Spectroscopy of tissue triglyceride composition : In vitro and in vivo studies

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.

Spectroscopy of tissue triglyceride composition : In vitro and in vivo studies

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.

Novel equation to determine the hepatic triglyceride concentration in humans by MRI: diagnosis and monitoring of NAFLD in obese patients before and after bariatric surgery

Background: Non-alcoholic fatty liver disease (NAFLD) is caused by abnormal accumulation of lipids within liver cells. Its prevalence is increasing in developed countries in association with obesity, and it represents a risk factor for non-alcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma. Since NAFLD is usually asymptomatic at diagnosis, new non-invasive approaches are needed to determine the hepatic lipid content in terms of diagnosis, treatment and control of disease progression. Here, we investigated the potential of magnetic resonance imaging (MRI) to quantitate and monitor the hepatic triglyceride concentration in humans. Methods: A prospective study of diagnostic accuracy was conducted among 129 consecutive adult patients (97 obesity and 32 non-obese) to compare multi-echo MRI fat fraction, grade of steatosis estimated by histopathology, and biochemical measurement of hepatic triglyceride concentration (that is, Folch value). Results: MRI fat fraction positively correlates with the grade of steatosis estimated on a 0 to 3 scale by histopathology. However, this correlation value was stronger when MRI fat fraction was linked to the Folch value, resulting in a novel equation to predict the hepatic triglyceride concentration (mg of triglycerides/g of liver tissue = 5.082 + (432.104 * multi-echo MRI fat fraction)). Validation of this formula in 31 additional patients (24 obese and 7 controls) resulted in robust correlation between the measured and estimated Folch values. Multivariate analysis showed that none of the variables investigated improves the Folch prediction capacity of the equation. Obese patients show increased steatosis compared to controls using MRI fat fraction and Folch value. Bariatric surgery improved MRI fat fraction values and the Folch value estimated in obese patients one year after surgery. Conclusions: Multi-echo MRI is an accurate approach to determine the hepatic lipid concentration by using our novel equation, representing an economic non-invasive method to diagnose and monitor steatosis in humans.

The triglyceride fatty acids from heart lipids of Puntius sarana and their variation with different sizes

The triglyceride fatty acid components from the heart lipid of Puntius sarana of different sizes have been characterized by thin-layer and gas liquid chromatography. Csub(10) to Csub(24) acids including both odd-numbered and branched chain acids were detected. The major constituents were ante-iso Csub(10), Csub(10), Csub(12:2), Csub(14), Csub(16), Csub(16:1),Csub(17), Csub(18) , Csub(18:1), Csub(18:2), Csub(18:3) and Csub(20:4) while twenty other acids were detected in lower proportion. The composition of these acids and their variation with size of fish have been investigated and discussed.

The application of calcined natural dolomitic rock as a solid base catalyst in triglyceride transesterification for biodiesel synthesis

Natural dolomitic rock has been investigated in the transesterification of C-4 and C-8 triglycerides and olive oil with a view to determining its viability as a solid base catalyst for use in biodiesel synthesis. XRD reveals that the dolomitic rock comprised 77% dolomite and 23% magnesian calcite. The generation of basic sites requires calcination at 900 degrees C, which increases the surface area and transforms the mineral into MgO nanocrystallites dispersed over CaO particles. Calcined dolomitic rock exhibits high activity towards the liquid phase transesterification of glyceryl tributyrate and trioctanoate, and even olive oil, with methanol for biodiesel production.

Chronic treatment with a stable obestatin analogue significantly alters plasma triglyceride levels but fails to influence food intake, fluid intake, body weight, or body composition in rats.

Obestatin (OB(1-23) is a 23 amino acid peptide encoded on the preproghrelin gene, originally reported to have metabolic actions related to food intake, gastric emptying and body weight. The biological instability of OB(1-23) has recently been highlighted by studies demonstrating its rapid enzymatic cleavage in a number of biological matrices. We assessed the stability of both OB(1-23) and an N-terminally PEGylated analogue (PEG-OB(1-23)) before conducting chronic in vivo studies. Peptides were incubated in rat liver homogenate and degradation monitored by LC-MS. PEG-OB(1-23) was approximately 3-times more stable than OB(1-23). Following a 14 day infusion of Sprague Dawley rats with 50 mol/kg/day of OB(1-23) or a N-terminally PEGylated analogue (PEG-OB(1-23)), we found no changes in food/fluid intake, body weight and plasma glucose or cholesterol between groups. Furthermore, morphometric liver, muscle and white adipose tissue (WAT) weights and tissue triglyceride concentrations remained unaltered between groups. However, with stabilised PEG-OB(1-23) we observed a 40% reduction in plasma triglycerides. These findings indicate that PEG-OB(1-23) is an OB(1-23) analogue with significantly enhanced stability and suggest that obestatin could play a role in modulating physiological lipid metabolism, although it does not appear to be involved in regulation of food/fluid intake, body weight or fat deposition.

Comparison of non-HDL cholesterol and waist circumference vs. triglyceride levels and waist circumference in predicting coronary heart disease risk

BACKGROUND: Dyslipidemia is recognized as a major cause of coronary heart disease (CHD). Emerged evidence suggests that the combination of triglycerides (TG) and waist circumference can be used to predict the risk of CHD. However, considering the known limitations of TG, non-high-density lipoprotein (non-HDL = Total cholesterol - HDL cholesterol) cholesterol and waist circumference model may be a better predictor of CHD. PURPOSE: The Framingham Offspring Study data were used to determine if combined non-HDL cholesterol and waist circumference is equivalent to or better than TG and waist circumference (hypertriglyceridemic waist phenotype) in predicting risk of CHD. METHODS: A total of3,196 individuals from Framingham Offspring Study, aged ~ 40 years old, who fasted overnight for ~ 9 hours, and had no missing information on nonHDL cholesterol, TG levels, and waist circumference measurements, were included in the analysis. Receiver Operator Characteristic Curve (ROC) Area Under the Curve (AUC) was used to compare the predictive ability of non-HDL cholesterol and waist circumference and TG and waist circumference. Cox proportional-hazards models were used to examine the association between the joint distributions of non-HDL cholesterol, waist circumference, and non-fatal CHD; TG, waist circumference, and non-fatal CHD; and the joint distribution of non-HDL cholesterol and TG by waist circumference strata, after adjusting for age, gender, smoking, alcohol consumption, diabetes, and hypertension status. RESULTS: The ROC AUC associated with non-HDL cholesterol and waist circumference and TG and waist circumference are 0.6428 (CI: 0.6183, 0.6673) and 0.6299 (CI: 0.6049, 0.6548) respectively. The difference in the ROC AVC is 1.29%. The p-value testing if the difference in the ROC AVCs between the two models is zero is 0.10. There was a strong positive association between non-HDL cholesterol and the risk for non-fatal CHD within each TO levels than that for TO levels within each level of nonHDL cholesterol, especially in individuals with high waist circumference status. CONCLUSION: The results suggest that the model including non-HDL cholesterol and waist circumference may be superior at predicting CHD compared to the model including TO and waist circumference.

Differential uptake of subfractions of triglyceride-rich lipoproteins by THP-1 macrophages

It is well known that raised plasma triglycerides (TG) are positively linked to the development of coronary heart disease. However, triglycerides circulate in a range of distinct lipoprotein subtractions and the relative atherogenicity of these subtractions is not clear. In this study, three fractions of triglyceride rich lipoprotein (TRL) were isolated from normolipidaemic males according to their differing Svedberg flotation (S-f) rates: chylomicron (CM, S-f > 400), very low-density lipoprotein (VLDL)-1 (S-f 60-400) and VLDL-2 (S-f 20-60). These fractions were incubated with THP-1 monocyte-derived macrophages for determination of cholesterol and TG accumulation, in the presence and absence of the lipoprotein lipase (LPL) inhibitor orlistat. Expression of LDL receptor related protein (LRP) and apolipoprotein B48 receptor (apoB48R) was also examined in both differentiating monocytes, and monocyte-derived macrophages, incubated with TRL. VLDL-I caused a significantly greater accumulation of TG within macrophages compared to VLDL-2. Binding studies also tended to show a greater preference for VLDL-1. No change in expression of LRP or apoB48R was observed in fully differentiated macrophages incubated with VLDL-1, VLDL-2 or CM, although a greater expression of LRP mRNA was observed in differentiating monocytes exposed to VLDL-1, compared to those incubated with CM or VLDL-2. TG loading in response to all three TRL fractions was blocked by orlistat, suggesting that it is likely that the major pathway for uptake of TG was hydrolysis by LPL. Calculations suggested that direct uptake of particles accounts for between 12 and 25% of total TAG uptake. In conclusion, THP monocyte-derived macrophages demonstrate a preference for VLDL-1, both through the LPL pathway and by direct uptake of whole particles. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Triglyceride-rich lipoproteins inhibit cholesterol efflux to apolipoprotein (apo) A1 from human macrophage foam cells

High circulating levels of triglyceride-rich lipoproteins (TGRL) represent an independent risk factor for coronary artery disease. Here, we show that TGRL inhibit the efflux of cholesterol from 'foam cell' macrophages to lipid-poor apolipoprotein (apo) A1, and may thereby inhibit arterial reverse cholesterol transport and promote the formation of atherosclerotic lesions. Human (THP-1) monocyte-derived macrophages were pre-incubated (48h) with acetylated low-density lipoprotein (AcLDL) to provide a foam cell model of cholesterol efflux to apoA1. Pre-incubation of macrophage 'foam cells' with TGRL (0-200 mug/ml, 0-24 h) inhibited the efflux of exogenously radiolabelled ([H-3]), endogenously synthesised ([C-14]) and cellular cholesterol mass to lipid-poor apoA1, but not control medium, during a (subsequent) efflux period. This inhibition is dependent upon the length of prior exposure to, and concentration of, TGRL employed, but is independent of changes in intracellular triglyceride accumulation or turnover of the cholesteryl ester pool. Despite the negative impact of TGRL on cholesterol efflux, major proteins involved in this process-namely apoE, ABCA1, SR-B1 and caveolin-1-were unaffected by TGRL pre-incubation, suggesting that exposure to these lipoproteins inhibits an alternate, and possibly novel, anti-atherogenic pathway. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

Influence of apoA-V gene variants on postprandial triglyceride metabolism: impact of gender

Although apolipoprotein AN (apoA-V) polymorphisms have been consistently associated with fasting triglyceride (TG) levels, their impact on postprandial lipemia remains relatively unknown. In this study, we investigate the impact of two common apoA-V polymorphisms (-1131 T>C and S19W) and apoA-V haplotypes on fasting and postprandial lipid metabolism in adults in the United Kingdom (n = 259). Compared with the wild-type TT, apoA-V -1131 TC heterozygotes had 15% (P = 0.057) and 21% (P = 0.002) higher fasting TG and postprandial TG area under the curve (AUC), respectively. Significant (P = 0.038) and nearly significant (P = 0.057) gender X genotype interactions were observed for fasting TG and TG AUC, with a greater impact of genotype in males. Lower HDL-cholesterol was associated with the rare TC genotype (P = 0.047). Significant linkage disequilibrium was found between the apoA-V -1131 T>C and the apoC-III 3238 C>G variants, with univariate analysis indicating an impact of this apoC-III single nucleotide polymorphism (SNP) on TG AUC (P = 0.015). However, in linear regression analysis, a significant independent association with TG AUC (P = 0.007) was only evident for the apoA-V -1131 T>C SNP, indicating a greater relative importance of the apoA-V genotype.