o and I, the thoracic duct was cannulated on

Transcription

1 GASTROENTEROLOGY Copyright 1967 by The Williams & Wilkins Co. Vol. 52, No.1 Printed in U.S.A. EFFECT OF AMINOPTERIN ON THE ABSORPTION OF FAT INTO THE LYMPH OF UNANESTHETIZED RATS T. G. REDGRAVE, M.B.B.S, AND W. J. SIMMONDS, M.B.B.S., D.PHIL. (OXON) Department of Physiology, University of Western Australia, Nedlands, Western Australia In the intestine, arrest of mitosis by radiation or drugs produces malabsorption and morphological changes resembling those in sprue. 1-4 After folic acid antagonists, morphological changes have been followed with light and electron microscopy.5-12 There is evidence for defective absorption of xylose 13,14 and medium chain fats,15 but malabsorption of long chain fats has not been conclusively shown. The morphological changes after aminopterin given in a single, fairly large dose have been well described by Millingtion et al. 12 They also demonstrated defective absorption of long chain fats but had difficulty in correlating this with the morphological events because of technical problems in the methods they used.1 6 Repeated measurements of fat absorption into lymph during steady fat infusion into the duodenum offered a prospect of following more closely the absorptive pattern after mitotic arrest and during recovery. This might then allow mucosal function to be correlated with the age of Received June 20, Accepted August 11, Address requests for reprints to: Dr. T. G. Redgrave, Department of Physiology, The Uni \'ersity of Western Australia, N edlands, Western Australia. This investigation was carried out with the aid of grants from the National Health and Medical Research Council of Australia and the Medical Research Grants Committee, The University of Western Australia. Dr. Redgrave was an Australian and New Zealand Life Insurance Medical Research Fellow and, during the earlier part of this work, was the recipient of a Saw Medical Research Fellowship. The technical assistance of Miss V. Orton is gratefully acknowledged. 54 the cells and their progression up the villus. This paper reports the initial stages of such an investigation which has demonstrated a clear and reproducible sequence of changes in fat absorption following a relatively small dose of aminopterin. The defect has been shown to be mucosal in origin, and a preliminary correlation between the age of mucosal cells and their ability to absorb fat is possible. Methods General procedure. Male albino rats weighing 200 to 220 g were used. Aminopterin, 250 p.g per rat, was injected intramuscularly at 10 AM on day O. It was given in 0.2 ml of NaOH solution, ph 10 to 11. On various days before or after aminopterin (from -2 days to day 3), the thoracic duct was cannulated under ether anesthesia with fine plastic tubing l7 following an overnight fast. At the same time, a fine silicone rubber tube was inserted through a stab incision in the muscular part of the stomach and passed through the pylorus into the duodenum. A fine stitch held the tip of the tube at the level of the entrance of the bile duct. Both tubes were exteriorized through the right side of the abdomen. In some animals, a fine polythene cannula was passed into the right jugular vein, filled with saline, exteriorized, and plugged. All animals were placed in a restraint cage and allowed to drink 0.64% N aci ad libitum until some hours before absorption was tested, but food was withheld. Fluid was steadily infused by the duodenal cannula at 1.5 ml per hour from operation until the animal was sacrificed. Except during absorption tests, NaCI, 150 mm, and KCI, 4 mm, were infused. Two absorption tests were carried out on each animal, on the second and third postoperative days. Thus for animals tested on day o and I, the thoracic duct was cannulated on day -2, for tests on day 1 and 2, cannulation

2 January 1967 AMINOPTERIN AND FAT ABSORPTION IN RATS 55 was performed on day -1, and so on. For tests on day 6, however, operation was performed on day 2 or 3 to avoid operation at the peak of aminopterin effect, and these animals were therefore subjected to the stress of fasting and loss of lymph for longer periods than the rest. On test days, fasting lymph was collected for 1 hr while intraduodenal infusion of saline continued. A priming dose of finely emulsified olive oil containing 100 fleq of esterified fatty acid in 0.2 ml of fluid was given by the duodenal cannula, and then the emulsion was steadily infused at the rate of 150 fleq of esterified fatty acid in 1.5 ml per hour for S hr. Lymph was collected hourly in graduated heparinized centrifuge tubes. After 8 hr, the fat infusion was replaced by saline at 1.5 ml per hour, lymph was collected overnight, and a second test was performed on the next day followed by a final overnight collection of lymph. Some representative animals were sacrificed at the completion of 8-hr fat infusion for histological studies. Sections of upper jejunum just beyond the ligament of Treitz were taken for paraffin and frozen sections. Demonstration of a micellar phase. Animals were prepared with intraduodenal tubes only and maintained postoperatively as described for the lymph fistula rats. On the third day after aminopterin (48 hr after operation), these animals were infused with the olive oil emulsion for 4 hr. Then under light ether anesthesia, the intestine was ligated at the pylorus and the ileocecal junction, removed, and washed briefly on the outside with saline. The undiluted contents were then collected, by opening the lumen, and incubated at 70 C for 10 min to destroy lipase activity.!s A pooled sample from several rats was then centrifuged at 120,000 g for 8 hr in a Spin co model E ultracentrifuge at 30 C. The clear infranatant phase and the oil phase at the top of the tube were recovered and extracted by the method of Blankenhorn and Ahrens.!O Qualitative class analysis of the lipid was performed by thin layer chromatography. Absorption of artificial micellar solutions. Sodium taurocholate solutions and phosphate buffer, ph 6.3, 0.15 M with respect to Na+ ions, were prepared as described by Hofmann."" Oleic acid and glyceryl mono-oleate in a molar ratio of 3: 1 were weighed out and taken up in the bile salt solution. The final concentration of bile salt was 20 mm and of lipid approximately 15 mm. Micellar solubilization was hastened by sonifying, and the solutions were finally filtered through 0.22 fl pore size membrane filters to yield a clear solution. Absorption of these solutions was measured on day 3 after aminopterin by the same procedure as for olive oil emulsion, except that the micellar fat solution was infused at 3 ml per hour intraduodenally. Olive oil emulsion. The preparation Lipomul for intravenous lipid infusion was generously supplied by the Upjohn Company, Kalamazoo, Michigan, or else prepared by us according to their formulation. Before use it was diluted 1: 5 with the infusion solution. Absorption of intact disaccharide. Intraduodenal cannulas were inserted into rats 1 day after aminopterin, and these animals and the controls were maintained in restraint cages with only salt solution to drink. On the third day after aminopterin, the animals were given 2 ml of 0.25 M sucrose slowly via the intraduodenal cannula, and then after 1 hr they were anesthetized and blood was taken from the abdominal aorta with a heparinized syringe for determination of plasma glucose'! before and after incubation of the plasma with an equal volume of 0.1 N HCI at 50 C for 10 min. In some other rats, urine was collected for several hours after the intraduodenal administration of sucrose or lactose, either as it was voided or by means of a cannula inserted into the bladder at the same time as the duodenum was cannulated. The urine was taken to dryness by lyophilization or in a rotary evaporator, and the residue was dissolved in a small volume of pyridine. Thin layer chromatography was then used to identify sugars in the urine. Mitotic counts. After completion of the 8-hr fat infusion in representative rats, segments of upper small intestine were fixed in Bouin's fluid. Paraffin sections 8 fl thick were used to ascertain the proportion of nuclei in mitosis to resting nuclei by counting at a magnification of 500. For each group of animals, 2000 cells were counted. Only cells in obvious mitosis were scored, so that cells in early prophase and late telophase were probably missed. This may explain absolute values for control animals lower than those reported by Leblond and Stevens" and Bertalanffy.23 For comparison between groups in this investigation, the figures are meaningful. Analytical. Thin layer chromatography of lipids was performed on preactivated l layers of Silica Gel G with tank saturation at room temperature. Solvents used were of analytical grade or redistilled. The lipids were detected by heating after spraying with 10% ethanolic phosphomolybdic acid.

3 56 REDGRA VE AND SIMMONDS Vol. 52, No. -/ DAYl ' - ' DAY DAY3.-. DAY4 '-' DAY DAY RECOVERY OF FAT IN ()( LYMPH ~ E q J H r. 5 FIG 1. /.-._ }-'-'--' / i FIG / /. ;./ _0/0-0_0--0 /./0 ~ y O.. _.. 10C FAT CONe. ~ E q. f m l. LYMPH VOLUME ml/hr "'.,...-'" / _0_ / _0_0_./' 0 Lo _._.-. ~ i e-._._._ : : - o ~ o : : : : : : : : : ~ O : ; : ;.._O--o_o ", -, : : : : O _ e-e--.;;; " ,, OL ~ L ~ HOURS HOURS FIG. 1. Fat absorption and lymph flow on days 1, 2, and 3 after aminopterin. On day 1, 24 hr after aminopterin, absorption increases rapidly and is maintained during the period of fat infusion. On day 2 absorption is severely depressed and declines during the course of the infusion, and on day 3 there is no increase in fat recovered in the lymph. The fat concentration in the lymph is decreased on days 2 and 3, and lymph flow is decreased by the administration of the fat on these days, compared with the increase in lymph flow over fasting values on day 1. FIG. 2. Fat absorption and lymph flow on days 4, 5, and 6 after aminopterin. On day 4 no fat is absorbed during the period of fat infusion. Days 5 and 6 show a marked recovery in absorption, and this is accompanied by an increase in fat concentration in the lymph. Lymph flow on day 4 is depressed by the administration of fat, but on days 5 and 6 flow is sustained. Thin layer chromatography of sugars was carried out on 250-p. layers of Kieselguhr G impregnated with sodium acetate and activated at 100 C for 30 min'" Development was carried out in ethyl acetate-isopropanol-water, 65: 23 : 12 (v Iv Iv), to 12 cm with tank sa turation at 4 C. The sugars were detected with anisaldehyde-sulphuric acid spray." Esterified fat in the lymph was measured by the method of Stern and Shapiro:' Nonesterified fatty acids in lymph were measured by the colorimetric method of Mosinger.26 Intestinal disaccharidase activity was estimated by the method of Sols and de la Fuente." Assay was performed after an overnight fast on mucosal scrapings from control rats and from rats that had received aminopterin 3 days previously. Results Fat absorption and gross pathology. A consistent pattern of changes in fat absorption was demonstrated by the technique of steady duodenal perfusion. From day 1 after aminopterin to day 3, absorption declined to minimal values (fig. 1), remained low on day 4, and recovered rapidly on days 5 and 6 (fig. 2). On day 1 after aminopterin, fat absorption and lymph flow were indistinguishable from control values in rats not

4 January 1967 AMINOPTERIN AND FAT ABSORPTION IN RATS 57 TABLE 1. Relation of mitotic count to fat absorption and fasting lymph a Days after aminopterin Control Mitotic count (%),,,,,,,,,, hr absorption (% of administered dose).. '..., hr absorption (% of administered dose)., Fat output in fasting lymph (jleq/hr),..., Fasting lymph flow (ml),,, Gastric dilation b.,.. ',, Diarrhea c, ,.,, Villus structured,...,...,... N A A A A N N- a Results are the means of from 4 (day 6) to 12 (control) experiments, except for mitotic counts which were performed on representative animals only. Values for percentage of absorption are corrected for endogenous fat in the lymph, which was assumed to remain constant during the test. b 0, no dilation; +, dilation; ++, gross dilation. t Diarrhea was spontaneous. 0, no diarrhea; +, diarrhea. d N, normal; A, abnormal (see figs. 9 to 13) ABSORPTION 50 (%OF INFUSED DOSE) CONTROL DAYS AFTER AMINOPTERIN FIG. 3. Twenty-four hour absorption on days 1 to 6 after aminopterin. The recovery of fat in the lymph is expressed as a percentage of the infused dose after correction for endogenous fasting levels of fat in the lymph. The endogenous fat is assumed to remain constant during the period under study. The shaded areas represent the portion of the 24-hr recovery absorbed overnight, after the completion of the fat infusion. On day 1 the pattern and amount of absorption are similar to controls. On day 2 total recovery is depressed, and no further fat is absorbed overnight. Very little fat is absorbed on days 3 and 4. On day 5 recovery is marked, and a large additional amount of fat is recovered overnight. On day 6 the 8-hr absorption increases further, but little more is recovered overnight for reasons discussed in the text. injected. On day 2, absorption was grossly impaired. Fat output in lymph and lymph flow declined from the third hour of fat infusion, and the total 24-hr recovery of fat, including 16 hr overnight, was only 31 % of that infused in the 8-hr test period, compared with 91% in controls and 92% on day 1 (table 1). On day 3, little or no fat was absorbed. Lymph flow declined progressively during fat infusion, so that a negligible increa8e in fat output occurred although there was a small increase in the concentration of fat in the lymph. On day 4 (fig. 2), absorption was still virtually nil, but a considerable recovery was apparent on day 5 with a sustained lymph flow and a sustained increase in

5 r;s REDGRA VE AND SIMMONDS Vol. 52, No.1 I so RECOVERY OF FAT IN ()( LYMPH ~ E q. / H r. I FAT CONC. ~ E q. f m l. LYMPH VOLUME ml/hr. 5 oc I ~. il i/! I ~ : : : : :. -.;::::::- O_! o ~ O. : : : : : :.! ~ -===-OJ DAY 2 concentration of fat III lymph. However, absorptive capacity was still depressed, as shown by the absorption of only 43% of the infused dose during the S-hr test period (fig. 3). This was similar to the performance on day 2, but, unlike day 2, the output of fat in lymph was sustained and a further 23% of the dose was absorbed overnight. On day 6, absorption during the period of fat infusion was further improved, approaching normal values (64% compared with 74%, table 1). Little extra fat was recovered overnight (fig. 3), probably because the day 6 rats were debilitated, having been subjected to fasting and loss of lymph for longer periods than the day 5 rats, for reasons explained under "Methods." During the period of malabsorption, ~ : : : : : : :.. / o""--i--.! chylomicrons were present when the : : : : : : : "! I ~ lymph was examined by phase contrast microscopy. The small amount of fat present in the lymph was shown by thin layer chromatography to be principally triglyceride. On day 3, nonesterified fatty acid in the lymph accounted for less than 10% of the fat when measured by the method of Mosinger.26 The concentration.0- -"""'i""-' / = i : : : : : : : OJ : ; and /. output - i ~ of fat in fasting lymph fell 3 HOURS NO ALBUMIN INfUSED.-. WITH I.V. ALBUMIN INFUSION FIG. 4. The effect of intravenous albumin on fat absorption on day 2 after aminopterin. The infusion of albumin intravenously during the period of fat administration does not improve fat absorption as measured by recovery of fat in the lymph. There is an improvement in lymph flow sustained during most of the fat infusion, but the concentration of fat in the lymph decreases, resulting in no net improvement in fat recovery. The slight over-all improvement in absorption compared with figure 1 is due to all animals in this series being tested 3 days postoperatively (see text). and rose with the decline and improvement of intestinal function (figs. 1 and 2, table 1). A notable feature which has been commented upon by previous workers was the disturbance of gastrointestinal motility and fluid balance following mitotic inhibition. Even on day 1, when fat absorption was normal, gastric dilation was apparent. By day 2 the stomach was very dilated and filled with watery fluid, while the small intestine, which was also dilated, was filled with bile-stained fluid and unabsorbed fat. Diarrhea was evoked by fat infusion on day 2 but was spontaneous by day 3. It was thought that fluid loss into the intestine might have contributed to the malabsorption of fat by reducing plasma volume and depressing intestinal blood flow and lymph formation. Therefore, a group of rats were given 5% human serum albumin intravenously at 0.56 ml per hour on day 2 while fat absorption was tested in the usual way. In the group receiving albumin, lymph flow during the first 5 hr of testing was increased by 10 to 20%, (P < 0.05 at hours 2 and 4, P < 0.01 at hour 5), compared with rats not receiving albumin, but fat absorption was not improved (fig. 4). The decline in lymph flow on days 2 to 4 (figs. 1 and 2) was clearly related to the administration of fat, because during the fasting periods lymph flow on these days was greater than in control rats (table 1). There is evidence that the presence of bile in the intestine aggravates the enteropathy following X-irradiation. 28 In 4

6 January 1967 AMINOPTERIN A ND FAT ABSORPTION IN RA TS 59 FIG. 5. Thin layer chromatograph of oil. and micellar phase of intestinal content. s, Standard mixture (Harmel TLC reference mixture no. 2), mg, monoglyceride, pi, phospholipid, el, cholesterol, fa, free fatty acid, tg, triglyceride, cl-e, cholesterol ester. Developing solvent hexane-diethyl ether-acetic acid, 80:20:2 (v/v/v). The micellar phase contains fatty acid, cholesterol, and monoglyceride (see fig. 6). The oil phase contains triglyceride and diglyceride as well as monoglyceride, fatty acid, and cholesterol ester. This system does not resolve diglyceride from cholesterol or monoglyceride from phospholipid. animals with bile fistulae, the average volume of intestinal contents at the end of 8-hr saline infusion on day 3 was 2.35 ml, while in 4 animals without bile fistulae the average volume was 1.8 m!. All animals developed diarrhea and dilation of the stomach and intestine. These preliminary results are not taken as excluding a role of biliary constituents in the enteropathy following radiomimetic drugs, but further work is clearly needed. Millington et au 6 found that fat feeding aggravated the enteropathy and general clinical deterioration after aminopterin. In the present experiments, the procedure was designed so that half of the animals tested on a given day after aminopterin had received a fat infusion on the preceding day, while the other half were receiving fat for the first time and were tested again on the following day. Comparison of these subgroups revealed no deterioration in performance of rats which had received a previous dose of fat. In fact, on day 2 absorption was slightly better if the animals had received a fat infusion on the preceding day. It will be noted that the dose of aminopterin and of fat were less than those employed by Millington et au 6 Indirect evidence of an adverse effect of fat feeding was provided by the decline in lymph flow during the latter part of fat infusion on days 2, 3, and 4. Presumably, this was due to depletion of intestinal tissue fluid by increased fluid loss into the intestine. The histological changes following aminopterin will not be presented in detail since they were consistent with those described by Millington et ai.12 and others. 1, 2, 5-10 The clinical and pathological findings are correlated with fat absorption and mitotic rate in table 1. It will be noted that the enteropathy was fully de-

7 60 REDGRA VE AND SIMMONDS Vol. 62, No.1 veloped on day 2, whereas absorption reached minimal values later, on days 3 and 4. Also, mitosis was reestablished on day 4, but improvement in fat absorption was not apparent until day 5. The significance of these findings will be discussed later. Micellar lipid. There was no evidence that defective lipolysis contributed to malabsorption after aminopterin. Intes- FIG. 6. Thin layer chromatograph of micellar phase of intestinal content. fa, fatty acid, dg, diglyceride, el, cholesterol, mg, monoglyceride, m, micellar phase. Solvent system, amylacetateheptane-acetic acid, 30:70:1 (v/ v/v). This system resolves diglyceride and cholesterol and monoglyceride from phospholipid and confirms the absence of diglyceride from the micellar phase. A trace of phospholipid in the micellar phase remains at the origin. tinal contents collected during fat infusion on day 3, as previously described, gave a micellar phase containing fatty acid, monoglyceride, and cholesterol (figs. 5 and 6). Also, enzyme assay showed pancreatic lipase in considerable amounts. Finally, infusion of micellar solutions of lipid (bile salts, monoglyceride, and fatty acid) on day 3 produced no increase in lymphatic output of fat (fig. 7). Lymphatic absorption during 8-hr infusion was nil compared with 60% of the infused lipid for the same solution in normal rats These results seem to localize the defect to the absorptive cells. Disaccharide hydrolysis and absorption. While pancreatic lipolysis in the lumen was unaffected by aminopterin, the epithelial disaccharidase activity was severely depressed (table 2). There was evidence for absorption of intact disaccharide. The plasma glucose was measured 1 hr after 2 ml of 0.25 M sucrose were given intraduodenally in 4 normal rats and in 4 aminopterin-treated animals on day 3. The control values before and after mild acid hydrolysis (0.1 N HCI, 50 C, 10 min) were 178 mg per 100 ml. In the aminopterintreated group, the values were 139 and 160 mg per 100 ml, the mean increase for the four plasma samples being statistically significant (P < 0.02). Absorption of disaccharide molecules was confirmed by thin layer chromatography of uncontaminated urine (fig. 8). Discussion The measurement of lymphatic absorption during steady infusion into the duodenum of pre-emulsified fat seems to be a useful way of mapping the decline and recovery of fat absorption after aminopterin. It avoids variations in the absorptive load associated with delayed stomach emptying after an oral dose of fat. It defines the changes with time more precisely than does a fecal balance study. Thus it was possible to obtain a consistent and reproducible pattern of malabsorption and recovery while using smaller doses of aminopterin and fat than in previous studies. 13

8 January 1967 AMINOPTERIN AND FAT ABSORPTION IN RATS '--x. 61 LYMPH VOLUME 1 ML.... x 'x -III x Infusion - Level ~ FAT RECOVERY 30- )JEq./Hr , x.-- x... x 0 L control rats HOURS. aminopterin day 3 FIG. 7. Absorption of micellar fat. During the infusion of an artificial micellar solution, control rats show an increasing output of the infused lipid in the lymph to attain a level approximating that of the micellar lipid infused. On day 3 after aminopterin, there is no increase in output of fat in the lymph. This confirms that the defect in absorption is mucosal rather than lipolytic in origin. After aminopterin, lymph flow is reduced during the infusion of micellar lipid. Aminopterin, like X-irradiation, produces gross disturbances of gastrointestinal functions other than absorption, and there are morphological changes in villi and epithelial cells, in addition to arrest of mitosis (figs. 9 to 13). Morphological studies in the present experiments were less intensive than in the detailed study of Millington et ap2 It was remarkable that the distorted epithelium seen on day 2 (fig. 10) was still capable of considerable fat absorption. Also, during recovery it seemed that absorption returned to normal more rapidly than mucosal architecture. It did not appear that the disparity between histological and physiological damage TABLE 2. Disaccharidase activity of mucosal homogenates Aminopterin Day 3 b Control rats C Sucrose ± ± <0.001 Lactose ± ± <0.01 : a Results expressed as mean ± standard error. Units are micromoles of disaccharide hydrolyzed per minute per gram (wet weight) of mucosa. Comparison by Student's t-test. b n = 4. en = 5. could be attributed to portions of mucosa unaffected by aminopterin in the early P

9 62 REDGRA VE AND SIMMONDS Vol. 52, No.1 FIG. 8. Thin layer chromatograph of urinary s ugars after sucrose administration. Urinary sugars have been chromatographed from an untreated (U) control rat and an aminopterintreated (T) rat on day 3, before and after (U1, T 1) intraduodenal sucrose administration. S, sucrose standard. The e xcretion of unchanged sucrose in the urine of the aminopteriutreated rat illustrates the absorption of molecular disaccharide. stages or recovering more quickly than the rest in the later stages. Evidence suggests that, compared with the cellular defect, disturbances in motility, intraluminal digestion, or intestinal fluid balance do not contribute greatly to malabsorption of fat. In rats, inhibition of motility does not necessarily reduce fat absorption. 29,30 The intravenous albumin, containing albumin equivalent to about half of the normal circulating amount, should have improved the intestinal circulation by expanding plasma volume, and, in fact, lymph flow was improved for the first 5 hr of infusion. Nevertheless, no increase in fat absorption occurred. Other possible effects of disturbed fluid and protein metabolism were not explored. The demonstration of a micellar lipid phase of intestinal contents at the height of the malabsorption and failure to absorb a micellar solution of fat indicate that the defect is predominantly cellular; decrease in disaccharidase levels provides further support for cellular dysfunction. The disturbance of intestinal fluid balance following aminopterin merits further study. In this, as in disturbances of motil-

10 January 1967 AMINOPTERIN AND FAT ABSORPTION IN RATS 63 ity, the effects of antimitotic drugs closely resemble those of X-irradiation. Sullivan et ap8 have suggested in the case of X irradiation that there is breakdown of an intestinal barrier to water exchange. They considered that the effect was probably not attributable to bacterial invasion and found that diversion of bile reduced diarrhea and discharge of epithelial mucus. Preliminary trials in the present experiments did not suggest that diversion of bile greatly influenced enteropathy after aminopterin. Histologically, there was edema of the intestinal mucosa, and, physiologically, the decrease in lymph flow during fat infusion suggested that digestion products further altered mucosal hemodynamics and fluid balance. Whether epithelial mucin restrains water movement between mucosa and lumen ;28 whether a cellular or noncellular barrier breaks down after aminopterin and X-irradiation; and whether the disturbances of fluid balance in the intestine are due to abnormal egress of fluid into the lumen, to abnormal access of chemical or microbial irritants from the lumen to the mucosa, or to delayed cellular damage from drugs or X-rays are all questions for future experimentation. The primary aim of the present experiments was to see if a fairly close correlation was possible between changes in fat absorption, on the one hand, and the arrest and subsequent recovery of mitosis after aminopterin with consequent decline and recovery of the population of mature cells on the villi on the other hand. A consistent physiological pattern was established, and some preliminary correlations with cellular changes are possible. First, while mitosis in the crypts was promptly inhibited, fat absorption remained normal for at least 32 hr after aminopterin (first 24 hr plus 8-hr infusion). For cells in rat jejunum, the a verage time from origin in crypts to shedding from villus tip has been estimated to be approximately 30.4 hr23 or 37.7 hr for rat duodenum. 22 This indicates that intestinal cells must retain their capacity to absorb fat until they are almost due to be extruded from the villi. Second. while mitosis recovered m the crypts on day 4, absorption was still considerably depressed 24 hr later, on day 5. Progressive improvement from day 5 to day 6 was shown by the considerable output of fat overnight on day 5 and an almost normal absorption rate during the infusion period on day 6. Reasons for failure to recover completely by day 6 have been previously discussed. The epithelial cells have been demonstrated to undergo enzymic and morphological differentiation as they progress up the villus. 3! An apparent gradient of function down the villus has been observed frequently during morphological studies of fat absorption (e.g.,32) but it is possible that morphological evidence of accumulation of lipid in these cells may not be a FIGS. 9 to 13. These photomicrographs of rat jejunum illustrate the morphological changes following inhibition of mitosis with aminopterin and the subsequent regenerative process. The sections, taken after an absorptive study in each case, are stained with hematoxylin and eosin, and all are magnified X 160. FIG. 9. Section of normal gut from a control rat.

11 FIG. 10. Section of gut on day 2. The crypts are severely atrophic, and the villi are shortened and covered with a flattened epithelium. There is a diffuse cellular infiltration of the submucosa. FIG. 11. Section of gut on day 4. The crypts show early recovery with the reappearance of mitosis. The villi are still severely stunted and covered with abnormal mucosal cells. FIG. 12. Section of gut on day 5. Further recovery is now apparent in the crypts with abundant mitotic figures, and the regenerated cells are commencing to migrate onto the villi. The villi are recovering in height, but cells at the tip are vacuolated. FIG. 13. Section of gut on day 6. There is now a return to apparently normal morphology of the crypts and the villi, and normal mucosal architecture is restored. 64

12 January 1967 AMINOPTERIN AND FAT ABSORPTION IN RATS 65 true guide to functional capacity. The maximal lipid accumulation frequently seen at the tip of the villus may be related to pre-extrusion degenerative changes in these cells, such as those reported by Curran and Creamer. 33 The present experiments suggest that aminopterin should provide a useful tool in relating absorptive capacity to the age of the epithelial cell and its stage of progress up the villus. Attention is now being concentrated on the relatively rapid changes between 32 and 48 hr after aminopterin and between the latter part of day 4 and the end of day 5. Attempts at close correlation between absorptive capacity and the age profile of cells covering the villi would be ill founded if there were too great a dispersion between villi in the rate of migration and shedding of cells or between crypts in recovery of mitosis after inhibition by aminopterin. The histological findings so far do not suggest that such variability is sufficient to complicate interpretation of results. Summary The effect of aminopterin on fat absorption into the lymph of unanesthetized rats has been studied at various times after the administration of the drug. A consistent effect on fat absorption has been shown, and this has been related to the age of the mucosal cells. After inhibition of mitosis with aminopterin, fat absorption remains normal for at least 32 hr, and then there follows a period of severe malabsorption which lasts for 2 days. This malabsorption has been shown to be mucosal in origin. Mitosis in the crypts recommences on the fourth day after aminopterin, but fat absorption does not begin to recover until about 24 hr later. The results indicate that a preliminary correlation of mucosal cell age to fat absorption is possible. Newly arisen cells appear to be unable to absorb fat until they are about 24 hr old, and then they retain this ability until they are due to be shed from the tips of the villi. REFERENCES 1. Woll, E., and J. J. Oleson The effects of a folic acid antagonist (Aminopterin) on albino rats. A study in the pathogenesis of sprue. Brit. J. Exp. Path. 391: Trier, J. S Morphological alterations induced by methotrexate in the mucosa of the human proximal intestine. II. Electron microscopic observations. Gastroenterology 43: Bond, V. P Effects of radiation on intestinal absorption. Amer. J. Clin. Nutr. 12: Quastler, H Effects of irradiation on intestinal mucosal cell population. Fed. Proc.9191: Vitale, J. J., N. Zamcheck, J. DiGiorgio, and D. M. Hegsted Effects of aminopterin administration on the respiration and morphology of the gastrointestinal mucosa of rats. J. Lab. Clin. Med. 43: Wynn-Williams, A Light and electron microscopy studies of the effects of 4- aminopteroylglutamic acid on the mucous membrane of the small intestine of the rat. Gut 2: Trier, J. S Morphologic alterations induced by methotrexate in the mucosa of the human proximal intestine. I. Serial observations by light microscopy. Gastroenterology 42: Dustin, P Lesions cellulaires provoquees par les acides 4-aminopteroyl-glutamiques chez la souris. Rev. Hematol. 5: Schiraldi, 0., and R. Marano Enteropatia acuta in ratti trattati con antimetaboliti (aminopterina). Rass. Fisiopat. Clin. Ter. 33: Baserga, A., and M. Morsiani Nonregenerative enteropathy. Rass. Med. (Milan) 35: Rybak, B. J Electron microscopic studies of intestinal lesions. I. Aminopterin induced lesions in mice. Gastroenterology 42: Millington, P. F., J. B. Finean, O. C. Forbes, and A. C. Frazer Studies of the effects of aminopterin on the small intestine of rats. I. The morphologic changes following a single dose of aminopterin. Exp. Cell Res. 28: Small, M. D., R. L. Cavanagh, L. Gottlieb, P. L. Colon, and N. Zamcheck The effect of aminopterin on the absorption of xylose from the rat intestine. Amer. J. Dig. Dis. 4: Butterworth, C. E., E. Perez-Santiago, J. Martinez-De-Jesus, and R. Santini Studies on the oral and parenteral administration of D(+) xylose. New Eng. J. Med. 261: Valdivieso, U. D., and A. D. Schwabe

13 66 REDGRA VE AND SIMMONDS Vol. 52, No.1 Alteration of intestinal epithelium and medium chain fat absorption (abstr,). Clin. Res. 13: Millington, P. F., O. C. Forbes, J. B. Finean, and A. C. Frazer Studies of the effects of aminopterin on the small intestine of rats. II. Fat absorption defects following a single intramuscular injection. Exp. Cell Res. 28: Bollman, J. L., J. C. Cain, and J. H. Grindlay Techniques for the collection of lymph from the liver, small intestine or thoracic duct of the rat. J. Lab. Clin. Med. 33: Hofmann, A. F., and B. Borgstrom The intraluminal phase of fat digestion in man: the lipid content of the micellar and oil phases of intestinal content obtained during fat digestion and absorption. J. Clin. Invest. 43: Blankenhorn, D. H., and E. H. Ahrens, Jr Extraction, isolation and identification of hydrolytic products of triglyceride digestion in man. J. BioI. Chern. 212: Hofmann, A. F The function of bile salts in fat absorption. Biochem. J. 89: Kingsley, G. R, and G. Getchell Direct ultramicro glucose oxidase method for determination of glucose in biologic fluids. Clin. Chern. 6: Leblond, C. P., and C. E. Stevens The constant renewal of the intestinal epithelium in the albino rat. Anat. Rec. 100: Bertalanffy, F. D Mitotic rates and renewal times of the digestive tract epithelia in the rat. Acta Anat. 40: Stahl, E., and U. Kaltenbach Dunnschicht-chromatographie. VI. Mitteilung. Spurenanalyse von Zuckergemischen auf Kieselgur G-Schichten. J. Chromatogr. 5: Stern, I., and B. Shapiro A rapid and simple method for the determination of esterified fatty acids and for total fatty acids in blood. J. Clin. Path. 6: Mosinger, F Photometric adaptation of Dole's microd t ermination of free fatty acid. J. Lipid Res. 6: Sols, A., and G. de la Fuente Hexokinase and other enzymes of sugar metabolism in the intestine. Meth. Med. Res. 9: Sullivan, M. F., E. V. Hulse, and R H. Mole The mucus-depleting action of bile in the small intestine of the irradiated rat. Brit. J. Exp. Path. 46: Simmonds, W. J The relationship between intestinal motility and the flow and rate of fat output in thoracic duct lymph in unanaesthetised rats. Quart. J. Exp. PhysioI. 42: Bennett, S., P. Shepherd, and W. J. Simmonds The effect of alterations in intestinal motility induced by morphine and atropine on fat absorption in the rat. Aust. J. Exp. BioI. Med. Sci. 40: Padykula, H. A Recent functional interpretations of intestinal morphology. Fed. Proc. 21: Ladman, A. J., H. A. Padykula, and E. W. Strauss A morphological study of fat transport in the normal human jejunum. Amer. J. Anat. 112: Curran, R C., and B. Creamer Ultrastructual changes in some disorders of the small intestine associated with malabsorption. J. Path. Bact. 86: 1-8.