Glycocyamine   中文名称: 胍基乙酸

Precursor for creatine

胍乙酸（GAA）是一种内源性动物组织（包括肌肉和神经）的氨基酸衍生物，已被报道可增强肌肉性能。微小RNA（miRNA）是一种转录后调节因子，在营养物介导的肌发生中起着关键作用。然而，GAA对肌源性分化和骨骼肌生长的影响，以及miRNA在这些过程中的潜在调节机制尚未阐明。在本研究中，我们研究了GAA对C2C12细胞和小鼠增殖、分化和生长的影响。结果表明，GAA显著抑制成肌细胞增殖，下调细胞周期蛋白D1（CCND1）和细胞周期蛋白依赖性激酶4（CDK4）mRNA表达，上调细胞周期蛋白激酶抑制剂1A（P21）mRNA的表达。我们还证明，GAA处理刺激肌源分化1（MyoD）和肌生成素（MyoG）mRNA表达，导致肌管融合率增加。同时，补充GAA通过增加总肌球蛋白重链（MyHC）蛋白水平、肌管厚度和腓肠肌横截面积来促进肌管生长。此外，小RNA测序显示，在补充GAA后，共有8种miRNA，包括miR-133a-3p和miR-1a-3p簇，显示出差异表达。为了进一步研究miR-133a-3p和miR-1a-3p在GAA诱导的骨骼肌生长中的作用，我们将miR-133a-3p和模拟miR-1a-3c转染到肌管中，肌管也诱导肌肉生长。通过生物信息学和双荧光素酶报告子系统，确定了miR-133a-3p和miR-1a-3p的靶基因。这两种miRNA被证明通过抑制靶基因表达来调节Akt/mTOR/S6K信号通路。总之，这些发现表明，补充GAA可以通过miR-133a-3p和miR-1a-3p诱导的AKT/mTOR/S6K信号通路的激活来促进成肌细胞分化和骨骼肌生长[2]。

胍乙酸（GAA）在肝脏和肾脏中由精氨酸和甘氨酸合成，随后被S-腺苷甲硫氨酸甲基化形成肌酸。进行了四项生物测定，以确定GAA作为日粮中Arg替代品的能力。在为期9天的电池饲养试验中，肉鸡被喂食缺乏精氨酸的右旋酪蛋白（0.88%精氨酸）或玉米-玉米副产品豆粕（1.0%精氨酸。由于添加了精氨酸、GAA或肌酸，体重增加和G:F增加（P<0.01），葡萄糖-酪蛋白饮食中的精氨酸明显不足。添加GAA的最佳水平为日粮的0.12%，但当添加到Arg充足的日粮中时，该水平的GAA或1.0%的creatin-H（2）O并不能改善生长性能。第二项测定证实了在补充l-Arg和GAA的2×2因子安排中的最佳Arg水平。使用基于玉米、玉米谷蛋白粉、带可溶性的蒸馏干谷物和豆粕的实用型饮食，添加0.25%精氨酸、0.12%GAA或0.15%肌酸·H（2）O可使G:F有类似的改善（P<0.05）。这些结果表明，添加0.12%的GAA，如肌酸，在喂食精氨酸缺乏饮食的幼鸡中产生一致的生长反应。为了提供GAA作为膳食精氨酸替代物的能力的进一步证据，在不存在或存在0.12%GAA的情况下，用7个分级剂量的精氨酸补充右旋酪蛋白饮食（总共14个饮食）。观察到体重增加的二次型（P<0.01）反应和对补充Arg的G:F反应。在不存在和存在0.12%GAA的情况下，估计了类似的补充Arg需求，但与Arg充足的饮食相比，当添加到Arg缺乏的饮食中时，GAA引起G:F的更大改善（P<0.05）。总之，这些数据表明，GAA可以作为幼鸡膳食精氨酸的有效替代品[1]。

小鼠骨骼肌细胞系C2C12在生长培养基（GM）中培养，生长培养基含有高糖DMEM，补充10%胎牛血清、100000单位/L青霉素钠和100 mg/L硫酸链霉素，温度为37°C，湿度为5%CO2。用含有高糖DMEM和2%马血清的分化培养基（DM）诱导C2C12成肌细胞8天。GAA以20mmol/L的最终浓度可溶于GM或DM。雷帕霉素（Rap）以100nmol/L的最终浓度在DMSO中稀释；DMSO在培养物中的最终浓度小于0.1%[2]。

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细胞增殖比率测定。[2]
通过细胞计数试剂盒-8（CCK-8）和EdU细胞增殖测定试剂盒检测C2C12细胞的增殖率。将C2C12细胞接种在96孔细胞培养板中，并在含有0mM、5mM、10mM或20mM GAA的GM中维持48小时；每12小时更换一次培养基。对于CCK-8，向培养基中加入10μL CCK-8试剂，1小时后，在450nm处测量光密度。对于EdU细胞增殖测定，用不同浓度的GAA处理细胞，当细胞达到约80%汇合时，将10μM EdU加入培养基中并孵育2小时。根据制造商的说明进行EdU染色；DAPI对细胞核进行染色。染色的细胞通过Olympus IX53显微镜成像。

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蛋白质印迹分析。[2]
简言之，将细胞接种在六孔细胞培养板中。分化4天后，如所述将细胞与Rap或GAA孵育48小时。然后将细胞在PBS中漂洗三次，并在含有1mM PMSF和0.02%蛋白酶磷酸酶抑制剂的200μL RIPA裂解缓冲液中裂解。对于组织，将25mg腓肠肌在500μL RIPA裂解缓冲液中匀浆，该缓冲液含有1mM PMSF和0.02%蛋白酶磷酸酶抑制剂。然后，将均化的液体在4°C下振荡30分钟，并通过12000×g离心15分钟从悬浮液中去除不溶性物质。使用BCA蛋白质测定法定量总蛋白质浓度。用10%十二烷基硫酸钠（SDS）聚丙烯酰胺电泳凝胶分离等分试样50μg蛋白质悬浮液后，使用Trans-Blot Turbo转移系统将蛋白质转移到聚偏二氟乙烯（PVDF）膜上，然后在室温下用含0.1%吐温20的Tris缓冲盐水中的5%（w/v）无脂肪干牛奶封闭2小时。然后将PVDF膜与所示抗体在4°C下孵育过夜，然后与第二抗体在室温下孵育1小时。使用ChemiDoc MP成像系统测量蛋白质表达，并将其标准化为微管蛋白表达。

Animals and Muscle Collection[2]
The Kunming male mice (n = 30) at 3 weeks of age were purchased from DASHUO Medical Laboratory Animal Center. The mice were given free access to food and water under constant 12 h light and 12 h dark cycle at a temperature of 23 °C ± 3 °C and relative humidity of 70% ± 10% throughout experimental period. After one week of acclimatization, the mice were randomly divided into three groups (n = 10 in each group) and supplemented with different concentrations of GAA (0%, 1% and 2%), dissolved in drinking water for 8 weeks. At the end of the experiment, serum was collected and the gastrocnemius muscle was taken for further testing. The serum was stored at −80 °C, the muscle stored in liquid nitrogen.

Metabolism / Metabolites
Guanidino acetic acid (GAA) is synthesized in the liver and kidney from Arg and Gly and subsequently methylated by S-adenosylmethionine to form creatine. The conversion of guaniodacetate to creatinine in the liver causes a depletion of methyl groups. This causes homocysteine levels to rise, which has been shown to produce cardiovascular and skeletal problems.

Toxicity Summary
Uremic toxins such as guanidinoacetic acid are actively transported into the kidneys via organic ion transporters (especially OAT3). Increased levels of uremic toxins can stimulate the production of reactive oxygen species. This seems to be mediated by the direct binding or inhibition by uremic toxins of the enzyme NADPH oxidase (especially NOX4 which is abundant in the kidneys and heart) (A7868). Reactive oxygen species can induce several different DNA methyltransferases (DNMTs) which are involved in the silencing of a protein known as KLOTHO. KLOTHO has been identified as having important roles in anti-aging, mineral metabolism, and vitamin D metabolism. A number of studies have indicated that KLOTHO mRNA and protein levels are reduced during acute or chronic kidney diseases in response to high local levels of reactive oxygen species (A7869)

[1]. Dietary guanidino acetic acid is an efficacious replacement for arginine for young chicks. Poult Sci. 2013 Jan;92(1):171-7.

Guanidinoacetic acid is the N-amidino derivative of glycine. It has a role as a human metabolite, a mouse metabolite, a nutraceutical, a rat metabolite and a bacterial metabolite. It is a conjugate acid of a guanidinoacetate. It is a tautomer of a guanidinoacetic acid zwitterion.
Glycocyamine has been reported in Citrus reticulata, Prunus persica, and other organisms with data available.
Glycocyamine is a naturally-occurring derivative of glycine and a metabolic precursor of creatine. Glycocyamine, also known as guanidinoacetic acid (GAA), is catalyzed by guanidinoacetate N-methyltransferase to form creatine. Creatine, in phosphate form, helps supply energy to muscle cells for contraction. After intense effort, when adenosine triphosphate (ATP) deposits are depleted, creatine phosphate donates phosphate groups toward the fast synthesis of ATP. Dietary supplementation with GAA may improve muscle wasting associated with cancer and other chronic diseases.
Guanidinoacetic acid is a uremic toxin. Uremic toxins can be subdivided into three major groups based upon their chemical and physical characteristics: 1) small, water-soluble, non-protein-bound compounds, such as urea; 2) small, lipid-soluble and/or protein-bound compounds, such as the phenols and 3) larger so-called middle-molecules, such as beta2-microglobulin. Chronic exposure of uremic toxins can lead to a number of conditions including renal damage, chronic kidney disease and cardiovascular disease.
Guanidoacetic acid is a metabolite in the Urea cycle and metabolism of amino groups, and in the metabolic pathways of several amino acids. This includes glycine, serine, threonine, arginine and proline metabolism. Guanidinoacetic acid is also a precursor of creatine, an essential substrate for muscle energy metabolism.

C3H7N3O2

117.12

117.053

C, 30.77; H, 6.03; N, 35.88; O, 27.32

352-97-6

763

Typically exists as white to off-white solids at room temperature

1.6±0.1 g/cm3

294.2±42.0 °C at 760 mmHg

300 °C(lit.)

131.7±27.9 °C

0.0±1.3 mmHg at 25°C

1.596

-1.85

99.2

3

3

2

8

116

0

O([H])C(C([H])([H])/N=C(\N([H])[H])/N([H])[H])=O

BPMFZUMJYQTVII-UHFFFAOYSA-N

InChI=1S/C3H7N3O2/c4-3(5)6-1-2(7)8/h1H2,(H,7,8)(H4,4,5,6)

2-(diaminomethylideneamino)acetic acid

AI3 17119; AI3-17119; glycocyamine; 352-97-6; guanidinoacetic acid; Guanidineacetic acid; 2-Guanidinoacetic acid; Guanidoacetic acid; N-amidinoglycine; Betacyamine; Glycocyamine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

1M HCl : 50 mg/mL (~426.95 mM)
H2O : ~5 mg/mL (~42.69 mM)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
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