Essential Fatty Acid Deficiency Associates with Growth Faltering and Environmental Enteric Dysfunction in Children.

Environmental enteric dysfunction (EED) is characterized by intestinal inflammation, malabsorption and growth-faltering in children with heightened exposure to gut pathogens. The aim of this study was to characterize serum non-esterified fatty acids (NEFA), in association with childhood undernutrition and EED, as potential biomarkers to predict growth outcomes. The study comprised a cohort of undernourished rural Pakistani infants (n = 365) and age-matched controls followed prospectively up to 24 months of age. Serum NEFA were quantified at ages 3-6 and 9 months and correlated with growth outcomes, serum bile acids and EED histopathological biomarkers. Serum NEFA correlated with linear growth-faltering and systemic and gut biomarkers of EED. Undernourished children exhibited essential fatty acid deficiency (EFAD), with low levels of linoleic acid and total n-6 polyunsaturated fatty acids, compensated by increased levels of oleic acid and increased elongase and desaturase activities. EFAD correlated with reduced anthropometric Z scores at 3-6 and 9 months of age. Serum NEFA also correlated with elevated BA and liver dysfunction. Essential fatty acid depletion and altered NEFA metabolism were highly prevalent and associated with acute and chronic growth-faltering in EED. The finding suggests that targeting early interventions to correct EFAD and promote FA absorption in children with EED may facilitate childhood growth in high-risk settings.

Cholestasis impairs gut microbiota development and bile salt hydrolase activity in preterm neonates.

Cholestasis refers to impaired bile flow from the liver to the intestine. In neonates, cholestasis causes poor growth and may progress to liver failure and death. Normal bile flow requires an intact liver-gut-microbiome axis, whereby liver-derived primary bile acids are transformed into secondary bile acids. Microbial bile salt hydrolase (BSH) enzymes are responsible for the first step, deconjugating glycine- and taurine-conjugated primary bile acids. Cholestatic neonates often are treated with the potent choleretic bile acid ursodeoxycholic acid (UDCA), although interactions between UDCA, gut microbes, and other bile acids are poorly understood. To gain insight into how the liver-gut-microbiome axis develops in extreme prematurity and how cholestasis alters this maturation, we conducted a nested case-control study collecting 124 stool samples longitudinally from 24 preterm infants born at mean 27.2 ± 1.8 weeks gestation and 946 ± 249.6 g, half of whom developed physiologic cholestasis. Samples were analyzed by whole metagenomic sequencing, in vitro BSH enzyme activity assays optimized for low biomass fecal samples, and quantitative mass spectrometry to measure the bile acid metabolome. In extremely preterm neonates, acquisition of the secondary bile acid biosynthesis pathway and BSH genes carried by Clostridium perfringens are the most prominent features of early microbiome development. Cholestasis interrupts this developmental pattern. BSH gene abundance and enzyme activity are profoundly reduced in cholestatic neonates, resulting in decreased quantities of unconjugated bile acids. UDCA restores total fecal bile acid levels in cholestatic neonates, but this is due to a 522-fold increase in fecal UDCA. A majority of bile acids in early development are atypical positional and stereo-isomers of bile acids. We report novel associations linking isomeric bile acids and BSH activity to neonatal growth trajectories. These data highlight deconjugation of bile acids as a key microbial function that is acquired in early neonatal development and impaired by cholestasis.

The impact of gastrointestinal dysmotility on the aerodigestive microbiome of pediatric lung transplant recipients.

BACKGROUND: Delayed gastric emptying has been associated with increased graft rejection, although the mechanism of this association is not known. This study aims to investigate the interrelationship between delays in gastrointestinal motility and the diversity and composition of gastric, oropharyngeal, and lung microbiomes in pediatric lung transplant recipients. METHODS: We prospectively recruited 23 pediatric lung transplant recipients and 98 pediatric patients with respiratory symptoms undergoing combined endoscopy and bronchoscopy. Gastric, oropharyngeal, and bronchoalveolar lavage samples were collected for 16S sequencing. Gastric samples were also analyzed for bile composition using liquid chromatography. RESULTS: Patients who underwent lung transplantation had significantly reduced alpha diversity in gastric and oropharyngeal sites compared with patients with respiratory symptoms. This reduction in alpha diversity was especially evident in gastric samples in patients with delayed gastric emptying defined as abnormal gastric emptying on nuclear scintigraphy or as an elevation in gastric bile concentration (p ≤ 0.05). Whereas monocolonies were seen in the lungs of patients who underwent transplantation, these were not the same microbes seen in the stomach; the microbial overlap between lung and gastric samples within patients was low, and data indicated high individual variation between lung transplant recipients. Other contributors to reduced alpha diversity included antibiotics in combination with proton pump inhibitors, especially in gastric and oropharyngeal samples. CONCLUSIONS: Lung transplant recipients have reduced microbial diversity in gastric fluid (GF) and oropharynx compared with patients who did not undergo lung transplantation. The decreased alpha diversity in GF may be associated with dysmotility.

Risk Factors for Bile Aspiration and its Impact on Clinical Outcomes.

INTRODUCTION: Bile reflux may cause for lung allograft rejection, yet there are no studies that determine (i) the relationship between gastric and lung bile concentrations, (ii) whether bile is present in lungs of nontransplant patients, (iii) the relationship between gastric dysmotility and lung bile, (iv) the impact of reflux therapies on lung bile, and (v) whether lung bile worsens outcomes in nontransplant patients. This study will address these gaps in the literature. METHODS: We prospectively recruited lung transplant (LTX) patients and nontransplant patients with respiratory symptoms (RP) and collected paired gastric and lung samples. Bile concentration and composition of samples was assessed using liquid chromatography-mass spectrometry. Bile results were compared with clinical parameters, including the presence of esophagitis, gastric dysmotility, and/or pathologic gastroesophageal reflux. RESULTS: Seventy patients (48 RP and 22 LTX) were recruited. Overall, 100% of gastric and 98% of bronchoalveolar lavage samples contained bile. The mean gastric bile concentrations in RP and LTX patients were 280 ± 703 nmol/L and 1,004 ± 1721 nmol/L, respectively (P = 0.02). There was no difference in lung bile concentrations between RP (9 ± 30 nmol/L) and LTX (11 ± 15 nmol/L, P = 0.7). Patients with delayed gastric emptying had higher lung bile concentrations (15.5 ± 18.8 nmol/L) than patients with normal gastric emptying (4.8 ± 5.7 nmol/L, P = 0.05) independently of reflux burden. Proton pump inhibitor use increased the proportion of unconjugated gastric bile acids. High lung bile concentrations were associated with an increased risk of hospitalization and longer hospital stays in RP patients (P < 0.05). DISCUSSION: Lung bile is almost universally present in symptomatic patients, and higher concentrations are associated with poorer respiratory outcomes.

Utilizing centralized biorepository samples for biomarkers of cystic fibrosis lung disease severity.

BACKGROUND: Circulating biomarkers reflective of lung disease activity and severity have the potential to improve patient care and accelerate drug development in CF. The objective of this study was to leverage banked specimens to test the hypothesis that blood-based biomarkers discriminate CF children segregated by lung disease severity. METHODS: Banked serum samples were selected from children who were categorized into two extremes of phenotype associated with lung function ('mild' or 'severe') based on CF-specific data and were matched on age, gender, CFTR genotype, and P. aeruginosa infection status. Targeted inflammatory proteins, lipids, and discovery metabolite profiles were measured in these serum samples. RESULTS: The severe cohort, characterized by a lower CF-specific FEV1 percentile, had significantly higher circulating concentrations of high sensitivity C-reactive protein, serum amyloid A, granulocyte colony stimulating factor, and calprotectin compared to the mild cohort. The mild cohort tended to have higher serum linoleic acid concentrations. The metabolite arabitol was lower in the severe cohort while other CF relevant metabolic pathways showed non-significant differences after adjusting for multiple comparisons. A sensitivity analysis to correct for biased estimates that may result from selecting subjects using an extremes of phenotype approach confirmed the protein biomarker findings. CONCLUSIONS: Circulating inflammatory proteins differ in CF children segregated by lung function. These findings serve to demonstrate the value of maintaining centralized, high quality patient derived samples for future research, with linkage to clinical information to answer testable hypotheses in biomarker development.

Accurate mass and retention time library of serum lipids for type 1 diabetes research.

Dysregulated lipid species are linked to various disease pathologies and implicated as potential biomarkers for type 1 diabetes (T1D). However, it is challenging to comprehensively profile the blood specimen lipidome with full structural details of every lipid molecule. The commonly used reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS)-based lipidomics approach is powerful for the separation of individual lipid species, but lipids belonging to different classes may still co-elute and result in ion suppression and misidentification of lipids. Using offline mixed-mode and RPLC-based two-dimensional separations coupled with MS/MS, a comprehensive lipidomic profiling was performed on human sera pooled from healthy and T1D subjects. The elution order of lipid molecular species on RPLC showed good correlations to the total number of carbons in fatty acyl chains and total number of double bonds. This observation together with fatty acyl methyl ester analysis was used to enhance the confidence of identified lipid species. The final T1D serum lipid library database contains 753 lipid molecular species with accurate mass and RPLC retention time uniquely annotated for each of the species. This comprehensive human serum lipid library can serve as a database for high-throughput RPLC-MS-based lipidomic analysis of blood samples related to T1D and other childhood diseases. Graphical abstract.

Off-line mixed-mode liquid chromatography coupled with reversed phase high performance liquid chromatography-high resolution mass spectrometry to improve coverage in lipidomics analysis.

The confident identification and in-depth profiling of molecular lipid species remain to be a challenge in lipidomics analysis. In this work, an off-line two-dimensional mixed-mode and reversed-phase liquid chromatography (RPLC) method combined with high-field quadrupole orbitrap mass spectrometer (Q Exactive HF) was developed to profile lipids from complex biological samples. In the first dimension, 22 different lipid classes were separated on a monolithic silica column with elution order from neutral to polar lipids. A total of 13 fractions were collected and run on a RPLC C30 column in the second dimension for further separation of the lipid molecular species based on their hydrophobicity, with the elution order being determined by both the length and degree of unsaturation in the fatty-acyl chain. The method was applied to analyze lipids extracted from rat plasma and rat liver. Fatty acid methyl ester analysis by gas chromatography-mass spectrometry was used to identify the fatty acyls from total lipid extracts, which provided a more confident identification of the lipid species present in these samples. More than 800 lipids were identified in each sample and their molecular structures were confidentially confirmed using tandem mass spectrometry (MS/MS). The number of lipid molecular species identified in both rat plasma and rat liver by this off-line two-dimensional method is approximately twice of that by one-dimensional RPLC-MS/MS employing a C30 column. This off-line two-dimensional mixed-mode LC-RPLC-MS/MS method is a promising technique for comprehensive lipid profiling in complex biological matrices.

Comprehensive untargeted lipidomic analysis using core-shell C30 particle column and high field orbitrap mass spectrometer.

The goal of untargeted lipidomics is to have high throughput, yet comprehensive and unambiguous identification and quantification of lipids. Novel stationary phases in LC separation and new mass spectrometric instruments capable of high mass resolving power and faster scanning rate are essential to achieving this goal. In this work, 4 reversed phase LC columns coupled with a high field quadrupole orbitrap mass spectrometer (Q Exactive HF) were thoroughly compared using complex lipid standard mixture and rat plasma and liver samples. A good separation of all lipids was achieved in 24min of gradient. The columns compared include C30 and C18 functionalization on either core-shell or totally porous silica particles, with size ranging from 1.7 to 2.6µm. Accucore C30 column showed the narrowest peaks and highest theoretical plate number, and excellent peak capacity and retention time reproducibility (<1% standard deviation). As a result, it resulted in 430 lipid species identified from rat plasma and rat liver samples with highest confidence. The high resolution offered by the up-front RPLC allowed discrimination of cis/trans isomeric lipid species, and the high field orbitrap mass spectrometer afforded the clear distinction of isobaric lipid species in full scan MS and the unambiguous assignment of sn-positional isomers for lysophospholipids in MS/MS. Taken together, the high efficiency LC separation and high mass resolving MS analysis are very promising tools for untargeted lipidomics analysis.

A new SPE/GC-fid method for the determination of cholesterol oxidation products. Application to subcutaneous fat from Iberian dry-cured ham.

A new method for the isolation and analysis of cholesterol oxidation products (COPs) using solid phase extraction (SPE) and silica columns was developed using gas chromatography-flame ion detection (GC-FID). The method comprises of saponification and liquid-liquid extraction of the unsaponifiable fraction prior to the isolation and derivatization of the COPs to trimethylsilyl ethers. The COPs used in this study are cholestane-5α-6α-epoxide, cholestane-3ß-5α-6ß-triol, 25-hydroxycholesterol and 5-cholesten-3ß-ol-7-one. In order to identify the COPs fraction a GC-ion-trap-mass spectrometry experiment were conducted using authentic standards to verify the presence of the COPs. The method was effective at rapidly separating the COPs (25 min run). Calibration curves were linear with the LODs and LOQs bellow 0.03 and 0.07 mgkg(-1) for all cases, respectively. This methodology gave a total recovery for every compound that was used in the study. Betulin was used as an internal standard to monitor the recovery. The method was validated with a standard mixture of COPs. The method has been applied to characterize the COP fraction of subcutaneous fat from Iberian dry-cured ham. Cholestane-5α-6α-epoxide, cholestane-3ß-5α-6ß-triol, 25-hydroxycholesterol and 5-cholesten-3ß-ol-7-one have been identified for the first time in these samples.

Feasibility of use of fatty acid and triacylglycerol profiles for the authentication of commercial labelling in Iberian dry-cured sausages.

In the present study, fatty acid and triacylglycerol profiles were used to evaluate the possibility of authenticating Iberian dry-cured sausages according to their label specifications. 42 Commercial brand 'chorizo' and 39 commercial brand 'salchichón' sausages from Iberian pigs were purchased. 36 Samples were labelled Bellota and 45 bore the generic Ibérico label. In the market, Bellota is considered to be a better class than the generic Ibérico since products with the Bellota label are manufactured with high quality fat obtained from extensively reared pigs fed on acorns and pasture. Analyses of fatty acids and triacylglycerols were carried out by gas chromatography and a flame ion detector. A CP-SIL 88 column (highly substituted cyanopropyl phase; 50 m × 0.25 mm i.d., 0.2 µm film thickness) (Varian, Palo Alto, USA) was used for fatty acid analysis and a fused silica capillary DB-17HT column (50% phenyl-50% methylpolysiloxane; 30 m × 0.25 mm i.d., 0.15 µm film thickness) was used for triacylglycerols. Twelve fatty acids and 16 triacylglycerols were identified. Various discriminant models (linear quadratic discriminant analyses, logistic regression and support vector machines) were trained to predict the sample class (Bellota or Ibérico). These models included fatty acids and triacylglycerols separately and combined fatty acid and triacylglycerol profiles. The number of correctly classified samples according to discriminant analyses can be considered low (lower than 65%). The greatest discriminant rate was obtained when triacylglycerol profiles were included in the model, whilst using a combination of fatty acid and triacylglycerol profiles did not improve the rate of correct assignation. The values that represent the reliability of prediction of the samples according to the label specification were higher for the Ibérico class than for the Bellota class. In fact, quadratic and Support Vector Machine discriminate analyses were not able to assign the Bellota class (0%) when combined fatty acids and triacylglycerols were included in the model. The use of fatty acid and triacylglycerol profiles to discriminate Iberian dry-cured sausages in the market according to their labelling information is unclear. In order to ensure the genuineness of Iberian dry-cured sausages in the market, identification of fatty acid and triacylglycerol profiles should be combined with the application of quality standard traceability techniques.

Subcutaneous fat triacylglycerols profile from Iberian pigs as a tool to differentiate between intensive and extensive fattening systems.

Triacylglycerols of subcutaneous fat of Iberian pigs reared on two different feeding systems, extensive and intensive, have been determined by gas chromatography with a flame ionization detector. Analyses were performed on a column coated with a bonded stationary phase (50% phenyl-50% methylpolysiloxane) with hydrogen as the carrier gas. Lipids were extracted by melting the subcutaneous fat in a microwave oven and then filtering and dissolving in hexane. A total amount of 1995 samples from several campaigns were considered. Palmitoyl-stearyl-oleoyl glycerol and palmitoyl-dioleoyl glycerol were the most abundant triacylglycerols found in the samples. A study on the discriminating power of the triacylglycerols to differentiate samples according to the pig feeding system was performed. By using the triacylglycerols as chemical descriptors, principal component analysis, linear discriminant analysis, and soft independent modeling of class analogy were applied. Dioleoyl-linoleoyl glycerol and oleoyl-dilinoleoyl glycerol were the most discriminating variables. Variable-variable plots of these two glycerols allow separation of the samples according to their content.

A new high-performance liquid chromatographic method with evaporative light scattering detector for the analysis of phospholipids. Application to Iberian pig subcutaneous fat.

A new method for the analysis of phospholipids by normal-phase HPLC is described using a silica column. Addition of ammonia and triethylamine to a gradient based on chloroform/methanol/water promoted a good and rapid separation of phospholipid classes (20 min run). The use of an evaporative light scattering detector permitted an accurate analysis of a mixture of phospholipids. Calibration curves were linear within different range for each phospholipid class. The LOD and LOQ obtained were below 0.03 and 0.05 mg kg⁻¹ for all cases, respectively. Besides, a new method for the separation of phospholipids from total lipids before HPLC analysis by a solid-phase extraction (SPE) with Si cartridges has been developed. This methodology gave a good recovery ranging from 97 to 117%. The method was validated with a standard mixture of phospholipids. This method has been applied to characterize the phospholipid fraction of subcutaneous fat from Iberian pig. Cardiolipin, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and sphingomyelin have been described for first time in these samples. The fatty acid composition of the different phospholipid classes and their HPLC electrospray ionization mass spectrometry have been used for characterizing the molecular species present in each one.

Volatile hydrocarbon profile of Iberian dry-cured hams. A possible tool for authentication of hams according to the fattening diet.

The aims of this work were to carry out a comprehensive study of the volatile hydrocarbons of 34 Iberian dry-cured hams and to evaluate the efficiency of these compounds for discriminating hams according to the fattening system: "Montanera" (B) and "Cebo" (C). The samples of hams were obtained by mincing the semimembranosus and semitendinosus muscles from slices of dry-cured ham. The analyses were carried out by gas chromatography-mass spectrometry with a polar capillary column and after a previous extraction by Purge and Trap method. Forty-three volatile hydrocarbons were identified, 26 of them for the first time in Iberian dry-cured ham. Only five compounds showed significant differences between the two types of hams. Among the 33 volatile hydrocarbons, 22 of them allowed a complete discrimination of the two groups of hams according the fattening system.

Authentication of fattening diet of goat kid according to their fatty acid profile from perirenal fat.

Fatty acids of forty-two samples of perirenal fat of goat kids reared on three different feeding systems: goat milk (B), milk replacer (R) and milk-based starter (F) have been analyzed by Gas Chromatography flame ionization detector. The lipids were extracted by melting of perirenal fat in a microwave oven. The fat was then filtered and dissolved in hexane. This analysis was performed on a column (100 m x 0.25 mm i.d. and 0.25 microm film thickness) coated with a polar stationary phase HP-88 and flame ionization detector was used. Hydrogen (25 psi inlet constant pressure) was used as carrier gas. Programmed temperature was kept at 175 degrees C and held isothermally for 10 min, and was then raised to 205 degrees C at a rate of 3 degrees C/min and held isothermally for 10 min. By using the fatty acids as chemical descriptors, pattern recognition techniques were applied to differentiate between goat milk, milk replacer and milk-based starter fattening diet of goat kid. C18:2 and C18:3 acids were found to be the most differentiating variables.

Determination of ent-kaurene in subcutaneous fat of Iberian pigs by gas chromatography multi-stage mass spectrometry with the aim to differentiate between intensive and extensive fattening systems.

This work presents a gas chromatography multi-stage mass spectrometry (GC-MS(3)) method for the determination of ent-kaurene in subcutaneous fat of Iberian pig, present in adipose tissue of animals due to pasture ingestion (extensive fattening system). The method comprises a saponification and a liquid-liquid extraction of the unsaponifiable fraction, followed by an isolation of the hydrocarbon fraction by high performance liquid chromatography (HPLC) and analysis by GC-MS(3) (ion trap) with electronic ionization. The GC-MS(3) analysis allows the isolation and characterization of specific fragments from the original (MS(1)) molecular structure, and particularly, those fragments originated from the precursor ion (m/z=229) characteristic of ent-kaurene. The MS/MS product fragment m/z=213 is used as a further precursor fragment giving rise to a MS(3) spectrum specific for ent-kaurene. The limit of detection of the MS(3) technique is lower than 0.2 microg kg(-1) and a linear regression has been found between 0.2 and 112 microg kg(-1). This method is applicable for the determination of the fattening system of the Iberian pig.

Changes in the fatty acid and triacylglycerol profiles in the subcutaneous fat of Iberian ham during the dry-curing process.

In this study, we have evaluated the changes that occur in the profiles of total fatty acids and triacylglycerols during the dry-curing process (730 days) of Iberian ham. The subcutaneous adipose tissues of six hams obtained from three Iberian pigs fed on acorns were analyzed periodically during the processing time (from the raw to the dry-cured samples), including postsalting, drying, and ripening stages. The environmental conditions were also registered. The curing process significantly decreased (p < 0.01) the relative percentages of total polyunsaturated fatty acids, including C18:2n-6 and C18:3n-3 and, therefore, significantly increased (p < 0.05) the level of monounsaturated fatty acids. The triglycerides containing 0-2 double bonds showed an increase during the curing process. On the contrary, the more unsaturated ones (3-5 double bonds) suffered a significant decrease. We have postulated that these changes could also be due to polymerization and oxidation reactions that affect the triacylglycerols and besides the fatty acids. In general, most fatty acids and triacylglycerols reversed the trend by about 500-600 days of processing.

Changes in the concentrations of free fatty acid, monoacylglycerol, and diacylglycerol in the subcutaneous fat of Iberian ham during the dry-curing process.

Changes in diacylglycerols, monoacylglycerols, and free fatty acid composition of subcutaneous fat of six Iberian hams during the dry-cured process were investigated. In addition, an analytical method for simultaneous quantification of diacylglycerols, monoacylglycerols, and free fatty acid by solid-phase extraction-gas chromatography was developed. The different molecular species of free fatty acids, monoacylglycerols, and diacylglycerols and 1,2- and 1,3-isomers of diacylglycerols have been described for the first time in this type of sample. A logarithmic increase of the 1,3-diacylglycerol profile throughout the processing time has been found, reaching a balance value of 62% around 500 days. The formation of diacylglycerol isomers takes place, although the 1,3-/1,2-diacylglycerol ratio increases during the process to 1.65 due to isomerization of the 1,2-form toward the 1,3-form. The profiles of monoacyl- and diacylglycerols and free fatty acids follow the same trend. The experimental values of free fatty acid are greater than theoretical prediction, probably due to phospholipid and monoacylglycerol hydrolysis.