Predictive modeling for identification of older adults with high utilization of health and social services.

AIM: Machine learning techniques have demonstrated success in predictive modeling across various clinical cases. However, few studies have considered predicting the use of multisectoral health and social services among older adults. This research aims to utilize machine learning models to detect high-risk groups of excessive health and social services utilization at early stage, facilitating the implementation of preventive interventions. METHODS: We used pseudonymized data covering a four-year period and including information on a total of 33,374 senior citizens from Southern Finland. The endpoint was defined based on the occurrence of unplanned healthcare visits and the total number of different services used. Input features included individual's basic demographics, health status and past usage of healthcare resources. Logistic regression and eXtreme Gradient Boosting (XGBoost) methods were used for binary classification, with the dataset split into 70% training and 30% testing sets. RESULTS: Subgroup-based results mirrored trends observed in the full cohort, with age and certain health issues, e.g. mental health, emerging as positive predictors for high service utilization. Conversely, hospital stay and urban residence were associated with decreased risk. The models achieved a classification performance (AUC) of 0.61 for the full cohort and varying in the range of 0.55-0.62 for the subgroups. CONCLUSIONS: Predictive models offer potential for predicting future high service utilization in the older adult population. Achieving high classification performance remains challenging due to diverse contributing factors. We anticipate that classification performance could be increased by including features based on additional data categories such as socio-economic data.

Ceramides and Phosphatidylcholines Associate with Cardiovascular Diseases in the Elderly.

BACKGROUND: The ceramide- and phospholipid-based cardiovascular risk score (CERT2) has been found to predict the risk for cardiovascular disease (CVD) events, especially cardiovascular mortality. In the present study, our aim was to estimate the predictive ability of CERT2 for mortality of CVD, coronary artery disease (CAD), and stroke in the elderly and to compare these results with those of conventional lipids. METHODS: We conducted a prospective study with an 18-year follow-up period that included a total of 1260 participants ages ≥64 years. Ceramides and phosphatidylcholines were analyzed using a LC-MS. Total cholesterol and triglycerides were performed by enzymatic methods and HDL cholesterol was determined by a direct enzymatic method. Concentrations of LDL-cholesterol were calculated according to the Friedewald formula. RESULTS: A higher score of CERT2 was significantly associated with higher CVD, CAD, and stroke mortality during the 18-year follow-up both in unadjusted and adjusted Cox regression models. The unadjusted hazard ratios (HRs) of CERT2 (95% CI) per SD for CVD, CAD, and stroke were 1.72 (1.52-1.96), 1.76 (1.52-2.04), and 1.63 (1.27-2.10), respectively, and the corresponding adjusted HRs (95% CI) per SD for CERT2 were 1.48 (1.29-1.69), 1.50 (1.28-1.75), and 1.41 (1.09-1.83). For conventional lipids, HRs per SD were lower than for CERT2. CONCLUSIONS: The risk score CERT2 associated strongly with CVD, CAD, and stroke mortality in the elderly, while the association between these events and conventional lipids was weak.

Development and validation of a ceramide- and phospholipid-based cardiovascular risk estimation score for coronary artery disease patients.

AIMS: Distinct ceramide lipids have been shown to predict the risk for cardiovascular disease (CVD) events, especially cardiovascular death. As phospholipids have also been linked with CVD risk, we investigated whether the combination of ceramides with phosphatidylcholines (PCs) would be synergistic in the prediction of CVD events in patients with atherosclerotic coronary heart disease in three independent cohort studies. METHODS AND RESULTS: Ceramides and PCs were analysed using liquid chromatography-mass spectrometry (LC-MS) in three studies: WECAC (The Western Norway Coronary Angiography Cohort) (N = 3789), LIPID (Long-Term Intervention with Pravastatin in Ischaemic Disease) trial (N = 5991), and KAROLA (Langzeiterfolge der KARdiOLogischen Anschlussheilbehandlung) (N = 1023). A simple risk score, based on the ceramides and PCs showing the best prognostic features, was developed in the WECAC study and validated in the two other cohorts. This score was highly significant in predicting CVD mortality [multiadjusted hazard ratios (HRs; 95% confidence interval) per standard deviation were 1.44 (1.28-1.63) in WECAC, 1.47 (1.34-1.61) in the LIPID trial, and 1.69 (1.31-2.17) in KAROLA]. In addition, a combination of the risk score with high-sensitivity troponin T increased the HRs to 1.63 (1.44-1.85) and 2.04 (1.57-2.64) in WECAC and KAROLA cohorts, respectively. The C-statistics in WECAC for the risk score combined with sex and age was 0.76 for CVD death. The ceramide-phospholipid risk score showed comparable and synergistic predictive performance with previously published CVD risk models for secondary prevention. CONCLUSION: A simple ceramide- and phospholipid-based risk score can efficiently predict residual CVD event risk in patients with coronary artery disease.

New evidence from plasma ceramides links apoE polymorphism to greater risk of coronary artery disease in Finnish adults.

apoE, a key regulator of plasma lipids, mediates altered functionalities in lipoprotein metabolism and thus affects the risk of coronary artery disease (CAD). The significance of different apoE polymorphisms remains unclear; although the ε4 allele is clearly associated with increased cholesterol levels (which inform CAD risk), direct studies about apoE polymorphisms on CAD risk and development have yielded controversial results. Furthermore, certain species of ceramides-complex lipids abundant in plasma LDL-are markers of increased risk of myocardial infarction and cardiovascular death. Using a high-throughput MS approach, we quantified 30 molecular plasma ceramide species from a cohort of 2,160 apoE-genotyped (rs7412, rs429358) young adults enrolled in the population-based Cardiovascular Risk in Young Finns Study. We then searched this lipidome data set to identify new indications of pathways influenced by apoE polymorphisms and possibly related to CAD risk. This approach revealed a previously unreported association between apoE polymorphism and a consistently documented high-risk CAD marker, Cer(d18:1/16:0). Compared with the apoE ε3/3 reference group, plasma levels of apoE ε4 were elevated and those of apoE ε2 were lowered in all subjects without evidence of apoE-by-sex interactions. apoE associated with seven ceramides that are connected to atherogenically potent macrophages and/or lipoprotein particles; these associations could indicate a plausible linkage between apoE polymorphism and ceramide metabolism, leading to adverse plasma LDL metabolism and atherogenesis. In conclusion, new evidence from plasma ceramides links apoE polymorphism with an increased risk of CAD and extends our understanding of the role of apoE in health and disease.

Susceptibility of low-density lipoprotein particles to aggregate depends on particle lipidome, is modifiable, and associates with future cardiovascular deaths.

Aims: Low-density lipoprotein (LDL) particles cause atherosclerotic cardiovascular disease (ASCVD) through their retention, modification, and accumulation within the arterial intima. High plasma concentrations of LDL drive this disease, but LDL quality may also contribute. Here, we focused on the intrinsic propensity of LDL to aggregate upon modification. We examined whether inter-individual differences in this quality are linked with LDL lipid composition and coronary artery disease (CAD) death, and basic mechanisms for plaque growth and destabilization. Methods and results: We developed a novel, reproducible method to assess the susceptibility of LDL particles to aggregate during lipolysis induced ex vivo by human recombinant secretory sphingomyelinase. Among patients with an established CAD, we found that the presence of aggregation-prone LDL was predictive of future cardiovascular deaths, independently of conventional risk factors. Aggregation-prone LDL contained more sphingolipids and less phosphatidylcholines than did aggregation-resistant LDL. Three interventions in animal models to rationally alter LDL composition lowered its susceptibility to aggregate and slowed atherosclerosis. Similar compositional changes induced in humans by PCSK9 inhibition or healthy diet also lowered LDL aggregation susceptibility. Aggregated LDL in vitro activated macrophages and T cells, two key cell types involved in plaque progression and rupture. Conclusion: Our results identify the susceptibility of LDL to aggregate as a novel measurable and modifiable factor in the progression of human ASCVD.

Plasma concentrations of molecular lipid species predict long-term clinical outcome in coronary artery disease patients.

We investigated the associations of ten previously identified high risk molecular lipid species and three ceramide ratios with the occurrence of major adverse cardiac events (MACEs) during a median follow-up of 4.7 years in patients with coronary artery disease (CAD). Between 2008 and 2011, 581 patients underwent diagnostic coronary angiography or percutaneous coronary intervention for stable angina pectoris (SAP) or acute coronary syndrome (ACS). Blood was drawn prior to the index procedure and lipid species were determined. The primary endpoint was the occurrence of a MACE, comprising all-cause mortality, nonfatal ACS, or unplanned coronary revascularization. The secondary endpoint comprised all-cause mortality or nonfatal ACS. During a median follow-up of 4.7 [IQR: 4.2-5.6] years, 155 patients (27%) had MACEs. In multivariable analyses, Cer(d18:1/16:0) concentration was associated with MACEs {hazard ratio 2.32; 95% CI [1.09-4.96] per natural logarithm (ln) (pmol/ml) P = 0.030} after adjustment for cardiac risk factors, clinical presentation, statin use at baseline, and admission nonHDL cholesterol level. Furthermore, after multivariable adjustment, concentrations of Cer(d18:1/16:0), Cer(d18:1/20:0), Cer(d18:1/24:1), and their ratios to Cer(d18:1/24:0) were associated with the composite endpoint death or nonfatal ACS. The data together show the circulating ceramide lipids we investigated here are associated with adverse cardiac outcome during long-term follow-up independent of clinical risk factors.

Ceramide stearic to palmitic acid ratio predicts incident diabetes.

AIMS/HYPOTHESIS: Ceramide lipids have a role in the development of insulin resistance, diabetes and risk of cardiovascular disease. Here we investigated four ceramides and their ratios to find the best predictors of incident diabetes. METHODS: A validated mass-spectrometric method was applied to measure Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0) and Cer(d18:1/24:1) from serum or plasma samples. These ceramides were analysed in a population-based risk factor study (FINRISK 2002, n = 8045), in a cohort of participants undergoing elective coronary angiography for suspected stable angina pectoris (Western Norway Coronary Angiography Cohort [WECAC], n = 3344) and in an intervention trial investigating improved methods of lifestyle modification for individuals at high risk of the metabolic syndrome (Prevent Metabolic Syndrome [PrevMetSyn], n = 371). Diabetes risk score models were developed to estimate the 10 year risk of incident diabetes. RESULTS: Analysis in FINRISK 2002 showed that the Cer(d18:1/18:0)/Cer(d18:1/16:0) ceramide ratio was predictive of incident diabetes (HR per SD 2.23, 95% CI 2.05, 2.42), and remained significant after adjustment for several risk factors, including BMI, fasting glucose and HbA1c (HR 1.34, 95% CI 1.14, 1.57). The finding was validated in the WECAC study (unadjusted HR 1.81, 95% CI 1.53, 2.14; adjusted HR 1.39, 95% CI 1.16, 1.66). In the intervention trial, the ceramide ratio and diabetes risk scores significantly decreased in individuals who had 5% or more weight loss. CONCLUSIONS/INTERPRETATION: The Cer(d18:1/18:0)/Cer(d18:1/16:0) ratio is an independent predictive biomarker for incident diabetes, and may be modulated by lifestyle intervention.

Ovarian tumours of different histologic type and clinical stage induce similar changes in lipid metabolism.

BACKGROUND: Previous results obtained from serum samples of late-stage, high-grade serous ovarian carcinoma patients showed large alterations in lipid metabolism. To validate and extend the results, we studied lipidomic changes in early-stage ovarian tumours. In addition to serous ovarian cancer, we investigated whether these changes occur in mucinous and endometrioid histological subtypes as well. METHODS: Altogether, 354 serum or plasma samples were collected from three centres, one from Germany and two from Finland. We performed lipidomic analysis of samples from patients with malignant (N = 138) or borderline (N = 25) ovarian tumours, and 191 controls with benign pathology. These results were compared to previously published data. RESULTS: We found 39 lipids that showed consistent alteration both in early- and late-stage ovarian cancer patients as well as in pre- and postmenopausal women. Most of these changes were already significant at an early stage and progressed with increasing stage. Furthermore, 23 lipids showed similar alterations in all investigated histological subtypes. CONCLUSIONS: Changes in lipid metabolism due to ovarian cancer occur in early-stage disease but intensify with increasing stage. These changes occur also in other histological subtypes besides high-grade serous carcinoma. Understanding lipid metabolism in ovarian cancer may lead to new therapeutic and diagnostic alternatives.

Circulating Ceramides Predict Cardiovascular Outcomes in the Population-Based FINRISK 2002 Cohort.

OBJECTIVE: Ceramides are molecular lipids implicated in apoptosis, inflammation, obesity, and insulin resistance. An earlier study reported that ceramides were associated with fatal outcome among patients with coronary heart disease. Here, we examined whether ceramides are associated with major adverse cardiovascular events (MACEs) among apparently healthy individuals. APPROACH AND RESULTS: FINRISK 2002 is a population-based risk factor survey, which recruited men and women aged 25 to 74 years. The cohort was followed up until the end of 2014. We quantified 4 circulating ceramides, Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0), and Cer(d18:1/24:1), in 8101 serum samples by a targeted liquid chromatography-tandem mass spectrometry assay. Primary outcome of interest was incident MACE (n=813). Secondary analyses were performed for MACE death (n=116) without previous nonfatal MACE and for recurrent MACE (n=226) among survivors of a previous incident MACE. We used Cox proportional hazard models adjusted for the Framingham covariates to determine the association of ceramides with the outcomes. Of the ceramide species, Cer(d18:1/18:0) had the strongest association with incident MACE and the highest unadjusted hazard ratio of 1.31 (95% confidence interval, 1.21-1.41), which remained significant at 1.21 (95% confidence interval, 1.11-1.33) after Framingham risk factor adjustments. The hazard ratios were generally stronger for recurrent and fatal events than for first events. Clinical net reclassification improvement was 7.5% (P=6.9×10-5) for Cer(d18:1/18:0). CONCLUSIONS: Distinct serum ceramides are associated with the risk of incident MACE in apparently healthy individuals. These results should encourage more detailed analyses of ceramides in cardiovascular pathobiology and suggest new biomarkers of MACE risk.

Plasma ceramides predict cardiovascular death in patients with stable coronary artery disease and acute coronary syndromes beyond LDL-cholesterol.

AIMS: The aim was to study the prognostic value of plasma ceramides (Cer) as cardiovascular death (CV death) markers in three independent coronary artery disease (CAD) cohorts. METHODS AND RESULTS: Corogene study is a prospective Finnish cohort including stable CAD patients (n = 160). Multiple lipid biomarkers and C-reactive protein were measured in addition to plasma Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0), and Cer(d18:1/24:1). Subsequently, the association between high-risk ceramides and CV mortality was investigated in the prospective Special Program University Medicine-Inflammation in Acute Coronary Syndromes (SPUM-ACS) cohort (n = 1637), conducted in four Swiss university hospitals. Finally, the results were validated in Bergen Coronary Angiography Cohort (BECAC), a prospective Norwegian cohort study of stable CAD patients. Ceramides, especially when used in ratios, were significantly associated with CV death in all studies, independent of other lipid markers and C-reactive protein. Adjusted odds ratios per standard deviation for the Cer(d18:1/16:0)/Cer(d18:1/24:0) ratio were 4.49 (95% CI, 2.24-8.98), 1.64 (1.29-2.08), and 1.77 (1.41-2.23) in the Corogene, SPUM-ACS, and BECAC studies, respectively. The Cer(d18:1/16:0)/Cer(d18:1/24:0) ratio improved the predictive value of the GRACE score (net reclassification improvement, NRI = 0.17 and ΔAUC = 0.09) in ACS and the predictive value of the Marschner score in stable CAD (NRI = 0.15 and ΔAUC = 0.02). CONCLUSIONS: Distinct plasma ceramide ratios are significant predictors of CV death both in patients with stable CAD and ACS, over and above currently used lipid markers. This may improve the identification of high-risk patients in need of more aggressive therapeutic interventions.

Fish oil and krill oil differentially modify the liver and brain lipidome when fed to mice.

BACKGROUND: Marine food is an important source of omega-3 fatty acids with beneficial health effects. Oils from marine organisms have different fatty acid composition and differ in their molecular composition. Fish oil (FO) has a high content of eicosapentaenoic and docosahexaenoic acids mainly esterified to triacylglycerols, while in krill oil (KO) these fatty acids are mainly esterified to phospholipids. The aim was to study the effects of these oils on the lipid content and fatty acid distribution in the various lipid classes in liver and brain of mice. METHODS: Mice were fed either a high-fat diet (HF), a HF diet supplemented with FO or with KO (n = 6). After six weeks of feeding, liver and brain lipid extracts were analysed using a shotgun and TAG lipidomics approach. Student t-test was performed after log-transformation to compare differences between study groups. RESULTS: Six weeks of feeding resulted in significant changes in the relative abundance of many lipid classes compared to control mice. In both FO and KO fed mice, the triacylglycerol content in the liver was more than doubled. The fatty acid distribution was affected by the oils in both liver and brain with a decrease in the abundance of 18:2 and 20:4, and an increase in 20:5 and 22:6 in both study groups. 18:2 decreased in all lipid classes in the FO group but with only minor changes in the KO group. Differences between the feeding groups were particularly evident in some of the minor lipid classes that are associated with inflammation and insulin resistance. Ceramides and diacylglycerols were decreased and cholesteryl esters increased in the liver of the KO group, while plasmalogens were decreased in the FO group. In the brain, diacylglycerols were decreased, more by KO than FO, while ceramides and lactosylceramides were increased, more by FO than KO. CONCLUSION: The changes in the hepatic sphingolipids and 20:4 fatty acid levels were greater in the KO compared to the FO fed mice, and are consistent with a hypothesis that krill oil will have a stronger anti-inflammatory action and enhances insulin sensitivity more potently than fish oil.

Monounsaturated fatty acids in serum triacylglycerols are associated with response to neoadjuvant chemotherapy in breast cancer patients.

Changes in cellular lipid metabolism are a common feature in most solid tumors, which occur already in early stages of the tumor progression. However, it remains unclear if the tumor-specific lipid changes can be detected at the level of systemic lipid metabolism. The objective of this study was to perform comprehensive analysis of lipids in breast cancer patient serum samples. Lipidomic profiling using an established analytical platform was performed in two cohorts of breast cancer patients receiving neoadjuvant chemotherapy. The analyses were performed for 142 patients before and after neoadjuvant chemotherapy, and the results before chemotherapy were validated in an independent cohort of 194 patients. The analyses revealed that in general the tumor characteristics are not reflected in the serum samples. However, there was an association of specific triacylglycerols (TGs) in patients' response to chemotherapy. These TGs containing mainly oleic acid (C18:1) were found in lower levels in those patients showing pathologic complete response before receiving chemotherapy. Some of these TGs were also associated with estrogen receptor status and overall or disease-free survival of the patients. The results suggest that the altered serum levels of oleic acid in breast cancer patients are associated with their response to chemotherapy.

15-Hydroxyprostaglandin dehydrogenase associates with poor prognosis in breast cancer, induces epithelial-mesenchymal transition, and promotes cell migration in cultured breast cancer cells.

Breast cancer is the most frequent cancer and the leading cause of cancer-related deaths in women worldwide. The prognosis of breast cancer is tightly correlated with the degree of spread beyond the primary tumour. Arachidonic acid (AA) and prostaglandin E(2) (PGE(2)) are known to regulate tumour metastasis enabling epithelial-mesenchymal transition (EMT). However, the detailed role of 15-hydroxyprostaglandin dehydrogenase (HPGD), the key enzyme degrading prostaglandin E(2) , remains unclear in breast cancer. Here, we show that HPGD mRNA is overexpressed in a subset of clinical breast cancers compared to normal breast tissue samples and that high HPGD mRNA expression associates with poor prognosis. Immunohistochemical staining of primary breast cancer and lymph node metastasis tissue samples confirmed high HPGD protein expression in 20% of the samples, as well as associated HPGD expression with aggressive characteristics, such as increased risk of disease relapse and shorter disease-free survival. Results from cultured cells indicated abundant HPGD expression in highly metastatic breast cancer cells, and impairment of HPGD expression using RNA interference led to a significant decrease in transforming growth factor-ß signalling, in cellular arachidonic acid levels as well as in cell migration. Furthermore, gene expression microarray analysis followed by quantitative RT-PCR validation showed that HPGD silencing decreased aryl hydrocarbon receptor signalling and induced mesenchymal-epithelial transition. In conclusion, our results indicate that HPGD is highly expressed in metastatic and aggressive breast cancer and promotes EMT and migration in breast cancer cells.

Characterization, bioinformatic analysis and dithiocarbamate inhibition studies of two new α-carbonic anhydrases, CAH1 and CAH2, from the fruit fly Drosophila melanogaster.

Carbonic anhydrases (CAs) are essential and ubiquitous enzymes. Thus far, there are no articles on characterization of Drosophila melanogaster α-CAs. Data from invertebrate CA studies may provide opportunities for anti-parasitic drug development because α-CAs are found in many parasite or parasite vector invertebrates. We have expressed and purified D. melanogaster CAH1 and CAH2 as proteins of molecular weights 30kDa and 28kDa. CAH1 is cytoplasmic whereas CAH2 is a membrane-attached protein. Both are highly active enzymes for the CO2 hydration reaction, being efficiently inhibited by acetazolamide. CAH2 in the eye of D. melanogaster may provide a new animal model for CA-related eye diseases. A series of dithiocarbamates were also screened as inhibitors of these enzymes, with some representatives showing inhibition in the low nanomolar range.

The Novel Ceramide- and Phosphatidylcholine-Based Risk Score for the Prediction of New-Onset of Hypertension.

Ceramides and other sphingolipids are implicated in vascular dysfunction and inflammation. They have been suggested as potential biomarkers for hypertension. However, their specific association with hypertension prevalence and onset requires further investigation. This study aimed to identify specific ceramide and phosphatidylcholine species associated with hypertension prevalence and onset. The 2002 FINRISK (Finnish non-communicable risk factor survey) study investigated the association between coronary event risk scores (CERT1 and CERT2) and hypertension using prevalent and new-onset hypertension groups, both consisting of 7722 participants, over a span of 10 years. Ceramide and phosphatidylcholine levels were measured using tandem liquid chromatography-mass spectrometry. Ceramide and phosphatidylcholine ratios, including ceramide (d18:1/18:0), ceramide (d18:1/24:1), phosphatidylcholine (16:0/16:0), and the ratio of ceramide (d18:1/18:0)/(d18:1/16:0), are consistently associated with both prevalence and new-onset hypertension. Ceramide (d18:1/24:0) was also linked to both hypertension measures. Adjusting for covariates, CERT1 and CERT2 showed no-longer-significant associations with hypertension prevalence, but only CERT2 predicted new-onset hypertension. Plasma ceramides and phosphatidylcholines are crucial biomarkers for hypertension, with imbalances potentially contributing to its development. Further research is needed to understand the underlying mechanisms by which ceramides will contribute to the development of hypertension.

Integration of metabolomics and expression of glycerol-3-phosphate acyltransferase (GPAM) in breast cancer-link to patient survival, hormone receptor status, and metabolic profiling.

Changes in lipid metabolism are an important but not well-characterized hallmark of cancer. On the basis of our recent findings of lipidomic changes in breast cancer, we investigated glycerol-3-phosphate acyltransferase (GPAM), a key enzyme in the lipid biosynthesis of triacylglycerols and phospholipids. GPAM protein expression was evaluated and linked to metabolomic and lipidomic profiles in a cohort of human breast carcinomas. In addition, GPAM mRNA expression was analyzed using the GeneSapiens in silico transcriptiomics database. High cytoplasmic GPAM expression was associated with hormone receptor negative status (p = 0.013). On the protein (p = 0.048) and mRNA (p = 0.001) levels, increased GPAM expression was associated with a better overall survival. Metabolomic analysis by GC-MS showed that sn-glycerol-3-phosphate, the substrate of GPAM, was elevated in breast cancer compared to normal breast tissue. LC-MS based lipidomic analysis identified significantly higher levels of phospholipids, especially phosphatidylcholines in GPAM protein positive tumors. In conclusion, our results suggest that GPAM is expressed in human breast cancer with associated changes in the cellular metabolism, in particular an increased synthesis of phospholipids, the major structural component of cellular membranes.

Remodeling of central metabolism in invasive breast cancer compared to normal breast tissue - a GC-TOFMS based metabolomics study.

BACKGROUND: Changes in energy metabolism of the cells are common to many kinds of tumors and are considered a hallmark of cancer. Gas chromatography followed by time-of-flight mass spectrometry (GC-TOFMS) is a well-suited technique to investigate the small molecules in the central metabolic pathways. However, the metabolic changes between invasive carcinoma and normal breast tissues were not investigated in a large cohort of breast cancer samples so far. RESULTS: A cohort of 271 breast cancer and 98 normal tissue samples was investigated using GC-TOFMS-based metabolomics. A total number of 468 metabolite peaks could be detected; out of these 368 (79%) were significantly changed between cancer and normal tissues (p<0.05 in training and validation set). Furthermore, 13 tumor and 7 normal tissue markers were identified that separated cancer from normal tissues with a sensitivity and a specificity of >80%. Two-metabolite classifiers, constructed as ratios of the tumor and normal tissues markers, separated cancer from normal tissues with high sensitivity and specificity. Specifically, the cytidine-5-monophosphate / pentadecanoic acid metabolic ratio was the most significant discriminator between cancer and normal tissues and allowed detection of cancer with a sensitivity of 94.8% and a specificity of 93.9%. CONCLUSIONS: For the first time, a comprehensive metabolic map of breast cancer was constructed by GC-TOF analysis of a large cohort of breast cancer and normal tissues. Furthermore, our results demonstrate that spectrometry-based approaches have the potential to contribute to the analysis of biopsies or clinical tissue samples complementary to histopathology.

Brain phenotype of carbonic anhydrase IX-deficient mice.

Preliminary observations have suggested mild behavioral changes and a morphological disruption of brain histology in 1.5-year-old carbonic anhydrase IX (CA IX)-deficient (Car9 (-/-)) mice. These findings led us to design a 1-year follow-up study in which the behavior and brain histology of Car9 (-/-) and wild-type mice were monitored. Morphological analysis revealed vacuolar degenerative changes in the brains of Car9 (-/-) mice. The changes became visible at the age of eight to ten months. Behavioral tests showed that the Car9 (-/-) mice exhibited abnormal locomotor activity and poor performance in a memory test. To further identify the transcriptomic responses to CA IX deficiency in the brain, genome-wide cDNA microarray analyses were performed. Thirty-one and 37 genes were significantly up- or down-regulated, respectively, in the brain of Car9 (-/-) mice compared to the wild-type mice. Functional annotation revealed that the genes with increased expression were involved in several processes, such as RNA metabolism, and the genes with reduced expression were implicated in other important processes, including the regulation of cellular ion homeostasis. Notably, the biological processes "behavior" and "locomotory behavior" were the two prominent terms overrepresented among the down-regulated genes, which is consistent with the behavioral phenotype. These results suggest that CA IX may directly or indirectly play novel functions in brain tissue. Furthermore, the brain phenotype of Car9 (-/-) mice seems to be age-dependent. The results indicate that the functional changes precede the microscopic alterations in the brains of Car9 (-/-) mice.

Crystal structure of the catalytic domain of the tumor-associated human carbonic anhydrase IX.

Carbonic anhydrase (CA) IX is a plasma membrane-associated member of the alpha-CA enzyme family, which is involved in solid tumor acidification. It is a marker of tumor hypoxia and a prognostic factor in several human cancers. An aberrant increase in CA IX expression in chronic hypoxia and during development of various carcinomas contributes to tumorigenesis through at least two mechanisms: pH regulation and cell adhesion control. Here we report the X-ray structure of the catalytic domain of CA IX in complex with a classical, clinically used sulfonamide inhibitor, acetazolamide. The structure reveals a typical alpha-CA fold, which significantly differs from the other CA isozymes when the protein quaternary structure is considered. Thus, two catalytic domains of CA IX associate to form a dimer, which is stabilized by the formation of an intermolecular disulfide bond. The active site clefts and the PG domains are located on one face of the dimer, while the C-termini are located on the opposite face to facilitate protein anchoring to the cell membrane. A correlation between the three-dimensional structure and the physiological role of the enzyme is here suggested, based on the measurement of the pH profile of the catalytic activity for the physiological reaction, CO(2) hydration to bicarbonate and protons. On the basis of the structural differences observed between CA IX and the other membrane-associated alpha-CAs, further prospects for the rational drug design of isozyme-specific CA inhibitors are proposed, given that inhibition of this enzyme shows antitumor activity both in vitro and in vivo.