精胺丁二酸合成酶(argininosuccinate synthetase, ASS)廣泛存在於人體內，尤其大量表現於肝臟與腎臟中。ASS在尿素循環、多胺(polyamine)及肌酸酐生合成中扮演重要的角色，也參與製造一氧化氮的精胺酸-瓜胺酸循環中。成人發作第二型瓜胺酸血症(adult-onset type II citrullinemia, CTLN2)的致病原因為位於粒線體內膜上維持胞內氧化還原平衡的天門冬胺酸-谷胺酸載體(檸檬酸)的缺乏，而且其肝臟中的ASS活性降低。然而，在另一檸檬酸缺乏的疾病：新生兒肝內膽汁淤積症(neonatal intrahepatic cholestasis caused by citrin deficiency, NICCD)，病人肝臟ASS蛋白質含量差異大。NICCD患者常伴隨著代謝性異常的症狀，如半乳糖血症，而且一部分NICCD患者會在長大後發展為CTLN2，但目前原因尚不清楚。臨床上NICCD病人在使用無乳糖配方奶粉後，可以明顯改善他們的臨床症狀。先前已有研究指出，細胞培養在含有半乳糖的培養基中會使細胞內還原壓力增加。因此，本研究目的為探討肝細胞培養於非葡萄糖培養基的代謝壓力及ASS變化。首先建立使用液相層析串聯質譜儀(liquid chromatography-tandem mass spectrometry, LC-MS/MS) 測定ASS活性、以及精胺酸與其代謝物的方法；探討半乳糖、果糖與低濃度葡萄糖(9 mM葡萄糖)對於有無檸檬酸抑制之肝細胞中細胞狀態、ASS活性、細胞內還原壓力以及精胺酸代謝的變化。此外，我們也嘗試周邊淋巴球ASS活性的測定，並與肝臟ASS活性做對照。我們利用小鼠肝細胞AML12，經Slc25a13-shRNA轉染以抑制檸檬酸表現，並以含有半乳糖、果糖或低濃度之葡萄糖的培養基進行培養。培養在半乳糖、果糖或低濃度葡萄糖培養之肝細胞，其ASS活性會降低(154.7, 140.0 and 159.8 v.s 203.1 mU/g)、培養基上清液乳酸對丙酮酸比值增加(64.5, 63.7 and 101.1 v.s 53.3)，而且細胞質中NADH濃度升高(44.6, 40.8 and 52.0 v.s 22.5 nmol/g)。經西方墨點法測量發現檸檬酸的抑制並不影響ASS蛋白質量。然而，檸檬酸抑制的肝細胞培養在半乳糖、果糖或9 mM葡萄糖中，其粒線體膜電位會降低、ASS活性降低、乳糖對丙酮酸比值增加，且NADH濃度升高。這些細胞再給予過氧化氫的刺激會造成細胞存活率下降，而且，培養在半乳糖、果糖或低濃度葡萄糖中會使肝細胞對過氧化氫的傷害有加劇的現象。而我們也發現，乳酸對丙酮酸比值與ASS活性呈現負相關(r=-0.647, p<0.001)。在代謝變化的部分，給予細胞不同含糖的培養基中，我們發現細胞內精胺酸及賴胺酸不會受影響；但是，asymmetry dimethylarginine (ADMA)、symmetry dimethylarginine (SDMA)及瓜胺酸則會上升。給予丙酮酸或甘胺酸皆可以有效回復ASS活性；而N-乙醯基半胱胺酸、丙酮酸或甘胺酸也都可以回復NADH濃度(p<0.05)以及經過過氧化氫刺激對肝細胞加劇的傷害和存活率。本研究中建立之ASS活性測定方法可以成功應用於活化之周邊淋巴球，參考活性範圍為88 ± 24 mU/g。總結，本研究已建立使用LC-MS/MS測定ASS活性以及精胺酸相關代謝之方法，其結果顯示肝細胞在非葡萄糖培養基內，其還原壓力會上升，ASS活性會下降；而給予丙酮酸、甘胺酸或N-乙醯基半胱胺酸可以有效回復。

Argininosuccinate synthetase (ASS) is a ubiquitous enzyme with most abundant levels in liver and kidney. ASS plays a key role in pathways of urea cycle, polyamine synthesis, creatinine synthesis and arginine-citrulline cycle for NO production. Decreased liver-specific ASS activity was found in adult-onset type II citrullinemia (CTLN2) patients, which is due to the deficiency of citrin, an aspartate-glutamate carrier on the inner membrane of mitochondria to maintain cellular redox states. However, ASS activity was at variable levels in neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). Metabolic disorders, such as galactosemia, are shown in NICCD patients. Some NICCD patients progress to CTLN2 decades later with unknown mechanism. Lactose-restricted formula improved clinical symptoms in NICCD patients. Moreover, increased cytosolic reducing power was found in cells grown in galactose-containing culture medium. Therefore, this study aimed to establish two liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the measurements of ASS activity, and arginine and its related metabolites, and to investigate the impact of galactose, fructose and low glucose (9 mM glucose) on reducing power, ASS activity and cellular stress in primary hepatocytes with or without citrin-knockdown. In addition, we examined the feasibility of ASS activity measurement in peripheral lymphocytes, and the comparison with liver ASS activity. Mice hepatocytes, AML12, were transfected with Slc25a13-shRNA to knockdown citrin and cultured in medium with or without galactose, fructose or low glucose. Decreased ASS specific activity was found in hepatocytes cultured in galactose- (154.7 ± 8.0 mU/g), fructose- (140.0 ± 9.2 mU/g) or low glucose-containing medium (159.8 ± 11.4 mU/g), compared with 18 mM glucose medium (203.1 ± 3.0 mU/g). Increased lactate/pyruvate ratio in culture media was also found in 9 mM glucose + 9 mM galactose or fructose, or 9 mM glucose only (64.5, 63.7 and 101.1 v.s 53.3), as well as cytosolic NADH concentrations (44.6, 40.8 and 52.0 v.s 22.5 nmol/g). Citrin knockdown did not alter ASS protein levels measured by western bloting. However, glucose insufficient culture medium caused impaired mitochondrial membrane potential (MMP) in citrin-KD cells, as well as the decreased ASS activity, increased lactate/pyruvate ratio and increased NADH concentration. The above changes were similar with AML12 cells without citrin-KD. Moreover, hepatocytes cultured in glucose insufficient medium displayed increased degree of vulnerability to H2O2, as indicated by decreased MMP and cell viability. The lactate/pyruvate ratio and ASS activity showed a negative correlation (r=-0.647, p<0.001). Despite of the changes in ASS activity and cytosolic reducing power, the intracellular arginine and lysine did not change, but citrulline, asymmetry dimethylarginine (ADMA), and symmetry dimethylarginine (SDMA) were increased in AML12 cultured with glucose insufficient medium. Glucose insufficiency-induced changes of ASS activity were restored by the treatment of pyruvate or glycine, and the enhanced NADH, impaired MMP reduced cell viability were restored by N-acetylcysteine (NAC), pyruvate or glycine. The ASS1 mRNA expression did not change in AML12 cells cultured with glucose insufficient medium. The established ASS activity measurement was applied to activated lymphocytes, with 88 ± 24 mU/g in normal subjects (N=10). Taken together, the method for measuring ASS activity by LC-MS/MS was established. Our results indicate that glucose-insufficiency induced reducing power which may be associated with the decrease of ASS activity from NICCD progression to CTLN2, and pyruvate, glycine or NAC treatment would reverse the changes.

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