| 1. |
- Fernandez, Celine, et al.
(författare)
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Metabolomic and proteomic analysis of a clonal insulin-producing beta-cell line (INS-1 832/13).
- 2008
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 7:1, s. 400-411
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Tidskriftsartikel (refereegranskat)abstract
- Metabolites generated from fuel metabolism in pancreatic beta-cells control exocytosis of insulin, a process which fails in type 2 diabetes. To identify and quantify these metabolites, global and unbiased analysis of cellular metabolism is required. To this end, polar metabolites, extracted from the clonal 832/13 beta-cell line cultured at 2.8 and 16.7 mM glucose for 48 h, were derivatized followed by identification and quantification, using gas chromatography (GC) and mass spectrometry (MS). After culture at 16.7 mM glucose for 48 h, 832/13 beta-cells exhibited a phenotype reminiscent of glucotoxicity with decreased content and secretion of insulin. The metabolomic analysis revealed alterations in the levels of 7 metabolites derived from glycolysis, the TCA cycle and pentose phosphate shunt, and 4 amino acids. Principal component analysis of the metabolite data showed two clusters, corresponding to the cells cultured at 2.8 and 16.7 mM glucose, respectively. Concurrent changes in protein expression were analyzed by 2-D gel electrophoresis followed by LC-MS/MS. The identities of 86 spots corresponding to 75 unique proteins that were significantly different in 832/13 beta-cells cultured at 16.7 mM glucose were established. Only 5 of these were found to be metabolic enzymes that could be involved in the metabolomic alterations observed. Anticipated changes in metabolite levels in cells exposed to increased glucose were observed, while changes in enzyme levels were much less profound. This suggests that substrate availability, allosteric regulation, and/or post-translational modifications are more important determinants of metabolite levels than enzyme expression at the protein level.
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| 2. |
- Hallgard, Elna, et al.
(författare)
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Addition of Various Carbohydrates to Beef Burgers Affects the Formation of Heterocyclic Amines during Frying
- 2004
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Ingår i: Journal of Agricultural and Food Chemistry. - : American Chemical Society (ACS). - 0021-8561 .- 1520-5118. ; 52:25, s. 7561-7566
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Tidskriftsartikel (refereegranskat)abstract
- The influence of the addition of carbohydrates with different physicochemical properties on weight loss and formation of heterocyclic amines (HAs) during the frying of beef burgers was examined. Furthermore, the capability of carbohydrates to bind HAs was tested. Beef burgers containing 1.5% NaCl and 0.3% tripolyphosphate (reference), with the addition of 1.5% carbohydrate, were fried for 5 min at 200 C in a double-sided pan fryer. The beef burgers were analyzed for HAs with solid phase extraction and liquid chromatography/mass spectrometry. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP), and 9H-pyrido[3,4-b]indole (Norharman) were detected in all of the beef burgers. The addition of carbohydrates affected both the weight loss and the formation of HAs during cooking. The formation of HAs could be correlated to depend on both the weight loss and the type of the added carbohydrate. Of the 11 different carbohydrates tested, raw potato starch was most capable of inhibiting the formation of HAs, while potato fiber gave the lowest weight loss and a comparably low amount of PhIP. Wheat bran and potato fiber were found to reversibly bind HAs. It is concluded that adding small amounts of certain carbohydrates may be a simple and effective way of reducing the amount of HAs and can easily be applied in households and commercial preparations of beef burgers. Keywords: Heterocyclic amines; beef burger; frying; carbohydrates; water holding; PhIP; MeIQx; 4,8-DiMeIQx; Harman; Norharman
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| 3. |
- Hallgard, Elna, et al.
(författare)
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Effect of high water-holding capacity on the formation of heterocyclic amines in fried beefburgers.
- 2003
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Ingår i: Journal of Agricultural and Food Chemistry. - : American Chemical Society (ACS). - 0021-8561 .- 1520-5118. ; 51:15, s. 4472-4477
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Tidskriftsartikel (refereegranskat)abstract
- Mutagenic/carcinogenic heterocyclic amines (HAs) are formed in the crust during the cooking of meat. The influence of cooking loss, time, and temperature on the formation of HAs was investigated in fried beefburgers. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) were identified in all samples. The amounts of PhIP, MeIQx, and 4,8-DiMeIQx increased with increasing cooking time, and this effect was significant for all three HAs. The pan temperature had a significant effect on the formation of PhIP and 4,8-DiMeIQx. The addition of NaCl/sodium tripolyphosphate to the beefburgers reduced the cooking loss and decreased the formation of PhIP, MeIQx, and 4,8-DiMeIQx. This decrease was significant for MeIQx and 4,8-DiMeIQx. The results clearly show that it is possible to modify cooking practices to minimize the formation of HAs.
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| 4. |
- Hallgard, Elna, et al.
(författare)
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Heterocyclic amine formation during frying of frozen beefburgers
- 2008
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Ingår i: International Journal of Food Science & Technology. - : Wiley. - 0950-5423 .- 1365-2621. ; 43:1, s. 62-68
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Tidskriftsartikel (refereegranskat)abstract
- The formation of heterocyclic amines (HCAs) was studied during frying of beefburgers with different fat contents (6.7%, 16.1% and 39%). Beefburgers were fried from the frozen state for 60, 90, 120 s, and until the centre temperature had reached 72 °C (approximately 150 s) in a double-sided pan fryer. The beefburgers were analysed for HCAs with solid-phase extraction and LC/MS detection, and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 1-methyl-9H-pyrido[3,4-b]indole (Harman) and 9H-pyrido[3,4-b]indole (Norharman) were detected in all samples. The concentrations of HCAs ranged between 0 and 2.3 ng g−1. The HCAs concentrations were fitted to a first-order reaction model. The amounts of HCAs in beefburgers fried from the frozen state were in the same range as in beefburgers that have reached room temperature before frying. Furthermore, it was found that the formation of HCAs is not only concentration-controlled but also mass transport-controlled and that kinetic models stated in earlier studies fit relatively well our analysed values on HCAs in fried beefburgers.
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| 5. |
- Hallgard, Elna
(författare)
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Modifying Cooking Practices to Reduce the Formation of Heterocyclic Amines
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Commonly cooked meat dishes contain heterocyclic amines (HAs) at ng/g levels. HAs are animal carcinogens, form DNA adducts in human tissues, and have been associated with increased risk of cancer in epidemiological studies, and it is thus recommended that human intake be decreased. The aim of this investigation was to find ways of modifying cooking practices to reduce the formation of HAs. Various factors that influence the formation of HAs during cooking of chicken breasts and beefburgers were investigated. The factors studied included cooking method, time, temperature, heat transfer, weight loss during cooking, the chemical composition of beefburgers and antioxidants. The conditions for the cooking experiments were similar to those employed in normal domestic, restaurant and industrial cooking. The analysis of HAs was focused on MeIQx, 4,8-DiMeIQx, PhIP, Harman and Norharman, as these compounds are considered to be the most abundant HAs in cooked meat. The concentrations ranged from undetectable amounts to 30 ng/g. Cooking temperature was shown to be an important factor, especially during frying, since large concentrations of HAs can be formed at high temperatures. Avoiding over-cooking of meat seems to be another important way to decrease the content of HAs. Reducing the weight loss during cooking may be a means of decreasing the formation of HAs, and this correlation is probably due to the transport of water-soluble precursors to the meat surface. This transport can be lowered by the addition of water-holding ingredients, such as common salt or potato starch. Addition of carbohydrates may also affect the formation of HAs chemically. Frying in oil containing antioxidants may also provide a way of reducing the amount of HAs formed, although the amounts of antioxidants and the effect of storage must be taken into consideration. A kinetic model was used to describe the formation of HAs and this can be further developed to predict the formation of HAs. The results from this research can be used to design cooking equipment and processes and to form a basis for guidelines to consumers, restaurants and industry, on how to obtain a product with good sensoric properties and minimised contents of HAs. Such recommendations may decrease the human intake of HAs. The data on HAs in ordinary cooked meat can be compiled into a database for intake estimates, epidemiological studies and for future risk assessment.
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| 6. |
- Krus, Ulrika, et al.
(författare)
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Pyruvate dehydrogenase kinase 1 controls mitochondrial metabolism and insulin secretion in INS-1 832/13 clonal beta-cells
- 2010
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 429, s. 205-213
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Tidskriftsartikel (refereegranskat)abstract
- Tight coupling between cytosolic and mitochondrial metabolism is key for GSIS (glucose-stimulated insulin secretion). In the present study we examined the regulatory contribution of PDH (pyruvate dehydrogenase) kinase 1, a negative regulator of PDH, to metabolic coupling in 832/13 clonal beta-cells. Knockdown of PDH kinase 1 with siRNA (small interfering RNA) reduced its mRNA (>80 %) and protein level (>40 %) after 72 h. PDH activity, glucose-stimulated cellular oxygen consumption and pyruvate-stimulated mitochondrial oxygen consumption increased 1.7- (P < 0.05), 1.6- (P < 0.05) and 1.6-fold (P < 0.05) respectively. Gas chromatography/MS revealed an altered metabolite profile upon silencing of PDH kinase 1, determined by increased levels of the tricarboxylic acid cycle intermediates malate, fumarate and alpha-ketoglutarate. These metabolic alterations were associated with exaggerated GSIS (5-fold compared with 3.1-fold in control cells; P < 0.01). Insulin secretion, provoked by leucine and dimethylsuccinate, which feed into the tricarboxylic acid cycle bypassing PDH, was unaffected. The oxygen consumption and metabolic data strongly suggest that knockdown of PDH kinase 1 in beta-cells permits increased metabolic flux of glucose-derived carbons into the tricarboxylic acid cycle via PDH. Enhanced insulin secretion is probably caused by increased generation of tricarboxylic acid cycle-derived reducing equivalents for mitochondrial electron transport to generate ATP and/or stimulatory metabolic intermediates. On the basis of these findings, we suggest that PDH kinase 1 is an important regulator of PDH in clonal beta-cells and that PDH kinase 1 and PDH are important for efficient metabolic coupling. Maintaining low PDH kinase I expression/activity, keeping PDH in a dephosphorylated and active state, may be important for beta-cells to achieve the metabolic flux rates necessary for maximal GSIS.
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| 7. |
- Santos, F J, et al.
(författare)
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Analysis of heterocyclic amines in food products: interlaboratory studies
- 2004
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Ingår i: Journal of Chromatography. B. - : Elsevier BV. - 1873-376X .- 1570-0232. ; 802:1, s. 69-78
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Tidskriftsartikel (refereegranskat)abstract
- A feasibility study and two interlaboratory exercises on the determination of selected heterocyclic amines (HAs) in beef extract, organised in the framework of a European project, are presented. The aim of these exercises was to improve the quality of the laboratories and to evaluate the performance of a standardised analytical method and also the methods currently used by each of the participants for the analysis of these compounds. Three lyophilised portions of a commercial beef material previously spiked with HAs at different concentration levels ranging from 10 to 75 ng g(-1) were used as laboratory reference materials (lot A, B and C). Firstly, a feasibility study was carried out using a test standard solution and the beef extract (lot A), which contained only five HAs. Then, two interlaboratory exercises were carried out using the laboratory reference materials lot B and lot C, containing 10 selected HAs at two different concentration levels, 75 and 10ng/g, respectively. The results obtained by all participant laboratories using the proposed method showed satisfactory agreement and the CV(%) between-laboratories obtained were from 8.3 to 24.1% for lot B and from 8.7 to 44.5% for lot C. The standardised method evaluated in these collaborative studies is therefore proposed for the analysis of HAs in food material. Moreover, LC-MS is recommended as the most suitable technique for the analysis of a large number of HAs in food samples. (C) 2003 Elsevier B.V All rights reserved.
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