Magnesium bioavailability from mineral water. A study in adult men.

OBJECTIVE: To assess magnesium enteral absorption from a magnesium-rich mineral water. DESIGN: Experimental study. SETTING: Department of Nuclear Medicine, Brugmann Hospital, Brussels, Belgium. SUBJECTS: Ten healthy male volunteers in the age range 25-42 y. INTERVENTION: Each subject completed two sessions in a random order. At one session, they received an oral load of 300 ml of water (containing 1.2 mmol Mg), traced with (28)Mg, and at the other session they received an intravenous injection of (28)Mg, in order to take into account the metabolism of endogenous magnesium. The dietary consumption was further noted on a weekly diary. RESULTS: The mean bioavailability was 59.1% (s.d.+/-13.6). Magnesium absorption and age were significantly inversely correlated (r=-0.68, P=0.035). CONCLUSION: Magnesium-rich mineral water is a reliable source of magnesium. Our observation of decreased magnesium absorption with age deserves further investigations. SPONSORSHIP: The study was sponsored by SEV, Bourg la Reine, France.

The anti-HIV pentameric pseudopeptide HB-19 is preferentially taken up in vivo by lymphoid organs where it forms a complex with nucleolin.

The HB-19 pseudopeptide 5[Kpsi(CH(2)N)PR]-TASP, psi(CH(2)N) for reduced peptide bond, is a specific inhibitor of HIV infection in different CD4(+) cell lines and in primary T-lymphocytes and macrophages. It blocks virus-particle attachment to permissive cells by binding and forming a stable complex with nucleolin expressed on the cell surface. Here, we have investigated the tissue distribution of the tritiated HB-19 by using beta-radio imager whole-body mapping in rats. A rapid, selective, and stable distribution and accumulation of the systematically administered HB-19 was demonstrated within the spleen, liver, bone, and kidney as soon as 5 min following its administration. No apparent uptake of HB-19 occurred in the brain and the muscle tissue. Interestingly and despite its rapid clearance from the blood, at 24 h postexposure a significant proportion of HB-19 was still recovered from target organs, of which 16-37% could be accounted for intact pseudopeptide. The elimination of HB-19 mainly occurred by renal glomerular filtration and most of the excreted radioactivity appeared to be HB-19 metabolites. Finally, injection of the biotin-labeled HB-19 pseudopeptide but not its control counterpart allowed the recovery of the HB-19-nucleolin complex from the liver, spleen, thymus, and bone marrow, thus indicating that the in vivo molecular target of HB-19 is surface nucleolin. Our results demonstrate the preferential uptake and stability of HB-19 in lymphoid organs that are the site of HIV propagation.

Characterization of immunoreactive acetyl-Ser-Asp-Lys-Pro in human plasma and urine by liquid chromatography-electrospray mass spectrometry.

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.

Immunoassays for the detection of nicergoline and its metabolites in human plasma.

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.

Microdialysis study of bromocriptine and its metabolites in rat pituitary and striatum.

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.

Improvement of in vivo stability of phosphodiester oligonucleotide using anionic liposomes in mice.

Antisense phosphodiester oligonucleotides (ODN) are unstable in biological fluids due to nuclease-mediated degradation and therefore cannot be used in most antisense therapeutic applications. We describe here an in vitro and in vivo stabilization of a 15 mer phosphodiester sequence using anionic liposomes. Two formulations have been studied: DOPC/OA/CHOL and DOPE/OA/CHOL (pH-sensitive liposomes). Our in vitro findings reveal the same stabilization effect in mouse plasma for both anionic liposomes. In vivo investigation showed a great protective effect for both formulations after intravenous administration to mice. By contrast with in vitro results, a higher protection of ODN was observed with DOPC/OA/CHOL liposomes compared to the DOPE/OA/CHOL formulation. The latter was degraded in blood (75% of the injected dose at 5 min) probably due to interactions with blood components, and the remaining (25% at 5 min) was distributed mostly to the liver and spleen. DOPC liposomes were remarkably stable in blood and were distributed more slowly to all studied organs (liver, spleen, kidneys and lungs). Intact ODN was still observed in some organs (liver, spleen, lungs), but not in blood, 24 hours after DOPC liposome administration. These results suggest that this antisense strategy using carrier systems may be applicable to the treatment of diseases involving the reticuloendothelial system.

A sensitive amphotericin B immunoassay for pharmacokinetic and distribution studies.

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.

Real-time monitoring of the hybridization reaction: application to the quantification of oligonucleotides in biological samples.

We describe here a competitive hybridization assay using TRACE technology which can be used for real-time monitoring of oligonucleotide hybridization. This assay quantifies all kinds of oligonucleotides in biological fluids without extraction. The assay makes use of two different probes and involves a fluorescent transfer process. As fluorescence measurements are not destructive, they can be sequentially repeated, thereby allowing comparison of the hybridization kinetics and binding strength of chemically modified backbone oligonucleotides (>0.5 nM) in biological media. The assay was validated for pharmacokinetic analysis of phosphodiester and phosphorothioate oligonucleotides in plasma and in different organs (liver, kidneys, lungs, spleen) at low concentrations (0.4 mg/kg, corresponding to clinical doses). Respective sensitivities for phosphodiester and phosphorothioate were 0.2 and 0.8 pmol/ml in plasma and 2 and 8 pmol/g in tissues, which allow to recover intact phosphorothioate sequences in some organs even after 24 h.

Effect of chronic magnesium supplementation on magnesium distribution in healthy volunteers evaluated by 31P-NMRS and ion selective electrodes.

AIMS: The role of magnesium (Mg) intake in the prevention and treatment of diseases is greatly debated. Mg biodistribution after chronic Mg supplementation was investigated, using state-of-the-art technology to detect changes in free ionized Mg, both at extra- and intracellular levels. METHODS: Thirty young healthy male volunteers participated in a randomised, placebo (P)-controlled, double-blind trial. The treated group (MgS) took 12 mmol magnesium lactate daily for 1 month. Subjects underwent in vivo 31P-NMR spectroscopy and complete clinical and biological examinations, on the first and last day of the trial. Total Mg was measured in plasma, red blood cells and 24 h urine ([Mg]U ). Plasma ionized Mg was measured by ion-selective electrodes. Intracellular free Mg concentrations of skeletal muscle and brain tissues were determined noninvasively by in vivo 31P-NMR at 3T. NMR data were automatically processed with the dedicated software MAGAN. RESULTS: Only [Mg]U changed significantly after treatment (in mmol/24 h, for P, from 4.2+/-1.4 before to 4.1+/-1.3 after and, for MgS, from 3.9+/-1.1 before to 5. 1+/-1.1 after, t=2.15, P=0.04). The two groups did not differ, either before or after the trial, in any other parameter, whether clinical, biological or in relation with the Mg status. CONCLUSIONS: Chronic oral administration of Mg tablets to young healthy male volunteers at usual pharmaceutical doses does not alter Mg biodistribution. This study shows that an adequate and very complete noninvasive methodology is now available and compatible with the organization of clinical protocols which aim at a thorough evaluation of Mg biodistribution.

A new specific enzyme immunoassay allowing an efficient pharmacokinetic evaluation of gamma-cyclodextrin after intravenous administration to rats.

PURPOSE: Because of its ability to form complexes with drugs, gamma-cyclodextrin is of great potential value in pharmaceutical formulations. The biological fate of y-cyclodextrin must therefore be considered in safety evaluation, using sensitive and specific methods applicable to biological fluids. METHODS: Antibodies were raised against gamma-cyclodextrin, allowing the development of a new enzyme immunoassay. The analytical characteristics of this assay were evaluated. Rats were given a single intravenous 25 mg/kg dose of gamma-cyclodextrin. Plasma and urine samples were collected and assayed. RESULTS: This new enzyme immunoassay was sensitive (limit of detection close to 94 pg/mL) and suitable for quantification of gamma-cyclodextrin in urine and plasma after methanol extraction. The use of different linear and cyclic compounds demonstrated the high specificity of the assay. After i.v. administration, the concentration of gamma-cyclodextrin rapidly decreased in the plasma while the molecule was probably distributed into the tissues. Although urinary elimination predominates, only 50% of the injected gamma-cyclodextrin was recovered in urine, suggesting enzymatic degradation and/or tissular storage. CONCLUSIONS: This assay may provide important information on the fate of gamma-cyclodextrin inclusion complexes dedicated to drug-delivery using various modes of administration (oral, parenteral, transmucosal or dermal).

Pharmacokinetic analysis of 6-monoamino-beta-cyclodextrin after intravenous or oral administration to rats using a specific enzyme immunoassay.

We have developed a highly sensitive enzyme immunoassay for 6-monoamino-beta-CD (mono(6-amino-6-deoxy)cyclomaltoheptaose) and its parent compound (beta-CD) with a detection limit in the 100 pg/mL range. The polyclonal antibodies obtained are highly specific for the beta-cyclodextrin core and do not recognize other cyclic cyclodextrins (i.e., alpha- and gamma-CD) or linear analogues. This enzyme immunoassay can be used to quantify 6-monoamino-beta-CD in rat urine and plasma. Using this immunoassay, we have evaluated the main pharmacokinetic parameters of 6-monoamino-beta-CD after iv administration to the rat of a 25 mg/kg dose. Since this method is strictly specific to the native beta-CD form, we have demonstrated that the molecule rapidly disappeared from plasma but is probably distributed in the tissues. The urinary route appears as the predominant way of elimination since almost all the administered drug is recovered in urine. Finally, analysis of the same molecule after oral administration to the rat (25 mg/kg) demonstrates low plasma levels and that about 1% of the administered dose is excreted in urine. These experiments demonstrate the high stability of the beta-CD core irrespective of the method of administration. This immunological method could provide relevant information on the fate of beta-CD and some derivatives for drug delivery using different modes of administration (oral, parenteral, transmucosal, or dermal).

Development and in vivo assessment of a transdermal system for physostigmine.

A copolymer was developed as a transdermal (TD) system for physostigmine. The loading was carried out with a solution of physostigmine (PHY) base (20 mg/ml) in water/ethanol: 80/20 (v/v) at 40 degrees C for 3 h. The PHY load was 5.3 mg/cm2 (n = 3). Desorption carried out in vitro showed that 70% was desorbed during the first 6 h. More than 50% of the PHY was degraded within 45 min in skin homogenate. The TD was tested in vivo in rabbits during a 24 h experiment. PHY was quantified using a validated HPLC method. AUC0-24 h was 245.2 +/- 337.2 h.ng/ml. The mean pad flux reached 4.6 +/- 6.3 micrograms/cm2 from 0 to 24 h and, 24 h after the application of the pad, 110 micrograms/cm2 of PHY had been passed through the skin. After removed of the patch, plasma concentrations first increased from 15.8 +/- 28.6 ng/ml (at 24 h) to 21.4 +/- 36.7 ng/ml, then decreased with an elimination half-life of 0.7 +/- 0.2 h. AChE inhibition percentages increased from 6.5 +/- 2.3% to 16.0 +/- 27.7%. In vitro and in vivo studies in rabbit have shown that this system is suitable for further investigations in order to obtain a possible carrier for PHY therapy.

Magnesium isotopic abundance measurement in humans: comparison of two mass spectrometric methods.

Pharmacokinetic studies using stable isotopes of magnesium as tracers need to determine the isotopic abundance in biological media by means of mass spectrometry. Of mass spectrometric techniques, electronic impact-mass spectrometry (EI-MS) and inductively coupled plasma-mass spectrometry (ICP-MS) can be used. We have measured the isotopic abundance in plasma and urinary samples and compared the precision and accuracy of these two methods. Graphical representations showed that mean differences were close to 0, there was no obvious relationship between the difference and the mean, and no systematic bias was evidenced at low or high isotopic abundance. This was shown for isotopic abundance of either 25 Mg and 26 Mg. EI-MS is able to measure magnesium isotopic abundance with an intra-day precision between 0.14 and 0.45 per cent and with an inter-day precision between 0.20 and 1.23 per cent. ICP-MS exhibited an intra-day precision between 0.01 and 0.06 per cent and an inter-day precision between 0.01 and 0.15 per cent. Our results showed that, despite the similar isotopic abundances in biological samples obtained with the two methods, a large difference in precision clearly favours ICP-MS in studies of magnesium behaviour using stable isotopes.

High plasma level of N-acetyl-seryl-aspartyl-lysyl-proline: a new marker of chronic angiotensin-converting enzyme inhibition.

The acute administration of the angiotensin-converting enzyme (ACE) inhibitor captopril to healthy subjects transiently increases 5.5-fold the plasma levels of a natural stem-cell regulator, N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP). The aim of this study was to measure plasma Ac-SDKP levels during chronic treatment with all types of ACE inhibitors and to assess its relevance as a marker of ACE inhibition. Plasma levels of Ac-SDKP were blindly determined in age- and sex-matched hypertensive patients either treated (ACEI group, n=27) or not (non-ACEI group, n=23) with an ACE inhibitor for more than 1 month. Geometric mean [range] of plasma Ac-SDKP levels were significantly higher in the ACEI group (3.78 [1.48 to 14.5] pmol/mL) than in the non-ACEI group, with no overlap between the groups (0.75 [0.36 to 1.22] pmol/mL, P<.0001). The measurement of Ac-SDKP in plasma discriminated all the patients of the ACEI group, whereas the simultaneous determination of either in vitro (using hippuryl-histidine-leucine as substrate) or in vivo (angiotensin II/angiotensin I ratio) ACE activity failed to identify nine and five cases, respectively. We conclude that Ac-SDKP accumulates in plasma during chronic ACE inhibitor treatment. The long-term consequences of Ac-SDKP accumulation are unknown. The reliability of plasma Ac-SDKP measurement makes it the best marker of chronic ACE inhibition, which can help to verify patients' compliance to ACE inhibitor treatment.

An enzyme immunoassay for rat growth hormone: validation and application to the determination of plasma levels and in vitro release.

A competitive enzyme immunoassay for rat growth hormone (rGH) has been developed using polyclonal anti-rGH antibodies and an acetylcholinesterase (EC 3.1.1.7.) enzymatic tracer coupled covalently with rGH. The assay was performed in 96-well microtiter plates coated with rabbit polyclonal anti-goat immunoglobulin antibodies. Molecular sieve filtration and Western blot analysis revealed a single immunoreactive peak for rat plasma or pituitary extracts. Cross-reactivity with other rat pituitary hormones or human GH was less than 1%. Assay of samples in a concentration range of 0.7 to 69 ng/ml by enzyme immunoassay and radioimmunoassay were well correlated (r = 0.87 and 0.85 respectively for plasma and culture medium samples). Intra- and inter-assay variations in plasma were 4 (n = 24) and 14% (n = 9) respectively. Minimal detectable amounts of rGH were 0.6 ng/ml. A two-site immunometric assay also developed with the same antibodies allowed a detection threshold of 0.25 ng/ml.

Targets for SMR1-pentapeptide suggest a link between the circulating peptide and mineral transport.

The submandibular rat 1 protein (SMR1) is selectively processed at pairs of basic amino acid residues in a tissue- and sex-specific manner. We have mapped peripheral targets for the final secretory maturation product of SMR1, the pentapeptide QHNPR, by examining in vivo the tissue distribution of the radiolabeled peptide using beta-radio imager whole body autoradiography. The characteristics of tissue uptake allowed specific binding sites at physiological peptide concentrations to be identified within the renal outer medulla, bone and dental tissue, glandular gastric mucosa, and pancreatic lobules. Direct evidence that pentapeptide binding sites are localized in selective portions of the male rat nephron, within the S3, S2, and S1 segments of the proximal tubules, was obtained. In bone tissue the pentapeptide exclusively accumulates within the trabecular bone remodeling unit, and in dental tissue it concentrates within the tubules of the dentinal rat incisor. In relation to male rat-specific behavioral characteristics, our data suggest that the circulating androgen-regulated SMR1-derived pentapeptide is primarily involved in the modulation of mineral balance between at least four systems: kidney, bone, tooth, and circulation.

Metabolite involvement in bromocriptine-induced prolactin inhibition in rats.

Bromocriptine (BCT) is a dopamine D2 receptor agonist used for the treatment of Parkinson's disease and hyperprolactinemic disorders. After oral administration, BCT is metabolized into mono- or dihydroxylated metabolites. To study how these metabolites influence parent drug pharmacodynamics, we administered BCT to rats intravenously (1 mg/kg i.v.) and orally (10 mg/kg p.o.) and measured the inhibition of prolactin secretion. Despite similar areas under the curve for BCT, the duration of the effect was 36 h after oral and only 18 h after intravenous administration. Pharmacokinetic/pharmacodynamic models were used to correlate the concentration of BCT in the effect compartment with the lowering of prolactin. One of these models (effect compartment model) showed that the effective concentration (EC50) at the site of action was much lower after oral (0.56 nM) than after intravenous administration (3.68 nM). In contrast, the EC50 values based on BCT metabolite data were in the same range for both administrations. These observations suggested the activity of one or more BCT metabolites. To confirm this hypothesis, hydroxylated metabolites of BCT (produced in vitro by rat liver microsomes) were administered i.v. (100 microg/kg) in rats. We found that monohydroxylated BCT was able to lower prolactin secretion like BCT. Dihydroxylated metabolites, as well as monohydroxylated metabolites, were effective in reducing in vitro prolactin secretion. Because we demonstrated that the concentration of hydroxylated metabolites after oral administration is 55-fold that of BCT, it can be concluded that BCT activity in the pituitary after oral administration is mediated by its metabolites.

A competitive enzyme hybridization assay for plasma determination of phosphodiester and phosphorothioate antisense oligonucleotides.

An enzyme competitive hybridization assay was developed and validated for determination of mouse plasma concentrations of a 15mer antisense phosphodiester oligodeoxyribonucleotide and of two phosphorothioate analogs. Assays were performed in 96-well microtiter plates. The phosphodiester sense sequence was covalently bound to the microwells. The 5'-biotinylated antisense sequence was used as tracer. The principle of the assay involves competitive hybridization of tracer and antisense nucleotide to the solid phase-immobilized sense oligonucleotide. Solid phase- bound tracer oligonucleotide was assayed after reaction with a streptavidin-acetylcholinesterase conjugate, using the colorimetric method of Ellman. As in competitive enzyme immunoassays, coloration was inversely related to the amount of analyte initially present in the sample. The limit of quantification was 900 pM for phosphodiester antisense oligonucleotide using a 100 microl volume of plasma without extraction. Cross-reactivity was negligible after a four base deletion in either the 3'or 5'position. The assay was simple and sensitive, suitable for in vitro screening of oligonucleotide hybridization potency in biological fluids and for measuring the plasma pharmacokinetics of phosphorothioate and phosphodiester sequences.

Effect of variability of plasma interferences on the accuracy of drug immunoassays.

Most immunoassays applied to drugs in human plasma do not use an extraction of analyte. To compensate for interferences due to plasma proteins or salts, standards are prepared in drug-free plasma. Because the concentration of plasma components varies from one subject to another, it is likely that the drug-free plasma is not representative of the potential interference in each plasma. Using two immunoassays, for a steroid (nomegestrol acetate) and a heptapeptide (BN 52080), the authors have shown that tracer binding to the antibody may vary significantly between plasma from different subjects. Intersubject variability of tracer-antibody binding was 21.6% (coefficient of variation for 25 subjects) for nomegestrol acetate. When the same plasma were spiked with the steroid at a concentration corresponding to the central part of the standard curve, the recovery was between 39 and 215%. Intersubject variability in tracer binding was lower (7.7%) for the peptide immunoassay, but still affected accuracy. The authors show that this problem is common to direct immunoassays for other drugs and must be solved in assay development.