Gary J. Duncan

Identification of potential serum biomarkers of inflammation and lipid modulation that are altered by fish oil supplementation in healthy volunteers.

Long chain n‐3 polyunsaturated fatty acids (n‐3 LCPUFA) lower risk of coronary heart disease (CHD), but mechanisms are not well understood. We used proteomics to identify human serum proteins that are altered by n‐3 LCPUFA. Such proteins could identify pathways whereby they affect CHD. Eighty‐one healthy volunteers entered a double blind randomised trial to receive 3.5u2005g of fish oil or 3.5u2005g of high oleic sunflower oil daily. Serum was collected before and after 6u2005wk of intervention. Serum was analysed by proteomics using 2‐DE. Proteins that were differentially regulated were identified by MS. We also analysed serum apolipoprotein A1 (apo A1), high‐density lipoprotein (HDL) particle size and haptoglobin. Serum levels of apo A1, apo L1, zinc‐α‐2‐glycoprotein, haptoglobin precursor, α‐1‐antitrypsin precursor, antithrombin III‐like protein, serum amyloid P component and haemopexin were significantly downregulated (all p<0.05) by fish oil compared with high oleic sunflower oil supplementation. Fish oil supplementation caused a significant shift towards the larger, more cholesterol‐rich HDL2 particle. The alterations in serum proteins and HDL size imply that fish oil activates anti‐inflammatory and lipid modulating mechanisms believed to impede the early onset of CHD. These proteins are potential diagnostic biomarkers to assess the mechanisms whereby fish oil protects against CHD in humans.

Disruption of lipid metabolism in the liver of the pregnant rat fed folate-deficient and methyl donor-deficient diets.

The importance of folic acid and the methionine cycle in fetal development is well recognised even though the mechanism has not been established. Since the cycle is active in the maternal liver, poor folate status may modify hepatic metabolism. Pregnant rats were fed diets deficient in folic acid (-F) or in three key methyl donors, folic acid, choline and methionine (-FLMLC) and the maternal liver was analysed on day 21 of gestation. Two-dimensional gel electrophoresis of soluble proteins identified differentially abundant proteins, which could be allocated into nine functional groups. Five involved in metabolic processes, namely, folate/methionine cycle, tyrosine metabolism, protein metabolism, energy metabolism and lipid metabolism, and three in cellular processes, namely, endoplasmic reticulum function, bile production and antioxidant defence. The mRNA for sterol regulatory element-binding protein-1c and acetyl-CoA carboxylase-1 (fatty acid synthesis) were decreased by both -F and -FLMLC diets. The mRNA for PPARalpha and PPARgamma and carnitine palmitoyl transferase (fatty acid oxidation) were increased in the animals fed the -FLMLC diets. Changes in the abundance of proteins associated with intracellular lipid transport suggest that folate deficiency interferes with lipid export. Reduced fatty acid synthesis appeared to prevent steatosis in animals fed the -F diet. Even with increased oxidation, TAG concentrations were approximately three-fold higher in animals fed the -FLMLC diet and were associated with an increase in the relative abundance of proteins associated with oxidative stress. Fetal development may be indirectly affected by these changes in hepatic lipid metabolism.

Rat metallothionein-2 containsNα-acetylated and unacetylated isoforms

Mammalian metallothioneins (MT), are characteristically Nα‐acetylated and the presence of an unblocked N‐terminus has not previously been reported. On‐line capillary electrophoresis‐electrospray mass spectrometry of hepatic MT‐2 from rats injected with zinc revealed two isoforms differing by a mass equivalent to that of a single acetyl group. The lower mass component constituted > 20% of total MT‐2 protein and both MT‐2 isoforms were separated by reversed‐phase high‐performance liquid chromatography. The identity of each fraction was confirmed by matrix‐assisted laser desorption ionisation mass spectrometry, and amino acid analysis and N‐terminal sequencing revealed that the lower mass isoform was unblocked at the N‐terminus and had an amino acid composition and sequence which is characteristic of rat MT‐2. Thus the complementary techniques of mass spectrometry and N‐terminal sequencing demonstrated conclusively that purified MT‐2 from zinc‐treated rats contains an unacetylated isoform. We propose that the cotranslational acetylation of rat MT‐2 may under some circumstances be inefficient compared to that in other nonrodent species, where we have detected only trace levels of unacetylated MT isoforms.

Aorta protein networks in marginal and acute zinc deficiency

Human zinc deficiency is a global problem and may influence the development of cardiovascular disease. Our objective was to determine Zn deficiency affected pathways and protein interactions in rat aorta and their likely influence on stress‐induced atherogenesis. In two separate studies, rats were given diets acutely (<1u2005mg Zn/kg) or marginally (6u2005mg Zn/kg) deficient in Zn. Both studies included Zn adequate controls (35u2005mg Zn/kg) and the acute deficiency study included a pair‐fed group. After 6u2005wk, proteins from thoracic aorta were separated by 2‐DE. Proteins affected by zinc deficiency were identified by principal component analysis. Multiple correlations of identified proteins indicated protein networks of related function. Proteins clusters decreased in zinc deficiency were related to fatty acid and carbohydrate metabolism. Structurally related proteins, including zyxin and over nine transgelin 1 proteins, were either increased or decreased by acute and marginal deficiencies. PKCα was significantly decreased in Zn deficiency suggesting that Zn may regulate the phosphorylation of target proteins. Zn deficiency‐related changes in structural, carbohydrate and fatty acid‐related proteins may be disadvantageous for maintaining vascular health and are consistent with a protective role for zinc in the development of atherosclerosis.

Profiling of mitochondrial associated proteins from rat colon

Mitochondrial dysfunction, damage and mutations of mitochondrial proteins give rise to a range of ill understood patterns of disease. Although there is significant general knowledge of the proteins and the functional processes of the mitochondria, there is little knowledge of difference about how mitochondria respond and how they are regulated in different organs and tissues. Proteomic profiling of mitochondria and associated proteins involved in mitochondrial regulation and trafficking within cells and tissues has the potential to provide insights into mitochondrial dysfunction associated with many human diseases. The rat colon mitoproteome analysis presented here provides a useful tool to assist in identification and interpretation of mitochondrial dysfunction implicated in colon pathogenesis. 2DPAGE followed by LC/MS/MS was used to identify 430 proteins from mitochondrial enriched fractions prepared from rat colon, resulting in 195 different proteins or approximately 50% of the resolved proteins being identified as multiple protein expression forms. Proteins associated with the colon mitoproteome were involved in calcium binding, cell cycle, energy metabolism and electron transport chain, protein folding, protein synthesis and degradation, redox regulation, structural proteins, signalling and transporter and channel proteins. The mitochondrial associated proteins identified in this study of colon tissue complement and are compared with other recently published mitoproteome analyses from other organ tissues, and will assist in revealing potentially organ specific roles of the mitochondria and organ specific disease associated with mitochondrial dysfunction. J. Cell. Biochem. 103: 78–97, 2008.

DNA/europium ion interaction by matrix-assisted laser desorption time-of-flight mass spectrometry

The interaction of DNA and lanthanide ions using Matrix assisted laser desorption time of flight mass spectrometry. 2, 4, 6 hydroxy acetophenone was the matrix used, and Europium chloride was the lanthanide salt used to interact with Adenine and Guanine containing synthetic oligonucleotides. The MALDI analysis permitted the detection of DNA/Europium adducts and also provided information on the stoichiometry of the interaction.