Quantitative determination of saturated-, monounsaturated- and polyunsaturated fatty acids in pork adipose tissue with non-destructive Raman spectroscopy

The composition of dietary fat has received increased attention during the recent years because it influences human health. Seventy seven samples from pork adipose tissue and melted fat from the same tissue were measured with Raman spectroscopy. Gas chromatography analysis was conducted as reference. Iodine values (IV) ranged from 58.2 to 90.4 g iodine added per 100 g fat. Polyunsaturated fatty acids (PUFA) ranged from 7.8% to 31.7% and monounsaturated fatty acids (MUFA) from 35.2% to 51.5% of total fatty acids. When applied on pre-processed spectra of melted fat, partial least square regression (PLSR) with cross-validation gave a correlation coefficient (R) = 0.98, and root mean square error of cross-validation (RMSECV) = 1.4 for IV, using 3 PLS factors in the model. PUFA gave R = 0.98 and RMSECV = 1.0% of total fatty acids, using 5 PLS factors. MUFA were predicted with R = 0.96 and RMSECV = 1.0% of total fatty acids, using 9 PLS factors. On adipose tissue a model with 3 PLS factors gave R = 0.97 and RMSECV = 1.8 for IV. For PUFA, a model with 3 PLS factors gave R = 0.95 and RMSECV = 1.5% of total fatty acids. For MUFA a model with 6 PLS factors gave R = 0.91 and RMSECV = 1.5% of total fatty acids. The results indicate the feasibility to use Raman spectroscopy as a rapid and non-destructive method to determine IV, PUFA, MUFA and saturated fatty acids (SFA) measured directly on pork adipose tissue and in melted fat from the same tissue.     

Effect of marine oils supplementation on coagulation and cellular activation in whole blood

A study was performed to explore the effects of supplemental intake of various marine oils known to be part of the Eskimo diet. Healthy men and women (134) were randomly selected to consume 15 mL/d of oil from blubber of seal, cod liver, seal/cod liver, blubber of Minke whale, or no oil for ten weeks. Total cholesterol was unchanged in the oil groups, whereas high density lipoprotein cholesterol increased 7% in the seal/cod liver oil (CLO) group (P<0.05) and 11% in the whale oil group (P<0.005). Triacylglycerol was significantly reduced in the CLO group only. The concentration of prothrombin fragment 1+2 was reduced 25% (P<0.05) after whale oil supplementation. No change in fibrinogen or factor VIIc was detected. Tumor necrosis factor generation in lipopolysaccharide (LPS)-stimulated blood was 30% reduced after whale oil (P<0.05), but was unaffected by intake of seal or CLO. The LPS-induced tissue factor activity in monocytes was reduced to a significant degree only in the seal/CLO group (34%) and whale oil group (35%) (P<0.05). The most dramatic change in thromboxane B₂ in LPS-stimulated blood was seen after whale oil intake with 44% reduction (P<0.01). Supplementation of a regular diet with a combination of seal oil and CLO and especially with whale oil seems to have beneficial effects on several products thought to be associated with cardiovascular and thrombotic diseases.     

Lipids rich in phosphatidylethanolamine from natural gas-utilizing bacteria reduce plasma cholesterol and classes of phospholipids: A comparison with soybean oil

We compared the effects of three different high-lipid diets on plasma lipoproteins and phospholipids in mink (Mustela vison). The 18 mink studied were fed one of the three diets during a 25-d period in a parallel group design. The compared diets had 0,17, and 67% extracted lipids from natural gas-utilizing bacteria (LNGB), which were rich in PE. The group with 0% LNGB was fed a diet for which the lipid content was 100% soybean oil. The total cholesterol, LDL cholesterol, and HDL cholesterol of animals consuming a diet with 67% LNGB (67LNGB-diet), were significantly lowered by 35, 49, and 29%, respectively, and unesterified cholesterol increased by 17% compared with the animals fed a diet of 100% lipids from soybean oil (SB-diet). In addition, the ratio of LDL cholesterol to HDL cholesterol was 27% lower in mink fed the 67LNGB-diet than those fed the SB-diet. When the mink were fed the 67LNGB-diet, plasma PC, total phospholipids, lysoPC, and PI were lowered significantly compared with the mink fed a SB-diet. Plasma total cholesterol was correlated with total phospholipids as well as with PC (R=0.8, P<0.001). A significantly higher fecal excretion of unesterified cholesterol, cholesteryl ester, PC, lysoPC, and PE was observed in the 67LNGB-fed mink compared with the SB-fed mink. We conclude that phospholipids from the 67LNGB-diet decreased plasma lipoprotein levels, the LDL/HDL cholesterol ratio, and plasma phospholipid levels, especially lysoPC and PC, compared with the highly unsaturated soybean oil. Our findings indicate that the decrease of plasma cholesterol is mainly caused by a specific mixture of phospholipids containing a high level of PE, and not by the dietary FA composition. The lack of significant differences in the level of plasma PE due to the diets indicates that most of the PE from LNGB has been converted to PC in the liver. Thus, plasma cholesterol may at least be partly regulated by phospholipid methylation from PE to PC in the liver.     

Enhanced incorporation of n−3 fatty acids from fish compared with fish oils

This work was undertaken to study the impact of the source of n−3 FA on their incorporation in serum, on blood lipid composition, and on cellular activation. A clinical trial comprising 71 volunteers, divided into five groups, was performed. Three groups were given 400 g smoked salmon (n=14), cooked salmon (n=15), or cooked cod (n=13) per week for 8 wk. A fourth group was given 15 mL/d of cod liver oil (CLO) (n=15), and a fifth group served as control (n=14) without supplementation. The serum content of EPA and DHA before and after intervention revealed a higher rise in EPA and DHA in the cooked salmon group (129% rise in EPA and 45% rise in DHA) as compared with CLO (106 and 25%, respectively) despite an intake of EPA and DHA in the CLO group of 3.0 g/d compared with 1.2 g/d in the cooked salmon group. No significant changes were observed in blood lipids, fibrinogen, fibrinolysis, or lipopolysaccharide (LPS)-induced tissue factor (TF) activity, tumor necrosis factor-α (TNFα), interleukin-8 (IL-8), leukotriene B₄ (LTB₄), and thromboxane B₂ (TxB₂) in whole blood. EPA and DHA were negatively correlated with LPS-induced TNFα, IL-8, LTB₄, TxB₂, and TF in whole blood. In conclusion, fish consumption is more effective in increasing serum EPA and DHA than supplementing the diet with fish oil. Since the n−3 FA are predominantly in TAG in fish as well as CLO, it is suggested that the larger uptake from fish than CLO is due to differences in physiochemical structure of the lipids.     

Antibody affinity maturation using bacterial surface display

A quantitative system for screening combinatorial single-chain Fv (scFv) antibody libraries was developed utilizing surface display on Escherichia coli and fluorescence-activated cell sorting (FACS). This system was employed to isolate clones with high-affinity to a fluorescently-labeled hapten from libraries constructed by randomizing heavy and light-chain residues in the anti-digoxin 26-10 derived antibody, scFv(dig). The use of flow cytometry enabled the detection of rare library members directly in heterogeneous populations and the optimization of selection conditions prior to sorting. A heavy-chain mutant having wild-type affinity (KD = 0.91+/-0.22 nM) and an expected representation frequency of less than 1 x 10(6), was selected to homogeneity after three rounds utilizing increasingly stringent selection conditions. The isolated clone possessed two distinct point mutations relative to the wild-type DNA sequence, yet still coded for the wild-type amino acid sequence, suggesting that the wild-type residues may be optimal at the randomized positions. An affinity improved clone (KD = 0.30+/-0.05 nM), having a dissociation constant approximately threefold lower than the wild-type antibody, was isolated from a smaller light-chain library in a single sorting step. Flow cytometry was shown to be a simple and rapid method for the determination of the relative hapten dissociation rate constants of selected clones without requiring subcloning. The relative rate constants estimated by FACS were confirmed by producing the scFv antibodies in soluble form and measuring hapten binding kinetics by surface plasmon resonance (SPR). These results demonstrate that E.coli surface display, coupled with quantitative selection and analysis using FACS, has the potential to become a powerful tool for rapid isolation and characterization of desirable mutants from large polypeptide libraries.      

Natural honey prevents ischaemia-reperfusion-induced gastric mucosal lesions and increased vascular permeability in rats

OBJECTIVE: It has been proposed that natural honey may contain a 'sucralfate-like' substance. Recent studies have shown that sucralfate affords protection against ischaemia-reperfusion-induced injuries in the rat stomach. Therefore, the effect of honey was studied on ischaemia-reperfusion-induced gastric lesions, intraluminal bleeding, vascular permeability and non-protein sulphhydryls (NP-SH) in the rat stomach. METHODS: Rats were subjected to 30 min of gastric ischaemia in the presence of 100 mM HCl and reperfusion period of 60 min. Intraluminal bleeding was assessed macroscopically and the gastric lesions were graded microscopically under an inverted microscope. Vascular permeability was quantified by measuring spectrophotometrically the extravasated Evans blue dye in the stomach. NP-SH levels were measured spectrophotometrically. A luminol-dependent chemiluminescence method was used to assess antioxidant effects of honey in vitro. RESULTS: There were significantly more gastric lesions, more severe intraluminal bleeding, more leakage of Evans blue and depletion of NP-SH during the reperfusion period as compared to controls. Pre-treatment with honey (0.078-0.625 g/kg, orally) or dimethyl sulphoxide (0.02-0.08 g/kg, intraperitoneally) 30 min before the ischaemia-reperfusion dose-dependently reduced the gastric lesions and intraluminal bleeding and decreased the vascular permeability. Furthermore, honey reversed the ischaemia-reperfusion-induced depletion of NP-SH levels and inhibited the luminol-dependent chemiluminescence induced in a cell-free xanthine-xanthine oxidase system. CONCLUSION: These results suggest that gastric protection by honey may be a result of its antioxidant effect. It is suggested that this property of honey may be due to the presence of a 'sucralfate-like' substance.