Effects Of Glutamine And Alanyl-glutamine On Small Intestinal Epithelial Cell Proliferation And Gut Immunity In Early-weaned Piglets

A high performance liquid chromatography method for determining the concentration of free Glutamine (Gin) and Alanyl-Glutamine (Ala-Gin) in feed additive was established in this study. We have investigated the effect of Gin or Ala-Gin on the intestinal epithelial cell and mesenteric lymph node (MLN) cell proliferation in vitro and performance and blood biochemincal index of the 28 d weaned piglets. These provide a scientific data that Gin or Ala-Gin was supplemented in the early-weaned piglets diet as immunity promoter to alleviate early-weaned stress. The main results are followed.1. we have investigated the high performance liquid chromatography method for determining the concentration of free Gin and Ala-Gin in feed additive. Gin was derived with o-phthaldialdehyde (OPA) and diluted with phosphate solution buffer (50mmol/L) containing 6% acetonitrile on reverse phase CIS column. The derivation was detected at excitaion wave 230nm,emission wave 389nm with fluorescence detector. The result was showed as follows: calibration on curves for Gin was linear from 200 moI/L to 6400 mol/L; linear formula is Y=1793.4X+274241,r=0.9999 and CV was 1.39%. Ala-Gin was derived with o-phthaldialdehyde (OPA) and diluted with phosphate solution buffer (12.5mmol/L, pH 4.35) containing 15% acetonitrile on reverse phase CIS column. The derivation was detected at excitaion wave 338nm,emission wave 450nm with fluorescence detector. The result was showed as follows: calibration on curves for Ala-Gin was linear from 6.25 to lOOmmol/L; linear formula is Y=28186X-9905.1,r=0.9997and CV was 5.33%; The recovery was 98%. The concent of free Gin and Ala-Gin in Biocom are 21.1% and 2.65% respectively. We draw a conclusion from the experiment that the method is simple, quick and efficient and is suit to determining the concentration of free Gin and Ala-Gin in feed additive.2. To investigate the effect of Gin and Ala-Gin on the proliferation of piglet intestinal epithelial cells, we use collagenase/dispase digestion. The cells were cultured in DMEM (non-Gin) containing different concentration of Gin or Ala-Gin at 37 for 72h.Cell proliferation was determined by MTT colormetric assay. A large number of viable and pure pig intestinal epithelial cells were separated rapidly by collagenase XI /dispase Idigestion.The rapid proliferation was presented as the concentration of Gin exceeding 0.5mmol/L. The significant promotion was presented at 0.25mmol/L Ala-Gln.The maximal proliferation rate wds appeared at 2mmol/L Ala-Gin. There is no significant difference between the same concentration of Gin and Ala-Gin (p>0.05). These results suggest that Gin and Ala-Gin improve the proliferation of piglet's intestinal epithelial cells and have similar effects.3. To investigate the effect of Gin and Ala-Gin on MLN cell proliferation of early-weaned piglets we use lymphocyte proliferation response in vitro. The jejunal and ileal MLN were dissected from the piglet weaned at 28 day and lymphocytes were prepared in sterile, the cells were plated in the RPMI 1640 (non-Gin) containing different concentration Gin of Ala-Gin with or without PHA and cultured at 37 for 72h .Cell proliferation was determined by MTT colormetric assay. The maximal stimulation index (SI) was presented as the concentration of Gin is 2 mmol/L and that of Ala-Gin is 1 mmol/L. The SI did not increase with the increase of the concentration of Gin or Ala-Gin. The results suggest that Gin and Ala-Gin are both promote the proliferation of MLN with PHA-stimulated and have sililar effect in the appropriate concentration.4. Forty-two 28 day old weaned piglets (LandranceX Yorkshire, 8.37 ?.13kg BW) were selected and divided into 3 groups with 2 repetitions and included 28 day experiment period to study the effect of Gin and Biocom (a protein source containing Ala-Gin) on the growth performance and blood biochemical index of the weaned piglet. The result showed that daily weight gain, feed intake, feed conversion, diarrhea rate and serum total protein was no difference with Gin o...