Development of the digestive system: comparative animal studies1

Transcription

1 Development of the digestive system: comparative animal studies1 Elsie M Widdowson, FRS, CBE Although all the organs and tissues of the body grow and develop in an orderly fashion in relation to the body as a whole, many of them are capable of particularly rapid growth in response to the stimulus of additional demand. This may occur even after the body has ceased to grow, and examples are the heart, the lungs, the liver, the kidneys, the gastrointestinal tract and skeletal muscles. Hypertrophy of these organs in a mature animal or man is not brought about by an increase in the number of functional units, but by an increase in the size of those already there. The number of nephrons in the kidney, of alveoli in the lungs, and of fibers in muscle, for example, is fixed early in life and cannot be increased, though each pre-existing unit can be expanded considerably in size. It has been known for some time that the gastrointestinal tract may enlarge in response to an increased food intake, for example during lactation in the rat (1). The number of villi in the intestinal mucosa does not increase, but those that are there become longer, and cell proliferation speeds up (2). Some years ago I began to think about the response of the gastrointestinal tract to its first real test, when it has to deal with the colostrum that reaches it, sometimes within minutes, after the animal is born. The digestive tract has been growing and developing before birth, and in fact a great deal of detailed morphological work has been done on the development of the digestive tract of the human fetus. Some of this work was described at a Ross Conference held in 1976 on Gastrointestinal Development and Neonatal Nutrition, and at a Ciba Foundation Conference in 1979 on Development of Mammalian Absorptive Processes (3, 4, 5). The intestine has functioned in a small way before birth in that it has absorbed water and dissolved substances from the swallowed amniotic fluid. But colostrum and milk are very different from amniotic fluid, containing as they do nutrients never before encountered-milk proteins, fat and lactose, all of which require considerably more processing than water. The whole well-being of the newborn animal and baby depends on the gastrointestinal tract being functionally capable of digesting these nutrients and absorbing the products of digestion. Anyone who has tried to handle the intestine of a newborn rat must be astonished that so fragile an organ can deal with sufficient material to enable the animal to double its weight in less than a week. Composition of colostrum and later milk differs in composition from later milk in many ways. Table 1 shows the concentration of total protein, fat and lactose in colostrum secreted during the first 24 hours and in mature milk of four species. The greatest difference between colostrum and mature milk is in protein, and the concentration of protein falls very rapidly indeed (Fig 1). Much of the protein in early cobstrum is present as soluble whey proteins, of which the major part consists of immunoglobulins. In some species, for example man, immunoglobulin A predominates; in others, for example the ruminants, immunogbobulin G forms the largest part. This is linked yith the permeability of the intestine to macromolecules at the time of birth. Before birth the intestine of all species is permeable to macromolecules and these are absorbed by pinocytosis (6). At the time of birth the barrier to protein uptake in the intestine is not completely mature in any species studied, From the Department of Medicine, University of Cambridge. 384 The American Journal of Clinical Nutrition 41: FEBRUARY 1985, pp Printed in USA 1985 American Society for Clinical Nutrition

2 DEVELOPMENT DIGESTIVE SYSTEM: ANIMALS 385 but it is more fully developed in some species than in others. In some species, for example man, the maternal -y globulins which provide the newborn with passive systemic immunity have been transferred to the fetus from the maternal circulation before birth, and the intestine of the fullterm newborn infant is relatively impermeable to protein. In other species, for exmple ruminants, and the pig, -y globulins are not transferred to the fetus before birth; the colostrum contains these antibodies and the intestine is permeable to them, so that they are absorbed, and within 2 or 3 days the concentration of them in the serum of the newborn reaches adult levels. Then the intestinal tract closes and this prevents further passage of proteins. Rodents are different again. They receive part of their passive immunity from intrauterine transport of maternal antibodies and part from selective intestinal uptake of -y globulins after birth. This goes on for 20 days, and the interesting thing is that the process is selective-the y globulins are absorbed but not other whey proteins such as albumen (6). I mentioned that the major protein in human cobostrum is -y globulin; not y globulin as in these other species. The first cobstrum secreted has about 12 g/loo ml IGA (7). By Day 5 this has fallen to 0.5 g/100 ml; and in mature milk the concentration of IGA is about 0.1%. Immunogbobulin A acts in the intestine, and limits the multiplication of bacterial and viral antigens within the digestive tract. At least three quarters of the IGA is excreted as protein in the feces. Human cobostrum, and to a smaller extent later milk, contain other substances important TABLE 1 Protein, fat and carbohydrate in colostrum (first 24 hours) and mature milk (g/loo ml) Woman Cow Mare Sow Protein Fat Lactoae E z w I- 0 a: a Concenfration of Protein in and 0 I Cow D Human DAYS AFTER BIRTH Mature FIG 1. Concentration of protein in colostrum and milk. for the infant, for example lactoferrin which binds iron, and makes it unavailable to E. coli in the intestine and inhibits bacterial growth. It contains other nutrient binding proteins, for example for zinc, vitamin Bl2 and folate, which are thought to act in a similar way to lactoferrin. These binder proteins are present also in the cobostrum of the pig and of ruminants, and in these species also they act in the gut, binding the micronutrients and so making them unavailable to bacteria (8). Other differences between cobostrum and mature milk are in sodium, vitamin A and carotenoids and vitamin Bb2. The comparatively high concentration of sodium in the first food the newborn mammal receives seems at first sight to be a rather strange provision of nature in view of the immaturity of the kidney in the neonatal period. The ionic composition of the fluid in the nonlactating mammary gland resembles that of plasma. In the lactating gland there is an active pump on the apical membrane, which pumps sodium out of the secretion. This process uses energy, as does the synthesis of lactose from glucose. The energy available depends on mammary blood flow, which is lower in the early stages of lactation than later. Less energy is available for the production of the first secretion, cobostrum, and this is more like plasma insofar as sodium is

3 386 WIDDOWSON TABLE 2 Weights of bodies of pigs (g) (8-13 animals in each group) When killed At birth After death corrected for GI contents Gain per 24 h At birth hunfed h suckled days suckled concerned, and it contains less lactose than later milk (9). The extra sodium is probably not of any benefit to the newborn animal, but it is an inevitable part of the initiation of lactation and does it no harm. Human colostrum contains 2-3 times as much vitamin A and 8 times as much carotenoids as mature milk (10). For cows the cobostrum contains 6 times as much vitamin A and 12 times as much carotenoids as milk on the 20th day. Similar differences have been found for sheep, goats and pigs. Some of the vitamin A in cobostrum is absorbed by the intestine of the newborn animal, for it has been shown that there is a rapid rise in the concentration of both vitamin A and carotenoids in the plasma of the newborn (in calves for example, to 5 and 10 times their pre-feeding values respectively). The concentration of vitamin Bl2 is five times higher in human cobostrum than in later milk (11), and there is a similar difference in the cobostrum and milk of the cow and goat (12). After weaning, these species synthesize vitamin Bb2 in the rumen, but during the suckling period they depend on an external source of the vitamin. The development of the digestive tract in response to the first feeding In order to study the development of the digestive tract in response to the first feed after birth, I chose piglets as my experimental animals. Their intestines are easier to handle than those of newborn rats, and pigs have large litters, so the young in a litter can be divided among several experimental groups. The stage of development at birth is somewhat similar to that of man, though the rate of growth is much faster, and piglets begin to feed directly after birth; in fact the first to be born are often feeding before the last has been delivered. We used 38 piglets from 8 litters, all weighing over 1 kg at birth, and the members of each litter were divided into 4 groups. One or two members of each litter were killed at birth before being fed. A second one or two were removed before they had fed and were given 20 ml of water by stomach tube every 4 hours for 24 hours. This meant spending the night with the piglets, but we were quite used to that. All the remainder of the litter were put back to the sow and allowed to suckle normally. One or two were killed after 24 hours, along with those that had received only water, and the remainder were killed after 10 days. Table 2 shows the mean weights of the animals in the four groups, at birth and at the time of killing (13). The mean weights were satisfactorily similar at birth. The suckled piglets gained weight during the first 24 hours; those unfed but receiving water lost a little. The gain per 24 h was considerably greater between days 1 and 9 than it was during the first day. Table 3 shows the weight of the stomach. It increased in weight by 28% during the first 24 h and 23% per 24 h over the next 9 days. By 10 days it weighed 3#{189} times as much as it had done at birth. It has been reported in the infant also that during the first 24 h the stomach grows more rapidly than the body as a whole. Table 4 gives the same information about the duodenum. It also gained weight very rapidly, gaining 42% of its weight at birth during the first 24 hours. It went on growing fast-more rapidly than the stomach, so its weight at 10 days was nearly 5 times the weight at birth. Table 5 shows the weight of the jejunum. Here the growth is even more remarkable, particularly during the first 24 hours when it increased in weight by 70%. Then the rate of growth slowed down and TABLE 3 Weight of stomach g Weight Gain per 24 h At birth h unfed h suckled days suckled

4 DEVELOPMENT DIGESTIVE SYSTEM: ANIMALS 387 TABLE 4 Weight of duodenum g TABLE 6 Weight of ileum g Gain per 24 h At birth h unfed h suckled days suckled Weight Gain per 24 h At birth h unfed h suckled days suckled the weight at 10 days was about 4 times the weight at birth. The response of the ileum to the first food is similar to that of the jejunum (Table 6), a very rapid gain in weight during the first 24 hours and then a slower gain for the next 9 days so that, like the jejunum, the weight at 10 days was 4 times what it was at birth. The large intestine also grew rapidly, but at a steady rate over the first 10 days (Table 7). All parts of the intestine grew in length as well as in weight. Table 8 shows the mean values for the jejunum, which added 22% to its length in 24 hours, and the ileum (Table 9) grew in length by 24%. Thus all parts of the digestive tract respond to food by growing much more rapidly than the body as a whole, and the growth of the jejunum and ileum are particularly remarkable. We separated the jejunal mucosa from the muscle and found that, although both parts grew, the mucosa gained weight more rapidly so that its weight doubled in 24 hours (Table 10). We analyzed the mucosa for nitrogen and found that this, and hence the protein, more than doubled during this short period of time. The amount of DNA in the whole jejunal mucosa also increased from 46.4 mg at birth, to 65.2 mg at 24 hours, but this increase was proportionally less than the protein. We did not examine the intestine histologically, so I cannot say whether there was any increase in the number of villi, or whether all the growth was brought about by lengthening of the villi already there at birth. We TABLE 5 Weight of jejunum g did, however, look at the secretion of mucus from the goblet cells (14). At birth the piglet has goblet cells in each part of its small intestine, some of which are ready to discharge and some of which are immature. The mature goblet cells discharged their mucm in response to food during the first 24 hours and a new generation of cells developed. This process was much more active in the duodenum and ileum than in the jejunum, and it did not take place in piglets that were given water instead of cobostrum. I first presented these results at a Ross Symposium in Dr Heird from New York was there, and he was inspired to make a study on another species-beagle puppies. He found, as we did, with these animals there was a rapid increase in weight of the intestinal mucosa of suckled puppies during the first 24 hours and an increase in the amounts of protein and DNA (15). Some of their puppies were fed artificial bitch milk, presumably based on cows milk. These puppies gained as much body weight as those that were suckled, but there was no appreciable growth of the intestinal mucosa. This led the authors to suggest that cobostrum contains a growth factor specific for intestinal mucosa. Both the pig and the dog are species in which immunogbobulin G in the cobostrum is transferred through the gut during the first 1-2 days after birth. I was a little worried that the increase in protein we observed in the jejunal mucosa of these two species might be partly accounted for by ICC in process TABLE 7 Weight of large intestine g Gain per 24 h At birth h unfed h suckled lodayssuckled Weight Gain per 24 h At birth h unfed h suckled days suckled

5 388 WIDDOWSON TABLE 8 Length of jejunum cm TABLE 10 Weight of jejunal mucosa and of protein in it Length Gain per 24 h At birth hunfed hsuckled lodayssuckled of being absorbed. We showed many years ago that the newborn piglet absorbs so much protein in this way that in 24 hours it was able to incrase the total globulin in its plasma from 0.9 to 3.6 g/100 ml (16). At the same time it increased its plasma volume by 30%, and the total amount of globulin absorbed and added to its plasma during the first 24 hours amounted to 3.6 g or 150 mg an hour or 25 mg a minute. In the later experiments the increase in total protein in the jejunal mucosa in 24 hours was 1.4 g. I do not know anything about the rate of absorption or how much globulin was passing through the intestine at any one time, and we did not separate the proteins in the mucosa. Because of this difficulty I decided to repeat the study on another species and I chose the rabbit because it, like the human baby, receives its passive immunity before birth. The rabbit, however, has a rather unusual way of suckling its young. The doe builds a nest and lines it with fine hair plucked from her abdomen. This hair, under the influence of estrogens, becomes loose. The litter is completely covered inside the nest, and the doe visits them only once a day, usually at a regular time, and spends 3-5 minutes suckling her young, during which time they take sufficient milk to last them for the next 24 hours. How long after birth the young have their first feed is variable. It may be within a few hours, but more often it is between 16 and 20 hours (17). In a previous study (18) we weighed the stomachs and small and lrge intestines of rabbits at birth and at 3 dars, and we found TABLE 9 Length of ileum cm Length Gain per 24 h At birth hunfed hsuckled days suckled Weight Gain per 24 It Weight of mucosa g At birth h unfed h suckled days suckled Protein in mucosa mg At birth h unfed hsuckled days suckled that the stomach had increased in weight 2#{189} times during the first 3 days. The rapid increase in weight of the stomach is in line with the functional demands placed upon it, for by 3 days it has to hold milk weighing a quarter as much as the young animal it is nourishing. The clotted milk is gradually passed from the stomach, which acts as a reservoir, into the duodenum over the next 24 hours. The inrease in weight of the stomach over the first 24 hours was much smaller (19), and this was probably because no food had entered it until towards the end of the time. The small and large intestines increased in weight considerably during the first 24 hours, which suggests that possibly the stomach of the rabbit enlarges in response to the load put upon it, whereas the small intestine responds to quite small amounts of colostrum and the growth factor in it. I do not propose to say much about enzymes as Dr Kretchmer is covering this aspect of development. However I should like to make three points. First, it has sometimes been reported that activity of the digestive enzymes falls after birth. The conventional method of expressing enzymes measured in the intestinal mucosa is as so-called specific activity-that is, activity per unit weight of protein. But when protein in the mucosa is 2In reply to the question why the colostral immunoglobulins are not broken down by the digestive enzymes, Dr Kretchmer made two suggestions. They are glycoproteins and it is possible that the carbohydrate protects them from digestion. Alternatively, and more likely, it may be that their amino acid sequence, and particularly the position of the aromatic amino acid is such that the peptide linkage between carboxyl and amino groups cannot be broken down by pepsin or chymotrypsin.

6 DEVELOPMENT DIGESTIVE SYSTEM: ANIMALS 389 increasing as rapidly as it is in piglets and puppies during the first 24 hours, this method of expression can be quite misleading. Table 11 illustrates this for lactase. When expressed per mg protein the lactase activity fell from 347 to 208 units, but in fact the total lactase activity of the jejunal mucosa rose from 290 to 443 units (13). Heird and Hansen (15) made a similar observation on their puppies. My second point about digestive enzymes concerns lipase. We have known for some long time that the activity of pancreatic lipase is low in the newborn period and it was a puzzle how young infants and animals could break down the large amounts of fat in cobostrum and milk. Then in the early 1970 s Margit Hamosh came along with her lingual lipase, and she summarized her work at the Ciba Symposium (20). She showed that in both the newborn rat and the human infant lingual serous glands, or von Ebner s glands, secrete a potent lipase that hydrolyzes triglycerides to a mixture of di- and monoglycerides, glycerol and free fatty acids. Sucking probably facilitates secretion and the enzyme is swallowed with the milk and acts in the stomach. It is independent of bile salts and has a low ph optimum. There is some secretion of the enzyme before birth, but secretion rises very rapidly indeed in response to food. Suckling ruminants too secrete lingual lipase and in fact studies had been made on calves 20 years earlier, to find out how they were able to digest the fat of milk, when after weaning they have very little fat in their diets (21, 22). The enzyme was called pregastric esterase. Its activity was shown to be high in young calves, to decline sharply when the animals were weaned, and to be greatly enhanced by sucking. There seems no doubt that lingual lipase is quantitatively the most important lipase for the digestion of fat by the sucking infant and animal. But there is TABLE 11 Effect of feeding on the activity of lactase in the jejunal mucosa Activity units/g protein Total activity units At birth h unfed h suckled days suckled TABLE 12 Protein in organs and muscles of newborn piglets mg Birth 24hunfed 24hsuckled Quadriceps muscle Gastrocnemius muscle Kidneys Liver Heart Lungs Spleen Pancreas yet another lipase, which seems peculiar to man and possibly the gorilla. This is a lipase contained in the milk, and bile salts are essential for its activity (23). Why primates, at any rate the higher ones, should be blessed in this way is an aspect of evolution we know nothing about. My third point about enzymes concerns the proteolytic ones. How do the immunoglobulins, both A and G, in cobostrum manage to escape digestion? I can find very little in the literature about this. Some protein must be broken down in the intestine and the amino acids used for growth, for even during the first 24 hours after birth the internal organs and skeletal muscles of the pig grew, and the amount of protein in them increased (24) (Table 12). This was true of all parts of the body we studied-the skeletal muscles, the kidneys, the liver, the heart, the lungs, the spleen and the pancreas. I do not know whether the proteolytic enzymes in the intestine can selectively break down casein and leave the whey proteins undigested, or whether proteolytic enzyme activity is low during the first 24 hours. The pancreas of the piglets increased in weight more rapidly than any other organ in response to cobstrum. There was a rapid incorporation of protein but no significant change in DNA. The magnitude of the change in proteinan increase of over 60% in 24 hours-suggests that it was the acini of the serous cells that were growing so fast in response to food. We made no measurements of the tryptic activity of pancreatic secretion during that time. There are many things still to be done and to be discovered about the adaptation of the digestive tract to the first food after birth. Some aspects of it have become highly specialized, the enzymes involved in membrane

7 390 WIDDOWSON and intraluminal digestion, for example, and the role of a variety of hormones in controlling the development and function of the gut. I shall leave these confidently to both Dr Aynsley-Green and Dr Kretchmer B References 1. Fell BF, Smith KA, Campbell RM. Hypertrophic and hyperplastic changes in the alimentary canal of the lactating rat. J Path Bact l963;85:l Cairnie AB, Bentley RE. Cell proliferation studies in the intestinal epithelium of the rat. Hyperplasia during lactation. Exp Cell Res l964;l8: Moxey PC. Morphological development of the human fetal small intestine. In: Gastrointestinal Development and Neonatal Nutrition, Report of the Seventy-second Ross Conference on Pediatric Research. Columbus, OH: Ross Laboratories, 1977: Trier is, Moxey PC. Morphogenesis of the small intestine during fetal development. In: Development of Mammalian Absorptive Processes. Amsterdam: Ciba Foundation Symposium 40 (NS), Excerpta Medica, 1979: Moog F. The differentiation and redifferentiation of the intestinal epithelium and its brush border membrane. In: Development of Mammalian Absorptive Processe& Amsterdam: Ciba Foundation Symposium 70 (NS), Excerpta Medica, 1979: Walker WA. Gastrointestinal host defence: importance of gut closure in control of macromolecular transport. In: Development of Mammalian Absorptive Processes. Amsterdam: Ciba Foundation Symposium 70 (NS), Excerpta Medica, 1979: McClelland DBL, McGrath J, Samson RR. Antimicrobial factors in human milk Acta Paediat Scand 1978; Supplement Ford JE. Some observations on the possible nutritional significance of vitamin B12 and folate binding proteins in milk. Brit J Nutr 1974;3l: Linzell JL, Peaker M. Mechanism of milk secretion. Physiol Rev 197l;5 1: Moore T. Vitamin A. Amsterdam: Elsevier, Samson RR, McClelland DBL. Vitamin Bl2 in human colostrum and milk. Acta Paediat Scand l980;69: Gregory ME. The microbiological assay of vitamin Bl2 in the milk of different animal species. Brit J Nutr l954;8: Widdowson EM, Colombo VE, Artavanis CA. Changes in the organs of pigs in response to feeding for the first 24 hours after birth. II. The digestive tract. Biol Neonate l97623:272-8l. 14. Stoddart RW, Widdowson EM. Changes in the organs of pigs in response to feeding for the first 24 hours after birth. III. Fluorescence histochemistry of the carbohydrates in the intestine. Biol Neonate : Heird WC, Hansen IH. Effect of colostrum on growth of intestinal mucosa. Pod Res l977;l 1: McCance RA, Widdowson EM. The effect of cobstrum on the composition and volume of the plasma of newborn piglets. J Physiol l959;l45: Venge 0. The influence of nursing behaviour and milk production on early growth in rabbits. Anim Behav l953;ll: Davies is, Widdowson EM, McCance RA. The intake of milk and the retention of its constituents while the newborn rabbit doubles its weight. Brit J Nutr 1964;l8: Hall RA, Widdowson EM. Response of the organs of rabbits to feeding during the first days after birth. Biol Neonate 1979;35:l Hamosh M. The role of lingual lipase in neonatal fat digestion. In: Development of Mammalian Absorptive Processes. Amsterdam: Ciba Foundation Symposium 70 (NS), Excerpta Medica, 1979: Ramsey HA, Wise GH, Tove SB. Esterolytic activity of certain alimentary tissues from cattle in different age groups. J Dairy Sci 1956;39:l Grosskopf JFW. Studies on salivary lipase in young ruminants. Onderstepoort J Vet Res l965;32: Hall BM. Lipid biochemistry of human milk. PhD Thesis, University of London, Widdowson EM, Crabb DE. Changes in the organs of pigs in response to feeding for the first 24 hours after birth. I. The internal organs and muscles. Biol Neonate l976;28:26l-7 1.