A longitudinal study of the blood and urine metabolome of Vipera berus envenomated dogs.

Envenomation of dogs by the common European adder (Vipera berus) is associated with high morbidity. The cytotoxic venom of Vipera berus contains enzymes with the potential to cause acute kidney injury, among other insults, however robust biomarkers for such effects are lacking. A prospective observational follow-up study of naturally envenomated dogs and controls was conducted to fill knowledge gaps regarding canine Vipera berus envenomation, attempt to identify novel biomarkers of envenomation and related kidney injury, and elucidate potential long-term effects. Blood and urine samples were analyzed with a global metabolomics approach using liquid chromatography-mass spectrometry, uncovering numerous features significantly different between cases and controls. After data processing and feature annotation, eight features in blood and 24 features in urine were investigated in order to elucidate their biological relevance. Several of these are associated with AKI, while some may also originate from disturbed fatty acid ß-oxidation and soft tissue damage. A metabolite found in both blood and a venom reference sample may represent identification of a venom component in case dogs. Our findings suggest that envenomated dogs treated according to current best practice are unlikely to suffer permanent injury.

Method Development for Omics Analyses using Schirmer Strips.

PURPOSE: The aim of this article was to investigate whether Schirmer strips gathered during clinical dry eye examinations can be prepared for omics analyses in a standardized way, to adjust for variations in tear volume and enable two separate omics analyses from the same sample. In addition, the intention was to investigate whether fluorescein dye instillation in the eyes gave bias effects on metabolomic analysis. METHODS: Twelve samples from six individuals, with normal or reduced tear production, were collected. Half of the samples were harvested after instillation of fluorescein in the eye. Each strip was divided in half along the length and prepared with a new method for extracting tear content from the Schirmer strip. The new method was established to compensate for different dilutions of metabolites in varying Schirmer strip wetting levels when using identical extraction volume for all samples. Metabolomic data were compared in samples with and without fluorescein dye and Schirmer strips ranging from 1 to 35 mm wetting levels using a global LC-MS method. RESULTS: All samples were successfully analyzed with an average of ∼350 relevant features detected per sample after using both positive and negative electrospray ionization mode, despite low tear volumes in some samples and that only one half of the Schirmer strips were used. Principal component analysis plots and heatmaps revealed no bias effects of fluorescein dye presence or different Schirmer strip values when using the proposed method. CONCLUSION: A high number of relevant metabolomic features can be extracted from longitudinally cut halves of Schirmer strips, which may enable analyses with more than one omics modality from the same sample. With the pre-analytical method described, Schirmer strips can be used for metabolomic analyses even in cases of very low or high tear volume with or without fluorescence.

Tear and Saliva Metabolomics in Evaporative Dry Eye Disease in Females.

Accurate diagnosis of dry eye disease (DED) is challenging, and even today there is no gold standard biomarker of DED. Hypothesis-free global metabolomic studies of tears from DED patients have great potential to discover metabolites and pathways affected in the pathophysiology of DED, and to identify possible future biomarkers. These metabolites and biomarkers could be important for diagnosing and monitoring disease as well as for new therapeutic targets and strategies. As DED is associated with dry mouth, this study aimed to perform metabolomic analyses of tears and saliva from patients with decreased tear film break-up time but normal Schirmer test, and age-matched controls with both tear production and stability within physiological range. We applied strict inclusion criteria to reduce sampling bias in the metabolomic analyses and selected only age-matched females with Schirmer test values between 10-15 mm/5 min. The tear film analysis arm included 19 patients (with tear film break-up time 0-5 s) and 12 controls (with tear film break-up time 10-30 s), while the salivary analysis arm consisted of a subset which included 18 patients and six controls. Metabolomic analyses were performed using liquid chromatography and high-resolution mass spectrometry. Analyses using a global database search detected a total of 56 metabolites in tear samples that were significantly different between the groups. Of these, several have known associations with DED. These metabolites are present in meibum and have anti-oxidative characteristics or associations with the ocular microbiome, and altered concentrations suggest that they may play a significant role in DED associated with decreased tear film stability. In saliva, hypotaurine levels were lower among patients with tear film instability. In this pilot study, we found different levels of several metabolites in patients with decreased tear film break-up time that may have associations with DED. Future studies are required to replicate our findings and clarify the exact roles of these metabolites.

APOE Polymorphism Is Associated with Changes in the Kynurenine Pathway.

BACKGROUND: APOE polymorphism and the Kynurenine pathway (KP) are associated with many disorders, but little is known about associations between APOE polymorphism and the KP. This study explored the associations between the KP and APOE polymorphism in disorders associated with APOE polymorphism and changes in the KP. METHODS: Subjects with morbid obesity before and after bariatric surgery (numbers 139 and 95, respectively), depression (number 49), and unspecified neurological symptoms (number 39) were included. The following grouping of the APOE genotypes was used: E2 = ɛ2ɛ2 + ɛ2ɛ3, E3 = ɛ3ɛ3 + ɛ2ɛ4, and E4 = ɛ3ɛ4 + ɛ4ɛ4. The KP metabolites Tryptophan, Kynurenine, Kynurenic acid, Quinolinic acid, and Xanthurenic acid were quantified in serum. RESULTS: The main findings were a significant positive association between E3 and Quinolinic acid (difference between E3 and E2E4: 12.0 (3.5; 18.6) ng/mL); p = 0.005), and a negative association between E4 and Kynurenine (difference between E4 and E2E3: -31.3 (-54.2; -3.2) ng/mL; p = 0.008). Quinolinic acid has been ascribed neurotoxic and inflammatory effects, and Kynurenine is a marker of inflammation. CONCLUSIONS: The findings indicate that APOE polymorphism might cause changes in the KP that contribute to the disease. Inflammation could be the link between APOE and the KP.

Dried blood spot analysis with liquid chromatography and mass spectrometry: Trends in clinical chemistry.

Dried blood spot samples are simple to prepare and transport, enabling safe and accessible diagnostics, both locally and globally. We review dried blood spot samples for clinical analysis, focusing on liquid chromatography-mass spectrometry as a versatile measurement tool for these samples. Dried blood spot samples can provide information for, for example, metabolomics, xenobiotic analysis, and proteomics. Targeted analyses of small molecules are the main application of dried blood spot samples and liquid chromatography-mass spectrometry, but emerging applications include untargeted metabolomics and proteomics. Applications are highly varied, including analyses related to newborn screening, diagnostics and monitoring of disease progression and treatment effects of virtually any disease, as well as studies into the physiology and effects of diet, exercise, xenobiotics, and doping. A range of dried blood spot products and methods are available, and applied liquid chromatography-mass spectrometry instrumentation is varied with regard to liquid chromatography column formats and selectivity. In addition, novel approaches such as on-paper sample preparation (e.g., selective trapping of analytes with paper-immobilized antibodies) are described. We focus on research papers published in the last 5 years.

Ex Vivo Pig Eye Model for Studying Diffusion of Acylcarnitines with Different Physicochemical Properties.

Purpose: To explore the diffusion capacities between the anterior and vitreous chambers in a novel ex vivo pig eye model using a mix of stable isotope-labeled acylcarnitines representing metabolites with different physical and chemical properties, and analysis using mass spectrometry (MS). Methods: Enucleated pig eyes were injected in the anterior or vitreous chamber of the eye with a stable isotope-labeled acylcarnitine mix (free carnitine, C2, C3, C4, C8, C12, and C16-having an increasing size and hydrophobicity in that order). Samples were collected from each chamber at 3, 6, and 24 h postincubation for analysis using MS. Results: After injection into the anterior chamber, the concentration of all acylcarnitines increased in the vitreous chamber over the observation period. After injection in the vitreous chamber, the acylcarnitines diffused to the anterior chamber with the highest concentration observed at 3 h postinjection, followed by a decrease in concentration possibly due to an elimination from the anterior chamber despite continued diffusion from the vitreous chamber. C16, the most hydrophobic and longest chain molecule, showed slower diffusion in both experimental settings. Conclusion: We hereby show a distinct diffusion pattern of molecules with different molecular size and hydrophobicity within and between the anterior and vitreous chamber. This model can be useful for optimizing choices and design of therapeutic molecules with higher retaining or depot properties into the two chambers of the eye for future intravitreal, intracameral, and topical treatment purposes.

A global metabolomics minefield: Confounding effects of preanalytical factors when studying rare disorders.

A common challenge when studying rare diseases or medical conditions is the limited number of patients, usually resulting in long inclusion periods as well as unequal sampling and storage conditions. The main purpose of this study was to demonstrate the challenges when comparing samples subject to different preanalytical conditions. We performed a global (commonly referred to as "untargeted") liquid chromatography-high resolution mass spectrometry metabolomics analysis of blood samples from cases of sudden infant death syndrome and controls stored as dried blood spots on a chemical-free filter card for 15 years at room temperature compared with the same blood samples stored as whole blood at -80°C before preparing new dried blood spots using a chemically treated filter card. Principal component analysis plots distinctly separated the samples based on the type of filter card and storage, but not sudden infant death syndrome versus controls. Note that, 1263 out of 5161 and 642 out of 1587 metabolite features detected in positive and negative ionization mode, respectively, were found to have significant 2-fold changes in amounts corresponding to different preanalytical conditions. The study demonstrates that the dried blood spot metabolome is largely affected by preanalytical factors. This emphasizes the importance of thoroughly addressing preanalytical factors during study design and interpretation, enabling identification of real, biological differences between sample groups whilst preventing other factors or random variation to be falsely interpreted as positive results.

Global Metabolomics Discovers Two Novel Biomarkers in Pyridoxine-Dependent Epilepsy Caused by ALDH7A1 Deficiency.

Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive developmental and epileptic encephalopathy caused by pathogenic variants in the ALDH7A1 gene (PDE-ALDH7A1), which mainly has its onset in neonates and infants. Early diagnosis and treatment are crucial to prevent severe neurological sequelae or death. Sensitive, specific, and stable biomarkers for diagnostic evaluations and follow-up examinations are essential to optimize outcomes. However, most of the known biomarkers for PDE lack these criteria. Additionally, there is little discussion regarding the interdependence of biomarkers in the PDE-ALDH7A1 metabolite profile. Therefore, the aim of this study was to understand the underlying mechanisms in PDE-ALDH7A1 and to discover new biomarkers in the plasma of patients using global metabolomics. Plasma samples from 9 patients with genetically confirmed PDE-ALDH7A1 and 22 carefully selected control individuals were analyzed by ultra high performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). Two novel and reliable pyridoxine-independent diagnostic markers, 6-hydroxy-2-aminocaproic acid (HACA) and an isomer of C9H11NO4, were identified. Furthermore, a possible reaction mechanism is proposed for HACA. This study demonstrates the capability of global metabolomics in disease screening to detect established and novel biomarkers.

Changes in circulating sirtuin 1 after bariatric surgery.

BACKGROUND AND AIMS: Obesity is associated with chronic inflammation and oxidative stress. Weight loss after bariatric surgery improves the inflammatory state and risk of cardiovascular disease. Improvement in metabolic dysfunction might be associated with changes in the activity of sirtuin 1 (SIRT1) and we aimed to investigate the effect of bariatric surgery on its circulating levels. METHODS AND RESULTS: This is a sub-study of a prospective cohort study, including 110 subjects with morbid obesity. The surgical procedure was either laparoscopic Roux-en-Y gastric bypass (RYGB) or sleeve gastrectomy (SG). Blood was sampled at inclusion and six and 12 months after surgery. SIRT1 was measured in EDTA plasma with an enzyme-linked immunosorbent assay. The mean age in the population was 43 years, 80% were women and mean body mass index (BMI) was 38.8 kg/m2. RYGB and SG were performed in 89 and 21 subjects, respectively. SIRT1 concentration was significantly reduced from baseline to six and 12 months after surgery, with mean values (SD) 156.8 (82.6), 119.5 (65.6) and 94.9 (45.6) ng/mL, respectively, (p ≤ 0.002, all), accompanied by significant reductions in C-reactive protein (CRP), BMI and triglycerides from inclusion (p < 0.001, all). Type of surgery did not differently modify SIRT1 levels (p = 0.09). CRP and triglycerides were both positively predictive of SIRT1 levels (p ≤ 0.001, both). CONCLUSION: SIRT1 concentration was significantly lower six and 12 months after bariatric surgery. CRP and triglycerides independently predicted SIRT1 levels, suggesting that reduction in SIRT1 levels might not intrinsically be related to weight reduction, but to improvement in metaflammation.

The Role of Tryptophan Dysmetabolism and Quinolinic Acid in Depressive and Neurodegenerative Diseases.

Emerging evidence suggests that neuroinflammation is involved in both depression and neurodegenerative diseases. The kynurenine pathway, generating metabolites which may play a role in pathogenesis, is one of several competing pathways of tryptophan metabolism. The present article is a narrative review of tryptophan metabolism, neuroinflammation, depression, and neurodegeneration. A disturbed tryptophan metabolism with increased activity of the kynurenine pathway and production of quinolinic acid may result in deficiencies in tryptophan and derived neurotransmitters. Quinolinic acid is an N-methyl-D-aspartate receptor agonist, and raised levels in CSF, together with increased levels of inflammatory cytokines, have been reported in mood disorders. Increased quinolinic acid has also been observed in neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and HIV-related cognitive decline. Oxidative stress in connection with increased indole-dioxygenase (IDO) activity and kynurenine formation may contribute to inflammatory responses and the production of cytokines. Increased formation of quinolinic acid may occur at the expense of kynurenic acid and neuroprotective picolinic acid. While awaiting ongoing research on potential pharmacological interventions on tryptophan metabolism, adequate protein intake with appropriate amounts of tryptophan and antioxidants may offer protection against oxidative stress and provide a balanced set of physiological receptor ligands.

Glioblastoma microenvironment contains multiple hormonal and non-hormonal growth-stimulating factors.

BACKGROUND: The growth of malignant tumors is influenced by their microenvironment. Glioblastoma, an aggressive primary brain tumor, may have cysts containing fluid that represents the tumor microenvironment. The aim of this study was to investigate whether the cyst fluid of cystic glioblastomas contains growth-stimulating factors. Identification of such growth factors may pave the way for the development of targeted anti-glioblastoma therapies. METHODS: We performed hormone analysis of cyst fluid from 25 cystic glioblastomas and proteomics analysis of cyst fluid from another 12 cystic glioblastomas. RESULTS: Glioblastoma cyst fluid contained hormones within wide concentration ranges: Insulin-like growth factor 1 (0-13.7 nmol/L), insulin (1.4-133 pmol/L), erythropoietin (4.7-402 IU/L), growth hormone (0-0.93 µg/L), testosterone (0.2-10.1 nmol/L), estradiol (0-1.0 nmol/L), triiodothyronine (1.0-11.5). Tumor volume correlated with cyst fluid concentrations of growth hormone and testosterone. Survival correlated inversely with cyst fluid concentration of erythropoietin. Several hormones were present at concentrations that have been shown to stimulate glioblastoma growth in vitro. Concentrations of erythropoietin and estradiol (in men) were higher in cyst fluid than in serum, suggesting formation by tumor or brain tissue. Quantitatively, glioblastoma cyst fluid was dominated by serum proteins, illustrating blood-brain barrier leakage. Proteomics identified several proteins that stimulate tumor cell proliferation and invasiveness, others that inhibit apoptosis or mediate adaption to hypoxia and some that induce neovascularization or blood-brain barrier leakage. CONCLUSION: The microenvironment of glioblastomas is rich in growth-stimulating factors that may originate from the circulation, the tumor, or the brain. The wide variation in cyst fluid hormone concentrations may differentially influence tumor growth.

Circulating Lipoproteins in Subjects with Morbid Obesity Undergoing Bariatric Surgery with Gastric Bypass or Sleeve Gastrectomy.

The efficacy of various bariatric procedures on the mitigation of the obese dyslipidemia remains debated, and the impact of these measures on lipoprotein(a) (Lp(a)) levels is unknown. In this study we aimed to compare the two most commonly used procedures: gastric bypass (RYGB) and sleeve gastrectomy (SG). Adult patients with morbid obesity were assigned to receive either RYGB or SG. The levels of non-HDL cholesterol, LDL/HDL-ratio and Lp(a) at examinations conducted 6 and 12 months postoperatively were determined and compared to preoperative levels to estimate the efficacy of the two surgical methods. All results 6 and 12 months after surgery were used in the comparisons with the preoperative results. A linear mixed regression model for repeated analyses was used. The Lp(a) and the non-HDL cholesterol levels were considerably reduced in the RYGB group, in contrast to the minor changes in the SG group. In addition, the LDL/HDL ratio was significantly more reduced in the RYGB group when compared to the SG group. Conclusively, RYGB was found to be more efficient than SG for the mitigation of obese dyslipidemia, including preoperative high Lp(a)-levels. This might have important individual and societal implications, especially regarding the potential to reduce the risk of cardiovascular disease and the related societal costs.

Metabolomics - a new biochemical golden age for personalised medicine. / Metabolomikk ­ ny biokjemisk gullalder for persontilpasset medisin.

Thousands of different molecules can be measured in one drop of blood. Metabolomics can detect 'all' the small molecular substances in any sample material. This is the biochemistry equivalent of whole genome sequencing in genetics. The technology can take personalised medicine to the next level.

APOE Polymorphism and Endocrine Functions in Subjects with Morbid Obesity Undergoing Bariatric Surgery.

BACKGROUND: Obesity is an interplay between genes and the environment, including lifestyle. The genetics of obesity is insufficiently understood. Apolipoprotein E (APOE) genetic polymorphism has been associated with a wide range of disorders. Knowing that some APOE alleles are associated with obesity and endocrine disorders that are common in obesity, the present study aimed at exploring associations between APOE polymorphisms and endocrine functions in subjects with obesity undergoing bariatric surgery. METHODS: Analyses of hormones in blood collected before and one year after bariatric surgery were examined. The APOE alleles were grouped as follows: E2 = ε2ε2 + ε2ε3; E3 = ε3ε3 + ε2ε4; E4 = ε3ε4 + ε4ε4. The APOE groups were analysed as nominal and ordered groups (E2-E3-E4) with a linear mixed model to predict the hormonal effects of the groups. RESULTS: Forty-nine women (79%) and thirteen (21%) men with a mean age of 47.7 (SD 8.5) years were included in the study. The adiponectin level was significantly lower (p < 0.05) in the E2 group compared with the E4 group. Adiponectin and cortisol were positively and negatively associated, respectively, with the ordered APOE groups. CONCLUSIONS: The ordered APOE groups E2-E3-E4 were significantly associated with high and low levels of adiponectin and cortisol, respectively. The findings indicate APOE-mediated effects on body weight and metabolic functions in subjects with morbid obesity.

Saliva Metabolomics in Dry Mouth Patients with Head and Neck Cancer or Sjögren's Syndrome.

The etiology of dry mouth conditions is multi-faceted. Patients radiated after head and neck cancer (HNC) and those with primary Sjögren's syndrome (pSS) share many of the same symptoms despite different causes. With the aim of better understanding the pathophysiology and biochemical processes behind dry mouth with different etiologies, we investigated the metabolic profile of 10 HNC patients, 9 pSS patients and 10 healthy controls using high-performance liquid chromatography-high resolution mass spectrometry (HPLC-MS) metabolomics. Principal component analysis (PCA) revealed different metabolic profiles when comparing all subjects included in the study. Both patient groups showed higher ratios of several pyrimidine nucleotides and nucleosides when compared to controls. This finding may indicate that purinergic signaling plays a role in dry mouth conditions. Moreover, significantly increased levels of DL-3-aminoisobutyric acid were found in HNC patients when compared to controls, and a similar tendency was observed in the pSS patients. Furthermore, a dysregulation in amino acid metabolism was observed in both patient groups. In conclusion, metabolomics analysis showed separate metabolic profiles for HNC and pSS patients as compared to controls that could be useful in diagnostics and for elucidating the different pathophysiologies. The demonstrated dysregulation of pyrimidine nucleotides and levels of metabolites derived from amino acids in the patient groups should be studied further.

A pioneer study on human 3-nitropropionic acid intoxication: Contributions from metabolomics.

The neurotoxin 3-nitropropionic acid (3-NPA) is an inhibitor of succinate dehydrogenase, an enzyme participating both in the citric acid cycle and the mitochondrial respiratory chain. In human intoxications, it produces symptoms such as vomiting and stomach ache in mild cases, and dystonia, coma, and sometimes death in severe cases. We report the results from a liquid chromatography-Orbitrap mass spectrometry metabolomics study mapping the metabolic impacts of 3-NPA intoxication in plasma, urine, and cerebrospinal fluid (CSF) samples of a Norwegian boy initially suspected to suffer from a mitochondrial disease. In addition to the identification of 3-NPA, our findings included a large number of annotated/identified altered metabolites (80, 160, and 62 in plasma, urine, and CSF samples, respectively) belonging to different compound classes, for example, amino acids, fatty acids, and purines and pyrimidines. Our findings indicated protective mechanisms to attenuate the toxic effects of 3-NPA (e.g., decreased oleamide), occurrence of increased oxidative stress in the patient (such as increased free fatty acids and hypoxanthine) and energy turbulence caused by the intoxication (e.g., increased succinate). To our knowledge, this is the first case of 3-NPA intoxication reported in Norway and the first published metabolomics study of human 3-NPA intoxication worldwide. The unexpected identification of 3-NPA illustrates the importance for health care providers to consider intake-related intoxications during diagnostic evaluations, treatment and follow-up examinations for neurotoxicity and a wide range of metabolic derangements.

Reply to Paredes et al. Comment on "Aaseth et al. Circulating Lipoproteins in Subjects with Morbid Obesity Undergoing Bariatric Surgery with Gastric Bypass or Sleeve Gastrectomy. Nutrients 2022, 14, 2381".

Hereby, we thank Silvia Paredes et al. [...].

Bridging the Polar and Hydrophobic Metabolome in Single-Run Untargeted Liquid Chromatography-Mass Spectrometry Dried Blood Spot Metabolomics for Clinical Purposes.

Dried blood spot (DBS) metabolite analysis is a central tool for the clinic, e.g., newborn screening. Instead of applying multiple analytical methods, a single liquid chromatography-mass spectrometry (LC-MS) method was developed for metabolites spanning from highly polar glucose to hydrophobic long-chain acylcarnitines. For liquid chromatography, a diphenyl column and a multi-linear solvent gradient operated at elevated flow rates allowed for an even-spread resolution of diverse metabolites. Injecting moderate volumes of DBS organic extracts directly, in contrast to evaporation and reconstitution, provided substantial increases in analyte recovery. Q Exactive MS settings were also tailored for sensitivity increases, and the method allowed for analyte retention time and peak area repeatabilities of 0.1-0.4 and 2-10%, respectively, for a wide polarity range of metabolites (log P -4.4 to 8.8). The method's performance was suited for both untargeted analysis and targeted approaches evaluated in clinically relevant experiments.

Effects of nutrition therapy on growth, inflammation and metabolism in immature infants: a study protocol of a double-blind randomized controlled trial (ImNuT).

BACKGROUND: Current nutritional management of infants born very preterm results in significant deficiency of the essential fatty acids (FAs) arachidonic acid (ARA) and docosahexaenoic acid (DHA). The impact of this deficit on brain maturation and inflammation mediated neonatal morbidities are unknown. The aim of this study is to determine whether early supply of ARA and DHA improves brain maturation and neonatal outcomes in infants born before 29 weeks of gestation. METHODS: Infants born at Oslo University Hospital are eligible to participate in this double-blind randomized controlled trial. Study participants are randomized to receive an enteral FA supplement of either 0.4 ml/kg MCT-oil™ (medium chain triglycerides) or 0.4 ml/kg Formulaid™ (100 mg/kg of ARA and 50 mg/kg of DHA). The FA supplement is given from the second day of life to 36 weeks' postmenstrual age (PMA). The primary outcome is brain maturation assessed by Magnetic Resonance Imaging (MRI) at term equivalent age. Secondary outcomes include quality of growth, incidence of neonatal morbidities, cardiovascular health and neuro-development. Target sample size is 120 infants (60 per group), this will provide 80% power to detect a 0.04 difference in mean diffusivity (MD, mm2/sec) in major white matter tracts on MRI. DISCUSSION: Supplementation of ARA and DHA has the potential to improve brain maturation and reduce inflammation related diseases. This study is expected to provide valuable information for future nutritional guidelines for preterm infants. TRIAL REGISTRATION: Clinicaltrials.gov ID: NCT03555019 . Registered 4 October 2018- Retrospectively registered.

APOE - a genetic marker of comorbidity in subjects with morbid obesity.

BACKGROUND: In population-based studies, the genetic variability of the APOE E alleles have been associated with health outcomes. Health problems are common in subjects with obesity. This study explored associations between the APOE E alleles and comorbidity in subjects with morbid obesity. METHODS: The study included consecutive subjects referred for evaluation of bariatric surgery with morbid obesity (defined as BMI > 40 or > 35 kg/m2 with complications related to obesity). The subjects followed a conservative weight loss program for 6 months before surgery and had a follow-up visit 12 months after surgery. Demographic data and a set psychosomatic scores (musculoskeletal pain, WHO-5 Well-Being Index, Rosenberg Self-Esteem Scale, Hopkins Symptom Check-list 10; Epworth Sleepiness Scale, and Fatigue Severity Scale) were collected, and blood samples were analysed for haematological and biochemical parameters and APOE alleles. RESULTS: One hundred and forty subjects (men/women: 32 (23%)/108 (77%) with mean age 43.0 (SD 8.7) years and BMI 42.1 (SD 3.8) kg/m2 were included. One hundred and eight and 92 subjects had data after conservative treatment and 12 months after surgery, respectively. The prevalence of the APOE alleles were: E2E2: 1 (0.7%), E2E3: 13 (9.3%), E2E4: 4 (2.9%), E3E3: 71 (50.7%), E3E4: 47 (33.6%), and E4E4: 4 (2.9%). The prevalence rates were as anticipated in a Norwegian population. The weight loss during conservative treatment and after bariatric surgery was independent of E allele variability. E2 was associated with a significant or clear trend toward improvement of all psychosomatic disorders. There was a significant fall in CRP during the two treatment periods with weight loss. E2 and E4 were significantly associated with high and low CRP, respectively, but no associations were seen between CRP and comorbidity. CONCLUSIONS: The most marked finding was the association between E2 and improvement of all psychosomatic disorders. The positive and negative associations between CRP and E2 and E4, respectively, could indicate effects on inflammation and immunological reactions.