Determination of 2-hydroxyflutamide in human plasma by high-performance liquid chromatography and its application to pharmacokinetic studies

Flutamide is a potent antiandrogen used for the treatment of prostatic cancer. Flutamide undergoes extensive first-pass metabolism to the pharmacologically active metabolite 2-hydroxyflutamide. A simple, sensitive, precise, accurate and specific HPLC method, using carbamazepine as the internal standard, for the determination of 2-hydroxyflutamide in human plasma was developed and validated. After addition of the internal standard, the analytes were isolated from human plasma by liquid–liquid extraction. The method was linear in the 25 to 1000 ng/ml concentration range (r>0.999). Recovery for 2-hydroxyflutamide was greater than 91.4% and for internal standard was 93.6%. The limit of quantitation was 25 ng/ml. Inter-batch precision, expressed as the relative standard deviation (RSD), ranged from 4.3 to 7.9%, and accuracy was better than 93.9%. Analysis of 2-hydroxyflutamide concentrations in plasma samples from 16 healthy volunteers following oral administration of 250 mg of flutamide provided the following pharmacokinetic data (mean±SD): C_max, 776±400 ng/ml; AUC_0–∞, 5368±2689 ng h/ml; AUC_0–t, 5005±2605 ng h/ml; T_max, 2.6±1.6 h; elimination half-life, 5.2±2.0 h.     

Determination of celecoxib in human plasma by normal-phase high-performance liquid chromatography with column switching and ultraviolet absorbance detection

A method is described for the determination of celecoxib in human plasma. Samples were extracted using 3M Empore membrane extraction cartridges and separated under normal-phase HPLC conditions using a Nucleosil-NO₂ (150×4.6 mm, 5 μm) column. Detection was accomplished using UV absorbance at 260 nm. The HPLC method included a column switching procedure, in which late eluting compounds were diverted to waste, to reduce run-time to 12 min. The assay was linear in the concentration range of 25–2000 ng/ml when 1-ml aliquots of plasma were extracted. Recoveries of celecoxib were greater than 91% over the calibration curve range. Intraday precision and accuracy for this assay were 5.7% C.V. or better and within 2.3% of nominal, respectively. The assay was used to analyze samples collected during human clinical studies.     

Determination of a HIV protease inhibitor (DMP 450) in animal and human plasma by solid-phase extraction and high-performance liquid chromatography

Extraction of DMP 450 from plasma was performed with C₂ solid-phase extraction columns, using 0.1 M ammonium acetate in 90% methanol to elute DMP 450. The extraction recovery over the range of 10 to 10 000 ng/ml averaged 81.0, 96.2, 77.4, 95.2 and 68.0% from rat, dog, monkey, chimpanzee (25–10 000 ng/ml) and human plasma, respectively. HPLC analysis was carried out with a C₁₈ column and a mobile phase of acetonitrile, methanol and 30 mM potassium phosphate (pH 3), the composition dependent on the type of plasma being analyzed, and monitored at a wavelength of 229 nm. Intra-day and inter-day coefficients of variation were less than 9.9 and 12.9%, respectively. Absolute differences were less than 11.5%.     

High-throughput, semi-automated determination of a cyclooxygenase II inhibitor in human plasma and urine using solid-phase extraction in the 96-well format and high-performance liquid chromatography with post-column photochemical derivatization-fluorescence detection

Compound I, 5-chloro-3-(4-methanesulfonylphenyl)-6′-methyl-[2,3′]bipyridinyl, has been found to be a specific inhibitor of the enzyme cyclooxygenase II (COX II). The anti-inflammatory properties of this compound are currently being investigated. HPLC assays for the determination of this analyte in human plasma and human urine have been developed. Isolation of I and the internal standard (II) was achieved by solid-phase extraction (SPE) in the 96-well format. A C₈ SPE plate was used for the extraction of the drug from human plasma (recovery >90%) while a mixed-mode (C₈/Cation) SPE plate was used to isolate the analytes from human urine (recovery approximately 71%). The analyte and internal standard were chromatographed on a Keystone Scientific Prism-RP^® guard column (20×4.6 mm) connected to a Prism-RP^® analytical column (150×4.6 mm), using a mobile phase consisting of 45% acetonitrile in 10 mM acetate buffer (pH=4); the analytes eluted at retention times of 5.2 and 6.9 min for I and II, respectively. Compounds I and II were found to form highly fluorescent products after exposure to UV light (254 nm). Thus, the analytes were detected by fluorescence (λ_ex=260 nm, λ_em=375 nm) following post-column photochemical derivatization. Eight point calibration curves over the concentration range of 5–500 ng/ml for human plasma and human urine yielded a linear response (R²>0.99) when a 1/y weighted linear regression model was employed. Based on the replicate analyses (n=5) of spiked standards, the within-day precision for both assays was better than 7% C.V. at all points on the calibration curve; within-day accuracy was within 5% of nominal at all standard concentrations. The between-run precision and accuracy of the assays, as calculated from the results of the analysis of quality control samples, was better than 8% C.V. and within 8% of nominal. I was found to be stable in human plasma and urine for at least 8 and 2 months, respectively. In addition, the human plasma assay was semi-automated in order to improve sample throughput by utilizing a Packard liquid handling system and a Tom-Tec Quadra 96 SPE system. The precision and accuracy of the semi-automated procedure were comparable to the manual procedure. Over 5000 clinical samples have been analyzed successfully using these methods.     

A new reliable chemiluminescence immunoassay (CLIA) for prostaglandin E2 using enhanced luminol as substrate

A sensitive and reliable chemiluminescence immunoassay suitable for the quantitative determination of prostaglandin E₂ (PGE₂) has been developed using 96 well microtiter plates (MTP). The assay is based on a competitive reaction between a highly specific monoclonal anti-PGE₂ antibody (mouse), free antigen and solid phase bound antigen. The MTP was first coated with a bovine serum albumin (BSA)-PGE₂ conjugate. Then, after preincubating, the anti-PGE₂ antibody (Ab) and the analyte were added. The remaining amount of free antibody was captured by the solid phase bound BSA-PGE₂ conjugate. The monoclonal antibody captured on the MTP was determined using biotinylated antimouse-Ab and a complex of avidin and biotin-labelled horseradish peroxidase (HRP). Substrate for HRP was the cyclic diacyl hydrazide compound luminol, enhanced by p-iodophenol. Photons emitted during the reaction were measured using a photomultiplier tube. The assay has been validated with assay buffer and human plasma over a concentration range of 10–50,000 pglml. The lower limit of quantification is 100 pglml (2 pglwell) and 150 pglml (3 pglwell) for buffer and plasma, respectively. The intea-day coefficients of variation (CV) for the range of 100–50,000 pglml are 3.2–8.9% (buffer) and 4.2–17.7% (plasma) and inter-day CV are 2.9–19.8% (buffer) and 3.6–21.2% (plasma). The method can be used for quantification of PGE2 in biological fluids like plasma and suction blister fluid.     

Simultaneous determination of all-trans-, 13-cis-, 9-cis-retinoic acid and their 4-oxo-metabolites in plasma by high-performance liquid chromatography

A gradient reversed-phase high-performance liquid chromatographic technique is described for the easy separation and quantification of some retinoids; all-trans-retinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid and their corresponding 4-oxometabolites, in plasma. The method involved a diethyl ether-ethyl acetate (50:50, v/v) mixture extraction at pH 7 with acitretin and 13-cis-acitretin as internal standards. A Nova-Pak C₁₈ steel cartridge column was used. The mobile phase was methanol-acetonitrile (65:35, v/v) and 5% tetrahydrofuran (solvent A) and 2% aqueous acetic acid (solvent B) at 1 ml/min. The gradient composition was (only the percentages of solvent B are mentioned): I, 25% solvent B at the time of injection; II, 12% solvent B at 11 min until 30 min; III, 25% solvent B and maintenance of 25% solvent B for 10 min until a new injection. Total time between injections was 40 min. Detection was by absorbance at 350 nm. The precision calculated for plasma concentrations ranging from 2 to 250 ng/ml was better than 15% and the accuracy was less than 12%. The linearity of the method was in the range of 2 to 400 ng/ml of plasma. The limit of quantification was 2 ng/ml for each of the compounds. The HPLC method was applied to plasma specimens collected from animals receiving single dose administrations of all-trans-retinoic acid, 13-cis-retinoic acid and 9-cis-retinoic acid.     

Development of a sensitive and quantitative HPLC-FLD method for the determination of obestatin in human plasma

Obestatin is a gastrointestinal system peptide. The quantification of this peptide is conventionally performed using immunological techniques. In this study, a selective and sensitive HPLC method coupled with fluorescence detection for the quantitation of obestatin in human plasma was developed and validated. The separation was obtained on a C₁₈ (4.6 × 100 mm, 3.5-μm particles) column using a mobile phase composed of acetonitrile and water, both including 0.1% trifluoroacetic acid. The developed method was found to be linear in the concentration range of 20 to 1000 ng/mL, with a coefficient of determination of 0.9982. The precision results were less than 10%, and the accuracy results were between 92% and 107%. The detection and quantification limit values were obtained as 2.8 and 9.4 ng/mL, respectively. Analyte solutions were found stable for 24 h at room temperature, three freeze–thaw cycles, and 2 weeks at −20°C. The developed method was successfully used for the quantification of obestatin in human plasma samples. In conclusion, the developed method is sensitive and specific for measuring the plasma concentrations of obestatin.     

Determination of haloperidol and its reduced metabolite in human plasma by liquid chromatography-mass spectrometry with electrospray ionization

A sensitive and accurate liquid chromatographic-electrospray mass spectrometric (LC-ES-MS) method for the determination of haloperidol (H) and reduced haloperidol (RH) in human plasma is presented, using chlorohaloperidol as the internal standard. A 2-ml volume of plasma subjected to basic (NaOH) extraction, acid (HCl) back-extraction, acid wash and basic (NaOH) re-extraction. The extraction solvent was hexane-isoamyl alcohol (99:1, v/v) for the whole procedure. A Nucleosil C₁₈ column (150×1 mm) was used for high-performacne liquid chromatography, together with 2 mM HCOONH₄-acetonitrile (55:45, v/v; pH 3.0) as the mobile phase. For each drug, four characteristic ions were monitored. Linearity was assessed in the ranges 0.1–50 and 0.25–50 ng/ml for H and RH, respectively. Recoveries were 58 and 70% and detection limits were 0.075 and 0.100 ng/ml for H and RH, respectively. Correlation coefficients were better than 0.999 for both compounds. R.S.D.s for repeatability and reproducibility at 0.25 ng/ml were 11.1 and 8.5% for H and 9.4 and 11.2% for RH, respectively. One of the main advantages of (LC-ES-MS) over other detection systems is the increase in selectivity obtained by monitoring three ions of confirmation for each of the drugs.     

High-performance liquid chromatographic determination of levodropropizine in human plasma with fluorometric detection

The present describes a new high-performance liquid chromatographic method with fluorescence detection for the analysis of levodropropizine [S-(−)-3-(4-phenylpiperazin-1-yl)-propane-1,2-diol] (Levotuss), an anti-tussive drug, in human serum and plasma. A reversed-phase separation of levodropropizine was coupled with detection of the native fluorescence of the molecule, using excitation and emission wavelengths of 240 nm and 350 nm respectively. The analytical column was packed with spherical 5 μm poly(styrene-divinylbenzene) particles and the mobile phase was 0.1 M NaH₂PO₄ pH 3-methanol (70:30, v/v), containing 0.5% (v/v) tetrahydrofuran. For quantitation, p-methoxylevodropropizine was used as the internal standard. Samples of 200 μl of either serum or plasma were mixed with 200 μl of 0.1 M Na₂HPO₄ pH 8.9 and extracted with 5 ml of chloroform-2-propanol (9:1, v/v). The dried residue from the organic extract was redissolved with distilled water and directly injected into the chromatograph. The limit of detection for levodropropizine, in biological matrix, was about 1–2 ng/ml, at a signal-to-noise ratio of 3. The linearity was satisfactory over a range of concentrations from 3 to 1000 ng/ml (r² = 0.99910); within-day precision tested in the range 5–100 ng/ml as well as day-to-day reproducibility proved acceptable, with relative standard deviations better than 1% in most cases. Interferences from as many as 91 therapeutic or illicit drugs were excluded.     

Simultaneous determination of epirubicin, doxorubicin and their principal metabolites in human plasma by high-performance liquid chromatography and electrochemical detection

A high-performance liquid chromatographic method with electrochemical detection has been developed for the simultaneous determination of epirubicin, 13-S-dihydroepirubicin, doxorubicin and 13-S-dihydrodoxorubicin in human plasma. An aliquot of 200 μl plasma, spiked with internal standard, was extracted by solid-phase extraction using polymeric adsorbent columns. Chromatography was performed using a C₁₈ reversed-phase column with a mobile phase consisting of water–acetonitrile (71:29, v/v) containing 0.05 M Na₂HPO₄ and 0.05% v/v triethylamine adjusted to pH 4.6 with citric acid. Linearity of the method was obtained in the concentration range of 1–500 ng/ml for all the analytes. Analytical recoveries of the analytes ranged from 89 to 93%. The assay can be used for the simultaneous determination of the four analytes, or for epirubicin and its metabolite or doxorubicin and its metabolite, using the other parent drug as an internal standard. The method was applied to analyze human plasma samples from patients treated with epirubicin using doxorubicin as an internal standard.     

Determination of plasma bromvalerylurea and its main metabolite by a simple high-performance liquid chromatographic method and quantitation of bromide by energy dispersive X-ray spectrometry in carbon tetrachloride-intoxicated rats

In the present study, small volumes of plasma were used for the measurement of bromvalerylurea (BVU), its metabolite, 3-methylbutyrylurea (MVU), and bromide in carbon tetrachloride (CCl₄)-treated rats by HPLC–UV and energy dispersive X-ray spectrometry. A liquid–liquid extraction system was also investigated. BVU and MVU were extracted from 100 μl plasma samples in a single-step involving deproteination with 1 M hydrochloric acid using ethenzamide as internal standard. Samples were separated by HPLC in an acetonitrile–8 mM potassium dihydrogenphosphate buffer (35:65, v/v) mobile phase at a flow-rate of 0.4 ml/min on a 15 cm octadecylsilyl column at room temperature. Analytes were detected at a wavelength of 210 nm. The limits of quantitation for BVU, MVU and bromide are 0.1, 0.1 and 50 μg/ml, respectively. The intra-day accuracies over the range of concentrations were 95.8 to 121.1%, 97.2 to 119.7% and 96.2 to 105.8% for BVU, MVU and bromide, respectively. The inter-day accuracies were 97.7 to 115.1%, 98.3 to 111.6% and 98.3 to 102.9% for BVU, MVU and bromide, respectively. The absolute recoveries using tert.-butyl methyl ether are 96–98% for BVU and 95–98% for MVU. The decline in the plasma concentrations of BVU in olive oil-treated rats fitted a one-compartment model and the plasma MVU level reached a peak at around 1.5–2 h and then decreased gradually. The elimination of BVU in CCl₄ (1 ml/kg)-treated rats was delayed and MVU production was less than that in the olive oil-treated group. However, there was no difference in the plasma levels of bromide between CCl₄-treated rats and control rats.     

Gas chromatographic–mass spectrometric determination of α-ketoisocaproic acid and [H7]α-ketoisocaproic acid in plasma after derivatization with N-phenyl-1,2-phenylenediamine

A method for determination of α-ketoisocaproic acid (KIC) and [4,5,5,5,6,6,6-²H₇]α-ketoisocaproic acid ([²H₇]KIC) in rat plasma was developed using gas chromatography–mass spectrometry-selected ion monitoring (GC–MS-SIM). [5,5,5-²H₃]α-Ketoisocaproic acid ([²H₃]KIC) was used as an analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. The keto acids were extracted by cation-exchange chromatography using BondElut SCX cartridge and derivatized with N-phenyl-1,2-phenylenediamine to form N-phenylquinoxalinone derivatives. Quantitation was performed by SIM of the respective molecular ions at m/z 278, 281 and 285 for the derivatives of KIC, [²H₃]KIC and [²H₇]KIC on the electron impact method. The limit of detection was found to be 70 fmol per injection (S/N=3) and the limit of quantitation for [²H₇]KIC was around 50 nM in rat plasma. Endogenous KIC concentrations in 50 μl of rat plasma were measured with relative intra- and inter-day precision of 4.0% and 3.3%, respectively. The intra- and inter-day precision for [²H₇]KIC spiked to rat plasma in the range of 0.1 to 10 μM gave good reproducibility with relative standard deviation (RSD) of 6.5% and 5.4%, respectively. The intra- and inter-day relative errors (RE) for [²H₇]KIC were less than 6.4% and 3.8%, respectively. The method was applied to determine the plasma concentration of [²H₇]KIC after an intravenous administration of [²H₇]KIC in rat.     

Increased production of tumor necrosis factor and prostaglandin E2 by monocytes in cancer patients and its unique modulation by their plasma

Summary We investigated the role of monocytes in the production of tumor necrosis factor (TNF) and prostaglandin E₂ (PGE₂) in 77 cancer patients with malignancies of the digestive tract, using 30 normal individuals and 18 noncancer patients as controls. Monocytes were incubated with lipopolysaccharide for 20 h, and TNF production and PGE₂ production were analyzed by bioassays. Elevated levels of TNF (>512 U/ml) and PGE₂ (>8 ng/ml) production were demonstrated in many cancer patients when these factors were induced in the medium with 10% fetal bovine serum. The elevated level of TNF was seen to be restricted for the most part to patients with malignancies. Thus, 51 out of 59 cancer patients (86%), consisting of 44 primary cancer patients and 15 recurrent cancer patients, showed an increased level of TNF. In contrast, almost all of 18 postoperative cancer patients showed TNF levels comparable to those of normal individuals. Furthermore, 16 primary cancer patients were also demonstrated to have reduced levels of TNF production by monocytes after curative operation. When 10% cancer-patient plasma was added to the induction culture, TNF production by monocytes was drastically suppressed in the cancer patients. Interestingly, the same addition of plasma induced a prominent enhancement of PGE₂ production in the cancer patients. The plasma of noncancer patients did not modulate production of these factors. No TNF activity was found in the plasma of cancer patients, but such plasma did contain an increased level of PGE₂ (100–300 pg/ml). Although PGE₂ (>2ng/ml) was able to suppress TNF production by monocytes, the addition of 10% plasma PGE₂ was not enough to induce suppression. An unknown factor(s) in the plasma of cancer patients may uniquely modulate the elevated TNF and PGE₂ production in these patients.     

Gas chromatographic–mass spectrometric determination of α-ketoisocaproic acid and [2H7]α-ketoisocaproic acid in plasma after derivatization with N-phenyl-1,2-phenylenediamine

A method for determination of α-ketoisocaproic acid (KIC) and [4,5,5,5,6,6,6-²H₇]α-ketoisocaproic acid ([²H₇]KIC) in rat plasma was developed using gas chromatography–mass spectrometry-selected ion monitoring (GC–MS-SIM). [5,5,5-²H₃]α-Ketoisocaproic acid ([²H₃]KIC) was used as an analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. The keto acids were extracted by cation-exchange chromatography using BondElut SCX cartridge and derivatized with N-phenyl-1,2-phenylenediamine to form N-phenylquinoxalinone derivatives. Quantitation was performed by SIM of the respective molecular ions at m/z 278, 281 and 285 for the derivatives of KIC, [²H₃]KIC and [²H₇]KIC on the electron impact method. The limit of detection was found to be 70 fmol per injection (S/N=3) and the limit of quantitation for [²H₇]KIC was around 50 nM in rat plasma. Endogenous KIC concentrations in 50 μl of rat plasma were measured with relative intra- and inter-day precision of 4.0% and 3.3%, respectively. The intra- and inter-day precision for [²H₇]KIC spiked to rat plasma in the range of 0.1 to 10 μM gave good reproducibility with relative standard deviation (RSD) of 6.5% and 5.4%, respectively. The intra- and inter-day relative errors (RE) for [²H₇]KIC were less than 6.4% and 3.8%, respectively. The method was applied to determine the plasma concentration of [²H₇]KIC after an intravenous administration of [²H₇]KIC in rat.     

Determination of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma by reversed-phase liquid chromatography with ultraviolet detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantitation of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma, using clozapine as internal standard. After sample alkalinization with 1 ml of NaOH (2 M) the test compounds were extracted from plasma using diisopropyl ether–isoamylalcohol (99:1, v/v). The organic phase was back-extracted with 150 μl potassium phosphate (0.1 M, pH 2.2) and 60 μl of the acid solution was injected into a C₁₈ BDS Hypersil analytical column (3 μm, 100×4.6 mm I.D.). The mobile phase consisted of phosphate buffer (0.05 M, pH 3.7 with 25% H₃PO₄)–acetonitrile (70:30, v/v), and was delivered at a flow-rate of 1.0 ml/min. The peaks were detected using a UV detector set at 278 nm and the total time for a chromatographic separation was about 4 min. The method was validated for the concentration range 5–100 ng/ml. Mean recoveries were 98.0% for risperidone and 83.5% for 9-hydroxyrisperidone. Intra- and inter-day relative standard deviations were less than 11% for both compounds, while accuracy, expressed as percent error, ranged from 1.6 to 25%. The limit of quantitation was 2 ng/ml for both analytes. The method shows good specificity with respect to commonly prescribed psychotropic drugs, and it has successfully been applied for pharmacokinetic studies and therapeutic drug monitoring.     

High-performance liquid chromatography of the aromatase inhibitor, letrozole, and its metabolite in biological fluids with automated liquid-solid extraction and fluorescence detection

An analytical method for the determination of letrozole (CGS 20 267) in plasma and of letrozole and its metabolite, CGP 44 645, in urine is described. Automated liquid-solid extraction of compounds from plasma and urine was performed on disposable 100-mg C₈ columns using the ASPEC system. The separation was achieved on an ODS Hypersil C₁₈ column using acetonitrile-phosphate buffer, pH 7, as the mobile phase at a flow-rate of 1.5 ml/min. A fluorescence detector was used for the quantitation. The excitation and emission wavelengths were 230 and 295 nm, respectively. The limits of quantitation (LOQ) of letrozole in plasma and in urine were 1.40 nmol/l (0.4 ng/ml) and 2.80 nmol/l, respectively. The respective mean recoveries and coefficient of variation (C.V.) were 96.5% (9.8%) in plasma and 104% (7.7%) in urine. The LOQ of CGP 44 645 in urine was 8.54 nmol/l (2 ng/ml). The mean recovery was 108% (6.3%). The compounds were well separated from co-extracted endogenous components and no interferences were observed at the retention times of compounds. The sensitivity of this method for letrozole in plasma should be sufficient for kinetic studies in humans with single doses of 0.5 mg and possibly less.     

Determination of cimetidine in human plasma by high-performance liquid chromatography following liquid-liquid extraction

A new method is described for the determination of cimetidine in human plasma. The drug and internal standard (ranitidine) were separated on a Nucleosil C₁₈ 5 μm (25 × 4.6 mm I.D.) column using a mobile phase of acetonitrile-phosphate buffer, pH 6.2 (25:75, v/v) containing 2.5 g/l heptane sulphonic acid. The mobile phase was delivered at a flow-rate of 0.9 ml/min, detection was by ultraviolet absorption at 228 nm and concentrations were calculated on the basis of peak areas. The drugs were extracted from alkaline plasma into ethyl acetate using a salting out procedure which involved the addition of 100 ml of a saturated solution of K₂CO₃ to each 250-μl plasma aliquot. The method was validated over the concentration ranges 50–3000 ng/ml and 100–7000 ng/ml for two separate studies. Mean coefficients of variation were less than 6% for both intra- and inter-assay in both studies and recoveries varied between 71 and 81%. The method was successfully applied to the determination of cimetidine in plasma for a pharmacokinetic study.     

Maltobionic acid enhances intestinal absorption of calcium and magnesium in rats

ABSTRACT

In Experiment 1, the effects of calcium maltobionate (MBCa) on calcium and magnesium absorption were examined using male rats. Four diets were designed in which 25%, 50%, and 100% of calcium carbonate (CaCO₃, Control) were substituted with MBCa and were designated as MBCa-25, MBCa-50, and MBCa-100, respectively. The cecal concentration of short-chain fatty acids was significantly higher in groups MBCa-50 and MBCa-100; however, pH of cecal contents did not significantly differ among the groups. Retention rates of calcium and magnesium were significantly higher in all MBCa groups as compared to the Control. In Experiment 2, the efficiency of calcium absorption was compared using everted sacs of jejunum and ileum with CaCO₃ and MBCa as calcium sources. More calcium from MBCa was absorbed as the concentration of calcium increased in comparison to CaCO₃. It was concluded that MBCa is a better calcium source than CaCO₃ in terms of both calcium retention and absorption.

Abbreviations: ANOVA: analysis of variance; Ca: Calcium; CaCO_3: calcium carbonate; ICP-OES: Inductivity coupled plasma optical emission spectrometer; Mg: magnesium; MBCa: calcium maltobionate; OCPC: o-cresolphthalein complexone; SCFAs: short-chain fatty acids; SE: standard error; TRPM6: transient receptor potential melastatin 6.     

Effect of a low-carbohydrate diet on plasma and sweat ammonia concentrations during prolonged nonexhausting exercise

The purpose of this investigation was to examine the effect of low body glycogen stores on plasma ammonia concentration and sweat ammonia excretion during prolonged, nonexhausting exercise of moderate intensity. On two occasions seven healthy untrained men pedalled on a cycle ergometer for 60 min at 50% of their predetermined maximal O₂ uptakes ( _max) firstly, following 3 days on a normal mixed diet (N-diet) (60% carbohydrates, 25% fat and 15% protein) and secondly, following 3 days on a low-carbohydrate diet (LC-diet) (less than 5% carbohydrates, 50% fat and 45% protein) of equal energy content. Blood was collected from the antecubital vein immediately before, at 30th and at 60th min of exercise. Sweat was collected from the hypogastric region using gauze pads. It was shown that plasma ammonia concentrations after the LC-diet were higher than after the N-diet at both the 30th and 60th min of exercise. Sweat ammonia concentration and total ammonia loss through the sweat were also higher after the LC-diet. The higher ammonia concentrations in plasma and sweat after the LC-diet would seem to indicate an increased ammonia production, which may be related to reduced initial carbohydrate stores.