Development of a sensitive direct competitive chemiluminescent enzyme immunoassay for gentamicin based on the construction of a specific single-chain variable fragment-alkaline phosphatase fusion protein.

A sensitive chemiluminescent enzyme immunoassay (CLEIA) was established for the determination of gentamicin (GEN) residue levels in animal tissue. This assay is based on a fusion protein of single-chain variable fragment (scFv) and alkaline phosphatase (AP). Initially, VL and VH derived from anti-gentamicin monoclonal antibody were linked by a short peptide to construct a scFv. Subsequently, the constructed scFv sequence was accessed into the pLIP6/GN vector, and a soluble scFv-AP fusion protein was generated. The scFv-AP fusion protein was used to develop a direct competitive CLEIA (dcCLEIA) for the determination of gentamicin. In the dcCLEIA, the half inhibitory concentration (IC50) and limit of detection (LOD) were 1.073 ng/mL and 0.380 ng/mL, respectively. The average recoveries of gentamicin spiked in animal tissue samples ranged from 78% to 96%. These results showed a strong correlation with ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The above results suggest that the anti-GEN scFv-AP fusion protein is suitable for detecting gentamicin residues in edible animal tissues.

Dietary sn-2 palmitic triacylglycerols reduced faecal lipids, calcium contents and altered lipid metabolism in Sprague-Dawley rats.

In this study, the impact of dietary sn-2 palmitic triacylglycerol (sn-2 PTAG) on faecal lipids, calcium excretion and lipid metabolic alternation was investigated in Sprague-Dawley (SD) rats fed with high-fat diet containing either palm olein (PO, sn-2 palmitic acid (PA) of 14.8%), sn-2 PTAG50 (sn-2 PA of 56.4%) or sn-2 PTAG70 (sn-2 PA of 72.4%), respectively. After 4-week feeding period, SD rats fed with sn-2 PTAGs showed reduced faecal soap fatty acids, neutral lipid and calcium excretion compared to those of PO-fed rats, whereas a significant difference was only observed for the sn-2 PTAG70-fed rats (p < .05). Moreover, dietary sn-2 PTAG70 also showed a significant effect on decreasing serum triacylglycerol (TAG) level, reducing perirenal adipocyte size and regulating lipid metabolism in small intestine and perirenal adipose tissue of SD rats. Significantly increased mRNA levels of genes involved in intestinal lipid anabolism as well as lipid catabolism were both observed in the sn-2 PTAG70-fed rats (p < .05). Meanwhile, dietary sn-2 PTAG70 also significantly up-regulated lipolysis, mitochondrial fatty acid oxidation and thermogenesis-related gene and protein levels in perirenal adipose tissue, which might be correlated with the reduced perirenal adipocyte size. Taken together, our findings indicated that sn-2 PTAG70 may have some beneficial effects on intestinal lipid utilisation and lipid metabolic activity for energy supply in visceral adipose tissue.

[Determination of 12 prohibited veterinary drug residues in pig urine by ultra high performance liquid chromatography-tandem mass spectrometry].

A method was established for the simultaneous detection of 12 prohibited veterinary drugs, including ß2-receptor agonists, nitrofuran metabolites, nitroimidazoles, chlorpromazine, and chloramphenicol, in pig urine. The sample was pretreated by enzymolysis, acid hydrolysis/derivatization, and liquid-liquid extraction combined with solid-phase extraction. Detection was performed using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Ammonium acetate solution (0.2 mol/L, 4.5 mL) and ß-glucuronidase/aryl sulfatase (40 µL) were added to the sample, which was subsequently enzymolized at 37 ℃ for 2 h. Then, 1.5 mL of 1.0 mol/L hydrochloric acid solution and 100 µL of 0.1 mol/L o-nitrobenzaldehyde solution were added to the sample. The mixture was incubated at 37 ℃ for 16 h, and the analytes were extracted with 8 mL of ethyl acetate by liquid-liquid extraction. The lower aqueous phase obtained after extraction was extracted and purified using a mixed cation-exchange solid-phase extraction column. The extracts were combined, the extraction solution was blow-dried with nitrogen, and the residue was redissolved for determination. The samples were analyzed under multiple-reaction monitoring mode with both positive and negative electrospray ionization, and quantified using an isotope internal standard method. The correlation coefficients (r) of the 12 compounds were >0.99. The limits of detection (LODs) and quantification (LOQs) of chloramphenicol were 0.05 and 0.1 µg/L, respectively, and the LODs and LOQs of the other compounds were 0.25 and 0.5 µg/L, respectively. The mean recoveries and RSDs at 1, 2, and 10 times the LOQ were 83.6%-115.3% and 2.20%-12.34%, respectively. The proposed method has the advantages of high sensitivity, good stability, and accurate quantification; thus, it is suitable for the simultaneous determination of the 12 prohibited veterinary drug residues in pig urine.

The triacylglycerol structure and composition of a human milk fat substitute affect the absorption of fatty acids and calcium, lipid metabolism and bile acid metabolism in newly-weaned Sprague-Dawley rats.

In this study, the effect of sn-2 palmitic triacylglycerols (sn-2 palmitic TAGs) and the ratio between the two major sn-2 palmitic TAGs (OPL to OPO ratio) in a human milk fat substitute (HMFS) on growth, fatty acid and calcium absorptions, and lipid and bile acid metabolic alterations was investigated in Sprague-Dawley rats. After 4 weeks of high-fat feeding, rats fed with the HMFS containing a sn-2 palmitic acid content of 57.87% and an OPL to OPO ratio of 1.4 showed the lowest TAG accumulation in their livers and hypertrophy of perirenal adipocytes, compared to the groups fed with fats containing a lower sn-2 palmitic acid content or a lower OPL to OPO ratio. Meanwhile, synergistically improved absorption of fatty acids and calcium and increased levels of total bile acids (BAs), especially for the tauro-conjugated BAs (TCDCA, TUDCA, TαMCA, TßMCA, TDCA and TωMCA), were observed in rats by both increasing the sn-2 palmitic acid content and the OPL to OPO ratio in HMFS. In addition, the levels of total BAs and tauro-conjugated BAs were negatively correlated with serum TAG, TC, and LDL-c levels and positively correlated with HDL-c levels according to Spearman's correlation analysis (P < 0.05). Collectively, these findings present new nutritional evidence for the potential effects of the TAG structure and composition of a human milk fat substitute on the growth and lipid and bile acid metabolism of the host in infancy.

Total Sn-2 Palmitic Triacylglycerols and the Ratio of OPL to OPO in Human Milk Fat Substitute Modulated Bile Acid Metabolism and Intestinal Microbiota Composition in Rats.

In this study, the impact of sn-2 palmitic triacyclglycerols (TAGs) in combination with their ratio of two major TAGs (1-oleoyl-2-palmitoyl-3-linoleoylglycerol (OPL) to 1,3-dioleoyl-2-palmitoylglycerol (OPO)) in human milk fat substitute (HMFS) on bile acid (BA) metabolism and intestinal microbiota composition was investigated in newly-weaned Sprague-Dawley rats after four weeks of high-fat feeding. Compared to those of control group rats, HMFS-fed rats had significantly increased contents of six hepatic primary BAs (CDCA, αMCA, ßMCA, TCDCA, TαMCA and TßMCA), four ileal primary BAs (UDCA, TCA, TCDCA and TUDCA) and three secondary BAs (DCA, LCA and ωMCA), especially for the HMFS with the highest sn-2 palmitic acid TAGs of 57.9% and OPL to OPO ratio of 1.4. Meanwhile, the inhibition of ileal FXR-FGF15 and activation of TGR5-GLP-1 signaling pathways in HMFS-fed rats were accompanied by the increased levels of enzymes involved in BA synthesis (CYP7A1, CYP27A1 and CYP7B1) in the liver and two key thermogenic proteins (PGC1α and UCP1) in perirenal adipose tissue, respectively. Moreover, increasing sn-2 palmitic TAGs and OPL to OPO ratio in HMFS also altered the microbiota composition both on the phylum and genus level in rats, predominantly microbes associated with bile-salt hydrolase activity, short-chain fatty acid production and reduced obesity risk, which suggested a beneficial effect on host microbial ecosystem. These observations provided important nutritional evidence for developing new HMFS products for infants.

[Determination of 3-methyl-quinoxaline-2-carboxylic acid residue in pork by high performance liquid chromatography-tandem mass spectrometry].

A method was developed for the simultaneous quantitative and confirmatory determination of 3-methylquinoxaline-2-carboxylic acid (MQCA) residues in pork by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The sample was hydrolyzed with 0.3 mol/L hydrochloric acid solution, and MQCA was extracted by water bath oscillation. MQCA was first extracted with acetonitrile and ethyl acetate and then re-extracted from the extraction solution with 0.1 mol/L sodium hydroxide solution. Then MQCA was purified by an anion exchange solid phase extraction column. The chromatographic separation was performed using an Agilent Eclipse Plus C18 column (50 mm×3.0 mm, 1.8 µm). The quantitative analysis was performed by the matrix matching addition method. The correlation coefficient of MQCA in the range of 1.0-50 µg/L was greater than 0.99. The recovery was 90.5%-119.6% at the spiked levels of 0.5, 1.0 and 5.0 µg/kg, and the relative standard deviation was 3.14%-4.22%. The method can be used for the rapid quantitative determination of 3-methylquinoxaline-2-carboxylic acid residues in pork.

[Determination of chlorate and perchlorate in milk power by high-performance liquid chromatography-tandem mass spectrometry].

An analytical method based on high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed for detecting chlorate and perchlorate residues in milk power. Chlorate and perchlorate in milk power were extracted using a 0.1% (v/v) formic acid-acetonitrile solution. The extract was centrifuged at 10000 r/min for 10 min, and the supernatant was cleaned up on a PRiME HLB column. Separation of chlorate and perchlorate was performed on an ion-exchange column (Thermo Scientific Acclaim TRINITY P1, 50 mm×2.1 mm, 3 µm) by gradient elution using acetonitrile and 20 mmol/L ammonium acetate solution as the mobile phase. The analytes were identified by MS/MS. Quantification was achieved using internal standards. Chlorate and perchlorate demonstrated good linearity in the ranges of 2.0-40.0 and 1.0-20.0 µg/L, respectively, with correlation coefficients (r2) greater than 0.999. The limits of quantification (LOQs) of chlorate and perchlorate were found to be 15.0 and 7.5 µg/kg, respectively. The recoveries of chlorate and perchlorate ranged from 89.24% to 107.85% at the three spike levels of 30.0, 60.0, and 120.0, and 15.0, 30.0, and 60.0 µg/kg, respectively, with relative standard deviations (RSDs) ranging from 3.15% to 10.42% (n=6). This method is convenient, rapid, accurate, and efficient, thus demonstrating its suitability for use in the determination of chlorate and perchlorate in milk power.

A simplified sample pretreatment for the rapid determination of 22 ß-agonist residues in swine muscle and liver tissues by ultra-high-performance liquid chromatography tandem mass spectrometry.

A reliable ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous identification and quantification of 22 ß-agonist residues in swine muscle and liver tissues. A special focus was given to sample the pretreatment procedures and was crucial for an accurate and reliable analytical method. With regard to the high complexity of food matrices, the extraction solvent, time, and temperature, as well as clean-up cartridges, were optimized to improve the extraction efficiency and reduce matrix effects. The UHPLC-MS/MS method was further validated by determining the linearity (R2 ≥ 0.9900), sensitivity (limit of detection varying from 0.05 µg/kg to 0.8 µg/kg, limit of quantitation ranging from 0.2 µg/kg to 2.5 µg/kg), recovery (ranging from 76.4 to 111.7%) and precision (≤22.9%) in swine muscle and liver tissues. The UHPLC-MS/MS method was proven to be suitable for both screening and the confirmatory determination of 22 ß-agonist residues in swine muscle and liver tissues.

The impact of dietary sn-2 palmitic triacylglycerols in combination with docosahexaenoic acid or arachidonic acid on lipid metabolism and host faecal microbiota composition in Sprague Dawley rats.

Sn-2 palmitic acid triacylglycerols (sn2PA fat) and polyunsaturated fatty acids are thought to influence the metabolic status and intestinal bacterial population of the host. In this study, the impact of sn2PA fat in combination with DHA or ARA in the diet on lipid metabolism in the liver and faecal microbiota composition were investigated in rats fed diets containing sn2PA fat, 90% sn2PA fat + 10% DHA oil (wt%), or 90% sn2PA fat + 10% ARA oil (wt%). Tissue fatty acid composition was measured using gas chromatography (GC), whereas the faecal microbial composition was assessed using 16S rRNA high-throughput sequencing technology. In addition, faecal short-chain fatty acids (SCFA) were analyzed using ion chromatography. The results showed that sn2PA fat in combination with DHA or ARA significantly reduced liver triacylglyceride (TG) content compared with the sn2PA fat only group. Moreover, the supplementation with sn2PA fat in combination with DHA or ARA significantly promoted the growth of Lactobacillus in the feces at the genus level. On the other hand, the growth of the opportunistic pathogen Desulfovibrio was significantly inhibited by sn2PA fat in combination with ARA compared with the sn2PA fat group. In addition, sn2PA fat in combination with DHA or ARA significantly increased total SCFA concentration in the faeces, suggesting a beneficial effect on host intestinal health.

Gypenosides Reduced the Risk of Overweight and Insulin Resistance in C57BL/6J Mice through Modulating Adipose Thermogenesis and Gut Microbiota.

This study investigated whether and how gypenosides from jiaogulan tea at 100 and 300 mg/kg/day levels could reduce the development of overweight and insulin resistance in C57 BL/6J mice fed a high-fat diet in 12 weeks. The 300 mg/kg/day gypenosides supplement significantly reduced final body weight, plasma total cholesterol, and homeostasis model assessment-estimated insulin resistance (HOMA-IR) index by 19.9%, 40%, and 36%, respectively, compared with the high-fat diet control group. Gypenosides also increased brown adipocyte tissue activity and white adipose tissue browning. The expression of genes involved in mitochondrial activity and fatty acid ß-oxidation were also increased in both brown and white adipocyte tissues. In addition, gypenosides at 100 and 300 mg/kg/day levels decreased the ratio of Firmicutes to Bacteroidetes by 20% and 58.6%, respectively, and increased Akkermansia muciniphila abundance in the gut microbiota.

Characterisation of Fecal Soap Fatty Acids, Calcium Contents, Bacterial Community and Short-Chain Fatty Acids in Sprague Dawley Rats Fed with Different sn-2 Palmitic Triacylglycerols Diets.

The structure of dietary triacylglycerols is thought to influence fatty acid and calcium absorption, as well as intestinal microbiota population of the host. In the present study, we investigated the impact of palmitic acid (PA) esterified at the sn-2 position on absorption of fatty acid and calcium and composition of intestinal microorganisms in rats fed high-fat diets containing either low sn-2 PA (12.1%), medium sn-2 PA (40.4%) or high sn-2 PA (56.3%), respectively. Fecal fatty acid profiles in the soaps were measured by gas chromatography (GC), while fecal calcium concentration was detected by ICP-MS. The fecal microbial composition was assessed using a 16S rRNA high-throughput sequencing technology and fecal short-chain fatty acids were detected by ion chromatograph. Dietary supplementation with a high sn-2 PA fat significantly reduced total fecal contents of fatty acids soap and calcium compared with the medium or low sn-2 PA fat groups. Diet supplementation with sn-2 PA fat did not change the entire profile of the gut microbiota community at phylum level and the difference at genera level also were minimal in the three treatment groups. However, high sn-2 PA fat diet could potentially improve total short-chain fatty acids content in the feces, suggesting that high dietary sn-2 PA fat might have a beneficial effect on host intestinal health.