Comparative Biochemistry and Physiology Part A 135 (2003) 425–433 Effects of inhibition gastric acid secretion on arterial acid–base status during digestion in the toad Bufo marinus Johnnie B. Andersena,*, Denis V. Andradeb, Tobias Wanga of Zoophysiology, Institute of Biological Sciences, University of Aarhus, Universitetsparken Building 131, bDepartment de Zoologia, Universidade Estadual Paulista, Rio Claro SP 13506-900, Brazil Received 18 December 2002; received in revised form 8 April 2003; accepted 9 April 2003 Abstract
Digestion affects acid–base status, because the net transfer of HCl from the blood to the stomach lumen leads to an HCO3 levels in both extra- and intracellular compartments. The increase in plasma w tide, is particularly pronounced in amphibians and reptiles, but is not associated with an increased arterial pH, becauseof a concomitant rise in arterial PCO2 caused by a relative hypoventilation. In this study, we investigate whether thepostprandial increase in PaCO2 of the toad Bufo marinus represents a compensatory response to the increased plasmaw or a state-dependent change in the control of pulmonary ventilation. To this end, we successfully prevented the alkaline tide, by inhibiting gastric acid secretion with omeprazole, and compared the response to that of untreated toadsdetermined in our laboratory during the same period. In addition, we used vascular infusions of bicarbonate to mimicthe alkaline tide in fasting animals. Omeprazole did not affect blood gases, acid–base and haematological parameters infasting toads, but abolished the postprandial increase in plasma w and the rise in arterial PCO2 that normally peaks 48 h into the digestive period. Vascular infusion of HCO3 , that mimicked the postprandial rise in plasma HCO3 , led to a progressive respiratory compensation of arterial pH through increased arterial PCO2. Thus, irrespective of whether the metabolic alkalosis is caused by gastric acid secretion in response to a meal or experimental infusion ofbicarbonate, arterial pH is being maintained by an increased arterial PCO2. It seems, therefore, that the elevated PCO2,occuring during the postprandial period, constitutes of a regulated response to maintain pH rather than a state-dependentchange in ventilatory control.
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Keywords: Toad; B. marinus; Digestion; Acid–base status; Alkaline tide; Gastric acid secretion; Omeprazole; Ventilatory control 1. Introduction
. Digestion causes a rise in metabolicrate, the ‘specific dynamic action of food’ (SDA), Many ectothermic vertebrates eat large meals at infrequent intervals and the ensuing digestion is presence of food in the stomach stimulate a net HCl secretion from the blood to the stomach lumenthat leads to an increase in HCO3 , the so-called ‘alkaline tide’, is 1095-6433/03/$ - see front matter ᮊ 2003 Elsevier Science Inc. All rights reserved.
doi:10.1016/S1095-6433(03)00108-9 J.B. Andersen et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 425–433 pronounced in amphibians and reptiles, but the immersion into a 1.0 g ly1 benzocaine solution (ethyl p-amino benzoate Sigma᭨ E 1501), and surgery started when the corneal reflex disap- peared. The right femoral artery was occlusively cannulated through an incision in the leg, and the acterised, therefore, by a metabolic alkalosis that catheter was secured to the back of the animal by is almost fully compensated by a respiratory aci- three or four sutures. The surgery normally lasted dosis apparently caused by a relative hypoventila- less than 30 min and all toads regained normal righting reflexes within 30 min after being placed under running tap water. All toads were treated qualitatively similar respiratory compensations of with enrofloxacin (Baytril; 2 mg kgy1, i.p.) to the alkaline tide have been observed in mammals prevent infections. When the toads had regained normal reflexes, each individual animal was trans- ferred to an experimental chamber (40=30=20 the regulation of ventilation during digestion is cm) containing wet paper towels and a dry area.
geared at maintaining pH rather than PCO2. How- These containers were maintained within a climatic ever, because digestion is associated with large chamber at a constant temperature of 25 8C, the metabolic increments, it is possible that the rise in arterial PCO2 (PaCO2) simply reflects an ineffective ventilatory compensation to the increased meta- bolic rate, leading to an un-regulated maintenanceof pH. Alternatively, it is possible that the increased PaCO2 during digestion is caused byinduction of a more relaxed state with low respon- 2.2.1. Effects of omeprazole on blood gas compo- siveness to ventilatory stimuli during the postpran- To inhibit gastric acid secretion, omeprazole was To study whether toads regulate pHa or PaCO2 given orally to six fasting toads prior to the during digestion, we measured acid–base parame- experiments. Omeprazole was dissolved in meth- ters of animals, where gastric acid secretion was ylcellulose (1.5%) and administrated through a inhibited by the specific proton-pump inhibitor soft rubber tubing inserted into the stomach omeprazole. Omeprazole has been previously through the mouth. A dose of 0.06 mg kgy1 (2 shown to uncouple Hq and Cly secretion in the ml of 28 mg kgy1 pr kg toad) omeprazole was gastric mucosa in the frog Rana catesbeiana applied daily over 4 days before cannulation, and a final dose was administered a few hours before formed on the marine toad (Bufo marinus), which has been extensively studied with regards to its A blood sample from fasting animals was with- acid–base regulation and from which we have data drawn 24 h after surgery, as we have previously shown that arterial blood gases and acid–base parameters of B. marinus have stabilised at thistime , and analysed 2. Materials and methods
immediately (see below). Then, the animals wereforce-fed rat pups amounting to 7.0"0.3% of body mass. Subsequent blood samples were taken 24and 48 h after feeding.
Toads, Bufo marinus (Linnaeus, 1758) of unde- A group of un-treated toads, where blood sam- termined sex and body masses between 230–522 ples were taken at the same time into the digestive g (355"24 g, mean"1 S.E.M.) were obtained period, were included for comparison. These data from Lemberger (Oshkosh, WI, USA) and kept at the University of Aarhus for several months. The toads were kept at 23–28 8C in large containers during the same period as those described in the with access to running water and dry areas and present study and using the same batch of toads fed mealworms daily. Toads were anaesthetised by J.B. Andersen et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 425–433 2.2.2. Effects of bicarbonate injection on arterial response compared to untreated animals. The SDA response of untreated B. marinus was used from a A control blood sample was withdrawn 24 h previous study using the same batch of animals after surgery. Then, bicarbonate was injected as a 1.5 to 3.5 ml bolus, depending on the mass of the animal, of 1 mol ly1 NaHCO3 giving a final bonate injection, a one-way ANOVA for repeated concentration of 6.9"0.04 mmol kgy1 toad. Blood measures was employed. All differences among samples were withdrawn 1, 2, 6, 12 and 24 h after means were assessed by a SNK post-hoc test. The level of significance was chosen at the P-0.05level. All data in text and figures are presented as 3. Blood gas analysis
Arterial blood was analysed for oxygen tension 4. Results
(PaO2), pH, haematocrit, blood haemoglobin con-centration (wHb x 4.1. Arterial acid–base status during digestion carbon dioxide content of plasma (wCO x). PaO and pHa were measured with Radiometer (Copen-hagen, Denmark) electrodes maintained in a BMS In the untreated control toads, digestion was 3 electrode set-up at 25 8C while displaying the associated with a 12 mmol ly1 increase in plasma output on a Radiometer PHM 73. Haematocrit was determined in duplicate as the fractional red cell volume after centrifugation (12 000 rpm for 3 so that arterial pH did not change during conversion to cyanmethaemoglobin, applying a millimolar extinction coefficient of 10.99 at 540 Arterial acid–base status of fasting animals was not significantly affected by omeprazole treatment affected. Thus, in the omeprazole-treated toads, change significantly during digestion when com- bers were maintained at 40 8C. Haemoglobin bound oxygen (HbO2) was calculated as wO x remained relatively constant and significantly low- er than in the untreated toads throughout the digestive period . The difference between omeprazole-treated and untreated animals becomes (HbO2sat) was calculated as: HbO2ywHbx, under even more apparent when depicting arterial acid– the assumption that all Hb was functional. Arterial base parameters in a Davenport diagram .
carbon dioxide tension (PaCO2) was calculated The omeprazole-treated animals show a minor respiratory disturbance, whereas the untreated ani- mals show a metabolic alkalosis, which is com- pensated by a respiratory acidosis, thereby keeping Assuming that the carbonate concentration Digestion was not associated with changes in blood oxygen levels and haematological parame- 4.2. Acid–base status after vascular injections ofbicarbonate Significant effects of digestion were found by the use of a one-way ANOVA for repeated meas- Vascular injection of bicarbonate caused a sig- ures. A two-way ANOVA was employed to iden- tify significant effects of omeprazole on the SDA 26.5"1.37 to 36.0"1.77 mmol ly1 1 h after J.B. Andersen et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 425–433 was associated with a substantial increase in pHafrom 7.74"0.03 to 7.91"0.02, at unchangedPaCO .
2 However, as seen in the Davenport diagram , the metabolic alkalosis was progressivelycompensated by an increased PaCO2, (i.e. a respi-ratory acidosis). Plasma w icantly elevated 24 h after the infusion, while pHawas fully compensated. The bicarbonate injectionhad no effect on blood oxygen levels and haema-tological parameters .
5. Discussion
Our aim of this study was to investigate whether the increased PaCO2 during digestion in Buforepresents a compensatory response to increasedplasma w HCO3 , (i.e. the alkaline tide) or whether the increased PaCO2 represents a state-dependentchange in the control of pulmonary ventilation. Tothis end, we successfully prevented the alkalinetide by inhibiting gastric acid secretion with ome-prazole and, in addition, vascular infusions ofbicarbonate mimicked the alkaline tide in fastinganimals.
5.1. Effects of digestion on arterial blood gases inB. marinus In untreated toads, digestion was associated with of the simultaneous increase in PaCO2, pHa didnot change during digestion. A similar respiratorycompensation of the postprandial metabolic alka-losis, (i.e. the alkaline tide) has been observed inall amphibians and reptiles where blood sampleshave been obtained from undisturbed animalsusing indwelling catheters Fig. 1. Arterial acid–base parameters in the toad Bufo marinusbefore and during digestion. The circles show the response of untreated control animals, while the response of omeprazole- treated toads is shown by the squares. Fasting values are pre- marinus and other amphibians and reptiles is sented as open symbols. (a) arterial pH; (b) plasma numerically larger than in mammals. This is par- tially due to the smaller meal size ingested by tension, PaCO2. Means that are different from the fasting valueare marked with an asterisk, while significant effects of ome- mammals and a consequence of a more regular prazole treatment are marked with a dagger. Data are presented feeding pattern where gastric acid secretion is as mean "1 S.E.M. (Ns6 in each group).
continuously countered by pancreatic base secre-tion to the small intestine. Furthermore, the mam- infusion . There was no significant reduc- malian kidney responds effectively to metabolic tion during the next 24 h. This increase in plasma acid–base disturbances and the alkaline tide is rapidly reduced by increased base output in the to changes elicited by digestion in untreated toads case in amphibians where transport of acid–base J.B. Andersen et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 425–433 Fig. 2. Davenport diagram showing plasma w and arterial pH during fasting and digestion in the toad Bufo marinus. Animals treated with omeprazole are shown with the squares and untreated control animals are shown by circles. The Davenport diagram includestwo in vitro non-bicarbonate buffer lines (dotted lines, bNB) determined by and isobars for the partial pressureof CO2 in arterial blood (PaCO2, curved lines). Data are presented as mean "1 S.E.M. (Ns6 in each group).
relevant ions over the bladder and kidney is less 5.2. Acid–base regulation after inhibition of gas- effective than the mammalian kidney (see tric acid secretion with omeprazole . Inthe present experiments this is revealed by the mechanism, which is the final step in the secretory infusion: less than half of the extra bicarbonate, process of the ATP-driven proton pump, and inhib- present in the plasma 1 h after infusion, had been its both basal and meal-stimulated secretion of removed 24 h into the experiment . In R. catesbeiana, transepithelial acid–base exchange is . Arterial blood gases and haematological parameters of fasting omeprazole-treated toads were not significantly different from untreated Table 1Effects of digestion on arterial blood gases and haematological parameters in omeprazole-treated and untreated toads (Bufo marinus) 2 ), oxygen tension (PaO2), haemoglobin oxygen saturation (HbO2sat), haematocrit (Hct), haemoglobin 4 ), mean cellular haemoglobin concentration, plasma pH, plasma carbon dioxide (PaCO2 ) and bicarbonate concen- Values are mean "1 S.E.M (Ns6 in each group) J.B. Andersen et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 425–433 remains uncertain whether acid–base status offasting animals is affected by omeprazole. Inmammals, omeprazole is considered to be veryspecific and without side effects (e.g. and arterial acid–base parameters of fastingrats are not affected by omeprazole (Wang, Norlenand Haakanson, unpublished). Almost half of theomeprazole-treated toads vomited within 48 h afterforce feeding, and while their blood gas composi-tion did not differ from those completing digestion(data not shown), we excluded these animals fromthe study. It is likely that inhibition of gastric acidsecretion impaired the digestive ability and stimu-lated the emetic reflex, and secondary adverseeffects of omeprazole cannot be ruled out.
in omeprazole-treated toads, which indicates aneffective inhibition of the proton pump of theparietal cells in the gastric mucosa. The inhibitionof the alkaline tide by omeprazole is consistentwith the postprandial increase in plasma w being caused by a rise in plasma strong iondifference, as protons and chloride are secretedinto the stomach lumen. Omeprazole also inhibitedthe postprandial rise in plasma w The inhibition of the postprandial rise in plasma respiratory acidosis reflects a ventilatory compen-sation to maintain pHa. 48 h into the postprandialperiod, omeprazole had completely abolished theincreased PaCO2. This indicates that the relativehypoventilation during the postprandial period is aregulated response that act to maintain pHa bymodulating PaCO2. A similar conclusion wasreached in experiments on B. constrictor, whereomeprazole fully abolished the increase in PaCO2 Fig. 3. Effects of a bicarbonate injection at 0h (6.9"0.04 mmol kgy1) on arterial acid–base parameters in the toad Bufo tilatory regulation of pHa, rather than PaCO2, is marinus. Open symbol denotes pre-injected, whereas closed further supported by the observation that vascular symbols denotes post-injected animals. (a) arterial pH; (b) bicarbonate infusion led to an increased PaCO that re-established pHa at the control level 24 h bon dioxide tension, PaCO2. Significant differences from thepre-injected values are marked with an asterisk. Data are pre- after infusion, however, the response at 24 h after sented as mean "1 S.E.M. (Ns6).
feeding was less clear, because there was a tenden-cy, albeit not statistically significant, for an toads and were similar to previous studies on B. Our study cannot reveal, which chemoreceptors are involved in mediating ventilatory regulation of ever, appeared slightly higher in omeprazole-treat- pHa during the postprandial period, but it indicates ed animals, which was also observed in the snake that the overall modality of the chemoreceptors controlling ventilation, at fast and during digestion, given the lack of statistically significant effects, it is pHa and not PaCO2. The ventilatory response to J.B. Andersen et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 425–433 Fig. 4. Davenport diagram showing plasma w and arterial pH of fasting Bufo marinus before (open symbols) and after (closed symbols) a vascular injection of bicarbonate (6.9"0.04 mmol kgy1). The Davenport diagram includes two in vitro non-bicarbonatebuffer lines (dotted lines, bNB) determined by and isobars for the partial pressure of CO2 in arterial blood(PaCO2, curved lines). Data are presented as mean "1 S.E.M. (Ns6).
hypercapnia (the combination of increased PCO2 and reduced pH) of B. marinus is primarily driven permeability of the blood brain barrier to ions and by central chemoreceptors in the medulla CO2 has not been characterised in ectothermic vertebrates, but it is likely that the slow rate for central receptors are responsible for the postpran- the development of the alkaline tide allows for the dial response, it is required that metabolic acid– metabolic alkalosis to be transmitted from the base disturbances are transmitted from plasma to blood to the CSF. This may even be the case in the cerebrospinal fluid (CSF). In mammals, the mammals, since the small alkaline tide is associ- blood brain barrier separating blood from CSF, is ated with small respiratory compensations rather impermeable to ions while changes in PCO2 are readily transmitted between the two Table 2Effects of a bicarbonate injection (6.9"0.04 mmol kgy1) on arterial blood gases and haematological parameters in toads (Bufo marinus) 2 ), oxygen tension (PaO2), haemoglobin oxygen saturation (HbO2sat), haematocrit (Hct), haemoglobin 4 ), mean cellular haemoglobin concentration, plasma pH, plasma carbon dioxide (PaCO2 ) and bicarbonate concen- Values are mean "1 S.E.M (Ns6) J.B. Andersen et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 425–433 relevant ions between plasma and CSF could also American alligator, Alligator mississippiensis. J. Exp. Biol.
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Correspondence Infant mortality (per thousand livebirths) group on HIV in women and children. Mortality among infected and uninfectedAfrica: a pooled analysis. Lancet 2004; 364: Abrams EJ, Wiener J, Carter R, et al. Maternalhealth factors and early pediatric antiretroviraltherapy influence the rate of perinatal HIV-Idisease progression in children. AIDS , 2003; Table: Infant m

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