Quantifying apoprotein synthesis in rodents: coupling LC-MS/MS analyses with the administration of labeled water

Zhou, Haihong; Li, Wenyu; Wang, Shengping; Mendoza, Vivienne; Rosa, Raymond; Hubert, James; Herath, Kithsiri; McLaughlin, Theresa; Rohm, Rory J.; Lassman, Michael E.; Wong, Kenny K.; Johns, Douglas G.; Previs, Stephen F.; Hubbard, Brian K.; Roddy, Thomas P.

doi:10.1194/jlr.d021295

Cited by 23 publications

(44 citation statements)

References 37 publications

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“…As a proof of principle we applied this technique to estimate HDL turnover in genetically modified mice with a large range of HDLc levels. Measured ApoAI turnover rates were within the range of the values reported in the literature using different methods and consistent with the expectations based on genetic modifications in these animals 14, 35, 36 . For example, the human ApoAI FCR we measured in ApoAI transgenic mice of 11.1 ± 0.6 %/hr agrees well with the human ApoAI FCR values of 11 ± 1 %/hr obtained in similar mice by radioiodination and following turnover after i.v.…”

Section: Discussionsupporting

confidence: 88%

² H ₂ O-Based High-Density Lipoprotein Turnover Method for the Assessment of Dynamic High-Density Lipoprotein Function in Mice

Kasumov

Willard

et al. 2013

ATVB

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Objective High-density lipoprotein (HDL) promotes reverse cholesterol transport (RCT) from peripheral tissues to the liver for clearance. Reduced HDL-cholesterol (HDLc) is associated with atherosclerosis; however, as a predictor of cardiovascular disease, HDLc has limitations as it is not a direct marker of HDL functionality. Our objective was to develop a mass spectrometry based method for the simultaneous measurement of HDLc and ApoAI kinetics in mice using a single 2H2O tracer, and use it to examine genetic and drug perturbations on HDL turnover in vivo. Approach and Results Mice were given 2H2O in the drinking water and serial blood samples were collected at different time points. HDLc and ApoAI gradually incorporated 2H, allowing experimental measurement of fractional catabolic rates (FCR) and production rates (PR) for HDLc and ApoA1. ApoE−/− mice displayed increased FCR (p<0.01) and reduced PR of both HDLc and ApoAI (p<0.05) compared to controls. In human ApoAI transgenic mice, levels and PRs of HDLc and human ApoAI were strikingly higher than in wild type mice. Myriocin, an inhibitor of sphingolipid synthesis, significantly increased both HDL flux and macrophage-to-feces RCT, indicating compatibility of this HDL turnover method with the macrophage specific RCT assay. Conclusions 2H2O-labeling can be used to measure HDLc and ApoAI flux in vivo, and to assess the role of genetic and pharmacological interventions on HDL turnover in mice. Safety, simplicity, and low cost of the 2H2O-based HDL turnover approach suggest that this assay can be scaled for human use to study effects of HDL targeted therapies on dynamic HDL function.

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Section: Discussionsupporting

confidence: 88%

² H ₂ O-Based High-Density Lipoprotein Turnover Method for the Assessment of Dynamic High-Density Lipoprotein Function in Mice

Kasumov

Willard

et al. 2013

ATVB

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“…Movement of label from H 2 18 O into amino acids is rather slow (Zhou et al, 2012). Label moves rapidly from 2 H 2 O into amino acids and proteins (Mitra et al, 1976;Yang et al, 2010), but discrimination against 2 H 2 O can lead to changes in metabolite pools and fluxes, to changes in gene expression indicative of stress, and to inhibition of growth (Thomson et al, 1963;Sacchi and Cocucci, 1992;Kushner et al, 1999;Yang et al, 2010).…”

mentioning

confidence: 99%

Quantifying Protein Synthesis and Degradation in Arabidopsis by Dynamic ¹³CO₂ Labeling and Analysis of Enrichment in Individual Amino Acids in Their Free Pools and in Protein

et al. 2015

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ORCID IDs: 0000-0002-7658-0473 (H.I.); 0000-0001-8931-7722 (T.O.); 0000-0002-6113-9570 (R.S.).Protein synthesis and degradation represent substantial costs during plant growth. To obtain a quantitative measure of the rate of protein synthesis and degradation, we supplied 13 CO 2 to intact Arabidopsis (Arabidopsis thaliana) Columbia-0 plants and analyzed enrichment in free amino acids and in amino acid residues in protein during a 24-h pulse and 4-d chase. While many free amino acids labeled slowly and incompletely, alanine showed a rapid rise in enrichment in the pulse and a decrease in the chase. Enrichment in free alanine was used to correct enrichment in alanine residues in protein and calculate the rate of protein synthesis. The latter was compared with the relative growth rate to estimate the rate of protein degradation. The relative growth rate was estimated from sequential determination of fresh weight, sequential images of rosette area, and labeling of glucose in the cell wall. In an 8-h photoperiod, protein synthesis and cell wall synthesis were 3-fold faster in the day than at night, protein degradation was slow (3%-4% d 21), and flux to growth and degradation resulted in a protein half-life of 3.5 d. In the starchless phosphoglucomutase mutant at night, protein synthesis was further decreased and protein degradation increased, while cell wall synthesis was totally inhibited, quantitatively accounting for the inhibition of growth in this mutant. We also investigated the rates of protein synthesis and degradation during leaf development, during growth at high temperature, and compared synthesis rates of Rubisco large and small subunits of in the light and dark.

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“…The concentration of total circulating cholesterol was determined via enzymatic assay ( 19,20 ). The plasma level and fl ux of ApoB were quantifi ed by the LC-MS/MS method, as described previously ( 21 ).…”

Section: Calculations and Statistical Analysismentioning

confidence: 99%

Glucagon receptor antagonism induces increased cholesterol absorption

Guan

Yang

et al. 2015

Journal of Lipid Research

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Quantifying apoprotein synthesis in rodents: coupling LC-MS/MS analyses with the administration of labeled water

Cited by 23 publications

References 37 publications

² H ₂ O-Based High-Density Lipoprotein Turnover Method for the Assessment of Dynamic High-Density Lipoprotein Function in Mice

² H ₂ O-Based High-Density Lipoprotein Turnover Method for the Assessment of Dynamic High-Density Lipoprotein Function in Mice

Quantifying Protein Synthesis and Degradation in Arabidopsis by Dynamic ¹³CO₂ Labeling and Analysis of Enrichment in Individual Amino Acids in Their Free Pools and in Protein

Glucagon receptor antagonism induces increased cholesterol absorption

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Quantifying apoprotein synthesis in rodents: coupling LC-MS/MS analyses with the administration of labeled water

Cited by 23 publications

References 37 publications

2 H 2 O-Based High-Density Lipoprotein Turnover Method for the Assessment of Dynamic High-Density Lipoprotein Function in Mice

2 H 2 O-Based High-Density Lipoprotein Turnover Method for the Assessment of Dynamic High-Density Lipoprotein Function in Mice

Quantifying Protein Synthesis and Degradation in Arabidopsis by Dynamic 13CO2 Labeling and Analysis of Enrichment in Individual Amino Acids in Their Free Pools and in Protein

Glucagon receptor antagonism induces increased cholesterol absorption

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² H ₂ O-Based High-Density Lipoprotein Turnover Method for the Assessment of Dynamic High-Density Lipoprotein Function in Mice

² H ₂ O-Based High-Density Lipoprotein Turnover Method for the Assessment of Dynamic High-Density Lipoprotein Function in Mice

Quantifying Protein Synthesis and Degradation in Arabidopsis by Dynamic ¹³CO₂ Labeling and Analysis of Enrichment in Individual Amino Acids in Their Free Pools and in Protein