Clusters of adipose tissue dysfunction in adults with type 2 diabetes identify those with worse lipidomic profile despite similar glycaemic control.

AIMS: To investigate clusters of adipose tissue dysfunction, that is, with adipose tissue insulin resistance (ADIPO-IR) and large waist circumference (WC), identify a worse lipidomic profile characterised by a high proportion of lipids rich in saturated fatty acids (SFA). MATERIALS AND METHODS: Hierarchical clustering based on WC and ADIPO-IR (calculated as fasting plasma non-esterified fatty acids times fasting plasma insulin, FFA×INS), was performed in 192 adults with overweight/obesity and type 2 diabetes (T2D) treated with metformin (HbA1c = 7.8%). Free fatty acid composition and lipidomic profile were measured by mass spectrometry (GC-MS and LC-MSQTOF). Indexes of fatty acid desaturation (stearoyl-coA desaturase-1 activity, SCD116 = palmitoleic acid/palmitic acid and SCD118 = oleic acid/stearic acid) and of insulin resistance (HOMA-IR) were also calculated. RESULTS: Three clusters were identified: CL1 (ADIPO-IR = 4.9 ± 2.4 and WC = 96±7 cm, mean ± SD), CL2 (ADIPO-IR = 6.5 ± 2.5 and WC = 114 ± 7 cm), and CL3 (ADIPO-IR = 15.0 ± 4.7 and WC = 107 ± 8 cm). Insulin concentrations, ADIPO-IR, and HOMA-IR significantly increased from CL1 to CL3 (all p < 0.001), while fasting glucose concentrations, HbA1c, dietary lipids and caloric intake were similar. Moreover, CL3 showed significantly higher concentrations of monounsaturated free fatty acids, oleic and palmitoleic acids, triglycerides (TAG) rich in saturated FA and associated with de novo lipogenesis (i.e., TAG 46-50), higher SCD116, SCD118, ceramide (d18:0/18:0), and phosphatidylcholine aa(36:5) compared with CL1/CL2 (all p < 0.005). CONCLUSIONS: High ADIPO-IR and large WC identify a worse lipid profile in T2D characterised by complex lipids rich in SFA, likely due to de novo synthesis given higher plasma monounsaturated FFA and increased desaturase activity indexes. REGISTRATION NUMBER TRIAL: ID NCT00700856 https://clinicaltrials.gov.

GLUT4 localisation with the plasma membrane is unaffected by an increase in plasma free fatty acid availability.

BACKGROUND: Insulin-stimulated glucose uptake into skeletal muscle occurs via translocation of GLUT4 from intracellular storage vesicles to the plasma membrane. Elevated free fatty acid (FFA) availability via a lipid infusion reduces glucose disposal, but this occurs in the absence of impaired proximal insulin signalling. Whether GLUT4 localisation to the plasma membrane is subsequently affected by elevated FFA availability is not known. METHODS: Trained (n = 11) and sedentary (n = 10) individuals, matched for age, sex and body mass index, received either a 6 h lipid or glycerol infusion in the setting of a concurrent hyperinsulinaemic-euglycaemic clamp. Sequential muscle biopsies (0, 2 and 6 h) were analysed for GLUT4 membrane localisation and microvesicle size and distribution using immunofluorescence microscopy. RESULTS: At baseline, trained individuals had more small GLUT4 spots at the plasma membrane, whereas sedentary individuals had larger GLUT4 spots. GLUT4 localisation with the plasma membrane increased at 2 h (P = 0.04) of the hyperinsulinemic-euglycemic clamp, and remained elevated until 6 h, with no differences between groups or infusion type. The number of GLUT4 spots was unchanged at 2 h of infusion. However, from 2 to 6 h there was a decrease in the number of small GLUT4 spots at the plasma membrane (P = 0.047), with no differences between groups or infusion type. CONCLUSION: GLUT4 localisation with the plasma membrane increases during a hyperinsulinemic-euglycemic clamp, but this is not altered by elevated FFA availability. GLUT4 appears to disperse from small GLUT4 clusters located at the plasma membrane to support glucose uptake during a hyperinsulinaemic-euglycaemic clamp.

A novel ABCD1 gene mutation causes adrenomyeloneuropathy presenting with spastic paraplegia: A case report.

RATIONALE: X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene leading to very long chain fatty acid (VLCFA) accumulation. The disease demonstrates a spectrum of phenotypes including adrenomyeloneuropathy (AMN). We aimed to identify the genetic basis of disease in a patient presenting with AMN features in order to confirm the diagnosis, expand genetic knowledge of ABCD1 mutations, and elucidate potential genotype-phenotype associations to inform management. PATIENT CONCERNS: A 29-year-old male presented with a 4-year history of progressive spastic paraplegia, weakness of lower limbs, fecal incontinence, sexual dysfunction, hyperreflexia, and positive Babinski and Chaddock signs. DIAGNOSES: Neuroimaging revealed brain white matter changes and spinal cord thinning. Significantly elevated levels of hexacosanoic acid (C26:0) and tetracosanoic acid (C24:0) suggested very long chain fatty acids (VLCFA) metabolism disruption. Genetic testing identified a novel hemizygous ABCD1 mutation c.249dupC (p.F83fs). These findings confirmed a diagnosis of X-linked ALD with an AMN phenotype. INTERVENTIONS: The patient received dietary counseling to limit VLCFA intake. Monitoring for adrenal insufficiency and consideration of Lorenzo's oil were advised. Genetic counseling and testing were offered to at-risk relatives. OUTCOMES: At present, the patient continues to experience progressive paraplegia. Adrenal function remains normal thus far without steroid replacement. Family members have undergone predictive testing. LESSONS: This case expands the known mutation spectrum of ABCD1-linked X-ALD, providing insight into potential genotype-phenotype correlations. A thoughtful diagnostic approach integrating clinical, biochemical and genetic data facilitated diagnosis. Findings enabled genetic counseling for at-risk relatives regarding this X-linked disorder.

Visceral Adipose Tissue: The Hidden Culprit for Type 2 Diabetes.

Type 2 diabetes (T2D) is a chronic metabolic disorder characterized by insulin resistance in various tissues. Though conventionally associated with obesity, current research indicates that visceral adipose tissue (VAT) is the leading determining factor, wielding more influence regardless of individual body mass. The heightened metabolic activity of VAT encourages the circulation of free fatty acid (FFA) molecules, which induce insulin resistance in surrounding tissues. Individuals most vulnerable to this preferential fat deposition are older males with ancestral ties to Asian countries because genetics and sex hormones are pivotal factors for VAT accumulation. However, interventions in one's diet and lifestyle have the potential to strategically discourage the growth of VAT. This illuminates the possibility that the expansion of VAT and, subsequently, the risk of T2D development are preventable. Therefore, by reducing the amount of VAT accumulated in an individual and preventing it from building up, one can effectively control and prevent the development of T2D.

Models of the Three-Component Bilayer of Stratum Corneum: A Molecular Simulation Study.

The construction of the stratum corneum (SC) is crucial to the problems of transdermal drug delivery. SC consists of the keratinocyte layers and the lipid matrix surrounding it. Among them, the lipid matrix is the barrier for many exogenous molecules, mainly composed of ceramides (CERs), free fatty acids (FFA), and cholesterol (CHOL). In this work, we developed single-component (CERs, CER-NS, and CER-EOS) and six three-component models, and each model was simulated by using the GROMOS-54A7 force field. Short-period phase (SPP) and long-period phase (LPP) systems were established separately, and area per lipid (APL), thickness, order of carbon chain (SCD), and density distribution were analyzed. The transition of CER-NS and CER-EOS in LPP was observed. The results of hydrogen bonds in the lipid systems indicated that a strong hydrogen-bond network was formed between the skin-lipid bilayers. Umbrella sampling method simulations were performed to calculate the free energy change of ethanol moving into the skin-lipid bilayer. The results revealed that ethanol molecules pulled some water molecules into the membrane when they passed through SPP-1. Our findings provided some insights and models of the stratum corneum that could be used for the subsequent mechanism of macromolecule permeation through membranes in drugs, cosmetics, and so on.

ß-Cell Insulin Resistance Plays a Causal Role in Fat-Induced ß-Cell Dysfunction In Vitro and In Vivo.

In the classical insulin target tissues of liver, muscle, and adipose tissue, chronically elevated levels of free fatty acids (FFA) impair insulin signaling. Insulin signaling molecules are also present in ß-cells where they play a role in ß-cell function. Therefore, inhibition of the insulin/insulin-like growth factor 1 pathway may be involved in fat-induced ß-cell dysfunction. To address the role of ß-cell insulin resistance in FFA-induced ß-cell dysfunction we co-infused bisperoxovanadate (BPV) with oleate or olive oil for 48 hours in rats. BPV, a tyrosine phosphatase inhibitor, acts as an insulin mimetic and is devoid of any antioxidant effect that could prevent ß-cell dysfunction, unlike most insulin sensitizers. Following fat infusion, rats either underwent hyperglycemic clamps for assessment of ß-cell function in vivo or islets were isolated for ex vivo assessment of glucose-stimulated insulin secretion (GSIS). We also incubated islets with oleate or palmitate and BPV for in vitro assessment of GSIS and Akt (protein kinase B) phosphorylation. Next, mice with ß-cell specific deletion of PTEN (phosphatase and tensin homolog; negative regulator of insulin signaling) and littermate controls were infused with oleate for 48 hours, followed by hyperglycemic clamps or ex vivo evaluation of GSIS. In rat experiments, BPV protected against fat-induced impairment of ß-cell function in vivo, ex vivo, and in vitro. In mice, ß-cell specific deletion of PTEN protected against oleate-induced ß-cell dysfunction in vivo and ex vivo. These data support the hypothesis that ß-cell insulin resistance plays a causal role in FFA-induced ß-cell dysfunction.

Levels of lipid-derived gut microbial metabolites differ among plant matrices in an in vitro model of colon fermentation.

This study explored differences in microbial lipid metabolites among sunflower seeds, soybeans, and walnuts. The matrices were subjected to in vitro digestion and colonic fermentation. Defatted digested materials and fiber/phenolics extracted therefrom were added to sunflower oil (SO) and also fermented. Targeted and untargeted lipidomics were employed to monitor and tentatively identify linoleic acid (LA) metabolites. Walnut fermentation produced the highest free fatty acids (FFAs), LA, and conjugated LAs (CLAs). Defatted digested walnuts added to SO boosted FFAs and CLAs production; the addition of fibre boosted CLAs, whereas the addition of phenolics only increased 9e,11z-CLA and 10e,12z-CLA. Several di-/tri-hydroxy-C18-FAs, reported as microbial LA metabolites for the first time, were annotated. Permutational multivariate analysis of variance indicated significant impacts of food matrix presence and type on lipidomics and C18-FAs. Our findings highlight how the food matrices affect CLA production from dietary lipids, emphasizing the role of food context in microbial lipid metabolism.

Lipidomics analysis unveils the dynamic alterations of lipid degradation in rice bran during storage.

Recent explorations into rice bran oil (RBO) have highlighted its potential, owing to an advantageous fatty acid profile in the context of health and nutrition. Despite this, the susceptibility of rice bran lipids to oxidative degradation during storage remains a critical concern. This study focuses on the evolution of lipid degradation in RBO during storage, examining the increase in free fatty acids (FFAs), the formation of oxylipids, and the generation of volatile secondary oxidation products. Our findings reveal a substantial rise in FFA levels, from 109.55 to 354.06 mg/g, after 14 days of storage, highlighting significant lipid deterioration. Notably, key oxylipids, including 9,10-EpOME, 12,13(9,10)-DiHOME, and 13-oxoODE, were identified, with a demonstrated positive correlation between total oxylipids and free polyunsaturated fatty acids (PUFAs), specifically linoleic acid (LA) and α-linolenic acid (ALA). Furthermore, the study provides a detailed analysis of primary volatile secondary oxidation products. The insights gained from this study not only sheds light on the underlying mechanisms of lipid rancidity in rice bran but also offers significant implications for extending the shelf life and preserving the nutritional quality of RBO, aligning with the increasing global interest in this high-quality oil.

Influences of oxidative stress and energy balance on pregnancy rates after the transfer of embryos to repeat-breeder Japanese Black cattle.

The purpose of this study is to reassess our previously reported findings on the diminished pregnancy rate of embryo transfer (ET) in summer for repeat-breeder (RB) cows compared with other seasons, with a focus on oxidative stress and energy balance. The study involved Japanese Black cattle, including 224 heifers (aged <3 years) and 278 (aged <9 years) cows with one or more deliveries, defined as RB animals. Evaluation of the effects of season on pregnancy rates revealed significantly lower rates in RB cows during summer compared with spring and autumn. Moreover, serum malondialdehyde (MDA) concentration in RB cows during summer was significantly higher than in winter, with no difference in RB heifers. Seasonal effects on serum nonesterified fatty acid (NEFA) concentration in both RB heifers and RB cows showed no significant differences. However, median MDA and NEFA concentrations in RB cows were significantly elevated compared with RB heifers, suggesting that factors contributing to low fertility should consider parity. Furthermore, our study indicated that RB cows were under oxidative stress, as demonstrated by significantly elevated MDA concentrations compared with normally reproducing cows in summer. Further investigation is necessary to gain a better understanding of these observations in the future.

Alterations of circulating free fatty acids in patients with pemphigus vulgaris.

Free fatty acids (FFA) have gained research interest owing to their functions in both local and systemic immune regulation. Changes in the serum levels of anti-inflammatory short chain fatty acids (SCFA), primarily derived from the gut microbiota, and pro-inflammatory medium (MCFA) and long (LCFA) chain fatty acids, derived from either the gut microbiota or the diet, have been associated with autoimmunity. Circulating FFA were retrospectively analysed by a gas chromatography-mass spectrometry method in the serum of 18 patients with pemphigus vulgaris (PV) at the baseline and 6 months (n = 10) after immunosuppressive treatments, and 18 healthy controls (HC). Circulating FFA were correlated with the Pemphigus Disease Area Index (PDAI) and serum concentrations of interferon-gamma (IFN-γ), Interleukin (IL)-17A, IL-5, IL-10 and IL-21. Principal Component analysis computed on FFA abundances revealed significant differences in the profile of SCFA (p = 0,012), MCFA (p = 0.00015) and LCFA (p = 0,035) between PV patients and HC, which were not significantly changed by immunosuppressive treatments. PV patients showed a significantly lower serum concentration of propionic (p < 0.0005) and butyric (p < 0.0005) acids, SCFA with anti-inflammatory functions, while hexanoic (p < 0.0005) and hexadecanoic (p = 0.0006) acids, pro-inflammatory MCFA and LCFA respectively, were over-represented. Treatments induced a significant decrease of hexanoic (p = 0.035) and a further increase of hexadecanoic (p = 0.046) acids. Positive correlations emerged between IFN-γ and acetic acid (Rho = 0.60), IFN-γ and hexanoic acid (Rho = 0.46), IL-5 and both hexadecanoic acid (Rho = 0.50) and octadecanoic acid (Rho = 0.53), butyric acid and PDAI (Rho = 0.53). PV was associated with a remarked imbalance of circulating FFA compared to HC. The serum alterations of SCFA, MCFA, and LCFA may contribute to promoting inflammation in PV. Deeper insights into the immunomodulatory functions of these molecules may pave the way for personalized dietary interventions in PV patients.

Prepartum behaviors as early indicators for postpartum energy associated biomarkers status in Holstein dairy cows.

Our main aim was to investigate the predictive value of prepartum behaviors such as total daily rumination (TDR), total daily activity (TDA) and dry matter intake (DMI) as early indicators to detect cows at risk for hyperketonemia (HYK), hypoglycemia (HYG) or high non-esterified fatty acid (NEFA) status in the first (wk1) and second week (wk2) postpartum. In a case control study, 64 Holstein cows were enrolled 3 weeks before the expected time of calving and monitored until 15 days in milk (DIM). Postpartum blood samples were taken at D3 and D6 for wk1 and at D12 and D15 for wk2 to measure beta-hydroxybutyrate, NEFA and glucose concentration. Ear-mounted accelerometers were used to measure TDR and TDA. DMI and milk yield were obtained from farm records. Relationships between the average daily rate of change in prepartum TDR (ΔTDR), TDA (ΔTDA), and DMI (ΔDMI) with postpartum HYK, HYG and NEFA status in wk1 and wk2 post-partum were evaluated using linear regression models. Models were adjusted for potential confounding variables, and covariates retained in the final models were determined by backward selection. No evidence was found to support the premise that prepartum ΔTDR, ΔTDA or ΔDMI predicted postpartum HYK, HYG or NEFA status in wk1 or in wk2. Overall, prepartum ΔTDR, ΔTDA and ΔDMI were not effective predictors of HYK, HYG or NEFA status in the first 2 weeks postpartum.

Characterization of Palm Fatty Acid Distillate, Diacylglycerol Regioisomers, and Esterification Products Using High-Performance Size Exclusion Chromatography.

High-performance size exclusion chromatography (HPSEC) equipped with an evaporative light scattering detector (ELSD) was utilized for characterization of palm fatty acid distillate (PFAD) and its esterified products, with a particular focus on lipid profiles and diacylglycerol (DAG) regioisomers. The separation of triacylglycerol (TAG), DAG, monoacylglycerol (MAG), and free fatty acid (FFA) was achieved through a single 100-Å Phenogel column, coupled with a 2-cm C18 guard, utilizing toluene/acetic acid (100:0.25, v/v) as the mobile phase. This separation was based on size sieving principles and the interactions between the hydroxyl group(s) and the Phenogel matrix. The limit of detection (LOD) and limit of quantification (LOQ) for the esterified PFAD products analyzed by this method fell within the range of 4.8-5.5 µg/mL and 14.7-16.7 µg/mL, respectively. Additionally, the same column, paired with a 2-cm silica guard and a mobile phase comprised of toluene/isooctane/acetic acid (35:65:0.15, v/v/v), was used for the characterization of DAG regioisomers within the esterified PFAD. LODs and LOQs for sn-1,3-DAG and sn- 1,2-DAG were determined to be 39.2 and 118.7 µg/mL, and 32.8 and 99.5 µg/mL, respectively. Investigation of esterified PFAD products prepared using 4% H2SO4 at 120°C. After 2 h, the analysis revealed the highest MAG content at 31.85%, accompanied by 51.54% DAG, 2.35% TAG, and a residual 14.27% FFA. Notably, as the reaction time extended, the MAG content decreased, while both DAG and TAG levels exhibited an increasing trend. Further examination of DAG regioisomers during PFAD esterification, under varying catalyst concentrations (2-10%) and reaction temperatures (80-140°C), demonstrated a significant increase in the percentage of sn-1,3-DAG, inversely correlated with the reduction in FFA from 2% H 2 SO 4 and 80°C onwards. Remarkably, the percentage of sn-1,2-DAG remained relatively stable regardless of changes in catalyst concentrations or temperatures, confirming its susceptibility to isomerization into the thermodynamically more stable sn-1,3-DAG form. This study provides valuable insights into the composition and behavior of esterified PFAD products.

Validation of an on-farm portable blood analyzer for quantifying blood analytes in dairy cows.

The periparturient period for dairy cows is a metabolically dynamic time period where the cow is adjusting from gestation to the onset of lactation. Metabolic disorders such as ketosis, hypocalcemia, and fatty liver occur during this time; however, tools to diagnose these diseases on-farm is limited. The need for compact metabolite quantification devices that can quantify metabolites on farm from whole blood samples is warranted. The purpose of this study was to validate a portable blood analyzer (PBA) by analyzing metabolites on privately owned dairy farms in southcentral Wisconsin. Additional tests were completed to determine if plasma metabolite quantification was similar to whole-blood quantification. Two phases were conducted on two separate farms to complete these analyses and data were analyzed by Bland-Altman plot and correlations. Metabolites quantified from whole blood samples included albumin, alanine and aspartate aminotransferases, ß-hydroxybutyrate, blood urea nitrogen, total calcium, cholesterol, creatinine kinase, γ-glutamyl transferase, glucose, magnesium, nonesterified fatty acids, phosphorous, and total protein and were analyzed in the lab after plasma separation to determine gold-standard laboratory concentrations. Across Phase 1 and 2, whole-blood PBA metabolite concentrations resulted in similar results compared to the laboratory assays. For plasma analyzed on the PBA, overall results were positively correlated, but robustness was dependent upon initial validation results indicating some metabolites are suitable for plasma quantification on the device. These results indicate that the PBA is a viable on-farm metabolite quantification tool that will be valuable for on-farm diagnosis of metabolic stress and dysfunction in transition dairy cows.

Identification of cheese rancidity-related lipases in Aspergillus oryzae AHU 7139.

The adjunct product with enzymatic activity from Aspergillus oryzae is beneficial for flavor enrichment in the ripened cheese. However, an excessive lipolytic reaction leads to the release of volatile free fatty acids. Accordingly, a strong off-flavor (i.e., rancidity) has been detected when A. oryzae AHU 7139 is used. To identify the rancidity-related lipase from this strain, we evaluated the substrate specificity and lipase distribution using five mutants cultured on a whey-based solid medium under different initial pH conditions. The results showed a higher diacylglycerol lipase activity than triacylglycerol lipase activity. Moreover, an initial pH of 6.5 for the culture resulted in higher lipolytic activity than a pH of 4.0, and most of the activity was found in the extracellular fraction. Based on the gene expression analysis by real-time polymerase chain reaction and location and substrate specificity, five genes (No. 1, No. 19, mdlB, tglA, and cutL) were selected among 25 annotated lipase genes to identify the respective knockout strains. Because ΔtglA and ΔmdlB showed an outstanding involvement in the release of free fatty acids, these strains were applied to in vitro cheese curd experiments. In conclusion, we posit that triacylglycerol lipase (TglA) plays a key role as the trigger of rancidity and the resulting diglycerides have to be exposed to diacylglycerol lipase (MdlB) to stimulate rancidity in cheese made with A. oryzae AHU 7139. This finding could help screen suitable A.oryzae strains as cheese adjuncts to prevent the generation of the rancid-off flavor.

Activation of free fatty acid receptors, FFAR1 and FFAR4, ameliorates ulcerative colitis by promote fatty acid metabolism and mediate macrophage polarization.

OBJECTIVE: To investigate the mechanism of action of fatty acid receptors, FFAR1 and FFAR4, on ulcerative colitis (UC) through fatty acid metabolism and macrophage polarization. METHODS: Dextran sulfate sodium (DSS)-induced mouse model of UC mice was used to evaluate the efficacy of FFAR1 (GW9508) and FFAR4 (GSK137647) agonists by analyzing body weight, colon length, disease activity index (DAI), and histological scores. Real-time PCR and immunofluorescence analysis were performed to quantify the levels of fatty acid metabolizing enzymes and macrophage makers. FFA-induced lipid accumulation in RAW264.7 cells was visualized by Oil Red O staining analysis, and cells were collected to detect macrophage polarization by flow cytometry. RESULTS: The combination of GW9508 and GSK137647 significantly improved DSS-induced UC symptoms, caused recovery in colon length, and decreased histological injury. GW9508 + GSK137647 treatment upregulated the expressions of CD206, lipid oxidation enzyme (CPT-1α) and anti-inflammatory cytokines (IL-4, IL-10, IL-13) but downregulated those of CD86, lipogenic enzymes (ACC1, FASN, SCD1), and pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α). Combining the two agonists decreased FFA-induced lipid accumulation and increased CD206 expression in cell-based experiments. CONCLUSION: Activated FFAR1 and FFAR4 ameliorates DSS-induced UC by promoting fatty acid metabolism to reduce lipid accumulation and mediate M2 macrophage polarization.

Profiling of Oxylipins as Markers of Oxidative Stress in Biological Samples.

Oxylipins are oxidized metabolites of polyunsaturated fatty acids (PUFAs). They represent a class of risk markers and/or therapeutic targets for diseases associated with inflammation, including cardiovascular disease and brain disorders. Because the biological activities of free PUFAs and oxylipins depend on their chemical structures and concentrations, monitoring PUFAs and oxylipin levels in biological systems is critical for understanding their roles in health and disease. Traditionally, accurate quantification of free PUFAs and oxylipins in biological samples was performed separately, as PUFAs are often 1000-fold more abundant than the derived oxidized fatty acids (oxylipins). This article describes a liquid chromatography multiple reaction monitoring tandem mass spectrometry method for the quantitative analysis of five free PUFAs and 88 oxylipins in various biological fluids, including plasma, platelet supernatants, and tissues. The same approach can also be used in conjunction with an alkaline hydrolysis step to quantify total oxylipins in fish oils. We observed that in some samples, linoleic acid levels in plasma and eicosapentaenoic acid and arachidonic acid levels in brain tissue were above the upper limit of quantification. To address this issue, we developed a data analysis method to obtain PUFA and oxylipin concentrations in these samples without additional sample preparation, thus significantly saving time and labor. © 2024 Wiley Periodicals LLC. Basic Protocol: Quantification of polyunsaturated fatty acids (PUFAs) and oxylipins using liquid chromatography multiple reaction monitoring tandem mass spectrometry Support Protocol 1: Preparation of internal standard mixed working solution Support Protocol 2: Preparation of standard mixed stock solution Support Protocol 3: Preparation of standard mixed working solution Alternate Protocol 1: Extraction and quantitation of free PUFAs and oxylipins from mouse brain tissue Alternate Protocol 2: Extraction and quantitation of total PUFAs and oxylipins from fish oil.

Untargeted metabolomic profiling reveals molecular signatures associated with type 2 diabetes in Nigerians.

BACKGROUND: Type 2 diabetes (T2D) has reached epidemic proportions globally, including in Africa. However, molecular studies to understand the pathophysiology of T2D remain scarce outside Europe and North America. The aims of this study are to use an untargeted metabolomics approach to identify: (a) metabolites that are differentially expressed between individuals with and without T2D and (b) a metabolic signature associated with T2D in a population of Sub-Saharan Africa (SSA). METHODS: A total of 580 adult Nigerians from the Africa America Diabetes Mellitus (AADM) study were studied. The discovery study included 310 individuals (210 without T2D, 100 with T2D). Metabolites in plasma were assessed by reverse phase, ultra-performance liquid chromatography and mass spectrometry (RP)/UPLC-MS/MS methods on the Metabolon Platform. Welch's two-sample t-test was used to identify differentially expressed metabolites (DEMs), followed by the construction of a biomarker panel using a random forest (RF) algorithm. The biomarker panel was evaluated in a replication sample of 270 individuals (110 without T2D and 160 with T2D) from the same study. RESULTS: Untargeted metabolomic analyses revealed 280 DEMs between individuals with and without T2D. The DEMs predominantly belonged to the lipid (51%, 142/280), amino acid (21%, 59/280), xenobiotics (13%, 35/280), carbohydrate (4%, 10/280) and nucleotide (4%, 10/280) super pathways. At the sub-pathway level, glycolysis, free fatty acid, bile metabolism, and branched chain amino acid catabolism were altered in T2D individuals. A 10-metabolite biomarker panel including glucose, gluconate, mannose, mannonate, 1,5-anhydroglucitol, fructose, fructosyl-lysine, 1-carboxylethylleucine, metformin, and methyl-glucopyranoside predicted T2D with an area under the curve (AUC) of 0.924 (95% CI: 0.845-0.966) and a predicted accuracy of 89.3%. The panel was validated with a similar AUC (0.935, 95% CI 0.906-0.958) in the replication cohort. The 10 metabolites in the biomarker panel correlated significantly with several T2D-related glycemic indices, including Hba1C, insulin resistance (HOMA-IR), and diabetes duration. CONCLUSIONS: We demonstrate that metabolomic dysregulation associated with T2D in Nigerians affects multiple processes, including glycolysis, free fatty acid and bile metabolism, and branched chain amino acid catabolism. Our study replicated previous findings in other populations and identified a metabolic signature that could be used as a biomarker panel of T2D risk and glycemic control thus enhancing our knowledge of molecular pathophysiologic changes in T2D. The metabolomics dataset generated in this study represents an invaluable addition to publicly available multi-omics data on understudied African ancestry populations.

Sex-Specific Effects of Long-Term Antipsychotic Drug Treatment on Adipocyte Tissue and the Crosstalk to Liver and Brain in Rats.

Antipsychotic drug (APD) medication can lead to metabolic dysfunctions and weight gain, which together increase morbidity and mortality. Metabolically active visceral adipose tissue (VAT) in particular plays a crucial role in the etiopathology of these metabolic dysregulations. Here, we studied the effect of 12 weeks of drug medication by daily oral feeding of clozapine and haloperidol on the perirenal fat tissue as part of VAT of male and female Sprague Dawley rats in the context of complex former investigations on brain, liver, and blood. Adipocyte area values were determined, as well as triglycerides, non-esterified fatty acids (NEFAs), glucose, glycogen, lactate, malondialdehyde equivalents, ferric iron and protein levels of Perilipin-A, hormone-sensitive-lipase (HSL), hepcidin, glucose transporter-4 (Glut-4) and insulin receptor-ß (IR-ß). We found increased adipocyte mass in males, with slightly higher adipocyte area values in both males and females under clozapine treatment. Triglycerides, NEFAs, glucose and oxidative stress in the medicated groups were unchanged or slightly decreased. In contrast to controls and haloperidol-medicated rats, perirenal adipocyte mass and serum leptin levels were not correlated under clozapine. Protein expressions of perilipin-A, Glut-4 and HSL were decreased under clozapine treatment. IR-ß expression changed sex-specifically in the clozapine-medicated groups associated with higher hepcidin levels in the perirenal adipose tissue of clozapine-treated females. Taken together, clozapine and haloperidol had a smaller effect than expected on perirenal adipose tissue. The perirenal adipose tissue shows only weak changes in lipid and glucose metabolism. The main changes can be seen in the proteins examined, and probably in their effect on liver metabolism.

Free Fatty Acid Determination in Broccoli Tissues Using Liquid Chromatography-High-Resolution Mass Spectrometry.

Broccoli (Brassica oleracea L. var. italica Plenck) is a widely consumed vegetable, very popular due to its various nutritional and bioactive components. Since studies on the lipid components of broccoli have been limited so far, the aim of the present work was the study of free fatty acids (FFAs) present in different broccoli parts, aerial and underground. The direct determination of twenty-four FFAs in broccoli tissues (roots, leaves, and florets) was carried out, using a liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method in a 10 min single run. Linolenic acid was found to be the most abundant FFA in all different broccoli parts in quantities ranging from 0.76 to 1.46 mg/g, followed by palmitic acid (0.17-0.22 mg/g) and linoleic acid (0.06-0.08 mg/g). To extend our knowledge on broccoli's bioactive components, for the first time, the existence of bioactive oxidized fatty acids, namely hydroxy and oxo fatty acids, was explored in broccoli tissues adopting an HRMS-based lipidomics approach. 16- and 2-hydroxypalmitic acids were detected in all parts of broccoli studied, while ricinoleic acid was detected for the first time as a component of broccoli.

Determination of free fatty acids in edible oil based on hollow mesoporous silica nanoparticles.

In our study, ammoniated hollow mesoporous silica nanoparticles (NH2-HMSN) with uniform diameter and stable structure were successively prepared via SiO2 core hard template method. Fourier transformed infrared spectroscopy revealed that amino group was effectively modified. Adsorption experiments showed that adsorption capacity of NH2-HMSN towards free fatty acids (FFAs) was superior to aminated mesopores or silica microspheres. Following through optimization of extraction conditions, FFAs from edible oil samples were successfully gathered by NH2-HMSN and showed favorable linearities (0.2-90 µg g-1), remarkably low limit of detections (0.03-0.15 nmol g-1), acceptable recoveries (85.08-96.82 %) and relatively accurate precisions (1.64-4.99 %). In comparison to existing adsorbent, NH2-HMSN could be successfully prepared via the chemical reaction of common raw materials under normal pressure and temperature. Furthermore, NH2-HMSN with hollow and mesoporous structure was more effective than the current adsorbents aimed at FFAs analysis in aspect of surface area and adsorption capacity.