Br. J. Surg. Vol. 64 (1977)851-856
Ammonia absorption from the canine colon after portacaval shunt I. D. HARRISON, A. H A R D Y SMITH AND R. SHIELDS* SUMMARY
Ammonia absorption was studied from Thiry- Vella colon loops in 6 dogs. Four underwent an end-to-side portacaval shunt and it was shown that absorption of ammonia from the colon significantly increased postoperatively. Absorption of ammonia from various solutions was also measured before and after portacaval shunt and it was shown that absorption was increased from a high p H solution and from a solution with a high bicarbonate content and reduced from a low p H , low osmolality and high osmolality solution. Ifincreased deposits of stainable iron were demonstrated in the livers of dogs following portacaval shunt.
AMMONIA was one of the first compounds to be incriminated in the pathogenesis of portosystemic encephalopathy and it is still held to be the ‘key cerebral toxin’ (Walker and Schenker, 1970). The concentration of ammonia in the blood, however, is not consistently related to the clinical severity of encephalopathy, and quite severe coma can occur when the concentration of ammonia is within the normal range (Sherlock, 1958). Although there is, asfyet, no agreement as to the precise nature of the compound or compounds causing encephalopathy, we suggest that it is reasonable to regard ammonia as representative of this compound or compounds because of the similarity between the known physicochemical properties of ammonia and those of the actual causative agent suggested by clinical observation and the results of clinical treatment. Recently, in this laboratory the faecal concentration of ammonia was shown to be grossly elevated in cirrhotic patients before portacaval shunt (Sein et al., 1974). After portacaval shunt, however, the concentration of ammonia fell to near normal levels, accompanied by a gross elevation in the concentration of ammonia in the blood. This suggested that absorption of ammonia may be enhanced following a shunt. It was therefore decided to study the effect of a portacaval shunt on ammonia absorption from the canine colon. In addition, the absorption of ammonia from solutions of varying physicochemical composition was investigated, since it is known that ammonia absorption can be influenced by intraluminal pH (Castell and Moore, 1971), the concentration of bicarbonate ions in the lumen and also intraluminal osmolality (Swales et al., 1970).
Materials and methods Preparation of animals Under general anaesthesia and with strict aseptic technique, Thiry-Vella loops of colon were fashioned in each of 6 Labrador dogs weighing from 14 to 22-5 kg. The longest segment consistent with a safe, tension-free colon anastomosis was established, the average length being 12 cm. After operation, the dogs were allowed water as soon as they were conscious and after 24 hours soups were permitted, followed by a normal diet on the fourth day. Absorption tests were begun 10-14 days following operation.
Fig. 1. Method of producing an end-to-side portacaval shunt. IVC, Inferior vena cava; PV, portal vein.
Fig. 2. Dog colon loop with cannula system. After the initial series of absorption experiments an end-toside portacaval shunt was performed in 4 dogs and a sham operation was carried out in 2, selected randomly. The portal vein and inferior vena cava were approximated using a Satinsky partially occluding clamp and a side-to-side anastomosis was constructed using standard vascular techniques. The portal vein between the anastomosis and the liver was then ligated to produce an end-to-side portacaval shunt, thus completely depriving the liver of its portal venous supply (Fig. 1). The pressures in both the portal vein and the inferior vena cava were measured before and after portacaval shunt using strain gauge manometers, with the level at the anterior surface of the dorsal vertebra being taken as the zero reference point. For the sham operation an identical incision was made, the vessels being mobilized and the Satinsky clamp being applied as described above, but no anastomosis was made. After 10-14 days, absorption tests were repeated and these post-shunt results were compared with those obtained before the shunt. Before each absorption experiment and operation blood was analysed to assess the concentration of ammonia and electrolytes and to determine the packed cell volume and osmolality. At the completion of the study a laparotomy was performed in each dog at which the patency of the portacaval anastomosis was confirmed and the presence of any intraperitoneal
* Department of
Surgery, University of Liverpool.
I. D. Harrison et al.
852
Table I: PHYSICAL AND CHEMICAL PROPERTIES OF VARIOUS TEST SOLUTIONS Solution Standard Ammonia (mmol/l) Sodium (mmol/l) Potassium (mmol/l) Chloride (mmol/l) Total CO, (mmol/l) PH Osmolality (mmol/kg) Mannitol (g/l)
25 119 3.4 144 2 7.0 275 0
High bicarbonate 25 123 3.4 115 37 7.0 278 0
Table 11: EFFECT O F PORTACAVAL SHUNT ON AMMONIA ABSORPTION Ammonia absomtion (umol cm-a h-l) Doe No. 1
2 3
Mean
Pre-shunt 12.051- 1.39 ( n = 18) 10.72f0.99 ( n = 12) 12321-0.77 (n = 6) 11.611 1.33
Post-shunt 15.90& 1.70 (n = 18) 14.31 12.71 (n = 17) 16.08* 1.99 (n = 6) 15.27 1- 2.3 1
High PH
Low PH
25 123 3.4 141 2 9.0 273 0
25 119 3.4 142 2 4.5 270 0
High osmolality 25 118 3.4 142 2 7.0 316 18
Low osmolality 25 63 3.4 86 2 7.0 164 0
emptied and the aspirate centrifuged before analysis. The radioactivity of the aspirate was determined. The loop was then washed with a further 100 ml of unlabelled test solution before the next 20-min absorption period. In any series of experiments 6 20-min tests were performed. Test solutions (Table I ) In 5 animals (dogs 1, 2, 3, 5 and 6) the test solution was isotonic with plasma and of neutral pH and no bicarbonate was added (this was designated 'standard' solution). In a further animal (dog 8) absorption of ammonia was studied both before and after portacaval shunt, using several test solutions with a high bicarbonate concentration, high pH, low pH, high osmolality or low osmolality.
n, Number of test periods studied. All values are meanrts.d.
Table 111: EFFECT OF SHAM OPERATION ON AMMONIA ABSORPTION Ammonia absomtion (umol cm-, h-l) Doe No. Preoo. Poston. 5 1001 f1.07 10.16rt2.06 ( n = 12) (n= 12) 6 12.80f 1.86 10.33f 1.84 (n= 11) (n= 11) Mean 11.34f2.04 10.24%1.92
n, Number of test periods studied. All values are meanfs.d. sepsis or peptic ulceration determined. The colon loop was excised, opened along its antemesenteric border and the dimensions measured to relate the rate of absorption to the mucosal surface area. Samples of the colonic loop, the remaining colon and the liver were removed for microscopic examination. Measurement of absorption With the animal lying supine, two Foley balloon catheters were inserted, one into each stoma of the colon loop. The two catheters were joined within the loop by a perforated plastic cage (Fig.2). The lumen of one catheter (size 14 F) was used for instillation and removal of the test solution, and the lumen of the other catheter (size 28 F) was used for the introduction of a Radiometer pH probe. The tip of the pH probe lay within the plastic cage to monitor the reaction of the luminal contents continuously. The balloons of the Foley catheters were gently inflated to prevent leakage of solution from the loop. At the commencement of an absorption test the loop was rinsed with 1 litre of sodium chloride solution (0.9 per cent w/v) which had been brought to pH 7 with 0-1 mol/l sodium hydroxide and which was maintained at 37 "C in a water bath Thereafter, the loop was washed with 100ml of the appropriate test solution. The loop was then emptied as completely as possible before instilling exactly 22 ml of test solution to which had been added 0.5 pCi of Wr-EDTA nonabsorbed volume marker. By a repeated process of removal and reinstillation of the loop contents, complete mixing was performed over 30 s after which a 2-ml aliquot was withdrawn and its radioactivity counted to determine the precise initial volume of the loop contents. The remaining solution was left in the loop, being mixed once at 10 min. At 20 min the loop was
Analytical methods The ammonia concentration of the aspirates and test solutions was measured by the indophenol blue reaction, produced by mixing the sample with a solution of phenol, sodium nitroprusside, sodium hypochlorite and sodium hydroxide (Fawcett and Scott, 1960). The intensity of the blue colour reaction was then measured using a Unicam SP 600 spectrophotometer with a red photocell at 625 nm. All samples were analysed in duplicate and a reagent blank read. The concentration of bicarbonate was measured as total CO, using the Van Slyke technique (Harleco CO, apparatus). Osmolality was determined by depression of the freezing point, using an Osmette S automatic osmometer. The plasma ammonia was measured after isolation by selective absorption on to a strongly acidic cation-exchange resin and production of an indophenol blue reaction as described previously. Concentrations of sodium and potassium were determined by flame photometry. The radioactivity of the solutions was measured by gamma counting in a well-type scintillation counter. The pH of the test solutions was determined using a Radiometer pH probe. Calculation of results The rate of net water absorption was calculated from changes in the concentration of a non-absorbed volume marker ( W r EDTA). The rate of net ammonia absorption was calculated from the following equation:
where MA is the net absorption of ammonia during the test (pmol cm-* 20 min-l); Cois the initial ammonia concentration; C, is the final ammonia concentration (after time t ) ; S is the surface area of the colon loop (cm,); RI is the radioactivity of the test solution introduced into the loop (counts ml-l min-l); Rt is the radioactivity of the loop contents on completion of the test (counts ml-I min-I); RD is the radioactivity of the loop content after the initial mixing phase. Statistical analysis Analysis of the results obtained from dog 8, in which the absorption of ammonia from solutions of differing physicochemical composition was studied, was carried out using Student's t test. The results in the remaining dogs, using the standard test solution before and after both portacaval shunt and a sham operation, were compared by analysis of variance.
Ammonia absorption from canine colon
853
Table LV: EFFECT O F OSMOLALITY ON AMMONIA ABSORPTION Ammonia absorption ( p o l cm-a h-l) Solution High osmolality
Pre-shunt
Post-shunt
9’93*1‘54* }t= 4.30, p
Ammonia absorption from the canine colon after portacaval shunt I. D. HARRISON, A. H A R D Y SMITH AND R. SHIELDS* SUMMARY
Ammonia absorption was studied from Thiry- Vella colon loops in 6 dogs. Four underwent an end-to-side portacaval shunt and it was shown that absorption of ammonia from the colon significantly increased postoperatively. Absorption of ammonia from various solutions was also measured before and after portacaval shunt and it was shown that absorption was increased from a high p H solution and from a solution with a high bicarbonate content and reduced from a low p H , low osmolality and high osmolality solution. Ifincreased deposits of stainable iron were demonstrated in the livers of dogs following portacaval shunt.
AMMONIA was one of the first compounds to be incriminated in the pathogenesis of portosystemic encephalopathy and it is still held to be the ‘key cerebral toxin’ (Walker and Schenker, 1970). The concentration of ammonia in the blood, however, is not consistently related to the clinical severity of encephalopathy, and quite severe coma can occur when the concentration of ammonia is within the normal range (Sherlock, 1958). Although there is, asfyet, no agreement as to the precise nature of the compound or compounds causing encephalopathy, we suggest that it is reasonable to regard ammonia as representative of this compound or compounds because of the similarity between the known physicochemical properties of ammonia and those of the actual causative agent suggested by clinical observation and the results of clinical treatment. Recently, in this laboratory the faecal concentration of ammonia was shown to be grossly elevated in cirrhotic patients before portacaval shunt (Sein et al., 1974). After portacaval shunt, however, the concentration of ammonia fell to near normal levels, accompanied by a gross elevation in the concentration of ammonia in the blood. This suggested that absorption of ammonia may be enhanced following a shunt. It was therefore decided to study the effect of a portacaval shunt on ammonia absorption from the canine colon. In addition, the absorption of ammonia from solutions of varying physicochemical composition was investigated, since it is known that ammonia absorption can be influenced by intraluminal pH (Castell and Moore, 1971), the concentration of bicarbonate ions in the lumen and also intraluminal osmolality (Swales et al., 1970).
Materials and methods Preparation of animals Under general anaesthesia and with strict aseptic technique, Thiry-Vella loops of colon were fashioned in each of 6 Labrador dogs weighing from 14 to 22-5 kg. The longest segment consistent with a safe, tension-free colon anastomosis was established, the average length being 12 cm. After operation, the dogs were allowed water as soon as they were conscious and after 24 hours soups were permitted, followed by a normal diet on the fourth day. Absorption tests were begun 10-14 days following operation.
Fig. 1. Method of producing an end-to-side portacaval shunt. IVC, Inferior vena cava; PV, portal vein.
Fig. 2. Dog colon loop with cannula system. After the initial series of absorption experiments an end-toside portacaval shunt was performed in 4 dogs and a sham operation was carried out in 2, selected randomly. The portal vein and inferior vena cava were approximated using a Satinsky partially occluding clamp and a side-to-side anastomosis was constructed using standard vascular techniques. The portal vein between the anastomosis and the liver was then ligated to produce an end-to-side portacaval shunt, thus completely depriving the liver of its portal venous supply (Fig. 1). The pressures in both the portal vein and the inferior vena cava were measured before and after portacaval shunt using strain gauge manometers, with the level at the anterior surface of the dorsal vertebra being taken as the zero reference point. For the sham operation an identical incision was made, the vessels being mobilized and the Satinsky clamp being applied as described above, but no anastomosis was made. After 10-14 days, absorption tests were repeated and these post-shunt results were compared with those obtained before the shunt. Before each absorption experiment and operation blood was analysed to assess the concentration of ammonia and electrolytes and to determine the packed cell volume and osmolality. At the completion of the study a laparotomy was performed in each dog at which the patency of the portacaval anastomosis was confirmed and the presence of any intraperitoneal
* Department of
Surgery, University of Liverpool.
I. D. Harrison et al.
852
Table I: PHYSICAL AND CHEMICAL PROPERTIES OF VARIOUS TEST SOLUTIONS Solution Standard Ammonia (mmol/l) Sodium (mmol/l) Potassium (mmol/l) Chloride (mmol/l) Total CO, (mmol/l) PH Osmolality (mmol/kg) Mannitol (g/l)
25 119 3.4 144 2 7.0 275 0
High bicarbonate 25 123 3.4 115 37 7.0 278 0
Table 11: EFFECT O F PORTACAVAL SHUNT ON AMMONIA ABSORPTION Ammonia absomtion (umol cm-a h-l) Doe No. 1
2 3
Mean
Pre-shunt 12.051- 1.39 ( n = 18) 10.72f0.99 ( n = 12) 12321-0.77 (n = 6) 11.611 1.33
Post-shunt 15.90& 1.70 (n = 18) 14.31 12.71 (n = 17) 16.08* 1.99 (n = 6) 15.27 1- 2.3 1
High PH
Low PH
25 123 3.4 141 2 9.0 273 0
25 119 3.4 142 2 4.5 270 0
High osmolality 25 118 3.4 142 2 7.0 316 18
Low osmolality 25 63 3.4 86 2 7.0 164 0
emptied and the aspirate centrifuged before analysis. The radioactivity of the aspirate was determined. The loop was then washed with a further 100 ml of unlabelled test solution before the next 20-min absorption period. In any series of experiments 6 20-min tests were performed. Test solutions (Table I ) In 5 animals (dogs 1, 2, 3, 5 and 6) the test solution was isotonic with plasma and of neutral pH and no bicarbonate was added (this was designated 'standard' solution). In a further animal (dog 8) absorption of ammonia was studied both before and after portacaval shunt, using several test solutions with a high bicarbonate concentration, high pH, low pH, high osmolality or low osmolality.
n, Number of test periods studied. All values are meanrts.d.
Table 111: EFFECT OF SHAM OPERATION ON AMMONIA ABSORPTION Ammonia absomtion (umol cm-, h-l) Doe No. Preoo. Poston. 5 1001 f1.07 10.16rt2.06 ( n = 12) (n= 12) 6 12.80f 1.86 10.33f 1.84 (n= 11) (n= 11) Mean 11.34f2.04 10.24%1.92
n, Number of test periods studied. All values are meanfs.d. sepsis or peptic ulceration determined. The colon loop was excised, opened along its antemesenteric border and the dimensions measured to relate the rate of absorption to the mucosal surface area. Samples of the colonic loop, the remaining colon and the liver were removed for microscopic examination. Measurement of absorption With the animal lying supine, two Foley balloon catheters were inserted, one into each stoma of the colon loop. The two catheters were joined within the loop by a perforated plastic cage (Fig.2). The lumen of one catheter (size 14 F) was used for instillation and removal of the test solution, and the lumen of the other catheter (size 28 F) was used for the introduction of a Radiometer pH probe. The tip of the pH probe lay within the plastic cage to monitor the reaction of the luminal contents continuously. The balloons of the Foley catheters were gently inflated to prevent leakage of solution from the loop. At the commencement of an absorption test the loop was rinsed with 1 litre of sodium chloride solution (0.9 per cent w/v) which had been brought to pH 7 with 0-1 mol/l sodium hydroxide and which was maintained at 37 "C in a water bath Thereafter, the loop was washed with 100ml of the appropriate test solution. The loop was then emptied as completely as possible before instilling exactly 22 ml of test solution to which had been added 0.5 pCi of Wr-EDTA nonabsorbed volume marker. By a repeated process of removal and reinstillation of the loop contents, complete mixing was performed over 30 s after which a 2-ml aliquot was withdrawn and its radioactivity counted to determine the precise initial volume of the loop contents. The remaining solution was left in the loop, being mixed once at 10 min. At 20 min the loop was
Analytical methods The ammonia concentration of the aspirates and test solutions was measured by the indophenol blue reaction, produced by mixing the sample with a solution of phenol, sodium nitroprusside, sodium hypochlorite and sodium hydroxide (Fawcett and Scott, 1960). The intensity of the blue colour reaction was then measured using a Unicam SP 600 spectrophotometer with a red photocell at 625 nm. All samples were analysed in duplicate and a reagent blank read. The concentration of bicarbonate was measured as total CO, using the Van Slyke technique (Harleco CO, apparatus). Osmolality was determined by depression of the freezing point, using an Osmette S automatic osmometer. The plasma ammonia was measured after isolation by selective absorption on to a strongly acidic cation-exchange resin and production of an indophenol blue reaction as described previously. Concentrations of sodium and potassium were determined by flame photometry. The radioactivity of the solutions was measured by gamma counting in a well-type scintillation counter. The pH of the test solutions was determined using a Radiometer pH probe. Calculation of results The rate of net water absorption was calculated from changes in the concentration of a non-absorbed volume marker ( W r EDTA). The rate of net ammonia absorption was calculated from the following equation:
where MA is the net absorption of ammonia during the test (pmol cm-* 20 min-l); Cois the initial ammonia concentration; C, is the final ammonia concentration (after time t ) ; S is the surface area of the colon loop (cm,); RI is the radioactivity of the test solution introduced into the loop (counts ml-l min-l); Rt is the radioactivity of the loop contents on completion of the test (counts ml-I min-I); RD is the radioactivity of the loop content after the initial mixing phase. Statistical analysis Analysis of the results obtained from dog 8, in which the absorption of ammonia from solutions of differing physicochemical composition was studied, was carried out using Student's t test. The results in the remaining dogs, using the standard test solution before and after both portacaval shunt and a sham operation, were compared by analysis of variance.
Ammonia absorption from canine colon
853
Table LV: EFFECT O F OSMOLALITY ON AMMONIA ABSORPTION Ammonia absorption ( p o l cm-a h-l) Solution High osmolality
Pre-shunt
Post-shunt
9’93*1‘54* }t= 4.30, p
