabbits' tissues with variations in serum levels and duration of exposure☆

Abstract A factorial experiment involving 90 adult male New Zealand white rabbits was carried out to explore the effect of serum cholesterol levels and duration of exposure on the cholesterol contents of 15 different tissues. Individual serum cholesterol levels were maintained constant at fixed levels ranging from 29 to 2632 mg/100 ml among the groups for periods of 30, 60, and 90 days before being sacrificed. The relationships among these three variables could be expressed by various forms of regression equations. Cholesterol contents of pancreas and testis were proportional to serum cholesterol level only, while those of colon, spleen, heart, and kidney were related independently to serum cholesterol level and exposure time. Interaction of these two factors played the major role in determination of the cholesterol content of aorta and adipose tissue and to some extent the lung. A quadratic equation or curvilinear curve was found in the other five tissues. Cholesterol content of the skin was primarily affected by the interaction of serum cholesterol level and exposure time, that of adrenal and liver was determined by these two factors independently, whereas that of small intestine and muscle was affected by both of these factors and their interaction. Brain was the only tissue that did not show a change of its cholesterol content throughout the experiment.

Inhibitory Effect of Bile Acids on the Activity of Human β-Glucuronidase at Its Optimal pH

Abstract The effect of bile acids on β-glucuronidase activity was studied with centrifuged and dialyzed urines as the source of enzyme and phenolphthalein glucuronide as the substrate. The enzyme activity was measured at 56° in acetate buffer pH 5.2. All unconjugated bile acids tested showed no effect on the enzyme activity, primarily due to their low solubility in water. The glycine and taurine conjugates of cholic acid also showed no significant influence on the enzyme. Conjugated deoxycholic and chenodeoxycholic acids, on the other hand, were profoundly inhibitory to the enzyme within the physiological range of concentration in bile. Kinetic studies revealed that taurine conjugates of these two bile acids were pure competitive inhibitors of the enzyme for the substrate; whereas, their glycine conjugates exhibited a mixed competitive and noncompetitive inhibition. Since both urine and bile enzymes have an identical narrow pH activity curve, we conclude that pH and bile acids are two major determinants of β-glucuronidase activity in bile and their alteration may predispose to bilirubin pigment gallstone formation.

The role of the intestinal mucosa in cholesterol metabolism: its relation to plasma and luminal cholesterol.

Abstract Interrelationships of cholesterol in the intestinal mucosa, lumen, and plasma were studied in three groups of rats, i.e., fed a sterol-free diet, a 2% cholesterol diet, and a sterol-free diet followed by biliary diversion. All rats received an intracardiac injection of cholesterol-4- 14 C and were sacrificed sequentially. Amounts and specific activities (SA) of cholesterol in the plasma, mucosa, and luminal contents were determined and the following results obtained. (1) Plasma cholesterol SA was greater than mucosal cholesterol SA which in turn was greater than luminal cholesterol SA suggesting that there were two pools of cholesterol in the mucosa: one was active in cholesterol synthesis, while the other was not. (2) Without cholesterol feeding, 79% of the cholesterol in the active mucosa was derived from de novo synthesis which contributed 77% of the fecal cholesterol. Transport of cholesterol from mucosa to lumen was not merely by desquamation of epithelium but primarily by active secretion. (3) Cholesterol feeding did not affect the mucosal cholesterol content, nor its SA and transport to the lumen. (4) Expansion of the cholesterol pool in active mucosa and increase of luminal cholesterol content were found throughout the intestinal tract as a consequence of biliary diversion.

Studies on cholesterol metabolism in rats by application of D2O and mass spectrometry

Abstract The experiment was designed to study the dynamic aspects of cholesterol metabolism in rats by application of D 2 O and mass spectrometry. Thirty-six control rats were fed a cholesterol-free diet and 34 experimental rats were on a 2% cholesterol diet for 3 months and were then given a constant concentration (0 to 20%) of D 2 O in their drinking water. Deuterium in atoms percent of body water, serum, tissue and fecal cholesterol was determined. Daily fecal output of neutral and acid sterols was also measured. The results showed no change in serum and tissue cholesterol concentration in control and experimental groups in spite of the fact that the latter had a mean daily cholesterol absorption of 182 mg/kg and a dietary contribution of 76% of the body exchangeable cholesterol. The experimental group also exhibited a 2–3 fold shorter turnover time and a 40% suppression of endogenous synthesis which compensated perfectly for the intestinal influx of dietary cholesterol. Cholesterol metabolism was disturbed only when a high level (20%) of D 2 O was given to the animal.

Mode of Cholesterol Accumulation in Various Tissues of Rabbits with Moderate Hypercholesteremia

Summary A moderate hypercholesteremia (300 to 400 mg/100 ml) was induced and maintained in a group of New Zealand white rabbits. The animals were killed weekly up to 12 weeks and cholesterol contents of 15 tissues were determined. Muscle, fat, pancreases, and brain showed no increase of their cholesterol contents. Small increases were noted in testis, colon, and heart, but these were not related to time. Linear arithmetic increases of tissue cholesterol contents were found in adrenal, kidney, small intestine, and lung while aorta, liver, spleen, and skin exhibited an exponential accumulation of cholesterol over a 3-month period of moderate hypercholesteremia. The rapid and avid uptake of cholesterol by aorta may play a role in the pathogenesis of atherosclerosis.

Correlation of Kinetic Parameters of Cholesterol Metabolism with Serum Cholesterol Levels

The factors affecting individuals’ serum cholesterol levels are multiple and complicated. The present study was designed to determine a possible correlation of the individual serum cholesterol levels with various kinetic parameters of cholesterol metabolism in 15 healthy, white male subjects.

Identification and quantitation of dietary and fecal neutral sterols by mass spectrometry

Abstract A procedure is described for the identification and quantitation of dietary and fecal neutral sterols by the combination of thin-layer chromatography, mass spectrometry and Liebermann-Burchard colorimetry. The neutral sterols in the diet and feces were extracted by the method of Ho and Taylor and Abell el al. respectively. The extracted neutral sterols were then further separated from other compounds by thin-layer chromatography with silica gel H as adsorbent and a solvent system composed of light petroleum:ethyl ether:glacial acetic acid ( 80:20:1 , by volume). The isolated neutral sterols consist of stigmastanol, sitosterol, stigmasterol, campesterol, coprostanol and cholesterol. The relative quantitaty of each sterol in such a mixture can be measured from its mass spectrum. Their relative absorbance of the Liebermann-Burchard reaction can also be determined. Thus, the absolute quantity of each individual neutral sterol in a mixture can be computed from the data obtained from the mass spectrum and colorimetry of the sample. The method is accurate and reproducible and of great value in the study of cholesterol metabolism when the diet and feces contain a variety of plant sterols.

Sources of Cholesterol in the Intestinal Lymph in Rats Fed a Cholesterol-Free Diet

Summary The intestinal lymphatics were successfully cannulated in 10 adult male Sprague-Dawley rats fed a cholesterol-free diet; 40-60 days prior to cannulation, these animals received an intracardiac injection of a single dose of cholesterol-4-14C. The intestinal lymph was collected over a period of 6 hr. The animals were then sacrificed. The cholesterol and its specific activity in plasma, liver, and mucosa of various segments of gastrointestinal tract were measured and the sources of cholesterol in the intestinal lymph were determined by comparison of their relative specific activities. Of the total cholesterol in the intestinal lymph, 0.491 μM/hr, 81.5% or 0.398 μM/hr was derived from exchangeable cholesterol in the intestine considered to be the same pool as the plasma cholesterol, and 18.5% or 0.088 μM/hr was synthesized de novo in the intestinal mucosa. The latter accounted for 22% of the daily turnover of the plasma cholesterol. The authors express their appreciation to Miss Belma Mikkelson and Miss Janice Drummond for their technical assistance.