Evaluation of NMR predictors for accuracy and ability to reveal trends in 1 H NMR spectra of fatty acids.

Four different nuclear magnetic resonance (NMR) predictors have been evaluated for their ability to predict 600-MHz 1 H spectra of free fatty acids and fatty acid methyl esters of 20 common fatty acids. The predictors were evaluated on two main criteria: (1) their accuracy in direct prediction of the spectra (absolute accuracy) and (2) the ability to reveal trends or predict the change that occurs in the spectra as a result of a change in the fatty acid carbon chain, or by esterification of the free fatty acids to methyl esters (relative accuracy). The absolute accuracy in chemical shift prediction for fatty acids was good, compared with previous reports on a broader range of compounds. All four predictors had median prediction errors for chemical shifts of the signals in fatty acid methyl esters well below 0.1 ppm and as low as 0.015 ppm for one of the predictors. However, all predictors also had outliers with errors far above the upper interquartile range. In general, they also fail to reproduce trends of diagnostic value that were observed in the experimental data or properly predict the result of a minor change in molecular structure. All four predictors depend on experimental data from different origins. This may be a limiting factor for the relative accuracy of the predictors.

Effects of low doses of fish and milk proteins on glucose regulation and markers of insulin sensitivity in overweight adults: a randomised, double blind study.

PURPOSE: To examine whether supplementation with low doses of fish or milk proteins would affect glucose regulation and circulating lipid concentrations in overweight healthy adults. METHODS: Ninety-three overweight adults were assigned to receive 2.5 g protein/day from herring (HER), salmon (SAL), cod (COD) or milk (CAS, a casein-whey mixture as positive control) as tablets for 8 weeks. RESULTS: Seventy-seven participants were included in the analyses. HER and SAL did not affect glucose and insulin concentrations. COD significantly reduced within-group changes in 90 and 120 min postprandial glucose concentrations but changes were not different from HER and SAL groups. CAS supplementation significantly reduced the area under the curve for glucose concentrations (- 7%), especially when compared to SAL group, and reduced postprandial insulin c-peptide concentration (- 23%). Reductions in acetoacetate (- 24%) and ß-hydroxybutyrate (- 29%) serum concentrations in HER group were more prominent compared to SAL and COD groups, with no differences between fish protein groups for α-hydroxybutyrate. Serum concentrations of α-hydroxybutyrate (- 23%), acetoacetate (- 39%) and ß-hydroxybutyrate (- 40%) were significantly reduced within CAS group, and the decreases were significantly more pronounced when compared to SAL group. Serum lipid concentrations were not altered in any of the intervention groups. CONCLUSION: Findings indicate that 2.5 g/day of proteins from fish or milk may be sufficient to improve glucose regulation in overweight adults. The effects were most pronounced after supplementation with proteins from cod, herring and milk, whereas salmon protein did not affect any of the measurements related to glucose regulation. CLINICAL TRAIL REGISTRATION: This trial was registered at clinicaltrials.gov as NCT01641055.

Chromatographic efficiency of polar capillary columns applied for the analysis of fatty acid methyl esters by gas chromatography.

The chromatographic efficiency that could be achieved in temperature-programmed gas chromatography was compared for four capillary columns that are typically applied for analysis of fatty acid methyl esters (FAME). Three different carrier gases, hydrogen, helium and nitrogen, were applied. For each experiment, the carrier gas velocities and the temperature rates were varied with a full 9 × 3 design, with nine levels on the carrier gas velocity and temperature rates of 1, 2 or 3°C/min. Response surface methodology was used to create models of chromatographic efficiency as a function of temperature rate and carrier gas velocity. The chromatographic efficiency was defined as the inverse of peak widths measured in retention index units. The final results were standardized so that the efficiencies that could be achieved within a certain time frame, defined by the retention time of the last compound in the chromatogram, could be compared. The results show that there were clear differences in the efficiencies that could be achieved with the different columns and that the efficiency decreased with increasing polarity of the stationary phase. The differences can be explained by higher resistance to mass transfer in the stationary phase in the most polar columns.

High intake of fatty fish, but not of lean fish, improved postprandial glucose regulation and increased the n-3 PUFA content in the leucocyte membrane in healthy overweight adults: a randomised trial.

The prevalence of type 2 diabetes (T2D) is low in populations with a high fish intake; however prospective studies with fish intake have shown positive, negative or no association between fish intake and the risk for T2D. The aim of this study was to investigate the effects of high intake of lean or fatty fish on glucose tolerance, leucocyte membrane fatty acid composition and leucocyte function in overweight/obese adults. In this randomised clinical trial, sixty-eight healthy overweight/obese participants consumed 750 g/week of either lean or fatty fish as dinners, or were instructed to continue their normal eating habits but to avoid fish intake (control group), for 8 weeks. Energy and macronutrient intake and physical activity were not changed within the groups during the study period. High intake of fatty fish, but not of lean fish, significantly improved glucose regulation 120 min postprandially (P=0·012), but did not affect fasting glucose concentration. A smaller increase in fasting to 120 min postprandial insulin C-peptide concentration was seen after fatty fish intake (P=0·012). Lean fish increased the DHA content in leucocyte membranes (P=0·010), and fatty fish increased the total content of n-3 PUFA (P=0·00016) and reduced the content of n-6 PUFA (P=0·00057) in leucocyte membranes. Lean and fatty fish intake did not affect phagocytosis of bacteria ex vivo. The findings suggest that high intake of fatty fish, but not of lean fish, beneficially affected postprandial glucose regulation in overweight/obese adults, and may therefore prevent or delay the development of T2D in this population.

Application of gas chromatography/tandem mass spectrometry to determine a wide range of petrogenic alkylated polycyclic aromatic hydrocarbons in biotic samples.

RATIONALE: Due to the inherent toxicity of these compounds, analysis of petrogenic alkylated polycyclic aromatic hydrocarbons (PAHs) and heteroaromatic compounds in environmental samples is crucial. However, most existing methods are not sensitive enough when the analyte concentrations are low. Gas chromatography/tandem mass spectrometry (GC/MS/MS) is well suited to tackle this challenge, but methods are yet to be fully developed. METHODS: A gas chromatograph coupled with a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode was applied for the analysis. MRM transitions and analytical conditions were optimized for the cluster analysis of 24 different compound classes of alkylated PAHs and heteroaromatics present in crude oils. Further, the method was verified and applied to analyze the body burden of these compounds in crude oil-exposed haddock eggs. RESULTS: A comprehensive and sensitive method for the determination of C1-C4 alkylated naphthalenes, benzothiophenes, fluorenes, phenanthrenes, dibenzothiophenes, chrysenes, pyrenes and fluoranthenes was developed. The method showed good precision (RSD <15%), and the obtained concentrations were similar to those obtained by traditional GC/MS analysis. CONCLUSIONS: We demonstrate the successful application of this method towards crude oil-exposed fish eggs, where we are able to precisely measure total PAH body burden below 0.5 µg/g. The demonstrated method facilitates further miniaturization in sample preparation. Copyright © 2016 John Wiley & Sons, Ltd.

Dietary fish protein hydrolysates containing bioactive motifs affect serum and adipose tissue fatty acid compositions, serum lipids, postprandial glucose regulation and growth in obese Zucker fa/fa rats.

The world's fisheries and aquaculture industries produce vast amounts of protein-containing by-products that can be enzymatically hydrolysed to smaller peptides and possibly be used as additives to functional foods and nutraceuticals targeted for patients with obesity-related metabolic disorders. To investigate the effects of fish protein hydrolysates on markers of metabolic disorders, obese Zucker fa/fa rats consumed diets with 75 % of protein from casein/whey (CAS) and 25 % from herring (HER) or salmon (SAL) protein hydrolysate from rest raw material, or 100 % protein from CAS for 4 weeks. The fatty acid compositions were similar in the experimental diets, and none of them contained any long-chain n-3 PUFA. Ratios of lysine:arginine and methionine:glycine were lower in HER and SAL diets when compared with CAS, and taurine was detected only in fish protein hydrolysate diets. Motifs with reported hypocholesterolemic or antidiabetic activities were identified in both fish protein hydrolysates. Rats fed HER diet had lower serum HDL-cholesterol and LDL-cholesterol, and higher serum TAG, MUFA and n-3:n-6 PUFA ratio compared with CAS-fed rats. SAL rats gained more weight and had better postprandial glucose regulation compared with CAS rats. Serum lipids and fatty acids were only marginally affected by SAL, but adipose tissue contained less total SFA and more total n-3 PUFA when compared with CAS. To conclude, diets containing hydrolysed rest raw material from herring or salmon proteins may affect growth, lipid metabolism, postprandial glucose regulation and fatty acid composition in serum and adipose tissue in obese Zucker rats.

Metabolic markers in blood can separate prostate cancer from benign prostatic hyperplasia.

BACKGROUND: An individualised risk-stratified screening for prostate cancer (PCa) would select the patients who will benefit from further investigations as well as therapy. Current detection methods suffer from low sensitivity and specificity, especially for separating PCa from benign prostatic conditions. We have investigated the use of metabolomics analyses of blood samples for separating PCa patients and controls with benign prostatic hyperplasia (BPH). METHODS: Blood plasma and serum samples from 29 PCa patient and 21 controls with BPH were analysed by metabolomics analysis using magnetic resonance spectroscopy, mass spectrometry and gas chromatography. Differences in blood metabolic patterns were examined by multivariate and univariate statistics. RESULTS: By combining results from different methodological platforms, PCa patients and controls were separated with a sensitivity and specificity of 81.5% and 75.2%, respectively. CONCLUSIONS: The combined analysis of serum and plasma samples by different metabolomics measurement techniques gave successful discrimination of PCa and controls, and provided metabolic markers and insight into the processes characteristic of PCa. Our results suggest changes in fatty acid (acylcarnitines), choline (glycerophospholipids) and amino acid metabolism (arginine) as markers for PCa compared with BPH.

Optimizing the relationship between chromatographic efficiency and retention times in temperature-programmed gas chromatography.

A methodology that can maximise the chromatographic efficiency that can be achieved within a defined time frame in temperature-programmed gas chromatography is described. The efficiency can be defined as the inverse of peak widths measured in retention index units. This parameter can be described by a model similar to the van Deemter equation, which is expanded to account for the effect of the temperature rate in addition to the effect of carrier gas velocity. The model of efficiency is found by response surface methodology, where the temperature rates and the carrier gas velocities are systematically varied in the experiments. A second model that accurately explains the retention time of the last eluting compound can be found from the same experiments, and optimal conditions are found by combining the two models. The methodology has been evaluated with four capillary columns and three carrier gases, using fatty acid methyl esters as analytes. All experiments showed that there is a fairly linear decrease in efficiency with increasing temperature rates. At any temperature rate, optimal velocity is only marginally higher than the velocity that maximises chromatographic efficiency, since the carrier gas velocity has a limited effect on the retention times.

Discovery of polycyclic aromatic acid metabolites in fish exposed to the petroleum compounds 1-methylphenanthrene and 1,4-dimethylphenanthrene.

Most of the polycyclic aromatic hydrocarbons (PAHs) in petroleum are alkylated (alkyl PAHs), still the metabolism of these alkyl PAHs to the expected acid products (polycyclic aromatic acids; PAAs) has yet to be demonstrated in oil-exposed fish. Should these compounds be discovered in fish as they have in ragworm, rodents, and humans, they could present an indicative biomarker for assessing oil pollution. In this study, the ability to biotransform alkyl PAHs to PAAs was examined on Atlantic haddock (Melanogrammus aeglefinus). Exposure to phenanthrene, 1-methyphenanthrene or 1,4-dimethylphenanthrene was performed via intraperitoneal injection. An Ion Mobility Quadrupole Time-Of-Flight Mass Spectrometer (IMS-Q-TOF MS) was used in exploratory analysis of extracted bile samples. Acquisition of four-dimensional information by coupling liquid chromatography with the IMS-Q-TOF MS and in-silico prediction for feature prioritization in the data processing workflow allowed several tentative identifications with high degree of confidence. This work presents the first detection of PAAs in fish and suggests the importance of investigating alkyl PAHs in ecotoxicological studies of oil-polluted fish environments.

Minor compounds and potential interferents in gas chromatographic analyses of human serum fatty acids.

Fatty acids from 100 randomly selected human serum samples were esterified to fatty acid methyl esters and analyzed by gas chromatography with flame ionization detector. A subset of the 20 samples that spans the variation in the original set of 100 samples were thereafter analyzed by gas chromatography-mass spectrometry (GC-MS). The GC-MS data were acquired using capillary columns with two different stationary phases, BP20 (polyethylene glycol) and BPX70 (cyanopropyl polysilphenylene-siloxane). Equivalent chain lengths on the two columns are reported for 69 compounds that constituted more than 0.1% of the chromatographic area in at least one sample. Of these, 39 compounds were identified as regular fatty acid methyl esters. The remaining 30 compounds were decomposition products from cholesterol, dimethylacetals, three compounds that have been linked to poor kidney function, and 13 compounds that are currently unidentified. The retention index patterns showed that on both columns there were 16 compounds that were separated by less than 0.05 equivalent chain length units from the nearest neighbor, meaning that they were overlapping or poorly resolved. The relationship between the peak threshold level and the number of peaks found above the level predicts a dramatic increase in the number of peaks that have to be resolved if the threshold is lowered below 0.1%.

Enhancing EPA Content in an Arctic Diatom: A Factorial Design Study to Evaluate Interactive Effects of Growth Factors.

Microalgae with a high content of the omega-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are of great demand for microalgae-based technologies. An Arctic strain of the diatom Attheya septentrionalis was shown in previous experiments to increase its EPA content from 3.0 to 4.6% of dry weight (DW) in the nutrient-replete exponential phase and nutrient-depleted stationary phase, respectively. In the present study, a factorial-design experiment was used, to investigate this effect in more detail and in combination with varying salinities and irradiances. A mathematical model revealed that both growth phase and salinity, alone and in combination, influenced the EPA content significantly. Maximum EPA values of 7.1% DW were obtained at a salinity of 22 and after 5 days in stationary phase, and might be related to a decreased silica content, an accumulation of storage lipids containing EPA, or both. However, growth rates were lower for low salinity (0.54 and 0.57 d-1) than high salinity (0.77 and 0.98 d-1) cultures.

Intake of Baked Cod Fillet Resulted in Lower Serum Cholesterol and Higher Long Chain n-3 PUFA Concentrations in Serum and Tissues in Hypercholesterolemic Obese Zucker fa/fa Rats.

Increasing evidence indicates that lean fish consumption may benefit cardiovascular health. High cholesterol and low n-3 PUFA concentrations in serum are associated with an increased risk of coronary heart disease; therefore, it is of interest to investigate effects of cod intake on cholesterol and n-3 PUFAs in serum and tissues. Hypercholesterolemic obese Zucker fa/fa rats were fed diets containing 25% protein from baked cod fillet and 75% protein from casein (Baked Cod Diet), or casein as the sole protein source (Control Diet) for four weeks. Consuming Baked Cod Diet resulted in lower serum cholesterol and lower hepatic mRNA concentrations of HMG-CoA reductase and sterol O-acyltransferase-2 without affecting serum bile acid concentration, faecal excretion of cholesterol and bile acid, and hepatic concentrations of bile acids, cholesterol and cholesterol 7 alpha-hydroxylase mRNA when compared to Control Diet. Rats fed Baked Cod Diet had higher concentrations of n-3 PUFAs in serum, liver, skeletal muscle and adipose tissue. To conclude, baked cod fillet intake resulted in lower serum cholesterol, which was probably caused by lower endogenous cholesterol synthesis, and higher n-3 PUFA in serum and tissues in obese Zucker fa/fa rats. These findings support the evidence that lean fish consumption might benefit cardiovascular health.

Identification of estrogen-like alkylphenols in produced water from offshore oil installations.

Produced water released into the sea from oil installations contains a vast number of organic compounds. This work focuses on the analysis and identification of phenols in produced water, in particular long-chain para-substituted alkylphenols, which act as endocrine disruptors for marine biota. Some alkylphenol standards, unavailable commercially, have been synthesised and some compounds of interest identified. However, a complete identification is not possible since conventional GC techniques cannot achieve the desired degree of separation. An overview of the levels of the 52 known alkylphenols in produced water from nine oil installations in the North and Norwegian Seas has been made. The previously unidentified alkylphenols in produced water have been characterised by means of alkylphenol retention indices (APRI) and mass spectrometry, and their total amounts estimated for the same nine locations. Our results confirm the presence of naphthols and other as yet unidentified compounds in produced water, while thiophenols were not detected by the used technique.

Effects of baked and raw salmon fillet on lipids and n-3 PUFAs in serum and tissues in Zucker fa/fa rats​​​​​​​​​​​​​​​​​​​​.

Knowledge of the health impact of consuming heat-treated versus raw fish fillet is limited. To investigate effects of baked or raw salmon fillet intake on lipids and n-3 PUFAs in serum and tissues, obese Zucker fa/fa rats were fed diets containing 25% of protein from baked or raw salmon fillet and 75% of protein from casein, or casein as the sole protein source (control group) for four weeks. Salmon diets had similar composition of amino and fatty acids. Growth and energy intake were similar in all groups. Amounts of lipids and n-3 PUFAs in serum, liver and skeletal muscle were similar between rats fed baked or raw salmon fillet. When compared to the control group, rats fed baked salmon had lower serum total and LDL cholesterol and higher serum triacylglycerol levels. Both raw and baked salmon groups had lower HDL cholesterol level when compared to control rats. In conclusion, baking as a preparation method does not alter protein and fat qualities of salmon fillets, and intake of baked and raw salmon fillets gave similar effects on lipids and n-3 PUFAs in serum and tissues from rats.

Prediction of equivalent chain lengths from two-dimensional fatty acid retention indices.

A recently introduced two-dimensional fatty acid retention index system (2D-FARI) was used as basis for prediction of equivalent chain lengths (ECL) of fatty acid methyl esters (FAME) on a BPX-70 stationary phase. Models for the relationship between 2D-FARI data and ECL values of a calibration sample with 30 common fatty acids were established by a simple multivariate regression. The models were thereafter applied on 2D-FARI data for other FAMEs and used to predict the ECLs for these compounds. The 2D-FARI values for the fatty acids in the calibration sample are given by definition. Thus, the only information necessary to calculate the ECL value for a compound run under identical conditions as the calibration sample is the 2D-FARI values for the compound, which can be acquired from literature data. The method was validated with test sets analysed with different temperature and flow programs. ECLs of various marine FAME and trans isomers of Eicosapentaenoic and Docosahexaenoic acid were predicted with root mean squared error of prediction from 0.002 to 0.012 ECL units.

Prediction of gas chromatographic retention of polyunsaturated fatty acid methyl esters.

Multivariate regression models were applied to predict retention indices as equivalent chain lengths (ECL) for methylene-interrupted polyunsaturated fatty acids. Simple molecular descriptors, the chain length, the number of double bonds and the position of the double bond system, were used as predictors. The merits of different variable combinations were evaluated. For general models, it was necessary to include the distance from the double bond system to both the carbonyl group (Delta-position) and the methyl end of the fatty acid (n-position). The best accuracy was found for models including higher order terms of Delta and n. For models restricted to n-3 and n-6 isomers, it was not necessary to include the n-position among the variables. The highest residuals for the most accurate models were below 0.06 ECL units, and root mean square error of prediction was below 0.030. The ECL data was achieved by three different temperature programs on a cyanopropyl column.

Alkylphenol retention indices.

A novel type of retention indices for alkylphenols and related compounds are proposed. The alkylphenol retention indices (APRI) use para-substituted n-alkylphenols as reference series. APRI for para-n-alkylphenols are per definition equal to the number of carbon atoms in the alkyl substituent; the value for phenol is zero. Application of the APRI system with different types of derivatisation of the phenolic hydroxy group showed that the derivatisation has limited influence on these indices. Especially para-substituted alkylphenols gave APRI values that could be transferred with high accuracy from one type of derivative to another. By comparing results obtained with different gradients in temperature-programmed GC, it was also shown that APRI is less affected by chromatographic conditions than retention indices based on n-alkanes.

Validation of a one-step extraction/methylation method for determination of fatty acids and cholesterol in marine tissues.

A simple and fast direct extraction/methylation with methanolic hydrogen chloride was validated for determination of fatty acids (FA) in marine tissues. Three parameters: reaction time, temperature and presence of non-polar solvent, were studied by an experimental 2(3) full factorial design. The method was validated for five different types of samples; cod liver (high lipid content >60%, mainly triacylglycerol), cod muscle (low lipid content, approximately 1%, mainly phospholipids), cod plasma (lipid content, approximately 2%, mainly lipoprotein complex, high water amount), cod testis (lipid content approximately 3%, high levels of cholesterol), and herring muscle (lipid content approximately 7%). The one-step procedure for extraction/methylation of wet tissues was compared with the traditional procedure of extraction of the lipids by the Folch method (chloroform/methanol, 2:1, v/v), followed by methylation. The two methods gave similar FA profiles. The one-step extraction/methylation procedure gave a higher recovery of the total FA than the traditional procedure. Problems with carry-over peaks of cholesterol from previous samples were avoided by application of extra long GC temperature programs. The cholesterol decomposed to some degree under the preceding methanolysis step, giving several peaks in the chromatograms. The decomposition peaks were identified by mass spectrometry as cholestdienes originating from dehydration of cholesterol, a metylether of cholesterol and a cholesteryl chloride. These cholesterol artefacts can be used for quantitative determination of cholesterol in the samples. Standard samples of cholesterol were determined with high accuracy, (R(2)>0.99), and cholesterol in cod plasma was compared with good agreement (R(2)=0.97) to an enzymatic method.

Properties of trans isomers of eicosapentaenoic acid and docosahexaenoic acid methyl esters on cyanopropyl stationary phases.

The trans isomers of 5,8,11,14,17-eicosapentaenoic acid (EPA) and 4,7,10,13,16,19-docosahexaenoic acid (DHA) methyl esters were prepared by isomerisation with paratoluenesulfinic acid (PTSA) in dioxane. The isomers were fractionated by silver ion liquid chromatography with baseline resolution between the isomers with different number of trans double bonds. The fractions were analysed by GC-MS and the gas chromatographic properties of the EPA and DHA isomers with one and two trans double bonds were investigated on BPX-70 and SP-2560 cyanopropyl stationary phases. Different temperature and pressure programs were applied to introduce variations in retention indices of the isomers. The retention indices of all the trans isomers showed a strong linear correlation to the retention indices of the equivalent all-cis isomer, but the slopes for corresponding linear regression lines varied with the number of trans double bonds in the molecule. The regression lines were used to predict optimal conditions for the separation of trans isomers from the corresponding all-cis isomers. For DHA on BPX-70, and for EPA on both columns, it was possible to find windows where isomers with one trans double bond can be resolved from the corresponding all-cis isomers with R(s) > 1.0. In general, BPX-70 seems to have a more suitable selectivity for the analysis of these isomers than SP-2560. Two-dimensional fatty acid retention indices (2D-FARI) were found to be suitable for identification of trans geometry in polyunsaturated fatty acids (PUFA). Although there were substantial overlaps in the range of retention times between the all-cis isomers and isomers with one and two trans double bonds, 2D-FARI separated the isomers into distinct groups according to the number of trans double bonds.

Identification of fatty acids in gas chromatography by application of different temperature and pressure programs on a single capillary column.

A method for the identification of fatty acid methyl esters (FAME) based on the analysis of shifts in equivalent chain lengths (ECL) is described. The method is based on two-dimensional retention data achieved on one capillary column. Various temperature and pressure programs are applied on the same cyanopropyl column and the shifts in the ECL values are analysed by multivariate methods. The chain length, number of double bonds, and the double bond positions can be determined with high accuracy. The same procedure is suitable for determination of the number of trans and cis double bonds in trans fatty acids, and for detection of artefacts in fatty acid methyl ester chromatograms.