Plasma and hepatic free fatty acid, tricarboxylic acid cycle, and ketone bodies metabolic profiles in progressive Gao-Binge model
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摘要:
肝细胞内脂质蓄积是酒精性脂肪肝 (AFLD) 的重要病理学特征，其形成与游离脂肪酸 (FFAs)、三羧酸 (TCA) 循环、酮体代谢密切相关。为揭示 AFLD 中肝脏脂质蓄积与 FFAs、TCA 循环、酮体代谢的相关性。采用 C57BL/6N 小鼠构建慢性酒精喂养加急性酒精灌胃（Gao-Binge 模型）来模拟不同阶段的 AFLD，运用液相色谱-质谱联用技术 (LC-MS/MS) 检测小鼠肝组织和血浆中的 FFAs、TCA 循环中间产物和酮体水平，并进行 Pearson 相关性分析。研究结果显示，长短期 AFLD 小鼠模型中，血浆和肝脏中的总 FFAs、饱和FFAs、短链 FFAs 以及酮体 β-羟丁酸 (HDBT) 水平均显著增加，提示 FFAs 代谢谱在 Gao-Binge 模型下出现紊乱。此外，长短期模型中均发现乙酸 (AA)、2-甲基丁酸 (2-meBA)、HDBT 与肝脏损伤指标在血浆和肝脏样本中呈现出显著正相关性（以短期模型中血浆数据为例，r = 0.834、0.699、0.818, P<0.05），而丙酮酸 (PRA) 表现出显著负相关性 (r = −0.66, P<0.05)。结果表明，长短期 AFLD 小鼠模型中出现 FFAs、TCA 循环、酮体代谢紊乱，AA、2-meBA、HDBT、PRA 等代谢物可作为 AFLD 的潜在生物标志物，有助于该疾病的诊断和治疗。
Abstract:To investigate the correlation between hepatic lipid accumulation and the metabolic profiles of free fatty acids(FFAs), tricarboxylic acid (TCA) cycle, and ketone body in alcoholic fatty liver disease (AFLD), a chronic plus acute alcohol feeding model (Gao-Binge model) was employed using C57BL/6N mice to simulate different stages of AFLD. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to measure the levels of FFAs, TCA cycle intermediates, and ketone bodies in mouse liver tissue and plasma, followed by Pearson correlation analysis. The study revealed that both acute and chronic models showed significant increases in total FFAs, saturated FFAs and short-chain FFAs, as well as β-hydroxybutyric acid(HDBT) in plasma and liver, indicating FFA metabolic profile disturbances in the Gao-Binge model. Moreover, in both models, acetic acid (AA), 2-Methylbutyric acid (2-meBA), and HDBT displayed strong positive correlations with hepatic injury markers in plasma and liver samples (for instance, in the acute model plasma data, r = 0.834, 0.699, 0.818, P<0.05), while pyruvic acid (PRA) showed a strong negative correlation (r = −0.66, P<0.05). These findings suggest that FFAs, TCA cycle, and ketone body metabolism are disrupted in the alcoholic liver disease in mice model, and metabolites such as AA, 2-meBA, HDBT and PRA may serve as potential biomarkers for AFLD, which would be helpful in the diagnosis and treatment of this disease.
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Figure 1. Establishment of a short-term Gao-Binge alcohol-induced liver disease mouse model($\bar{x}\pm s $, control group: n = 6; Ethanol group: n = 8)
A: Average daily food intake; B: Daily weight change; C: Liver injury-related markers; D: Pathologic staining results*P<0.05, **P<0.01, ***P<0.001 vs control group
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Figure 2. Establishment of a long-term Gao-Binge alcohol-induced liver disease mouse model($ \bar{x} \pm s $, control group: n = 7; Ethanol group: n = 9)
A: Average daily food intake; B: Daily weight change; C: Liver injury-related markers; D: Pathologic staining results *P<0.05, **P<0.01, ***P<0.001 vs control group
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Figure 3. Changes in endogenous substances in short-term models
A: Levels of different classes of fatty acids in plasma; B: Levels of energy-related carboxylic acids in plasma; C: Specific levels of fatty acids in plasma; D: Levels of different classes of fatty acids in liver; E: Levels of energy-related carboxylic acids in liver; F: Specific levels of fatty acids in liver ($ \bar{{x}}\pm s $, control group: n = 6; Ethanol group: n = 8, *P<0.05, **P<0.01, ***P<0.001 vs control group) Total FFAs: Total free fatty acids; SCFA:Short-chain fatty acids; MCFA: Medium-chain fatty acids; LCFA: Long-chain fatty acids; VLCFA: Very-long-chain fatty acids; SFA: Saturated fatty acids; UFA: Unsaturated fatty acids; Total CA:Total carboxylic acids of energy-related; HDBT: β-Hydroxybutyric acid; CTA: Citric acid; MLA: Malic acid; PRA:Pyruvic acid; MVA: Mevalonic acid; LTA: Lactic acid; SCA: Succinic acid; FMA: Fumaric acid; AA: Acetic acid; PPA: Propionic acid; BA: Butyric acid; VA: Valeric acid; HXA: Hexanoic acid; iso-BA: Isobutyric acid; 2-meBA: 2-methylbutyric acid; iso-VA: Isovaleric acid; 3-mePTA: 3-methylpentanoic acid; OTA: Octanoic acid; DA: Decanoic acid; DDA: Dodecanoic acid; MRA: Myristic acid; PA: Palmitic acid; SA: Stearic acid; PMA: Palmitoleic acid; OA: Oleic acid; LA: Linolenic acid; LLA: Linoleic acid; ESA: Eicosenoic acid; ARA: Arachidonic acid; EPA: Eicosapentaenoic acid; LNA: Lignoceric acid; BHA: Behenic acid; EA: Erucic acid; DHA: Docosahexaenoic acid; NVA: Nervonic acid
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Figure 4. Changes in endogenous substances in long-term models
A: Levels of different classes of fatty acids in plasma; B: Levels of energy-related carboxylic acids in plasma; C: Specific levels of fatty acids in plasma; D: Levels of different classes of fatty acids in liver; E: Levels of energy-related carboxylic acids in liver; F: Specific levels of fatty acids in liver ($ \bar{x}\pm s $, control group: n = 7; Ethanol group: n = 9, *P<0.05, **P<0.01, ***P<0.001 vs control group)
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Figure 5. Pearson correlation analysis of liver injury biomarkers with FFAs, TCA cycle, and ketone body metabolic profiles
A: Plasma samples in short-term model; B: Liver samples in short-term model; C: Plasma samples in long-term model; D: Liver samples in long-term model *P<0.05, **P<0.01, ***P<0.001
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