Quantitation of serum pregnanediol-3-glucuronide level in different endocrine statuses by LC-MS/MS.
LC-MS/MS 法测定不同内分泌状态下血清 pregnanediol-3-glucuronide 水平
- 作者列表："Chen J","Hou H","Chen H","Luo Y","He Y","Wang H","Deng H","Zhang L","Zhang Y","Liu H","Liu Y","Wang A","Hu Q
:Pregnanediol-3-glucuronide (PdG) is the major terminal metabolite of progesterone, playing an important role in physiological processes, such as the female menstrual cycle, pregnancy (supports gestation), embryogenesis and maternal immune response of humans and other species. Hence, accurate measurement of PdG in serum/plasma is needed for the evaluation of progesterone production. However, such high-specificity determination of PdG is lacking in clinical sample detection. In this study, a highly sensitive and accurate LC-MS/MS method was firstly established for subsequent measurement of PdG in serum of three different female groups: thyroid cancer patients (TCs), healthy controls (HCs) and pregnant women. The factors affecting the sample preparation, MS/MS method, gradient elution program, selection of chromatographic column and internal standard (IS) have been optimized in this study. Compared with enzyme immunoassay (EIA) method, we used LC-MS/MS to shorten analysis time, increase sensitivity, raise specificity, simplify sample preparation, and reduce costs. As a result, the linear range of the method was from 0.38 to 100 ng/mL with a limit of quantification (LOD) of 0.01 ng/mL. Precision assays showed that relative standard deviation (RSD) was less than 10.6, accuracy was between 90.6 % and 110.4 %, and mean recovery was 103.4 %. In addition, the serum PdG/creatinine levels were significantly down-regulated in TCs and up-regulated in pregnant women versus HCs. Receive operating characteristic curve (ROC) analysis enabled to identify TC with a sensitivity of 83.3 %, specificity of 68.0 % and area under curve (AUC) of 0.781 (95 % CI: 0.684 to 0.879), and it enabled to identify pregnant women with a sensitivity of 94.7 %, specificity of 68.5 % and AUC of 0.811 (95 % CI: 0.732 to 0.890). Our results implied that an increase in female serum PdG/creatinine level might be associated with a risk of pregnancy, but serum PdG/creatinine decreasing might be related to a risk of TC.
: Pregnanediol-3-glucuronide (PdG) 是孕酮的主要代谢产物，在女性月经周期、妊娠 (支持妊娠) 、人类和其他物种的胚胎发生和母体免疫反应。因此，需要准确测量血清/血浆中的 PdG 来评价孕酮的产生。然而，临床样本检测中缺乏如此高特异性的 PdG 测定。本研究首次建立了一种高灵敏、准确的 LC-MS/MS 方法，用于甲状腺癌患者 (TCs) 、健康对照 (HCs) 和孕妇。本研究对影响样品制备的因素、 MS/MS 方法、梯度洗脱程序、色谱柱和内标 (IS) 的选择进行了优化。与酶免疫分析 (EIA) 方法相比，我们使用 LC-MS/MS 缩短了分析时间，增加了灵敏度，提高了特异性，简化了样品制备，降低了成本。结果，方法的线性范围为 0.38 ~ 100 ng/mL，定量限 (LOD) 为 0.01 ng/mL。精密度测定结果显示，相对标准偏差 (RSD) 小于 10.6，准确度在 90.6% ~ 110.4% 之间，平均回收率为 103.4%。此外，与 HCs 相比，血清 PdG/肌酐水平在 TCs 中显著下调，在孕妇中显著上调。接受操作特征曲线 (ROC) 分析，能够识别灵敏度为 83.3%，特异度为 68.0%，曲线下面积 (AUC) 为 0.781 (95% CI: 0.684 至 0.879) 的 TC, 它能够识别敏感性为 94.7% 的孕妇,68.5% 的特异性和 0.811 的 AUC (95% CI: 0.732 ~ 0.890)。我们的研究结果表明，女性血清 PdG/肌酐水平的升高可能与妊娠风险相关，但血清 PdG/肌酐降低可能与 TC 风险相关。
METHODS:OBJECTIVES:To assess the prevalence of Hashimoto thyroiditis (HT) in primary thyroid lymphoma (PTL) and whether it differs between mucosa-associated lymphoid tissue (MALT) lymphoma and diffuse large B-cell lymphoma (DLBCL). METHODS:Electronic databases were searched for studies assessing HT prevalence in PTL, based on antithyroid antibodies, clinical history, or pathology. Pooled prevalence of HT and its association with histotype (MALT or DLBCL) were calculated. RESULTS:Thirty-eight studies with 1,346 PTLs were included. Pooled prevalence results were 78.9% (any HT evidence), 65.3% (antithyroid antibodies), 41.7% (clinical history), and 64% (pathology). HT prevalence was significantly higher in MALT lymphoma than in DLBCL (P = .007) and in mixed DLBCL/MALT than in pure DLBCL (P = .002). CONCLUSIONS:Overall, 78.9% of patients with PTL have any HT evidence, but only half of these had been clinically followed. The difference in HT prevalence suggests that a subset of DLBCL may not derive from MALT lymphoma.
METHODS:Background Whether chronic lymphocytic thyroiditis (CLT) influences the risk of development and the progression of papillary thyroid cancer (PTC) remains uncertain. We investigated the effects of CLT on the clinicopathologic features and prognosis of PTC. Methods Two thousand nine hundred twenty-eight consecutive patients with PTC treated between 2009 and 2017 were divided into two groups: one with chronic lymphocytic thyroiditis and one without; 1174 (40%) of the patients had coincident CLT. Results In univariate analysis, CLT correlated positively with small tumor size, frequent extrathyroidal extension, multifocal diseases, and p53 but negatively with central lymph node (LN) metastasis and BRAF mutation. In multivariate analysis, CLT was associated with extrathyroidal extension and multifocal disease; however, it was not a prognostic factor for recurrence even though it was associated with two aggressive factors. Compared with patients with PTC alone, there were more retrieved central LNs in the PTC + CLT group, and these patients also underwent more invasive diagnostic tests such as fine needle aspiration cytology and frozen biopsy of LN. Conclusions The CLT patients with PTC had better behavior features and prognoses than did those with PTC alone despite frequent multifocality and extrathyroidal extension. However, precaution may be necessary to avoid performing invasive diagnostic procedures for lateral LN metastasis and to manage the patients appropriately.
METHODS::PTPN2 is one of the members of the protein Tyrosine Phosphatases (PTPs) family. To explore the promotive effect of upregulated PTPN2 induced by inflammatory response or oxidative stress on the progression of thyroid cancer. PTPN2 level in thyroid cancer tissues and cell lines was detected. Kaplan-Meier method was applied for evaluating the prognostic value of PTPN2 in thyroid cancer patients. After stimulation of inflammatory response (treatment of IFN-γ and TNF-α), or oxidative stress (treatment of H2O2), protein level of PTPN2 in K1 cells was measured by Western blot. Regulatory effects of PTPN2 on EdU-positive staining and Ki-67 positive cell ratio in K1 cells either with H2O2 stimulation or not were determined. PTPN2 was upregulated in thyroid cancer tissues and cell lines. Its level was higher in metastatic thyroid cancer patients than those of non-metastatic ones. High level of PTPN2 predicted worse prognosis of thyroid cancer. Treatment of either IFN-γ or TNF-α upregulated protein level of PTPN2 in K1 cells. Meanwhile, H2O2 stimulation upregulated PTPN2, which was reversed by NAC administration. With the stimulation of increased doses of H2O2, EdU-positive staining and Ki-67 positive cell ratio were dose-dependently elevated. Silence of PTPN2 attenuated proliferative ability and Ki-67 expression in K1 cells either with H2O2 stimulation or not. Inflammatory response or oxidative stress induces upregulation of PTPN2, thus promoting the progression of thyroid cancer.