Quantification of multiple steroid hormones in serum and human breast cancer tissue by liquid chromatography-tandem mass spectrometry analysis.

Introduction: Systemic and local steroid hormone levels may function as novel prognostic and predictive biomarkers in breast cancer patients. We aimed at developing a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous measurement of multiple, biologically pivotal steroid hormones in human serum and breast cancer tissue. Methods: The quantitative method consisted of liquid-liquid extraction, Sephadex LH-20 chromatography for tissue extracts, and analysis of steroid hormones by liquid-chromatography-tandem mass spectrometry. We analyzed serum and tissue steroid hormone levels in 16 and 40 breast cancer patients, respectively, and assessed their correlations with clinical parameters. Results: The method included quantification of nine steroid hormones in serum [including cortisol, cortisone, corticosterone, estrone (E1), 17ß-estradiol (E2), 17α-hydroxyprogesterone, androstenedione (A4), testosterone and progesterone) and six (including cortisone, corticosterone, E1, E2, A4, and testosterone) in cancer tissue. The lower limits of quantification were between 0.003-10 ng/ml for serum (250 µl) and 0.038-125 pg/mg for tissue (20 mg), respectively. Accuracy was between 98%-126%, intra-assay coefficient of variations (CV) was below 15%, and inter-assay CV were below 11%. The analytical recoveries for tissue were between 76%-110%. Tissue levels of E1 were positively correlated with tissue E2 levels (p<0.001), and with serum levels of E1, E2 and A4 (p<0.01). Tissue E2 levels were positively associated with serum E1 levels (p=0.02), but not with serum E2 levels (p=0.12). The levels of tissue E2 and ratios of E1 to A4 levels (an index for aromatase activity) were significantly higher in patients with larger tumors (p=0.03 and p=0.02, respectively). Conclusions: The method was convenient and suitable for a specific and accurate profiling of clinically important steroid hormones in serum. However, the sensitivity of the profile method in steroid analysis in tissue samples is limited, but it can be used for the analysis of steroids in breast cancer tissues if the size of the sample or its steroid content is sufficient.

Comprehensive multi-omics analysis of breast cancer reveals distinct long-term prognostic subtypes.

Breast cancer (BC) is a leading cause of cancer-related death worldwide. The diverse nature and heterogeneous biology of BC pose challenges for survival prediction, as patients with similar diagnoses often respond differently to treatment. Clinically relevant BC intrinsic subtypes have been established through gene expression profiling and are implemented in the clinic. While these intrinsic subtypes show a significant association with clinical outcomes, their long-term survival prediction beyond 5 years often deviates from expected clinical outcomes. This study aimed to identify naturally occurring long-term prognostic subgroups of BC based on an integrated multi-omics analysis. This study incorporates a clinical cohort of 335 untreated BC patients from the Oslo2 study with long-term follow-up (>12 years). Multi-Omics Factor Analysis (MOFA+) was employed to integrate transcriptomic, proteomic, and metabolomic data obtained from the tumor tissues. Our analysis revealed three prominent multi-omics clusters of BC patients with significantly different long-term prognoses (p = 0.005). The multi-omics clusters were validated in two independent large cohorts, METABRIC and TCGA. Importantly, a lack of prognostic association to long-term follow-up above 12 years in the previously established intrinsic subtypes was shown for these cohorts. Through a systems-biology approach, we identified varying enrichment levels of cell-cycle and immune-related pathways among the prognostic clusters. Integrated multi-omics analysis of BC revealed three distinct clusters with unique clinical and biological characteristics. Notably, these multi-omics clusters displayed robust associations with long-term survival, outperforming the established intrinsic subtypes.

18F-FACBC and 18F-FDG PET/MRI in the evaluation of 3 patients with primary central nervous system lymphoma: a pilot study.

BACKGROUND: This PET/MRI study compared contrast-enhanced MRI, 18F-FACBC-, and 18F-FDG-PET in the detection of primary central nervous system lymphomas (PCNSL) in patients before and after high-dose methotrexate chemotherapy. Three immunocompetent PCNSL patients with diffuse large B-cell lymphoma received dynamic 18F-FACBC- and 18F-FDG-PET/MRI at baseline and response assessment. Lesion detection was defined by clinical evaluation of contrast enhanced T1 MRI (ce-MRI) and visual PET tracer uptake. SUVs and tumor-to-background ratios (TBRs) (for 18F-FACBC and 18F-FDG) and time-activity curves (for 18F-FACBC) were assessed. RESULTS: At baseline, seven ce-MRI detected lesions were also detected with 18F-FACBC with high SUVs and TBRs (SUVmax:mean, 4.73, TBRmax: mean, 9.32, SUVpeak: mean, 3.21, TBRpeak:mean: 6.30). High TBR values of 18F-FACBC detected lesions were attributed to low SUVbackground. Baseline 18F-FDG detected six lesions with high SUVs (SUVmax: mean, 13.88). In response scans, two lesions were detected with ce-MRI, while only one was detected with 18F-FACBC. The lesion not detected with 18F-FACBC was a small atypical MRI detected lesion, which may indicate no residual disease, as this patient was still in complete remission 12 months after initial diagnosis. No lesions were detected with 18F-FDG in the response scans. CONCLUSIONS: 18F-FACBC provided high tumor contrast, outperforming 18F-FDG in lesion detection at both baseline and in response assessment. 18F-FACBC may be a useful supplement to ce-MRI in PCNSL detection and response assessment, but further studies are required to validate these findings. Trial registration ClinicalTrials.gov. Registered 15th of June 2017 (Identifier: NCT03188354, https://clinicaltrials.gov/study/NCT03188354 ).

Profiling of serum metabolome of breast cancer: multi-cancer features discriminate between healthy women and patients with breast cancer.

Introduction: The progression of solid cancers is manifested at the systemic level as molecular changes in the metabolome of body fluids, an emerging source of cancer biomarkers. Methods: We analyzed quantitatively the serum metabolite profile using high-resolution mass spectrometry. Metabolic profiles were compared between breast cancer patients (n=112) and two groups of healthy women (from Poland and Norway; n=95 and n=112, respectively) with similar age distributions. Results: Despite differences between both cohorts of controls, a set of 43 metabolites and lipids uniformly discriminated against breast cancer patients and healthy women. Moreover, smaller groups of female patients with other types of solid cancers (colorectal, head and neck, and lung cancers) were analyzed, which revealed a set of 42 metabolites and lipids that uniformly differentiated all three cancer types from both cohorts of healthy women. A common part of both sets, which could be called a multi-cancer signature, contained 23 compounds, which included reduced levels of a few amino acids (alanine, aspartate, glutamine, histidine, phenylalanine, and leucine/isoleucine), lysophosphatidylcholines (exemplified by LPC(18:0)), and diglycerides. Interestingly, a reduced concentration of the most abundant cholesteryl ester (CE(18:2)) typical for other cancers was the least significant in the serum of breast cancer patients. Components present in a multi-cancer signature enabled the establishment of a well-performing breast cancer classifier, which predicted cancer with a very high precision in independent groups of women (AUC>0.95). Discussion: In conclusion, metabolites critical for discriminating breast cancer patients from controls included components of hypothetical multi-cancer signature, which indicated wider potential applicability of a general serum metabolome cancer biomarker.

Diagnostic accuracy of anti-3-[18F]-FACBC PET/MRI in gliomas.

PURPOSE: The primary aim was to evaluate whether anti-3-[18F]FACBC PET combined with conventional MRI correlated better with histomolecular diagnosis (reference standard) than MRI alone in glioma diagnostics. The ability of anti-3-[18F]FACBC to differentiate between molecular and histopathological entities in gliomas was also evaluated. METHODS: In this prospective study, patients with suspected primary or recurrent gliomas were recruited from two sites in Norway and examined with PET/MRI prior to surgery. Anti-3-[18F]FACBC uptake (TBRpeak) was compared to histomolecular features in 36 patients. PET results were then added to clinical MRI readings (performed by two neuroradiologists, blinded for histomolecular results and PET data) to assess the predicted tumor characteristics with and without PET. RESULTS: Histomolecular analyses revealed two CNS WHO grade 1, nine grade 2, eight grade 3, and 17 grade 4 gliomas. All tumors were visible on MRI FLAIR. The sensitivity of contrast-enhanced MRI and anti-3-[18F]FACBC PET was 61% (95%CI [45, 77]) and 72% (95%CI [58, 87]), respectively, in the detection of gliomas. Median TBRpeak was 7.1 (range: 1.4-19.2) for PET positive tumors. All CNS WHO grade 1 pilocytic astrocytomas/gangliogliomas, grade 3 oligodendrogliomas, and grade 4 glioblastomas/astrocytomas were PET positive, while 25% of grade 2-3 astrocytomas and 56% of grade 2-3 oligodendrogliomas were PET positive. Generally, TBRpeak increased with malignancy grade for diffuse gliomas. A significant difference in PET uptake between CNS WHO grade 2 and 4 gliomas (p < 0.001) and between grade 3 and 4 gliomas (p = 0.002) was observed. Diffuse IDH wildtype gliomas had significantly higher TBRpeak compared to IDH1/2 mutated gliomas (p < 0.001). Adding anti-3-[18F]FACBC PET to MRI improved the accuracy of predicted glioma grades, types, and IDH status, and yielded 13.9 and 16.7 percentage point improvement in the overall diagnoses for both readers, respectively. CONCLUSION: Anti-3-[18F]FACBC PET demonstrated high uptake in the majority of gliomas, especially in IDH wildtype gliomas, and improved the accuracy of preoperatively predicted glioma diagnoses. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT04111588, URL: https://clinicaltrials.gov/study/NCT04111588.

Randomised controlled trial of exercise training during lactation on breast milk composition in breastfeeding people with overweight/obesity: a study protocol for the MILKSHAKE trial.

Breast milk from people with overweight/obesity may differ in composition compared with that from normal-weight people. Exercise training can modify breast milk composition in rodent models, with a beneficial impact demonstrated on the offspring's metabolism, but whether these findings translate to humans is unclear. This trial aims to determine the effect of an exercise intervention on breast milk composition and whether an exercise-induced modification of breast milk impacts the infants' growth and body composition. Effect of Exercise Training on Breastmilk Composition is a randomised, controlled trial with two parallel groups, one exercise group and one control group, with a 1:1 allocation. We will include a minimum of 62 exclusively breastfeeding participants, 6 weeks postpartum. The exercise intervention lasts 8 weeks and comprises 25 supervised endurance exercise sessions with moderate or high intensity. The primary outcome measure is the change in the relative concentration of the human milk oligosaccharide 3'sialyllactose in breast milk from baseline at 6 weeks postpartum to the end of the intervention period. Secondary outcomes include breast milk concentrations of other metabolites, cytokines, hormones and microRNA, maternal health outcomes, infant growth, infant gut microbiome and infant circulating microRNA. Maternal and infant outcomes will be measured before, during and after the intervention period, with a follow-up of the infants until they are 24 months old. Trial registration number NCT05488964.

The impact of pre-processing and disease characteristics on reproducibility of T2-weighted MRI radiomics features.

PURPOSE: To evaluate the reproducibility of radiomics features derived via different pre-processing settings from paired T2-weighted imaging (T2WI) prostate lesions acquired within a short interval, to select the setting that yields the highest number of reproducible features, and to evaluate the impact of disease characteristics (i.e., clinical variables) on features reproducibility. MATERIALS AND METHODS: A dataset of 50 patients imaged using T2WI at 2 consecutive examinations was used. The dataset was pre-processed using 48 different settings. A total of 107 radiomics features were extracted from manual delineations of 74 lesions. The inter-scan reproducibility of each feature was measured using the intra-class correlation coefficient (ICC), with ICC values > 0.75 considered good. Statistical differences were assessed using Mann-Whitney U and Kruskal-Wallis tests. RESULTS: The pre-processing parameters strongly influenced the reproducibility of radiomics features of T2WI prostate lesions. The setting that yielded the highest number of features (25 features) with high reproducibility was the relative discretization with a fixed bin number of 64, no signal intensity normalization, and outlier filtering by excluding outliers. Disease characteristics did not significantly impact the reproducibility of radiomics features. CONCLUSION: The reproducibility of T2WI radiomics features was significantly influenced by pre-processing parameters, but not by disease characteristics. The selected pre-processing setting yielded 25 reproducible features.

Sexual functioning, sexual enjoyment, and body image in Norwegian breast cancer survivors: a 12-year longitudinal follow-up study and comparison with the general female population.

BACKGROUND: Given the scarcity of evidence concerning the long-term sexual health of breast cancer (BC) survivors (BC-Pop), we aimed to assess how BC treatments affect short- and long-term sexual functioning, sexual enjoyment, and body image, and compare with aged-matched women in the Norwegian general population (F-GenPop). MATERIAL AND METHODS: The 349 patients in BC-Pop treated at Trondheim University Hospital in 2007-2014, were assessed in clinical controls at the hospital; before starting radiotherapy (T1, baseline), immediately after ending radiotherapy (T2), and after 3, 6, and 12 months (T3-T5), and at a long-term follow-up 7-12 years after baseline (T6). Meanwhile, F-GenPop included 2254 age-matched women in the Norwegian general population. The impact of BC treatment on sexual functioning was examined using a Linear Mixed Model. Sexual functioning, sexual enjoyment, and body image were assessed with the EORTC's QLQ-BR23 scales and compared between the populations in the four age groups (30-49, 50-59, 60-69, and 70+ years) using means with 95% confidence intervals and Student t-test. Linear regression, adjusted for age and comorbidity was applied to estimate individual scores. RESULT: BC survivors treated with mastectomy had overall lower sexual functioning than patients who had received breast-conserving surgery (p = 0.017). Although BC survivors treated with chemotherapy had lower sexual functioning than those treated without chemotherapy at T1-T5 (p = 0.044), both groups showed the same level of functioning at T6. BC-Pop exhibited significantly poorer sexual functioning (p < 0.001), lower sexual enjoyment (p < 0.05), and better body image (p < 0.001) than F-GenPop in all age groups. CONCLUSION: The impact of specific BC treatments on sexual functioning was modest; only mastectomy had a persistent negative influence. Nevertheless, all age groups in BC-Pop displayed significantly poorer sexual functioning than F-GenPop at both 12 months and up to 12 years after treatment.

Association of serum cortisol and cortisone levels and risk of recurrence after endocrine treatment in breast cancer.

Metabolic reprogramming in breast cancer involves changes in steroid hormone synthesis and metabolism. Alterations in estrogen levels in both breast tissue and blood may influence carcinogenesis, breast cancer growth, and response to therapy. Our aim was to examine whether serum steroid hormone concentrations could predict the risk of recurrence and treatment-related fatigue in patients with breast cancer. This study included 66 postmenopausal patients with estrogen receptor-positive breast cancer who underwent surgery, radiotherapy, and adjuvant endocrine treatment. Serum samples were collected at six different time points [before the start of radiotherapy (as baseline), immediately after radiotherapy, and then 3, 6, 12 months, and 7-12 years after radiotherapy]. Serum concentrations of eight steroid hormones (cortisol, cortisone, 17α-hydroxyprogesterone, 17ß-estradiol, estrone, androstenedione, testosterone, and progesterone) were measured using a liquid chromatography-tandem mass spectrometry-based method. Breast cancer recurrence was defined as clinically proven relapse/metastatic breast cancer or breast cancer-related death. Fatigue was assessed with the QLQ-C30 questionnaire. Serum steroid hormone concentrations measured before and immediately after radiotherapy differed between relapse and relapse-free patients [(accuracy 68.1%, p = 0.02, and 63.2%, p = 0.03, respectively, partial least squares discriminant analysis (PLS-DA)]. Baseline cortisol levels were lower in patients who relapsed than in those who did not (p < 0.05). The Kaplan-Meier analysis showed that patients with high baseline concentrations of cortisol (≥ median) had a significantly lower risk of breast cancer recurrence than patients with low cortisol levels (

Colorectal cancer detected by liquid biopsy 2 years prior to clinical diagnosis in the HUNT study.

BACKGROUND: Colorectal cancer (CRC) is often diagnosed in advanced stages. Circulating tumour DNA (ctDNA) has been proposed as an early diagnostic biomarker. However, as a screening tool, ctDNA has mainly been studied in selected populations at the time of clinical diagnosis. The aim of this study was to detect CRC by known ctDNA markers up to 2 years prior to clinical diagnosis. METHODS: In this case-control study, methylated ctDNA markers were detected in plasma samples from 106 healthy controls and 106 individuals diagnosed with CRC within 24 months following participation in The Trøndelag Health Study. RESULTS: The most specific single markers were BMP3, FLI1, IKZF1, SFRP1, SFRP2, NPTX2, SLC8A1 and VIM (specificity >70%). When combining these into a panel, the CRC sensitivity was 43% (95% CI 42.7-43.4) and the CRC specificity was 86% (95% CI 85.7-86.2). The findings were reproduced in an independent validation set of samples. CONCLUSIONS: Detection of known methylated ctDNA markers of CRC is possible up to 2 years prior to the clinical diagnosis in an unselected population resembling the screening setting. This study supports the hypothesis that some patients could be diagnosed earlier, if ctDNA detection was part of the CRC screening programme.

The association between circulating lipoprotein subfractions and lipid content in coronary atheromatous plaques assessed by near-infrared spectroscopy.

Background: Lipid content in coronary atheromatous plaques, measured by near-infrared spectroscopy (NIRS), can predict the risk of future coronary events. Biomarkers that reflect lipid content in coronary plaques may therefore improve coronary artery disease (CAD) risk assessment. Purpose: We aimed to investigate the association between circulating lipoprotein subfractions and lipid content in coronary atheromatous plaques in statin-treated patients with stable CAD undergoing percutaneous coronary intervention. Methods: 56 patients with stable CAD underwent three-vessel imaging with NIRS when feasible. The coronary artery segment with the highest lipid content, defined as the maximum lipid core burden index within any 4 mm length across the entire lesion (maxLCBI4mm), was defined as target segment. Lipoprotein subfractions and Lipoprotein a (Lp(a)) were analyzed in fasting serum samples by nuclear magnetic resonance spectroscopy and by standard in-hospital procedures, respectively. Penalized linear regression analyses were used to identify the best predictors of maxLCBI4mm. The uncertainty of the lasso estimates was assessed as the percentage presence of a variable in resampled datasets by bootstrapping. Results: Only modest evidence was found for an association between lipoprotein subfractions and maxLCBI4mm. The lipoprotein subfractions with strongest potential as predictors according to the percentage presence in resampled datasets were Lp(a) (78.1 % presence) and free cholesterol in the smallest high-density lipoprotein (HDL) subfractions (74.3 % presence). When including established cardiovascular disease (CVD) risk factors in the regression model, none of the lipoprotein subfractions were considered potential predictors of maxLCBI4mm. Conclusion: In this study, serum levels of Lp(a) and free cholesterol in the smallest HDL subfractions showed the strongest potential as predictors for lipid content in coronary atheromatous plaques. Although the evidence is modest, our study suggests that measurement of lipoprotein subfractions may provide additional information with respect to coronary plaque composition compared to traditional lipid measurements, but not in addition to established risk factors. Further and larger studies are needed to assess the potential of circulating lipoprotein subfractions as meaningful biomarkers both for lipid content in coronary atheromatous plaques and as CVD risk markers.

Lipoprotein subfraction profiling in the search of new risk markers for myocardial infarction: The HUNT study.

BACKGROUND: Traditional biomarkers used to measure risk of myocardial infarction (MI) only explain a modest proportion of the incidence. Lipoprotein subfractions have the potential to improve risk prediction of MI. AIM: We aimed to identify lipoprotein subfractions that were associated with imminent MI risk. METHODS: We identified apparently healthy participants with a predicted low 10-year risk of MI from The Trøndelag Health Survey 3 (HUNT3) that developed MI within 5 years after inclusion (cases, n = 50) and 100 matched controls. Lipoprotein subfractions were analyzed in serum by nuclear magnetic resonance spectroscopy at time of inclusion in HUNT3. Lipoprotein subfractions were compared between cases and controls in the full population (N = 150), and in subgroups of males (n = 90) and females (n = 60). In addition, a sub analysis was performed in participants that experienced MI within two years and their matched controls (n = 56). RESULTS: None of the lipoprotein subfractions were significantly associated with future MI when adjusting for multiple testing (p<0.002). At nominal significance level (p<0.05), the concentration of apolipoprotein A1 in the smallest high-density lipoprotein (HDL) subfractions was higher in cases compared to controls. Further, in sub analyses based on sex, male cases had lower lipid concentration within the large HDL subfractions and higher lipid concentration within the small HDL subfractions compared to male controls (p<0.05). No differences were found in lipoprotein subfractions between female cases and controls. In sub analysis of individuals suffering from MI within two years, triglycerides in low-density lipoprotein were higher among cases (p<0.05). CONCLUSION: None of the investigated lipoprotein subfractions were associated with future MI after adjustment for multiple testing. However, our findings suggests that HDL subfractions may be of interest in relation to risk prediction for MI, especially in males. This need to be further investigated in future studies.

Association of serum metabolome profile with the risk of breast cancer in participants of the HUNT2 study.

Background: The serum metabolome is a potential source of molecular biomarkers associated with the risk of breast cancer. Here we aimed to analyze metabolites present in pre-diagnostic serum samples collected from healthy women participating in the Norwegian Trøndelag Health Study (HUNT2 study) for whom long-term information about developing breast cancer was available. Methods: Women participating in the HUNT2 study who developed breast cancer within a 15-year follow-up period (BC cases) and age-matched women who stayed breast cancer-free were selected (n=453 case-control pairs). Using a high-resolution mass spectrometry approach 284 compounds were quantitatively analyzed, including 30 amino acids and biogenic amines, hexoses, and 253 lipids (acylcarnitines, glycerides, phosphatidylcholines, sphingolipids, and cholesteryl esters). Results: Age was a major confounding factor responsible for a large heterogeneity in the dataset, hence age-defined subgroups were analyzed separately. The largest number of metabolites whose serum levels differentiated BC cases and controls (82 compounds) were observed in the subgroup of younger women (<45 years old). Noteworthy, increased levels of glycerides, phosphatidylcholines, and sphingolipids were associated with reduced risk of cancer in younger and middle-aged women (≤64 years old). On the other hand, increased levels of serum lipids were associated with an enhanced risk of breast cancer in older women (>64 years old). Moreover, several metabolites could be detected whose serum levels were different between BC cases diagnosed earlier (<5 years) and later (>10 years) after sample collecting, yet these compounds were also correlated with the age of participants. Current results were coherent with the results of the NMR-based metabolomics study performed in the cohort of HUNT2 participants, where increased serum levels of VLDL subfractions were associated with reduced risk of breast cancer in premenopausal women. Conclusions: Changes in metabolite levels detected in pre-diagnostic serum samples, which reflected an impaired lipid and amino acid metabolism, were associated with long-term risk of breast cancer in an age-dependent manner.

Primary Treatment Effects for High-Grade Serous Ovarian Carcinoma Evaluated by Changes in Serum Metabolites and Lipoproteins.

High-grade serous ovarian carcinoma (HGSOC) is the most common and deadliest ovarian cancer subtype. Despite advances in treatment, the overall prognosis remains poor. Regardless of efforts to develop biomarkers to predict surgical outcome and recurrence risk and resistance, reproducible indicators are scarce. Exploring the complex tumor heterogeneity, serum profiling of metabolites and lipoprotein subfractions that reflect both systemic and local biological processes were utilized. Furthermore, the overall impact on the patient from the tumor and the treatment was investigated. The aim was to characterize the systemic metabolic effects of primary treatment in patients with advanced HGSOC. In total 28 metabolites and 112 lipoproteins were analyzed by nuclear magnetic resonance (NMR) spectroscopy in longitudinal serum samples (n = 112) from patients with advanced HGSOC (n = 24) from the IMPACT trial with linear mixed effect models and repeated measures ANOVA simultaneous component analysis. The serum profiling revealed treatment-induced changes in both lipoprotein subfractions and circulating metabolites. The development of a more atherogenic lipid profile throughout the treatment, which was more evident in patients with short time to recurrence, indicates an enhanced systemic inflammation and increased risk of cardiovascular disease after treatment. The findings suggest that treatment-induced changes in the metabolome reflect mechanisms behind the diversity in disease-related outcomes.

Effect of high-fat diet and morning or evening exercise on lipoprotein subfraction profiles: secondary analysis of a randomised trial.

We investigated the effect of a high-fat diet (HFD) on serum lipid subfractions in men with overweight/obesity and determined whether morning or evening exercise affected these lipid profiles. In a three-armed randomised trial, 24 men consumed an HFD for 11 days. One group of participants did not exercise (n = 8, CONTROL), one group trained at 06:30 h (n = 8, EXam), and one group at 18:30 h (n = 8, EXpm) on days 6-10. We assessed the effects of HFD and exercise training on circulating lipoprotein subclass profiles using NMR spectroscopy. Five days of HFD induced substantial perturbations in fasting lipid subfraction profiles, with changes in 31/100 subfraction variables (adjusted p values [q] < 0.05). Exercise training induced a systematic change in lipid subfraction profiles, with little overall difference between EXam and EXpm. Compared with CONTROL, exercise training reduced serum concentrations of > 20% of fasting lipid subfractions. EXpm reduced fasting cholesterol concentrations in three LDL subfractions by ⁓30%, while EXam only reduced concentration in the largest LDL particles by 19% (all q < 0.05). Lipid subfraction profiles changed markedly after 5 days HFD in men with overweight/obesity. Both morning and evening exercise training impacted subfraction profiles compared with no exercise.

Immunomodulatory Effects of Metformin Treatment in Pregnant Women With PCOS.

CONTEXT: Polycystic ovary syndrome (PCOS) is a common endocrine disorder associated with low-grade systemic inflammation and increased risk of pregnancy complications. Metformin treatment reduces the risk of late miscarriage and preterm birth in pregnant women with PCOS. Whether the protective effect of metformin involves immunological changes has not been determined. OBJECTIVE: To investigate the effect of metformin on the maternal immunological status in women with PCOS. METHODS: A post-hoc analysis was performed of two randomized controlled trials, PregMet and PregMet2, including longitudinal maternal serum samples from 615 women with PCOS. Women were randomized to metformin or placebo from first trimester to delivery. Twenty-two cytokines and C-reactive protein were measured in serum sampled at gestational weeks 5 to 12, 19, 32, and 36. RESULTS: Metformin treatment was associated with higher serum levels of several multifunctional cytokines throughout pregnancy, with the strongest effect on eotaxin (P < .001), interleukin-17 (P = .03), and basic fibroblast growth factor (P = .04). Assessment of the combined cytokine development confirmed the impact of metformin on half of the 22 cytokines. The immunomodulating effect of metformin was more potent in normal weight and overweight women than in obese women. Moreover, normoandrogenic women had the strongest effect of metformin in early pregnancy, whereas hyperandrogenic women presented increasing effect throughout pregnancy. CONCLUSION: It appears that metformin has immunomodulating rather than anti-inflammatory properties in pregnancy. Its effect on the serum levels of many multifunctional cytokines demonstrates robust, persisting, and body mass-dependent immune mobilization in pregnant women with PCOS.

Prediction of recurrence from metabolites and expression of TOP2A and EZH2 in prostate cancer patients treated with radiotherapy.

BACKGROUND: The dual upregulation of TOP2A and EZH2 gene expression has been proposed as a biomarker for recurrence in prostate cancer patients to be treated with radical prostatectomy. A low tissue level of the metabolite citrate has additionally been connected to aggressive disease and recurrence in this patient group. However, for radiotherapy prostate cancer patients, few prognostic biomarkers have been suggested. The main aim of this study was to use an integrated tissue analysis to evaluate metabolites and expression of TOP2A and EZH2 as predictors for recurrence among radiotherapy patients. METHODS: From 90 prostate cancer patients (56 received neoadjuvant hormonal treatment), 172 transrectal ultrasound-guided (TRUS) biopsies were collected prior to radiotherapy. Metabolic profiles were acquired from fresh frozen TRUS biopsies using high resolution-magic angle spinning MRS. Histopathology and immunohistochemistry staining for TOP2A and EZH2 were performed on TRUS biopsies containing cancer cells (n = 65) from 46 patients, where 24 of these patients (n = 31 samples) received hormonal treatment. Eleven radical prostatectomy cohorts of a total of 2059 patients were used for validation in a meta-analysis. RESULTS: Among radiotherapy patients with up to 11 years of follow-up, a low level of citrate was found to predict recurrence, p = 0.001 (C-index = 0.74). Citrate had a higher predictive ability compared with individual clinical variables, highlighting its strength as a potential biomarker for recurrence. The dual upregulation of TOP2A and EZH2 was suggested as a biomarker for recurrence, particularly for patients not receiving neoadjuvant hormonal treatment, p = 0.001 (C-index = 0.84). While citrate was a statistically significant biomarker independent of hormonal treatment status, the current study indicated a potential of glutamine, glutamate and choline as biomarkers for recurrence among patients receiving neoadjuvant hormonal treatment, and glucose among patients not receiving neoadjuvant hormonal treatment. CONCLUSION: Using an integrated approach, our study shows the potential of citrate and the dual upregulation of TOP2A and EZH2 as biomarkers for recurrence among radiotherapy patients.

Small LDL subfractions are associated with coronary atherosclerosis despite no differences in conventional lipids.

Lipoprotein subfractions currently represent a new source of cardiovascular disease (CVD) risk markers that may provide more information than conventional lipid measures. We aimed to investigate whether lipoprotein subfractions are associated with coronary atherosclerosis in patients without prior known CVD. Fasting serum samples from 60 patients with suspected coronary artery disease (CAD) were collected before coronary angiography and analyzed by nuclear magnetic resonance (NMR) spectroscopy. The severity of coronary atherosclerosis was quantified by the Gensini score (≤20.5 = nonsignificant coronary atherosclerosis, 20.6-30.0 = intermediate coronary atherosclerosis, ≥30.1 = significant CAD). Differences in lipoprotein subfractions between the three Gensini groups were assessed by two-way ANOVA, adjusted for statin use. Despite no differences in conventional lipid measures between the three Gensini groups, patients with significant CAD had higher apolipoprotein-B/apolipoprotein-A1 ratio, 30% more small and dense low-density lipoprotein 5 (LDL-5) particles, and increased levels of cholesterol, triglycerides, and phospholipids within LDL-5 compared with patients with nonsignificant coronary atherosclerosis and intermediate coronary atherosclerosis (P ≤ 0.001). In addition, the low-density lipoprotein (LDL) cholesterol/high-density lipoprotein cholesterol ratio, and triglyceride levels of LDL 4 were significantly increased in patients with significant CAD compared with patients with nonsignificant coronary atherosclerosis. In conclusion, small and dense lipoprotein subfractions were associated with coronary atherosclerosis in patients without prior CVD. Additional studies are needed to explore whether lipoprotein subfractions may represent biomarkers offering a clinically meaningful improvement in the risk prediction of CAD.

High-intensity exercise increases breast milk adiponectin concentrations: a randomised cross-over study.

Introduction: Adiponectin plays a role in glucose and fat metabolism and is present in human breast milk. It has been postulated that higher breast milk adiponectin concentrations may prevent rapid weight gain in infancy. Prior research indicates that circulating adiponectin increases acutely after endurance exercise, but no prior research has investigated the effect of exercise on breast milk adiponectin concentrations. The purpose of this randomised, cross-over study was to determine the acute effects of endurance exercise on adiponectin concentrations in human breast milk. Methods: Participants who were exclusively breastfeeding a 6-12 week-old term infant (N = 20) completed three conditions in the laboratory: (1) Moderate-intensity continuous training (MICT), (2) High-intensity interval training (HIIT), and (3) No activity (REST). At each condition, we collected breast milk at 07:00 h (before exercise/rest), 11:00 h (immediately after exercise/rest), 12:00 h (1 h after exercise/rest), and 15:00 h (4 h after exercise/rest) and determined adiponectin concentrations using enzyme-linked immunosorbent assay. We compared changes in adiponectin concentrations after MICT and HIIT, adjusted for the morning concentration on each test day, with those after REST, using paired t-tests. Results: Adiponectin concentrations increased 1 h after HIIT, from 4.6 (± 2.2) µg/L in the 07:00 h sample to 5.6 (± 2.6) µg/L. This change was 0.9 µg/L (95% confidence interval 0.3 to 1.5) greater than the change between these two timepoints in the REST condition (p = 0.025). There were no other statistically significant changes in adiponectin concentrations. Conclusion: HIIT may increase adiponectin concentrations in breast milk acutely after exercise. Further studies should determine the impact of exercise-induced elevations in breast milk adiponectin concentrations on growth and metabolism in infancy.