Isotope labeling is a classical method used to study metabolic processes in living organisms. We consider culturing mammalian cells under oxygen-sufficient conditions using glucose labeled with different $^{13}C$, and adding different exogenous molecules to regulate cellular metabolism. Below is a description of the corresponding experimental methods and expected results. Please select the correct one.
同位素标记是经典的用于研究生物体内的代谢过程的方法。我们考虑用不同$^{13}C$标记的葡萄糖在氧气充足的条件下培养哺乳动物细胞，并分别添加不同的外源分子调控细胞代谢。以下是关于相应实验方法和预期结果的描述，请选出其中正确的一项。
1. Cultivate cells with glucose labeled at the C4 position with <sup>13</sup>C, without adding any other exogenous small molecules, and the first carbon in pyruvate to be isotopically labeled will be the keto carbonyl carbon.
After pretreating cells with iodoacetate, the culture medium was removed. When cells were cultured with glucose labeled with <sup>13</sup>C at the C6 position, the final isotopic labeling remained on the aldehyde carbon of glyceraldehyde-3-phosphate or the phosphate carbon of dihydroxyacetone phosphate.
After pretreating the cells with sodium arsenate, the culture medium was removed. Cells were then cultured with glucose labeled with $^{13}C$ at the C3 position, without adding any other exogenous small molecules, resulting in pyruvate not being isotopically labeled.
When cells are cultured with glucose labeled at the C6 position using <sup>13</sup>C and a certain concentration of sodium fluoride is added to the culture medium, the final isotopic labeling is concentrated on the methylene carbon of phosphoenolpyruvate.
After culturing cells with glucose labeled at the C4 position with $^{13}C$, the cells were collected within a short period, and saturated fatty acids were isolated from the cells. The $^{13}C$ isotopic label could be detected in these fatty acids.
After culturing cells with glucose labeled at the C1 position with <sup>13</sup>C, isolating the intracellular ketone bodies and detecting the isotopic labeling, the isotopic label should appear in the methyl and methylene groups of 3-hydroxybutyrate and acetoacetate, as well as in the carbonyl carbon of acetone.
Cells were cultured with glucose labeled at the C6 position with <sup>13</sup>C, and a high concentration of fluoroacetate was added to the culture medium. This treatment did not affect the detection of isotopic labeling in oxaloacetate.
When cells are cultured with glucose labeled with $^{13}C$ at the C3 position, the isotopic label can be detected in the carbon dioxide produced by cellular metabolism after at least one round of the citric acid cycle.
When cells are cultured with glucose labeled with $^{13}C$ at the C6 position, a minimum of 3 rounds of the citric acid cycle are required to generate isotopically labeled carbon dioxide in the citric acid cycle.
None of the above statements are correct
1用$^{13}C$标记C4位的葡萄糖培养细胞，不加任何其他外源小分子，则丙酮酸中最先被同位素标记的碳为酮羰基碳。
用碘乙酸预处理细胞后，撤去培养液。用$^{13}C$标记C6位的葡萄糖培养细胞，则最终同位素标记停留在3-磷酸甘油醛的醛基碳或磷酸二羟基丙酮的磷酸基碳。
用砷酸钠预处理细胞后，撤去培养液。用$^{13}C$标记C3位的葡萄糖培养细胞，不加任何其他外源小分子，则丙酮酸不会被同位素标记。
用$^{13}C$标记C6位的葡萄糖培养细胞，并在培养液中加入一定浓度的氟化钠，则最终同位素标记集中在磷酸烯醇式丙酮酸的亚甲基碳。
用$^{13}C$标记C4位的葡萄糖培养细胞后，在短时间内收集细胞并分离细胞内的饱和脂肪酸，在这些脂肪酸中能检测到$^{13}C$的同位素标记。
用$^{13}C$标记C1位的葡萄糖培养细胞后，分离细胞内的酮体并检测同位素标记，则同位素标记应当出现在3-羟基丁酸、乙酰乙酸的甲基与亚甲基，和丙酮的酮羰基碳。
用$^{13}C$标记C6位的葡萄糖培养细胞，并在培养液中加入高浓度的氟乙酸，该处理不会影响草酰乙酸被检测到同位素标记。
用$^{13}C$标记C3位的葡萄糖培养细胞，最少需要经过1轮柠檬酸循环，就可以在细胞代谢产生的二氧化碳中检测到同位素标记。
用$^{13}C$标记C6位的葡萄糖培养细胞，最少需要经过3轮柠檬酸循环，才能在柠檬酸循环中产生有同位素标记的二氧化碳。
以上说法均不正确
First, the glycolysis process is examined. In glycolysis, the C1-C6 of glucose remain unchanged until the formation of fructose-1,6-bisphosphate. Subsequently, fructose-1,6-bisphosphate undergoes cleavage: carbons 1, 2, and 3 enter dihydroxyacetone phosphate (DHAP), with carbon 1 bonded to a phosphate group, carbon 2 as a carbonyl group, and carbon 3 bonded to a hydroxyl group; carbons 4, 5, and 6 enter glyceraldehyde-3-phosphate (G3P), with carbon 4 as an aldehyde group, carbon 5 bonded to a hydroxyl group, and carbon 6 bonded to a phosphate group. Under the action of triosephosphate isomerase, dihydroxyacetone phosphate isomerizes to glyceraldehyde-3-phosphate. This process does not involve the migration of the phosphate group, so carbon 1 retains the phosphate group, carbon 2 is bonded to a hydroxyl group, and carbon 3 remains an aldehyde group. Therefore, in glucose labeling, after the formation of glyceraldehyde-3-phosphate, the pairs 1/6, 2/5, and 3/4 are equivalent within their respective groups.  

<Checkpoint>[In isotopically labeled glucose, the labeling can be divided into three cases: 1/6, 2/5, and 3/4, where the two labels within each group are equivalent.](1)</Checkpoint>  

First, consider the 3/4 group, corresponding to options A, C, E, and H. The aldehyde group of glyceraldehyde-3-phosphate corresponds to the carboxyl group in pyruvate, carbon 5 corresponds to the keto carbonyl group, and carbon 6 corresponds to the methyl group. Thus, the isotope in pyruvate appears in the carboxyl group. Option A is incorrect.  

<Checkpoint>[Labeling glucose at C4 results in the isotope appearing in the carboxyl group of pyruvate; A is incorrect.](1)</Checkpoint>  

Sodium arsenate is an inhibitor of glyceraldehyde-3-phosphate oxidation, capable of uncoupling oxidation and phosphorylation. However, it is a reversible inhibitor, so glycolysis resumes normally after removal from the culture medium. Therefore, option C is incorrect.  

<Checkpoint>[Sodium arsenate inhibition is reversible, and glycolysis can resume normally after removal; C is incorrect.](1)</Checkpoint>  

<Checkpoint>[The isotope first appears in the carboxyl group of pyruvate.](1)</Checkpoint>  

When pyruvate is converted to acetyl-CoA, the carboxyl group is released as carbon dioxide. Thus, the isotope does not appear in downstream metabolic processes, making options E and H incorrect.  

<Checkpoint>[The isotopic label on the carboxyl group of pyruvate does not enter acetyl-CoA and thus will not appear in synthesized saturated fatty acids; E is incorrect.](1)</Checkpoint>  

<Checkpoint>[The isotope does not need to enter the citric acid cycle to produce isotopically labeled carbon dioxide via acetyl-CoA synthesis; H is incorrect.](1)</Checkpoint>  

Next, examine the 1/6 group, corresponding to descriptions 2, 4, 6, 7, 9, and 10. Iodoacetate is an irreversible inhibitor of GAPDH, and its inhibition cannot be reversed by removing the culture medium. Glycolysis ultimately halts at glyceraldehyde-3-phosphate (or dihydroxyacetone phosphate), with the isotopic label on the carbon bonded to the phosphate group. Option B is incorrect.  

<Checkpoint>[Iodoacetate is an irreversible inhibitor of GAPDH.](1)</Checkpoint>  

<Checkpoint>[The isotopic label in both compounds is located on the carbon bonded to the phosphate group; B is incorrect.](1)</Checkpoint>  

Sodium fluoride inhibits pyruvate kinase and enolase, causing glycolysis to halt at 2-phosphoglycerate rather than accumulating phosphoenolpyruvate. The isotopic label is on the methylene carbon, making option D incorrect.  

<Checkpoint>[Sodium fluoride inhibits pyruvate kinase and enolase.](1)</Checkpoint>  

<Checkpoint>[The isotopic label primarily remains at 2-phosphoglycerate; D is incorrect.](1)</Checkpoint>  

In acetyl-CoA, the methyl carbon carries the isotopic label, which subsequently enters the ketone body synthesis pathway. Three molecules of acetyl-CoA synthesize HMG-CoA, where the isotopic label appears on the α-carbon of the thioester, the β-methyl carbon, and the α-carbon of the carboxyl group. HMG-CoA is then cleaved to produce acetoacetate, with the isotopic label appearing on the methyl and methylene groups. Acetoacetate decarboxylates to form acetone, with the label on both methyl groups; or is reduced to 3-hydroxybutyrate, where the label remains on the methyl and methylene groups. Option F is incorrect.  

<Checkpoint>[In ketone bodies synthesized from methyl-labeled acetyl-CoA, the isotope is located on methyl or methylene groups; F is incorrect.](1)</Checkpoint>  

Fluoroacetate is converted to fluoroacetyl-CoA, which reacts with oxaloacetate to form fluorocitrate, inhibiting citrate synthase and blocking the citric acid cycle. Since isotopically labeled glucose requires glycolysis to produce acetyl-CoA, and large amounts of fluoroacetate can block the citric acid cycle early on, labeled acetyl-CoA hardly enters the cycle. Thus, oxaloacetate shows almost no isotopic labeling. Option G is incorrect.  

<Checkpoint>[Fluoroacetate-derived fluorocitrate inhibits citrate synthase.](1)</Checkpoint>  

<Checkpoint>[The citric acid cycle is blocked, so oxaloacetate will not show isotopic labeling.](1)</Checkpoint>  

Finally, examine isotopic transfer in the citric acid cycle. When the methyl group of acetyl-CoA carries an isotopic label, it appears on one of the methylene groups of succinate in the cycle. Note that succinate is a symmetric molecule; after hydration to malate and oxidation to oxaloacetate, the isotopic label may appear on either the carbonyl or methylene group of oxaloacetate. If the methylene group is labeled, in the next cycle, the label will appear on the carboxyl group of oxaloacetate. If the carbonyl group is labeled, in the next cycle, the label will appear on the hydroxyl carbon of citrate, return to the methylene group of succinate, and then reappear on the carbonyl or methylene group of oxaloacetate. Considering only the carboxyl group of oxaloacetate, one carboxyl group is released as CO₂ during the conversion of α-ketoglutarate to succinyl-CoA. Thus, option I is correct.  

<Checkpoint>[At least three rounds of the citric acid cycle are required: acetyl-CoA → methylene of oxaloacetate → carboxyl of oxaloacetate → CO₂, to transfer the isotope to CO₂; option I is correct.](1)</Checkpoint>
首先考察糖酵解过程，糖酵解中葡萄糖的C1-C6在生成1,6-二磷酸果糖为止都保持不变。随后1,6-二磷酸果糖发生裂解，1,2,3号碳进入磷酸二羟基丙酮，1号碳连磷酸基，2号碳为羰基，3号碳连羟基；4,5,6号碳进入3-磷酸甘油醛，4号碳为醛基，5号碳连羟基，6号碳连磷酸基.在磷酸丙糖异构酶的作用下，磷酸二羟丙酮异构化位3-磷酸甘油醛，该过程不涉及磷酸基的迁移，因此1号碳为磷酸基，2号碳连羟基，3号碳为醛基。故葡萄糖标记中，在形成3-磷酸甘油醛之后的过程里，1/6号，2/5号，3/4号组内是两两等价的。

<Checkpoint>[同位素标记的葡萄糖中，标记可以分为1/6，2/5，3/4三种情况，组内两种标记是等价的。](1)</Checkpoint>

首先考察3/4组，对应选项为A、C、E、H. 3-磷酸甘油醛的醛基在丙酮酸中对应羧基，5号碳对应酮羰基，6号碳对应甲基，因此丙酮酸中的同位素出现在羧基。A选项错误。

<Checkpoint>[标记葡萄糖的C4则同位素出现在丙酮酸的羧基，A错误](1)</Checkpoint>

砷酸钠是3-磷酸甘油醛氧化的抑制剂，能够解除氧化和磷酸化的偶联作用，但是为可逆抑制，撤去培养液后，糖酵解恢复正常，故C选项错误。

<Checkpoint>[砷酸钠的抑制是可逆的，在撤去后糖酵解能恢复正常，C错误](1)</Checkpoint>

<Checkpoint>[同位素会首先出现在丙酮酸的羧基](1)</Checkpoint>

丙酮酸转化成乙酰辅酶A，羧基以二氧化碳的形式离去，故同位素不会出现在下游的代谢过程中，选项E、H均错误。

<Checkpoint>[丙酮酸羧基的同位素标记不会进入乙酰辅酶A，故不会出现在细胞合成的饱和脂肪酸中，E错误](1)</Checkpoint>

<Checkpoint>[不需要进入柠檬酸循环，就能通过乙酰辅酶A的合成产生带有同位素标记的二氧化碳，H错误](1)</Checkpoint>

下面考察1/6组，对应描述为2，4，6，7，9，10.碘乙酸为GAPDH的不可逆抑制剂，撤去培养液不能逆转抑制效果。最终糖酵解终止在3-磷酸甘油醛（磷酸二羟基丙酮）处，同位素标记的是与磷酸基相连的碳，选项B错误。

<Checkpoint>[碘乙酸是GAPDH的不可逆抑制剂](1)</Checkpoint>

<Checkpoint>[同位素标记在两种化合物中都位于与磷酸基相连的碳，B错误](1)</Checkpoint>

氟化钠是丙酮酸激酶和烯醇化酶的抑制剂，因此糖酵解会停留在磷酸烯醇式丙酮酸之前的2-磷酸甘油酸处，而非集中在磷酸烯醇式丙酮酸。同位素标记的是亚甲基碳，选项D错误。

<Checkpoint>[氟化钠抑制丙酮酸激酶和烯醇化酶](1)</Checkpoint>

<Checkpoint>[同位素标记主要停留在2-磷酸甘油酸，D错误](1)</Checkpoint>

乙酰辅酶A中，甲基碳带有同位素标记，此后进入酮体合成途径，3分子乙酰辅酶A合成HMG-CoA，其中存在同位素标记的是硫酯α位碳、β位上取代的甲基的碳、和羧基α位碳。随后HMG-CoA裂解产生乙酰乙酸，同位素标记出现在甲基和亚甲基。乙酰乙酸脱羧产生丙酮，同位素标记位于两个甲基；乙酰乙酸还原为3-羟基丁酸，同位素位置不变，仍然在甲基和亚甲基。选项F错误。

<Checkpoint>[甲基标记的乙酰辅酶A合成的酮体分子中，同位素均位于甲基或者亚甲基处，F错误](1)</Checkpoint>

氟乙酸转化为氟乙酰CoA后，可以与草酰乙酸反应为氟柠檬酸，并抑制柠檬酸合酶，从而阻断柠檬酸循环。考虑到同位素标记的葡萄糖需要糖酵解产生乙酰辅酶A，而大量氟乙酸在一开始即能阻断柠檬酸循环，因此含有同位素的乙酰辅酶A几乎不能进入柠檬酸循环，故草酰乙酸中几乎不能检测到同位素的标记。选项G错误。

<Checkpoint>[氟乙酸转化成的氟柠檬酸会抑制柠檬酸合酶](1)</Checkpoint>

<Checkpoint>[柠檬酸循环被阻断，草酰乙酸中不会检测到同位素标记](1)</Checkpoint>

下面考察柠檬酸循环的同位素传递，乙酰辅酶A的甲基上有同位素标记，进入三羧酸循环后会出现在琥珀酸的其中一个亚甲基上。注意到琥珀酸是完全对称的分子，在水化为苹果酸并氧化为草酰乙酸后，同位素标记可能是在草酰乙酸的羰基或者亚甲基上。若亚甲基带有同位素，则在下一轮循环中，同位素会出现在草酰乙酸的羧基上。若羰基上带有同位素，则在下一轮循环中，同位素会出现在柠檬酸羟基碳上，并回到琥珀酸的亚甲基，然后再回到草酰乙酸的羰基或者亚甲基上。下面仅考虑草酰乙酸的羧基的同位素标记，则其中一个羧基会在α-酮戊二酸转化为琥珀酰辅酶A的过程中转变为二氧化碳。故选项I是正确的。

<Checkpoint>[最少需要三轮柠檬酸循环：乙酰辅酶A→草酰乙酸的亚甲基→草酰乙酸的羧基→二氧化碳，就可以把同位素传递到二氧化碳上；I选项正确](1)</Checkpoint>