Carbonic anhydrase IX as a marker for poor prognosis in soft tissue sarcoma.

PURPOSE: Hypoxia is associated with malignant progression and poor outcome in several human tumors, including soft tissue sarcoma. Recent studies have suggested that carbonic anhydrase (CA) IX is an intrinsic marker of hypoxia, and that CA IX correlates with poor prognosis in several types of carcinoma. The aim of this study was to quantify the extent of CA IX expression and to investigate whether CA IX is a marker for poor prognosis in soft tissue sarcoma patients at high risk of developing metastasis. EXPERIMENTAL DESIGN: Archival paraffin-embedded blocks were retrieved from 47 patients with deep, large, high-grade soft tissue sarcoma. Sections from two separate and representative tumor areas were immunostained for CA IX, and the CA IX-positive area fraction was quantified by image analysis, excluding areas of normal stroma and necrosis that were identified from serial H&E-stained sections. Patients were then subject to survival analysis. RESULTS: CA IX-positive area fractions of viable tumor tissue varied significantly between tumors (range, 0-0.23; median, 0.004), with positive membranous CA IX staining in 66% (31 of 47) of the tumors. Patients with CA IX-positive tumors had a significantly lower disease-specific and overall survival than patients with CA IX-negative tumors (P = 0.033 and P = 0.044, respectively). CONCLUSIONS: These data suggest that CA IX, a potential intrinsic marker of hypoxia, predicts for poor prognosis in patients with deep, large, high-grade soft tissue sarcoma. Larger studies are required to determine whether CA IX has independent prognostic value in this group of tumors.     

Carbonic anhydrase IX in bladder cancer

BACKGROUND:

The objective of this study was to evaluate the role of carbonic anhydrase IX (CAIX) in urothelial carcinoma of the bladder.

METHODS:

A tissue microarray was constructed that contained 724 tissue samples from 340 patients. Immunohistochemical staining was performed using the antibody MN‐75, the percentage of positive cells was evaluated, and their association with tumor (T) classification, grade, and survival was assessed.

RESULTS:

All normal urothelial tissue samples were negative for CAIX expression, whereas 71% of bladder cancers expressed CAIX. CAIX expression was higher in noninvasive (Ta) versus invasive (T1‐T4) tumors (P < .001), in low‐grade versus high‐grade bladder cancer (P < .001), and in metastases versus the corresponding primary tumor (P = .032). For patients with nonmuscle invasive carcinoma who underwent transurethral resection (TUR), higher CAIX expression was associated with poorer recurrence‐free survival (P = .001). In addition, for patients with T1 tumors who underwent TUR, higher CAIX expression conveyed a 6.5‐fold higher risk of progression into muscle‐invasive disease (P = .006). In patients who underwent cystectomy, higher CAIX expression was associated with worse overall survival (P = .003). Multivariate Cox models revealed that CAIX expression was the strongest, independent prognostic factor of recurrence‐free survival (hazard ratio, 2.29; P = .001) and overall survival (hazard ratio, 1.9; P < .001).

CONCLUSIONS:

CAIX was expressed differentially in noninvasive versus invasive tumors, in low‐grade versus high‐grade bladder cancer, and in primary tumors versus metastases. The current results indicated that CAIX is a strong predictor of recurrence, progression, and overall survival of patients with bladder cancer; and the integration of CAIX expression into conventional prognostic models significantly improved their predictive accuracy. The data suggest a tripartite role of CAIX as a diagnostic, prognostic, and therapeutic molecular marker in bladder cancer. Cancer 2009. © 2009 American Cancer Society.     

Carbonic anhydrase 9 in clear cell renal cell carcinoma: A marker for diagnosis,prognosis and treatment

Carbonic anhydrase 9 (CA9) is a transmembrane member of the carbonic anhydrase family. It catalyses the reversible hydration of carbon dioxide into bicarbonate and a proton, thus enabling tumour cells to maintain a neutral pH despite an acidic microenvironment. CA9 is not expressed in healthy renal tissue but is expressed in most clear cell renal cell carcinomas (CCRCC) through HIF-1α accumulation driven by hypoxia and inactivation of the VHL gene. CA9 expression can be detected in the tumour by immunohistochemistry (IHC), in blood and tissue by ELISA assay and RT-PCR. It has a 100% diagnostic specificity in solid renal tumours, while ELISA assays on aspiration fluids may help in atypical cysts. Blood-based assays, ELISA for CA9 antigen and RT-PCR for CA9 mRNA are promising for the prognosis and follow-up of localised CCRCC. In metastatic disease, high CA9 expression by IHC was reported to be a powerful prognostic marker with better survival and sensitivity to IL-2, but this is still debated. Almost no data are currently available on the association of CA9 expression and outcome to targeted drugs. The prognostic value of CA9 in CCRCC could be explained by the frequent VHL gene inactivation driving an early activation of the HIF pathway. The poorer prognosis associated with low CA9 expressing tumours could be due to the simultaneous overexpression of EGFR contributing to the activation of AkT and mTOR pathways. Targeting CA9 by inhibitors, radioimmunotherapy, monoclonal antibodies or vaccination is promising and offers new avenues for clinical research.     

Carbonic anhydrase IX in early-stage non-small cell lung cancer.

PURPOSE: Tumor hypoxia is associated with poor prognosis and increased tumor aggressiveness. Carbonic anhydrase (CA) IX, an endogenous marker for tumor hypoxia, catalyzes the hydration of carbon dioxide into carbonic acid and contributes to the pH regulation of tumor cells. Therefore, CA IX might allow tumors to acclimate to a hypoxic microenvironment, promoting tumor cell proliferation. We hypothesized that CA IX expression is related to tumor cell proliferation and poor disease-free survival in patients with early-stage non-small-cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: CA IX expression was measured in 75 resected NSCLC tumors to assess prognostic implications for disease-free survival. The relationship of CA IX expression with microvessel density (MVD) and proliferation (Ki-67) index was assessed via colocalization analysis. RESULTS: All patients had operable NSCLC (stage I, 58; stage II, 17). CA IX expression was present in 54 (72%) of 75 patients and was associated with tumor necrosis (P < 0.05). CA IX-positive tumor areas showed greater cell proliferation as measured by Ki-67 index (P < 0.05) and less MVD (P < 0.05) than did CA IX-negative areas in colocalization analysis. The percentage of CA IX-positive tumor cells was significantly related to postoperative recurrence and poor disease-free survival (P < 0.05). Ki-67 index and pathologic stage were also independent prognostic factors for worse disease-free survival (P < 0.05). CONCLUSIONS: CA IX expression of tumor cells may be an indicator for poor disease-free survival in early-stage NSCLC.     

Detection of an intrinsic marker in hypoxic cells

An autoradiographic method is presented for detecting, within a cell population, those cells which have been subjected to chronic hypoxia. No radioisotope is administered; rather the photographic emulsion is chemically reduced by intrinsic constituents of the cells. Hypoxic regions in the sandwich system, a multicellular in vitro tumor model, were detected in this manner. These regions were then compared with hypoxic sandwich regions as demonstrated by [3H]misonidazole labeling. Auxiliary studies, including studies on hypoxic monolayers, were consistent with the sandwich results. In all cases, the intracellular distribution of the chemographic grains was found to be cytosolic. Often the grains were clustered near the nucleus, perhaps in the region of the endoplasmic reticulum and the Golgi. We conclude that cells in a state of hypoxia and nutrient deprivation similar to that found in solid tumors retain a detectably altered biology for a significant period after reoxygenation. Therefore systematic methods of detecting previous hypoxia in histological tumor sections are feasible.     

Characterization of carbonic anhydrase IX (CA IX) as an endogenous marker of chronic hypoxia in live human tumor cells

PURPOSE: Published clinical studies provide conflicting data regarding the prognostic significance of carbonic anhydrase IX (CA IX) overexpression as an endogenous marker of tumor hypoxia and its comparability with other methods of hypoxia detection. We performed a systematic analysis of CA IX protein levels under various in vitro conditions of tumor hypoxia in HT 1080 human fibrosarcoma and FaDu human pharyngeal carcinoma cells. Because sorting of live CA IX positive cells from tumors provides a tool to study the radiosensitivity of chronically hypoxic cells, we modified and tested a CA IX flow cytometry protocol on mixed hypoxic/aerobic suspensions of HT 1080 and FaDu cells. METHODS AND MATERIALS: HT 1080 and FaDu cells were treated with up to 24 h of in vitro hypoxia and up to 96 h of reoxygenation. To test the effect of nonhypoxic stimuli, glucose and serum availability, pH and cell density were modified. CA IX protein was quantified in Western blots of whole-cell lysates. Mixed suspensions with known percentages of hypoxic cells were prepared for CA IX flow cytometry. The same mixtures were assayed for clonogenic survival after 10 Gy. RESULTS: Hypoxia-induced CA IX protein expression was seen after >6 h at < or =5% O(2), and protein was stable over 96 h of reoxygenation in both cell lines. Glucose deprivation abolished the hypoxic CA IX response, and high cell density caused CA IX induction under aerobic conditions. Measured percentages of CA IX-positive cells in mixtures closely reflected known percentages of hypoxic cells in HT 1080 and were associated with radioresistance of mixtures after 10 Gy. CONCLUSION: CA IX is a stable marker of current or previous chronic hypoxia but influenced by nonhypoxic stimuli. Except the time course of accumulation, all properties of this marker resembled our previous findings for hypoxia-inducible factor-1alpha. A modified flow cytometry protocol provided good separability of CA IX-negative and -positive cells in vitro and can be applied to sort chronically hypoxic cells from tumors.     

Carbonic anhydrase IX is highly expressed in hereditary nonpolyposis colorectal cancer.

Carbonic anhydrase (CA) II, CA IX, and CA XII are expressed in various neoplasias and have been linked to tumorigenesis. We examined their expression in three different groups of colorectal cancer [i.e., microsatellite stable (MSS), microsatellite instable (MSI), and hereditary nonpolyposis colorectal cancer (HNPCC)]. First, we analyzed gene expression profiles of 113 specimens by a microarray method to study the expression of various CA isozymes in the subgroups of colorectal cancer. The results indicated that mRNAs for CA II and CA XII are down-regulated and CA IX mRNA is up-regulated in all three tumor categories when compared with the normal tissue. The up-regulation of CA IX was greatest in the HNPCC group. For more information, 77 specimens were immunohistochemically stained to study the levels of CA II, CA IX, and CA XII. Immunohistochemical analyses further confirmed that the subgroups express CA II, CA IX, and CA XII differentially, and the HNPCC tumors express high levels of CA IX. Expression of these CAs did not correlate to Dukes stage or grade of differentiation. Our results show that CAs are differentially expressed in the subgroups of colorectal cancer, and CA IX expression seems to be very high in most cases of HNPCC. CA IX could be a potential diagnostic and therapeutic target in HNPCC.     

Carbonic anhydrase XII is a marker of good prognosis in invasive breast carcinoma

Hypoxia and pH influence gene expression in tumours, and it is becoming increasingly clear that the pattern of genes expressed by a tumour determines its growth and survival characteristics. Hypoxia-inducible factor-1 (HIF-1) is a key mediator of the cellular response to hypoxia and high HIF-1 expression has been identified as a poor prognostic factor in tumours. Recently, we identified the tumour-associated carbonic anhydrases (CA), CA9 and CA12 as hypoxia-inducible in tumour cell lines. Furthermore, we identified CA IX to be a poor prognostic factor in breast cancer. The aim of this study was to assess the prognostic significance of CA XII. CA XII expression was studied by immunohistochemistry in a series of 103 cases of invasive breast cancer and any association with recognised prognostic factors or relation with the outcome was examined. CA XII expression was present in 77 out of 103 (75%) cases and was associated with lower grade (P=0.001), positive estrogen receptor status (P<0.001), and negative epidermal growth factor receptor status (P<0.001). Furthermore, although CA XII expression was associated with an absence of necrosis (P<0.001), expression of CA XII in some high-grade tumours was induced in regions directly adjacent to morphological necrosis. Additionally, using univariate analysis, CA XII positive tumours were associated with a lower relapse rate (P=0.04) and a better overall survival (P=0.01). In conclusion, CA XII expression is influenced both by factors related to differentiation and hypoxia in breast cancer in vivo and CA XII expression is associated with a better prognosis in an unselected series of invasive breast carcinoma patients.     

Carbonic anhydrase IX expression predicts outcome of interleukin 2 therapy for renal cancer.

PURPOSE: Renal cancer response to interleukin 2 (IL-2) therapy and patient survival has been correlated with tumor histology and carbonic anhydrase IX (CAIX) expression. In an effort to confirm and expand these observations, we examined CAIX expression in pathology specimens from renal cancer patients who had previously received IL-2 therapy. EXPERIMENTAL DESIGN: Paraffin-embedded tissue sections of renal cancer were immunostained with the MN-75 monoclonal antibody to CAIX and expression levels were correlated with histologic findings and clinical outcome. RESULTS: Tissue specimens were obtained from 66 patients; 27 of whom (41%) had responded to IL-2-based therapy. Fifty-eight specimens were assessed as clear cell, with 56, 33, and 4 having alveolar, granular, and papillary features, respectively. Twenty-four (36%), 31 (47%), and 11 (17%) were classified into good, intermediate, and poor prognosis groups according to the Upton pathology model. Forty-one specimens (62%) had high CAIX expression. Twenty-one of 27 (78%) responding patients had high CAIX expressing tumors compared with 20 of 39 (51%) nonresponders (odds ratio, 3.3; P = 0.04). Median survival was prolonged (P = 0.04) and survival >5 years was only seen in high CAIX expressers. In patients with intermediate pathologic prognosis, all nine responders had high CAIX expression versus 11 of 22 nonresponders. A resultant group with good pathologic prognosis alone or with intermediate pathologic prognosis and high CAIX contained 26 of 27 (96%) responders compared with 18 of 39 (46%) nonresponders (odds ratio, 30; P < 0.01) and exhibited longer median survival (P < 0.01). CONCLUSIONS: CAIX expression seems to be an important predictor of outcome in renal cell carcinoma patients receiving IL-2-based therapy and may enhance prognostic information obtained from pathology specimens.     

Carbonic anhydrase IX, an endogenous hypoxia marker, expression in head and neck squamous cell carcinoma and its relationship to hypoxia, necrosis, and microvessel density.

Carbonic anhydrase IX (CA IX) is a transmembrane glycoprotein with an active extracellular enzyme site. We have shown previously that it was hypoxia inducible and may therefore be an endogenous marker of hypoxia. It is overexpressed in some tumors, particularly renal cell carcinoma. The aim of this study was to examine the expression and localization of CA IX in head and neck squamous cell carcinoma (HNSCC) and relate this to the location of tumor microvessels, angiogenesis, necrosis, and stage. Expression of CA IX was determined by immunoblotting in three HNSCC cell lines grown in normoxia and hypoxia (pO(2) 0.1%) and three paired tumor and normal tissue samples of HNSCC. Archived paraffin sections (79) of HNSCC were immunostained with antibodies to CA IX and CD34 to determine microvessel density (MVD). By double staining sections with CA IX and CD34, the distance between blood vessels and the start of CA IX expression and necrosis was calculated. CA IX was induced by hypoxia in all three HNSCC cell lines and overexpressed in HNSCC tumor tissue. Overexpression was localized to the perinecrotic area of the tumor on immunostaining, and the percentage area of the tumor expressing CA IX was significantly higher with more tumor necrosis (P = 0.001), a high MVD (P = 0.02), and advanced stage (P = 0.033) on univariate analysis and necrosis (P = 0.0003) and MVD (P = 0.0019) on multivariate analysis. The median distance between a blood vessel and the start of CA IX expression was 80 microm (range, 40-140 microm). CA IX is overexpressed in HNSCC because of hypoxia and is a potential biomarker for hypoxia in this tumor. Overexpression may help to maintain the intracellular pH, giving tumor cells a survival advantage and enhancing resistance to radiotherapy and chemotherapy. CA IX is a potential target for future therapy in HNSCC.     

Carbonic anhydrase IX expression, hypoxia, and prognosis in patients with uterine cervical carcinomas.

PURPOSE: Hypoxia is associated with adverse outcome for a number of solid tumors, including cervical carcinomas. Direct pO(2) measurement requires specialized equipment and expertise that is not generally available. Immunohistochemical measurement of intrinsic tissue markers of hypoxia is an alternative approach. Recent studies suggest that carbonic anhydrase IX (CA IX), which is regulated via hypoxia-inducible factor 1, is a useful intrinsic marker of tumor hypoxia. EXPERIMENTAL DESIGN: Biopsies were obtained from 110 patients with locally advanced cervical carcinoma treated with radiotherapy or chemoradiotherapy. Tissue sections were labeled using an immunofluorescence technique and CA IX expression in the viable tumor area measured using a semiautomated fluorescence image analysis technique. Results were compared with direct pO(2) values obtained using an Eppendorf probe and to patient outcome. Intratumoral heterogeneity of CA IX expression was examined in a subgroup of patient who underwent multiple biopsies. RESULTS: The median percentage of tumor area staining for CA IX was 3.56 (range, 0.01-58.85). CA IX staining did not correlate with the Eppendorf pO(2) measurements. Whereas the latter values were predictive of patient outcome, the CA IX levels were not. Measurement of CA IX in multiple biopsies indicated that intratumoral heterogeneity accounted for 41% of the total variance in the data set. CONCLUSIONS: In contrast to some recent studies, we did not find significant associations between CA IX expression and tumor pO(2) levels or patient outcome in locally advanced carcinomas of the cervix. Probable explanations relate to the problems of sampling error using single biopsies and the existence of biological factors other than hypoxia that influence CA IX levels.     

Matrix metalloproteinase-9 is a prognostic marker for patients with cervical cancer

Cervical cancer remains one of the most common malignancies in women. Previous study proved MMP-9 might be prognostic marker for multiple human malignancies. The present study was to investigate the protein expression of MMP-9 in cervical cancer and its association with clinicopathological characteristics as well as prognosis of patients. Cervical cancer specimens from 225 cases who had not received chemotherapy or radiotherapy prior to surgery were collected. Immunochemistry assays were utilized to investigate MMP-9 protein expression. Results showed that MMP-9 expression was increased in cervical cancer and associated with stromal invasion, FIGO stage, lymph node metastasis, and vascular invasion. Kaplan?CMeier analysis showed that patients with cervical cancer of positive MMP-9 staining tend to have worse overall survival. In multivariate analysis stratified for known prognostic variables, MMP-9 was proved to be an independent prognostic factor. The present study confirmed that MMP-9 expression in cervical cancer was an independent prognostic factor of patients, which might be a potential diagnostic and even therapeutic target of cervical cancer.     

Carbonic anhydrase IX expression and outcome after radiotherapy for muscle-invasive bladder cancer

AimsCarbonic anhydrase IX (CA IX) expression has been described as an endogenous marker of hypoxia in solid neoplasms. Furthermore, CA IX expression has been associated with an aggressive phenotype and resistance to radiotherapy. We assessed the prognostic significance of CA IX expression in patients with muscle-invasive bladder cancer treated with radiotherapy.Materials and methodsA standard immunohistochemistry technique was used to show CA IX expression in 110 muscle-invasive bladder tumours treated with radiotherapy. Clinicopathological data were obtained from medical case notes.ResultsCA IX immunostaining was detected in 89 (∼81%) patients. Staining was predominantly membranous, with areas of concurrent cytoplasmic and nuclear staining and was abundant in luminal and perinecrotic areas. No significant correlation was shown between the overall CA IX status and the initial response to radiotherapy, 5-year bladder cancer-specific survival or the time to local recurrence.ConclusionsThe distribution of CA IX expression in paraffin-embedded tissue sections seen in this series is consistent with previous studies in bladder cancer, but does not provide significant prognostic information with respect to the response to radiotherapy at 3 months and disease-specific survival after radical radiotherapy.     

Carbonic anhydrase IX expression, a novel surrogate marker of tumor hypoxia, is associated with a poor prognosis in non-small-cell lung cancer.

PURPOSE: To evaluate carbonic anhydrase (CA) IX as a surrogate marker of hypoxia and investigate the prognostic significance of different patterns of expression in non-small-cell lung cancer (NSCLC). METHODS: Standard immunohistochemical techniques were used to study CA IX expression in 175 resected NSCLC tumors. CA IX expression was determined by Western blotting in A549 cell lines grown under normoxic and hypoxic conditions. Measurements from microvessels to CA IX positivity were obtained. RESULTS: CA IX immunostaining was detected in 81.8% of patients. Membranous (m) (P =.005), cytoplasmic (c) (P =.018), and stromal (P <.001) CA IX expression correlated with the extent of tumor necrosis (TN). The mean distance from vascular endothelium to the start of tumor cell positivity was 90 micro m, which equates to an oxygen pressure of 5.77 mmHg. The distance to blood vessels from individual tumor cells or tumor cell clusters was greater if they expressed mCA IX than if they did not (P <.001). Hypoxic exposure of A549 cells for 16 hours enhanced CA IX expression in the nuclear and cytosolic extracts. Perinuclear (p) CA IX (P =.035) was associated with a poor prognosis. In multivariate analysis, pCA IX (P =.004), stage (P =.001), platelet count (P =.011), sex (P =.027), and TN (P =.035) were independent poor prognostic factors. CONCLUSION: These results add weight to the contention that mCA IX is a marker of tumor cell hypoxia. The absence of CA IX staining close to microvessels suggests that these vessels are functionally active. pCA IX expression is representative of an aggressive phenotype.     

Hypoxia-related carbonic anhydrase 9 induces serpinB9 expression in cancer cells and apoptosis in T cells via acidosis

Hypoxia is a common feature of solid tumors. However, the impact of hypoxia on immune cells within tumor environments remains underexplored. Carbonic anhydrase 9 (CA9) is a hypoxia-responsive tumor-associated enzyme. We previously noted that regardless of human CA9 (hCA9) expression, hCA9-expressing mouse renal cell carcinoma RENCA (RENCA/hCA9) presented as a “cold” tumor in syngeneic aged mice. This study delves into the mechanisms behind this observation. Gene microarray analyses showed that RENCA/hCA9 cells exhibited elevated mouse serpinB9, an inhibitor of granzyme B, relative to RENCA cells. Corroborating this, RENCA/hCA9 cells displayed heightened resistance to antigen-specific cytotoxic T cells compared with RENCA cells. Notably, siRNA-mediated serpinB9 knockdown reclaimed this sensitivity. In vivo tests showed that serpinB9 inhibitor administration slowed RENCA tumor growth, but this effect was reduced in RENCA/hCA9 tumors, even with adjunctive immune checkpoint blockade therapy. Further, inducing hypoxia or introducing the mouse CA9 gene upregulated serpinB9 expression, and siRNA-mediated knockdown of the mouse CA9 gene inhibited the hypoxia-induced induction of serpinB9 in the original RENCA cells. Supernatants from RENCA/hCA9 cultures had lower pH than those from RENCA, suggesting acidosis. This acidity enhanced serpinB9 expression and T cell apoptosis. Moreover, coculturing with RENCA/hCA9 cells more actively prompted T cell apoptosis than with RENCA cells. Collectively, these findings suggest hypoxia-associated CA9 not only boosts serpinB9 in cancer cells but also synergistically intensifies T cell apoptosis via acidosis, characterizing RENCA/hCA9 tumors as “cold.”     

CRISPR/Cas9敲除内源TCR增强TCR-T细胞对HPV16阳性宫颈癌SiHa细胞的杀伤

目的：基于CRISPR/Cas9基因编辑技术制备无内源TCR的TCR-T细胞并鉴定其在体外杀伤HPV16阳性宫颈癌SiHa细胞的功能。方法：培养健康志愿者外周血CD8⁺T细胞和Jurkat细胞,CRISPR/Cas9基因编辑技术敲除CD8⁺T、Jurkat细胞的TCR基因,制备过表达转基因TCR的重组慢病毒,在敲除内源性TCR的CD8⁺T和Jurkat细胞中用慢病毒过表达转基因TCR制备TCR-T细胞,多色FCM检测TCR-T细胞中TCR和CD3的表达水平,荧光素酶活性实验检测TCR-T细胞对HPV16阳性SiHa细胞的杀伤效率。结果：CRIPSR/Cas9基因编辑技术高效地敲除了外周血CD8⁺T细胞和Jurkat细胞中的TRAC和TRBC基因,敲除效率分别为(81.4±4.5)%(、98.5±0.07)%,制备的无内源TCR的TCR-T细胞高效表达转基因TCR,在外周血CD8⁺T和Jurkat细胞中表达率为(66.0±17.8)%、(97.3±2.6)%,敲除内源TRAC和T...     

Nitrotyrosine as a new marker for endogenous nitrosation and nitration.

A sensitive and selective method has been developed for analysing 3-nitrotyrosine (NTTYR), an exposure marker for exogenous and endogenous nitrosating or nitrating agents, in tissue and blood proteins by gas chromatography-thermal energy analysis. Using this method, a number of kinetic studies were carried out. Free and protein-bound tyrosine were reacted easily to yield NTTYR. The method was also applied to the study of NTTYR formation in vivo; a dose-dependent increase in NTTYR was seen in both plasma proteins and haemoglobin obtained from rats 24 h after intraperitoneal injection of various doses (0.5-2.5 mumol/rat) of tetranitromethane. Major urinary metabolites of NTTYR, given orally to rats, were isolated and identified as 3-nitro-4-hydroxyphenylacetic acid (NHPA) and 3-nitro-4-hydroxyphenyllactic acid. About 44% and 5% of the oral dose of NTTYR (100 micrograms/rat), respectively, was excreted as these metabolites. Some human urine samples were analysed for NHPA by gas chromatography-thermal energy analysis after ethyl acetate extraction and high-performance liquid chromatography purification; 2.8 +/- 2.3 (mean +/- SD; n = 11) micrograms/24 h, ranging from 0-7.9 micrograms/24 h, were detected (detection limit, 0.2 micrograms/l). In conclusion, NTTYR in proteins or its metabolites in urine could be readily analysed by gas chromatography-thermal energy analysis as a new, additional marker for endogenous nitrosation and nitration.