ABSTRACT
BACKGROUND: Differences between major allergens from distinct grass species remain to be investigated, both in terms of structure and antigenicity. METHODS: Group 1 and 5 allergens purified from five common Pooideae species were analysed by mass spectrometry (MS). Major histocompatibility complex (MHC) class II-restricted T cell epitopes were identified using predictive algorithms and human leucocyte antigen (HLA)-binding assays. CD4+ T cell reactivity and IgE binding were assessed based on the induction of CD154 expression in peripheral blood mononuclear cells and using competitive ELISA assays, respectively. RESULTS: MS analysis of group 5 pollen allergens reveals considerable intra- and inter-species variability in amino acid sequence, with 30-50 predominant isoforms found for each species. Differences in the amino acid sequence as well as N- and O-glycosylation contribute to the variability of group 1 allergens, yielding 5-10 main isoforms, depending on the species. Out of 14 MHC class II-restricted T cell epitopes identified within group 1, only one is conserved among the five grass species. Significant differences in binding affinities for HLA-DR molecules result in variable CD4+ T cell recognition of group 1 and 5 allergens purified from the various species. Up to 38% and 85% of patients exhibit seric IgE responses to species-restricted (or semi-restricted) epitopes associated with group 1 or 5 allergens, respectively. CONCLUSION: Major pollen allergens from distinct grass species bear both shared and species-restricted T and B cell immune epitopes. When compared with single extracts, a five grass pollen extract is thus more suitable for specific immunotherapy, as it contains a broader repertoire of the IgE epitopes to which patients are sensitized.
Subject(s)
Allergens/chemistry , Allergens/classification , Desensitization, Immunologic , Poaceae/chemistry , Poaceae/classification , Pollen/chemistry , Allergens/immunology , Allergens/isolation & purification , Amino Acid Sequence , Animals , Antigen-Antibody Reactions , Epitopes/chemistry , Epitopes/immunology , Humans , Immunoglobulin E/immunology , Leukocytes/immunology , Mice , Models, Molecular , Molecular Sequence Data , Peptide Mapping , Protein Conformation , Species Specificity , T-Lymphocytes/immunologyABSTRACT
The toxin ricin is a biological weapon that may be used for bioterrorist purposes. As a member of the group of ribosome-inactivating proteins (RIPs), ricin has an A-chain possessing N-glycosidase activity which irreversibly inhibits protein synthesis. In this paper, we demonstrate that provided appropriate sample preparation is used, this enzymatic activity can be exploited for functional ricin detection with sensitivity similar to the best ELISA and specificity allowing application to environmental samples. Ricin is first captured by a monoclonal antibody directed against the B chain and immobilized on magnetic beads. Detection is then realized by determination of the adenine released by the A chain from an RNA template using liquid chromatography coupled to tandem mass spectrometry. The immunoaffinity step combined with the enzymatic activity detection leads to a specific assay for the entire functional ricin with a lower limit of detection of 0.1 ng/mL (1.56 pM) after concentration of the toxin from a 500 microL sample size. The variability of the assay was 10%. Finally, the method was applied successfully to milk and tap or bottled water samples.
Subject(s)
Adenine/analysis , Bioterrorism , Chromatography, Affinity/methods , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/chemistry , Ricin/chemistry , Tandem Mass Spectrometry/methods , Chromatography, Liquid/methods , RNA/chemistry , Reproducibility of Results , Ricin/immunology , Sensitivity and SpecificityABSTRACT
In higher plants and some fungi, heavy metals induce the synthesis of chelating peptides known as phytochelatins (PCs). They are characterized by the general structure (gamma-Glu-Cys)n-Gly, but in some plant species, the C-terminal glycine can be replaced by serine, glutamine, glutamate or alanine, leading to iso-phytochelatins (iso-PCs). Although the distribution of iso-PCs is considered to differ from one species to another, we previously showed that Arabidopsis thaliana (A. thaliana) cells are able to synthesize most PC-related peptides (PCs and iso-PCs) described in the literature. We also observed an accumulation of the dipeptide gamma-glutamylcysteine (gamma-EC) when cadmium (Cd) (200 microM) was added to the culture medium, suggesting that either glutathione synthetase or glycine availability could be a limiting factor for the biosynthesis of PC-related peptides. In this context, the aim of the present work was to seek new insights into the regulation of PC synthesis by performing metabolic profiling using liquid chromatography-mass spectrometry. The levels of PC-related peptides and their precursors were measured in A. thaliana cells following Cd exposure. A range of doses (0, 50, 200 and 400 microM CdNO3) and kinetic studies (from 1 to 48 h) showed a dose threshold (50 microM CdNO3) and a lag time between the appearance of PCs and iso-PCs concomitant with the gamma-EC accumulation induced by Cd, occurring at cadmium concentrations above 50 microM. This accumulation was suppressed by supplementation of the culture medium with 25 mM glycine. Glycine supplementation had a limited impact on the concentrations of glutathione and PCs whereas the levels of most iso-PCs were significantly increased. Taken together, these results indicate that GSH is involved in the biosynthesis of the iso-PCs in vivo, and that the biosynthesis of PC-related peptides is limited by the availability of glycine in the presence of high cadmium concentrations.
Subject(s)
Arabidopsis Proteins/biosynthesis , Arabidopsis/metabolism , Glutathione/biosynthesis , Metals, Heavy/metabolism , Culture Media , Glutathione/chemistry , Glutathione/metabolism , Kinetics , Nitrogen/metabolism , Phytochelatins , Spectrometry, Mass, Electrospray IonizationABSTRACT
LC/ESI-MS/MS is a promising alternative to immunoassays in improving the analysis of recombinant therapeutic proteins in biological fluids for toxicity and pharmacokinetics purposes. To assess the sensitivity and validation issues associated with this technique, we use here as a model EPI-hNE4, a 56-amino acid recombinant protein, and demonstrate that a method based on tandem mass spectrometry combined with liquid chromatography and electrospray interface can reach sensitivity similar to that of ELISA but without its potential cross-reactivity. For this purpose, a triple quadrupole mass spectrometer operating in positive ion and single reaction monitoring mode with transition, m/z 1040 --> 1224.5, was used for selective peak detection. Particular issues related to the internal standard, i.e., elution and ionization patterns similar to the protein without stable isotope labeling, and to analytical interference due to endogenous binding antibodies were addressed. A limit of quantification in human or monkey plasma of 5 ng/mL was reached with a sample volume of 100 microL, corresponding to 40 fmol injected into the HPLC column. Intra- and interassay precision and accuracy were below 15%. No matrix effect was detected.
Subject(s)
Pancreatic Elastase/antagonists & inhibitors , Peptides/pharmacology , Protease Inhibitors/pharmacology , Recombinant Proteins/blood , Animals , Calibration , Chromatography, High Pressure Liquid/methods , Enzyme-Linked Immunosorbent Assay/methods , Haplorhini , Humans , Isotope Labeling , Reproducibility of Results , Sensitivity and Specificity , Tandem Mass Spectrometry/methods , Time FactorsABSTRACT
OBJECTIVE: Our objective was to investigate the potential for relevant pharmacotherapeutic interaction between cytochrome P4503A4 (CYP3A4)-inhibiting agents such as erythromycin and the dopamine agonist alpha-dihydroergocryptine (DHEC). METHODS: The study was carried out as a single-center, controlled, nonblinded, 2-way crossover clinical trial with randomly allocated period-balanced sequences, investigating two treatments of a single oral dose of 10 mg DHEC (on the morning of day 1), once administered alone (reference), once along with a 4-day treatment (days -2 to 1) of 500 mg erythromycin 3 times daily. Periods were separated by a washout of at least 14 days. Nine healthy white male volunteers, 22 to 42 years old, with a body weight range of 58 to 90 kg (body mass index, 20.2-25.1 kg x m(-2)) began the study. One subject discontinued prematurely, and 8 concluded the study in accordance with the study protocol. RESULTS: The plasma and urinary pharmacokinetics of DHEC and its metabolites were characterized by a large variability. Concomitant treatment with erythromycin led to respective increases of 9.5 (95% confidence interval [CI], 6.5 to 13.9) and 16.5 (95% CI, 8.7 to 31.5) times the maximum observed plasma drug concentration and the area under the time course of the plasma concentrations up to the last quantifiable concentration after dosing of unchanged DHEC (determined by radioimmunoassay). The 24-hour urinary excretion was on average 11 times larger (95% CI, 5.9 to 20.7). Qualitatively similar findings were recorded for the total of DHEC plus metabolites (as determined by enzyme immunoassay). CONCLUSIONS: The concomitant use of erythromycin or similarly CYP3A4-inhibiting agents along with direct dopaminergic agonists such as the ergoline DHEC may cause a clinically relevant increase in pharmacokinetic exposure, which may induce exaggerated dopaminergic effects.
Subject(s)
Cytochrome P-450 Enzyme Inhibitors , Dihydroergotoxine/urine , Dopamine Agonists/urine , Enzyme Inhibitors/pharmacology , Erythromycin/pharmacology , Mixed Function Oxygenases/antagonists & inhibitors , Administration, Oral , Adult , Analysis of Variance , Anti-Bacterial Agents/pharmacology , Area Under Curve , Cross-Over Studies , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/metabolism , Drug Administration Schedule , Enzyme Inhibitors/administration & dosage , Erythromycin/administration & dosage , Humans , Male , Mixed Function Oxygenases/metabolism , RadioimmunoassayABSTRACT
The tetrapeptide Acetyl-Ser-Asp-Lys-Pro (AcSDKP) has been shown to protect hematopoietic stem cells from the toxicity of anticancer chemotherapies. Since its pharmacological efficacy is limited by a rapid degradation by Angiotensin-I Converting Enzyme (ACE), AcSDKP analogs resistant to ACE have been synthesized. One of these compounds (AcSDKP-NH,) differs from the native AcSDKP by amidation of the C-terminus. Further evaluations of this molecule require an analytical method in order to characterize its pharmacokinetic profile. We report, here, the development of a highly specific and sensitive enzyme immunoassay (EIA) for AcSDKP-NH, thatdoes not cross-react with endogenous or exogenous AcSDKP. Using AcSDKP-NH2-acetylcholinesterase conjugate as a tracer, rabbit specific antiserum and microtiter plates coated with goat anti-rabbit immunoglobulins, this EIA allows the determination of AcSDKP-NH2 with limits of quantitation of 1 nM in mouse plasma and 100 pmol/g in tissues. Intra-day and inter-day coefficients of variations were less than 20%. The method was successfully applied to a pharmacokinetic study in order to compare plasma and tissue profiles of AcSDKP-NH2 and AcSDKP. Plasma AcSDKP-NH2 levels were found higher than those of AcSDKP, with AUCinf and Cmax values, respectively, 26- and 10-fold higher than that of AcSDKP.
Subject(s)
Amides/chemistry , Immunoenzyme Techniques/methods , Oligopeptides/analysis , Animals , Humans , Immune Sera , Mice , Mice, Inbred CBA , Oligopeptides/chemistry , Oligopeptides/pharmacokinetics , Protein Conformation , Reproducibility of Results , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
BACKGROUND: N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a natural inhibitor of pluripotent hematopoietic stem cell proliferation. Ac-SDKP plasma concentration is increased 5-fold after angiotensin-converting enzyme inhibition. Here we studied the effect of Ac-SDKP on monocyte/macrophage infiltration, fibroblast proliferation, and collagen deposition in the rat heart in renovascular hypertension. METHODS AND RESULTS: We investigated whether long-term Ac-SDKP administration would prevent left ventricular (LV) hypertrophy and interstitial collagen deposition in rats with 2-kidney, 1-clip (2K-1C) hypertension. Ac-SDKP (400 microgram. kg(-1). d(-1)) did not affect development of hypertension. Mean blood pressure was similar in rats with 2K-1C hypertension whether they were given vehicle or Ac-SDKP and was higher than in controls. Both LV weight and cardiomyocyte size were significantly increased in rats with 2K-1C hypertension compared with controls and were unaffected by Ac-SDKP. Proliferating cell nuclear antigen- and monocyte/macrophage-positive cells were increased in the LV of 2K-1C hypertensive rats; this increase was significantly blunted by Ac-SDKP (P<0.001). LV interstitial collagen fraction was also increased in 2K-1C hypertensive rats given vehicle (10.1+/-0.8%) compared with sham (5.3+/-0.1%, P<0.0001), and this increase was prevented by Ac-SDKP (5.4+/-0.4%, P<0.001). CONCLUSIONS: Ac-SDKP inhibited monocyte/macrophage infiltration, cell proliferation, and collagen deposition in the LV of hypertensive rats without affecting blood pressure or cardiac hypertrophy, suggesting that it may be partly responsible for the cardioprotective effect of angiotensin-converting enzyme inhibitors.
Subject(s)
Collagen/drug effects , Heart Ventricles/drug effects , Hypertension, Renovascular/metabolism , Oligopeptides/pharmacology , Animals , Blood Pressure/drug effects , Cell Division/drug effects , Collagen/metabolism , Dose-Response Relationship, Drug , Heart/drug effects , Heart Rate/drug effects , Heart Ventricles/metabolism , Hypertension, Renovascular/physiopathology , Hypertrophy, Left Ventricular/pathology , Macrophages/drug effects , Macrophages/pathology , Male , Monocytes/drug effects , Monocytes/pathology , Myocardium/pathology , Oligopeptides/blood , Rats , Rats, Sprague-Dawley , Time FactorsABSTRACT
The phosphinic peptide RXP 407 has recently been identified as the first potent selective inhibitor of the N-active site (domain) of angiotensin-converting enzyme (ACE) in vitro. The aim of this study was to probe the in vivo efficacy of this new ACE inhibitor and to assess its effect on the metabolism of AcSDKP and angiotensin I. In mice infused with increasing doses of RXP 407 (0.1--30 mg/kg/30 min), plasma concentrations of AcSDKP, a physiological substrate of the N-domain, increased significantly and dose dependently toward a plateau 4 to 6 times the basal levels. RXP 407 significantly and dose dependently inhibited ex vivo plasma ACE N-domain activity, whereas it had no inhibitory activity toward the ACE C-domain. RXP 407 (10 mg/kg) did not inhibit the pressor response to an i.v. angiotensin I bolus injection in mice. In contrast, lisinopril infusion (5 and 10 mg/kg/30 min) affected the metabolism of both AcSDKP and angiotensin I. Thus, RXP 407 is the first ACE inhibitor that might be used to control selectively AcSDKP metabolism with no effect on blood pressure regulation.
Subject(s)
Angiotensin I/metabolism , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Oligopeptides/antagonists & inhibitors , Oligopeptides/pharmacology , Peptidyl-Dipeptidase A/chemistry , Phosphinic Acids/pharmacology , Animals , Blood Pressure/drug effects , Dose-Response Relationship, Drug , Hydrolysis , Indicators and Reagents , Lisinopril/pharmacology , Male , Mice , Oligopeptides/blood , Peptidyl-Dipeptidase A/blood , Peptidyl-Dipeptidase A/drug effects , Time FactorsABSTRACT
In order to determine nicergoline pharmacokinetics after oral administration to humans, we have developed two radioimmunoassays, one directed against nicergoline and the other directed against known nicergoline metabolites. The assays were validated according to the recommendations of international regulatory agencies and their limits of quantification were 40 and 10 pg/ml, respectively. In order to further validate the methods, a chromatographic separation of immunoreactive entities was performed with samples from healthy volunteers who were given 15 mg of Sermion (nicergoline orally administered). Chromatographic determination of assay specificity showed that the metabolite radioimmunoassay recognised known nicergoline metabolites but also a new metabolite. Using the antibodies directed against nicergoline, we were unable to detect nicergoline in the human plasma. This suggests that nicergoline is absent in the circulation because of complete metabolism through its first-pass effect.
Subject(s)
Adrenergic alpha-Antagonists/blood , Immunoassay/methods , Nicergoline/blood , Adrenergic alpha-Antagonists/metabolism , Adult , Humans , Male , Nicergoline/metabolism , Pilot Projects , Reproducibility of ResultsABSTRACT
The tetrapeptide AcSDKP, a natural and specific substrate of angiotensin I-converting enzyme (ACE), is a negative regulator of hematopoiesis. AcSDKP has been measured in various biological media using an enzyme immunoassay (EIA), but its presence in human plasma and urine has not been formally established. By using immunoaffinity extraction and liquid chromatography-electrospray mass spectrometry, we demonstrate that AcSDKP-like immunoreactivity measured with EIA in plasma and urine samples from untreated, captopril- (an ACE inhibitor) and AcSDKP-treated subjects corresponds to AcSDKP. The present study confirms that AcSDKP is naturally present in human plasma and urine and that EIA is reliable for its measurement in such media.
Subject(s)
Chromatography, Affinity/methods , Oligopeptides/metabolism , Humans , Oligopeptides/blood , Oligopeptides/urine , Reference Values , Spectrometry, Mass, Electrospray Ionization/methodsABSTRACT
OBJECTIVE: The aim of this study was to evaluate the pharmacokinetic behavior of unchanged alpha-dihydroergocryptine (DHEC, Almirid, Desitin Arzneimittel GmbH, Hamburg, Germany, under licence of Polichem S.A., Luxembourg) and total DHEC (unchanged DHEC and pooled metabolites) in plasma and urine in patients with impaired hepatic function, following administration of single oral doses. METHODS: The study was carried out according to an open, uncontrolled, parallel-group design, investigating two study groups: patients with hepatic dysfunction, i.e. with evidence of stable cirrhosis (n = 10) and age- and sex-matched healthy subjects (n = 8). Each subject received a single dose of 20 mg DHEC. Blood samples were taken at specified intervals up to 72 h after dosing and urine was collected fractionally for 24 h. Concentrations of unchanged DHEC were determined by RIA and concentrations of total DHEC (unchanged and pooled metabolites) by EIA. RESULTS: The plasma and urinary pharmacokinetics of DHEC and its metabolites were characterized by large variability. In patients with impaired hepatic function, the geometric mean Cmax and AUC(0-infinity) values for unchanged DHEC were 571.3 pg/ml (CV: 0.87) and 4038 pg x h/ml (CV: 1.04) and were approximately 2 times (2.04, 95% CI: 0.93 to 4.46 and 2.11, 95% CI: 0.58 to 7.73 for Cmax and AUC(0-infinity), respectively) larger than those measured in age-matched healthy controls. The 24-hour urinary excretion was approximately 3 times (3.41, 95% CI: 0.95 to 12.21) higher in patients with hepatic dysfunction. Similar results were obtained for total DHEC. CONCLUSIONS: The results reflect an increased systemic exposure in patients with impaired hepatic function which is not due to a reduced urinary excretion/elimination or reduced renal clearance. The most likely mechanism involved is a reduction in pre-systemic biotransformation. The observed range of effects on the pharmacokinetics of DHEC in patients with compromized hepatic function does not suggest the need to revise the dosage recommendations, since treatment with DHEC is generally started with low doses and is slowly up-titrated according to the individual response and the occurrence of adverse effects. Nevertheless, lower maintenance doses are likely to be achieved.
Subject(s)
Dihydroergotoxine/pharmacokinetics , Dopamine Agonists/pharmacokinetics , Liver Diseases/metabolism , Adolescent , Adult , Aged , Area Under Curve , Dihydroergotoxine/blood , Dihydroergotoxine/urine , Dopamine Agonists/blood , Dopamine Agonists/urine , Female , Humans , Male , Middle AgedABSTRACT
1. Angiotensin I-converting enzyme (ACE) has two homologous active N- and C-terminal domains and displays activity towards a broad range of substrates. The tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) has been shown to be hydrolysed in vitro by ACE and to be a preferential substrate for its N-terminal active site. This peptide reversibly prevents the recruitment of pluripotent haematopoietic stem cells and normal early progenitors into the S-phase. 2. Angiotensin I-converting enzyme inhibitors, given as a single dose to normal subjects or during long-term treatment in hypertensive patients, result in plasma AcSDKP levels five- to six-fold higher and urine concentrations 40-fold higher than those of control subjects and/or patients. Thus, AcSDKP is a natural peptide hydrolysed by the N-terminal domain of ACE in vivo. In addition, ACE may be implicated in the process of haematopoietic stem cell regulation by permanently degrading this natural circulating inhibitor of cell entry into the S-phase. 3. Besides hydrolysis by ACE, the second very effective mechanism by which AcSDKP is cleared from plasma is glomerular filtration. Because of its high sensitivity and specificity, the measurement of AcSDKP in plasma and urine provides a valuable tool in screening specific inhibitors of the N-terminal domain of ACE and in monitoring ACE inhibition during chronic treatment. 4. The long-term consequences of AcSDKP accumulation are not known. During chronic ACE inhibition in rats, AcSDKP levels slightly increase in organs with high ACE content (kidneys, lungs). To significantly increase its concentration in target haematopoietic organs (the extracellular fraction of bone marrow), AcSDKP has to be infused on top of a captopril-based treatment. 5. A selective inhibitor of the N-domain of ACE in vitro and in vivo has been identified recently. The phosphinic peptide RXP 407 does not interfere with blood pressure regulation, but does increase, dose dependently, plasma concentrations of AcSDKP in mice, in contrast with lisinopril, which affects the metabolism of both AcSDKP and angiotensin I. N-Terminal-selective ACE inhibitors may be used to selectively control AcSDKP metabolism in target haematopoietic organs. This new therapeutic strategy may be of value for protecting haematopoietic cells from the toxicity of cancer chemotherapy.
Subject(s)
Oligopeptides/metabolism , Peptidyl-Dipeptidase A/metabolism , Angiotensin-Converting Enzyme Inhibitors/administration & dosage , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Animals , Humans , Hydrolysis/drug effects , Hypertension/drug therapy , Mice , Oligopeptides/blood , Oligopeptides/urine , Peptidyl-Dipeptidase A/chemistry , Protein Structure, Tertiary , Rats , Structure-Activity Relationship , Substrate SpecificityABSTRACT
While nephrologists often observe reduced hematocrit associated with inhibitors of angiotensin-converting enzyme (ACE), the basis for this effect is not well understood. We now report that two strains of ACE knockout mice have a normocytic anemia associated with elevated plasma erythropoietin levels. (51)Cr labeling of red cells showed that the knockout mice have a normal total blood volume but a reduced red cell mass. ACE knockout mice, which lack tissue ACE, are anemic despite having normal renal function. These mice have increased plasma levels of the peptide acetyl-SDKP, a possible stem cell suppressor. However, they also show low plasma levels of angiotensin II. Infusion of angiotensin II for 2 weeks increased hematocrit to near normal levels. These data suggest that angiotensin II facilitates erythropoiesis, a conclusion with implications for the management of chronically ill patients on inhibitors of the renin-angiotensin system.
Subject(s)
Anemia/blood , Angiotensin II/pharmacology , Erythropoiesis/drug effects , Peptidyl-Dipeptidase A/deficiency , Angiotensin II/blood , Animals , Blood Pressure/drug effects , Erythrocyte Indices , Female , Genotype , Hematocrit , Kidney/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Peptidyl-Dipeptidase A/blood , Peptidyl-Dipeptidase A/genetics , SystoleABSTRACT
Bromocriptine, a D2 receptor agonist, was administered intravenously (1mg/kg) to anesthetized rats. Microdialysis probes were implanted in the pituitary and the striatum, known sites of D2 agonist action. Bromocriptine and its metabolites were monitored in plasma and tissue dialysates for 4 h. Drug analyses were performed using two different enzyme immunoassays specific for untransformed bromocriptine or a pool of parent drug plus hydroxylated metabolites. The metabolites/parent drug ratio for areas under the curve was 5.5 in plasma and 1 in the pituitary. No metabolites could be detected in the striatum. Bromocriptine penetration was at least 10-fold greater in the pituitary than in the striatum. The kinetics of bromocriptine in the pituitary and striatum did not parallel those in plasma, indicating that the prolonged action of bromocriptine reported by other authors may be due to slow dissociation from receptors.
Subject(s)
Bromocriptine/metabolism , Corpus Striatum/metabolism , Dopamine Agonists/metabolism , Pituitary Gland/metabolism , Receptors, Dopamine D2/agonists , Animals , Male , Microdialysis , Rats , Rats, Sprague-DawleyABSTRACT
Angiotensin I-converting enzyme (ACE) has been shown to be involved in the catabolism of the tetrapeptide acetyl-Ser-Asp-Lys-Pro (AcSDKP). As AcSDKP is a physiological inhibitor of haematopoietic stem cell proliferation, we investigated the in vitro and in vivo effects of captopril, one of the specific inhibitors of ACE, on the proliferation of primitive haematopoietic cells. Regenerating bone marrow cells obtained from mice given one injection of cytosine arabinoside (100 mg/kg) as well as SA2 myeloid leukaemia cells were incubated in vitro for 24 h with 10-6 M captopril. Captopril significantly reduced the proportion of high proliferative potential colony-forming cells (HPP-CFC-1) in S-phase, whereas it had no effect on the proportion of SA2 leukaemic colony-forming cells in S-phase. When given in vivo to mice 1 h after 2 Gy gamma-irradiation or cytosine arabinoside (AraC) injection, captopril (100 mg/kg) was shown to prevent HPP-CFC-1 entry into S-phase induced by these cytotoxic treatments. The observed effects correlated with a reduction in ACE degradative activity and an increase in the level of endogenous AcSDKP both in the supernatants of captopril-treated bone marrow cells and in plasma of treated animals. The present findings suggest that AcSDKP might mediate the observed in vitro and in vivo inhibitory effects of captopril on primitive haematopoietic cell proliferation.
Subject(s)
Angiotensin-Converting Enzyme Inhibitors/pharmacology , Captopril/pharmacology , Hematopoietic Stem Cells/drug effects , Animals , Antimetabolites, Antineoplastic/pharmacology , Cell Division/drug effects , Cell Line , Cells, Cultured , Cytarabine/pharmacology , Interleukin-3/pharmacology , Leukemia, Myeloid/immunology , Leukemia, Myeloid/metabolism , Macrophage Colony-Stimulating Factor/pharmacology , Mice , Mice, Inbred Strains , Oligopeptides/metabolism , Peptidyl-Dipeptidase A/blood , Recombinant Proteins/pharmacology , S PhaseABSTRACT
Since currently used assays of amphotericin B (AMB) lack sensitivity or are not easily adaptable in all laboratories, we have developed an enzyme immunoassay for AMB in biological fluids and tissues. Antibodies to AMB were raised in rabbits after administration of an AMB-bovine serum albumin conjugate. The enzymatic tracer was obtained by coupling AMB via its amino group to acetylcholinesterase (EC 3.1.1.7). These reagents were used for the development of a competitive immunoassay performed on microtitration plates. The limit of quantification was 100 pg/ml in plasma and 1 ng/g in tissues. The plasma assay was performed directly without extraction on a minimal volume of 0.1 ml. The intra- and interassay coefficients of variation were in the range of 5 to 17%, and the recoveries were 92 to 111% for AMB added to human plasma. The assay was applied to a pharmacokinetic study with mice given AMB intraperitoneally at the dose of 1 mg/kg. The drug distribution in selected compartments (plasma, liver, spleen, lung, and brain) was monitored until 72 h after administration. In conclusion, our assay is at least 100-fold more sensitive than previously described bioassays or chromatographic determinations of AMB and may be useful in studying the tissue pharmacokinetics of new AMB formulations and in drug monitoring in clinical situations.
Subject(s)
Amphotericin B/pharmacokinetics , Immunoenzyme Techniques , Amphotericin B/administration & dosage , Amphotericin B/blood , Animals , Biological Assay , Humans , Mice , Rabbits , Reproducibility of Results , Sensitivity and Specificity , Tissue DistributionABSTRACT
The tetrapeptide N-Acetyl-Seryl-Aspartyl-Lysyl-Proline (AcSDKP) is a natural regulator of hematopoietic stem cell proliferation. The present study was aimed at investigating the presence and the role of AcSDKP in rat testis. Specific immunoreactivity was always observed in the interstitial tissue at all stages of testicular development and in elongated spermatids at 45 days of age and in adults. In accordance with the interstitial labeling, high AcSDKP levels were detected in Leydig cell and testicular macrophage culture media and cell extracts, as well as in the testicular interstitial fluid (TIF). Much lower concentrations were found in peritubular cells and Sertoli cells cultures, whereas very low concentrations were present in cultured spermatocytes and spermatids. In contrast to the slight degradation rate of AcSDKP observed in the spermatocyte and spermatid culture media, no catabolism of the peptide was seen in testicular somatic cell culture medium. Furthermore, the degradation rate of AcSDKP was much lower in TIF than in peripheral blood plasma. Despite the very strong evidence indicating that Leydig cells and testicular macrophages produce AcSDKP, the selective destruction of these cells did not result in any change in AcSDKP levels in TIF or in plasma. This suggests a compensatory mechanism ensuring constant levels of the peptide in TIF when interstitial cells are absent. Finally, in vitro, in the presence of AcSDKP, significantly more [(3)H]thymidine incorporation was found in A spermatogonia. In conclusion, this study establishes the presence of very high concentrations of AcSDKP in rat testis and demonstrates its Leydig cell and testicular macrophage origin. The presence of AcSDKP in the TIF and its stimulatory effect on thymidine incorporation in spermatogonia very strongly suggest its implication in the paracrine control of spermatogenesis.
Subject(s)
Oligopeptides/metabolism , Testis/cytology , Testis/metabolism , Animals , Cell Cycle/drug effects , Cell Extracts , Cells, Cultured , Culture Media, Conditioned/chemistry , Culture Techniques , Extracellular Space/chemistry , Half-Life , Leydig Cells/chemistry , Leydig Cells/cytology , Leydig Cells/metabolism , Macrophages/chemistry , Macrophages/cytology , Macrophages/metabolism , Male , Oligopeptides/analysis , Oligopeptides/pharmacology , Rats , Rats, Sprague-Dawley , Seminiferous Tubules/chemistry , Seminiferous Tubules/cytology , Seminiferous Tubules/drug effects , Seminiferous Tubules/metabolism , Sertoli Cells/chemistry , Sertoli Cells/cytology , Sertoli Cells/metabolism , Spermatozoa/chemistry , Spermatozoa/cytology , Spermatozoa/drug effects , Spermatozoa/metabolism , Testis/chemistry , Testis/growth & development , Thymidine/metabolismABSTRACT
OBJECTIVES: This study was designed to compare different proposed methods of assessing adherence with angiotensin-converting enzyme (ACE) inhibitor (ACEI) therapy in chronic heart failure. BACKGROUND: The use of ACEIs in chronic heart failure gives us a unique opportunity to assess a patient's adherence by measuring whether the expected biochemical effect of an ACEI is present in the patient's bloodstream. In fact, there are several different ways of assessing ACE in vivo: these are serum ACE activity itself, plasma N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), urine AcSDKP, plasma angiotensin I (AI), plasma angiotensin II (AII), or the AII/AI ratio. METHODS: Patients with chronic heart failure (n = 39) were randomized to regimens of ACEI nonadherence for one week, ACEI adherence for one week or two versions of partial adherence for one week, after which the above six tests were performed. RESULTS: All six tests significantly distinguished between full nonadherence for one week and full or partial adherence. Only plasma AcSDKP produced a significantly different result between partial adherence and either full adherence or full nonadherence for one week. In terms of their ability to distinguish full nonadherence from full adherence, plasma AcSDKP was 89% sensitive and 100% specific with an area under its ROC of 0.95. Corresponding figures for urine AcSDKP were 92%, 97% and 0.95 and for serum ACE they were 86%, 95% and 0.90. CONCLUSIONS: All six tests distinguished full nonadherence from all other forms of adherence. The rank order of performance was plasma AcSDKP, urine AcSDKP, serum ACE, AII/AI ratio and plasma AII followed by plasma AI.
Subject(s)
Angiotensin-Converting Enzyme Inhibitors/therapeutic use , Heart Failure/drug therapy , Lisinopril/therapeutic use , Treatment Refusal , Aged , Angiotensin I/blood , Angiotensin II/blood , Biomarkers/blood , Biomarkers/urine , Chronic Disease , Diuretics/therapeutic use , Drug Therapy, Combination , Echocardiography , Furosemide/therapeutic use , Heart Failure/diagnosis , Heart Failure/metabolism , Humans , Oligopeptides/blood , Oligopeptides/urine , Peptidyl-Dipeptidase A/blood , Radionuclide Ventriculography , Treatment Outcome , Treatment Refusal/statistics & numerical dataABSTRACT
The hemoregulatory peptide Acetyl-Ser-Asp-Lys-Pro (AcSDKP) has been reported to accumulate in plasma and urine after the oral administration of angiotensin-converting enzyme (ACE) inhibitors in humans. It is unknown whether such an accumulation also occurs in tissues. We administered captopril (3, 10, or 30 mg/kg) orally for 2 weeks to Wistar rats. In a second experiment, captopril (10 mg/kg) was administered for 9 days and was followed by a 1-h i.v. infusion of either AcSDKP (0.1 or 2 mg/kg) or saline on day 9. Captopril alone dose-dependently increased plasma AcSDKP by a factor of 3 to 5 and urine AcSDKP by a factor of 3. It slightly increased renal and pulmonary AcSDKP concentrations but did not affect AcSDKP concentrations in bone marrow and spleen. The combination of AcSDKP (2 mg/kg) and captopril gave very high AcSDKP concentrations in plasma and urine and increases in AcSDKP concentration by factors of 27 in kidney, 5.5 in lung, and 6.9 in the extracellular fraction of bone marrow. In contrast, no change was observed in the AcSDKP concentration in spleen and in the intracellular fraction of bone marrow. In conclusion, during chronic ACE inhibition in rats, AcSDKP levels slightly increased in organs with high ACE contents. No such increase occurred in hematopoietic organs. AcSDKP had to be combined with captopril to significantly increase its concentration in tissues other than the spleen. The possibility of pharmacologically increasing AcSDKP levels in the extracellular fraction of bone marrow may be of value for protecting hematopoietic cells from the toxicity of cancer chemotherapy.
Subject(s)
Angiotensin-Converting Enzyme Inhibitors/pharmacology , Captopril/pharmacology , Growth Inhibitors/metabolism , Oligopeptides/metabolism , Animals , Bone Marrow/drug effects , Bone Marrow/metabolism , Dose-Response Relationship, Drug , Growth Inhibitors/blood , Growth Inhibitors/urine , Kidney/drug effects , Kidney/metabolism , Lung/drug effects , Lung/metabolism , Male , Oligopeptides/blood , Oligopeptides/urine , Proteins/metabolism , Rats , Rats, Wistar , Tissue DistributionABSTRACT
Angiotensin I-converting enzyme (ACE) is a zinc metallopeptidase that plays a major role in blood pressure regulation. The demonstration that the hemoregulatory peptide acetyl-Ser-Asp-Lys-Pro (AcSDKP) is a natural and specific substrate of the N-active site of ACE suggests that this enzyme may have a new physiological role such as the modulation of hematopoietic stem cells. In vitro studies have shown that ACE inhibitors displayed various potencies in inhibiting the degradation of different natural or synthetic substrates of ACE, among which captopril inhibits AcSDKP hydrolysis more potently than angiotensin I hydrolysis. To look for this selectivity in vivo, we investigated the pharmacodynamic effect of increasing doses of captopril (0.01-10 mg/kg) during the 90 min after i.v. administration to spontaneously hypertensive rats. Plasma and urinary AcSDKP levels were measured. The renin-angiotensin system was evaluated by measurements of ACE activity in plasma samples, using the synthetic substrate Hip-His-Leu, by determinations of plasma renin concentrations and measurements of arterial blood pressure. The results showed that captopril (0.01-0.3 mg/kg) selectively inhibited AcSDKP hydrolysis, with limited effects on the renin-angiotensin system. AcSDKP levels in plasma and urine rose to a plateau 4 times the basal level for doses more than 0.3 mg/kg. All of the parameters reflecting the renin-angiotensin system were significantly affected at doses of 1 and 10 mg/kg. The present study therefore confirms that captopril can be used to protect hematopoietic stem cells during antitumor chemotherapy while having only a limited effect on cardiovascular homeostasis.