Analysis Of Non-esterified Fatty Acids Research Articles

Relationship between metabolic profile, diseases, productive and reproductive performance in high- producing Holstein cows in the postpartum period

The present study aimed to monitor Holstein cows in the postpartum period, and to evaluate the occurrence of clinical diseases and their relationship with metabolic profile, milk yield and composition, and reproductive performance. One hundred and five Holstein cows, 32 primiparous and 73 multiparous, from two dairy herds in Arapoti, Paraná State, were clinically monitored up to ten days after calving. The clinical occurrences diagnosed were: dystocia, metritis, retained placenta, clinical hypocalcemia, displaced abomasum, mastitis, pneumonia, and digital dermatitis. Blood samples were collected at one, two, five, and ten days postpartum for analysis of non-esterified fatty acids (NEFA), β-hydroxybutyrate (BHB), and total calcium. Individual milk yield was measured up to 100 days in milk (DIM), and the first test-day was evaluated for milk composition. Statistical analyses were conducted using the MIXED procedure of SAS, and the fixed effects of farm (A and B), parity (primiparous and multiparous), and occurrence of clinical diseases (sick and healthy cows) were included. Forty-eight cows (45.7%) had one or more clinical occurrences. In these sick cows, the BHB concentration at five DIM was higher (P = 0.06) than in healthy cows; 0.78 and 0.57 mmol L-1, respectively. Regarding milk yield, cows with one or more clinical occurrences had lower (P < 0.01) daily milk yield up to 30 DIM, and had a tendency of lower (P = 0.09) accumulated production up to 100 DIM than healthy cows. Sick cows had a tendency to show lower (P = 0.08) milk total solids content than healthy cows; 12.04 and 12.60%, respectively. There were no significant differences between sick and healthy animals in the remaining milk components, or the reproductive parameters assessed. The differences observed for days in the first artificial insemination (AI), AI number, and days open occurred due to the effect of the herd.

Effect of β-carotene supplementation to prepartum Holstein cows on colostrum quality and calf performance

β-Carotene, a pro-vitamin A carotenoid, acts as an antioxidant, able to scavenge free radicals to prevent oxidative damage. It has also been shown to increase rumen microbial production in vitro. When supplemented prepartum, β-carotene increased colostral fat and protein concentrations. The objective of this experiment was to evaluate the effects of supplementing 700 mg/d β-carotene (BC) daily for 4 wk prepartum on cow performance, colostrum, and performance in subsequent calves. Eighteen multiparous Holstein cows housed in a tiestall barn were blocked by expected calving date and randomly assigned to treatments at 4 wk prepartum. Blood samples were collected 3 times a week for analysis of nonesterified fatty acids, ketones, β-carotene, and IgG. Urine samples were also collected 3 times a week for analysis of creatinine and purine derivatives. Colostrum was collected within 90 min after parturition. Calves were removed from their dams before suckling, weighed within 30 min of birth, and received 4 L of maternal colostrum. Blood samples were collected from calves before colostrum administration (0 h) and at 24 h via jugular venipuncture for analysis of IgG concentration and apparent efficiency of IgG absorption. The 18 calves born were blocked based on treatments of dams. All calves were fed 449 g/d dry matter of milk replacer (20% crude protein, 20% fat) and an 18% crude protein textured starter and water ad libitum at 2 d of age until weaning at 42 d. There were no differences in any blood parameters of cows during the prepartum period. Supplemental BC increased the solids content of colostrum compared with control (22.89% control; 27.75% BC). Calves born from control fed cows had greater efficiency of IgG absorption than those born from BC supplemented cows (52.16% control; 39.50%, BC). Calves born from BC fed cows had greater feed efficiency (average daily gain/dry matter intake) compared with those born from control supplemented cows (0.33 for control; 0.44 for BC). These data indicate that although supplementing β-carotene to cows in the prepartum period negatively affects apparent efficiency of IgG absorption, it improved feed efficiency in calves.

Lipidomic analysis of non-esterified furan fatty acids and fatty acid compositions in dietary shellfish and salmon by UHPLC/LTQ-Orbitrap-MS

Lipids such as furan fatty acids (F-acids) are the valuable minor bioactive components of food such as fatty fish and plants. They are reported to have positive health benefits, including antioxidant and anti-inflammatory activities. Despite their importance, limited studies are focusing on F-acid determination in dietary seafood. This study aimed to identify and profile non-esterified F-acids and free fatty acids in total lipid extract of seafood such as shellfish and salmon. The lipidomic analysis using liquid chromatography-linear trap quadrupole-orbitrap mass spectrometry led to identifying seven types of free F-acids in shellfish (n = 5) and salmon (n = 4). The identified F-acids were confirmed by their high-resolution masses and acquired mass spectra. The relative concentrations of F-acids in shellfish range from 0.01 to 10.93 mg/100 g of the fillet, and in salmon, 0.01 to 14.21 mg/100 g of the fillet. The results revealed the highest abundance of F-acids in Sakhalin surf clam, Japanese scallop, and a fatty salmon trout. Besides, relative levels of saturated, monounsaturated, and polyunsaturated fatty acids (PUFAs) in these seafoods were compared with each other, suggesting basket clams and salmon trout to have significantly higher levels of PUFAs. The dietary seafoods enriched with F-acids and PUFAs may have possible health benefits. Hence, the applied technique could be a promising tool for rapid detection and analysis of non-esterified fatty acids in food.

Supplementation of nicotinic acid to prepartum Holstein cows increases colostral immunoglobulin G, excretion of urinary purine derivatives, and feed efficiency in calves

We evaluated the effects of incremental levels of unprotected nicotinic acid (NA) supplementation prepartum (0, 16, 32, or 48 g/d; CON, 16NA, 32NA, and 48NA, respectively) on colostrum yield and composition and cow and calf performance. Previous research indicated that 48 g/d of NA prepartum increased colostrum IgG concentration. Exact mechanisms for this increase are not clear. The effects of NA supplementation to prepartum cows on growth and performance of their calves have not been studied. Thirty-six multiparous Holstein cows housed in a tie-stall barn were blocked by expected calving date and randomly assigned to treatments at 4 wk prepartum. Blood samples were collected 3 times weekly for analysis of nonesterified fatty acids, ketones, and IgG. Urine samples were also collected 3 times weekly for analysis of creatinine and purine derivatives. Colostrum was collected within 90 min after parturition. Calves were removed from their dams before suckling, weighed within 30 min of birth, and received 4 L of maternal colostrum. The 38 calves born were blocked based on treatments of dams. All calves were fed 449 g dry matter (DM) of milk replacer (19.3% crude protein, 19.5% fat, DM basis) and a textured starter (41% starch, DM basis) at 2 d of age until weaning at 42 d, with water available ad libitum. Feeding NA resulted in linear decreases in DM intake in cows, but colostrum yield was not affected. Yield of metabolizable energy (ME) tended to change cubically, decreasing from control (CON) to 16NA, increasing from 16NA to 32NA, and decreasing from 32NA to 48NA. Concentration of IgG, protein, ash, and solids increased linearly with NA. Concentration of ME showed a tendency to increase quadratically with NA. Yield of IgG, fat, protein, and solids content increased quadratically with NA, while allantoin and total purine derivatives increased linearly. Calf 24-h IgG and apparent efficiency of absorption were not affected by NA. Calf ME intake from colostrum tended to increase quadratically with NA, but calf starter intake was not affected. Feed efficiency of calves increased quadratically with NA. Calf average daily gain changed cubically with NA, decreasing from CON to 16NA, increasing from 16NA to 32NA, and decreasing from 32NA to 48NA. Hip width gain, body length gain, and final body length changed cubically with NA, decreasing from CON to 16NA, increasing from 16NA to 32NA, and decreasing from 32NA to 48NA. Calf blood concentrations of ketones increased quadratically with NA. These data suggest that increasing levels of NA can be fed prepartum to increase colostral components and 32 g/d NA can improve calf performance.

Association among lipopolysaccharide, the transforming growth factor–beta superfamily, follicular growth, and transcription factors in spontaneous bovine ovarian cysts

The aim of this study was to investigate some of the growth and transcriptional factors originating from oocytes and granulosa cells in follicular fluid and to identify the relationships between the basic blood metabolite-metabolic hormones and intrafollicular lipopolysaccharide (LPS) concentrations. Thirty cows included in the study were allocated into 2 groups comprising 15 cows with healthy preovulatory follicles (cyclic cows) and 15 cows with confirmed cystic follicles. The ovaries and uteri of all cows were assessed by transrectal ultrasonographic examination. Blood serum samples were collected at 15, 25, 35, 45, and 55 d after calving for analysis of nonesterified fatty acids, β-hydroxybutyrate, insulin, glucose, IGF-I, ACTH, and cortisol. Ovaries and uteri were examined using transrectal ultrasound. Vaginal discharge was evaluated on the same days. Follicular fluid was also aspirated on days 35–55 from the healthy preovulatory follicles and cystic follicles using a transvaginal ovum pickup method. The densitometric levels of inhibin-α, growth and differentiation factor (GDF-9), bone morphogenetic protein (BMP-6), and GATA-4 and GATA-6 proteins were analyzed by the Western blotting technique; the concentrations of antimullerian hormone (AMH), IGF-I, estradiol-17 beta (E2), and progesterone (P4) were determined by ELISA; and the concentrations of LPS in the follicular fluid were measured by the Limulus amebocyte lysate test. The serum insulin, ACTH, and cortisol concentrations were higher in cystic cows than cyclic cows, but serum IGF-I concentrations were lower in cystic cows. The IGF-I concentrations of cystic follicular fluids were lower, whereas AMH levels were significantly greater than those of healthy preovulatory follicular fluids. The cystic follicles had significantly lower expression levels of GDF-9, BMP-6, GATA-4, and GATA-6; in contrast, inhibin-α expression and LPS concentrations were significantly higher than in healthy preovulatory follicles. The proportion of pathologic vaginal discharge within 25 d postpartum in cystic cows were higher than in the cyclic group. In conclusion, it is suggested that intrafollicular dysregulation of the transforming growth factor–β superfamily, growth, and transcriptional factors is affected by high intrafollicular LPS concentrations and systemic metabolic changes and these disturbances may be responsible for the generation of ovarian cysts.

Changes in Lipoproteins, Plasma Fatty Acid Profiles, and MicroRNA Following Four Months of Daily Almond Consumption (P08-098-19)

ObjectivesTo determine the effects of chronic almond consumption on blood lipoproteins, circulating fatty acids and microRNA profiles. MethodsThis study was a sixteen week two arm parallel randomized controlled trial. Participants received 20% of their daily calories from almonds or a calorie and fiber-matched control snack. Blood was collected at baseline, two months (midpoint), and four months (endpoint). Blood lipid panels were performed at all three time points and non-esterified fatty acid composition of fasting plasma was analyzed at the Western Human Nutrition Research Center. Total RNA was isolated from plasma samples and amplified by PCR using purchased microRNA primers, selected based on literature review and relevant biological pathways. Lipoprotein data were analyzed using repeated measures ANOVA and lipidomics data were compared using the standard least squares approach. ResultsTwenty eight subjects completed the study protocol. There were no significant differences within or between groups for total cholesterol, HDL, or triglycerides. Time by treatment effects were significant for LDL in the treatment group at endline (−13.2 ±6.5 mg/dL, P = 0.048). There were no differences in concentration or profile found in plasma fatty acid profile and these amounts were largely unchanged by diet treatment. In analysis of non-esterified fatty acids, there was a weak time by treatment effect for calendic acid (18:3, n-6) that is not significant after comparison for multiple corrections. microRNA is being investigated as a potential driver of this change in LDL and results are pending. ConclusionsThis pilot investigation found a small but significant difference in LDL concentrations from baseline following 4 months of daily almond consumption. Findings that there is no difference by treatment in non-esterified plasma fatty acids suggest that this difference is not driven through fatty acid signaling. Post-transcriptional regulation of gene expression through microRNA is a line of inquiry that is currently being explored. Funding SourcesAlmond Board of California, Jastro Shields Research Grant.

High-Throughput Quantitative Lipidomics Analysis of Nonesterified Fatty Acids in Plasma by LC-MS.

Nonesterified fatty acids are important biological molecules which have multiple functions such as energy storage, gene regulation, or cell signaling. Comprehensive profiling of nonesterified fatty acids in biofluids can facilitate studying and understanding their roles in biological systems. For these reasons, we have developed and validated a high-throughput, nontargeted lipidomics method coupling liquid chromatography to high-resolution mass spectrometry for quantitative analysis of nonesterified fatty acids. Sufficient chromatographic separation is achieved to separate positional isomers such as polyunsaturated and branched-chain species and quantify a wide range of nonesterified fatty acids in human plasma samples. However, this method is not limited only to these fatty acid species and offers the possibility to perform untargeted screening of additional nonesterified fatty acid species.

High-Throughput Quantitative Lipidomics Analysis of Nonesterified Fatty Acids in Human Plasma.

We present a high-throughput, nontargeted lipidomics approach using liquid chromatography coupled to high-resolution mass spectrometry for quantitative analysis of nonesterified fatty acids. We applied this method to screen a wide range of fatty acids from medium-chain to very long-chain (8 to 24 carbon atoms) in human plasma samples. The method enables us to chromatographically separate branched-chain species from their straight-chain isomers as well as separate biologically important ω-3 and ω-6 polyunsaturated fatty acids. We used 51 fatty acid species to demonstrate the quantitative capability of this method with quantification limits in the nanomolar range; however, this method is not limited only to these fatty acid species. High-throughput sample preparation was developed and carried out on a robotic platform that allows extraction of 96 samples simultaneously within 3 h. This high-throughput platform was used to assess the influence of different types of human plasma collection and preparation on the nonesterified fatty acid profile of healthy donors. Use of the anticoagulants EDTA and heparin has been compared with simple clotting, and only limited changes have been detected in most nonesterified fatty acid concentrations.

Considerations about the in situ derivatization and fractionation of EFA and NEFA in biological and food samples

Despite their important role in tissues, fluids and foods, the analysis of non-esterified fatty acids (NEFA) as methyl esters (NEFAME) is performed using expensive, cumbersome and time-consuming procedures that needs of isolation, fractionation and derivatization steps. However, Yi et al. [1] proposed a promising in situ, single-step procedure to analyze esterified fatty acids (EFA) and NEFA from a same sample on the basis that acylglycerols and free fatty acids can be derivatized using specific reactions. However, according to the data presented in this research work, some modifications need to be performed to increase the reliability of the method:•Increment of the transesterification performance by adding hexane to the reaction mixture, decreasing the time for the derivatization of acylglycerols from 10min to 3–4min and stopping the reaction with sulfuric acid.•Avoid cross-contamination of the NEFAME extract by adding 500μL of water after collection of EFA methyl esters (EFAME).•Samples are spiked with three internal standards: a triacylglycerol (to calculate the concentration of EFA), a free fatty acid (to calculate NEFA) and a FAME (to control isolation of FAME and cross-contamination).

Application of solid phase microextraction for quantitation of polyunsaturated fatty acids in biological fluids.

Development of a straightforward strategy for simultaneous quantitative analysis of nonesterified fatty acids (NEFA) species in biofluids is a challenging task because of the extreme complexity of fatty acid distribution in biological matrices. In this study, we present a direct immersion solid phase microextraction method coupled to a liquid chromatography-mass spectrometry platform (DI-SPME- HPLC-ESI -MS) for determination of unconjugated fatty acids (FA) in fish and human plasma. The proposed method was fully validated according to bioanalytical method validation guidelines. The LOD and LOQ were in the range of 0.5-2 and 5-12 ng/mL, respectively, with a linear dynamic range of 100 fold for each compound. Absolute and relative matrix effects were comprehensively evaluated and found to be in the acceptable range of 91-116%. The affinity constant (Ka) of individual FAs to protein albumin was determined to be 9.2 × 10(4) to 4.3 × 10(5) M(-1). The plasma protein binding (PPB%) was calculated and found to be in the range of 98.0-99.7% for different polyunsaturated fatty acids (PUFAs). The PUFAs under study were found at a high concentration range in fish plasma, whereas only a few were within quantification range in control human plasma. The method was successfully applied for monitoring PUFA changes during the operation in plasma samples obtained from patients undergoing cardiac surgery with the use of cardiopulmonary bypass (CPB). The most significant contribution induced by surgery was noticed in the concentration level of α-linolenic acid (18:3, ALA), arachidonic acid (20:4, AA), and docosahexanoic acid (22:6, DHA) soon after administration of CPB in all cases.

Analysis of non-esterified fatty acids in human samples by solid-phase-extraction and gas chromatography/mass spectrometry

The determination of the fatty acid (FA) profile of lipid classes is essential for lipidomic analysis. We recently developed a GC/MS-method for the analysis of the FA profile of total FAs, i.e. the totality of bound and unbound FAs, in any given biological sample (TOFAs). Here, we present a method for the analysis of non-esterified fatty acids (NEFAs) in biological samples, i.e. the fraction that is present as extractable free fatty acids. Lipid extraction is performed according to Dole using 80/20 2-propanol/n-hexane (v/v), with 0.1% H2SO4. The fatty acid-species composition of this NEFA-fraction is determined as FAME after derivatization with our GC/MS-method on a BPX column (Shimadzu). Validation of the NEFA-method presented was performed in human plasma samples. The validated method has been used with human plasma, cells and tissues, as well as mammalian body fluids and tissue samples. The newly developed solid-phase-extraction (SPE)-GC-MS method allows the rapid separation of the NEFA-fraction from a neutral lipid extract of plasma samples. As a major advantage compared to G-FID-methods, GC-MS allows the use of stable isotope labeled fatty acid precursors to monitor fatty acid metabolism.

Reproductive endocrinology and postweaning performance in the multiparous sow: Part 1. Influence of metabolic status during lactation

The metabolic status of the sow during lactation might influence reproductive endocrinology and the postweaning reproductive performance. With regard to the multiparous sow, previous studies addressing this topic are scarce and the results inconsistent. Blood samples were collected from 18 multiparous sows during lactation and after weaning for analysis of nonesterified fatty acids (NEFA), triglycerides, creatinine, urea progesterone, LH, and estradiol-17β. Based on the average preweaning NEFA levels the sows were divided into a “high” and a “low” catabolism group. The NEFA values were higher in the “high” group during each of the last 3 weeks of lactation. The levels of urea, creatinine and progesterone were similar (P>0.05) in the two groups throughout the study. Reproductive functions seemed equally inhibited during lactation in the two groups and there were no differences in postweaning reproductive performance. The results suggest that metabolic rate during lactation varies considerably between equally nourished multiparous sows but this has no influence on postweaning reproductive performance.

Influence of arachidonic acid on indices of phospholipase A2 activity in the human neutrophil.

The present studies were conducted to understand better the regulation of phospholipase A2 (PLA2)-dependent mobilization of lipid mediators by arachidonic acid (C20:4). After stimulation of human neutrophils, g.l.c./m.s. analysis of non-esterified fatty acids indicated that the quantity of C20:4 increased as a function of time after stimulation, from undetectable quantities to > 800 pmol/10(7) cells. In contrast with C20:4, the quantities of other free fatty acids such as oleic and linoleic were high in resting cells and did not change after stimulation. Some 15% of the C20:4 released from cellular lipids remained cell-associated. To examine the effect of C20:4 on its own release, neutrophils were exposed to [2H8]C20:4, to differentiate it by g.l.c./m.s. from naturally occurring C20:4. In A23187-stimulated neutrophils, low concentrations (5-10 microM) of [2H8]C20:4 added just before A23187 increased the quantity of C20:4 produced by the cell, whereas higher concentrations (30-50 microM) decreased the quantity of C20:4 released from phospholipids. As other measures of PLA2 activity, the effects of C20:4 on production of platelet-activity factor (PAF) and leukotriene B4 (LTB4) were assessed. C20:4 treatment just before stimulation of neutrophils blocked PAF and LTB4 production in a concentration-dependent manner (IC50 10-20 microM). The effect of C20:4 was not blocked by the cyclo-oxygenase inhibitor naproxine (10 microM), nor could it be mimicked by 1 microM LTB4, 5-hydroxyeicosa-6,8,11,14-tetraenoic acid (5HETE), 5-hydroperoxyeicosa-6,8,11,14-tetraenoic acid (5HPETE) or 15-hydroxyeicosa-5,8,11,13-tetraenoic acid (15HETE). The 5-lipoxygenase (5LO) inhibitor zileuton induced a concentration-dependent decrease in PAF, with a maximal effect of a 50% decrease at 10-50 microM. The decrease in PAF by the 5LO inhibitor could not be circumvented by addition of 1 microM 5HETE, 5HPETE and LTB4, and may be attributed to the capacity of zileuton to increase the quantity of C20:4 in A23187-treated neutrophils. The inhibitory effect of C20:4 (20-40 microM) on PAF production could be antagonized by the protein kinase C inhibitor staurosporine (30 nM), but not by inhibitors of protein kinase A, tyrosine kinase or calmodulin kinase II. Taken together, these data demonstrate that C20:4 is selectively released from membrane phospholipids of A23187-stimulated neutrophils, and this C20:4 may play an important role in regulating the mobilization of C20:4 by altering PLA2 activity.

Rapid analysis of non-esterified fatty acids as methyl esters from different biological specimens by gas chromatography after one-step esterification

A rapid gas chromatographic method for the determination of medium-chain and long-chain free fatty acids (C 14:0 to C 24:0 fatty acids) from different biological specimens is presented. After a rapid one-step transesterification method in methanol-acetyl chloride (50:1, v/v), fatty acid methyl esters were extracted into n-hexane and analysed on a 15-m Durabond-Wax column within a 12-min chromatographic run. The detection limit is 500 pg per injection.

Phospholipid interference and other variables in analysis of non-esterified fatty acids by copper soap formation

Phospholipid interference and other variables in analysis of non-esterified fatty acids by copper soap formation