FACTORS INFLUENCING GASTROINTESTINAL
ABSORPTION OF DRUGS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University
By
ANGEL LUIS IGLESIAS, B. S., M. S.
The Ohio State University
1958
Approved by
.ege of Pharmacy ACKNOWLEDGEMENTS
The author wishes to make grateful acknowledgement to Dr. John W. Nelson, Professor, College of Pharmacy, and to
Dr. Arthur Tye, Professor, College of Pharmacy, for constant counsel and advice in the fulfillment of this work.
ii TABLE OF CONTENTS
Page
INTRODUCTION ------1
Methods Used in the Study of Absorption ______1 Factors Involved in Absorption ______9
EXPERIMENTAL PROCEDURE ------22
Objectives ______22 Extraction and Analysis ______23 Experiment I. Absorption Rates of Phenobarbital (5 Mg. Dose) ______30 Experiment II. Absorption Rates (25 Mg. Dose) _ _ - L3 Experiment III. Drug Retained by Intestinal Wall _ _ 53 Experiment IV. Drug Retained by Stomach Wall _ - - 65 Experiment V. Effect of Total Volume of Fluid - - - 77 Experiment VI. Use of Alcohol as Solvent ____-•-81 Experiment VII. Effect of Olive Oil ______83 Experiment VIII. Effect of Glucose ______85 Experiment IX. Effect of Sucrose 89 Experiment X. Effect of Feeding ______91 Experiment XI. Effect of Atropine ______94 Experiment XII. Effect of Stress ______97 Experiment XIII. Effect of Bentonite Magma _____ 99 Experiment XIV. Effect of Glucosamine Hydrochloride - 101 Experiment XV. Effect of Poloxalkol ______103 Experiment XVI. Absorption from the Stomach _ _ _ _ 106 Experiment XVTI. Absorption from the Large Intestine 115
DISCUSSION 118
SUMMARY AND CONCLUSIONS ------123
BIBLIOGRAPHY 125
iii LIST OF TABLES
TABLE PAGE
1 Per Cent Phenobarbital Absorbed in 5 Minutes From a 5 Mg. Dose ______33
2 Per Cent Phenobarbital Absorbed in 10 Minutes From a 5 Mg. Dose 34
3 Per Cent Phenobarbital Absorbed in 15 Minutes From a 5 Mg. Dose - - --- ______35
4 Per Cent Phenobarbital Absorbed in 20 Minutes From a 5 Mg. Dose ------36
5 Per Cent Phenobarbital Absorbed in 25 Minutes From a 5 Mg. Dose ______37
6 Per Cent Phenobarbital Absorbed in 30 Minutes From a 5 Mg. Dose 38
7 Per Cent Phenobarbital Absorbed in 45 Minutes From a 5 Mg. Dose 39
8 Per Cent Phenobarbital Absorbed in 60 Minutes From a 5 Mg. Dose ------40
9 Per Cent Phenobarbital Absorbed in 75 Minutes From a 5 Mg. Dose 41
10 Per Cent Phenobarbital Absorbed from a 5 Mg. Dose After Different Intervals ------42
11 Per Cent Phenobarbital Absorbed in 5 Minutes From a 25 Mg. Dose ______46
12 Per Cent Phenobarbital Absorbed in 10 Minutes From a 25 Mg. Dose ------47
13 Per Cent Phenobarbital Absorbed in 20 Minutes From a 25 Mg. Dose ------_____ 48
14 Per Cent Phenobarbital Absorbed in 30 Minutes From a 25 Mg. Dose 49
15 Per Cent Phenobarbital Absorbed in 45 Minutes From a 25 Mg. Dose ------50
iv LIST OF TABLES (Continued) TABLE PAGE
16 Per Cent Phenobarbital Absorbed in 60 Minutes From a 25 Mg. Dose 51
1? Per Cent Phenobarbital Absorbed From a 25 Mg. Dose at Different Intervals ------52
18 Per Cent of a 5 Mg. Dose of Phenobarbital Retained By Intestinal Wall After 5 Minutes ______55
19 Per Cent of a 5 Mg. Dose of Phenobarbital Retained By Intestinal Wall After 10 Minutes ______56
20 Per Cent of a 5 Mg. Dose of Phenobarbital Retained By Intestinal Wall After 15 Minutes ______57
21 Per Cent of a 5 Mg. Dose of Phenobarbital Retained By Intestinal Wall After 20 Minutes ______58
22 Per Cent of a 5 Mg. Dose of Phenobarbital Retained By Intestinal Wall After 25 Minutes ______59
23 Per Cent of a 5 Mg. Dose of Phenobarbital Retained By Intestinal Wall After 30 Minutes ______60
24 Per Cent of a 5 Mg. Dose of Phenobarbital Retained By Intestinal Wall After 45 Minutes ______6l
25 Per Cent of a 5 Mg. Dose of Phenobarbital Retained By Intestinal Wall After 60 Minutes ______62
26 Per Cent of a 5 Mg. Dose of Phenobarbital Retained By Intestinal Wall After 75 Minutes ______63
27 Per Cent Phenobarbital Retained by Intestinal Wall From a 5 Mg. Dose ______64
28 Per Cent of a 5 Mg. Dose of Phenobarbital Retained By Stomach Wall After 5 Minutes ______67
29 Per Cent of a 5 Mg. Dose of Phenobarbital Retained By Stomach Wall After 10 Minutes _ 68
30 Per Cent of a 5 Mg. Dose of Phenobarbital Retained By Stomach Wall After 15 Minutes 69
v LIST OF TABLES (Continued)
TABLE PAGE
31 Per Cent of a 5 Mg. Dose of Phenobarbital Retained By Stomach Wall After 20 Minutes 70
32 Per Cent of a 5 Mg. Dose of Phenobarbital Retained By Stomach Wall After 25 Minutes ------71
33 Per Cent of a 5 Mg. Dose of Phenobarbital Retained By Stomach Wall After 30 Minutes 72
3h Per Cent of a 5 Mg. Dose of Phenobarbital Retained By Stomach Wall After h5 Minutes ------73
35 Per Cent of a 5 Mg. Dose of Phenobarbital Retained by Stomach Wall After 60 Minutes ______7b
36 Per Cent of a 5 Mg. Dose of Phenobarbital Retained By Stomach Wall After 75 Minutes 75
37 Per Cent Phenobarbital Retained by Stomach Wall From a 5 Mg. Dose - - ______76
38 Effect of Total Volume of Fluid cn Absorption (5 c.c.) 79
39 Effect of Total Volume of Fluid on Absorption (15 c.c.) 80
h0 Effect of Alcohol on Absorption of Phenobarbital - - - 82
hi Effect of Olive Oil on Absorption of Phenobarbital _ _ 8h
h2 Effect of Glucose on Absorption of Phenobarbital - _ 88
h3 Effect of Sucrose on Absorption of Phenobarbital - - 90
hh Absorption by Starved Rats 92
h5 Absorption by Fed Rats ______93
h6 Effect of Atropine on Absorption of Phenobarbital - - $6
h7 Effect of Stress on Absorption of Phenobarbital - - - 98
h8 Effect of Bentonite Magma on Absorption of Phenobarbital ______100
h9 Effect of Glucosamine on Absorption of Phenobarbital 102
50 Effect of Poloxalkol on Absorption of Phenobarbital lOh vi LIST OF TABLES (Continued)
TABLE PAGE
51 Changes in Per Cent Phenobarbital Absorbed From a 10 Mg. Dose in Twenty Minutes Produced by Varying the Solvent or Administering a Second Substance ______--- ______105
52 Absorption of Phenobarbital From Stomach (10 Min.) 108
53 Absorption of Phenobarbital From Stomach (20 Min.) 109
54 Absorption of Phenobarbital From Stomach (30 Min.) 110
55 Absorption of Phenobarbital From Stomach (45 Min.) 111
56 Absorption of Phenobarbital From Stomach (60 Min.) 112
57 Absorption of Phenobarbital From Stomach (90 Min.) 113
58 Absorption of Phenobarbital From Stomach (120 Min.) 114
59 Absorption From the Large Intestine (Blank Determinations) ______n 6
60 Absorption From the Large Intestine ______117
vii LIST OF FIGURES
FIGURE PAGE
1 Standard Curve for Phenobarbital Concentration Versus Optical Density ______29
2 Absorption From the Gastrointestinal Tract of the Rat Using a 5 Mg. Dose of Phenobarbital - 32
3 Absorption From the Gastrointestinal Tract of the Rat Using a 25 Mg. Dose of Phenobarbital _ 45
4 Drug Retained by Intestinal Mucosa ______54
5 Drug Retained by Stomach Mucosa ______66
viii INTRODUCTION
Methods Used in the Study of Absorption
For the last eighty years physiologists, and more recent ly pharmacologists and biochemists, have sought to explain the mechanisms by which substances, including foods and therapeutic agents, are transferred from the lumen of the alimentary canal to the circulating blood stream. Interest in the subject has increased since evidence has accumulated for the hypothesis that this trans fer in many cases is not simply mediated by physical phenomena alone, but also by biological activity within the mucosa lining the gastro intestinal tract. It is not unusual to find names like Cori, Van
Slyke, Kalckar and others, along with those of physiologists and pharmacologists like Starling, von Mering, and Sollmann in the literature dealing with studies on absorption.
The most serious difficulty that is met when making studies on absorption in the problem of obtaining an animal prepara tion which will approach, as close as possible those conditions under which a normal, intact animal will absorb foodstuffs or drugs in a truly physiological manner.
The use of loops of excised intestines is an old, but still very much used method. Such loops can be used for the passage of substances out of the lumen into a Ringers or Tyrodo solution bathing them on the outside, where the substance being absorbed can be determined (1, 2, 3> 4). This method is far from approach ing truly physiological conditions. The.intestinal mucosa, like the serosa, is very sensitive to handling, changes in temperature, moisture, alterations in circulation etc. Circulation is of the greatest importance in a normal process of absorption and it cannot be expected to be normal even when the utmost care is exerted in the excision of the loops.
Recently, excised loops have been used in the study of actively transported substances. These are everted before using, turning the mucosal side to the outside. This is done so that the extremely active epithelium of the mucosa will be exposed directly to the oxygen bubbled into the bathing fluid. Here, the transfer is from the outside fluid to the serosal side inside the loop. Even with the more efficient oxygenation claimed, this method is at least as far apart from physiological conditions as the preceding one. The manual handling to which the loop of intestine is subjected during aversion must necessarily damage the villi lining the mucosa (1, 5 , 6).
Another method widely used is the perfusion of loops of intestine in situ, by cannulating at two sites, one for introducing the fluid, the other for recovering it and analysing the contents
(7, 8, 9. 10, 11, 12, 13, 14). This is done on anesthetized animals, the perfusion being done after the animal recovers from the surgical procedure. Although this method has the advantage of permitting more than one determination to be made on the same animal, it is not truly physiological since the effect of the anesthetic and the surgical procedure cannot be discounted. Besides, the solution which is being perfused passes through the intestine in a few min utes , while such a solution fed to the animal in a normal manner would take several hours in transit. Neither is the emptying time of the stomach considered in this method when absolute rates of absorption are studied.
A modification of the preceding method having no ad vantages and the disadvantage that only one determination can be made on one animal, is that of the closed loop. The segment of intestine is closed at both ends, left in situ, the substance in jected into the loop, and after a definite interval of time, the loop is removed and the residual substance determined (15, 1 6 , 1 7 ,
18, 19, 20, 1). In another method loops of intestine in situ are perfused with solutions containing the substances studied and at the same time, the vessels supplying the intestinal wall are perfused by Ringer's solution introduced by a cannula in the coeliac artery and collecting it by a cannula in the portal vein. In this way, not only the residual drug can be determined in the perfusate from the intestinal loop, but also the concentration of drug in the portal circulation (21, 22, 23, 2*1-, 25).
Another method is the preparation of fistulae or pouches.
These are segments of stomach or intestine whereby the contents and secretions are diverted to the outside through an opening in the abdominal wall. The segments are isolated from the rest of the gastrointestinal tract, whose continuity is insured by anastomosing the cut ends. There are many types of fistulae, among which the better known are Pavlov’s, Thiry, Vella, (or a combination of the last two); Priestley and Mann; and Crocker-Markowitz (26). The advantage of this method is that an unlimited number of determina tions can be made on the same animal while it survives, but there are a number of disadvantages, among which the most serious is that one can never be sure that the process of absorption going on in the fistula is identical with those taking place in the rest of the gastrointestinal tract. Another is the difficulty of preventing leakage (27, 28, 29, 30, 31, 32, 33, 3*+, 35. 36, 37, 38, 39).
In another type of fistula, there is no opening to the outside, and the substance to be studied is injected into it and removed by aspiration after definite intervals of time. The segment of intestine is isolated, preserving its blood and nervous supply, and placed below the skin of the abdomen in such a position that it is easy to reach with a needle. The cut ends of the rest of the intestine are anastomosed end to end. These pouches are apt to be filled in most cases with a nearly solid mass of cholesterol, epithel ial cells and many other substances secreted by the mucosa (1 , hO, hi, h2).
Evidence of the unreliability of these kinds of prepara tions is shown by the differences in rates of absorption observed by Rohse when using different types of loops and fistulae in the
study of the same drug under the same conditions (39)• The method which approaches most closely physiological conditions, is probably the one introduced by Carl F. Cori in 1925. for the study of the absorption of sugars, and used by many workers since then (h3, U4, h.5, k6, k7, *4-8, *+9. 50, 51. 52, 53). The sub stance to be studied is fed to the unanesthetized, intact, starved animal, by stomach tube. After a desired interval of time, the animal is killed by a blow on the head, the abdomen is opened, the gastrointestinal tract ligated at the cardiac end of the esophagus and at whatever level of the intestine the worker desires. The gut is then slit open longitudinally, the contents washed out carefully and collected and the amount of drug remaining unabsorbed determined.
The amount absorbed is calculated by difference. Besides determin ing residual drug in the contents of the gut, the amounts in the different tissues and organs of the animal can be determined if proper methods of extraction and analysis are available for the substance. Under these conditions the animal is absorbing under a truly physiological state, there is no interference with the circu lation of the intestinal wall, or with the epithelium of the mucosa, the time measured during an absorption period is the required one under normal conditions to reach that stage of absorption, and the effects of an anesthetic are avoided. The only disadvantages are the time required for each determination, and the fact that one animal is consumed for only one determination.
Another widely used method is the determination of the concentration of a substance in the blood after it is given orally. 6
This is accomplished by taking samples of blood from different
sites in the circulation. Whatever the site used to draw the
samples, the concentration of the substance determined in this way does not give a true picture f absorption from the gastro
intestinal tract. Even when the sample is drawn from the portal
circulation, the substance under study may have undergone in its passage through the intestinal wall, changes in its structure which wo\’ld, in many cases make the procedure for its determination use less (39. 5^, 55* 56, 50)* The mucosa of the stomach and intestine have been shown to retain considerable amounts of many substances while absorption is taking place (57, 5 8 , 59). If the sample is taken from the general circulation after passing the liver, the majority of substances studied will have undergone changes of vary ing degrees (61, 62). These changes give rise in most cases to metabolites whose structure is not known exactly, precluding the development of methods of extraction and analysis dependable enough to account for all of the original drug given. As an example, we have the great number of barbiturates used in therapy. Not a single one can be recovered completely after absorption, either in the form of their metabolites or when partly excreted unchanged.
Another unreliable factor of this blood-sampling method is that the distribution of a substance in the animal body may not be truly reflected by its concentration in the blood. An example is the preferential deposition of many drugs in lipoid tissue
(60, 63). 7
Even if the metabolites from a given substance can be determined by the same procedure used for the original substance, they may not be extractable to the same degree by the same sol vents (64, 65). For these reasons, blood sampling should be used only for qualitative determinations at the most.
Besides these general methods, there are others used in special cases in studying absorption. Among these are the following.
1. The concentration of a substance in the lymph is determined. The lymph is usually collected from the cysterna chyli, rather than from the thoracic duct, to insure that it comes directly from the lacteals in the intestine. This method is used mainly but not exclusively in the study of the absorption of lipids (6 6 , 67).
2. The feces are collected during an absorption period and the quantity of unabsorbed drug is determined.(68, 69). This procedure, besides having the difficulty of extracting and separating the substance from the many chemical compounds found in fecal matter does not take into account the action of the bacterial flora.
3. Differences in weight after administration and ab sorption of fluid are estimated. In this method, an ingenious procedure is desfribed for weighing the abdomen in an intact animal
(7 0 , 71)* But it is based upon the assumption that during the peak of water absorption, this is held mostly by muscular tissue and that no great amounts get into the kidneys or bladder during that period. If this assumption is not valid, the method is useless since in weighing the abdomen for loss of water from the gastro intestinal tract, one would be weighing water already absorbed and present in the kidneys and bladder.
4. Chylomicron counts and curves are studied during absorption of fats (72).
5. The absorption of fast in patients with chylothorax is studied. This is a rare anomaly where the pleural cavity in one or both lungs is filled with chyle (73).
6 . Markers or visible substances like phenol red are used to follow the absorption and transit of substances in the alimentary canal (74, 7 5 , 7 6 . 77).
7. Radioactive isotopes are used (67, 78, 79» 80, 81,
82, 83, 84). This is probably the most accurate procedure in ab sorption studies and it can be combined with most of the other methods. At present it is somewhat limited by the restricted avail ability of isotopic substances that could be used.
8 . The bile is examined for content of the absorbed sub stance. This of course does not give a true picture of absorption but rather an indication of the amount of the substance absorbed which is excreted in the bile (85, 93)* The same thing can be said of the examination of urine.
9. Absorption is studied by following pharmacologic action (8 6 , 87, 88). This method is extremely good for qualitative work because of the ease with which, in many cases, the symptoms brought about by the action of the drug can be followed, and the fact that the animal, in many cases, after a proper rest, can be used repeatedly. However, its use in quantitative work has so many limitations that they make it almost useless. Here, not only the animal variation in relation to absorption, but also variation in relation to pharmacologic action must be taken into account as well as the factors of quantity of drug destroyed before reaching a specific site of action, and the quantity of drug excreted in the feces without being absorbed.
10, The intestinal mucosa and villi are examined micro scopically for changes during absorption. This use of histological preparations is not of the nature of quantitative studies, but it might give information about the forces governing absorption (8 9 ,
90, 91. 92).
Factors Involved in Absorption
Besides the transfer of drugs through the membrane of the mucosal cells, studies in absorption must consider the passage of these substances through the membranes lining the capillaries also, since they must be removed from the extracellular compartments be tween the two before reaching the blood stream. So the nature of these membranes and their permeability characteristics are of primary importance as a factor.
The first to speculate on the nature of these barriers was Overton, who postulated the mechanisms by which a number of compounds penetrated cells by passive diffusion at rates which depend on their solubility in fat-like solvents. His belief that the membrane of living cells is fat-like in nature is rather gen erally accepted, at least in part, to the present day. But it is evident that many small molecules, even if lipid insoluble, can penetrate cells, so Overton's hypothesis has been modified to in clude a membrane which is not entirely a lipid barrier, but rather is interspersed with small holes through which small lipid insol uble molecules can pass. But something more than this passive barrier must account for the preferential passage of some molecules as compared to the passage of other very similar molecules of the same size. The presence of cellular "carriers" has been advanced as a theory to explain why galactose is transferred faster than glucose or sodium faster than potassium. If Overton's hypothesis is accepted, then, only the undissociated molecules of weak organic electrolytes could pass this kind of barrier readily. At a physio logical hydrogen ion concentration, these substances are present partly as dissociated and partly as undissociated molecules, there fore the hydrogen ion concentration of the media in which they are dissolved and their dissociation constantw will be of importance in determining the rate at which they are absorbed. It is obvious, then, that their rate of absorption will vary at different levels of the gastrointestinal tract since the pH of the various levels has been proved to be quite different (37) • Recently, it has been found that basic drugs will pass from plasma into the stomach, their passage regulated mainly by their basicity, while acid drugs will pass with difficulty (9h), only the very weak acids being found in detectable amounts. The reverse would be true, then, in the passage from the stomach to plasma. if we assume an oily layer as a barrier.
An acidic drug would be largely in the unionized form in the low pH medium of the gastric juice, and thus acceptable to the oily barrier; a basic drug would be largely in the ionized form in this same med ium and thus unacceptable to the barrier. The reverse would be true in the case of the relatively high pH of the plasma. The inter change in the intestine would be different since the difference in pH of the intestinal contents and plasma is not so sharp as in the case of the stomach. And yet, absorption rates of many substances which would be favored by the pH of the stomach are found to be as high or higher in the high pH medium of the intestine. This could be explained by the difference in time which the substance stays in the intestine, by the difference in area of absorbing surface between the two organs, or by other factors which might play a more important role than partition coefficients. There is, also, a limiting factor in the interchange of substances from one side of the membrane to the other, the blood flow through the walls.
The attempt to alter absorption rates significantly by changing the pH of the gastrointestinal tract is not an easy thing to accomplish if physiological states are desired at the same time.
It is difficult to maintain such a change for a significant length of time in an otherwise normal animal. It can be done by continuous perfusion of isolated segments of the gut, but not by feeding the 12
solutions to the intact animal, in which recovery to normal pH
would occur in a relatively short time. Besides, in the stomach,
the continuous secretion of acid in the secreting tubules would
make it next to impossible to change to a desired pH at the
vicinity of the absorbing surface.
Most of the work done on this subject of the lipoid
barrier in the gut has been under conditions quite different from
those of a physiologically intact animal. Quoting Brodie on this
problem:
it must be made clear that the experiments were designated to delineate the general characteristics of the boundary between the intestinal lumen and plasma and not to yield definite criteria for the absorption of drugs as administer ed therapeutically The results do not indicate whether those substances which were absorbed relatively poorly would be also absorbed poorly in the intact animal. In these experiments, the drug solution raced through the intestines in seven minutes, compared with the several hours that a drug might remain in the lumen when used therapeutically (9*0 .
The time that a drug stays in the gastrointestinal tract
is one of the most important factors in absorption, and this in
turn depends very much on the emptying time of the stomach. Var
iations in emptying time have been found to be very great in
different animals of the same species (77)• The emptying time
depends on many factors, such as the volume of stomach contents when the substance under study is given, the motility of the
stomach, this in turn is influenced through nervous mechanisms,
and probably by the chemical nature of the substances introduced
OW, 95 . 76 , 96 , 97). 13
Circulation in the gastric and intestinal walls, as men tioned before, is of paramount importance in the process of absorp tion. Under normal circumstances, this circulation does not change appreciably from animal to animal nor is it altered by the action of the great majority of drugs; but a change that could alter the rate of absorption might be expected with the administration of those drugs which have a direct action on the vessels, such as vasoconstrictors or vasodilators or those acting through the vaso motor center in the brain (22, 23, 98i 99. 2k). The study of the effect which local changes in circulation have on absorption is made extremely difficult by the fact that a gross change in pressure as measured from the general circulation would not necessarily parallel a change in circulation in the capillaries supplying the intestinal wall, because of the complexity of the compensatory mechanisms present in the animal body.
Another factor of importance in absorption through the membranes of the gastrointestinal tract is their state of nutrition expecially in regards to oxygen. Experiments on the effect of anoxemia on absorption of water have shown that a decrease in oxygen pressure to a limit of 12 per cent increased absorption (20). In other experiments, after a short period of oxygen lack, fluid fil tered at about four times the normal rate and the permeability to proteins was increased (100). The effect on permeability has been studied for other substances such as calcium, vitamin D, potassium ions, sodium ions, etc. (25, 52, 101, 102, 103). These experiments In
tend to indicate active transport mechanisms, although they do
not necessarily prove them, since the presence or absence of these
substances might alter the osmotic balance, or exert stimulating
or depressing influence on the villi (101, 89)» or alter circula
tion, etc.
There is not much doubt that two of the forces most
surely involved in absorption are filtration and diffusion. The main law involved in the first is that the rate of filtration
depends on the difference in hydrostatic pressure on the two sides
of the membrane, while in the second, the exchange of molecules does not need any difference in pressure, but is solely due to the kinetic energy of the molecules. In the case of filtration, the
substances allowed to pass depend on the size of the pores of the membrane and the size of the molecules passing. It would require
a membrane resembling an artificial porous one, and whose pores would be small enough not to allow the passage of large protein molecules, but of a size sufficient to allow the transfer of water
and other lipid insoluble molecules. Laminar flow is assumed and the usual laws applied, that is, that the rate of flow is propor tional to the gradient of hydrostatic pressure across the membrane, inversely proportional to the viscosity of the liquid and independent of temperature except insofar as temperature alters viscosity (104).
In the case of diffusion, the solubility of the substance in ques tion in the colloids making up the membrane, is to be considered.
And not only the solubility in these colloids, but also the solubility in the liquids which will bring the substance in con
tact with the membrane, that is, the solubility, first in the fluids
of the gastric and intestinal contents, second, in the fluids of
the extra-cellular spaces. The remarkable ability of the gastro
intestinal tract to increase the solubility of a great number of
substances, indicates special mechanisms for this function (l).
Besides the cholic acids, soaps of higher fatty acids, and many other common substances possessing this property, there are others in the tissues capable of increasing the solubility of quinine, diphenylamine and many other substances. Water extracts of tissues of dogs and rabbits and especially intestinal juice show powerful solvent action. This action has been explained by some as a surface activity phenomenon, but others have pointed out that in optically active systems, there is always a change in optical activity follow ing solution, indicating the formation of real molecular compounds.
Besides, there is a marked degree of specificity - not all the sur face active substances act as solubilizing agents (1 ).
When osmosis is considered as a factor in absorption, it is next to impossible to predict or to measure accurately its effects, since at any given time there are so many different substances in solution in the interior of the cells and in the blood, that their influence would be purely a matter of speculation.
It has been suggested that electric forces play a part in absorption, and an electric current has been recorded passing from the serosal to the mucosal side of the frog's intestine (1 ). But all asymmetric membranes show such potential differences whether living or not. Nevertheless, the electrical charge of
the membrane has an influence on permeability, and at least in part, absorption from the intestine could be a process of electro dialysis (l). The presence of a difference in potential might
account for many cases of unexplained absorption against a concen tration gradient (106, 107). The concentration gradient tends to make concentration equal on both sides of the barrier, electrical potential tends to make it unequal, but both forces may work in the same direction.
Surface activity has already been mentioned and has been mostly studied in connection with fats, but the effect on the absorp tion of other substances has also been investigated (68, 7 2 , 108,
109). Results have been of a contradictory nature in many cases.
Tween 80 and Tween 20, saponins, and aerosols have been employed.
Specificity has been encountered in some cases, absorption enhanced, decreased, or not affect in other cases.
Hydration of colloids present in the membrane has been pointed out as an important factor in absorption (1). Substances might change cell permeability by changing the state of hydration of the colloids; those electrolytes which cause dehydration (shrinking) would diminish absorption, those which cause swelling would increase absorption. Some authors even claim that this is the chief mechanism of absorption, the imbibition of water and salts dissolved in it, 17
and the subsequent carrying away of the fluid by capilarity to lymphatics and blood vessels. This would describe the wall of the gut as a sort of sponge.
Subject to a great deal of controversy is the role of villi in the process of absorption. Many ascribe to the villi a pumping movement which would have a profound effect on absorption, while others deny this entirely (89* 90, 91* 101, 1). In these studies, the effect of different substances on the motility of the villi has been noted, and the response to thermal, mechanical and nervous stimulation, and irritants such as cantharides, thymol, menthol, alcohol, peppermint oil, croton oil, etc. Of these, pepper mint oil increased activity to the highest degree, while croton oil was the least active. Stimulation of the peripheral ends of the splanchnics produced a vigorous reaction with differences in the various segments of the intestine, being sharpest in the duodenal segment, but cutting the splanchnics did not abolish villi move ments. Epinephrine by intravenous injection produced the same effects as splanchnic stimulation. Local applications of solutions of glucose, weak alkali, bile, and physiological salt solutions increased activity in fasting dogs. The alternating shortening and extension has been found to be independent of persist-alsis.
Application of dilute hydrochloric acid checks the movements; 10 per cent alcohol stimulates, then depresses; atropine intravenously stops the movements. Stimulating effects of electrolytes decreased in the order: K, Na, Ca, Mg in the duodenum; K, Na, Ca, Mg in 18 jejunum and ileum; Na = Fe at all levels; PO^ = SO^ at all levels.
Some workers have found no relation between degree of activity and the rate of absorption, and that the central lacteal is not emptied by the contraction of the villus. This would mean that the move ment of the villi is not of the nature of a pumping mechanism as suggested by early workers. But mention should be made of the conditions under which these studies were made. A loop of intes tine is isolated, opened longitudinally and stretched between clamps for microscopical observation of the villi activity while in con tact with the substance under study in a solution bathing the mucosa.
This procedure was not identical or simultaneous with those record ing absorption, in which the loops were not slit open. These con siderations could be used against the validity of the conclusions reached.
The concept of an active transport of substances across the gastrointestinal barrier, involving mechanisms which require the expenditure of energy derived from metabolic processes seems to be favorably supported by the weight of experimental evidence at hand, although a number of workers deny it. The exact nature of these mechanisms, their degree of specificity toward different sub stances , and their exact role in the sum total of the forces bring ing about absorption are still questions to be answered with the exclusion of any doubt, even in the case of the absorption of sugars, which are probably the substances most intensely studied. The find ings that galactose, with a molecule so similar to glucose is absorbed one and one tenth times as fast, that fructose is absorbed half
as fast, mannose one-fifth as fast and arabinose one-tenth as
fast suggest a specific mechanism for absorption (2, h9). That
the absorption of glycine and alamine is dependent of the absolute
amount or the concentration in the intestine; that a mixture of the
two amino acids tends to give a mutual inhibition of absorption
(5 0 ); the effect of the presence of glucose on the absorption of
galactose (5 1 ), the reduction in the absorption rate of glucose
in a vitamin B complex deficient animal (52); the effect of
anoxemia on the absorption of a number of different substances
(1 5 , 20); the fact that water from solutions of glucose or sodium
chloride is absorbed even when the intra-intestinal pressure is
diminished considerably below atmospheric pressure (11); that chlo
ride moves into the blood against a concentration gradient and
when certain substances are added in very small amounts (HgS, NaF,
NaCN) the phenomenon is abolished or diminished, while other sub
stances like sulfate increase it (102); all these, if they do not
prove an active transport, at least suggest it strongly. It is
significant that cyanide, fluoride, and sulfide, interfere with
oxidation reduction systems. It has been found also that some sub
stances like 2 , 4-dinitrophenol, an oxidation catalyst, accelerate
the flow of chloride against a concentration gradient. In an exper
iment with glucose and xylose solutions (3 5 ) > glucose and water were absorbed much more readily than xylose and water under normal
conditions, but when the membrane was poisoned by monoiodoacetic 20
acid, the absorption in both cases was identical, pointing to a
destruction of a specific mechanism for the absorption of glucose.
The findings that hexokinase participates directly in sugar ab
sorption and with phosphatase functions in a phosphorylation-
dephosphorylation cycle (110); the inhibitory effect of phloridzin
on phosphorylation (111) and its effect on intestinal absorption
of glucose using very low concentrations (75 ) - all point to
specific mechanisms of transfer. Water has been found to be ab sorbed against an adverse osmotic pressure difference of 400 to 700
centimeters of water, the process depending on the simultaneous absorption of glucose (3). No absorption of water occurs unless fluid in the lumen contains glucose; while urea, creatine and sorbitol are not absorbed unless water is being absorbed at the same time. Numerous reports on the active transport of amino acids can be found in the literature (5, 6 , 55» 112). Methionine is trans ferred against a concentration gradient under aerobic conditions but no active transfer occurs anaerobically. Isolated intestine transfers the L but not the D- isomers of histidine and phenylalanine against a concentration gradient, and cyanide inhibits L - histidine transfer.
These and many other instances of transfer through the gastrointestinal mucosa can be accounted for satisfactorily only by assuming an active transport system, since they occur under con ditions contrary to the laws usually used to explain absorption. 21
In conclusion it is evident that for any given sub stance to be absorbed, many different forces are involved, in some cases one factor predominating over others, but filtration and diffusion seem to play an essential role in all cases. EXPERIMENTAL PROCEDURE
Objectives
The chief object of the present work was to find a
relation between the fate of a substance given orally to an in
tact animal under conditions as near a physiological state as
possible and the process of absorption in general. It was real
ized that this is very difficult to accomplish by using a single
drug, since from a review of the literature on absorption, it is
seen that many different forces come into play, which may vary
in their role according to the type of drug which is being absorbed.
An attempt was made to select a drug whose structure would
be modified as little as possible in the animal body, so that its
determination and that of its metabolites would present the least
number of difficulties and a reasonably complete recovery could
be accomplished in its extraction. For this purpose, phenobarbital was chosen, since its extraction and that of its metabolites can
be accomplished by using the same solvents under practically the
same conditions, and the amount recovered by the methods used is relatively high. As the analytical methods used are also applica ble to the metabolites studied so far, a barbiturate was finally
adopted (6h, 65 , 113, llh, 115 , 116 , 117, 118, 129, 130). 23
To ensure uniform doses and avoid loss in the process of administration to the animals, the soluble sodium salt was used. In this way uniformity of solution was attained without chancing the unreliability of suspensions where part of the dose might remain in the syringe when given to the animal.
Extraction and Analysis
Animals used throughout were healthy albino male rats, ranging in weight from 90 to 200 grams.
The method used for studying absorption was one introduced by Cori in 1925 (^3)• Rats were starved for h8 hours, but had free access to water during that period. The presence of solid matter in the gut was avoided as much as possible in order to get uniform absorption and in order to have the least possible amount of foreign substances, which might interfere with the analysis of the gastro intestinal contents. For this same reason, precautions were taken to avoid coprophagy, and when evidence of the animal having eaten his feces was found, the animal was discarded. The rats were weighed at the time the drug was administered. A solution of the drug was introduced into the stomach by a Frankfeldt hemorrhoidal needle
(made by Becton, Dickinson and Company, Rutherford, N.J.) with the thin end cut off, and bent to such an angle that it would slide easily along the esophagus. This was fixed to a syringe holding the solution of the drug. It is easy to introduce the drug down to the cardiac region of the esophagus without hurting the animal, and ih without going into the trachea. (Rubber catheters, sometimes used for this purpose, can go into the lungs.) The desired inter val of time for absorption was allowed to elapse and then the animal was killed by a blow on the head. Immediately after, the abdomen was opened by a midline incision, the gastrointestinal tract was ligated at the distal end of the esophagus and at the ileocecal valve, thus including the stomach and small intestine.
This was removed carefully and the mesentery disengaged manually.
Stomach and intestine were then slit open longitudinally and the contents washed out carefully with the smallest possible amount of distilled water, the washings collected and then extracted for residual drug in the manner to be described presently.
When absorption from the stomach alone was to be observed, the animal was anesthetized very lightly with ether, the abdomen was opened, and the pyloric end of the stomach was ligated, taking care to interfere as little as possible with the blood supply to the stomach wall, and avoiding the tearing of the mesenteric attach ment and the pancreas. The abdomen was then closed with wound clips and the animal left to recover from the anesthetic, which, because of the light anesthesia used, took only a few minutes. The drug was given then as described above and the desired interval waited, after which the animal was killed, the abdomen opened again by removing the clips and the stomach removed. This was opened and the contents washed out and extracted. 25
When absorption from the large intestine alone was
studied, the animal was anesthetized lightly with ether, the ab
domen was opened, the ileocecal valve was ligated and also the rectum near the anal opening. The solution of the drug was then in jected into the colon by means of a hypodermic needle. The abdomen was then closed with wound clips, the desired time interval for ab
sorption allowed, after which the animal was killed, the abdomen opened again and the large intestine removed. This was opened and the contents washed out, collected and extracted.
In Cori's method a coefficient of absorption or Cori's
coefficient, as it is cometimes called, is calculated by the follow ing formula:
q _ A x 100 W x T
Where A is the total amount of substance absorbed in a given period,
W is the weight of the rat in grams after fasting ^8 hours and T is the period in hours (4-3). This is equivalent to the amount of sub stance absorbed per 100 grams of body weight in one hour. Because of the necessity of using animals of different weights it is im portant that the absorbing surface be proportional to the body weight, i.e. the quotient:
Intestinal Surface Amount Absorbed ■ ■ or — .. - ■ ■ Body Weight Body Weight is a constant. Experiments have shown a proportionality between amount absorbed and body weight for animals in an active period of growth and without excessive fat deposits (119)• 26
Since the quantity of drug absorbed was calculated by the difference between the amount given and the residual amount found in the gastrointestinal tract, it was desirable to check the results by determining directly the amount of drug and metabolites in the tissues of the entire animal. For this purpose, after the stomach and intestine were removed, the animal was skinned, the pelt was discarded, and the rest of the animal was ground finely by passing several times through a meat chopper, mixing everything intimately. An aliquot of this mixture was extracted and the amount of drug present determined. When the time allowed for absorption was long, the animal was placed in a metabolism cage after giving the drug, and the urine and feces collected and added to the tissue mixture before extraction.
The extraction of the tissues and gastrointestinal contents was made by a modification of Goldbaum's method for barbiturates
(118). The principle involved is to extract the drug with an organic solvent, and subsequently remove it from the organic solvent with alkali. The concentration of the barbiturate in the solution of alkali is then determined by the intensity of the absorption in the ultraviolet. Barbiturates are characterized by intense ultraviolet absorption, with a maximum at 255 millimicrons and a minimum at 235 millimicrons. At a wave length of 255 millimicrons, concentration bears a linear relationship to the optical density, up to at least a concentration of 20 micrograms per ml. (Fig. 1). Significant read ings are obtained with concentrations as low as 2 micrograms per ml. 27
The samples of intestinal contents were measured for approximate volume, one half as much .05 M phosphate buffer, pH 6 was added and twice as much chloroform, measured accurately. This was mixed and shaken for at least five minutes. Whenever emulsions were formed, these were broken by centrifugation. The mixture was transferred to a separatory funnel, the chloroform layer filtered through paper and an aliquot of 10 ml. was taken. To this was added enough 1 N NaOH solution as to make the final concentration of bar biturate fall approximately inside the range of 5 to 25 micrograms per ml. After shaking for at least three minutes in a separatory funnel, the mixture was allowed to separate, the chloroform layer was removed and discarded. The NaOH solution was shaken and centri fuged to remove traces of chloroform and then examined in a Beckmann
Quarts Spectrophotometer, Model D.U., using silica absorption cells, at a wave length of 255 millimicrons, using 1 N NaOH solution as a blank. A standard curve was prepared using different dilutions of phenobarbital sodium in IN NaOH (Fig. 1).
For tissues, a weighed aliquot amounting to about 5 grams was taken with 10 ml. of the phosphate buffer, 30 ml. of chloroform was added and the mixture thoroughly mixed and shaken, and then the same procedure was used as for the intestinal contents. For the determination of the amount of drug retained by the gastric and intestinal walls during absorption,the stomach and intestine separate ly, after emptying, were washed, minced and crushed finely, 5 ml. 28
of phosphate buffer and 20 ml. of chloroform were addeded and
then the procedure described for tissues was followed.
Using the same methods of extraction and analysis, but adding the drug directly to intestinal contents and tissues after the animal was killed, the drug was recovered in amounts up to 96 per cent.
A correction was necessary for each reading on the basis of readings given by solutions in NaOH of the contents of intestine and of tissues made by an identical procedure of extraction, but using no drug. This correction amounted to 1.7 micrograms per ml. expressed as phenobarbital. The same procedure used by Goldbaum gave the following results (118): blood - 1.3 micrograms per ml; liver - 1.9 micrograms per ml; kidney - 1.7 micrograms per ml.; brain - 1.7 micrograms per ml.; muscle - 1.7 micrograms per ml.
The figures obtained by the difference between drug given and drug found unabsorbed were used in the drawing of graphs and the discussion of each experiment, rather than the figures obtained by direct measurement of the quantities found in tissues, because more and greater variations between animals were found when the last were used. Figure 1
Relation Between Optical Oenaity at 255 millimicrons and Phenobarbital Concentration
900
&oo
Q o
&oo
%oo
loo
t o
Micrograms per ml. 30
Experiment I. Absorption Rates of Phenobarbital, £ Mg. Dose
The drug was given in 5 mg. doses, (5 c.c. of a solution
containing 1 mg. per c.c.) and absorption periods of 5» 10, 15, 20,
25 , 30, 45, 60 and 75 minutes allowed. Ten animals were used for
each period. No method of clarification was used for the final solu
tions, rather, those which came out colored were discarded. The re sults, (Tables 1 to 10 and Figure 2) show that when percentage ab sorption is plotted against time, a straight line is obtained for the first fifteen minutes, until 70 per cent absorption has occurred.
After that per cent absorption increases at a lower rate. The same thing is observed if Cori's coefficients are plotted against time.
Cori's coefficients were found to be as follows:
5 minutes - .021
10 " - .022
15 " - .017
20 " - .019
25 " - .010
30 » - .008
45 " - .005
60 " - .004
75 " - .002
Based on these results, the conclusion could be reached that the mucosa of the gastrointestinal tract becomes saturated with the drug after a time, an equilibrium being established between the drug coming 31 in from the lumen and that given off to the general circulation.
The results obtained in the determination of the quantity of drug retained by the gastrointestinal wall seem to bear out this assump tion. From this, it could be said that for this type of drug the rate of absorption is dependent up to a certain limit, on the con centration in the lumen and that in the blood, the limitation being governed by the capacity of the mucosa to retain the drug. Another interpretation would explain this saturation by assuming an active transport in which the mechanism involved is able to handle a more or less constant quantity of drug as a maximum at any given time. 32
Figure 2
Absorption from the Gastrointestinal Tract of the Rat Using a 5 mg. Dose of Phenobarbital
\ ■ TTT j | ' ' : ! 1 i : i • - . _ . :■ '; . • : * • T : "! ' • ' ' 1 ' ' ! " : ! ' ; j • , ! ; . : : ' l
70
to
tS 7 S Time (Minutes) TABLE 1
PER CENT PHENOBARBITAL ABSORBED IN 5 MINUTES FROM A 5 MILLIGRAM DOSE
Wt. of Micrograms Micrograms % Absorbed 7o Absorbed Rat in Recovered From Per Cent Recovered (Direct (By Grams G.I. Tract Unabsorbed From Tissues Measurement) Difference)
180 1600 32.0 3145 62.9 68.0
170 1500 30.0 3035 60.7 70.0
175 2050 41.0 2645 52.9 59.0
165 1955 39.1 2910 58.2 60.9
170 1890 37.8 2865 57.3 62.2
180 2000 40.0 2795 55.9 60.0
185 1900 38.0 2875 57.5 62.0
190 1380 27.6 3395 67.9 72.4
165 2350 47.0 2955 59.1 53.0
170 1470 29.4 3460 69.2 70.6
AVERAGES 36.2 60.2 63.8 TABLE 2
PER GENT PHENOBARBITAL ABSORBED IN 10 MINUTES FROM A 5 MILLIGRAM DOSE
Wt. of Micrograms Micrograms 7. Absorbed Absorbed Rat in Recovered From Per Cent Recovered (Direct (By Grams G.I. Tract Unabsorbed From Tissues Measurement) Difference)
80 990 19.8 3740 74.8 80.2
90 1140 22.8 3565 71.3 77.2
110 1050 21.0 3610 72.2 79.0
115 920 18.4 3920 78.4 81.6
90 710 14.2 3740 74.8 85.3
120 1190 23.8 3525 70.0 76.2
105 1000 20.0 3340 66.8 80.0
105 930 18.6 3665 73.3 81.4
125 870 17.4 4105 82.1 82.6
110 1110 22.2 3655 73.1 77.8
AVERAGES 19.8 71.7 80.2 TABLE 3
PER CENT PHENOBARBITAL ABSORBED IN 15 MINUTES
FROM A 5 MILLIGRAM DOSE
Wt. of Micrograms Micrograms % Absorbed % Absorbed Rat in Recovered From Per Cent Recovered (Direct (By Grains G.I. Tract Unabsorbed From Tissues Measurement) Difference)
105 550 11.0 4100 82.0 89.0
110 510 10.2 4130 82.6 89.8
125 470 9.4 5185 103.7 90.6
110 615 12.3 4210 84.2 87.7
120 520 10.4 4310 86.2 89.6
120 430 8.6 4265 85.3 91.4
115 580 11.6 4080 81.6 88.4
105 470 9.4 4170 83.4 90.6
110 415 8.3 4230 84.6 91.7
105 510 10.2 4220 84.4 89.8
AVERAGES 10.1 85.8 89.8 TABLE 4
PER CENT PHENOBARBITAL ABSORBED IN 20 MINUTES FROM A 5 MILLIGRAM DOSE
Wt. of Micorgrams Micrograms % Absorbed % Absorbed Rats in Recovered From Per Cent Recovered (Direct (By Grams G.I. Tract Unabsorbed From Tissues Measurement) Difference)
150 505 10.1 4300 86.0 89.9
140 470 9.4 4390 87.8 90.6
120 435 8.7 4310 86.2 91.3
130 410 8.2 4360 87.2 91.8
75 320 6.4 4340 86.8 93.7
85 345 6.9 4300 86.0 93.1
120 370 7.4 4355 87.1 92.6
140 405 8.1 4360 87.2 91.9
135 340 6.8 4510 90.2 93.2
120 360 7.2 4430 85.6 92.8
AVERAGE 7.9 87.0 92.1 TABLE 5
PER CENT PHENOBARBITAL ABSORBED IN 25 MINUTES
FROM A 5 MILLIGRAM DOSE
Wt. of Micrograms Micrograms % Absorbed 7, Absorbed Rat in Recovered From Per Cent Recovered (Firect (By Grams G.I. Tract Unabsorbed From Tissues Measurement) Difference)
100 405 8.1 4310 86.2 91.9
90 280 5.6 4595 91.9 94.4
110 405 8.1 4480 89.6 91.9
110 300 6.0 4215 84.3 94.0
95 280 5.6 4590 91.8 94.4
110 260 5.2 4490 89.8 94.8
115 315 6.3 4425 88.5 93.7
100 355 7.9 4365 87.3 92.1
115 235 4.7 4495 89.9 95.3
110 375 7.5 4420 88.4 92.5
AVERAGE 6.5 88.8 93.5 TABLE 6
PER GENT PHENOBARBITAL ABSORBED IN 30 MINUTES
FROM A 5 MILLIGRAM DOSE
Wt. of Micrograms Micrograms 7o Absorbed % Absorbed Rat in Recovered From Per Cent Recovered (Direct (By Grains G.I. Tract Unabsorbed From Tissues Measurement) Difference)
110 380 7.6 4500 90.0 92.4
110 310 6.2 4555 91.1 93.8
105 315 6.3 4490 89.8 93.7
100 290 5.8 4365 87.3 94.2
120 275 5.7 4505 90.1 94.3
110 295 5.9 4455 89.1 94.1
115 350 7.0 4375 87.5 93.0
120 370 7.4 4370 87.4 92.6
125 280 5.6 4450 89.0 94.4
110 375 7.5 4310 86.2 92.5
AVERAGE 6.5 88.8 93.5 TABLE 7
PER CENT PHENOBARBITAL ABSORBED IN 45 MINUTES FROM A 5 MILLIGRAM DOSE
Wt. of Micrograms Micrograms Absorbed % Absorbed Rat in Recovered From Per Cent Recovered (Direct (By Grains G.I. Tract Unabsorbed From Tissues Measurement) Difference)
130 270 5.4 4530 90.6 94.6
105 305 6.1 4570 91.4 93.9
115 260 5.2 4575 91.5 94.8
120 240 4.8 4430 88.6 95.2
120 185 3.7 3970 79.4 96.3
105 260 5.2 4425 88.5 94.8
110 280 5.6 4635 92.7 94.4
105 355 7.1 4450 89.0 92.9
100 235 4.7 4530 90.6 95.3
125 245 4.9 4475 89.5 95.1
AVERAGE 5.3 89.8 94.7 TABLE 8
PER CENT PHENOBARBITAL ABSORBED IN 60 MINUTES FROM A 5 MILLIGRAM DOSE
Wt. of Micrograms Micrograms V* Absorbed % Absorbed Rat in Recovered From Per Cent Recovered (Direct (By Grams G.I. Tract Unabsorbed From Tissues Measurement) Difference)
95 225 4.5 4480 89.6 95.5
120 235 4.7 4490 89.8 95.3
100 250 5.0 4450 89.0 95.0
135 220 4.4 4310 86.2 95.6
120 195 3.9 4405 88.1 96.1
105 145 2.9 4580 91.6 97.1
105 200 4.0 4505 90.1 96.0
120 260 5.2 4435 88.7 94.8
125 205 4.1 4405 88.1 95.9
100 230 4.6 4535 90.7 95.4
AVERAGE 4.3 89.2 95.7 TABLE 9
PER CENT PHENOBARBITAL ABSORBED IN 75 MINUTES FROM A 5 MILLIGRAM DOSE
Wt. of Micrograms Micrograms % Absorbed % Absorbed Rat in Recovered From Per Cent Recovered (Direct (By Grams G.I. Tract Unabsorbed From Tissues Measurement) Difference)
150 80 1.6 4655 93.1 98.4
135 105 2.1 4575 91.5 97.9
120 95 1.9 4675 93.5 98.1
115 95 1.9 4655 93.1 98.1
145 165 3.3 4710 94.2 96.7
120 120 2.4 4575 91.5 97.6
125 155 3.1 4535 90.7 96.9
130 85 1.7 4755 95.1 98.3
145 65 1.3 4735 94.7 98.7
140 135 2.7 4830 96.6 97.3
AVERAGES 2.2 93.4 97.8 42
TABLE 10
PER CENT PHENOBARBITAL ABSORBED FROM A FIVE MILLIGRAM DOSE AFTER DIFFERENT INTERVALS
Average Increase Time Per Cent in Per Cent Value of Level of (Min.) Absorbed Absorbed ,rt” Significance
5 63.8
10 80.2 16.4 7.69 99.9
15 89.8 9.6 9.89 99.9
20 92.1 2.3 4.18 99.0
25 93.5 1.4 2.54 95.0
30 93.5 0 - -
45 94.7 1.2 4.61 99.0
60 95.7 1.0 12.50 99.9
75 97.8 2.1 30.00 99.9 Experiment II, Absorption Rates with 25 Mg. Doses
The same procedure was followed as in the preceding ex periment, but using now, in each case, 5 c.c. of a solution contain ing 5 mg. per c.c. The results (Tables 11 to 17 and Figure 3*) show that absorption followed the same pattern, but in terms of percentage the saturation point was reached at a lower level. Tak ing the twenty minutes period as an example, only 81.7 per cent was absorbed from a 25 mg. dose while 92.1 per cent was absorbed from the 5 mg. dose. Of course a larger total amount of drug was ab sorbed, but the results show that concentration in the lumen has some effect on absorption rates.
Cori's coefficients were:
5 minutes - .100
10 " - .060
20 " - .033
30 " - .02h
h5 " - .012
60 " - .013
As in the preceding experiment it is seen that plotting either per cent absorption or Cori's coefficients against time, does not give a straight line. If absorption were independent of concentration in the lumen, then Cori's coefficient would be the same at all times for the same quantity of the same drug given. The results of other workers on the effect of concentration on absorption vary with different drugs, in some cases concentration altering rate of absorption, in others absorption being independent of concen tration (10, 46, 47). This suggests that the factors involved in absorption do not play a uniform role when different drugs are involved. Per Cent Absorbed too f bopin rm h Gastrointestinal the from Absorption rc o te a Uiga2 m. dose mg. 25 a Using Rat the of Tract ie (Minutes) Time Figure3
TABLE 11
PER CENT PHENOBARBITAL ABSORBED IN 5 MINUTES FROM A 25 MILLIGRAM DOSE
Wt. o Micrograms Micrograms % Absorbed Rat ii Recovered From Per Cent Recovered (Direct Grams G.I. Tract Unabsorbed From Tissues Measurement)
210 10020 40.0 14660 58.6
140 9540 38.1 15370 61.4
180 10200 40.8 14560 58.2
180 8880 35.5 14790 59.1
180 8100 32.4 14080 56.3
170 8700 34.8 15450 61.8
200 9720 38.8 14670 58.6
175 11010 44.0 14160 56.6
190 8580 34.5 15920 63.6
165 10140 40.5 14050 56.2
37.9 59.0 TABLE 12
PER CENT PHENOBARBITAL ABSORBED IN 10 MINUTES FROM A 25 MILLIGRAM DOSE
Wt. of Micrograms Micrograms % Absorbed % Absorbed Rat in Recovered From Per Cent Recovered (Direct (By Grams G.I. Tract Unabsorbed From Tissues Measurement) Difference)
210 6640 26.5 16120 64.4 73.5
220 5100 20.4 17855 71.4 79.6
180 7080 28.3 16305 65.2 71.7
140 7440 29.7 16070 64.2 71.3
150 6670 26.6 15940 63.7 73.4
190 7120 28.4 15465 61.8 71.6
170 6660 26.6 18065 72.2 73.4
180 5910 23.6 18010 72.0 76.4
170 7010 28.0 15335 61.3 72.0
160 7280 29.0 17300 69.2 71.0
AVERAGES 26.6 66.5 73.4 TABLE 13
PER CENT PHENOBARBITAL ABSORBED IN 20 MINUTES FROM A 25 MILLIGRAM DOSE
Wt. of Micrograms Micrograms 7» Absorbed % Absorbed Rat in Recovered From Per Cent Recovered (Direct (By Grams G.I. Tract Unabosrbed From Tissues Measurement) Difference)
200 3240 12.9 19420 77.6 87.1
140 5100 20.8 19835 79.3 79.2
160 5600 22.8 21275 85.1 77.2
210 3840 15.3 18070 72.2 74.7
220 6200 24.8 18560 74.2 75.2
180 3255 130 19820 79.2 87.0
170 4550 18.2 18815 75.2 81.8
220 4620 18.4 21070 84.2 81.6
150 5085 20.3 19120 76.4 79.7
180 4105 16.4 20220 80.8 83.6
AVERAGES 18.3 78.4 81.7 TABUS 14
PER CENT PHENOBARBITAL ABSORBED IN 30 MINUTES FROM A 25 MILLIGRAM DOSE
Wt. o Micrograms Micrograms °L Absorbed Rat i Recovered From Per Cent Recovered (Direct Grams G.I. Tract Unabsorbed From Tissues Measurement)
100 3970 15.8 22560 90.2
190 1280 5.1 19900 79.6
150 2640 10.5 19825 79.3
210 5700 22.8 21375 85.5
180 6110 24.4 21830 87.3
190 1840 7.3 21740 86.9
180 2010 8.0 21285 85.1
200 2080 8.3 21135 84.5
170 3710 14.8 20870 83.4
150 2740 10.9 22005 88.0
12.8 85.0 TABLE 15
PER CENT PHENOBARBITAL ABSORBED IN 45 MINUTES FROM A 25 MILLIGRAM DOSE
Wt. of Micrograms Micrograms % Absorbed V. Absorbed Rat in Recovered From Per Cent Recovered (Direct (By Grams G.I. Tract Unabsorbed From Tissues Measurement) Difference)
160 1920 7.6 21630 86.5 92.4
190 2280 9.1 21456 85.8 90.9
150 2310 9.2 21190 84.7 90.8
210 1840 7.3 21760 87.0 92.7
180 1970 7.8 19070 76.3 92.2
190 1920 7.6 20840 83.3 92.4
180 1940 7.5 21965 87.8 92.5
200 2130 8.5 20490 81.9 91.5
210 2040 8.1 21130 84.5 91.9
160 1990 7.9 / 21270 85.1 92.1
AVERAGES 8.1 84.3 91.9 TjABLE 16
i PER CENT PHENOBARBITAL ABSORBED IN 60 MINUTES FROM A 25 MILLIGRAM DOSE
Wt. of Micrograms Micrograms % Absorbed 7<, Absorbed Rat in Recovered From Per Cent Recovered (Direct (By Grams G.I. Tract Unabsorbed From Tissues Measurement) Difference)
140 1140 4.5 21840 87.3 95.5
160 1210 4.8 20935 83.7 95.2
200 960 3.8 22830 91.3 96.2
180 1220 4.8 23855 95.4 95.2
150 1280 5.! 19310 77.2 94.9
180 860 3.4 21675 86.7 96.6
190 1120 4.4 , 22290 89.1 95.6
210 1040 4.0 20930 83.7 96.0
180 1255 5.0 20120 80.5 95.0
190 890 3.6 21915 87.7 96.4
AVERAGES 4.3 86.3 95.7 52
TABLE 17
PER CENT PHENOBARBITAL ABSORBED FROM A TWENTY-FIVE MILLIGRAM DOSE AFTER DIFFERENT INTERVALS
Average Increase Time Per Cent in Per Cent Value of Level of (Min.) Absorbed Absorbed iltl< Significance
5 62.1
10 73.4 11.3 8.01 99.9
20 81.7 8.3 5.03 99.9
30 87.2 5.5 2.19 -
45 91.9 4.7 2.27 95.0
60 95.7 3.8 13.50 99.9 Experiment III. Drug Retained by Intestinal Wall
The results, shewn in Tables 18 to 27 and Figure if-, indicate a rapid uptake to 17*7 per cent of the drug given (5 mg.) in the first five minutes, to a relatively constant level of from
6 to 8 per cent in the next sixty minutes, after which it begins to fall off rapidly. This could be taken to mean that the mucosa takes up drug rapidly at the start, when there is a high concen tration in the lumen and none in the blood. When the concentration in the blood begins to build up,, the amount in the mucosa begins to stabilize itself and establish an equilibrium between the con centration in the lumen and that of the blood. When the concentra tion in the lumen falls to a level which is probably below that of the blood, the mucosa begins to lose drug rapidly.
Or, if we look upon the mucosa acting as a sponge (1) and we assume that the absorption of fluid is exactly parallel to the absorption of solute, then at the start the dry "sponge" will take a great volume of liquid, but eventually will become saturated and the rate of uptake will be decreased. The fact that the walls of the gastrointestinal tract retain quantities of drug during ab sorption which are out of proportion to their weight when compared to the quantities in other tissues, has been observed by a number of investigators (5^» 56, 57, 58* 59, 80). Whether this phenonenon is due only to the solubility of the drug in the lipids of the cell barrier, whether it is a storage mechanism, as some have suggested, or whether the formation of complexes with elements in the cell is the explanation remains to be determined. Per Cent o i Drug Retained by Intestinal Mucosa Intestinal by Retained Drug ie (Minutes)Time Figure 4 Figure 7 S 55
TABLE 18
PER CENT OF A 5 MILLIGRAM DOSE OF PHENOBARBITAL RETAINED BY INTESTINAL WALL AFTER 5 MINUTES
Wt. of Rat Micrograms in Grams Retained % Retained
180 905 18.1
170 1000 20.0
175 980 19.6
165 870 17.4
170 430 8.6
180 785 15.7
185 970 19.4
190 915 18.3
165 1045 20.9
170 925 18.5
AVERAGE 17.7 56
TABLE 19
PER CENT OF A 5 MILLIGRAM DOSE OF PHENOBARBITAL RETAINED BY INTESTINAL WALL AFTER 10 MINUTES
Wt. of Rat Micrograms in Grains Retained 7a Retained
80 320 6.4
90 510 10.2
110 410 8.2
115 470 9;4
90 390 7.8
120 325 6.5
105 340 6.8
105 360 7.2
125 470 9.4
110 445 8.9
AVERAGE 8.1 57
TABLE 20
PER CENT OF A 5 MILLIGRAM DOSE OF PHENOBARBITAL RETAINED BY INTESTINAL WALL AFTER 15 MINUTES
Wt. of Rat Micrograms in Grams Retained % Retained
105 400 8.0
110 480 8.4
125 325 6.5
110 500 10.0
120 405 8.1
120 520 10.4
115 510 10.2
105 380 7.6
110 295 5.9
105 435 8.7
AVERAGE 8.4 58
TABLE 21
PER CENT OF A 5 MILLIGRAM DOSE OF PHENOBARBITAL RETAINED BY INTESTINAL WALL AFTER 20 MINUTES
Wt. of Rat Micrograms in Grams Retained % Retained
150 400 8.0
140 450 9.0
120 460 9.2
132 350 7.0
75 1050 21.0*
65 600 12.0
120 450 9.0
140 400 8.0
135 295 5.9
120 420 8.4
AVERAGE 7.7
* Not included in average. 59
TABLE 22
PER CENT OF A 5 MILLIGRAM DOSE OF PHENOBARBITAL RETAINED BY INTESTINAL WALL AFTER 25 MINUTES
Wt. of Rat Micrograms in Grains Retained % Retained
100 450 9.0
90 435 8.7
110 415 8.3
110 390 7 .8
95 455 9.1
110 335 6.7
115 395 7.9
100 405 8.1
115 395 7.9
110 415 8.3
AVERAGE 8.2 60
TABLE 23
PER CENT OF A 5 MILLIGRAM DOSE OF PHENOBARBITAL RETAINED BY INTESTINAL WALL AFTER 30 MINUTES
Wt. of Rat Micrograms in Grams Retained % Retained
110 505 . 10.1
110 360 7.2
105 330 6.6
100 225 4.5
120 455 9.1
110 470 9.4
115 410 8.2
120 365 7.3
125 345 6.9
110 360 7.2
AVERAGE 7.6 61
TABLE 24
PER CENT OF A 5 MILLIGRAM DOSE OF PHENOBARBITAL RETAINED BY INTESTINAL WALL AFTER 45 MINUTES
Wt. of Rat Micrograms in Grams Retained % Retained
130 330 6.6
105 355 7.1
115 350 7.0
120 260 5.2
120 370 7.4
105 350 7.0
110 325 6.5
105 320 6.4
100 260 5.3
125 290 5.8
AVERAGE 6.4 62
TABLE 25
PER CENT OF A 5 MILLIGRAM DOSE OF PHENOBARBITAL RETAINED BY INTESTINAL WALL AFTER 60 MINUTES
Wt. of Rat Micrograms in Grams Retained 7 Retained
95 390 7.8
120 350 7.0
100 225 4.5
135 245 4.9
120 340 6.8
105 355 7.1
105 270 5.4
120 305 6.1
125 355 7.1
100 285 5.7
AVERAGE 6.2 63
TABUE 26
PER CENT OF A 5 MILLIGRAM DOSE OF PHENOBARBITAL RETAINED BY INTESTINAL WALL AFTER 75 MINUTES
Wt. of Rat Micrograms in Grams Retained 7„ Retained
150 265 5.3
135 250 5.0
120 210 4.2
115 255 5.1
145 190 3.8
120 295 5.9
125 220 4.4
130 195 3.9
145 250 5.0
140 240 4.8
AVERAGE 4.7 64
TABLE 27
PER CENT PHENOBARBITAL RETAINED BY INTESTINAL WALL FROM A FIVE MILLIGRAM DOSE AT DIFFERENT INTERVALS
Average Decrease Time Per Cent in Per Cent Value of Level of (Min.) Retained Retained "t" Significance
5 17.7
10 8.1 9.6 8.20 99.9
15 8.4 .3 .48 --
20 7.7 .7 .89 --
25 8.2 .5 .74
30 7.6 .6 1.07 --
45 6.4 1.2 2.14 --
60 6.2 .2 .49 --
75 4.8 1.5 3.84 99.9 Experiment IV. Drug Retained, by Stomach Wall
The results obtained (Tables 28 to 37 and Figure 5*) were similar to those obtained with the intestinal wall, with the difference that lower percentages of drug were retained, explain able by the smaller absorbing surface in the case of the stomach.
And the faster depletion of drug from the gastric wall is explained by the relatively short emptying time of the stomach as compared to the time it stays in the intestine. At the end of an hour probably most of the drug remaining unabsorbed has passed into the intestine. Per Cent 4 £ 8 Drug Retained by Stomach Mucosa Stomach by Retained Drug ie (Minutes) Time iue 5 Figure
7S 67
TABLE 28
PER CENT OF A 5 MILLIGRAM DOSE OF PHENOBARBITAL RETAINED BY STOMACH WALL AFTER 5 MINUTES
Wt. of Rat Micrograms in Grams Retained % Retained
180 420 8.4
170 440 8.8
175 455 9.1
165 490 9.8
170 410 8.2
180 395 7.9
185 505 10.1
190 485 9.7
165 520 10.4
170 465 9.3
AVERAGE 9.2 TABLE 29
PER CENT OF A 5 MILLIGRAM DOSE OF PHENOBARBITAL RETAINED BY STOMACH WALL AFTER 10 MINUTES
Wt. of Rat Micrograms in Grains Retained 7. Retained
80 220 4.4
90 405 8.1
110 150 3.0
115 250 5.0
90 200 4.0
120 300 6.0
105 300 6.0
105 255 5.1
125 235 4.7
110 260 5.2
AVERAGE 5.2 TABLE 30
PER CENT OF A 5 MILLIGRAM DOSE OF PHENOBARBITAL RETAINED BY STOMACH WALL AFTER 15 MINUTES
Wt. of Rat Micrograms in Grams Retained 7„ Retained
105 150 _ 3.0
110 200 4.0
125 160 3.2
110 260 5.2
120 205 4.1
120 245 4.9
115 220 4.4
105 190 3.8
110 260 5.2
105 235 4.7
AVERAGE 4.3 70
TABLE 31
PER CENT OF A 5 MILLIGRAM DOSE OF PHENOBARBITAL RETAINED BY STOMACH WALL AFTER 20 MINUTES
Wt. of Rat Micrograms in Grams Retained % Retained
150 340 6.8
140 240 4.8
120 300 6.0
132 210 4.2
75 240 4.8
85 350 7.0
120 305 6.1
140 260 5.2
135 210 0 4 '2 120 295 5.9
AVERAGE 5.5 71
TABLE 32
PER CENT OF A 5 MILLIGRAM DOSE OF PHENOBARBITAL RETAINED BY STOMACH WALL AFTER 25 MINUTES
Wt. of Rat Micrograms. in Grams Retained % Retained
100 255 5.1
90 240 4.8
110 265 5.3
110 280 5.6
95 255 5.1
110 245 4.9
115 300 6.0
100 195 3.9
115 240 4.8
110 265 5.3
AVERAGE 5.1 7J2
TABLE 33
PER CENT OF A 5 MILLIGRAM DOSE OF PHENOBARBITAL RETAINED BY STOMACH WALL AFTER 30 MINUTES
Wt. of Rat Micrograms in Grams Retained 7« Retained
110 335 6.7
110 190 3.8
105 200 4.0
100 40 0,8 *
120 305 6.1
110 185 3.7
115 245 4.9
120 180 3.6
125 255 5.1
110 220 4.4
AVERAGE 4.7
* Not included in average. TABLE 34
PER CENT OF A 5 MILLIGRAM DOSE OF PHENOBARBITAL RETAINED BY STOMACH WALL AFTER 45 MINUTES
Wt. of Rat Micrograms in Grams Retained % Retained
130 185 3.7
105 265 5.3
115 195 3.9
120 205 4.1
120 200 4.0
105 255 5.1
110 260 5.2
105 185 3.7
100 210 4.2
125 180 3.6
AVERAGE 4.3 7*+
, TABLE 35
PER CENT OF A 5 MILLIGRAM DOSE OF PHENOBARBITAL RETAINED BY STOMACH WALL AFTER 60 MINUTES
Wt. of Rat Micrograms in Grams Retained % Retained
95 55 1.1
120 40 0.8
100 55 1.1
135 100 2.0
120 80 1.6
105 70 1.4
105 35 0.7
120 60 1.2
125 45 0.9
100 70 1.4
AVERAGE 1.0 75
TABLE 36
PER CENT OF A 5 MILLIGRAM DOSE OF PHENOBARBITAL RETAINED BY STOMACH WALL AFTER 75 MINUTES
Wt. of Rat Micrograms in Grams Retained 7. Retained
150 40 0.8
135 30 0.6
120 60 1.2
115 40 0.8
145 20 0.4
120 45 0.9
125 35 0.7
130 55 1.1
145 10 0.2
140 35 0.7
AVERAGE 0.7 76
TABLE 37
PER CENT PHENOBARBITAL RETAINED BY STOMACH WALL FROM A FIVE MILLIGRAM DOSE AT DIFFERENT INTERVALS
Average Decrease Time Per Cent in Per Cent Value of Level of (Min.) Retained Retained 11 tU Significance
5 9.2
10 5.2 4.0 8.00 99.9
15 4.3 .9 1.82 --
20 5.5 1.2 3.07 95.0
25 5.1 .4 1.14 --
30 4.7 .4 1.11 --
45 4.3 .4 1.09 __
60 1.0 3.3 13.75 99.9
75 0.7 .3 1.87 — — Experiment V, Effect of Total Volume of Fluid
A 10 mg. dose was given to each animal, but varying the
total amount of solvent, (water). In one group, the total fluid
given was 5 c.c., while in the other it was 15 c.c. The time
allowed for absorption was twenty minutes in both cases. The re
sults (Tables 38 and 39) confirm the assumption that an increase
in hydrostatic pressvtre would increase absorption. This is assum
ing that the solvent and solute are absorbed simultaneously in exact parallelism. The per cent of drug absorbed increased from 82.8 per
cent to 89 per cent. Although the increase is not great, neither
is the increase in volume. A greater volume was not tried, because the relatively small size of the stomach of the rat would not permit a greater volume of fluid to be introduced at a time. Besides, un due distention of the walls of the stomach would introduce the factor of changes in permeability of the membranes. Whether varia tions in pressure in the lumen are factors in the absorption of a given drug depends, it would seem, on the parallelism between absorp tion of solvent and that of solute. It has been found in studies on water absorption that it is absorbed at rates proportional to the concentration of certain solutes (3), that certain substances fail to be absorbed in the absence of a concurrent absorption of water (3), but at the same time sane evidence has been obtained that the absorp tion of water is an active process which can take place against an adverse pressure. The presence of glucose in the lumen has also been shown to have a decided effect on water absorption. In the series of experiments following, a wide varia tion in the volume of fluid remaining unabsorbed has been ob served, while the variations in absorption of phenobarbital have been not too great. This tends to show no definite parallelism between absorption of water and that of solutes. TABLE 38
EFFECT OF TOTAL VOLUME OF FLUID ON ABSORPTION
10 MG. DOSE IN 5 cc. OF WATER 20 MINUTES
Wt. of Micrograms Micrograms 7. 7. Rat in Recovered From Per Cent Recovered Absorbed Absorbed Grains G .I. Trac t Unabsorbed From Tissues (Direct) (By Difference)
120 1980 19.8 7610 76.1 80.2
110 2280 22.8 7160 71.6 77.2
110 1320 13.2 8000 80.0 86.8
130 1140 11.4 8090 80.9 88.6
100 1090 10.9 8420 84.2 89.1
110 1560 15.6 8070 80.7 84.4
90 2040 20.4 7100 71.0 79.6
100 2080 20.8 7340 73.4 79.2
110 1930 19.3 7160 71.6 80.7
110 1770 17.7 7180 71.8 82.3
AVERAGES 17.2 76.1 82.8 TABLE 39
EFFECT OF TOTAL VOLUME OF FLUID ON ABSORPTION
10 MG. DOSE IN 15 cc. OF WATER 20 MINUTES
Wt. of Micrograms Micrograms % % Rat in Recovered From Per Cent Recovered Absorbed Absorbed Grams G.I. Tract Unabsorbed From Tissues (Direct) (By Difference)
120 960 9.6 8310 83.1 90.4
130 1150 11.5 7940 79.4 88.5
110 960 9.6 8140 81.4 90.4
140 1080 10.8 8120 81.2 89.2
130 1120 11.2 8090 80.9 88.8
130 970 9.7 8170 81.7 90.3
120 1290 12.9 7860 78.6 87.1
100 1040 10.4 8640 86.4 90.6
140 1210 12.1 8020 80.2 87.9
110 1250 12.5 8050 80.5 87.5
AVERAGES 11 81.3 89 Experiment VI. Use of Alcohol as Solvent
If the hypothesis of a lipoid membrane in the gastro intestinal tract is accepted, then changing the composition of the lumen’s content in such a way that the partition coefficient will be altered will have a definite effect on the rate of absorption.
For this purpose, alcohol was used as the solvent, since if pheno- barbital was to be absorbed in the undissociated form, the partition coefficient between the two phases, lipoid and lumen content, would be lowered if compared with that between a lipoid phase and an aqueous phase in the lumen. Unfortunately the concentration of alcohol that could be used without producing an anesthetic effect on a rat or altering the permeability of the membrane by its local action is relatively low. Even with the amount used, 5 c.c. of 15 per cent alcohol as solvent for 10 mgs. of phenobarbital, some anesthetic action was observed in some animals. The results are shown in Table ^-0. It is seen that the average absorption for a twenty minute period was decreased from 82.8 to 78. The difference is too small to permit any conclusions, taking into consideration that animal variation might have been entirely responsible for the difference observed. TABLE 40
EFFECT OF ALCOHOL ON ABSORPTION OF PHENOBARBITAL
5 cc OF 15% ALCOHOL AS SOLVENT 10 MG DOSE 20 MINUTES
Wt. of Micrograms Micrograms % % Rat in Recovered From Per Cent Recovered Absorbed Absorbed Grams G.I. Tract Unabsorbed From Tissues (Direct) (By Difference)
140 2160 21.6 7210 72.1 78.4
160 1980 19.8 7290 72.9 80.2
130 1200 12.0 7460 74.6 88.0
180 2280 22.8 7140 71.4 77.2
200 1800 18.0 8000 80.0 82.0
190 2400 24.0 7120 71.2 76.0
170 1920 19.2 7370 73.7 80.8
160 2820 28.2 6930 69.3 71.8
140 2700 27.0 6880 68.8 73.0
130 2700 27.0 6930 69.3 73.0 AVERAGES 21.9 72.3 78.1
00 ro Experiment VII. Effect of Olive Oil
Having the same objective in mind as in the preceding
experiment, olive oil was used instead of alcohol. Three c.c. of
olive oil were given immediately after 2 c.c. of a solution of phenobarbital sodium containing 5 mgs. per c.c. The results, after twenty minutes absorption time, are shown on Table hi. They show
an average absorption of 68.5 per cent as compared to 82.8 per cent when water alone was used. The difference of lh.3 per cent is quite great when the small amount of oil used is considered. It is felt that this decrease in absorption cannot be entirely due to a reduc tion in the emptying time of the stomach brought about by the oil, since when the intestine was opened after twenty minutes, a relative ly large amount of the oil was found even in the lower reaches of the ileum. And unless the absorption of glycerides is accomplished by a preferential mechanism by which the intestinal mucosa might partially exclude other substances while glyceride absorption is taking place, no other explanation but an alteration of the partition
coefficient seems feasible. If the pharmacologic action of sub
stances like thiopental are affected by its preferential deposition
in fatty tissue, by the same reasoning, an increased solubility in
an oil-rich lumenal content would decrease its rate of transfer to the circulation across a lipoid barrier (60, 63, 120, 121). TABLE 41
EFFECT OF OLIVE OIL ON ABSORPTION OF PHENOBARBITAL
3 cc. OLIVE OIL AND 2 cc. PHENOB. SOD. SOL. (10 MGS.) 20 MINUTES
Wt. Micrograms Micrograms 7. % of Recovered From Per Cent Recovered Absorbed Absorbed Rat G.I. Tract Unabsorbed From Tissues (Direct) (By Difference)
140 3825 38.3 5620 56.2 61.7
200 4860 48.6 4830 48.3 51.4
130 2880 28.8 6490 64.9 71.2
110 1980 19.8 7460 74.6 80.2
170 2880 28.8 6750 67.5 71.2
170 2835 28.4 6510 65.1 71.6
150 2880 28.8 6800 68.0 71.2
140 2925 29.3 6170 61.7 70.7
150 3015 30.2 6190 61.9 69.8
160 3420 34.2 6030 60.3 65.8
AVERAGES 31.5 62.9 68.5 Experiment VIII. Effect of Glucose
The effect of different substances, especially those commonly found in biological fluids, on the absorption of each other and that of foreign substances has been found to vary greatly
(33* 34, 46, 5 0 , 51* 102, 14). As one of the most common sub stances to be found in such cases is glucose, it was decided to study its effect on phenobarbital absorption. Another reason for selecting glucose is the known potentiating effect exerted by it on barbiturate anesthesia (122).
A 10 mg. dose of phenobarbital was given in 5 c.c. of a
40 per cent solution of glucose and the usual twenty minutes were allowed for absorption. The average per cent of absorption was
62.4 per cent which shows a difference of 20.4 per cent from the average absorption of 82.8 when water was used alone (Table 42).
This difference is higher than that obtained in the next experiment with sucrose in the same concentration, or that obtained with rats recently fed with common rat rations having a full stomach when the drug was given. This seems to point to a specific action of glucose on the absorption of phenobarbital. The study of osmore ceptor mechanisms in the stomach and intestine which seem to control the emptying time of the stomach (95) tends to show that these are more concerned by the volume of the meal introduced than by the nature of its constituents, except, of course, in those cases where the constituent may act specifically on the nervous control of the 86 stomach motility or the pyloric sphincter. So it is relatively safe to assume that there would not be any difference in emptying time produced by two substances like glucose and sucrose, thus ruling out this effect as the cause of the difference in absorp tion.
Two explanations then seem plausible to account for this difference. One, that the gastrointestinal mucosa is in the process of an active transport, and even partially metabolizing glucose, and for this reason its ability to cope with the transport of a foreign substance like phenobarbital is partially impaired. This necessarily would not have to mean that phenobarbital is transported actively, but the set up of the cell might be altered in such a way as to be less favorable for the absorption of any substance other than glucose even if this absorption is brought about merely by physical forces. The faster absorption of glucose and some amino acids by a living membrane in contrast to their absorption by a poisoned membrane, with the reverse being true in most cases of substances not essential in the economy of tissues, would point to the gastrointestinal wall as a barrier in the full sense of the word, and not indifferent to the nature of the substance to be transported across it. That is, there may not be specific mechanisms to carry a majority of substances across, but it seems there are mechanisms to restrain free passage of such substances. The other explanation for the decrease in absorption might be the formation of a complex less readily absorbed than phenobarbital itself or not absorbed at all, but not so stable as to stay long enough in the intestine and be eliminated in the feces. TABLE 42
EFFECT OF GLUCOSE ON ABSORPTION OF PHENOBARBITAL
10 MGS. PHENOBARBITAL IN 5 cc. 40% GLUCOSE SOL. 20 MINUTES
Wt. Micrograms Micrograms % % of Recovered From Per Cent Recovered Absorbed Absorbed Rat G.I. Tract Unabsorbed From Tissues (Direct) (By Difference)
140 3420 34.2 6030 60.3 65.8
120 3540 35.4 6050 60.5 64.6
150 4140 41.4 5710 57.1 58.6
160 2480 24.8 6740 67.4 75.2
130 . 4680 46.8 5050 50.5 53.2
130 3840 38.4 5670 56.7 61.6
140 4140 41.4 5140 51.4 58.6
120 3480 34.8 5980 59.8 65.2
160 4440 44.4 4970 49.7 55.6
130 3420 34.2 5890 58.9 65.8
AVERAGES 37.6 57.3 62.4 Experiment IX. Effect of Sucrose
In order to study further the apparent specific action of glucose on absorption, the effect of a substance like sucrose, which is not absorbed or metabolized as such, was determined and compared to that of glucose.
The same procedure was used as in the preceding experi ment, but with a hO per cent solution of sucrose. Table h3 shows a decrease in average absorption of 9 per cent which is less than one half that obtained with glucose. If "metabolic interference," or the formation of a complex with glucose is to be credited, the inference drawn from this result is obvious, sucrose having to be hydrolyzed before it is utilized, has then a lesser influence than glucose. TABLE 43
EFFECT OF SUCROSE ON ABSORPTION OF PHENOBARBITAL
10 MGS. PHENOBARBITAL IN 5 cc. 40% SUCROSE SOL. 20 MINUTES
Wt. Micrograms Micrograms % 7. of Recovered From Per Cent Recovered Absorbed Absorbed Rat G.I. Tract Unabsorbed From Tissues (Direct) (By Difference)
150 3480 34.8 5940 59.4 65.2
140 1800 18.0 7860 78.6 82.0
160 2760 27.6 6730 67.3 72.4
160 1980 19.8 7250 72.5 80.2
170 3840 38.4 5900 59.0 61.6
145 1800 18.0 7740 77.4 82.0
160 2880 28.8 6740 67.4 71.2
155 2820 28.2 6390 63.9 71.8
130 2280 22.8 7170 71.7 77.2
180 2530 25.8 6950 69.5 74.2
AVERAGES 26.2 68.7 73.8 91
Experiment X. Effect of Feeding (Full Stomach)
The absorption of 10 mg. doses of phenobarbital during ten minutes by recently fed rats was compared to that of rats starved for 48 hours (Tables'44 and 45). The average absorption for starved rats was 82.8 per cent while that for fed rats was
67.9 per cent, a difference of 14.9 per cent. It is impossible in this case to point out to what extent each of the factors that may be involved in decreasing the rate of absorption, is respon sible. The volume of the food mass in the stomach may act on the alleged osmoreceptors in altering the emptying time of the stomach or part of the drug might be delayed mechanically in coming in contact with absorbing surfaces, etc. TABLE 44
ABSORPTION BY STARVED RATS
10 MG. DOSE 20 MINUTES STARVED RATS
Wt. Micrograms Micrograms 7. 7. of Recovered From 7. Recovered Absorbed Absorbed Rat G.I. Tract Unabsorbed From Tissues (Direct) (By Difference)
90 855 7.6 5720 87.2 91.4
100 1980 19.8 7400 74.0 80.2
100 1800 18.0 7680 76.8 82.0
90 2250 22.5 7290 72.9 77.5
90 1440 14.4 7940 79.4 85.6
95 2025 20.3 7160 71.6 79.7
110 1710 17.1 7710 77.1 82.9
100 2030 20.3 7180 71.8 79.7
95 1420 14.2 8000 80.0 85.8
100 1720 17.2 7750 77 5 82.8
AVERAGES 17.2 76.8 82.8 TABLE 45
ABSORPTION BY FED RATS
10 MG. DOSE 20 MINUTES FED RATS
Wt. Micrograms Micrograms 7. % of Recovered From % Recovered Absorbed Absorbed Rat G.I. Tract Unabsorbed From Tissues (Direct) (By Difference)
100 2565 26.7 6890 68.9 73.3
80 3195 32.0 6500 65.0 68.0
90 3600 36.0 5930 59.3 64.0
100 3635 36.4 6020 60.2 65.6
110 2835 28.4 6650 66.5 71.6
100 2655 2 6.6 6740 67 .4 73.3
110 3330 33.3 6010 60.1 66.7
110 2655 26.6 6980 69.8 73.3
100 4275 42.8 5430 54.3 57.2
110 3420 34.2 6030 60.3 65.8 AVERAGES 32.1 63.2 67.9 Experiment XI. Effect of Atropine
The role of autonomic innervation in the control of absorption has been studied by many investigators. The results obtained in the majority of cases have been of a contradictory nature. The results are complicated by the fact that stimulation or blocking of the vagus or splanchnic may affect various factors at the same time, which modify absorption in an opposite sense.
They may modify the circulation in the gastrointestinal wall at the same time that they alter the permeability of the membrane, or the motility of the mucosa and sphincters and these effects might not act in the same direction in increasing or decreasing absorption. Another difficulty is the unpredictable nature of the responses obtained when autonomic stimulation or block, either direct or by means of drugs, is used where the gastrointestinal tract is concerned (23. 24, 44). Drugs used to mimic or block autonomic innervation have been reported to show decided changes in absorption in some cases, in others practically none. In one report both vagotomy and splanchnotomy produced increases in ab sorption of over 60 per cent and 90 per cent respectively (14).
In the present experiment 2 mgs. of atropine sulfate was injected intraperitoneally and immediately after, 10 mgs. of phenobarbital was given orally and 20 minutes allowed for absorption. Table 46 shows little alteration in the average absorption. It is to be concluded that when absorption from the entire gastrointestinal tract is considered autonomic innervation by acting on different sites at the same time, in some cases in favor, in others against absorption, tends to render results which balance each other. TAB IE 46
EFFECT OF ATROPINE ON ABSORPTION OF PHENOBARBITAL
2 MGS. ATROPINE SULFATE INTRAPERITONEALLY 20 MINUTES 10 MGS. PHENOBARBITAL ORALLY
Wt. of Micrograms Micrograms % % Rat in Recovered From % Recovered Absorbed Absorbed Grams G.I. Tract Unabsorbed From Tissues (Direct) (By Difference)
160 1935 19.4 7420 74.2 80.6
160 1110 11.1 7850 78,5 88.9
140 1920 19.2 7610 76.1 80.8
170 1265 12.7 7660 76.6 83.3
130 1170 11.7 8120 81.2 88.3
180 1420 14.2 7940 79.4 85.8
170 1840 18.4 7670 76.7 81.6
1.50 1350 13.5 7890 78.9 86.5
160 1710 17.1 7640 76.4 82.9
140 1790 17.1 7570 75.7 82.9
AVERAGES 15.5 77.4 84.5 Experiment XII. Effect of Stress
The purpose of this experiment was to find if the effect of an emotional disturbance on absorption presented a uniform pattern.
A 10 mg. dose was given to each rat, and immediately the animal was placed in a glass jar full of water so that they would have to swam constantly for 20 minutes, after which the con tents of the gastrointestinal tract and the tissues were examined.
The results (Table 47) vary so much from animal to animal that no average was calculated for the 10 animals used. Absorption varied from 39 per cent in the lowest case to 84.1 per cent in the highest.
Although it is seen that stress tends to decrease rate of absorp tion, there is no definite pattern. In some animals it remains almost normal, in others it decreases to less than half. It is probable that the effect is due to a contraction of the pyloric sphincter, this varying widely with the degree of excitement or fear experienced by each animal. TABLE 47
EFFECT OF STRESS ON ABSORPTION OF PHENOBARBITAL
10 MG. DOSE 20 MINUTES wt. Micrograms Micrograms 7. % of Recovered From 7. Recovered Absorbed Absorbed Rat G.I. Tract Unabsorbed From Tissues (Direct) (By Difference)
150 2115 21.2 7250 72.5 78.8
155 3510 35.1 6040 60.4 64.9
140 1785 17.9 7970 79.7 82.1
170 5085 50.9 4620 46.2 49.1
160 1620 16.2 7700 77.0 83.8
200 3840 38.4 5740 57.4 61.6
170 1590 15.9 7810 78.1 84.1
160 4210 42.1 5300 53.0 57.9
145 6100 61.0 3430 34.3 39.0
150 2240 22.4 7080 70.8 77.6
\o CO Experiment XIII. Effect of Bentonite Magma
The effect of adsorbents in the absorption of different substances has been reported in most cases as producing a decrease in the rate of absorption. The effect could be the result of various factors such as the mechanical entrainment of the sub stance in the solid particles of the adsorbent by real adsorption, or even by the formation of molecular complexes with the insoluble non-adsorbable adsorbent. Their effect has been studied, especially in relation to such substances as vitamins in which loss of part of the small doses might result in producing an avitaminosis (123,
124).
In the present experiment bentonite magma was used, feed ing 3 c.c. to each rat and immediately after giving 2 c.c. of a solution of phenobarbital sodium containing 5 mg* per c.c. A de crease of absorption from a normal average of 82.8 per cent to
74.7 per cent was observed (Table 48). TABLE 48
EFFECT OF BENTONITE MAGMA ON ABSORPTION OF PHENOBARBITAL 3 cc. OF BENTONITE MAGMA 2 cc. OF PHENOB. SOD. CONTAINING 10 MGS. 20 MINUTES
Wt. Micrograms Micrograms % 7. of Recovered From % Recovered Absorbed Absorbed Rat G.I. Tract Unabscrbed From Tissues (Direct) (By Difference)
150 2250 22.5 7080 70.8 77.5
150 1980 19.8 7090 70.9 80.2
160 3015 30.2 6460 64.6 69.8
140 2835 28.4 6650 66.5 71.6
130 4185 41.9 5610 56.1 58.1
130 1980 19.8 7270 72.7 80.2
140 2250 22.5 7140 71.4 77.5
130 2340 23.4 7020 70.2 7 6.6
180 1935 19.4 7700 77.0 80.6
160 2475 24.8 7010 70.1 75.2
AVERAGES 25.3 69.0 74.7 100 Experiment IV. Effect of Glucosamine Hydrochloride
In view of the fact that glucosamine has been found to be concerned in cellular chemistry and structure, and its recent use in enhancing antibiotic absorption and activity (125, 126, 127,
128, 131), it was thought to be advisable to study its effect on the absorption of phenobarbital from the gastrointestinal tract.
A dose of 10 mgs. of phenobarbital was accompanied by
500 mgs. of glucosamine hydrochloride. A decrease of the average absorption from 82.8 to 70 per cent was observed (Table 49). This is not in accord with the reported enhancement of absorption of tetracyclines. TABLE 49
EFFECT OF GLUCOSAMINE ON ABSORPTION OF PHENOBARBITAL
10 MG. DOSE PHENOB. 500 MG. GLUCOSAMINE HCL 10 MINUTES wt. Micrograms Micrograms % 7. of Recovered From 7. Recovered Absorbed Absorbed Rat G.I. Tract Unabsorbed From Tissues (Direct) (By Difference)
110 2925 29.3 6490 64.9 70.7
115 2070 20.7 7280 72.8 79.3
110 3330 33.3 6310 63.1 66.7
90 4410 44.1 5040 50.4 55.9
110 3735 37.4 5850 58.5 62.6
110 2025 20.3 7360 73.6 79.7
90 1845 18.5 7550 75.5 81.5
95 3105 31.1 6410 64.1 68.9
110 3420 34.2 5980 59.8 65.8
110 3105 31.1 6230 62.3 68.9
AVERAGES 30.0 64.6 70.0 102 Experiment XV. Effect of Polykol (Poloxalkol)
The effect of surface active agents on absorption has been mentioned earlier in the introduction. In this experiment
Polykol Drops (a brand of Poloxalkol, an oxyethylene oxypropylene polymer marked by the Upjohn Company) was used. One half c.c. containing 100 mgs. of poloxalkol was given orally followed by
10 mgs. of phenobarbital. The results show little effect on absorption with a slight increase from 82.8 per cent to 85.^ per cent (Table 50). TABLE 50
EFFECT OF POLYKOL (POLOXALKOL) ON ABSORPTION OF PHENOBARBITAL
Wt. Micrograms Micrograms % 7. of Recovered From 7. Recovered Absorbed Absorbed Rat G .I. Tract Unabsorbed From Tissues (Direct) (By Difference)
110 1373 13.7 8120 81.2 86.3
130 1868 18.7 7690 76.9 81.3
130 918 9.2 8460 84.6 90.8
110 1738 17.4 7190 71.9 82.6
110 1440 14.4 8030 80.3 85.6
120 1463 14.6 7870 78.7 85.4
110 1620 16.2 8010 80.1 83.8
115 1280 12.8 8250 82.5 87.2
120 1455 14.6 8090 80.9 85.4
120 1390 13.9 8180 81.8 86.1
AVERAGES 14.6 79.9 85.4 +?0T 105
TABLE 51
CHANGE IN PER CENT PHENOBARBITAL ABSORBED FROM A TEN MILLIGRAM DOSE IN TWENTY MINUTES PRODUCED BY VARYING THE SOLVENT OR ADMINISTERING A SECOND SUBSTANCE
Solvent or Increase Substance Per Cent or Value of Level of Added Absorbed Decrease Mtn Significance
Control (10 Mg.in 5 cc. of water) 82.8
10 Mg. in 15 cc. of water 89.0 -6.2 4.49 99.0
Alcohol 78.1 -4.7 2.30 95.0
Olive Oil 68.5 -14.3 6.13 99.9
Glucose 62.4 -20.4 8.53 99.9
Sucrose 73.8 -9.0 3.44 99.0
Atropine 84.5 +1.7 1.04 --
Bentonite 74.7 -8.1 3.19 95.0
Glucosamine 70.0 -12.8 4.75 95.0
Polykol 85.4 +2.6 1.67 --
Fed Rats 67.9 -14.9 7.09 99.9 Experiment XVI. Absorption from the Stomach
Except for a few isolated cases reporting absorption from the stomach,, the general belief for many years has been that this organ absorbs foodstuffs and drugs poorly, if at all. But in recent years the number of reports showing an unmistakable power of absorption by the gastric mucosa increases constantly.
The method used in this experiment to measure absorption from the stomach cannot be said to be one reflecting physiological conditions. Tying the pylorus checks the normal emptying of the stomach so that the absorption measured in a given time from a given dose is not the same as that which would result from the same dose in the same interval under normal conditions. The ligation of the pylorus seems to alter the normal power of absorption of the stomach, probably in part by interference with normal circulation of the stomach wall. This can be appreciated by the congested area seen some distance below the ligature, along the duodenum, and above along the pylorus. For these reasons, the present determinations are meant to show only the potential capacity of the gastric mucosa in absorbing, and not to measure the actual rate of absorption.
The absorption of a large number of different substances, including barbiturates, has been demonstrated from the stomach using indirect methods (10, 38, 5^« 76, ??, 86, 93 > 132, 133). In the present study, a 15 mg. dose was given after preparing the animal as described previously. Absorption was determined after 10, 20,
30, ^5. 60, 90 and 120 minutes (Tables 52 to 58). It can be seen that absorption is rapid during the first 10 minutes but it seems that after an initial peak, the capacity of absorption is somewhat lost, and only about 14 per cent is absorbed in the next two hours.
This could be accounted for by the interference with the circula tion and by the effect of the surgical procedure, and not by an actual saturation of the absortive capacity of the stomach. TABLE 52
ABSORPTION OF PHENOBARBITAL FROM THE STOMACH
15 MG. DOSE 10 MINUTES wt. Micrograms Micrograms 7. % of Recovered 7o Recovered Absorbed Absorbed Rat From Stomach Unabsorbed From Tissues (Direct) (By Difference)
160 8700 58.0 5860 39.1 42.0
160 9100 60.6 5670 37.8 39.4
140 8640 57.6 . 5760 38.4 42.4
200 8720 58.1 6000 40.0 41.9
120 9260 61.7 5280 35.2 38.3
150 8510 56.7 5505 36.7 43.3
140 9310 62.0 4950 33.0 38.0
180 9120 60.8 5175 34.5 39.2
AVERAGES 59.4 36.8 40.6 108 TABLE 53
ABSORPTION OF PHENOBARBITAL FROM THE STOMACH
15 MG. DOSE 20 MINUTES
wt. Micrograms Micrograms % % of Recovered % Recovered Absorbed Absorbed Rat From Stomach Unabsorbed From Tissues (Direct) (By Difference)
180 8220 54.8 6105 40.7 45.2
130 8160 54.4 6150 41.0 45.6
160 8110 54.0 5940 39.6 46.0
160 7980 53.2 5835 38.9 46.8
150 7830 52.2 6315 42.1 47.8
180 8070 53.8 5955 39.7 46.2
170 8180 54.5 6045 40.3 45.5
200 8760 58.4 5310 35.4 41.6
AVERAGES 54.3 39.7 45.7 TABLE 54
ABSORPTION OF PHENOBARBITAL FROM THE STOMACH
30 MINUTES wt. Micrograms Micrograms 7. 7. of Recovered 7. Recovered Absorbed Absorbed Rat From Stomach Unabsorbed From Tissues (Direct) (By Difference)
190 7840 52.2 6300 42.0 47.8
180 8200 54.6 5640 37.6 45.4
170 8120 54.1 6165 41.1 45.9
150 8320 55.4 5670 37.8 44.6
180 8110 54.2 5445 36.3 55.8
170 8010 53.4 5520 36.8 5 6.6
150 7630 50.8 6255 41.7 49.2
190 7970 53.1 6030 40.2 46.9
AVERAGES 53.4 39.2 46.6 110 TABLE 55
ABSORPTION OF PHENOBARBITAL FROM THE STOMACH
45 MINUTES
Wt. Micrograms Micrograms % % of Recovered 7. Recovered Absorbed Absorbed Rat From Stomach Unabsorbed From Tissues (Direct) (By Difference)
160 8120 54.1 6015 40.1 45.9
190 7790 51.9 6120 40.8 48.1
160 7920 52.8 6240 41.6 47.2
170 7810 52.0 5835 38.9 48.0
180 9040 60.2 5100 34.0 39.8
200 7230 48.2 7095 47.3 51.8
210 9020 60.1 5580 37.2 39.9
190 8390 55.9 6105 40.7 44.1
AVERAGES 54.4 40.0 45.6 111 TABLE 56
ABSORPTION OF PHENOBARBITAL FROM THE STOMACH
60 MINUTES
Wt. Micrograms Micrograms % 7. of Recovered 7. Recovered Absorbed Absorbed Rat From Stomach Unabsorbed From Tissues (Direct) (By Difference)
180 7910 52.7 6270 41.8 47.3
140 7640 50.9 6240 41.6 49.1
160 7095 47.3 7350 49.0 52.7
190 7370 49.1 7230 48.2 50.9
200 7530 50.2 5210 41.4 49.8
210 7280 48.5 6990 46.6 51.5
170 8010 53.4 6045 40.3 46.6
150 7820 52.1 6015 40.1 47.9
AVERAGES 50.8 43.6 49.2 TABLE 57
ABSORPTION OF PHENOBARBITAL FROM THE STOMACH
90 MINUTES
Wt. Micrograms Micrograms 7. 7o of Recovered 7o Recovered Absorbed Absorbed Rat From Stomach Unabsorhed From Tissues (Direct) (By Difference)
140 6920 46.1 7350 49.0 53.9
190 8020 53.4 5730 38.2 46.6
180 7290 48.6 5985 39.9 51.4
200 8140 54.2 5340 35.6 45.8
160 7360 49.0 6195 41.3 51.0
170 6540 40.9 7380 49.2 59.1
140 6710 44.7 5820 38.8 55.3
150 8830 51.5 5685 37.9 48.5
AVERAGES 48.5 41.2 51.5 TABLE 58
ABSORPTION OF PHENOBARBITAL FROM THE STOMACH
120 MINUTES
Wt. Micrograms Micrograms % % of Recovered % Recovered Absorbed Absorbed Rat From Stomach Unabsorbed From Tissues (Direct) (By Difference)
180 6780 45.2 6720 44.8 54.8
160 6980 46.5 6600 44.0 53.5
160 7010 46.7 6375 42.5 53.3
150 7240 48.2 5970 39.8 51.8
210 7020 46.8 6225 41.5 53.2
190 6410 42.7 7695 51.3 57.3
170 6670 44.4 7230 48.2 55.6
150 6530 43.5 7290 48.6 56.5
AVERAGES 45.5 45.1 54.5 115
Experiment XVII. Absorption from the Large Intestine
Blank determinations for the readings of optical densi ties given by the contents of the large intestine were made, ob
taining an average of 6.8 micrograms per c.c. on the basis of
phenobarbital .values (Table 59). As can be seen from the table, wide variations are observed in the values given by the feces of
different animals.
As with stomach studies the method used does not measure
the actual rate of absorption under normal conditions, but rather
the potentiality of the large intestine as an absorbing organ.
Another difficulty in this case is the distention produced by in troducing even small volumes of solution in an already full large
intestine.
The series of experiments by Goldschmidt on absorption from the colon (29, 3 0 * 31* 3 2 , 33* 3*0 seem to indicate that there is not much difference, at least potentially, between absorption from the large and small intestine. In the present study, the results show no great difference either (Table 60). TABLE 59
ABSORPTION FROM THE LARGE INTESTINE. BLANK DETERMINATIONS WITH CONTENTS OF COLON NO DRUG GIVEN
Wt. of Micrograms per C.C. Rat (On Phenob. Basis)
140 5.0
200 10.2
160 8.6
170 5.5
165 8.6
150 4.0
190 6.1
185 6.3
AVERAGE 6.8 TABLE 60
ABSORPTION OF PHENOBARBITAL FROM LARGE INTESTINE
10 MG. DOSE 20 MINUTES
Wt. Micrograms Micrograms % 7/» of Recovered 7. Recovered Absorbed Absorbed Rat From Colon Unabsorbed From Tissues (Direct) (By Difference)
160 1262 12.6 8110 81.1 87.4
130 1622 16.2 7680 76.8 83.8
150 1007 10.0 8460 84.6 90.0
130 1007 10.0 8090 80.9 90.0
180 2057 20.6 7230 12.3 79.4
150 1517 15.2 7890 78.9 84.8
160 1197 12.0 8250 82.5 88.0
130 1982 19.8 7360 73.6 80.2
140 2132 21.3 7310 73.1 78.7 CO r-- 150 1232 12.3 8250 82.5 r-.
AVERAGES 15.0 78.6 85.0 DISCUSSION
Both the shape of the curves obtained when per cent phenobarbital absorbed is plotted against time, and the fact that the stomach and intestinal walls retain a fairly constant amount of drug during absorption, tend to suggest diffusion as one of the predominant factors in the process. At the same time they tend to confirm the hypothesis of a lipid rich gastrointestinal barrier. The curves show a sharp rise the first thirty minutes, after which absorption is slowed significantly, the rate remain ing low for the rest of the absorption period. In the case of the stomach and intestinal walls, they take up a relatively high per cent of drug the first five minutes, then drop to a fairly constant per cent throughout the rest of the absorption period, until sixty minutes have elapsed in the case of the intestinal wall and forty- five minutes in the case of the stomach wall, when the amount of drug retained begins to drop sharply again. These observations would be explained by a system in which the drug is transferred by diffusion from the lumen of the alimentary canal to a lipid rich mucosa, then to plasma and from here distributed to the tissues.
The rapid absorption at the start of the period and the high intake of drug by the mucosa is explained by the fact that at the beginning there is a high concentration of drug in the lumen, and none in the mucosa and plasma. After a time, the mucosa becomes
118 119 saturated with drug, concentration in the lumen diminishes, while it increases in plasma. At this time rate of absorption is limit ed by the speed by which the drug is taken up by the tissues, therefore it slows down. And finally, when the concentration of the drug in the lumen has reached a value below that in the mucosa, the lipids in the mucosal wall begin to lose drug rapidly. This is seen first in the case of the stomach wall, since the stomach empties in a relatively short time, while the decrease in the in testinal wall takes a longer time because the drug stays much longer in the intestine.
If this mechanism of absorption is accepted, the partition coefficient of the system comprising the fluids in the lumen, the lipids in the mucosa and the fluids in plasma will have a signifi cant bearing on the rate of absorption of drugs which are soluble in the lipid barrier. And in the case of weak organic acids and bases, their dissociation constants and the pH of the physiological media will determine to a high degree their rate of absorption.
This is supported by the results obtained when alcohol and olive oil were used as solvents for the drug. The distributing system was altered in the sense that having a fluid in the alimentary canal in which the undissociated molecules of phenobarbital are soluble, the tendency of the drug to be preferentially partitioned to the lipids of the mucosa was diminished, and as a result absorp tion was decreased. 120
But it is difficult to consider diffusion as the only factor involved when the results obtained by giving glucose, sucrose or the normal rat’s diet are fed together with the drug.
The decrease in the rate of absorption, especially when glucose is used, suggest other mechanisms involved. These would be either an increase in osmotic pressure in the lumen working against simple filtration or interference with the process of diffusion in the cells of the mucosa by a preferential transport of a metabolite like glucose. Or in the case of glucose both factors could be involved. Where the process of filtration is involved, any sub stance present in the lumen which would increase the osmotic pressure would also decrease filtration. But if specific transport mechanisms are assumed for some metabolites, the presence of these in the lumen may very well engage the cells of the mucosa in pro cesses which could hinder the otherwise unobstructed process of diffusion, this by changing the composition or distribution of the cell contents.
The role of nervous control on absorption is difficult to evaluate due to the diversity of factors influenced by stimulation or blocking of autonomic innervation. Changes in circulation in the intestinal capillary bed, in motility of the stomach or in testine, in the emptying time of the stomach due to relaxation or contraction of the pyloric sphincter, etc. may occur at the same time and work in opposite directions increasing or decreasing 121
absorption. Thus it was not surprising to find no significant
change in absorption rate after the administration of atropine,
showing that the different influences on structures related to
absorption were balanced to produce no net effect.
Nervous effects controlled from the higher centers,
however, showed profound effects as evidenced by the results ob
tained when the animals were subjected to stress, although these were highly irregular. The pronounced decrease in absorption seen
in some animals is probably due to prolonged emptying time of the
stomach caused by contraction of the pyloric sphincter.
The delay in absorption of phenobarbital caused by
glucosamine is difficult to explain, unless a difficultly absorbed
complex is formed, or unless a specific transfer mechanism is
assumed for glucosamine, as in the case of glucose, interfering with the normal diffusion processes. Further work is indicated
on the remarkable retention of water caused by glucosamine.
The effect of circulating disturbances on absorption is manifested by the extremely reduced rate of absorption when the pylorus is ligated. It is evident from the appearance of the regions above and below the ligature, that the blood supply to the
stomach wall has been altered. As a consequence absorption is
checked almost completely after the first 10 minutes. This is in
accordance with the system of transfer proposed before, the
diffusion from lumen, to mucosa, to plasma. When the distribution
of the drug from plasma to tissues is interfered with the movement from lumen to mucosa and thence to plasma must obviously de
crease.
In conclusion, three processes can be postulated for
-absorption in general; filtration, diffusion and' active trans
port by enzyme systems. Physiological variations would play a
minor role in modifying these processes because normally these
variations are not large enough to impair a physiological process.
Only pathological changes in permeability of the mucosa, circu
latory stasis or prolonged spasm of the sphincters in the aliment
ary canal would alter significantly rates of absorption. On the
other hand, the nature of the substances absorbed and the condi
tions under which they are present in the lumen may alter consid
erably the three processes by introducing changes in osmotic pres
sure, hydrostatic pressure, partition coefficients, molecular size,
metabolic interference, etc. SUMMARY AND CONCLUSIONS
1. After making a survey of the methods used in studying absorp
tion from the gastrointestinal tract the conclusion was
reached that the method used by Cori in the study of the ab
sorption of sugars was the one which approached best, truly
physiological conditions.
2. As a check of the results obtained by using this method, it
was thought advisable to make direct determinations on the
tissues of the entire animal,
3. For the analysis of barbiturate both of the unabsorbed portion
in intestinal contents and absorbed portion in tissues, the
spectrophotometric procedure of Goldbaum, with modifications,
was used after testing the linear relationship between optical
density readings and concentration within certain concentra
tion range.
4. Results tend to show that in the case of phenobarbital -
a) Absorption is rapid in the first twenty minutes of the absorption period then slows to a nearly constant level for the next sixty or seventy-five minutes.
b) Abs rption is governed to a certain extent by the concen tration of the drug in the lumen.
c) During the period between the initial high absorption peak and the last stages, the gastric and intestinal mucosa retain an amount of drug, which is relatively high when their weight is considered.
123 1 Zk d) When the effect of total volume of fluid was studied, the results were inconclusive, but tended to show an effect on rate of absorption. e) Solvents like alochol or fatty substances were found to alter absorption, probably by changing partition coefficients. f) Glucose and, to a lesser extent, sucrose, altered absorption rates. g) The presence of food in the gastrointestinal tract was found to decrease absorption of the drug in a significant degree. h) Atropine by the intraperitoneal route did not significantly alter rate of absorption. i) Bentonite magma decreased absorption; Foloxalkol, a surface active agent, increased it to a small degree. j) Glucosamine hydrochloride decreased absorption k) Effect of stress on absorption was highly irregular, but tended to show a decrease in a number of animals.
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Iglesias, was born in San Lorenzo, Puerto Rico, January 5, 1911* I received my elementary and high school education in the public schools of Puerto Rico. % undergraduate training was obtained in the College of Pharmacy of the University of Puerto Rico, where I received the Bachelor of Science degree in Pharmacy. I received the Master of Science degree in the Philadelphia College of Pharmacy and Science in 1950. I have been an Assistant Professor in the College of Pharmacy of the University of Puerto Rico from 19^5 to this date.