Characterisation of glucose-induced protein fragments among the order Enterobacterales using matrix-assisted laser desorption ionization-time of flight mass spectrometry.

To characterise the glucose-induced protein fragments by MALDI-TOF MS analysis, we compared data for samples from Escherichia coli cultured in media with or without glucose. Characteristic peaks were observed in the presence of glucose, and MS/MS revealed Asr-specific fragments. The amino acid sequences of the fragments suggested sequence-specific proteolysis. Blast-analysis revealed that numerous Enterobacterales harbored genes encoding Asr as well as E. coli. Here, we analysed 32 strains from 20 genera and 25 species of seven Enterobacterales families. We did not detect changes in the mass spectra of four strains of Morganellaceae lacking asr, whereas peaks of Asr-specific fragments were detected in the other 28 strains. We therefore concluded that the induction of Asr production in the presence of glucose is common among the Enterobacterales, except for certain Morganellaceae species. In members of family Budviciaceae, unfragmented Asr was detected. Molecular genetic information suggested that the amino acid sequences of Asr homologs are diverse, with fragments varying in number and size, indicating that Asr may serve as a discriminative biomarker for identifying Enterobacterales species.

Investigating the cause of hemolysis in patients supported by a pulsatile ventricular assist device.

A survey conducted by Abiomed, Inc. revealed that 10 of 60 patients who received ventricular assistance via the AB5000 ventricular assist device (VAD) experienced hemolysis. The present study was conducted to investigate which factors influence hemolysis under pulsatile-flow VADs such as the AB5000. We compared the specificity of the AB5000 and its driving console with those of the NIPRO-VAD and VCT50χ under severe heart failure conditions using a mock circulatory system with a glycerol water solution. We used the mock circuit with bovine blood to confirm which pump conditions were most likely to cause hemolysis. In addition, we measured the shear velocity using particle image velocimetry by analyzing the seeding particle motion for both the AB5000 and NIPRO-VAD under the same conditions as those indicated in the initial experiment. Finally, we analyzed the correlation between negative pressure, exposure time, and hemolysis by continuously exposing fixed vacuum pressures for fixed times in a sealed device injected with bovine blood. Applying higher vacuum pressure to the AB5000 pump yielded a larger minimum inlet pressure and a longer exposure time when the negative pressure was under - 10 mmHg. The plasma-free hemoglobin increased as more negative pressure was driven into the AB5000 pump. Moreover, the negative pressure interacted with the exposure time, inducing hemolysis. This study revealed that negative pressure and exposure time were both associated with hemolysis.

Development of a flow rate monitoring method for the wearable ventricular assist device driver.

Our research institute has been working on the development of a compact wearable drive unit for an extracorporeal ventricular assist device (VAD) with a pneumatically driven pump. A method for checking the pump blood flow on the side of the drive unit without modifying the existing blood pump and impairing the portability of it will be useful. In this study, to calculate the pump flow rate indirectly from measuring the flow rate of the driving air of the VAD air chamber, we conducted experiments using a mock circuit to investigate the correlation between the air flow rate and the pump flow rate as well as its accuracy and error factors. The pump flow rate was measured using an ultrasonic flow meter at the inflow and outflow tube, and the air flow was measured using a thermal mass flow meter at the driveline. Similarity in the instantaneous waveform was confirmed between the air flow rate in the driveline and the pump flow rate. Some limitations of this technique were indicated by consideration of the error factors. A significant correlation was found between the average pump flow rate in the ejecting direction and the average air flow rate in the ejecting direction (R2 = 0.704-0.856), and the air flow rate in the filling direction (R2 = 0.947-0.971). It was demonstrated that the average pump flow rate was estimated exactly in a wide range of drive conditions using the air flow of the filling phase.

Trans-octadecenoic Acid Positional Isomers Have Different Accumulation and Catabolism Properties in Mice.

Trans fatty acids (TFA) are considered risk factors for cardiovascular disease (CVD), while the details of distribution and metabolism of the individual isomers are not clear. Here we investigated the accumulation and catabolic rate of TFA positional isomers of octadecenoic acid (18:1) in mice. ICR mice were fed deuterium- and [1-(13)C] stable isotope-labeled trans-9-18:1 (9t-18:1*), trans-10-18:1 (10t-18:1*), or trans-11-18:1 (11t-18:1*) for 2 or 4 weeks, or a TFA mixture (9t-18:1*, 10t-18:1*, and 11t-18:1*) for 3 weeks. Analysis of whole-body tissues by gas chromatography-chemical ionization mass spectrometry revealed the highest 9t-18:1* levels in the heart. Significant differences in the accumulation of the respective trans-18:1 were observed in the heart and erythrocytes, where 9t- > 11t- > 10t-18:1*, but no significant difference was observed in the liver or white adipose tissue (WAT). Mice fed on 11t-18:1 demonstrated accumulation of endogenously synthesized conjugated linoleic acid in the liver, WAT, and heart, but any other metabolites were not found in other groups. Furthermore, we analyzed catabolic rates of single-dose-administered trans-18:1* isomers into [(13)C]-labeled CO2 using isotope-ratio mass spectrometry, and the 10t-18:1*catabolic rate was significantly higher than those of 9t- and 11t-18:1*. We found that the accumulation and catabolism of trans-18:1 positional isomers varied in these mice. Differential accumulation in tissues suggests that individual TFA positional isomers may play different roles in human health.

Differential effects of triacylglycerol positional isomers containing n-3 series highly unsaturated fatty acids on lipid metabolism in C57BL/6J mice.

The present study investigated the effects of binding position of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to triacylglycerol (TAG) on lipid metabolism in C57BL/6J mice. Mice were treated with pure TAG positional isomers, including 1,2(2,3)-dipalmitoyl-3(1)-eicosapentaenoyl glycerol, 1,3-dipalmitoyl-2-eicosapentaenoyl glycerol, 1,2(2,3)-dipalmitoyl-3(1)-docosahexaenoyl glycerol, and 1,3-dipalmitoyl-2-docosahexaenoyl glycerol. Compared to DHA bound to the α-position of TAG, DHA bound to the ß-position more effectively inhibited fatty acid synthetic enzymes and cholesterol-metabolism enzymes and thus reduced TAG and cholesterol concentrations in the serum and liver. EPA bound to the α-position of TAG, but not EPA bound to the ß-position of TAG, significantly decreased hepatic cholesterol concentrations. Additionally, EPA bound to the α-position of TAG increased the ratio of PGI2 to TXA2 to a higher degree than EPA bound to the ß-position. These results suggested that the binding position of EPA and DHA to TAG affected TAG and cholesterol metabolism as well as eicosanoid production in C57BL/6J mice.

Generation of Tetracosahexaenoic Acid in Benthic Marine Organisms.

Tetracosahexaenoic acid (THA, 24:6n-3) has been shown to have the strongest ability to suppress accumulation of lipids in HepG2 cells among well-known n-3 highly unsaturated fatty acids, such as EPA and DHA. In this study, a method for mass production of THA was investigated using distributions of THA and DHA in thirty-two marine organisms, such as starfishes, right-eyed flounders, shellfishes, and sharks. The fatty acid composition of the marine organisms was analyzed using GC-FID and THA was detected in starfish, right-eyed flounder, and shark. Furthermore, the ratio of DHA and THA (DHA/THA) in each sample was calculated using chromatogram peak area of GC-FID, and the value was found to be lower than 1 in some starfishes. As a result, THA was thought to be synthesized in the starfishes. In contrast, the value of DHA/THA for right-eyed flounder and sharks was greater than 1. The THA accumulation in right-eyed flounder was considered to be because of the starfishes that the flounder consumes as part of its diet. DHA is synthesized from THA by beta-oxidation in peroxisomes, in the Sprecher's shunt. The high accumulation of THA observed in the flounder would be caused by the decreasing enzyme activation due to beta-oxidation in the peroxisomes of the starfishes. Understanding the differences in THA between aquatic species could also potentially allow us to understand why THA is generated in marine animals.

Regiospecific Distribution of trans-Octadecenoic Acid Positional Isomers in Triacylglycerols of Partially Hydrogenated Vegetable Oil and Ruminant Fat.

It is revealed that binding position of fatty acid in triacylglycerol (TAG) deeply relates to the expression of its function. Therefore, we investigated the binding positions of individual trans-octadecenoic acid (trans-C18:1) positional isomers, known as unhealthy fatty acids, on TAG in partially hydrogenated canola oil (PHCO), milk fat (MF), and beef tallow (BT). The analysis was carried out by the sn-1(3)-selective transesterification of Candida antarctica Lipase B and by using a highly polar ionic liquid capillary column for gas chromatography-flame ionization detection. Trans-9-C18:1, the major trans-C18:1 positional isomer, was selectively located at the sn-2 position of TAG in PHCO, although considerable amounts of trans-9-C18:1 were also esterified at the sn-1(3) position. Meanwhile, trans-11-C18:1, the major isomer in MF and BT, was preferentially located at the sn-1(3) position. These results revealed that the binding position of trans-C18:1 positional isomer varies between various fats and oils.

Abundances of Triacylglycerol Positional Isomers and Enantiomers Comprised of a Dipalmitoylglycerol Backbone and Short- or Medium-chain Fatty Acids in Bovine Milk Fat.

Bovine milk fat (BMF) is composed of triacylglycerols (TAG) rich in palmitic acid (P), oleic acid (O), and short-chain or medium-chain fatty acids (SCFAs or MCFAs). The composition and binding positions of the fatty acids on the glycerol backbone determine their physical and nutritional properties. SCFAs and MCFAs are known to characteristically bind to the sn-3 position of the TAGs in BMF; however, there are very few non-destructive analyses of TAG enantiomers binding the fatty acids at this position. We previously reported a method to resolve the enantiomers of TAGs, binding both long-chain saturated fatty acid and unsaturated fatty acid at the sn-1 and 3 positions, in palm oil, fish oil, and marine mammal oil using chiral HPLC. Here, we further developed a method to resolve several TAG enantiomers containing a dipalmitoyl (PP) glycerol backbone and one SCFA (or MCFA) in BMF. We revealed that the predominant TAG structure in BMF was homochiral, such as 1,2-dipalmitoyl-3-butyroyl-sn-glycerol. This is the first quantitative determination of many TAG enantiomers, which bind to a SCFA or MCFA, in BMF was evaluated simultaneously. Furthermore, the results indicated that the amount ratios of the positional isomers and enantiomers of TAGs consisting of a dipalmitoyl (PP) glycerol backbone and SCFA (or MCFA), resembled the whole TAG structures containing the other diacylglycerol backbones consisting of P, O, myristic acid, and/or stearic acid in BMF.

Editorial comment: feasibility of central gastrectomy for gastric cancer.

BACKGROUND: Central gastrectomy (CG) for gastric cancer was developed to preserve pyloric function and maintain a large gastric volume. Whether this procedure is feasible for limited cases of gastric cancer is unclear. METHODS: On the basis of Union Internationale Contre le Cancer TNM classification, pathologic characteristics, perioperative parameters, and long-term results, we analyzed 100 patients who underwent CG. RESULTS: Pathologic findings included T1 (tumor depth, mucosal or submucosal) in 82 patients and T2 (muscularis propria or subserosal) in 18 patients. Mean number of dissected lymph nodes was 17.3, and pathologic N1 (node metastasis, 6 or less) was found in 14 patients. There were no operative deaths, but 5 patients had postoperative complications: anastomotic leakage in 1, severe gastric stasis in 2, ischemic gastric ulcer in 1, and intra-abdominal bleeding in 1. No patient had a cancer recurrence in a mean follow-up of 49 months. New early gastric cancer was detected in 3 patients during follow-up endoscopic examination. The 5-year cumulative survival was 0.97. One year after CG, 63 patients had early satiety after food intake. Mean ratio of 1-year postoperative/preoperative body weight was 95%. CONCLUSIONS: Central gastrectomy with sufficient node dissection resulted in good long-term survival and minimal postoperative weight loss. CG is a safe and useful procedure for selected patients with gastric cancer, although close follow-up for recurrence and a more precise analysis on physiologic states is needed.

Characterization of cis- and trans-octadecenoic acid positional isomers in edible fat and oil using gas chromatography-flame ionisation detector equipped with highly polar ionic liquid capillary column.

In this study, the characterisation of all cis- and trans-octadecenoic acid (C18:1) positional isomers in partially hydrogenated vegetable oil (PHVO) and milk fat, which contain several cis- and trans-C18:1 positional isomers, was achieved by gas chromatography-flame ionisation detector equipped with a highly polar ionic liquid capillary column (SLB-IL111). Prior to analysis, the cis- and trans-C18:1 fractions in PHVO and milk fat were separated using a silver-ion cartridge. The resolution of all cis-C18:1 positional isomers was successfully accomplished at the optimal isothermal column temperature of 120 °C. Similarly, the positional isomers of trans-C18:1, except for trans-6-C18:1 and trans-7-C18:1, were separated at 120 °C. The resolution of trans-6-C18:1 and trans-7-C18:1 isomers was made possible by increasing the column temperature to 160 °C. This analytical method is suitable for determining the cis- and trans-C18:1 positional isomers in edible fats and oils.

Comparison of the lipid-lowering effects of four different n-3 highly unsaturated fatty acids in HepG2 cells.

The effects on lipid metabolism of four different n-3 highly unsaturated fatty acids (n-3HUFA) including eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid (DPA, 22:5n-3), docosahexaenoic acid (DHA, 22:6n-3), and tetracosahexaenoic acid (THA, 24:6n-3) were compared in the HepG2 cell model. None of the n-3HUFAs affected the viability of the cells. THA exerted the strongest suppression on the synthesis of triacylglycerol and cholesteryl ester (ChE), and the order of the strength of suppression was found to be THA > DHA > DPA > EPA. The mRNA level of fatty acid synthase was suppressed by the n-3HUFAs and the order of the strength of suppression by n-3HUFAs was the same in both triacylglycerol and ChE synthesis. These findings support previous animal test results using EPA, DPA, and DHA. In conclusion, both the number of carbon atoms and double bonds in an n-3HUFA structure has an effect on lipid metabolism in HepG2 cells.

Simple method for the quantification of milk fat content in foods by LC-APCI-MS/MS using 1,2-dipalmitoyl-3-butyroyl-glycerol as an indicator.

We have developed a simple method for the quantification of milk fat in foods using 1,2-dipalmitoyl-3-butyroyl-glycerol (PPBu) as an indicator of milk fat content by high-performance liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry. The separation of the triacylglycerol positional isomer, 1,3-dipalmitoyl-2-butyroyl-glycerol (PBuP) and PPBu, was achieved using an octacocyl silylation (C28) column, and multiple reaction monitoring was employed. The milk fat contents in butter, butter-blended margarine, and butter cookies were quantified using two different sample preparation methods. In the first method (Method A), the lipid in the food was extracted with organic solvents and used for the preparation of a sample solution. In the other method (Method B), the sample solution was prepared by dissolving the food in organic solvents; the PPBu content in the fat and oil was corrected by the lipid content in the food obtained by the rapid NMR method. The calibration curve of standard PPBu was a first-order equation over the range of 1-250 µg/mL. The recovery rates of PPBu spiked into butter evaluated by Methods A and B were 99.9-105.0% and 106.5-110.1%, respectively. PBuP was not detected in milk fat. The milk fat contents in blends of butter and margarine determined by the method developed in this study were equivalent to the contents calculated with the butyric acid (Bu) method using GC-FID. The milk fat contents in butter-blended margarine analyzed by Methods A and B were almost the same. The PPBu content in blends of butter and margarine was correlated with the butter content. Thus, we succeeded in developing an efficient method for the rapid quantification of milk fat content and the detection of milk fat adulteration.

Quantification of triacylglycerol molecular species in cocoa butter using high-performance liquid chromatography equipped with nano quantity analyte detector.

Triacylglycerol (TAG) molecular species were quantified through high-performance liquid chromatography (HPLC) equipped with a nano quantity analyte detector (NQAD). TAG standard compounds, i.e., 1,3-dipalmitoyl-2-oleoylglycerol (ß-POP), 1-palmitoyl-2-oleoyl-3-stearoyl-rac-glycerol (ß-POS), and 1,3-distearoyl-2-oleoylglycerol (ß-SOS), and natural cocoa butter were used for analyses. NQAD gave the first order equation passing through the origin for all TAG standard compounds. TAG molecular species in cocoa butter were quantified using the calibration curves and the obtained values were almost the same as the reported ones of conventional cocoa butter. Furthermore, a recovery test was also carried out and the values were almost 100. Therefore, HPLC-NQAD can be successfully used for the quantification of TAG molecular species in natural fats and oils.

Resolution behavior of cis- and trans-octadecenoic acid isomers by AOCS official method using SP-2560 column.

The gas chromatography-flame ionization detector equipped with a higher polarity column (i.e., SP-2560) has often been used for the quantification of trans-fatty acids in food. In particular, AOCS Ce 1h-05, the official method of the American Oil Chemists' Society (AOCS), is a highly effective method to separate the isomers of trans-fatty acids. In this study, the resolution behavior and the response factors of cis- and trans-octadecenoic acid methyl ester (C18:1-ME) isomers separated by the AOCS Ce 1h-05 method were investigated, and the contents of each cis- and trans-C18:1-ME isomer in partially hydrogenated vegetable oil (PHVO) and milk fat were quantified by using the calibration curves obtained for the respective isomers. The relative response factors for the trans- and cis-C18:1-ME isomers against the internal standard heneicosanoic acid methyl ester (C21:0-ME) were 1.031 ± 0.040 (mean ± SD) and 0.990 ± 0.032, respectively. The relative response factors of trans-isomers tend to be higher than those of cis-C18:1-ME isomers. The peaks of cis-4-C18:1-ME, cis-5-C18:1-ME, cis-6-C18:1-ME, cis-7-C18:1-ME, cis-8-C18:1-ME, and cis-9-C18:1-ME isomers overlapped with those of trans-C18:1-ME isomers. Both PHVO and milk fat contained many types of cis- and trans-C18:1 isomers, and the total contents of the trans-C18:1 isomer in PHVO and milk fat were 28.01 g and 3.62 g per 100 g oil, respectively. When the trans-C18:1-ME isomer was separated from the cis-C18:1-ME by using a silver-ion cartridge column before the analyses, the total contents of the trans-C18:1 isomer in PHVO and milk fat were 23.03 g and 2.78 g per 100 g oil, respectively. The difference in the trans-C18:1 isomer content between the two methods was ascribed to the partial overlapping of cis-isomer peaks with the peaks of trans-C18:1-ME isomers, in the chromatogram.

Actual ratios of triacylglycerol positional isomers and enantiomers comprising saturated fatty acids and highly unsaturated fatty acids in fishes and marine mammals.

It has been previously shown that the positional isomers of triacylglycerol (TAG) containing palmitic acid (P) and highly unsaturated fatty acids (HUFAs) such as DHA (D) and EPA (E) vary between fishes and marine mammals. However, it has not yet been understood why in marine mammals HUFAs are located only at the α position when two palmitic acid chains combine, and not in fishes. In order to gain further understanding of the biosynthetic pathways involved in the formation of these asymmetric TAGs, we investigated whether the HUFA in the TAG of marine mammals exists predominantly at the sn-1 or sn-3 position. We examined the TAG positional isomers and enantiomers in marine organisms in detail. As a result, while PDP and PEP were not detected, sn-PPD and sn-PPE were found in abundance in marine mammals. For fishes, on the other hand, PDP, PEP, sn-PPD, and sn-PPE were all identified. In the case of TAGs that contain two HUFAs and one palmitic acid, marine mammals were rich in DPD and EPE whereas fishes were rich in sn-PDD and sn-PEE.

Actual ratio of triacylglycerol positional isomers in milk and cheese.

Actual ratios of triacylglycerol (TAG) positional isomers in human, rat, and cow milk fat and cow, buffalo, goat, and sheep cheese fat were analyzed using HPLC-UV-atmospheric pressure chemical ionization-MS/MS system equipped with an octacosyl silylation column or polymeric ODS column. We substituted cheese fats for milk fats in parts of our study because milks from ruminants, with the exception of cows, are difficult to get in Japan. The actual ratio of ß-PPC (the TAG consisting of two palmitic acids (P) and one capric acid (C), with the palmitic acid located at the ß position) and ß-PCP in human milk was different from those in ruminants, with more than half of the medium-chain fatty acids located at the ß position even though other fats possessed it mainly at the α position. Palmitic acid was mainly located at the ß position for human milk and rat milk; however, the location in ruminant cheese fat was mainly at the α position. The location of fatty acids is thought to be very important for infant nutrition. Particularly, the location of palmitic acid in case of human milk and of medium-chain fatty acids in case of ruminant milk was very characteristic and is considered to be very important to the fatty acids in milk fat.

Enantiomeric separation of asymmetric triacylglycerol by recycle high-performance liquid chromatography with chiral column.

In our previous studies, we employed recycle HPLC for the separation of triacylglycerol (TAG)-positional isomers (PIs). In this study, a recycle HPLC system equipped with a polysaccharide-based chiral column was applied to the enantiomeric separation of some asymmetric TAGs having straight-chain C16-C18 acyl residues. As a result, 1,2-dipalmitoyl-3-oleoyl-rac-glycerol (rac-PPO), 1,2-dioleoyl-3-palmitoyl-rac-glycerol (rac-OOP), and 1,2-dipalmitoyl-3-linoleoyl-rac-glycerol (rac-PPL) were resolved into their respective enantiomers. However, neither 1,2-dioleoyl-3-linoleoyl-rac-glycerol (rac-OOL), consisting of only unsaturated fatty acids, nor 1,2-dipalmitoyl-3-stearoyl-rac-glycerol (rac-PPS), consisting of only saturated fatty acids, was resolved. These results suggest that the asymmetric TAGs, used in this study, having both a palmitic acid moiety and an oleic acid (or a linoleic acid) moiety at the sn-1 or sn-3 positions are resolved by the chiral column. This new chiral separation method can be used in combination with atmospheric pressure chemical ionization mass spectrometry to determine the sn-OOP/sn-POO ratio in palm oil. This method is applicable for the chiral separation of asymmetric TAGs in palm oil.

Rapid separation of triacylglycerol positional isomers binding two saturated Fatty acids using octacocyl silylation column.

The rapid separation of a triacylglycerol positional isomer (TAG-PI) pair was examined via high-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry using an octacocyl silylation (C28) column. A TAG-PI pair binding two palmitic acids and one fatty acid that structurally differs from palmitic acid was separated at 10°C and 15°C using acetone as the mobile phase. However, the TAG-PI pair binding two unsaturated fatty acids and one saturated fatty acid was not separated by the C28 column. The results indicate that the structures of the two palmitic acids (saturated fatty acids) and the other fatty acid at the α or ß position in TAG play an important role in the separation of the TAG-PI pair, and that the structure of the fatty acid needs to be considerably different from that of the palmitic acid, specifically in terms of the chain length or the location of the double bond.