163
Early Human Development, 31 (1992) 163-166 Elsevier Scientific Publishers Ireland Ltd. EHD 01352
Gastric secretory function in the developing human stomach Eric J. Kelly a,Keith G. Brownleeb and Simon J. Newellb ‘Department
of Anatomy,
University of Leeds and bDepartment of Paediatrics St. James’ University Hospital, Lee& (UK)
and Child Health,
(Received 14 July 1992; revision received 1 September 1992; accepted 2 September 1992)
Little data exists regarding the activity of gastric parietal cells in the very immature infant. Therefore we have examined the developing human stomach for the presence and location of parietal cells, using both standard histological methods and antibodies to the H+/K+ ATPase (proton pump) and intrinsic factor, in 35 fetuses (ranging from 13-28 weeks) and in five infants (2-21 weeks). Parietal cell activity was noted in the body, antrum and pyloric regions in all the fetal specimens examined. However, this activity was much more limited in the infant specimens. We have noted that from the end of the first trimester parietal cells are present in a mature, functional form with the potential to secrete both gastric acid and intrinsic factor. Key words: stomach; development; parietal cells; G-cells; fetus
Introduction The secretion of hydrochloric acid and intrinsic factor are important gastric functions. Acid is required both for the initiation of digestion and its role in the luminal immune system and intrinsic factor is essential for the absorbtion of the dietary cobalamin necessary for erythropoiesis. These functions are essential components of the newborn infant, most notably in the immature, preterm, infant where the provision of adequate early nutrition is of paramount importance [l]. These infants have greater protein requirements [2] and are at higher risk of macrocytic anaemia, than their term counterparts [3]. An understanding of the ontogeny of digestion is therefore central to our approach to enteral nutrition in the premature infant. Correspondence to: Eric J. Kelly, The Neonatal Unit, St. James’ University Hospital, Leeds LS9 7TF, UK.
0378-3782/92/$05.00 0 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
164
Gastric acid has been demonstrated during the first hour of life in both term and preterm infants from 33 weeks gestation [4]. Conclusive data, however, are not available for acid secretion in more immature infants in whom nutrition currently presents its greatest challenge. There have also been no previously published studies of intrinsic factor secretion in newborn infants. Immunohistochemistry now allows the localisation of components of parietal cells using monoclonal antibodies to specific parietal cell antigens. Combining this with conventional histological techniques we aimed to observe the distribution of parietal cells with advancing gestation and then to determine the presence of the hydrogenpotassium ATPase and intrinsic factor synthesis. Method Thirty-five fetal and five infant stomachs were obtained from the University Departments of Pathology and Obstetrics and Gynaecology, St. James’s University Hospital, Leeds. The specimens resulted from therapeutic abortions, miscarriages and cot deaths. Fetal gestation was calculated in weeks since the last menstrual period and confirmed by antenatal ultrasound assessment and fetal foot length measurements and ranged from 13 to 28 weeks (Table I), Infants who were studied following unexplained sudden infant death were all born at term and were 2, 4, 10, 13 and 21 weeks old, respectively. The specimens were obtained as close to death as possible and immersed in buffered formalin for a minimum of 4 days to ensure complete fixation. The distal pylorus and the anatomical antro-corpeal boundary (indicated by the point where the nerve of Latarjet crosses the lesser curvature) was marked with indian ink on both sides of the specimens. Strips were cut to include carpal, antral and pyloric mucosa from each stomach, these underwent standard histological processing and sections 8 pm thick were cut and stained using haematoxylin and eosin and the peroxidase anti-peroxidase technique of Stemberger [5]. Specific murine monoclonal antibodies were used to bind to intrinsic factor and the hydrogen-potassium ATP-ase (Dr. Smolka, University of South Carolina, Charleston), the second antibodies were raised in goat and complemented with rabbit peroxidase anti-peroxidase complex (Sigma, Poole). The sections were stained with 3,3 diamino benzidine and counterstained with methyl green. A negative control was performed for all sections in which the primary antibody was omitted. All sections were viewed under a Leitz Dialux 20 EB microscope using x 50 and x 100 objectives.
TABLE I The maturity of the fetuses studied. Gestation (weeks)
13
15
16
18
19
20
21
22
23
24
25
26
21
28
No. of specimens
1
1
2
2
6
4
5
3
2
1
2
1
I
1
16.5
All of the specimens had intact gastric mucosa which allowed the identification of parietal cells, these were detected on all haematoxylin and eosin stained specimens, from fetuses of 13 weeks to term infants with a post-natal age of 21 weeks. There was, however, a large difference in the distribution of parietal cells between the fetal and infant specimens. In all fetal specimens parietal cells extended from the body, through the antrum and into the pyloric region of the stomach. In the five infant stomachs extension of parietal cells into the antrum and pylorus was more limited (Table II). In two infants parietal cells were limited to the body, in two, parietal cells were found in the antrum and in one, the oldest, parietal cells were noted to extend into the pyloric mucosa. All specimens stained positive for intrinsic factor indicating that it is being synthesised by the parietal cell from at least 13 weeks gestation and for hydrogenpotassium ATP-ase indicating that it too is present from at least the end of the first trimester. The location of both antigens corresponded precisely with the location of parietal cells seen with haematoxylin and eosin staining. None of the negative controls showed any staining following the application of 3,3 diamino benzidine.
The most important finding of this study is that the parietal cell, with its specialised function of acid and intrinsic factor secretion, is present from 13 weeks of gestation with the functional capacity to synthesise intrinsic factor and with the hydrogen-potassium ATPase, a large transmembrane protein complex responsible for hydrogen ion secretion [6], in place. Previous investigators have noted that parietal cells are present in fetal stomachs from 11 gestational weeks [7] as differentiated epithelial cells which resemble adult parietal cells in both structure and histochemical staining [8]. In 1971, using a radioimmunoassay technique, Schwarz and Weber [9] suggested that intrinsic factor may be present in fetal stomach extracts from at least 13 weeks of gestation. We have shown, using immunohistochemistry, not that the framework
TABLE II Parietal cell localisation in the five infant stomachs examined. Age (weeks)
Body
Antrum
Pylorus
2 4 10 13 21
+ + + + +
+ + +
+
The presence of parietal cells is indicated by +, the absence by -.
166
for intrinsic factor production is in place, but that it is being synthesised by the parietal cell from the end of the first trimester, indicating that the fetus is potentially capable of intrinsic factor secretion and therefore absorbing cobalamin for erythropoiesis during the second and third trimesters. As less than 5% of secreted intrinsic factor needs to bind vitamin B t2 and be absorbed for adequate erythropoiesis it seems likely that the newborn infant is able to adequately absorb oral vitamin Br2 [lo]. There is great debate as to when the infant first produces gastric acid, in premature infants acid secretion has not been demonstrated at less than 33 weeks gestation [4]. However, as long ago as 1929, Lucas Keene and Hewer found that hydrochloric acid was present in the contents of fetal stomachs above 19 gestational weeks and in stomach extracts from 24 gestational weeks [l 11. Only the oldest infant showed a similar distribution of parietal cells to the fetal specimens, the other four agreed with the ‘typical’ adult distribution [ 121 indicating that a change in location may occur in the third trimester. The cause of this alteration is unknown but may be due to neuronal or hormonal influences. Our results indicate that even the most immature infant has the mechanisms necessary for acid and intrinsic factor secretion and future studies in premature infants ought to confirm this. Acknowledgement The authors would like to thank Dr Adam Smolka, Department of Anatomy, University of South Carolina, Charleston, South Carolina for the generous gift of antibody to both intrinsic factor and hydrogen-potassium ATPase. References 1
Lucas, A., Morley, R. and Cole, T.J. (1990): Early diet in pretetm babies and developmental status at 18 months. Lancet, 35, 1477-1481. 2 ESPGAN (1987): Committee on nutrition of the pre-term infant. Nutrition and feeding of the preterm infant. Acta Paediatr. Scand., Suppl., 330. 3 Stockman, ILL, J.A. (1977): Anemia of prematurity. Clin. Perinatol., 4, 239-257. 4 Euler, A.R., Bryne, W.J., Meis, P.J., Leake, R.D. and Ament, M.E. (1979): Basal and pentagastrin stimulated acid secretion in newborn human infants. Paediatr. Res., 13, 36-37. 5 Stemberger, L.A. (1979): Immunocytochemistry, 2nd edn., Wiley, New York. 6 Smolka, A., Helander, H.F. and Sachs, G.(1983): Monoclonal antibodies against gastric H+ + K+ ATPase. Am. J. Physiol., 245, G589-596. 7 Salenius, P. (1962): On the ontogenesis of the human gastric epithelial cells. Acta. Anat. (Basel), 46, l-76. 8 Normura, Y. (1966): On the submicroscopic morphogenesis of parietal cells in the gastric gland of the human foetus. Z. Anat. Entwicklungsgeschichte (Berlin), 125, 316-322. 9 Schwarz, M. and Weber, J. (1971): Gastric intrinsic factor in the human foetus. Stand. J. Gastroenterol., Suppl., 9, 57-59. 10 Molin, D.L. (1959): Radioactive vitamin Bts in the study of blood diseases. Br. Med. Bull., 15, 8-18. II Lucas Keene, M.F. and Hewer, E.E. (1929): Digestive enzymes in the human foetus. Lancet, i, 767-769. 12 Naik, K.S., Lagopoulos, M. and Primrose, J.N. (1990): Distribution of antral G-cells in relation to the parietal cells of the stomach and anatomical boundaries. Clin. Anat., 3, 17-24.
Early Human Development, 31 (1992) 163-166 Elsevier Scientific Publishers Ireland Ltd. EHD 01352
Gastric secretory function in the developing human stomach Eric J. Kelly a,Keith G. Brownleeb and Simon J. Newellb ‘Department
of Anatomy,
University of Leeds and bDepartment of Paediatrics St. James’ University Hospital, Lee& (UK)
and Child Health,
(Received 14 July 1992; revision received 1 September 1992; accepted 2 September 1992)
Little data exists regarding the activity of gastric parietal cells in the very immature infant. Therefore we have examined the developing human stomach for the presence and location of parietal cells, using both standard histological methods and antibodies to the H+/K+ ATPase (proton pump) and intrinsic factor, in 35 fetuses (ranging from 13-28 weeks) and in five infants (2-21 weeks). Parietal cell activity was noted in the body, antrum and pyloric regions in all the fetal specimens examined. However, this activity was much more limited in the infant specimens. We have noted that from the end of the first trimester parietal cells are present in a mature, functional form with the potential to secrete both gastric acid and intrinsic factor. Key words: stomach; development; parietal cells; G-cells; fetus
Introduction The secretion of hydrochloric acid and intrinsic factor are important gastric functions. Acid is required both for the initiation of digestion and its role in the luminal immune system and intrinsic factor is essential for the absorbtion of the dietary cobalamin necessary for erythropoiesis. These functions are essential components of the newborn infant, most notably in the immature, preterm, infant where the provision of adequate early nutrition is of paramount importance [l]. These infants have greater protein requirements [2] and are at higher risk of macrocytic anaemia, than their term counterparts [3]. An understanding of the ontogeny of digestion is therefore central to our approach to enteral nutrition in the premature infant. Correspondence to: Eric J. Kelly, The Neonatal Unit, St. James’ University Hospital, Leeds LS9 7TF, UK.
0378-3782/92/$05.00 0 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
164
Gastric acid has been demonstrated during the first hour of life in both term and preterm infants from 33 weeks gestation [4]. Conclusive data, however, are not available for acid secretion in more immature infants in whom nutrition currently presents its greatest challenge. There have also been no previously published studies of intrinsic factor secretion in newborn infants. Immunohistochemistry now allows the localisation of components of parietal cells using monoclonal antibodies to specific parietal cell antigens. Combining this with conventional histological techniques we aimed to observe the distribution of parietal cells with advancing gestation and then to determine the presence of the hydrogenpotassium ATPase and intrinsic factor synthesis. Method Thirty-five fetal and five infant stomachs were obtained from the University Departments of Pathology and Obstetrics and Gynaecology, St. James’s University Hospital, Leeds. The specimens resulted from therapeutic abortions, miscarriages and cot deaths. Fetal gestation was calculated in weeks since the last menstrual period and confirmed by antenatal ultrasound assessment and fetal foot length measurements and ranged from 13 to 28 weeks (Table I), Infants who were studied following unexplained sudden infant death were all born at term and were 2, 4, 10, 13 and 21 weeks old, respectively. The specimens were obtained as close to death as possible and immersed in buffered formalin for a minimum of 4 days to ensure complete fixation. The distal pylorus and the anatomical antro-corpeal boundary (indicated by the point where the nerve of Latarjet crosses the lesser curvature) was marked with indian ink on both sides of the specimens. Strips were cut to include carpal, antral and pyloric mucosa from each stomach, these underwent standard histological processing and sections 8 pm thick were cut and stained using haematoxylin and eosin and the peroxidase anti-peroxidase technique of Stemberger [5]. Specific murine monoclonal antibodies were used to bind to intrinsic factor and the hydrogen-potassium ATP-ase (Dr. Smolka, University of South Carolina, Charleston), the second antibodies were raised in goat and complemented with rabbit peroxidase anti-peroxidase complex (Sigma, Poole). The sections were stained with 3,3 diamino benzidine and counterstained with methyl green. A negative control was performed for all sections in which the primary antibody was omitted. All sections were viewed under a Leitz Dialux 20 EB microscope using x 50 and x 100 objectives.
TABLE I The maturity of the fetuses studied. Gestation (weeks)
13
15
16
18
19
20
21
22
23
24
25
26
21
28
No. of specimens
1
1
2
2
6
4
5
3
2
1
2
1
I
1
16.5
All of the specimens had intact gastric mucosa which allowed the identification of parietal cells, these were detected on all haematoxylin and eosin stained specimens, from fetuses of 13 weeks to term infants with a post-natal age of 21 weeks. There was, however, a large difference in the distribution of parietal cells between the fetal and infant specimens. In all fetal specimens parietal cells extended from the body, through the antrum and into the pyloric region of the stomach. In the five infant stomachs extension of parietal cells into the antrum and pylorus was more limited (Table II). In two infants parietal cells were limited to the body, in two, parietal cells were found in the antrum and in one, the oldest, parietal cells were noted to extend into the pyloric mucosa. All specimens stained positive for intrinsic factor indicating that it is being synthesised by the parietal cell from at least 13 weeks gestation and for hydrogenpotassium ATP-ase indicating that it too is present from at least the end of the first trimester. The location of both antigens corresponded precisely with the location of parietal cells seen with haematoxylin and eosin staining. None of the negative controls showed any staining following the application of 3,3 diamino benzidine.
The most important finding of this study is that the parietal cell, with its specialised function of acid and intrinsic factor secretion, is present from 13 weeks of gestation with the functional capacity to synthesise intrinsic factor and with the hydrogen-potassium ATPase, a large transmembrane protein complex responsible for hydrogen ion secretion [6], in place. Previous investigators have noted that parietal cells are present in fetal stomachs from 11 gestational weeks [7] as differentiated epithelial cells which resemble adult parietal cells in both structure and histochemical staining [8]. In 1971, using a radioimmunoassay technique, Schwarz and Weber [9] suggested that intrinsic factor may be present in fetal stomach extracts from at least 13 weeks of gestation. We have shown, using immunohistochemistry, not that the framework
TABLE II Parietal cell localisation in the five infant stomachs examined. Age (weeks)
Body
Antrum
Pylorus
2 4 10 13 21
+ + + + +
+ + +
+
The presence of parietal cells is indicated by +, the absence by -.
166
for intrinsic factor production is in place, but that it is being synthesised by the parietal cell from the end of the first trimester, indicating that the fetus is potentially capable of intrinsic factor secretion and therefore absorbing cobalamin for erythropoiesis during the second and third trimesters. As less than 5% of secreted intrinsic factor needs to bind vitamin B t2 and be absorbed for adequate erythropoiesis it seems likely that the newborn infant is able to adequately absorb oral vitamin Br2 [lo]. There is great debate as to when the infant first produces gastric acid, in premature infants acid secretion has not been demonstrated at less than 33 weeks gestation [4]. However, as long ago as 1929, Lucas Keene and Hewer found that hydrochloric acid was present in the contents of fetal stomachs above 19 gestational weeks and in stomach extracts from 24 gestational weeks [l 11. Only the oldest infant showed a similar distribution of parietal cells to the fetal specimens, the other four agreed with the ‘typical’ adult distribution [ 121 indicating that a change in location may occur in the third trimester. The cause of this alteration is unknown but may be due to neuronal or hormonal influences. Our results indicate that even the most immature infant has the mechanisms necessary for acid and intrinsic factor secretion and future studies in premature infants ought to confirm this. Acknowledgement The authors would like to thank Dr Adam Smolka, Department of Anatomy, University of South Carolina, Charleston, South Carolina for the generous gift of antibody to both intrinsic factor and hydrogen-potassium ATPase. References 1
Lucas, A., Morley, R. and Cole, T.J. (1990): Early diet in pretetm babies and developmental status at 18 months. Lancet, 35, 1477-1481. 2 ESPGAN (1987): Committee on nutrition of the pre-term infant. Nutrition and feeding of the preterm infant. Acta Paediatr. Scand., Suppl., 330. 3 Stockman, ILL, J.A. (1977): Anemia of prematurity. Clin. Perinatol., 4, 239-257. 4 Euler, A.R., Bryne, W.J., Meis, P.J., Leake, R.D. and Ament, M.E. (1979): Basal and pentagastrin stimulated acid secretion in newborn human infants. Paediatr. Res., 13, 36-37. 5 Stemberger, L.A. (1979): Immunocytochemistry, 2nd edn., Wiley, New York. 6 Smolka, A., Helander, H.F. and Sachs, G.(1983): Monoclonal antibodies against gastric H+ + K+ ATPase. Am. J. Physiol., 245, G589-596. 7 Salenius, P. (1962): On the ontogenesis of the human gastric epithelial cells. Acta. Anat. (Basel), 46, l-76. 8 Normura, Y. (1966): On the submicroscopic morphogenesis of parietal cells in the gastric gland of the human foetus. Z. Anat. Entwicklungsgeschichte (Berlin), 125, 316-322. 9 Schwarz, M. and Weber, J. (1971): Gastric intrinsic factor in the human foetus. Stand. J. Gastroenterol., Suppl., 9, 57-59. 10 Molin, D.L. (1959): Radioactive vitamin Bts in the study of blood diseases. Br. Med. Bull., 15, 8-18. II Lucas Keene, M.F. and Hewer, E.E. (1929): Digestive enzymes in the human foetus. Lancet, i, 767-769. 12 Naik, K.S., Lagopoulos, M. and Primrose, J.N. (1990): Distribution of antral G-cells in relation to the parietal cells of the stomach and anatomical boundaries. Clin. Anat., 3, 17-24.
