In this experiment the volumetric contribution of the meal, buccal, gastric and Brunner’s gland secretions were not accounted for. During digestion there is an inconsistent flux of fluid from the stomach into the small intestine, from the small intestine into systemic circulation and from the small into the large intestine. As a result there will be significant changes in concentrations of enzymes and bile throughout the small intestine as the water content of digesta decreases from the proximal to distal small intestine, e.g. water is absorbed from the small intestine of the rat at a rate of 182 µl/cm/hr (Fisher, 1955). Hence, the principal focus was ensuring that the ratios of pancreatic enzymes, and bile, to the succus entericus were physiological at a given time point. This rationale was used to assess how bile and pancreatin affected BB enzyme activities (temporal assay) (Chapter 6) and these ratios were used to solubilise BBMV-bound enzymes in order to assess their kinetics at different pH’s (Chapter 7).
4.3.1 Estimating the volume and concentration of pancreatin from in
vivo data
The volume of pancreatic secretions was calculated for the first hour of the post-prandial period. The volume of pancreatic secretions was taken from the volume of secretin induced fluid collected from the cannulated pancreatic ducts of human participants, i.e. 248 µl/kg/5 min (Domschke et al., 1977). This volume was extrapolated for a 70 kg person, equating to 208.32 ml/hr (Domschke et al., 1977). The concentration of pancreatic
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enzymes was based on published data that quantified the amylolytic activity of pancreatic juice; again collected via a cannula from the pancreatic ducts of humans during the post- prandial period (Ekmekcioglu, 2002), i.e. 55 U amylase activity are secreted per kg every 15 min following consumption of a 160 kcal meal (Ekmekcioglu, 2002). Extrapolating this equates to 15400 U or 63910 USP (1 U = 4.15 USP) of amylase activity per hour post-prandially for a 70 kg person. Pancreatin contains no less than 25 USP per mg and our pancreatin containing 8 x USP (US Pharmacopeia) pancreatin (p7545) contains 200 USP per mg. Hence, 63910 USP was equivalent to 355.19 mg of pancreatin/hr. Divided by the hourly secretion the concentration is calculated to be 1.53 mg of pancreatin per ml of buffer 2. This was in keeping with works, which show that pigs secrete 210 ml of pancreatic secretions per hour in the post-prandial period (Thaela et al., 1995), and that humans secrete 1-1.5 L of pancreatic fluid per day (DeSesso and Jacobson, 2001) For the purposes of this work porcine pancreatin was used to simulate pancreatic enzymes.
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Calculation for pancreatin concentration
Need 15400 U of amylase from porcine pancreatin
Amylase: 4.15 USP = 1 U; Lipase 1 USP = 1 U (Littlewood et al., 2006)
Pancreatin (Sigma Aldrich, P7545) 1mg of 8x USP = 25 USP units of amylase x 8 = 200 USP units per mg.
Need 63910 USP
Pancreatin: 200 USP units of amylolytic activity per mg of pancreatin: 63910/200
Need 319.55mg of pancreatin per hour of secretion
319.55mg/208.32ml pancreatic secretions = 1.53398 mg/ml.
Table 7. The ratio and concentration of small intestinal digestive secretions used to emulate small intestinal conditions
# the concentration of the BBMV and aqueous preparations were equivalent to a starting concentration of 5 mg of mucosal scrapings per millilitre buffer.
4.3.2 Estimating the volume and concentration of bile from in vivo
data
Like pancreatic exocrine secretions biliary fluid is continuously secreted and admixed with chyme exiting the stomach. Hence, estimates were based on the first hour of secretion. This method was modified to use crude bile salts, which are a composite of bile
Micellar bile preparation Pancreatic enzyme preparation BBMV and aqueous preparations Initial concentration (mg/ml) 20 (Begley et al., 2005) 1.53 (Ekmekcioglu, 2002) 5# Final concentration (mg/ml) 0.72 0.39 4 Volume (ml) 29 (Kararli, 1995) 208 (Domschke et al., 1977) 572 (Mosenthal, 1911, de Beer et al., 1935) Ratio 0.04 0.26 0.71
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acids in their natural concentrations, and so are most likely to reflect the range of bile salts present in vivo.
To make the biliary solution dried porcine bile salts were reconstituted with decanoic acid and salts into a micellar solution (Lentle et al., 2012). Porcine bile extract was added at concentration of 20 mg/ml (Begley et al., 2005) in a volume derived from published work of 0.415 (range 0.10-0.93) ml/kg/day (Kararli, 1995), and extrapolated to a hourly rate of 29 ml/hr.
Calculations for the preparation of micellar bile solution
Human bile secretion 29 ml/hr (Kararli, 1995)
580 mg porcine bile extract per 29 ml or 20 mg/ml (Begley et al., 2005)
6 mM KCl (74.55 g/mol) = 0.0089 g/20ml (Lentle et al., 2012)
120 mM NaCl (58.44g/mol) =0.14025 g/20 ml(Lentle et al., 2012)
20 mg/ml Bile acids = 0.4 g/20ml
Decanoic acid (172.26 g/mol) = 0.0034452 g/20 ml (Lentle et al., 2012)
Add bile acids, salts, and decanoic acid to deoinised water and stir for 1 hour at 40 ºC
4.3.3 Estimating the volume and concentration of BB MV secretions
from in vivo data
The volume and concentration of BBMV cannot be realistically determined in vivo due to variation along the length of the small intestine, and difficulty in determining the dilution of BBMV. Hence, BBMV may be more dilute in the intestinal lumen (McConnell et al., 2009) or more concentrated in the peri-apical space (luminal area surrounding the tips of microvilli), and in mucus secretions overlying the small intestinal surface. Moreover, all sites are difficult to harvest in vivo during the post-prandial phase. The total
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volume of small intestinal BBMV secretions was estimated from published estimates of canine succus entericus secretions collected from Thiry Vela loops. Thiry Vela loops are small intestinal segments that are surgically isolated, cannulated and externalised, which leave an innervated section of the small intestine, which is not subject to admixture with gastric and pancreatic contents, from which succus entericus can be collected. The continuity of the small intestine was maintained via anastomosis. The isolated intestinal segments maintained blood supply and are innervated, so are responsive to normal neural and enterokine stimulation (de Beer et al., 1935, Mosenthal, 1911). These works are in general agreement that ~35ml of BB succus entericus is secreted per hour of the post- prandial phase from Thiry Vella loops of 25-50 cm length (de Beer et al., 1935, Mosenthal, 1911) in canines with a mean weight of 12.84 kg ( i.e. 2.73 ml/kg/hr) (Mosenthal, 1911). The canine small intestine is approximately 225 - 290 cm long (Kararli, 1995), and the volumes secreted from the Thiry Vella loop differ greatly between intestinal segments, individuals and sampling points (Mosenthal, 1911, de Beer et al., 1935) so extrapolating, section for section, to match those secreted normally is difficult. Hence, it was assumed that each section of the small intestine secreted 35 ml of succus entericus totalling 105 ml/hour (8.18 ml/kg/cm) (Mosenthal, 1911). Extrapolated for a 70kg human this would be 572 ml/hour. With the estimated average daily secretions of succus entericus in humans of 1-2 litres per day (DeSesso and Jacobson, 2001) this estimate seems reasonable if associated with one meal.
Since there is no published data on the activity of BBMV enzymes in vivo, in the absence of biliopancreatic secretions, the concentration of BBMV used for the assay of BB enzymes was chosen to produce enzyme activities that could be measured by colorimetric assay.
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4.3.4 Estimating the volume and concentration of the aqueous
fraction
Described here is the rationale for the volume and concentration of the BB aqueous enzyme preparation. The aqueous fraction was recovered during the preparation and isolation of the BBMV fraction. It is likely to contain immature BB enzymes from the enterocyte, soluble BB enzymes and enzymes solubilised during the preparation of the BBMV fraction. There are no reliable estimates for the concentration of the aqueous fraction in the succus entericus. Since, enterocytes migrate to the tip of microvilli and are shed into the peri-apical space it is likely their contents are dispersed. Without appropriate data this fraction was diluted in the manner of the BBMV fraction.
4.3.5 Rationale for the admixture of component fractions
Bile and/or pancreatin were added to the BBMV and BB aqueous in the ratios outlined in table 7. These enzyme admixtures were either used for assay immediately or were held at 37 ᴼC for 1 hour or 2 hours prior to adding the substrate. This enabled the time at which enzyme activities were modified by bile and pancreatin to be determined. Controls included enzyme and no substrate, substrate and no enzyme, heat treated enzyme and substrate, pancreatin ± bile, and pancreatic α-amylase as a positive control. In the latter the concentration of α-amylase was that which has been reported in pancreatin, i.e. 15400U (Ekmekcioglu, 2002) or 7.39 mg/ml.