Hugo E. Gallo-Torres

Obligatory role of bile for the intestinal absorption of vitamin E.

Normal, white female rats subjected to cannulation of the abdominal thoracic duct have been utilized for a study on the essentiality of biliary and pancreatic secretions for the intestinal absorption of vitamin E. In all animals the thoracic duct lymph was collected. Some rats had the enterohepatic circulation undisturbed and in others bile or pancreatic juice or both were drained to the exterior by appropriate catheters in the common bile duct. On the first postoperative day, the animals received intragastrically an emulsion containing protein, carbohydrate, monoolein, 2 mg ofd,l-α-tocopheryl acetate plus 50 μC ofd,l-α-tocopheryl-1’,2’-3H-acetate. The appearance of radioactive α-tocopherol and its derivatives was determined in lymph, hourly, after emulsion administration. The obligatory role of bile in intestinal absorption ofd,l-α-tocopheryl-1’,2’-3H-acetate has been established. Pancreatic juice seems to be necessary for the hydrolysis of the vitamin E acetate ester. The simultaneous infusion of bile and pancreatic juice promotes absorption of about 10% of the administered dose into the lymph. A chromatographic separation of the radioactive vitamin E fractions revealed that most of the vitamin E, which is actively transfered from the intestinal lumen to the lymph, is nonesterified. An oxidation product of α-tocopherol, presumably itsp-quinone, appears in small amounts in the lymph, but almost no labeled α-tocopheryl acetate could be detected under these experimental conditions.

Distribution and metabolism of two orally administered esters of tocopherol

A comparison of the distribution of total radioactivity in rat tissue lipids after the oral administration of d,1-3,4-3H2-α-tocopheryl nicotinate and d,1-α-tocopheryl-1’,2’-3H2-acetate in equimolar concentrations has demonstrated that there is considerable variation in the concentration of vitamin E in organs at different times after dosing. A higher total radioactivity was found in the tissues of animals receiving α-tocopheryl nicotinate than after α-tocopheryl acetate 12 hr after feeding with an emulsion, but not at most other time intervals studied. These findings indicate that the tissue uptake of vitamin E after oral dosage with nicotinate ester is, perhaps, poorer than that occurring after feeding with tocopheryl acetate, or that α-tocopheryl nicotinate has a faster turnover than the acetate ester. Although total radioactivity in the blood and liver of those animals dosed with α-tocopheryl acetate varied slightly with time, there was a high peak of radioactivity at 12 hr after dosage with nicotinate ester. In both groups of rats, the adrenals, ovaries, adipose tissue and heart appeared to extract vitamin E from the blood for a period of up to 48 hr postabsorptively. Metabolic products of tocopherol detected by glass-fiber paper chromatography were found in both instances. This analysis revealed that, when orally administered, both α-tocopheryl nicotinate and α-tocopheryl acetate are extensively metabolized by the tissues of the rat. The metabolite most abundantly occurring under these conditions was α-tocopheryl quinone. In the adrenal glands, however, the most highly labeled compound was unesterified tocopherol, which increased with time and comprised up to 90% of the chromatographed radioactivity. From the data obtained, it can be assumed that the adrenal tissue plays a definite role in the metabolism of vitamin E.A comparison of the distribution of total radioactivity in rat tissue lipids after the oral administration of d,1-3,4-3H2-α-tocopheryl nicotinate and d,1-α-tocopheryl-1’,2’-3H2-acetate in equimolar concentrations has demonstrated that there is considerable variation in the concentration of vitamin E in organs at different times after dosing. A higher total radioactivity was found in the tissues of animals receiving α-tocopheryl nicotinate than after α-tocopheryl acetate 12 hr after feeding with an emulsion, but not at most other time intervals studied. These findings indicate that the tissue uptake of vitamin E after oral dosage with nicotinate ester is, perhaps, poorer than that occurring after feeding with tocopheryl acetate, or that α-tocopheryl nicotinate has a faster turnover than the acetate ester. Although total radioactivity in the blood and liver of those animals dosed with α-tocopheryl acetate varied slightly with time, there was a high peak of radioactivity at 12 hr after dosage with nicotinate ester. In both groups of rats, the adrenals, ovaries, adipose tissue and heart appeared to extract vitamin E from the blood for a period of up to 48 hr postabsorptively. Metabolic products of tocopherol detected by glass-fiber paper chromatography were found in both instances. This analysis revealed that, when orally administered, both α-tocopheryl nicotinate and α-tocopheryl acetate are extensively metabolized by the tissues of the rat. The metabolite most abundantly occurring under these conditions was α-tocopheryl quinone. In the adrenal glands, however, the most highly labeled compound was unesterified tocopherol, which increased with time and comprised up to 90% of the chromatographed radioactivity. From the data obtained, it can be assumed that the adrenal tissue plays a definite role in the metabolism of vitamin E.

Methodology for the determination of bioavailability of labeled residues

Methods are described for the determination of bioavailability of lipo- and hydro- soluble compounds in the rat. These procedures involve catheterization of the portal vein and/or intestinal lymphatics to study absorption in unanesthetized animals. Catheterization of the common bile duct prevents recycling of materials through the entero-porto-hepato-biliary circulation (EPHBC). Steady-state conditions are ensured by constant infusion of bile or a solution of bile acids into the stomach or duodenum. The techniques and physiological considerations discussed in detail here have resulted in new proposed animal preparations, of value in the accurate determination of the bioavailability of labeled residues ingested by the second species. Since intake must be adequate to permit meaningful conclusions, the concentration of radioactivity in the harvested homogenized tissue is increased by lyophilization. The lyophilized material is compressed into pellets of adequate size. The animals, kept in restraining cages to prevent coprophagy, are allowed to eat the labeled residue spontaneously, if necessary, for 48 and even 72 hr. Bile, urine, and feces are collected for a sufficient length of time to allow quantitative excretion of the labeled residue, if no absorption takes place. Collection of these excreta must be complete. It is essential to account for most-if not all-of the radioactivity administered. Data so obtained allow an accurate balance between intake and excretion of the labeled residue. The presence of radioactivity in the intestinal wall, carcass, liver, and urine is indicative of absorption. The appearance of radioactivity in the bile also indicates absorption, in addition to suggesting that the compound(s) may undergo EPHBC. The extent of recovery of the administered radioactivity in the luminal contents and feces indicates the extent to which the labeled residue is not bioavailable. Evaluation of data obtained with these new animal models should permit corresponding upward adjustments of the minimum levels of residues allowable in the tissue of animals intended for human consumption.

Prostaglandins and the Protection of the Gastroduodenal Mucosa in Humans: A Critical Review

M ucosal protection ([MP] “cytoprotection”) owes its inception largely to the demonstration by Robert and co-workers1’2 that it was possible to protect the mucosa of the rat from the injurious effects of a variety of noxious agents. These compounds included 6N HC1, iN NaOH, boiling water, absolute ethanol, and nonsteroidal antiinflammatory drugs (NSAIDs).’ Although cytoprotection was born from nonphysiologic experimentation, it is now being used by clinical researchers applying the knowledge of MP in a more physiologic fashion to the human subject. The clinical researcher has the difficult task of experimenting on human subjects, using ideas obtained largely from studies in rats. Thus, MP is a subject in a phase of very rapid advance, and a critical review over a broad clinical front is particularly appropriate at the moment. An immense preclinical literature base has accumulated since the pioneer work by Robert’s group. Recently, a very complete and useful account on the subject of protection of the intestinal mucosal has appeared.3’4 In humans, the bulk of information on MP has been obtained from studies of the upper gastrointestinal (CI) tract. Accordingly, the scope of this review will be restricted to data obtained from the stomach and duodenum. The main objective is to provide the reader with an up-to-date background regarding the interpretation of MP data.

Techniques for the in vivo catheterization of the portal vein in the rat.

Summary Techniques are reported for the catheterization of the rat portal vein. These methods permit drawing of blood samples without pain in unanesthetized animals under near physiological conditions. These procedures can be used for the assessment of the rate of intestinal absorption via the portal vein and/or the infusion of materials into the portal bloodstream.

A biliary metabolite of ascorbic acid in normal and hypophysectomized rats

Abstract 1. 1. A metabolite of ascorbic acid was studied in bile-duct cannulated normal and hypophysectomized rats. [1- 14 C]Ascorbic acid was given by gastric intubation either in an emulsion containing triolein, carbohydrates and proteins, or in an aqueous solution. 2. 2. The kinetics of the biliary excretion of radioactive material were followed. The rate of excretion was dependent upon the concomitant dietary components and on the nature of the solution infused in the upper part of the common bile-duct. 3. 3. Peak excretion occurred 4–7 h after administration of ascorbic acid in the emulsion and 2–3 h after giving the vitamin in water. 4. 4. Infusion of either rat bile or of a solution of taurocholate stimulated the excretion of the metabolite to a similar extent (0.75 and 0.65% of the dose excreted during the first 24 h, respectively). Infusion of an isotonic salt solution reduced the total excretion to 0.4%. The results after administration of ascorbic acid in water were as follows: 0.5, 0.45 and 0.25%, respectively. 5. 5. A lower excretion (0.04% of the given dose during the first 10 h) was observed in hypophysectomized rats after administration of [1- 14 C]ascorbic acid in the emulsion and during infusion of taurocholate solution. 6. 6. Electrophoretic as well as chromatographic data suggest that ascorbic acid-2-sulfate accounts for the radioactive material in the bile. No labelled ascorbic acid could be detected. 7. 7. The existence of an enterohepatic circulation for this metabolite is discussed. A role of taurocholate is postulated for the absorption of this metabolite from the intestinal tract.

An ultramicrotechnique for the detection and separation of small molecular weight peptides from amino acids

Abstract An ultramicroprocedure is described for determining picomole concentrations of small peptides and amino acids in microliter quantities of plasma. A micro Cu-Sephadex G-25 column is used to separate the small molecular weight peptides from the amino acids. The procedure is relatively simple, rapid, reliable, accurate, and very sensitive. The limits of sensitivity depend upon the specific activity of the dipeptides or amino acids used; increasing the specific activity gives greater sensitivity.

A method for the bioassay of antisecretory activity in the conscious rat with acute gastric fistula: Studies with cimetidine, somatostatin, and the prostaglandin E2 analog Ro 21-6937

Abstract A model consisting of an acute gastric fistula has been developed for the bioassay of gastric secretory activity in conscious rats. The gastric secretions are collected by gravity via a cannula in the most dependent site of the glandular stomach. The biliary and pancreatic secretions are collected by means of a catheter in the common bile duct. Hydration is maintained by intravenous infusion of saline. This method is ultrasensitive, since it can detect changes in the hydrogen ion concentration in gastric secretions elicited by microgram quantities of inhibitory compounds. The usefulness of this biological preparation is illustrated in studies with compounds of very different chemical structure, such as cimetidine, somatostatin, and the prostaglandin E 2 analog Ro 21-6937/000. These compounds are all effective inhibitors of basal acid secretory activity in the rat, but the PGE 2 analog is considerably more potent than either somatostatin or cimetidine.

Biliary, fecal and uninary excretion of [3H]-prostaglandins in the rat☆

The profiles of biliary, fecal and urinary excretion of tritium labeled prostaglandins (PGs) of differing biological activity were investigated in the rat. The PGs (10 micrograms/kg: 2 to 50 microCi/rat, in 1 ml polyethylene glycol-400) were administered intragastrically. Excretion data were expressed as a percentage of the total administered radioactivity. For the orally administered PGs 11R-methyl-16R-fluoro-15R-hydroxy-9-oxoprosta-ci s-5-trans-13-dienoic acid and its methyl ester, excretion was equally divided between urine and feces. The fecal and urinary profile of excretion of 3H after prostacyclin (PGI2) was similar to that following administration of 11R, 16, 16-trimethyl-15R-hydroxy-9-oxoprosta-cis-5-trans-13-dienoic acid (trimoprostil), a PG with antisecretory-antiulcer potential. However, PGI2 was very poorly absorbed from the intestine, while the absorption of trimoprostil was very efficient. Biliary excretion, with little entero-porto-hepatic biliary circulation, was the main route of elimination of trimoprostil, thereby resulting in rapid elimination of drug-related products and diminishing the potential for systemic liability in the rat.

The gastrointestinal absorption, tissue distribution, urinary excretion and metabolism of N-(2-aminoethyl)-glycine (AEG) in the rat

Abstract Radiolabeled N-(2-aminoethyl)-glycine (AEG) was synthesized and various aspects of its bioavailability were evaluated. AEG was rapidly and completely taken up by the small intestine of the rat. It was quickly absorbed into the portal vein. Most of the absorption took place during the first hour, with a peak at 30 min. Entry of this compound into the intestinal mucosal cell may be by a mechanism not involving active transport. Of many organs examined, only the liver took up significant amounts of AEG. The latter neither crossed the brain barrier nor was metabolized. Total urinary excretion (as intact AEG) averaged 80% of the administered dose within 4 hours and nearly 100% by 10 hours. Excluding the neutral-acidic amino acids and ammonia, AEG represented >99% of the ninhydrin positivity in the urine. AEG is thus an example of a substance which is rapidly and totally absorbed, as well as quickly and completely excreted.