A simple HPLC method for plasma level monitoring of mitotane and its two main metabolites in adrenocortical cancer patients

Mitotane (o,p'-DDD or (1,1-dichloro-2-[o-chlorophenyl]-2-[p-chlorophenyl]ethane, DDD) is the drug of choice for non-resectable and metastatic adrenocortical carcinomas (ACC). Measurement of mitotane and metabolites, o,p'-DDE (1,1-dichloro-2-[p-chlorophenyl]-2-[o-chlorophenyl]ethene, DDE) and o,p'-DDA (1,1-[o,p'-dichlorodiphenyl] acetic acid, DDA) provides a better understanding of mitotane pharmacokinetics and pharmacodynamics. We have developed a simple, robust and efficient high performance liquid chromatography (HPLC) method to measure mitotane and its two main metabolites, DDE and DDA. The method involves a single ethanol extraction of mitotane, DDE, DDA, and an internal standard (int std) p,p'-DDD (1,1-dichloro-2,2-bis(p-chlorophenyl)ethane) with an extraction efficiency of 77-88%. All compounds are measured simultaneously using a reversed-phase phenyl HPLC column with an isocratic elution of mobile phase at a flow rate of 0.6 ml/min followed by UV detection at λ 226 nm. Inter and intra-day validation demonstrates good reproducibility and accuracy. Limits of quantitation are 0.2 μg/ml for mitotane and DDE, and 0.5 μg/ml for DDA. The method has been evaluated in plasma from 23 patients on mitotane therapy, revealing DDA concentrations 1-18 times higher than the parent compound.     

Inhibition mechanism of reconstituted cytochrome P-450scc-linked monooxygenase system by antimycotic reagents and other inhibitors.

The effects of various antimycotic reagents and some other reagents on a cytochrome P-450-linked monooxygenase system were investigated with respect to the activities of NADPH-ferricyanide reductase. NADPH-cytochrome c reductase of NADPH-adreno-ferredoxin reductase from NADPH to cytochrome c via adreno-ferredoxin, NADPH-cytochrome P-450-phenylisocyanide complex reductase, and the cholesterol side chain cleavage of the cytochrome P-450scc-linked monooxygenase system. No reagents inhibited the NADPH-ferricyanide reductase activity. Only cloconazole inhibited about 50% of NADPH-cytochrome c reductase activity. Cloconazole, econazole, clotrimazole, etomidate and ketoconazole inhibited both NADPH-cytochrome P-450-phenylisocyanide complex reductase and the side chain cleavage activity of cholesterol of the cytochrome P-450scc-linked monooxygenase system. Cloconazole, econazole, etomidate and ketoconazole behaved like non-competitive inhibitors for NADPH-cytochrome P-450-phenylisocyanide reductase activities and their Ki values were 10(-4)-10(-6) M. Cloconazole was a non-competitive inhibitor of NADPH-cytochrome c reductase and its Ki value was 8.3 x 10(-4) M. Cloconazole, clotrimazole, econazole, etomidate, ketoconazole and mitotane completely inhibited the side chain cleavage activity of cholesterol.     

Inhibition of intrinsic proteolytic activities moderates preanalytical variability and instability of human plasma

Human plasma and serum proteins are subject to intrinsic proteolytic degradation both during and after blood collection. By monitoring peptides, we investigated the stability of plasma and serum samples and the effects of anticoagulants and protease inhibitors on the plasma samples. Serum and plasma were subjected to time-course incubation, and the peptides (750-3200 Da) were extracted and analyzed with MALDI-TOF MS. Peptides of interest were further identified by MALDI-TOF/TOF MS and ESI-MS/MS analyses. Our observations indicate that plasma peptides are significantly different from serum peptides. Intrinsic proteases cause these differences between plasma and serum samples, as well as the differences among three plasma samples using either EDTA, sodium citrate, or heparin as the anticoagulant, which accounts for partial inhibitory effects on plasma proteolytic activities. Proteases and peptidases, including both aminopeptidases and carboxypeptidases, also cause time-dependent, sequential generation and digestion of the peptides in serum and all three plasmas, specifically during early sample collection and processing. Protease inhibitors within an EDTA-plasma-collection device inhibit both intrinsic plasma peptidases and proteases and moderate the time-dependent changes of the plasma peptides, including bradykinin, and complement C4- and C3- derived peptides. Our results suggest that mixing protease inhibitors immediately with blood during blood collection provides enhanced stabilization of the plasma proteome.     

A rapid simple method for the assay of renin in rabbit plasma

1. EDTA (10mm), 2,3-dimercaptopropan-1-ol (10mm) and chlorhexidine gluconate (0.005%, w/v) cause complete inactivation of plasma enzymes that degrade angiotensin I, but have no effect on the reaction of renin with its substrate. The reagents were termed the selective inhibitors. 2. Thus it is possible to measure renin in plasma by its ability to catalyse the release of angiotensin I. 3. Sterile plasma, treated with the selective inhibitors, is incubated with renin substrate (500-1000ng. of angiotensin content/ml.) at pH6 at 42 degrees for 6hr. 4. Under these conditions the reaction obeys first-order kinetics. Renin activity is calculated in terms of the percentage release of the angiotensin content/hr. 5. As described, the assay is sufficiently sensitive to measure renin in the plasma of all normal rabbits. By extending the length of the incubation, much lower activities can be measured.     

Pregnancy in a patient with adrenocortical carcinoma during treatment with Mitotane - a case report

We present the case of a female patient with virilising adrenocortical carcinoma treated surgically who conceived during adjuvant treatment with mitotane. We discuss the frequently erroneous routine treatment with oral hormonal contraception without thorough differential diagnosis in female patients with oligo-/amenorrhea and subsequent delay in the proper diagnosis of adrenocortical carcinoma.     

Aromatase inhibitors: assessment of biochemical efficacy measured by total body aromatase inhibition and tissue estrogen suppression

The implementation of aromatase inhibitors for treatment of early and metastatic breast cancer has been one of the major improvements in endocrine therapy of breast cancer. Measurement of endocrine effects of aromatase inhibition in vivo has been a major tool in the process of evaluating novel compounds. Biochemical efficacy of aromatase inhibitors in vivo may be determined from their effects on “total body aromatization” as well changes in plasma and tissue estrogen levels. Due to high sensitivity, tracer methods allowing calculation of whole body aromatase inhibition are still considered the gold standard. The method developed by our group in collaboration with the Royal Marsden Hospital and the results of this joint program are summarized and discussed. These studies allowed classification of the different aromatase inhibitors and their optimal dosage, selecting the best compounds for clinical evaluation. In vivo total body aromatase assessment is a work-consuming method, allowing such studies to be conducted in a limited number of patients only. In contrast, plasma estrogen measurement is a cruder but simpler method, allowing screening of larger groups of patients. As plasma estrogens arise through passive diffusion of estrogens synthesized in different body compartments, plasma estrogens, as well as total body aromatase assessment, present a rough estimate of total body tissue estrogen production, and changes associated with treatment with aromatase inhibitors reflect the effects on tissue estrogen production in general. However, plasma estrogen levels do not correlate to breast cancer tissue estrogen levels. This is due to the endocrine autonomy of breast cancer tissue with significant local estrogen production in some tumors. Thus, direct measurement of intratumor estrogens is demanded to evaluate the effects of aromatase inhibitors in malignant target tissues. Our group has developed a highly sensitive HPLC-RIA for the simultaneous measurement of estrone, estradiol, and estrone sulfate in malignant breast tissue samples, and we are currently using this method to assess alterations in intratumor estrogen levels during treatment with different aromatase inhibitors.     

Hydrolysis and protection from hydrolysis of enkephalins in human plasma

Seven groups of enkephalin-degrading enzymes and three groups of inhibitors active on these enzymes were separated from human plasma. The activity of the enzymes in hydrolyzing enkephalins and of the inhibitors in protecting enkephalins from proteolysis was measured. Results obtained with the endogenous inhibitors were compared to those relative to synthetic inhibitors. Data obtained indicate that all enkephalin-degrading enzymes found in plasma are significantly inhibited by the endogenous substances present in this tissue. The inhibition of the different classes of plasma enzymes by two of the three groups of endogenous substances is quite uniform, while one group of inhibitors appears specific to dipeptidylpeptidases. Results obtained are discussed in terms of the functional role of the inhibitory substances and of the possible pharmacological implication of their presence in human plasma.     

In vitro effects of Echis carinatus venom on the human plasma proteome

Echis carinatus venom (EV) is a complex mixture of toxins that contribute to its lethality. EV proteolytic activity was analyzed by zymography, chromogenic assays, and SDS-PAGE. To understand the molecular mechanism of the envenomation, we investigated the in vitro effect of EV on human plasma proteins. We looked for EV protein substrates and their proteolytic fragments. We analyzed EV proteolytic activity on standard proteins such as prothrombin or fibrinogen. To set up the optimal EV:plasma protein ratio conditions, plasma was incubated with EV (treated plasma), depleted of abundant proteins, and subjected to SDS-PAGE. Samples from control and treated plasma were also analyzed by 2-DE/MALDI-TOF MS, leading to the identification of four classes of plasma proteins cleaved by EV: proteases, protease inhibitors, binding proteins, and transporters. EV mainly proteolyzes entire proteins but can also act on physiological fragments. In summary, the physiological effects of EV proteases involve other important processes in addition to blood coagulation; complement activation and hemoglobin metabolism are also affected. In particular, the cleavage of protease inhibitors appears to be the mechanism through which the venom neutralizes the body's defenses.     

Effects of low molecular weight plasma inhibitors of rainbow trout (Oncorhynchus mykiss) on human erythrocyte carbonic anhydrase-II isozyme activity in vitro and rat erythrocytes in vivo

The effects of low molecular weight plasma inhibitors from rainbow trout (Oncorhynchus mykiss) (RT) were investigated on the carbonic anhydrase enzyme (CA) activities in in vitro human and in in vivo Sprague-Dawley rat erythrocytes. The RT blood was used as extracellular fluid (plasma) source and plasma inhibitors were obtained by dialysis of the plasma. For the in vitro study, human carbonic anhydrase-II (HCA-II) isozyme was obtained by Sepharose 4B-L-tyrosine-sulfanylamide affinity chromatography with an overall purification of about 646-fold. The enzyme (specific activity of 7750 EU/mg protein) was obtained with a yield of 71.1% and SDS-PAGE showed a single band. From in vitro studies, the I50 value for RT plasma inhibitors obtained was 0.37 mg/ml. From in vivo studies on rat erythrocytes, CA activity was significantly inhibited by the inhibitors from the extracellular fluid of RT for up to 3 h (p < 0.05) following intraperitoneal administration.     

The proteasome inhibitor bortezomib depletes plasma cells and protects mice with lupus-like disease from nephritis

Autoantibody-mediated diseases like myasthenia gravis, autoimmune hemolytic anemia and systemic lupus erythematosus represent a therapeutic challenge. In particular, long-lived plasma cells producing autoantibodies resist current therapeutic and experimental approaches. Recently, we showed that the sensitivity of myeloma cells toward proteasome inhibitors directly correlates with their immunoglobulin synthesis rates. Therefore, we hypothesized that normal plasma cells are also hypersensitive to proteasome inhibition owing to their extremely high amount of protein biosynthesis. Here we show that the proteasome inhibitor bortezomib, which is approved for the treatment of multiple myeloma, eliminates both short- and long-lived plasma cells by activation of the terminal unfolded protein response. Treatment with bortezomib depleted plasma cells producing antibodies to double-stranded DNA, eliminated autoantibody production, ameliorated glomerulonephritis and prolonged survival of two mouse strains with lupus-like disease, NZB/W F1 and MRL/lpr mice. Hence, the elimination of autoreactive plasma cells by proteasome inhibitors might represent a new treatment strategy for antibody-mediated diseases.     

Natural proteinase inhibitors: blood coagulation inhibition and evolutionary relationships

1. Natural proteinase inhibitors are divided into polysaccharides, plasma proteinase inhibitors and natural non-plasma inhibitors. 2. Polysaccharides are antithrombin-III and heparin co-factor-II dependent or independent regarding their biological activity. Knowledge of the inhibitory mechanism at a molecular level was gained by the study of heparin. 3. Antithrombin-III, heparin-co-factor-II and alpha 2-macroglobulin are the most important plasma proteinase inhibitors involved in coagulation. alpha 2-macroglobulin has a particular inhibitory mechanism. 4. Non-plasma proteinase inhibitors were isolated from many species. They inhibit mainly the contact activation and fibrinolysis. 5. The evolutionary relationships are poorly understood.     

Vitronectin and its fragments purified as serum inhibitors of Staphylococcus aureus gamma-hemolysin and leukocidin, and their specific binding to the hlg2 and the LukS components of the toxins.

Staphylococcal gamma-hemolysin and leukocidin are bi-component cytolysins, consisting of LukF (or Hlg1)/Hlg2 and LukF/LukS, respectively. Here, we purified serum inhibitors of gamma-hemolysin and leukocidin from human plasma. Protein sequencing showed that the purified inhibitors of 62, 57, 50 and 38 kDa were the vitronectin fragments with truncation(s) of the C-terminal or both N- and C-terminal regions. The purified vitronectin fragments specifically bound to the Hlg2 component of gamma-hemolysin and the LukS component of leukocidin to form high-molecular-weight complexes with them, leading to inhibition of the toxin-induced lysis of human erythrocytes and human polymorphonuclear leukocytes, respectively. Intact vitronectin also showed inhibitory activity to the toxins. The ability of gamma-hemolysin and leukocidin to bind vitronectin and its fragments is a novel function of the pore-forming cytolysins.     

Serine proteinase inhibitors of seminal plasma of teleost fish: distribution of activity, electrophoretic profiles and relation to proteinase inhibitors of blood

Anti-proteinase activity has been found in seminal plasma of eight teleost fish species: brown trout, rainbow trout, brook trout, lake whitefish, bream, northern pike, Danube salmon and burbot. This activity correlated with seminal plasma protein and sperm concentrations. Using a mammalian (bovine) trypsin for detecting proteinase inhibitors it was found for the first time that there are species-specific electrophoretic profiles of anti-proteinase activity. One to three bands could be identified by this method. However, additional proteinase inhibitors could be identified by using fish (cod) trypsin. These inhibitors were detected in seminal plasma of salmonids and coregonids and have a slow migration rate. Fast-migrating proteinase inhibitors were present in rainbow, brown and brook trout, northern pike, whitefish and burbot. These inhibitors could be detected in brook and brown trout by using either trypsins. However, they were detected only with bovine trypsin in rainbow trout, northern pike, whitefish and burbot. These results suggest that multiple forms of serine proteinase inhibitors exist in seminal plasma of teleost fish and they differ in their affinity toward serine proteinases. Seminal plasma serine proteinase inhibitors of rainbow trout migrated during electrophoresis similarly to blood plasma proteinase inhibitors, and suggests that the two inhibitors may be similar or the same. Anti-proteinase specific activity was similar in blood and seminal plasma. Proteinase inhibitors of fish seminal plasma seem to be an important part of sperm physiology, possibly related to protection of spermatozoa. Staining for detection of serine proteinase inhibitors also allowed detection of presence of nonspecific esterase in seminal plasma of most species.     

Role of enzymes and inhibitors in leu-enkephalin metabolism in rabbit and human plasma

The hydrolysis of leucine enkephalin by the proteolytic enzymes present in human and rabbit plasma has been studied by kinetic and chromatographic techniques. Data obtained indicate the existence of noticeable intraspecific differences in the kinetics of leu-enkephalin degradation, and of formation of its hydrolysis by-products. The separation of the enzymes active on the substrate and of the inhibitors active on these enzymes evidences the existence of a species specific distribution of both groups of substances. Yet, the dissimilar kinetics of the substrate hydrolysis and of formation of its hydrolysis by-products appear to arise more from diversities in the competition between the enzymes present in plasma and in the role of inhibitors than from the differences in the enkephalin-degrading enzymes. It is suggested that differences observed may be related to the existence of species specific populations of the information-carrying plasma peptides.     

A more sensitive and specific radioenzymatic assay for catecholamines

This modification of the catechol-O-methyltransferase (COMT) based radioenzymatic assay for norepinephrine (NE) and epinephrine (E) improves sensitivity, selectivity and eliminates many inhibitors of COMT. Prior to assay, samples are extracted into heptane with diphenylborate, then into dilute acetic acid. This extraction procedure has an efficiency of 78% for NE but less than 2% for S-adenosylmethionine (SAM). The extraction procedure also excludes calcium and other COMT inhibitors present in urine, plasma and every tissue tested. This eliminates the requirement for individual standardization of tissue and urine samples. Sensitivity of the assay for NE and E is 10 and 6 pg/ml respectively in 1 ml of plasma. The intraassay coefficients of variation for NE and E are 4 and 13% and the interassay coefficients of variation for NE and E are 10 and 16% in a human plasma sample containing low catecholamine levels. The assay permits quantitation of plasma E levels that were undetectable in prior assays.     

Isolation, characterization and cDNA sequencing of a Kazal family proteinase inhibitor from seminal plasma of turkey (Meleagris gallopavo)

The turkey reproductive tract and seminal plasma contain a serine proteinase inhibitor that seems to be unique for the reproductive tract. Our experimental objective was to isolate, characterize and cDNA sequence the Kazal family proteinase inhibitor from turkey seminal plasma and testis. Seminal plasma contains two forms of a Kazal family inhibitor: virgin (Ia) represented by an inhibitor of moderate electrophoretic migration rate (present also in the testis) and modified (Ib, a split peptide bond) represented by an inhibitor with a fast migration rate. The inhibitor from the seminal plasma was purified by affinity, ion-exchange and reverse phase chromatography. The testis inhibitor was purified by affinity and ion-exchange chromatography. N-terminal Edman sequencing of the two seminal plasma inhibitors and testis inhibitor were identical. This sequence was used to construct primers and obtain a cDNA sequence from the testis. Analysis of a cDNA sequence indicated that turkey proteinase inhibitor belongs to Kazal family inhibitors (pancreatic secretory trypsin inhibitors, mammalian acrosin inhibitors) and caltrin. The turkey seminal plasma Kazal inhibitor belongs to low molecular mass inhibitors and is characterized by a high value of the equilibrium association constant for inhibitor/trypsin complexes.     

Simultaneous determination of functional coagulation factors and competitive (PIVKA-) inhibitors based on enzyme kinetics

For a plasma containing the competitive (PIVKA-) inhibitors induced by anticoagulant treatment the coagulation time t is related to the concentrations of functional coagulation factors S (substrates) and competitive inhibitors I by t = tmin + el/S + gamma I/S with tmin being the minimum possible coagulation time and e and gamma the sensitivities of the test procedure towards a change in the concentration of functional coagulation factors and competitive inhibitors, respectively. The calibration of the test procedure can be achieved by performing a series of dilutions on an inhibitor-free plasma (determination of tmin and e) and, after that, on a plasma of known inhibitor content (determination of gamma) in both cases recording the parametrizing straight line which results from multiplying the respective equation by S. The content of functional coagulation factors and competitive inhibitors in the plasmas of anticoagulated patients then can be determined simultaneously by treating the patient's plasma like in the calibration for gamma. The proposed method should allow the complete metrological characterization of thromboplastin time reagents without any need for reference thromboplastins.     

Gelatinases and serine proteinase inhibitors of seminal plasma and the reproductive tract of turkey (Meleagris gallopavo)

This study examined proteolytic enzymes and serine proteinase inhibitors in turkey seminal plasma with relation to their distribution within the reproductive tract and to yellow semen syndrome (YSS). Proteases of blood plasma, extracts from the reproductive tract, and seminal plasma were analyzed by gelatin zymography. We found a clear regional distribution of proteolytic enzymes in the turkey reproductive tract. Each part was characterized by a unique profile of serine proteolytic enzymes of molecular weights ranging from 29 to 88 kDa. The ductus deferens was found to be a site of very intense proteolytic activity. Two metalloproteases of 58 and 66 kDa were detected in all parts of the reproductive tract and seminal plasma. Using electrophoretic methods for detection of anti-trypsin activity, we found three serine proteinase inhibitors in turkey seminal plasma. Two inhibitors were found in the testis and epididymis and a third in the ductus deferens and seminal plasma. Blood plasma was characterized by the presence of two metalloproteinases and one serine proteinase inhibitor (of low migration rate) that were also detected in the reproductive tract. Amidase and anti-trypsin activities (expressed per gram of protein) differed for yellow and white seminal plasma. We concluded that turkey seminal plasma contains metalloproteases, serine proteinases, and serine proteinase inhibitors. The metalloproteases and one proteinase inhibitor are related to blood proteinases but the other two inhibitors and serine proteinases seem to be unique for the reproductive tract.     

Presenilins and gamma-secretase inhibitors affect intracellular trafficking and cell surface localization of the gamma-secretase complex components

The intramembranous cleavage of Alzheimer beta-amyloid precursor protein and the signaling receptor Notch is mediated by the presenilin (PS, PS1/PS2)-gamma-secretase complex, the components of which also include nicastrin, APH-1, and PEN-2. In addition to its essential role in gamma-secretase activity, we and others have reported that PS1 plays a role in intracellular trafficking of select membrane proteins including nicastrin. Here we examined the fate of PEN-2 in the absence of PS expression or gamma-secretase activity. We found that PEN-2 is retained in the endoplasmic reticulum and has a much shorter half-life in PS-deficient cells than in wild type cells, suggesting that PSs are required for maintaining the stability and proper subcellular trafficking of PEN-2. However, the function of PS in PEN-2 trafficking is distinct from its contribution to gamma-secretase activity because inhibition of gamma-secretase activity by gamma-secretase inhibitors did not affect the PEN-2 level or its egress from the endoplasmic reticulum. Instead, membrane-permeable gamma-secretase inhibitors, but not a membrane-impermeable derivative, markedly increased the cell surface levels of PS1 and PEN-2 without affecting that of nicastrin. In support of its role in PEN-2 trafficking, PS1 was also required for the gamma-secretase inhibitor-induced plasma membrane accumulation of PEN-2. We further showed that gamma-secretase inhibitors specifically accelerated the Golgi to the cell surface transport of PS1 and PEN-2. Taken together, we demonstrate an essential role for PSs in intracellular trafficking of the gamma-secretase components, and that selective gamma-secretase inhibitors differentially affect the trafficking of the gamma-secretase components, which may contribute to an inactivation of gamma-secretase.