Kudosrasvan koostumuksen analyysi spektroskopialla

Lipid analysis is commonly performed by gas chromatography (GC) in laboratory conditions. Spectroscopic techniques, however, are non-destructive and can be implemented noninvasively in vivo. Excess fat (triglycerides) in visceral adipose tissue and liver is known predispose to metabolic abnormalities, collectively known as the metabolic syndrome. Insulin resistance is the likely cause with diets high in saturated fat known to impair insulin sensitivity. Tissue triglyceride composition has been used as marker of dietary intake but it can also be influenced by tissue specific handling of fatty acids. Recent studies have shown that adipocyte insulin sensitivity correlates positively with their saturated fat content, contradicting the common view of dietary effects. A better understanding of factors affecting tissue triglyceride composition is needed to provide further insights into tissue function in lipid metabolism. In this thesis two spectroscopic techniques were developed for in vitro and in vivo analysis of tissue triglyceride composition. In vitro studies (Study I) used infrared spectroscopy (FTIR), a fast and cost effective analytical technique well suited for multivariate analysis. Infrared spectra are characterized by peak overlap leading to poorly resolved absorbances and limited analytical performance. In vivo studies (Studies II, III and IV) used proton magnetic resonance spectroscopy (1H-MRS), an established non-invasive clinical method for measuring metabolites in vivo. 1H-MRS has been limited in its ability to analyze triglyceride composition due to poorly resolved resonances. Using an attenuated total reflection accessory, we were able to obtain pure triglyceride infrared spectra from adipose tissue biopsies. Using multivariate curve resolution (MCR), we were able to resolve the overlapping double bond absorbances of monounsaturated fat and polyunsaturated fat. MCR also resolved the isolated trans double bond and conjugated linoleic acids from an overlapping background absorbance. Using oil phantoms to study the effects of different fatty acid compositions on the echo time behaviour of triglycerides, it was concluded that the use of long echo times improved peak separation with T2 weighting having a negligible impact. It was also discovered that the echo time behaviour of the methyl resonance of omega-3 fats differed from other fats due to characteristic J-coupling. This novel insight could be used to detect omega-3 fats in human adipose tissue in vivo at very long echo times (TE = 470 and 540 ms). A comparison of 1H-MRS of adipose tissue in vivo and GC of adipose tissue biopsies in humans showed that long TE spectra resulted in improved peak fitting and better correlations with GC data. The study also showed that calculation of fatty acid fractions from 1H-MRS data is unreliable and should not be used. Omega-3 fatty acid content derived from long TE in vivo spectra (TE = 540 ms) correlated with total omega-3 fatty acid concentration measured by GC. The long TE protocol used for adipose tissue studies was subsequently extended to the analysis of liver fat composition. Respiratory triggering and long TE resulted in spectra with the olefinic and tissue water resonances resolved. Conversion of the derived unsaturation to double bond content per fatty acid showed that the results were in accordance with previously published gas chromatography data on liver fat composition. In patients with metabolic syndrome, liver fat was found to be more saturated than subcutaneous or visceral adipose tissue. The higher saturation observed in liver fat may be a result of a higher rate of de-novo-lipogenesis in liver than in adipose tissue. This thesis has introduced the first non-invasive method for determining adipose tissue omega-3 fatty acid content in humans in vivo. The methods introduced here have also shown that liver fat is more saturated than adipose tissue fat.Ylipaino, metabolinen oireyhtymä ja niihin liittyvä tyypin 2 diabetes yleistyvät nopeasti ja pelkästään Suomessa on arvioitu olevan noin 450 000 tyypin 2 diabeetikkoa. Metabolisen oireyhtymän ja siihen liittyvän tyypin 2 diabeteksen arvellaan olevan seurausta kehon heikentyneestä vasteesta insuliinihormonin vaikutuksille eli ns. insuliinresistenssistä. Paljon rasvaa ja sokeria sisältävä ruokavalio altistaa painonnousulle ja insuliiniresesistenssin kehittymiselle. Aikaisempien tutkimusten perusteella paljon tyydyttyneitä ns. kovia rasvoja sisältävää ruokavaliota on pidetty haitallisena. Kovien rasvojen haitallisuudesta on kuitenkin esitetty eriäviä mielipiteitä ja aiheesta on käyty mediassa kiivasta keskustelua. Monityydyttämättömistä rasvoista erityisesti omega-3-rasvoja pidetään terveellisinä ja niitä myydään ravintolisävalmisteina. Ruokavalion omega-3-rasvojen vaikutusmekanismit ovat kuitenkin vielä epäselviä. Tutkimukset ruokavalion rasvan koostumuksen vaikutuksesta terveyteen ovat tuottaneet ristiriitaisia tuloksia. Ruokavalion tutkiminen on vaikeaa, sillä koehenkilöiden ruokavalion luotettava seuranta on käytännössä mahdotonta. Rasva kerääntyy kehossa pääosin ihonalaiseen rasvakudokseen, jonka on siksi ajateltu edustavan ruokavalion rasvan koostumusta. Äskettäin on kuitenkin havaittu, että ihonalaisen rasvakudoksen kovat rasvat liittyvät hyvään insuliinin vaikutukseen rasvakudoksessa. Metabolisessa oireyhtymässä rasvaa kertyy myös viskeraaliseen rasvakudokseen ja maksaan. Rasvan koostumus määritetään yleensä kudosnäytepaloista laboratorio-olosuhteissa. Viskeraalisen rasvakudoksen ja maksan rasvan tutkiminen vaatii siten kirurgista näytteenottoa vatsan sisäisistä kudoksista. Toimenpide on koehenkilölle hankala ja komplikaatioaltis, mikä on rajoittanut näiden erityisen haitallisten rasvakertymien tutkimista. Spektroskopiamenetelmät mahdollistavat kemiallisen analytiikan tuhoamatta näytettä. Infrapunaspektroskopia (FTIR) on laajasti käytetty kemiallinen analyysimenetelmä, jonka spektrit soveltuvat hyvin monimuuttuja-analyysiin. Magneettiresonassispektroskopia (MRS) on kliininen menetelmä, joka mahdollistaa (koehenkilöön) kajoamattoman kemiallisen analyysin magneettikuvauslaitteella. Tässä väitöskirjatyössä FTIR- ja MRS-menetelmiä laajennettiin ihmisen kudosrasvan koostumuksen analysointiin. FTIR-menetelmän käyttöä rasvakudoksen analyysissä rajoittaa spektrien analyyttisten piikkien huono erottuvuus toisistaan. Työssä parannettiin FTIR spektrien analyysiä soveltaen matemaattista monimuuttujamenetelmää. Työn tuloksena spektripiikkien erottelu parani ja niiden luotettava erottelu tuli mahdolliseksi. FTIR kykeni erottamaan rasvaspektristä trans- ja konjugoidut linolihapot, joiden analyysi perinteisillä menetelmillä on vaikeaa. MRS-menetelmää kehitettiin ja sovellettiin koehenkilöihin, joilla on metabolinen oireyhtymä. Uudella MRS-menetelmällä pystyttiin analysoimaan ihonalaista ja viskeraalista rasvakudosta ja maksaan kertynyttä rasvaa. Yllättävä havainto oli, että maksaan kertyvä rasva on tyydyttyneempää kuin rasvakudoksen rasva. Uudella menetelmällä pystyttiin myös ensimmäistä kertaa määrittämään ihmisen rasvakudoksen omega-3- rasvoja kajoamattomasti. Tässä väitöskirjatyössä kehitetyt kajoamattomat kudosrasvan analysointimenetelmät avaavat uusia mahdollisuuksia tutkia kudosrasvan koostumusta ja sen yhteyttä rasva-aineenvaihdunnan häiriöihin. Metabolisen oireyhtymän potilailla havaittu maksan rasvan korkea tyydyttyneisyys verrattuna rasvakudokseen saattaa olla seurausta muuttuneesta kudosten rasvahapposynteesistä

Body electrical loss analysis (BELA) in the assessment of visceral fat: a demonstration

Peer reviewe

Cardiac steatosis associates with visceral obesity in nondiabetic obese men.

Background: Liver fat and visceral adiposity are involved in the development of the metabolic syndrome (MetS). Ectopic fat accumulation within and around the heart has been related to increased risk of heart disease. The aim of this study was to explore components of cardiac steatosis and their relationship to intra-abdominal ectopic fat deposits and cardiometabolic risk factors in nondiabetic obese men. Methods: Myocardial and hepatic triglyceride (TG) contents were measured with 1.5 T magnetic resonance spectroscopy, and visceral adipose (VAT), abdominal subcutaneous tissue (SAT), epicardial and pericardial fat by magnetic resonance imaging in 37 men with the MetS and in 40 men without the MetS. Results: Myocardial and hepatic TG contents, VAT, SAT, epicardial fat volumes, and pericardial fat volumes were higher in men with the MetS compared with subjects without the MetS (P < .001). All components of cardiac steatosis correlated with SAT, VAT, and hepatic TG content and the correlations seemed to be strongest with VAT. Myocardial TG content, epicardial fat, pericardial fat, VAT, and hepatic TG content correlated with waist circumference, body mass index, high-density lipoprotein cholesterol TGs, very low-density lipoprotein-1 TGs, and the insulin-resistance homeostasis model assessment index. VAT was a predictor of TGs, high-density lipoprotein cholesterol, and measures of glucose metabolism, whereas age and SAT were determinants of blood pressure parameters. Conclusions: We suggest that visceral obesity is the best predictor of epicardial and pericardial fat in abdominally obese subjects. Myocardial TG content may present a separate entity that is influenced by factors beyond visceral adiposity

Mitochondria-related transcriptional signature is downregulated in adipocytes in obesity : a study of young healthy MZ twins

Low mitochondrial activity in adipose tissue is suggested to be an underlying factor in obesity and its metabolic complications. We aimed to find out whether mitochondrial measures are downregulated in obesity also in isolated adipocytes. We studied young adult monozygotic (MZ) twin pairs discordant (n = 14, intrapair difference Delta BMI ae 3 kg/m(2)) and concordant (n = 5, Delta BMI <3 kg/m(2)) for BMI, identified from ten birth cohorts of 22- to 36-year-old Finnish twins. Abdominal body fat distribution (MRI), liver fat content (magnetic resonance spectroscopy), insulin sensitivity (OGTT), high-sensitivity C-reactive protein, serum lipids and adipokines were measured. Subcutaneous abdominal adipose tissue biopsies were obtained to analyse the transcriptomics patterns of the isolated adipocytes as well as of the whole adipose tissue. Mitochondrial DNA transcript levels in adipocytes were measured by quantitative real-time PCR. Western blots of oxidative phosphorylation (OXPHOS) protein levels in adipocytes were performed in obese and lean unrelated individuals. The heavier (BMI 29.9 +/- 1.0 kg/m(2)) co-twins of the discordant twin pairs had more subcutaneous, intra-abdominal and liver fat and were more insulin resistant (p <0.01 for all measures) than the lighter (24.1 +/- 0.9 kg/m(2)) co-twins. Altogether, 2538 genes in adipocytes and 2135 in adipose tissue were significantly differentially expressed (nominal p <0.05) between the co-twins. Pathway analysis of these transcripts in both isolated adipocytes and adipose tissue revealed that the heavier co-twins displayed reduced expression of genes relating to mitochondrial pathways, a result that was replicated when analysing the pathways behind the most consistently downregulated genes in the heavier co-twins (in at least 12 out of 14 pairs). Consistently upregulated genes in adipocytes were related to inflammation. We confirmed that mitochondrial DNA transcript levels (12S RNA, 16S RNA, COX1, ND5, CYTB), expression of mitochondrial ribosomal protein transcripts and a major mitochondrial regulator PGC-1 alpha (also known as PPARGC1A) were reduced in the heavier co-twins' adipocytes (p <0.05). OXPHOS protein levels of complexes I and III in adipocytes were lower in obese than in lean individuals. Subcutaneous abdominal adipocytes in obesity show global expressional downregulation of oxidative pathways, mitochondrial transcripts and OXPHOS protein levels and upregulation of inflammatory pathways. The datasets analysed and generated during the current study are available in the figshare repository.Peer reviewe

Abdominal adipose tissue and liver fat imaging in very low birth weight adults born preterm : birth cohort with sibling-controls

Preterm birth at very low birth weight (VLBW, < 1500 g) is associated with an accumulation of cardiovascular and metabolic risk factors from childhood at least to middle age. Small-scale studies suggest that this could partly be explained by increased visceral or ectopic fat. We performed magnetic resonance imaging on 78 adults born preterm at VLBW in Finland between 1978 and 1990 and 72 term same-sex siblings as controls, with a mean age of 29 years. We collected T1-weighted images from the abdomen, and magnetic resonance spectra from the liver, subcutaneous abdominal adipose tissue, and tibia. The adipose tissue volumes of VLBW adults did not differ from their term siblings when adjusting for age, sex, and maternal and perinatal factors. The mean differences were as follows: subcutaneous - 0.48% (95% CI - 14.8%, 16.3%), visceral 7.96% (95% CI - 10.4%, 30.1%), and total abdominal fat quantity 1.05% (95% CI - 13.7%, 18.4%). Hepatic triglyceride content was also similar. VLBW individuals displayed less unsaturation in subcutaneous adipose tissue (- 4.74%, 95% CI - 9.2%, - 0.1%) but not in tibial bone marrow (1.68%, 95% CI - 1.86%, 5.35%). VLBW adults displayed similar adipose tissue volumes and hepatic triglyceride content as their term siblings. Previously reported differences could thus partly be due to genetic or environmental characteristics shared between siblings. The VLBW group displayed less unsaturation in subcutaneous abdominal adipose tissue, suggesting differences in its metabolic activity and energy storage.Peer reviewe

H-1-MRS of femoral red and yellow bone marrow fat composition and water content in healthy young men and women at 3 T

Objectives There is a discrepancy between studies suggesting that higher bone marrow fat saturation is associated with impaired health, and studies suggesting that erythropoiesis increases red bone marrow (RBM) fat saturation in young healthy individuals. Here, we seeked to elucidate these discrepancies by using long TE magnetic resonance spectroscopy (MRS) to study both yellow bone marrow (YBM) and RBM in the femur of healthy volunteers. Materials and methods Thirty-three young healthy volunteers (17 females), age range 20-31 years, underwent long TE H-1 MRS at 3.0 T of RBM and YBM fat composition in the left femur. The water content of the bone marrow depots was measured using short TE MRS. Results The female participants displayed a lower unsaturation in the sampled RBM volume (RBMV) than the males (P <0.01) without displaying a concomitant difference in YBM (P = 0.42). They also showed a higher water content and broader spectral linewidths in RBM (P = 0.04). The water content in RBM strongly associated with broader spectral linewidths (R = 0.887, P MUCH LESS-THAN 0.01) and inversely with RBMV fat unsaturation (R = - 0.365, P = 0.04). Discussion These results partly support the notion that females display higher rate of erythropoiesis and lower fat unsaturation in RBM.Peer reviewe

Upregulation of Early and Downregulation of Terminal Pathway Complement Genes in Sbcutaneous Adipose Tissue and Adipocytes in Acquired Obesity

Inflammation is an important mediator of obesity-related complications such as the metabolic syndrome but its causes and mechanisms are unknown. As the complement system is a key mediator of inflammation, we studied whether it is activated in acquired obesity in subcutaneous adipose tissue (AT) and isolated adipocytes. We used a special study design of genetically matched controls of lean and heavy groups, rare monozygotic twin pairs discordant for body mass index (BMI) [n = 26, within-pair difference (triangle) in body mass index, BMI >3 kg/m(2)] with as much as 18 kg mean triangle weight. Additionally, 14 BMI-concordant (BMIPeer reviewe

Abdominal obesity and circulating metabolites : A twin study approach

Objective. To investigate how obesity, insulin resistance and low-grade inflammation link to circulating metabolites, and whether the connections are due to genetic or environmental factors. Subjects and methods. Circulating serum metabolites were determined by proton NMR spectroscopy. Data from 1368 (531 monozygotic (MZ) and 837 dizygotic (DZ)) twins were used for bivariate twin modeling to derive the genetic (r(g)) and environmental (re) correlations between waist circumference (WC) and serum metabolites. Detailed examination of the associations between fat distribution (DEXA) and metabolic health (HOMA-IR, CRP) was performed among 286 twins including 33 BMI-discordant MZ pairs (intrapair BMI difference >= 3 kg/m(2)). Results. Fat, especially in the abdominal area (i.e. WC, android fat % and android to gynoid fat ratio), together with HOMA-IR and CRP correlated significantly with an atherogenic lipoprotein profile, higher levels of branched-chain (BCAA) and aromatic amino acids, higher levels of glycoprotein, and a more saturated fatty acid profile. In contrast, a higher proportion of gynoid to total fat associated with a favorable metabolite profile. There was a significant genetic overlap between WC and several metabolites, most strongly with phenylalanine (r(g) = 0.40), glycoprotein (r(g) = 0.37), serum triglycerides (r(g) = 0.36), BCAAs (r(g) = 0.30-0.40), HDL particle diameter (r(g) = -0.33) and HDL cholesterol (r(g) = -0.30). The effect of acquired obesity within the discordant MZ pairs was particularly strong for atherogenic lipoproteins. Conclusions. A wide range of unfavorable alterations in the serum metabolome was associated with abdominal obesity, insulin resistance and low-grade inflammation. Twin modeling and obesity-discordant twin analysis suggest that these associations are partly explained by shared genes but also reflect mechanisms independent of genetic liability. (C) 2015 Elsevier Inc. All rights reserved.Peer reviewe

Characterization of different fat depots in NAFLD using inflammation-associated proteome, lipidome and metabolome

Non-alcoholic fatty liver disease (NAFLD) is recognized as a liver manifestation of metabolic syndrome, accompanied with excessive fat accumulation in the liver and other vital organs. Ectopic fat accumulation was previously associated with negative effects at the systemic and local level in the human body. Thus, we aimed to identify and assess the predictive capability of novel potential metabolic biomarkers for ectopic fat depots in non-diabetic men with NAFLD, using the inflammation-associated proteome, lipidome and metabolome. Myocardial and hepatic triglycerides were measured with magnetic spectroscopy while function of left ventricle, pericardial and epicardial fat, subcutaneous and visceral adipose tissue were measured with magnetic resonance imaging. Measured ectopic fat depots were profiled and predicted using a Random Forest algorithm, and by estimating the Area Under the Receiver Operating Characteristic curves. We have identified distinct metabolic signatures of fat depots in the liver (TAG50:1, glutamate, diSM18:0 and CE20:3), pericardium (N-palmitoyl-sphinganine, HGF, diSM18:0, glutamate, and TNFSF14), epicardium (sphingomyelin, CE20:3, PC38:3 and TNFSF14), and myocardium (CE20:3, LAPTGF-beta 1, glutamate and glucose). Our analyses highlighted non-invasive biomarkers that accurately predict ectopic fat depots, and reflect their distinct metabolic signatures in subjects with NAFLD.Peer reviewe

Proton magnetic resonance spectroscopy in skeletal muscle: Experts' consensus recommendations

H-1-MR spectroscopy of skeletal muscle provides insight into metabolism that is not available noninvasively by other methods. The recommendations given in this article are intended to guide those who have basic experience in general MRS to the special application of H-1-MRS in skeletal muscle. The highly organized structure of skeletal muscle leads to effects that change spectral features far beyond simple peak heights, depending on the type and orientation of the muscle. Specific recommendations are given for the acquisition of three particular metabolites (intramyocellular lipids, carnosine and acetylcarnitine) and for preconditioning of experiments and instructions to study volunteers.Peer reviewe