The screening of inborn errors of metabolism in sick Chinese infants by tandem mass spectrometry and gas chromatography/mass spectrometry

BackgroundAnalyses of amino acid/acylcarnitines in dried blood spots (DBS) and organic acids in urine are the primary tests for inborn errors of metabolism (IEMs). Automated tandem mass spectrometry (MS/MS) and gas chromatography/mass spectrometry (GC/MS) can rapidly and simultaneously detect numerous metabolic compounds with high precision and sensitivity.MethodsThree thousand four hundred and twenty-nine DBSs and 2781 urine samples were collected from our hospital patients with suspected IEMs, and analyzed for amino acid/acylcarnitines and organic acids by MS/MS and GC/MS, respectively. The results were used in a coincidental survey to determine the efficacy of these methods for the diagnosis of IEMs.ResultsNineteen different types of IEMs were detected in 121 affected cases (1.95% of 6210 samples). There were 66.12% amino acid disorders, 29.75% organic acid disorders and 4.13% with fatty acid oxidation disorders. Conclusions: the sick infants tested in this study had high prevalence rates of neonatal intrahepatic cholestasis, methylmalonic acidemia, hyperphenylalaninemia, tyrosinemia type I, and urea cycle disorders.ConclusionThe combined use of MS/MS and GC/MS is an appropriate tool for screening of IEMs in sick infants.     

Improvement of sample throughput using fast gas chromatography mass-spectrometry for biochemical diagnosis of organic acid disorders

BACKGROUND: Gas chromatograph mass-spectrometric (GC/MS) method of analysis for urinary organic acids is used for the diagnosis of a variety of metabolic disorders. The method is time-consuming and does not allow for improvements in sample throughput. Although the sample preparation and the data processing have been improved, the long GC/MS analysis time still remains to be problematic. METHODS: The fast-GC/MS method, which utilizes a short microbore capillary GC column and fast temperature programming, was applied to the analysis for urinary organic acids. Urine samples obtained from 15 patients with 9 different disorders and 16 healthy controls were analyzed using conventional GC/MS and fast-GC/MS. RESULTS: Analysis cycle time was shortened from 1 h to 15 min. The automated data system uses retention indices determined by conventional-GC/MS for the identification of 134 organic acids. These retention indices can also be used in data obtained by fast-GC/MS. New fast-GC/MS method with the automated data system gave the same diagnostic results as conventional-GC/MS except for 1 healthy control. CONCLUSIONS: The combined system of fast-GC/MS and the automated data system will be powerful tools in clinical laboratories due to increased sample throughput and reduced analysis costs.     

A computerized classification technique for screening for the presence of breath biomarkers in lung cancer

A simple computer-based screening technique has been developed for classifying human expired air components into 16 chemical classes, based on empirical formulas. The sort procedure was developed to simplify the screening of the composition of expired air samples by sorting all components into chemical classes and classifying components at the greater than 75% and greater than 90% occurrence levels. Both occurrence-rate components are then evaluated as diagnostic markers in a discriminant function model for their ability to detect lung cancer. Of the 386 components detected in the gas chromatography/mass spectrometry (GC/MS) data files, 45 components were present at the greater than 75% occurrence level and 28 components at the greater than 90% occurrence level. Thus, this preliminary sort routine, performed by using a simple macro program installed into a standard personal-computer spread-sheet, greatly reduces the amount of data required for statistical treatment. Such a sort routine can also be applied as easily to other complex GC/MS data files for the purpose of data reduction.     

Influence of Honey on the Suppression of Human Low Density Lipoprotein (LDL) Peroxidation (In vitro)

The antioxidant activity of four honey samples from different floral sources (Acacia, Coriander, Sider and Palm) were evaluated with three different assays; DPPH free radical scavenging assay, superoxide anion generated in xanthine-xanthine oxidase (XOD) system and low density lipoprotein (LDL) peroxidation assay. The dark Palm and Sider honeys had the highest antioxidant activity in the DPPH assay. But all the honey samples exhibited more or less the same highly significant antioxidant activity within the concentration of 1mg honey/1 ml in XOD system and LDL peroxidation assays. The chemical composition of these samples was investigated by GC/MS and HPLC analysis, 11 compounds being new to honey. The GC/MS revealed the presence of 90 compounds, mainly aliphatic acids (37 compounds), which represent 54.73, 8.72, 22.87 and 64.10% and phenolic acids (15 compound) 2.3, 1.02, 2.07 and 11.68% for Acacia, Coriander, Sider and Palm honeys. In HPLC analysis, 19 flavonoids were identified. Coriander and Sider honeys were characterized by the presence of large amounts of flavonoids.     

Lipidomic Profiling of Sinus Mucosa from Patients with Chronic Rhinosinusitis

Sinusitis is a cause of significant morbidity, substantial healthcare costs, and negative effects on quality of life. The primary objective of this study is to characterize the previously unknown lipid profile of sinonasal mucosa from patients with chronic rhinosinusitis (CRS) and from controls. Sinus mucosa samples were analyzed from 9 CRS patients with concomitant nasal polyps, 11 CRS patients without polyps, and 12 controls. Ten lone polyp samples were also analyzed. Samples were subjected to a modified Bligh/Dyer lipid extraction, then high performance thin layer chromatography (HPTLC), combined gas chromatography/electron impact‐mass spectrometry (GC/EI‐MS), and flow‐injection/electrospray ionization‐tandem mass spectrometry (FI/ESI‐MS/MS). Data was analyzed for identification and profiling of major components. HPTLC revealed an array of species reflecting the lipid complexity of the samples. GC/EI‐MS revealed cholesterol and several fatty acids. FI/ESI‐MSMS revealed numerous lipid species, namely a host of phosphatidylcholines, phosphatidylethanolamines, ceramides and cholesteryl esters, but no detectable amounts of phosphatidyinositols or sulfated lipids. These results are a first step to uncover unique molecular biomarkers in CRS.     

NAD(P)‐dependent steroid dehydrogenase‐like protein and neutral cholesterol ester hydrolase 1 serve as novel markers for early detection of gastric cancer identified using quantitative proteomics

BackgroundGastric cancer (GC) is the third most common cause of cancer deaths worldwide. In the present study, we aimed to identify novel GC biomarkers by integrating isobaric tags of relative and absolute quantitation (iTRAQ) for aberrantly expressed proteins in GC patients.MethodsUsing stable isotope tags, we labeled an initial discovery group comprising four paired gastric cancer and adjacent gastric tissue samples, and subjected them to LC‐ESI‐MS/MS. We used a validation set comprising 129 paired gastric cancer and adjacent gastric tissues from patients and benign healthy controls to validate the candidate targets.ResultsWe identified two proteins, NAD(P)‐dependent steroid dehydrogenase‐like (NSDHL) and neutral cholesterol ester hydrolase 1 (NCEH1), that were significantly overexpressed in GC tissues. The sensitivity and specificity of NSDHL were 80.6% and 74.4%, respectively, in GC compared with a sensitivity of 25.6% in adjacent tissues and 24% in benign healthy controls. The area under the ROC curve (AUC) for NSDHL was 0.810 for GC detection. Overexpression of NSDHL in GC was significantly correlated with local tumor invasion. The sensitivity and specificity of NCEH1 were 77.5% and 73.6%, respectively, in GC compared with a sensitivity of 26.4% in adjacent tissues and 20% in benign controls. The AUC for NSDHL was 0.792. Overexpression of NCEH1 was significantly associated with tumor histological classification and local invasion. Moreover, a combined analysis of NSDHL and NCEH1 achieved a sensitivity and specificity of 85.7% and 83%, respectively, and the AUC was 0.872. The combined analysis of NSDHL and NCEH1 was significantly correlated with histological grade and TNM Ⅱ‐Ⅳ staging.ConclusionsiTRAQ‐labeled quantitative proteomics represents a powerful method to identify novel cancer biomarkers. The present study identified NSDHL and NCEH1 as useful biomarkers for screening, diagnosis, and prognosis of patients with gastric cancer.     

Plasma proteomics-based identification of novel biomarkers in early gastric cancer

BackgroundIdentification and treatment in the early stage can significantly improve the prognosis of gastric cancer (GC). However, to date, there is still no ideal biomarker that can be used for the screening of early stage GC (EGC). The proteomics supported by mass spectrometry offers more possibilities for discovering tumor biomarkers. The aim of this study was to explore candidate protein biomarkers for EGC screening with mass spectrometry and bioinformatics technology.MethodsPlasma samples were collected from 15 EGC patients and 15 healthy controls. After a selective immune-depletion to remove high abundance proteins, plasma samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with the tandem mass tags (TMT) labeling.ResultsA total of 2040 proteins were identified, and 11 proteins were found to be differentially expressed. The results of the logistic regression model and orthogonal signal correction-partial least squares discriminant analysis (OPLS-DA) model showed that the changed proteins identified by plasma proteomics could help distinguish EGC patients from healthy controls.ConclusionThe proteins identified by plasma proteomics using LC-MS/MS combined with TMT labeling could help distinguish EGC from healthy controls.     

Role of liquid chromatography-high-resolution mass spectrometry (LC-HR/MS) in clinical toxicology

Background. Gas chromatography (GC) and liquid chromatography (LC) coupled with mass spectrometry (MS) are widely used to confirm drug screening results and for urine screening in presumed intoxicated patients. These techniques are better suited to targeted analysis than to general unknown screening and, due to the complexity of testing, results are seldom available rapidly enough to contribute to the immediate care of the patient. High resolution (HR)/MS with time-of-flight (TOF) or orbitrap instruments offer potential advantages in clinical toxicology. Comparison of GC-MS, LC-MS/MS and LC-HR/MS. For unknown analyses, GC-MS and LC-MS/MS require comparison of full-scan spectra against preestablished libraries. Operation in full-scan mode greatly reduces sensitivity and some drugs present in low but significant concentrations may be missed. Selected ion monitoring (SIM) in GC/MS and selected reaction monitoring (SRM) in LC-MS/MS, where only targeted ions are monitored, increase sensitivity but require prior knowledge of what compound is to be measured. LC-HR/MS offers mass assignment with an accuracy of 0.001 atomic mass units (amu) compared with 1 amu in conventional MS. Tentative identification is thus directed to a very limited set of compounds (or even one unique compound) based on the exact molecular formula rather than a fragmentation pattern, since HR/MS can discriminate between compounds with the same nominal molecular mass. LC-MS/MS has clear advantages over GC/MS in ease and speed of sample preparation and the opportunities for its automation. LC-HR/MS is more suitable to clinical toxicology because the drugs present in a sample are rarely known a priori, and tentative identifications of unknowns can be made without the availability of a reference standard or a library spectrum. Blood can be used in preference to urine which is more relevant to the patient's current clinical situation. Methods. A literature search was conducted using PUBMED for clinical toxicology, adulterants in illicit drugs and herbal supplements, and case reports using LC-TOF/MS and LC-HR/MS. Only 42 papers in English were identified in these searches. LC-HR/MS in clinical toxicology. LC-HR/MS has been used to detect designer drugs, doping agents, (neurosteroids) and adulterants such as levamisole, a veterinary antihelmitic found in street cocaine, and pharmaceuticals in herbal medications marketed to contain only natural ingredients. LC-HR/MS has proved useful for cases where existing tests were unable to identify the cause of the intoxication. One patient suffered a drug-induced seizure which was originally thought to be caused by an herbal medication, but diphenhydramine was determined to be the culprit. In another, 5-oxoproline was identified as the cause of metabolic acidosis seen in chronic acetaminophen (paracetamol) use. LC-HR/MS has successfully identified medications that were mislabeled or misrepresented street drugs. In one case, medications sold as diazepam were determined to be glyburide instead. The identification of novel designer amines, stimulants found in "bath salts", and synthetic cannabinoids are well suited to LC-HR/MS. Dozens or even hundreds of possible compounds cannot realistically be tested on an individual basis by targeted LC-MS/MS or GC/MS analysis. Conclusions. LC-HR/MS offers unique opportunities for time-sensitive clinical analysis of blood samples from intoxicated patients and for comprehensive screening in a wide range of situations and materials. While the identification is not as definitive as that obtained by conventional fragmentation MS, the presumptive identification can be confirmed later with standards and spectral library matches. Optimum utilization of the presumptive diagnosis requires close collaboration between the laboratory analysts and their clinical counterparts.     

Role of liquid chromatography–high-resolution mass spectrometry (LC-HR/MS) in clinical toxicology

Background. Gas chromatography (GC) and liquid chromatography (LC) coupled with mass spectrometry (MS) are widely used to confirm drug screening results and for urine screening in presumed intoxicated patients. These techniques are better suited to targeted analysis than to general unknown screening and, due to the complexity of testing, results are seldom available rapidly enough to contribute to the immediate care of the patient. High resolution (HR)/MS with time-of-flight (TOF) or orbitrap instruments offer potential advantages in clinical toxicology. Comparison of GC-MS, LC-MS/MS and LC-HR/MS. For unknown analyses, GC-MS and LC-MS/MS require comparison of full-scan spectra against preestablished libraries. Operation in full-scan mode greatly reduces sensitivity and some drugs present in low but significant concentrations may be missed. Selected ion monitoring (SIM) in GC/MS and selected reaction monitoring (SRM) in LC-MS/MS, where only targeted ions are monitored, increase sensitivity but require prior knowledge of what compound is to be measured. LC-HR/MS offers mass assignment with an accuracy of 0.001 atomic mass units (amu) compared with 1 amu in conventional MS. Tentative identification is thus directed to a very limited set of compounds (or even one unique compound) based on the exact molecular formula rather than a fragmentation pattern, since HR/MS can discriminate between compounds with the same nominal molecular mass. LC-MS/MS has clear advantages over GC/MS in ease and speed of sample preparation and the opportunities for its automation. LC-HR/MS is more suitable to clinical toxicology because the drugs present in a sample are rarely known a priori, and tentative identifications of unknowns can be made without the availability of a reference standard or a library spectrum. Blood can be used in preference to urine which is more relevant to the patient's current clinical situation. Methods. A literature search was conducted using PUBMED for clinical toxicology, adulterants in illicit drugs and herbal supplements, and case reports using LC-TOF/MS and LC-HR/MS. Only 42 papers in English were identified in these searches. LC-HR/MS in clinical toxicology. LC-HR/MS has been used to detect designer drugs, doping agents, (neurosteroids) and adulterants such as levamisole, a veterinary antihelmitic found in street cocaine, and pharmaceuticals in herbal medications marketed to contain only natural ingredients. LC-HR/MS has proved useful for cases where existing tests were unable to identify the cause of the intoxication. One patient suffered a drug-induced seizure which was originally thought to be caused by an herbal medication, but diphenhydramine was determined to be the culprit. In another, 5-oxoproline was identified as the cause of metabolic acidosis seen in chronic acetaminophen (paracetamol) use. LC-HR/MS has successfully identified medications that were mislabeled or misrepresented street drugs. In one case, medications sold as diazepam were determined to be glyburide instead. The identification of novel designer amines, stimulants found in “bath salts”, and synthetic cannabinoids are well suited to LC-HR/MS. Dozens or even hundreds of possible compounds cannot realistically be tested on an individual basis by targeted LC-MS/MS or GC/MS analysis. Conclusions. LC-HR/MS offers unique opportunities for time-sensitive clinical analysis of blood samples from intoxicated patients and for comprehensive screening in a wide range of situations and materials. While the identification is not as definitive as that obtained by conventional fragmentation MS, the presumptive identification can be confirmed later with standards and spectral library matches. Optimum utilization of the presumptive diagnosis requires close collaboration between the laboratory analysts and their clinical counterparts.     

Gas chromatography for diagnosis and prognosis of destructive pancreatitis

One of modern and highly effective methods of diagnostics and differential diagnostics of infected pancreonecrosis is chromatographic technique, which allows for identification of anaerobic non-clostridial infection in the foci of pancreatic destruction by the presence of volatile fatty acids, the specific end products of anaerobic bacterial metabolism. Gas chromatography (GC) - mass spectrometry (MS) also make it possible to detect in peripheral blood of patients with pancreatitis certain metabolites (di-, polyamines, and aromatic amines) that are markers of tissue (protein structure) disintegration and the degree of pancreonecrosis, which is a valuable indicator of the degree of pancreonecrosis. The presence, according to GC - MS analysis, of natural inhibitors of transamidinase (compounds of thiourea and its metabolites, the group of mercaptopurines and mercapto-derivates of imidazole) in peripheral blood at the maximum level--0.71 to 0.78 mmol/l on days 7 to 10 upon the onset of the disease--is a prognostically favorable criterion. At the same time, the presence of the maximum level of anaerobic non-clostridial infection metabolite in peripheral blood 1.12 to 1.31 mmol/l on days 7 to 10 upon the onset of the disease--is prognostically infavorable. These chromatographic criteria in combination with clinical manifestations can be considered indications to surgical treatment of infected pancreonecrosis, and the prognosis of the disease can be based on them.     

ATP-生物荧光肿瘤药敏试验在筛选肺癌化疗药物中的应用

目的 探讨肿瘤体外药敏实验ATP生物荧光法（ATP-TCA）在肺癌化疗中的应用。方法 应用ATP-TCA对48例肺癌根治术标本、6例淋巴结活检标本、4例胸腔积液标本进行药敏检测。结果 ATP-TCA方法的可评估率为95％。健择（GEM）、诺维本（NVB）、阿霉素（ADM）分别与顺铂（DDP）组成两药联合方案（GP、NP、AP）。它们的敏感率分别为78％、64％、27％。化疗药物对肺癌的杀伤作用具有较强的个体差异性。结论 ATP-TCA是一种灵敏、可靠的肿瘤药敏感检测技术，可用于肺癌化疗中化疗药物的筛选。     

胃癌血清脂肪酸和脂肪酸酰胺水平的变化及其意义

目的:调查胃癌血清脂肪酸和脂肪酸酰胺的改变并探讨与胃癌相关的特征性小分子标记物。方法:利用GC/MS对30例胃癌和30例健康血清的脂肪酸和脂肪酸酰胺进行定量分析。结果:分析获得11种脂肪酸和3种脂肪酸酰胺,利用OPLS-DA建模可以较好地区分胃癌组和对照组,并且发现4种脂肪酸的变化具有统计学差异,8种脂肪酸对两组患者的区分作用具有较大的贡献(VIP值>1)。结论:本实验发现胃癌血清中脂肪酸和脂肪酸酰胺的代谢发生了紊乱,血清脂类对于该病的诊断具有很大的潜力。     

Urine 4-heptanone: a beta-oxidation product of 2-ethylhexanoic acid from plasticisers

4-Heptanone is a common volatile constituent of human urine and is of unknown origin. We hypothesised that it arises from in vivo beta-oxidation of 2-ethylhexanoic acid (EHA) from plasticisers, similar to formation of 3-heptanone from valproic acid. We investigated urine from individuals with normal and increased plasticiser exposure. Using GC/MS, solvent-extracted organic acids were analysed as trimethylsilyl (TMS) derivatives and heptanone with headspace solid-phase microextraction. We identified 3-oxo-2-ethylhexanoic acid, the beta-oxidation product of EHA, as an enol in all samples. This is the first report of its TMS mass spectrum. We also found 2-ethyl-1,6-hexanedioic acid and 5-hydroxyEHA, omega- and omega-1-oxidation products of EHA, respectively, and 2-ethylhexanoylglucuronide, but only in trace amounts in some plasticiser samples. These compounds have not been reported in human urine, nor has the TMS mass spectrum of 5-hydroxyEHA. The median concentrations of 3-oxoethylhexanoic acid and total 4-heptanone of seven plasticiser samples were around 30--175-fold higher than normal samples. 4-Heptanone was barely detectable and 3-oxoethylhexanoic acid was not increased in an eighth plasticiser sample, from a baby with deficiency of 2-methylbranched-chain acyl-CoA dehydrogenase. beta-Oxidation is a major catabolic pathway of EHA in man, and might be involved in the metabolism of other branched-chain drugs and environmental pollutants.     

Erythrocyte galactose 1-phosphate quantified by isotope-dilution gas chromatography-mass spectrometry

BACKGROUND: Measurements of alpha-D-galactose 1-phosphate (Gal-1-P) in erythrocytes are used to monitor the adequacy of dietary therapy in the treatment of galactosemia. We have devised a gas chromatography-mass spectrometry (GC/MS) isotope-dilution method for quantification of Gal-1-P. METHODS: We prepared trimethylsilyl (TMS) derivatives and used alpha-D-[2-(13)C]Gal-1-P as the internal standard for GC/MS. Results obtained with this method were compared with those determined by the established enzymatic method for samples from 23 healthy individuals (11 children and 12 adults), 9 suspected patients with galactosemia, 12 galactosemic patients on diet therapy, and 2 newly diagnosed toxic neonates. RESULTS: The method was linear up to 2.5 mmol/L with a lower limit of detection of 2.1 nmol (0.55 mg/L). Intra- and interassay imprecision (CVs) was 2.2-8.8%. In the 23 healthy individuals, values ranged from nondetectable to 9.2 micromol/L (2.4 mg/L of packed erythrocytes). Galactosemic patients on diet therapy had values of 10.9-45 mg/L of packed erythrocytes, whereas the newly identified patients had values of 166 and 373 mg/L. CONCLUSIONS: The GC/MS method is precise and useful over the wide range of concentrations needed to assess the galactose burden in patients with galactosemia.     

Determination of amphetamine,methamphetamine, MDA and MDMA in human hair by GC‐EI‐MS after derivatization with perfluorooctanoyl chloride

The aim of this study was to develop a quantitative gas chromatography mass spectrometry (GC‐MS) method to determine the classical amphetamines and their methylenedioxylated derivatives in human hair. The procedure involved liquid–liquid extraction of hydrolysed hair spiked with deuterated internal standards and direct derivatization with perfluorooctanoyl chloride. After evaporation of the organic phase and dissolution in butylacetate, the derivatized compounds were injected into a GC‐MS. Method validation results showed a linear range from 0.25 to 25?ng/mg for the target compounds: amphetamine (AM), methamphetamine (MA), methylenedioxyamphetamine (MDA) and methylenedioxymethamphetamine (MDMA or ecstasy). An intra‐day precision of 3–6?% RSD and an inter‐day precision of 3–17?% RSD were observed. Trueness was between 96?% and 106?% for the target compounds. The limit of detection ranged from 0.07 to 0.14?ng/mg and of quantification from 0.24 to 0.46?ng/mg, depending on compound. The method was applied on 40 authentic hair samples (segmented or pooled hair), of which 15 cases involved amphetamine and/or ecstasy. The hair concentrations ranged from LOD to 3.2?ng/mg of AM in 7 cases, to 0.4?ng/mg of MDA in 3 cases and to 5.9?ng/mg of MDMA in 13 cases. MA was only detected once at trace level. The method, including the derivatization procedure, is simple and robust with a sensitivity that is satisfactory for measurement of amphetamines and ecstasy in hair from abusers.     

Strategies towards simpler configuration and higher peak capacity with comprehensive multidimensional gas chromatography

Experimental and data analysis approaches in multidimensional gas chromatography (MDGC) comprising comprehensive multiple heart-cut (H/C) and comprehensive two dimensional GC (GC × GC) were developed with an example application illustrated for analysis of a technical glycol precursor sample. The GC × GC system employed a long ¹D (30 m) and a short ²D (5 m) column with a flow modulator and a Deans switch (DS) as a splitter; meanwhile. The H/C system was applied solely as a DS located between long ¹D (30 m) and ²D (60 m) columns without use of cryogenic trapping devices. The effects of injection time and ²D column flow in GC × GC and the impacts of H/C window and number of injections (total analysis time) in H/C analysis were investigated. The analysis performance for each condition was evaluated according to peak capacity and number of separated compounds. The continuum between the two techniques was then established via the relationship between analysis time and analysis performance. The separation performances were improved with longer analysis time so that the suitable condition was selected within this compromise. Under the selected conditions, volatile compounds in the technical glycol precursor sample were identified according to the match between the experimental MS spectra and first dimensional retention indices (¹I) with that from the NIST2014 database and literature. An hour analysis with GC × GC resulted in a total peak capacity of 798, number of separated peaks of 61 and average MS match score of 887 ± 35; meanwhile, the corresponding numbers were improved to be 9198, 107 and 898 ± 24, respectively, with the 25 h comprehensive H/C analysis.

Experimental and data analysis approaches in MDGC comprising comprehensive H/C and GC × GC were developed with an example application illustrated for analysis of a technical glycol precursor sample.     

吸烟肺癌患者口腔黏膜细胞全线粒体DNA获得性突变分析吸烟/口腔黏膜获得性突变与康复预防

目的了解肺癌患者口腔黏膜mtDNA获得性突变情况,探讨肺癌外细胞的线粒体基因突变与肺癌的关系和作为对肺癌预测的生物学指标的可能性。方法利用时相温度梯度凝胶电泳法对12例肺癌患者(吸烟者10例,非吸烟者2例)口腔黏膜细胞及匹配的血液细胞线粒体DNA进行突变筛选,然后进行测序分析。结果在大部分吸烟患者中(8/9),发现口腔黏膜具有两个以上的获得性突变,而在两个非吸烟的患者仅发现1个突变。在11例(11/12,92%)患者中共发现有26个获得性突变,15个突变发生在高变的D环区(58%),11个在mRNA区(42%)。除303～309位点具有长度不稳定外,未见其他微卫星不稳定和5kb的大范围缺失突变。结论吸烟的肺癌患者口腔黏膜细胞线粒体DNA具有高发生频率的获得性突变。     

肺鳞癌组织中208个肺癌相关基因的表达

目的 病理学上诊断同样为肺鳞癌的患者,其治疗疗效和愈后往往存在一定差异,本研究利用本实验室制作的 208个肺癌相关基因芯片,探讨了 7例肺鳞癌组织中基因表达的异质性,试图为患者的治疗和预后提供分子依据. 方法 提取 7例男性肺鳞癌患者(年龄在 55～ 65岁之间)癌组织的总 RNA并用 LD- PCR标记成探针,然后与含有 208个肺癌相关基因的 cDNA Microarray杂交,芯片用 ImaGene软件分析和处理数据. 结果 7例肺鳞癌组织之间 208个肺癌相关基因表达的相似性在 60.65% ～ 82.69%之间. 结论 本研究首次对肺鳞癌患者不同个体之间基因的表达进行研究并发现存在一定的差异,提示应注意癌症患者的个体化诊断和治疗.     

Detection and significance of serum protein markers of small-cell lung cancer

Currently, no satisfactory biomarkers are available to screen for small-cell lung cancer (SCLC). We applied a surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) ProteinChip system to detect 150 serum samples (including 54 SCLC patients, 24 non-small cell lung cancer [NSCLC] patients, 32 pneumonia patients, and 40 healthy individuals). The spectra data were analyzed by support vector machine (SVM) and potential biomarkers were chosen for the system training and used to construct diagnostic model. Pattern 1, constructed of four protein peaks with mass/charge (m/z) of 4,293 Da, 4,612 Da, 6,455 Da, and 7,582 Da, separated SCLC patients from the healthy individuals with a sensitivity of 88.9% and a specificity of 85.7%. This pattern performed significantly better than the current marker, neuron-specific enolase (NSE) (P<0.05). Pattern 2, constructed of protein peaks with mass/charge (m/z) of 2,764 Da and 1,7368 Da, separated SCLC from pneumonia with a sensitivity of 88.9% and a specificity of 91.7%. Pattern 3, constructed of another three protein peaks with m/z of 3,912 Da, 7,562 Da, and 13,777 Da, separated SCLC from NSCLC. The sensitivity and specificity were 83.3% and 75.0%, respectively. These results suggested that SELDI-TOF MS combined with support vector machine yields significantly higher sensitivity and specificity for the detection of serum protein of SCLC.     

False Positive Acetaminophen Levels Associated with Hyperbilirubinemia

Serum acetaminophen determination is frequently necessary in patients with hepatic failure. We observed two patients (#1, #2) with elevated serum total bilirubin levels (26.5 mg/dL and 40.1 mg/dL) who had multiple false positive acetaminophen levels using the kinetic method of the GDS Diagnostics enzymatic acetaminophen assay (GDS Diagnostics, Elkhart, IN). We investigated the magnitude, threshold, and linearity of this effect using the GDS Diagnostics assay and an EMIT acetaminophen assay on two other hyperbilirubinemic patients (#3, #4) and a commercial solubilized bilirubin standard. Samples were diluted using fresh frozen plasma, and acetaminophen levels were analyzed twice using the kinetic method of the GDS Diagnostic acetaminophen assay and twice with the EMIT assay. The absence of acetaminophen in all samples was verified by gas chromatography/mass spectroscopy (GC/MS). The kinetic GDS assay resulted in a positive acetaminophen assay (cutoff for a positive result = 10 mg/L) with patient #3, patient #4, and in the bilirubin standard when the total bilirubin levels were 28.2 mg/dL, 22.5 mg/dL, and 18.3 mg/dL, respectively. One sample was interpolated to give a positive acetaminophen reading when diluted to a total bilirubin concentration of 15 mg/L. None of the samples tested with GC/MS or the EMIT assay resulted in any detectable acetaminophen. In conclusion, caution must be taken utilizing the GDS Diagnostic assay for the quantification of acetaminophen with concomitant hyperbilirubinemia. Alternatives such as EMIT or GC/MS should be employed to assess acetaminophen levels in such patients.