U.‐H. Stenman

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.

Determination of urinary free cortisol by liquid chromatography-tandem mass spectrometry

Measurement of urinary free cortisol is clinically important in the diagnosis of Cushings syndrome. While liquid chromatography (LC) with UV detection provides much better specificity than immunologic methods, certain drugs cause interference. Detection by mass spectrometry (MS) is a potentially superior method. Our analysis utilizes 1u2005mL urine spiked with 6‐α‐methylprednisolone as internal standard. The samples were extracted with dichlormethane and the extract was washed, evaporated to dryness and analyzed by LC‐MS/MS operating in the negative mode after separation on a reversed‐phase C18 column. The calibration curves for analysis of urinary cortisol exhibited consistent linearity and reproducibility in the range of 10–400u2005nmol/L. Inter‐assay CVs were 4.0–7.6% at mean concentrations of 21–153u2005nmol/L. The detection limit was 1u2005nmol/L (signal‐to‐noise ratio=3). The mean recovery of cortisol added to urine ranged from 67% to 87% and that of the internal standard from 71% to 76%. The regression equation for the LC‐MS/MS (x) and HPLC (y) methods was: y=1.095x+8.0 (r=0.996; n=111). Drugs known to interfere with UV detection did not cause problems here. The sensitivity and specificity of the MS/MS method for urinary free cortisol offer advantages over HPLC with UV detection by eliminating drug interference. The higher equipment costs in comparison with HPLC methods using UV detection are balanced by higher throughput, thanks to shorter chromatographic run times.

Potentially hallucinogenic 5-hydroxytryptamine receptor ligands bufotenine and dimethyltryptamine in blood and tissues

Bufotenine and N,N‐dimethyltryptamine (DMT) are hallucinogenic dimethylated indolethylamines (DMIAs) formed from serotonin and tryptamine by the enzyme indolethylamine N‐methyltransferase (INMT) ubiquitously present in non‐neural tissues. In mammals, endogenous bufotenine and DMT have been identified only in human urine. The DMIAs bind effectively to 5HT receptors and their administration causes a variety of autonomic effects, which may reflect their actual physiological function. Endogenous levels of bufotenine and DMT in blood and a number of animal and human tissues were determined using highly sensitive and specific quantitative mass spectrometric techniques. A new finding was the detection of large amounts of bufotenine in stools, which may be an indication of its role in intestinal function. It is suggested that fecal and urinary bufotenine originate from epithelial cells of the intestine and the kidney, respectively, although the possibility of their synthesis by intestinal bacteria cannot be excluded. Only small amounts of the DMIAs were found in somatic or neural tissues and none in blood. This can be explained by rapid catabolism of the DMIAs by mitochondrial monoamino‐oxidase or by the fact that the dimethylated products of serotonin and tryptamine are not formed in significant amounts in most mammalian tissues despite the widespread presence of INMT in tissues.

Standardization of PSA determinations.

Standardization of determinations for prostate specific antigen (PSA) has become an important issue due to the widespread use of these determinations for prostate cancer screening. Standardization of this assay is complicated due to the occurrence of two major forms of PSA in serum, the free antigen and a complex between PSA and alpha 1-antichymotrypsin (PSA-ACT). These two forms of PSA are recognized differently by different antibodies, but by careful selection of antibodies, it is possible to design methods that measure each form equally. It is suggested, that standards for PSA and PSA-ACT should be prepared and established as international standards. Furthermore, reference methods should be established on the basis that these standards and carefully selected reference antibodies.

Determination of 17α‐hydroxyprogesterone in serum by liquid chromatography‐tandem mass spectrometry and immunoassay

17α‐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 0.5u2005ml serum spiked with 6‐α‐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 5–250u2005nmol/l. Interassay CVs were 8.5 and 9.2% at mean concentrations of 7.9 and 23u2005nmol/l, respectively. The detection limit was 1u2005nmol/l (signal‐to‐noise ratiou200a=u200a3). 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: yu200a=u200a0.87x+0.26 (ru200a=u200a0.97; nu200a=u200a100) and for a commercial RIA it was: yu200a=u200a1.32x+0.02 (ru200a=u200a0.97; nu200a=u200a26).

Quantification of prostate specific antigen mRNA levels in circulation after prostatic surgery and endocrine treatment by quantitative reverse transcription‐polymerase chain reaction

Various methods to detect prostatic cells in circulation have given conflicting results. This is probably because qualitative rather than quantitative methods have been used to detect mRNA from prostatic cells. A quantitative method has been developed based on reverse transcription‐polymerase chain reaction (RT‐PCR) for detection of prostate specific antigen (PSA) mRNA in peripheral blood. A competitive internal mRNA standard was used for quantification of absolute amounts of PSA mRNA. The detection limit of the assay was 7 copies of mRNA, and the highest level of circulating PSA mRNA in 88 control subjects was 25 copies per milliliter of blood. This method was used to study the influence of prostatic surgery and endocrine treatment on prostatic cells in the circulation of 56 patients undergoing biopsy, radical prostatectomy, transurethral resection of the prostate (TURP), orchiectomy, or androgen blockade. Blood samples were drawn before, during and up to 26u2005weeks after these procedures had been carried out. The highest level of PSA mRNA in controls was 25 copies per milliliter of blood. After RP, TURP or orchiectomy, PSA mRNA levels increased above this level in 27%, 29% and 25% of the samples, respectively. After prostate biopsy, two out of 15 patients became positive. PSA mRNA levels that were elevated by surgery became undetectable within 1–3u2005days. No significant correlation was found between PCR positivity and the clinical characteristics of the patients. It is concluded that the level of PSA mRNA in peripheral blood increases after prostatic surgery, indicating temporary dissemination of prostatic cells. However, preoperative levels do not correlate with serum PSA, stage or grade.

Development of novel peptide ligands modulating the enzyme activity of prostate-specific antigen

Prostate-specific antigen (PSA) is a serine proteinase produced mainly by epithelial cells of the prostate. Measurement of PSA in serum is widely used for diagnosis and monitoring of prostate cancer. The major problem of the PSA determination in early diagnosis is the high false positive rate due to benign prostatic hyperplasia, but the clinical accuracy can be improved by determining the proportions of various molecular forms of PSA. The main biological function of PSA is liquefaction of the seminal gel formed after ejaculation, but PSA has also been suggested to regulate invasiveness and metastatic potential of prostatic tumors. Thus, agents binding to and affecting the function of PSA have the potential to be used for diagnosis and therapy of prostate cancer. We have developed peptides specific for PSA by using cyclic phage display peptide libraries. After deducing the amino acid sequence of the peptides by sequencing the relevant part of phage genome, the peptides were expressed as glutathione- S -transferase (GST) fusion proteins or produced by chemical synthesis. The peptides were shown to bind to PSA specifically as indicated by lack of binding to other related serine proteinases. The binding of the peptides with PSA was strongly inhibited by monoclonal antibodies specific for free PSA and they did not bind to PSA-inhibitor complexes indicating that they bind close to the active site of the enzyme. Most of the peptides enhanced the enzyme activity of PSA against a chromogenic substrate. The affinity of the peptides could be increased by including Zn 2+ in the reaction mixture. These results show that peptides that bind to PSA and modulate its enzyme activity can be developed by phage display techniques. These peptides have the potential to be used for targeting of prostatic tumors and diagnostics of prostate cancer.

Characterization and determination of the complex between prostate-specific antigen and α1-protease inhibitor in benign and malignant prostatic diseases

Prostate-specific antigen (PSA) is a tissue-specific serine protease which forms complexes with protease inhibitors such as f 1 -antichymotrypsin and f 2 - macroglobulin. We have studied the interaction between PSA and f 1 -protease inhibitor (API) in vitro and found that 15% of the added PSA binds to API while the majority of API is cleaved between Met358 and Ser359 when PSA is incubated with a 5-fold excess of API at 37 C for 7 days. The complex between PSA and API (PSA- API) formed in vitro displays the same chromatographic behavior, molecular size and immunoreactivity as endogenous PSA- API occurring in serum, indicating that they are identical. PSA- API can be detected in serum by a time-resolved immunofluorometric assay (IFMA), in which a monoclonal antibody to PSA is used as a catcher and a polyclonal antibody to API labeled with a Eu-chelate is used as a tracer. Purified PSA- API formed in vitro is used as a calibrator. PSA- API in serum represents 1.0- 7.9% (median 2.4%) of total PSA (tPSA) in prostate cancer (PCa, n= 82) and 1.3- 12.2% (median 3.6%, p<0.01) in patients with benign prostatic hyperplasia (BPH, n= 66). The IFMA for PSA- API in serum is hampered by a variable background, which is caused by non-specific adsorption of the huge excess of API in serum to the solid phase. The background can be determined by an assay using the same tracer as in the IFMA for PSA- API but PSA-unrelated antibody on the solid phase. The background signal is subtracted from the PSA- API signal. The clinical utility of PSA- API in serum has been evaluated in PSA-positive subjects from the Finnish PCa screening trial. After subtraction of the background, the proportion of PSA- API in relation to tPSA is lower in PCa than in controls, 0.9% vs. 1.6%, respectively (p<0.001). Logistic regression analysis showed that the concentration of PSA- API was independent of the proportion of free PSA as a diagnostic variable among subjects with a tPSA of 4- 10 w g/l (p= 0.009). The probability of PCa calculated by logistic regression using the concentration of PSA- API and the proportion of free PSA in serum significantly improved cancer specificity at high sensitivity levels (85 - 95%) as compared to the proportion of free PSA alone.

Determination of human tumour necrosis factor-α (TNF-α) by time-resolved immunofluorometric assay

We have developed a ‘sandwich’-type time-resolved immunofluorometric assay (IFMA) for tumour necrosis factor alpha (TNF-α) using two monoclonal antibodies (mAb) and the streptavidin/biotin (SAB) system. In this simple and fast streptavidin/biotin IFMA (SAB-IFMA) we used streptavidin coated wells to which we added biotinylated mAb for 3h. After washing, the serum sample was added and incubated for 2h followed by washing. Another monoclonal europium-labelled tracer antibody was added and incubated for 1 h, the wells were washed and the fluorescence of Eu measured. We tested various assay conditions in order to optimize the assay for sensitivity and measuring range. Purification of the labelled antibody by hydrophobic interaction chromatography was found to be essential to improve sensitivity. With a sample volume of 50μl the detection limit was 6ngl−1 and the analytical range large, i.e. 10000-fold. The median concentration in serum from healthy subjects was 12ngl−1 and the reference range <39ngl−1. The mea...

Testicular cancer: The perfect paradigm for marker combinations

About 95% of all testicular cancers are germ cell tumors, which mostly contain several different histological types, i.e. embryonal carcinoma, syncytiotrophoblasts, yolk sac tumor, teratoma, and seminomatous components. These tissues express and secrete several fairly specific markers that can be determined in serum. The most important markers are alphafetoprotein (AFP), human chorionic gonadotropin (hCG) and lactic dehydrogenase (LDH). In non-seminomatous germ cell tumors (NSGCTs) the marker expression is dependent on the histological type; about 50% have elevated serum levels of hCG and 60% of AFP, while either marker is elevated in 90% of cases. LDH is elevated in half of the patients, but this finding can also be caused by many non-malignant diseases and by hemolysis. The utility of LDH can be improved by measurement of the LD-1 isoenzyme, which reflects amplification of the short arm of chromosome 12, a characteristic feature of germ cell tumors. About 10–20% of the patients with pure seminoma have elevated serum levels of hCG and 50% of LDH. The free beta subunit of hCG (hCGb) has been reported to be elevated in about 30% of the seminomas that do not express hCG. Very high marker concentrations are associated with adverse outcome and are used for staging of the disease. Detailed recommendations for treatment of germ cell cancer have recently been published [1]. Tumor markers are valuable for monitoring of many cancers, but they play an especially important role in the management of testicular cancer. As in choriocarcinoma, therapy is often initiated on the basis of increasing marker levels alone. However, while hCG alone is sufficient for the monitoring of patients with choriocarcinoma, combinations of several markers are essential for monitoring of testicular cancer [2]. Germ cell tumors comprise 95% of all testicular tumors and nearly all of them are malignant. Although they represent only 1% of all cancers, they are the most common solid