Determination of testosterone in serum by liquid chromatography-tandem mass spectrometry.

Commercial direct immunoassays for serum testosterone sometimes result in inaccuracies in samples from women and children, leading to misdiagnosis and inappropriate treatment. The diagnosis of male hypogonadism also requires an accurate testosterone assay method. We therefore developed a sensitive and specific stable-isotope dilution liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for serum testosterone at the concentrations encountered in women and children. Testosterone was extracted with ether-ethyl acetate from 250 microL or 500 microL of serum. Instrumental analysis was performed on an API 2000 tandem mass spectrometer in the multiple-reaction monitoring (MRM) mode after separation on a reversed-phase column. The MRM transitions (m/z) were 289/97 for testosterone and 291/99 for d(2) testosterone. The calibration curves exhibited consistent linearity and repeatability in the range 0.2-100 nmol/L. Interassay CVs were 4.2-7.6 % at mean concentrations of testosterone of 3.3-45 nmol/L. Total measurement uncertainty (U, k = 2) was 12.9 % and 13.4 % at testosterone levels of 2.0 nmol/L and 20 nmol/L, respectively. The limit of detection was 0.05 nmol/L (signal-to-noise ratio = 3) and the overall method recovery of testosterone was 95 %. Correlation (r) with our in-house extraction RIA was 0.98 and with a commercial RIA 0.92. Reference intervals for adult males and females in age groups 18-30, 31-50, 51-70 and over 70 years were established. Sensitivity and specificity of the LC-MS/MS method offer advantages over immunoassay and make it suitable for use as a high-throughput assay in routine clinical laboratories. The high equipment costs are balanced by higher throughput together with shorter chromatographic run times.

Determination of 17alpha-hydroxyprogesterone in serum by liquid chromatography-tandem mass spectrometry and immunoassay.

17Alpha-hydroxyprogesterone (17OHP) is the most important serum marker for congenital adrenal hyperplasia (CAH). 17OHP is usually measured by immunoassay but its detection by mass spectrometry (MS) is a potentially superior method. An LC-MS (liquid chromatography-mass spectrometry) method was developed which utilizes 0.5 ml serum spiked with 6-alpha-methylprednisolone (6-MP) or deuterated 17OHP (d8-IS) as the internal standard. The samples were extracted with ether/ethylacetate, and the extract was evaporated to dryness and analysed by LC-MS/MS operating in the positive mode after separation on a reversed-phase C18 column. The calibration curves for analysis of serum 17OHP exhibited consistent linearity and reproducibility in the range of 5-250 nmol/l. Interassay CVs were 8.5 and 9.2% at mean concentrations of 7.9 and 23 nmol/l, respectively. The detection limit was 1 nmol/l (signal-to-noise ratio=3). The mean recovery of 17OHP added to serum ranged from 76 to 89% and that of internal standards from 75 to 82%. The regression equation for the LC-MS/MS (x) and in-house radioimmunoassay (RIA) (y) methods was: y=0.87x+0.26 (r=0.97; n=100) and for a commercial RIA it was: y=1.32x+0.02 (r=0.97; n=26).

Serum samples from pancreatectomized patients contain trypsinogen immunoreactivity.

The concentrations of trypsinogen-1 and -2 in serum samples from patients who have undergone pancreatectomy were measured by highly sensitive and specific time-resolved immunofluorometric assays. The isoenzyme pattern was determined by ion-exchange chromatography and determination of immunoreactivity in the fractions. All samples contained trypsinogen-2, the mean level being one fifth of that in healthy controls. Trypsinogen-1 was detected in one of nine samples. In addition to the main trypsinogen isoenzymes, we observed in normal serum two trypsinogen isoenzymes previously found in mucinous ovarian cyst fluid. Our results suggest that trypsinogen is not exclusively expressed by the pancreas and certain tumors but that it also may be produced by normal extrapancreatic tissues. This should be considered when an assay of trypsinogen in serum is used for clinical purposes.

Optimization of a time-resolved immunofluorometric assay for tumor-associated trypsin inhibitor (TATI) using the streptavidin-biotin system.

We have developed two 'sandwich'-type time-resolved immunofluorometric assays (IFMA) for tumor-associated trypsin inhibitor (TATI) using monoclonal and polyclonal antibodies. In the standard assay the monoclonal antibody was immobilized onto the walls of polystyrene microstrip wells and the polyclonal reagent was labeled with a europium chelate. We tested various assay conditions in order to optimize the assay for sensitivity and measuring range. Purification of the labeled antibody by hydrophobic interaction chromatography was found to be the most important single factor affecting sensitivity. Assay sensitivity and range were also improved by acid treatment of the solid phase antibody. To improve the sensitivity further the streptavidin/biotin (SAB) system was incorporated into the IFMA technique. In this simple and fast streptavidin/biotin IFMA (SAB-IFMA) we used streptavidin-coated wells to which we added biotinylated monoclonal antibody and a serum or urine sample. After incubation for 1.5 h and washing, the polyclonal europium-labeled tracer antibody was added. After incubation for 1 h the wells were washed and the Eu fluorescence measured. The assay performance of the SAB-IFMA was compared to the standard IFMA and radioimmunoassay (RIA). The detection limit was 0.05 microgram/l and the analytical range 3000-fold. The mean analytical recovery was 101%. Other advantages of the SAB-IFMA were high sensitivity and the low amounts of monoclonal antibody required, only 1/50 of that used in the standard IFMA.

Tumor-associated trypsin participates in cancer cell-mediated degradation of extracellular matrix.

We have recently demonstrated that many cancer cell lines produce a novel trypsinogen isoenzyme called tumor-associated trypsinogen 2 (TAT-2). It was found during a search of the target protease for tumor-associated trypsin inhibitor (TATI). We now show that degradation of subendothelial cell extracellular matrix (ECM) by four different cell lines (COLO 205 colon carcinoma, K-562 erythroleukemia, CAPAN-1 pancreatic carcinoma, and HT 1080 fibrosarcoma) can be partially inhibited by TATI or neutralizing trypsin antibodies. When cells were cultured in serum-free medium on ECM, TATI and trypsin antibodies inhibited the release of immunoreactive fibronectin fragments from ECM by 47-54 and 40%, respectively. Degradation of isotopically labeled ([3H]serine, [3H]proline, and [35S]sulfate) ECM was also significantly prevented by TATI. At its maximum, it exerted a 57% inhibition on the degradation of [3H]serine-labeled ECM. Plasminogen added exogenously to the culture medium further potentiated the proteolysis of ECM. Interestingly, addition of enteropeptidase, an activator of TAT-2, also enhanced cell-mediated proteolysis as assessed by degradation of purified fibronectin coated onto the surface of wells. Immunoblot analysis showed that enteropeptidase-mediated proteolysis generated a pattern of fibronectin fragments similar to that obtained by digestion of purified fibronectin by TAT-2. These results demonstrate the existence of a proteolytic system in tumor cells which is dependent on the activation of TAT-2. We suggest that TAT-2 is involved in a protease cascade-stimulating tumor cell invasion and degradation of extracellular matrix.

Human colon carcinoma, fibrosarcoma and leukemia cell lines produce tumor-associated trypsinogen.

Previous studies have indicated that cyst fluid of ovarian tumors contains 2 trypsinogen isoenzymes, called tumor-associated trypsinogen-I (TAT-I) and trypsinogen-2 (TAT-2), the levels of which correlate with the degree of malignancy of the tumors. In addition, these cyst fluids contain large amounts of tumor-associated trypsin inhibitor (TATI), which is also expressed in many other human tumors. In the present study we examined the production of TAT-I, TAT-2 and TATI in 9 established tumor-cell lines. TAT-2 was produced by 5 cell lines. Its concentration in the conditioned medium of COLO 205 colon adenocarcinoma cells, K-562 erythroleukemia cells and fibrosarcoma cell lines HT 1080, 8387 and A 9733 was 460 micrograms/l, 9.8 micrograms/l, 21 micrograms/l, 8.8 micrograms/l and 0.24 micrograms/l, respectively. TAT-I was detectable in the conditioned medium of COLO 205 and HT 1080 cells at concentrations of 64 micrograms/l and 0.5 micrograms/l, respectively. TATI was detected only in the media of COLO 205 cells at a concentration of 23 micrograms/l. TAT-2 zymogen was purified from the conditioned medium of COLO 205 and HT 1080 cells by immunoaffinity chromatography. According to its aminoterminal amino acid sequence, a molecular mass of 28 kDa by SDS-PAGE, elution pattern in ion-exchange chromatography and ability to be activated by enteropeptidase, the zymogen is identical to that previously isolated from cyst fluid of ovarian tumors. In addition, we found that TAT-2 secretion could be down-regulated by dexamethasone in HT 1080 cells but not in COLO 205 cells. The abundant production of TAT-2 isoenzyme in different cancer cell lines suggests that it could contribute to the increased proteolytic activity of many human tumors.

Biology and function of tumor-associated trypsin inhibitor, TATI.

Tumor-associated trypsin inhibitor (TATI) is a 6,000 Daltons peptide, which is synthesized by several tumors and cell lines. TATI is identical to pancreatic secretory trypsin inhibitor (PSTI). This peptide is also produced by the mucosa of the gastrointestinal tract, where it is thought to protect the mucosal cells from proteolytic breakdown. Elevated serum and urine levels of TATI occur in connection with many types of cancer, especially mucinous ovarian cancer. Elevated levels may also occur in nonmalignant diseases, e.g. in pancreatitis, severe infections and tissue destruction. Thus TATI may behave as an acute phase reactant. Tumors producing TATI often express tumor-associated trypsinogen. Elevation of TATI in cancer and pancreatic disease is therefore associated with expression of trypsin, but such a connection has not been demonstrated in inflammatory disease. TATI can inhibit trypsin-mediated degradation of extracellular matrix by tumor cells. Therefore its role may be to control the activation of tumor-associated trypsinogen. TATI has also been shown to possess growth factor activity in vitro, but it is not known whether this is a physiological function.

Insulin-like growth factor-binding protein-1 inhibits binding of IGF-I on fetal skin fibroblasts but stimulates their DNA synthesis.

Insulin-like growth factor-binding protein-1 (IGFBP-1) was purified from human midtrimester amniotic fluid using monoclonal anti-IGFBP-1 affinity column. Two peaks were obtained in anion exchange chromatography. Both had the same molecular mass of 30 kDa. In monolayer cultures of fetal skin fibroblasts both forms of IGFBP-1 inhibited binding of [125I]IGF-I onto the cells, but amplified the IGF-I-stimulated [3H]thymidine incorporation into the same cells. Radiolabeled IGFBP-1 did not bind to the cells. No detectable IGFBP-1 was released into conditioned medium from the cells, and they contained no specific IGFBP-1 mRNA. Recently we found that the same IGFBP-1 preparation inhibits IGF-I-stimulated [3H]thymidine incorporation into human hyperstimulated granulosa cells. These results show that, depending on target cells, the same protein is capable of either stimulating or inhibiting DNA synthesis.

Time-resolved immunofluorometric assays for trypsinogen-1 and 2 in serum reveal preferential elevation of trypsinogen-2 in pancreatitis.

We have developed sensitive time-resolved immunofluorometric assays for the two trypsinogen isoenzymes, trypsinogen-1 and trypsinogen-2, which also are called cationic and anionic trypsinogen, respectively. The assays use monoclonal antibodies produced by immunization with tumor-associated trypsinogen that is isolated from mucinous ovarian cyst fluid. In each assay, one antibody is immobilized onto the walls of polystyrene microtiter strip wells and the other is labeled with an europium(III) chelate. The cross-reaction of each trypsinogen isoenzyme in the assay for the other isoenzyme is less than 1%. The detection limits are 0.1 micrograms/L for trypsinogen-1 and 0.3 micrograms/L for trypsinogen-2. In sera of healthy subjects and patients with extrapancreatic disease the concentration of trypsinogen-1 is higher (median, 21 micrograms/L) than that of trypsinogen-2 (median, 17 micrograms/L), but in acute pancreatitis the ratio is reversed. In acute pancreatitis the concentration of trypsinogen-2 is 50-fold higher than in controls, whereas the difference in trypsinogen-1 concentrations is only 15-fold. The corresponding difference in immunoreactive trypsin measured by a commercially available radioimmunoassay was also only 10-fold.

Cyst fluid of ovarian cancer patients contains high concentrations of trypsinogen-2.

We have determined the concentrations of two tumor-associated trypsinogen (TAT) isoenzymes, called TAT-1 and TAT-2, in human ovarian tumor cyst fluid using monoclonal antibody-based immunofluorometric assays specific for each isoenzyme. TAT-1 and TAT-2 are immunologically indistinguishable from the two pancreatic trypsinogen isoenzymes, cationic trypsinogen (-1) and anionic trypsinogen (-2). Our results show that of the two isoenzymes TAT-2 is the predominant form in cyst fluid and its concentrations are significantly higher than the levels of the trypsinogen isoenzymes in serum and in preovulatory follicular fluid from hyperstimulated ovaries. The median concentration of TAT-2 was higher in mucinous than in serous cyst fluid as has been found previously for the specific trypsin inhibitor, tumor-associated trypsin inhibitor. Most notably, in mucinous cyst fluids the median level of TAT-2 was higher in borderline and malignant (2640 micrograms/liter) than in benign cases (84 micrograms/liter). Also in serous cyst fluids the TAT-2 level was higher in borderline and malignant (median 345 micrograms/liter) than in benign cases (median 18 micrograms/liter). In fluids from other types of malignant ovarian carcinomas slightly elevated levels of TAT-2 were also observed (median 62 micrograms/liter). The identity of the trypsinogens was verified by isolating them by immunoaffinity chromatography using monoclonal antibodies. The increased levels in association with malignancy suggest that TAT is involved in ovarian tumor dissemination and breakage of tissue barriers.