半胱氨酸热原检查体外替代方法的建立及其与家兔法的比较研究

doi:10.12300/j.issn.1674-5817.2026.029

摘要/Abstract

摘要：

目的本研究尝试建立半胱氨酸的细菌内毒素检查法以作为半胱氨酸热原检测的一种体外替代方法，从而减少家兔在热原检测中的使用，保障实验动物福利。方法参考《中国药典》（2025年版）四部通则1142和1143、指导原则9251和9301章节，对3批半胱氨酸原料药进行热原及细菌内毒素检测的方法学研究。采用家兔热原检查法、单核细胞活化反应测定法检测半胱氨酸样品中潜在的致热原，并对其中1个批次样品单核细胞活化反应测定法的细胞活力进行测定。采用凝胶限度法、光度检测技术（微量动态显色法、动态显色法、动态浊度法）检测半胱氨酸样品的潜在细菌内毒素。结果采用两种热原检查法检测半胱氨酸时均存在假阴性；家兔热原检查结果中，样品+内毒素组均未出现阳性反应。单核细胞活化反应测定结果显示，半胱氨酸孵育后人外周血单个核细胞中白细胞介素-6的分泌受到抑制，回收率小于50%。采用凝胶限度法检测细菌内毒素时，在不超过最大有效稀释倍数的条件下，3批半胱氨酸样品均存在干扰。微量动态显色法的干扰试验结果显示，半胱氨酸样品浓度为1.05 mg/mL时，3批样品的回收率均在50%～200%范围内，能够排除样品对内毒素的干扰，方法可行。采用微量动态显色法、动态显色法、动态浊度法检测浓度为1.05 mg/mL半胱氨酸样品的细菌内毒素，回收率均在50%～200%范围内，3批半胱氨酸样品的细菌内毒素含量均符合限值规定。结论本研究建立了半胱氨酸的细菌内毒素检查法，且初步证明内毒素光度检测技术可替代热原检查法用于半胱氨酸细菌内毒素检查。

关键词: 半胱氨酸, 细菌内毒素检查法, 微量动态显色法, 热原检查法, 单核细胞活化反应测定法, 家兔

Abstract:

Objective This study attempts to establish a cysteine bacterial endotoxin test as an in vitro alternative method for cysteine pyrogen detection, so as to reduce the use of rabbits in pyrogen detection and ensure the welfare of experimental animals. Methods According to the Chinese Pharmacopoeia (2025 edition) Volume Ⅳ, General Chapters 1142 and 1143, and Guidelines 9251 and 9301, methodological studies on pyrogen and bacterial endotoxin testing were conducted on three batches of cysteine raw material. We used the pyrogen test (rabbit method, RPT) and the in vitro pyrogen test (monocyte activation test, MAT) to detect pyrogens in cysteine and assessed cell viability during the MAT for one batch. Bacterial endotoxin testing was performed using the gel-clot method (limit test) and the bacterial endotoxin photometric assay (microkinetic chromogenic assay, kinetic chromogenic assay, kinetic turbidimetric assay). Results Pyrogen testing of cysteine using the two pyrogen test methods yielded false-negative results. In the RPT, all the test articles spiked with endotoxin did not show a positive pyrogen response. The MAT results showed that cysteine inhibited interleukin-6 (IL-6) secretion by human peripheral blood mononuclear cells (hPBMCs) after incubation, with recovery rates below 50%. Interference was observed in bacterial endotoxin testing using the gel-clot method: all three batches showed interference at dilutions not exceeding the maximum valid dilution (MVD). In the interference test using the microkinetic chromogenic method, when cysteine was prepared at a concentration of 1.05 mg/mL, the recovery rates for all three batches were between 50% and 200%, indicating that the interference was eliminated and the method was feasible. The recovery rates measured by the microkinetic chromogenic assay, kinetic chromogenic assay and kinetic turbidimetric assay fell within the acceptable range of 50%–200% when the cysteine concentration was 1.05 mg/mL. The bacterial endotoxin levels in the three batches met the specified limits. Conclusion This study establishes a bacterial endotoxin assay for cysteine and preliminarily demonstrates that the photometric techniques can replace RPT for the bacterial endotoxin testing of cysteine.

Key words: Cysteine, Bacterial endotoxin test, Micro kinetic chromogenic assay, Pyrogen test, Monocyte activation test, Rabbits

中图分类号:

R917
R927.12

盖雅婷,林丽花. 半胱氨酸热原检查体外替代方法的建立及其与家兔法的比较研究[J]. 实验动物与比较医学, 2026, 46(5): 740-749. DOI: 10.12300/j.issn.1674-5817.2026.029.

GAI Yating,LIN Lihua. Establishment of In Vitro Alternative Methods for Cysteine Pyrogen Testing and Comparison with the Rabbit Method[J]. Laboratory Animal and Comparative Medicine, 2026, 46(5): 740-749. DOI: 10.12300/j.issn.1674-5817.2026.029.

图/表 6

参考文献 25

[1]	杨乾龙, 王静, 南韦肖, 等. 半胱氨酸的合成代谢及其生物学作用研究进展[J]. 饲料工业, 2022, 43(7): 19-22. DOI: 10.13302/j.cnki.fi.2022.07.003.
	YANG Q L, WANG J, NAN W X, et al. Research progress on the synthesis, metabolism and biological function of cysteine[J]. Feed Ind, 2022, 43(7): 19-22. DOI: 10.13302/j.cnki.fi.2022.07.003.
[2]	李兆周, 谢亚芳, 陶健, 等. 半胱氨酸荧光探针的构建及其在病理学检验中的应用[J]. 有机化学, 2025, 45(7): 2335-2349. DOI: 10.6023/cjoc202410024.
	LI Z Z, XIE Y F, TAO J, et al. Construction of cysteine fluorescent probes and their applications in pathological examination[J]. Chin J Org Chem, 2025, 45(7): 2335-2349. DOI: 10.6023/cjoc202410024.
[3]	RUSHWORTH G F, MEGSON I L. Existing and potential therapeutic uses for N-acetylcysteine: the need for conversion to intracellular glutathione for antioxidant benefits[J]. Pharmacol Ther, 2014, 141(2): 150-159. DOI: 10.1016/j.pharmthera.2013.09.006.
[4]	徐鑫荣, 俞雷, 张家留. 还原型谷胱甘肽和乙酰半胱氨酸对内毒素休克兔的保护作用[J]. 临床麻醉学杂志, 2001, 17(6): 318-321. DOI: 10.3969/j.issn.1004-5805.2001.06.008.
	XU X R, YU L, ZHANG J L. Protective effects of GSH or NAC on the endotoxine-shocked rabbits[J]. J Clin Anesth, 2001, 17(6): 318-321. DOI: 10.3969/j.issn.1004-5805.2001.06.008.
[5]	黄宣林, 赖俊, 张祥水, 等. N-乙酰半胱氨酸对细菌内毒素诱发急性肾损伤的保护作用及机制[J]. 齐齐哈尔医学院学报, 2023, 44(18): 1701-1705. DOI: 10.3969/j.issn.1002-1256.2023.18.001.
	HUANG X L, LAI J, ZHANG X S, et al. Protective effect and mechanism of N-acetylcysteine on acute kidney injury induced by bacterial endotoxins[J]. J Qiqihar Univ Med, 2023, 44(18): 1701-1705. DOI: 10.3969/j.issn.1002-1256.2023.18.001.
[6]	刘思华, 方昱, 洪启浩, 等. 单核细胞活化反应检查法在热原检测中的应用研究进展[J]. 中国新药杂志, 2025, 34(18): 1944-1951. DOI: 10.20251/j.cnki.1003-3734.2025.18.006.
	LIU S H, FANG Y, HONG Q H, et al. Advances in the application of monocyte activation test assay in the detection of pyrogens[J]. Chin J New Drugs, 2025, 34(18): 1944-1951. DOI: 10.20251/j.cnki.1003-3734.2025.18.006.
[7]	国家药典委员会.中华人民共和国药典(四部)[S].北京:中国医药科技出版社,2025.
	Chinese Pharmacopoeia Commission.Pharmacopoeia of the People's Republic of China(Volume Ⅳ)[S].Beijing:China Medical Science Press,2025.
[8]	方选, 于德志, 武刚毅. 乙酰半胱氨酸细菌内毒素检查法的建立[J]. 中国药品标准, 2022, 23(2): 220-223. DOI: 10.19778/j.chp.2022.02.019.
	FANG X, YU D Z, WU G Y. Establishment of bacterial endotoxin test method for acetylcysteine[J]. Drug Stand China, 2022, 23(2): 220-223. DOI: 10.19778/j.chp.2022.02.019.
[9]	盖雅婷, 王赟, 洪颖, 等. 清开灵注射液热原替代检查法对比研究[J]. 解放军药学学报, 2025, 38(1): 48-52. DOI: 10.3969/j.issn.1008-9926.2025.01.010.
	GAI Y T, WANG B, HONG Y, et al. Alternative pyrogen tests for Qingkailing injection[J]. Pharm J Chin People's Liberation Army, 2025, 38(1): 48-52. DOI: 10.3969/j.issn.1008-9926.2025.01.010.
[10]	European Directorate for the Quality of Medicines & HealthCare. European Pharmacopoeia 11.8[S]. Strasbourg: EDQM, 2025.
[11]	WANG C Y, LI Q Y, ZHOU P, et al. Bioprocess engineering, transcriptome, and intermediate metabolite analysis of L-serine high-yielding Escherichia coli W3110[J]. Microorganisms, 2022, 10(10): 1927. DOI: 10.3390/microorganisms10101927.
[12]	卞金玉, 张晓梅, 徐国强, 等. 途径工程改造谷氨酸棒杆菌合成L-半胱氨酸[J]. 食品与发酵工业, 2022, 48(20): 22-28. DOI: 10.13995/j.cnki.11-1802/ts.030796.
	BIAN J Y, ZHANG X M, XU G Q, et al. Pathway engineering modification of Corynebacterium glutamicum for L-cysteine synthesis[J]. Food Ferment Ind, 2022, 48(20): 22-28. DOI: 10.13995/j.cnki.11-1802/ts.030796.
[13]	AGUAYO-CERÓN K A, SÁNCHEZ-MUÑOZ F, GUTIERREZ-ROJAS R A, et al. Glycine: the smallest anti-inflammatory micronutrient[J]. Int J Mol Sci, 2023, 24(14): 11236. DOI: 10.3390/ijms241411236.
[14]	MAHDAVIFARD S, NAKHJAVANI M. Supplementation with cysteine improved metabolic syndrome in rats by increasing antioxidant potential in the liver and adipose tissue, as well as decreasing hepatic NF-κB expression[J]. Food Biosci, 2024, 62: 105123. DOI: 10.1016/j.fbio.2024.105123.
[15]	徐琳, 吴彦霖, 裴宇盛, 等. «中华人民共和国药典»(2025年版)注射剂安全性检查法应用指导原则修订解读[J]. 中国药物警戒, 2025, 22(11): 1258-1262. DOI: 10.19803/j.1672-8629.20250648.
	XU L, WU Y L, PEI Y S, et al. Interpretations of amendments to guidelines for applications of safety tests for injections in Chinese pharmacopoeia 2025 edition[J]. Chin J Pharmacovigil, 2025, 22(11): 1258-1262. DOI: 10.19803/j.1672-8629.20250648.
[16]	杨秀静, 许娇红, 张熠, 等. 单核细胞活化反应测定法在注射用曲克芦丁热原检测中的应用[J]. 中国食品药品监管, 2025(10): 108-113. DOI: 10.3969/j.issn.1673-5390.2025.10.011.
	YANG X J, XU J H, ZHANG Y, et al. Application of monocyte activation test in pyrogen detection of troxerutin for injection[J]. China Food Drug Adm Mag, 2025(10): 108-113. DOI: 10.3969/j.issn.1673-5390.2025.10.011.
[17]	刘思含, 裴宇盛, 谭德讲, 等. 热原与细菌内毒素检测法研究进展[J]. 中国药物警戒, 2025, 22(4): 361-367, 376. DOI: 10.19803/j.1672-8629.20240821.
	LIU S H, PEI Y S, TAN D J, et al. Research progress in detection methods of pyrogen and bacterial endotoxin[J]. Chin J Pharmacovigil, 2025, 22(4): 361-367, 376. DOI: 10.19803/j.1672-8629.20240821.
[18]	祝清芬, 冯巧巧, 肖英, 等. 复方氨基酸(15AA)双肽(2)注射液细菌内毒素检查方法研究[J]. 中国药房, 2016, 27(33): 4742-4744. DOI: 10.6039/j.issn.1001-0408.2016.33.45.
	ZHU Q F, FENG Q Q, XIAO Y, et al. Study on the test method for bacterial endotoxin in compound amino acids (15AA) and dipeptide (2) injection[J]. China Pharm, 2016, 27(33): 4742-4744. DOI: 10.6039/j.issn.1001-0408.2016.33.45.
[19]	彭玉, 肖斯婷, 曹春然. 门冬氨酸(供注射用)细菌内毒素检查方法学研究[J]. 中国药事, 2025, 39(3): 337-344. DOI: 10.16153/j.1002-7777.2024-08-0022.
	PENG Y, XIAO S T, CAO C R. Methodological study on bacterial endotoxin test for aspartic acid(for injection)[J]. Chin Pharm Aff, 2025, 39(3): 337-344. DOI: 10.16153/j.1002-7777.2024-08-0022.
[20]	王文佳, 陈志明, 张纳敏, 等. 凝胶法测定脂肪乳(10%)/氨基酸(15)/葡萄糖(20%)注射液中的细菌内毒素[J]. 中国药房, 2017, 28(18): 2576-2578. DOI: 10.6039/j.issn.1001-0408.2017.18.36.
	WANG W J, CHEN Z M, ZHANG N M, et al. Determination of bacterial endotoxin in fat emulsion(10%)/amino acid(15)/glucose(20%) injection by gel method[J]. China Pharm, 2017, 28(18): 2576-2578. DOI: 10.6039/j.issn.1001-0408.2017.18.36.
[21]	陈素珍, 陈琪, 曾秋敏, 等. 微量动态显色法用于细菌内毒素检测的可行性研究[J]. 中国药品标准, 2024, 25(6): 607-611. DOI: 10.19778/j.chp.2024.06.013.
	CHEN S Z, CHEN Q, ZENG Q M, et al. Feasibility study of micro kinetic-chromogenic method for bacterial endotoxin detection[J]. Drug Stand China, 2024, 25(6): 607-611. DOI: 10.19778/j.chp.2024.06.013.
[22]	洪颖, 翁鹭娜, 林丽花, 等. 氟维司群注射液前处理及细菌内毒素检查方法研究[J]. 中国药业, 2024, 33(6): 69-73. DOI: 10.3969/j.issn.1006-4931.2024.06.015.
	HONG Y, WENG L N, LIN L H, et al. Study on pre-treatment and bacterial endotoxin test of fulvestrant injection[J]. China Pharm, 2024, 33(6): 69-73. DOI: 10.3969/j.issn.1006-4931.2024.06.015.
[23]	江庆洋, 刘玲, 周丽. 复方氨基酸注射液(18AA)细菌内毒素检查法的建立[J]. 海峡药学, 2014, 26(10): 78-79. DOI: 10.3969/j.issn.1006-3765.2014.10.034.
	JIANG Q Y, LIU L, ZHOU L. Establishment of bacterial endotoxin test for compound amino acid injection (18AA)[J]. Strait Pharm J, 2014, 26(10): 78-79. DOI: 10.3969/j.issn.1006-3765.2014.10.034.
[24]	黄逯, 谢鹏. 盐酸赖氨酸(注射用)细菌内毒素检查法的研究[J]. 中国药业, 2024, 33(5): 73-76. DOI: 10.3969/j.issn.1006-4931.2024.05.017.
	HUANG L, XIE P. Bacterial endotoxin test of lysine hydrochloride(for injection)[J]. China Pharm, 2024, 33(5): 73-76. DOI: 10.3969/j.issn.1006-4931.2024.05.017.
[25]	王冲, 徐军田, 王祖璇, 等. 动态显色法检测谷氨酸中细菌内毒素含量[J]. 中国药学杂志, 2025, 60(3): 302-307. DOI: 10.11669/cpj.2025.03.013.
	WANG C, XU J T, WANG Z X, et al. Determination of bacterial endotoxin in glutamic acid by kinetic chromogenic assay[J]. Chin Pharm J, 2025, 60(3): 302-307. DOI: 10.11669/cpj.2025.03.013.

组别 Group	体重/kg Weight/kg	正常体温/℃ Normal temperature/℃	给药前最大升温/℃ Maximum temperature rise before drug administration/℃	给药前最大降温/℃ Maximum temperature reduction before drug administration/℃	升温总和/℃ Sum of temperature rise/℃	降温总和/℃ Sum of temperature reduction/℃
热原阳性对照组 Pyrogen positive control group	3.0	38.7	0.8	0.0	2.2	-0.1
	3.0	38.5	1.2	0.0
	3.2	38.9	0.2	-0.1
空白对照组 Blank control group	2.5	38.2	0.4	0.0	0.9	-0.2
	2.2	38.6	0.1	-0.2
	2.4	38.6	0.4	0.0
半胱氨酸（S202302014）组 Cysteine group（S202302014）	2.2	38.6	0.4	0.0	0.8	-0.4
	2.3	38.6	0.2	-0.4
	2.3	38.7	0.2	0.0
半胱氨酸（S202302014）+内毒素对照组 Cysteine （S202302014）+CSE group	2.2	38.6	0.2	0.0	0.7	-0.1
	2.1	38.2	0.5	0.0
	2.2	38.1	0.0	-0.1
半胱氨酸（S202302015）组 Cysteine （S202302015） group	2.8	39.1	0.1	-0.2	0.2	-0.5
	2.6	39.2	0.0	-0.3
	2.6	38.8	0.1	0.0
半胱氨酸（S202302015）+内毒素对照组 Cysteine （S202302015）+CSE group	2.8	39.0	0.0	-0.1	0.6	-0.6
	2.8	38.1	0.5	-0.3
	2.7	38.8	0.1	-0.2
半胱氨酸（S202302016）组 Cysteine （S202302016） group	2.6	38.9	0.0	-0.3	0.0	-0.6
	2.4	38.7	0.0	-0.3
	2.4	38.9	0.0	0.0
半胱氨酸（S202302016）+内毒素对照组 Cysteine （S202302016）+CSE group	3.0	38.7	0.0	-0.5	0.4	-1.0
	2.9	38.0	0.4	0.0
	3.0	38.5	0.0	-0.5

组别 Group	体重/kg Weight/kg	正常体温/℃ Normal temperature/℃	给药前最大升温/℃ Maximum temperature rise before drug administration/℃	给药前最大降温/℃ Maximum temperature reduction before drug administration/℃	升温总和/℃ Sum of temperature rise/℃	降温总和/℃ Sum of temperature reduction/℃
热原阳性对照组 Pyrogen positive control group	3.0	38.7	0.8	0.0	2.2	-0.1
	3.0	38.5	1.2	0.0
	3.2	38.9	0.2	-0.1
空白对照组 Blank control group	2.5	38.2	0.4	0.0	0.9	-0.2
	2.2	38.6	0.1	-0.2
	2.4	38.6	0.4	0.0
半胱氨酸（S202302014）组 Cysteine group（S202302014）	2.2	38.6	0.4	0.0	0.8	-0.4
	2.3	38.6	0.2	-0.4
	2.3	38.7	0.2	0.0
半胱氨酸（S202302014）+内毒素对照组 Cysteine （S202302014）+CSE group	2.2	38.6	0.2	0.0	0.7	-0.1
	2.1	38.2	0.5	0.0
	2.2	38.1	0.0	-0.1
半胱氨酸（S202302015）组 Cysteine （S202302015） group	2.8	39.1	0.1	-0.2	0.2	-0.5
	2.6	39.2	0.0	-0.3
	2.6	38.8	0.1	0.0
半胱氨酸（S202302015）+内毒素对照组 Cysteine （S202302015）+CSE group	2.8	39.0	0.0	-0.1	0.6	-0.6
	2.8	38.1	0.5	-0.3
	2.7	38.8	0.1	-0.2
半胱氨酸（S202302016）组 Cysteine （S202302016） group	2.6	38.9	0.0	-0.3	0.0	-0.6
	2.4	38.7	0.0	-0.3
	2.4	38.9	0.0	0.0
半胱氨酸（S202302016）+内毒素对照组 Cysteine （S202302016）+CSE group	3.0	38.7	0.0	-0.5	0.4	-1.0
	2.9	38.0	0.4	0.0
	3.0	38.5	0.0	-0.5

样品批号 Sample batch number	稀释倍数 Dilution factor	不加内毒素的实测值/（EU·mL^-1） Measured value without endotoxin/(EU·mL^-1)	加入0.06 EU/mL的内毒素 After addition of 0.06 EU/mL endotoxin		回收率/% Recovery rate/%
样品批号 Sample batch number	稀释倍数 Dilution factor		实测值/（EU·mL^-1） Measured value/(EU·mL^-1)	标准差 s	回收率/% Recovery rate/%
S202302014	5	<0.015 0	0.09	0.002	17.0
	10	<0.015 0	0.37	0.006	49.0
	20	<0.015 0	0.64	0.003	40.3
	25	<0.015 0	1.03	0.007	57.1
S202302015	30	<0.007 5	0.60	0.003	17.2
	40	<0.007 5	1.00	0.012	25.6
	45	<0.007 5	0.86	0.002	17.9
	50	<0.007 5	1.05	0.004	19.7
S202302016	30	<0.007 5	0.57	0.004	12.8
	40	<0.007 5	0.76	0.001	14.7
	45	<0.007 5	1.08	0.006	23.6
	50	<0.007 5	0.85	0.002	13.4

样品批号 Sample batch number	稀释倍数 Dilution factor	不加内毒素的实测值/（EU·mL^-1） Measured value without endotoxin/(EU·mL^-1)	加入0.06 EU/mL的内毒素 After addition of 0.06 EU/mL endotoxin		回收率/% Recovery rate/%
样品批号 Sample batch number	稀释倍数 Dilution factor		实测值/（EU·mL^-1） Measured value/(EU·mL^-1)	标准差 s	回收率/% Recovery rate/%
S202302014	5	<0.015 0	0.09	0.002	17.0
	10	<0.015 0	0.37	0.006	49.0
	20	<0.015 0	0.64	0.003	40.3
	25	<0.015 0	1.03	0.007	57.1
S202302015	30	<0.007 5	0.60	0.003	17.2
	40	<0.007 5	1.00	0.012	25.6
	45	<0.007 5	0.86	0.002	17.9
	50	<0.007 5	1.05	0.004	19.7
S202302016	30	<0.007 5	0.57	0.004	12.8
	40	<0.007 5	0.76	0.001	14.7
	45	<0.007 5	1.08	0.006	23.6
	50	<0.007 5	0.85	0.002	13.4

稀释倍数 Dilution factor	外加内毒素/（EU·mL^-1） Endotoxin spiking/（EU·mL^-1）	实测值/（EU·mL^-1） Measured value/（EU·mL^-1）	标准差 s	回收率/% Recovery rate/%	细胞存活率/% Cell survival rate /%
4	/	<0.03	/	/	/
4	0.06	<0.03	/	/	/
8	/	<0.03	/	/	59
8	0.06	<0.03	/	/	68
16	/	<0.03	/	4.67	/
16	0.06	0.53	0.002	4.67	/
30	/	<0.03	/	18.37	94
30	0.06	1.23	0.003	18.37	93
60	/	<0.03	/	18.91	/
60	0.06	2.52	0.003	18.91	/
120	/	<0.03	/	17.37	92
120	0.06	4.92	0.001	17.37	93