The influence of pH in proximal small bowel perfusion on 64Cu absorption in anaesthetised rats

Paul Tasker, FRCS, Harbans Sharma, MSc, PhD, DSc, Joan Braganza, DSc, FRCP, Maynard Case, BSc, PhD. Manchester Royal Infirmary.

INTRODUCTION: In the closed duodenal-jejunal loop, single shot doses of standard pH solutions had a very transitory effect on duodenal luminal pH.

METHOD: To produce a more persistent change in luminal pH at different levels, the duodenal-jejunal loop with the common duct still ligated was opened and two methods of fixing pH in the gut perfusion were investigated.

RESULTS: The first method was a pH-stat re-circulation technique using single rats. As an index of 64Cu absorption, 64Cu retention in liver, as a percentage of the input concentration, from unbuffered pH stat perfusion was 36.61 (±5.61)% (n=6) (% body count per g perfusate count) at pH 6 and 22.98 (±3.09)% (n=4) at pH 8 (Mean ±SD). The second method was a triple rat buffered single pass system providing perfusion in a steady state. Four levels of pH were studied, and the same trend was observed. From a MOPS, MES and HEPES (MMH) buffer with glucose liver 64Cu retention was 33.95 (±7.84)% (n=7) at pH5, 28.02 (±5.02)% (n=3) at pH 6, 20.54 (±3.65)% (n=5) at pH7, and 16.43 (±3.60) (n=7) at pH8. Carcass and total body absorption was also measured. Then using MMH buffer without glucose, 64Cu retention in the liver only was assessed: 22.58 (± 4.53)%(n=4) at pH 5, 19.08 (±4.03)%(n=5), at pH 6, 13.84 (±3.89)%(n=4) at pH7, and 10.73 (±2.95)%(n=6) at pH 8.

As gut length varies considerably even after taking a set length (17 cm), the stretched length and mucosal weight of gut was assessed at autopsy. As independent variables their sum was divided into the percentage absorption to give gut-related absorption. For method one total 64Cu absorption was 4.21 (±0.42) (% body count per g perfusate count per g (mucosa) plus cm (length of gut loop) (n=5) at pH6 and 2.75 (±0.32) (n=4) at pH 8 (P2 <0.001). 5.5% of the gut uptake at pH 6 and 4.1% at pH 8 absorbed leaving 95% in the gut mucosa. For method two total 64Cu absorption was 4.58 (±1.72) (n=6) at pH5, 4.15 (±0.16) (n=2) at pH6, 2.33 (±0.45) (n=4) at pH 7 and 2.32 (± 0.43) (n=7) at pH8 (P2<0.001). With total absorption a jump between pH 6 and pH7 was observed which might relate to the properties of the Cu(II). 3% of 64Cu uptake absorbed leaving 97% in the gut mucosa. Net effective absorption (mucosa to body transfer) was thus a rate-limiting process.

In the pH stat recirculation study, the ratio of mucosa over the initial perfusate specific activities (counts/g) was 6.5 at pH 6 and 5.5 at pH 8. In the single pass study, the ratio was 15.8 (±5.4), at pH 5, 11.5 (± 1.1), at pH 6, 6.1 (±2.2), at pH 7 and 8.8 (±8.3), at pH 8. The specific activity ratio was greater than five to 16 times the perfusate concentration depending on pH and gut preparation.

CONCLUSION: The pH effect had its main influence at the brush-border membrane or adjacent protein binding sites.  The more acid the pH the more copper was absorbed.


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