Application of C stable isotope labeling liquid chromatography–multiple reaction monitoring–tandem mass spectrometry method for determining intact absorption of bioactive dipeptides in rats

The liquid chromatography–multiple reaction monitoring–tandem mass spectrometry (LC–MRM–MS/MS) method using ¹³C stable isotope-labeled dipeptides was newly developed to simultaneously determine the absorption of three antihypertensive peptides (Val-Tyr, Met-Tyr, and Leu-Tyr) into blood of spontaneously hypertensive rats in one run-in assay. After extracting ¹³C-labeled peptides in blood sample with a C₁₈ cartridge, the extract was applied to a ¹³C monoisotopic transition LC–MRM–MS/MS system with d-Val-Tyr included as internal standard. An excellent separation of each dipeptide in LC was achieved at the elution condition of 5–100% methanol in 0.1% formic acid at a flow rate of 0.25 ml/min. The ¹³C-labeled peptides ionized by electron spray were detected in the positive ion mode within 15 min. The established method showed high reproducibility with less than 10% coefficient of variation as well as high accuracy of more than 85%. After the administration of a mixture containing the three ¹³C-labeled dipeptides to rats at each dose of 30 mg/kg, we could successfully determine the intact absorption of each ¹³C-labeled peptide with the maximal absorption amount of 1.1 ng/ml plasma for Val-Tyr by the proposed LC–MRM–MS/MS method.     

Improved Detection of Di-peptides by Liquid Chromatography-Tandem Mass Spectrometry with 2,4,6-Trinitrobenzene Sulfonate Conversion

Highly sensitive detection of small peptides at the pM level was achieved by liquid chromatography-multiple reaction monitoring-tandem mass spectrometry (LC-MRM-MS/MS) in combination with the 2,4,6-trinitrobenzene sulfonate (TNBS) conversion technique. Six di-peptides having Tyr at the C-terminal (i.e., Gly-Tyr, Val-Tyr, Met-Tyr, Glu-Tyr, Lys-Tyr and His-Tyr) were subjected to the TNBS-MRM analysis in this study. The TNBS conversion conditions of pH 8.0, 30 °C and 60-min incubation enabled the di-peptides to be successfully converted to a trinitrophenyl (TNP) form with the mass increment of +212 Da. The proposed TNBS-MRM method enabled di-peptide detection that was highly improved by a factor of 3–55 in signal-to-noise ratio due to increased hydrophobicity by the induced TNP moiety. The method also permitted highly sensitive detection of di-peptides with a detection limit of >54 pM (>1.35 fmol/injection), achieving high reproducibility (<5% coefficient of variation) and rapidity (<30 min) by LC-MRM-MS/MS.     

MIRACLE: mass isotopomer ratio analysis of U-13C-labeled extracts. A new method for accurate quantification of changes in concentrations of intracellular metabolites

First, we report the application of stable isotope dilution theory in metabolome characterization of aerobic glucose limited chemostat culture of S. cerevisiae CEN.PK 113-7D using liquid chromatography-electrospray ionization MS/MS (LC-ESI-MS/MS). A glucose-limited chemostat culture of S. cerevisiae was grown to steady state at a specific growth rate (mu)=0.05 h(-1) in a medium containing only naturally labeled (99% U-12C, 1% U-13C) carbon source. Upon reaching steady state, defined as 5 volume changes, the culture medium was switched to chemically identical medium except that the carbon source was replaced with 100% uniformly (U) 13C labeled stable carbon isotope, fed for 4 h, with sampling every hour. We observed that within a period of 1 h approximately 80% of the measured glycolytic metabolites were U-13C-labeled. Surprisingly, during the next 3 h no significant increase of the U-13C-labeled metabolites occurred. Second, we demonstrate for the first time the LC-ESI-MS/MS-based quantification of intracellular metabolite concentrations using U-13C-labeled metabolite extracts from chemostat cultivated S. cerevisiae cells, harvested after 4 h of feeding with 100% U-13C-labeled medium, as internal standard. This method is hereby termed "Mass Isotopomer Ratio Analysis of U-13C Labeled Extracts" (MIRACLE). With this method each metabolite concentration is quantified relative to the concentration of its U-13C-labeled equivalent, thereby eliminating drawbacks of LC-ESI-MS/MS analysis such as nonlinear response and matrix effects and thus leads to a significant reduction of experimental error and work load (i.e., no spiking and standard additions). By coextracting a known amount of U-13C labeled cells with the unlabeled samples, metabolite losses occurring during the sample extraction procedure are corrected for.     

Transepithelial transport and stability in blood serum of angiotensin-I-converting enzyme inhibitory dipeptides

The dipeptides Ala-Trp, Val-Phe, and Val-Tyr inhibit the angiotensin-I-converting enzyme. They are encrypted within the primary sequences of different food proteins, e.g. milk proteins. The angiotensin-I-converting enzyme inhibitory potency of these synthetic dipeptides was quantified using a spectrophotometric assay. The dipeptides showed no adverse effects on differentiated Caco-2 cells (model for human intestinal epithelium), as confirmed by transepithelial electrical resistance, microscopy and the activity of the brush-border enzyme dipeptidyl aminopeptidase IV. Furthermore, the transport of these bioactive dipeptides through intact Caco-2 monolayers and their stability to incubation in human blood serum has been demonstrated for the first time. Low molecular mass peptides represent the minimal structures required for angiotensin-I-converting enzyme inhibition which have a high potential bioavailability. Therefore, they may act as target peptides in enriched hydrolysates for the preparation of an angiotensin-I-converting enzyme inhibitory peptide and for the use in special formulations as functional foods/foods of specified health use.     

Rapid measurement of plasma free fatty acid concentration and isotopic enrichment using LC/MS

Measurements of plasma free fatty acids (FFA) concentration and isotopic enrichment are commonly used to evaluate FFA metabolism. Until now, gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) was the best method to measure isotopic enrichment in the methyl derivatives of ¹³C-labeled fatty acids. Although IRMS is excellent for analyzing enrichment, it requires time-consuming derivatization steps and is not optimal for measuring FFA concentrations. We developed a new, rapid, and reliable method for simultaneous quantification of ¹³C-labeled fatty acids in plasma using high-performance liquid chromatography-mass spectrometry (HPLC/MS). This method involves a very quick Dole extraction procedure and direct injection of the samples on the HPLC system. After chromatographic separation, the samples are directed to the mass spectrometer for electrospray ionization (ESI) and analysis in the negative mode using single ion monitoring. By employing equipment with two columns connected parallel to a mass spectrometer, we can double the throughput to the mass spectrometer, reducing the analysis time per sample to 5 min. Palmitate flux measured using this approach agreed well with the GC/C/IRMS method. This HPLC/MS method provides accurate and precise measures of FFA concentration and enrichment.     

Antihypertensive effects of Undaria pinnatifida (wakame) peptide on blood pressure in spontaneously hypertensive rats

We examined the angiotensin I-converting enzyme (ACE) inhibitory activity and antihypertensive effect of the hot water extract of wakame, Undaria pinnatifida. Ten dipeptides were isolated from the extract by several steps of chromatography, and their amino acid sequences were Tyr-His, Lys-Trp, Lys-Tyr, Lys-Phe, Phe-Tyr, Val-Trp, Val-Phe, Ile-Tyr, Ile-Trp, and Val-Tyr. Both single administration and repeated oral administration of synthetic Tyr-His, Lys-Tyr, Phe-Tyr, and Ile-Tyr significantly decreased blood pressure in spontaneously hypertensive rats.     

High incorporation of L-amino acids to cereulide, an emetic toxin from Bacillus cereus

Cereulide is a principal toxin causing emetic syndrome produced by Bacillus cereus. This paper deals with biosynthetic studies on this unusual cyclic depsipeptide toxin from 13C labeled L-amino acid precursors (Val, Leu, Ala) upon cultivation in synthetic media. The analyses were made at atomic level of the constituent amino- or oxy-acids through NMR and ESI-MS/MS spectroscopic methods on cereulide and its hydrolysate dipeptides. The incorporation of the 13C atom was 95% in each O-Val, O-Leu and L-Val, while 40% in D-Ala of cereulide.     

Development and evaluation of an efficient HPLC/MS/MS method for the simultaneous determination of pseudoephedrine and cetirizine in human plasma: application to phase-I pharmacokinetic study

A sensitive, simple and highly selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and evaluated to determine simultaneously the concentrations of pseudoephedrine and cetirizine in human plasma. The chief benefit of the present method is the minimal sample preparation, as the procedure is only one-step protein precipitation. Two drugs were separated on a C(8) column and analyzed by LC/MS/MS using positive electrospray ionisation (ESI). The method had a chromatographic run time of 12.0 min and a linear calibration curve over the concentration range of 1.0-800 ng/ml for pseudoephedrine and 1.0-400 ng/ml for cetirizine, respectively. The lower limit of quantification of the two drugs was 1.0 ng/ml, respectively. The intra- and inter-batch precisions were less than 9.7%. The method described herein has been first used to reveal the pharmacokinetic characters in healthy Chinese volunteers treated with oral administration of different dosages of cetirizine dihydrochloride and controlled-released pseudoephedrine hydrochloride compound tablet, and approached the influence of a standard meal on the extent and rate of absorption of the combination tablet.     

Determination of the active metabolite of prulifloxacin in human plasma by liquid chromatography-tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric method (LC-MS/MS) for the determination of ulifloxacin, the active metabolite of prulifloxacin, in human plasma is described. After sample preparation by protein precipitation with methanol, ulifloxacin and ofloxacin (internal standard) were chromatographically separated on a C(18) column using a mobile phase consisting of methanol, water and formic acid (70:30:0.2, v/v/v) at a flow rate of 0.5 ml/min and then were detected using MS/MS by monitoring their precursor-to-product ion transitions, m/z 350-->m/z 248 for ulifloxacin and m/z 362-->m/z 261 for ofloxacin, in selected reaction monitoring (SRM) mode. Positive electrospray ionization was used for the ionization process. The linear range was 0.025-5.0 microg/ml for ulifloxacin with a lower limit of quantitation of 0.025 microg/ml. Within- and between-run precision was less than 6.6 and 7.8%, respectively, and accuracy was within 2.0%. The recovery ranged from 92.1 to 98.2% at the concentrations of 0.025, 0.50 and 5.0 microg/ml. Compared with the reported LC method, the present LC-MS/MS method can directly determine the ulifloxacin in human plasma without any need of derivatization. The present method has been successfully used for the pharmacokinetic studies of a prulifloxacin formulation product after oral administration to healthy volunteers.     

Ultra sensitive method for the determination of 9-(2-phosphonylmethoxyethyl)adenine in human serum by liquid chromatography-tandem mass spectrometry

An ultra sensitive method for the direct measurement of 9-(2-phosphonylmethoxyethyl)adenine (PMEA), an antiviral agent for hepatitis B, in human serum using high performance liquid chromatography/tandem mass spectrometry (LC-MS/MS) has been developed. This method involves the addition of [13C]PMEA (contains 5 13C) as internal standard, the purification and enrichment by a MCX solid phase extraction (SPE) cartridge, and quantitative analysis using LC-MS/MS. The MS/MS is selected to monitor the m/z 272 --> 134 and m/z 277 --> m/z 139 transitions for PMEA and [13C]PMEA, respectively, using negative electrospray ionization. The MS/MS response is linear over a concentration of 0.1-10 ng/ml with a lower limit of quantitation (LLOQ) of 0.1 ng/ml. The mean inter-assay accuracy (%Bias) for quality control (QC) at 0.1, 0.25, 1.0, and 10 ng/ml are 10, 1.6, -0.8, and 0.0%, respectively. The mean inter-assay precision (%CV) for the corresponding QCs is 3.9, 3.8, 5.3, and 3.4%, respectively. The method has been used to determine PMEA concentration in human serum following a single oral administration of a PMEA pro-drug at dose of 10 and 30 mg.     

Fast detection of fluoroacetamide in body fluid using gas chromatography-mass spectrometry after solid-phase microextraction

A novel method for fast determination of fluoroacetamide, a kind of organic fluorine pesticide, in blood and urine samples was developed with acetamide as an internal standard using gas chromatography/mass spectrometry (GC/MS) after solid-phase microextraction (SPME) technique. The SPME was performed by immersing a PDMS fiber of 100 microm coating thickness in a sample solution for 25 min at 70 degrees C with (CH(3)CH(2))(4)NBr to improve the extraction efficiency. After a GC sample injection, the extracted fluoroacetamide was desorbed from the fiber for 4 min to perform the GC/MS detection with a HP-PLOT Q capillary column. The analytical conditions were optimized by examining systematically, the effects of experimental parameters on the ratio of characteristic ion peak areas of fluoroacetamide to acetamide. Under optimal conditions, the ratio was proportional to the concentration of fluoroacetamide ranging from 5.0 to 90 microg/ml with a detection limit of 1.0 microg/ml. The average recovery of fluoroacetamide in blood sample was 92.2%. The established method could be used for the fast and convenient measurement of fluoroacetamide in poisoned sample.     

LC-ESI-MS determination and pharmacokinetics of adrafinil in rats

A highly sensitive and specific liquid chromatography/tandem mass spectrometric (LC-MS/MS) method for investigating the pharmacokinetics of adrafinil in rats was developed. Rat serum pretreated by solid-phase extraction (SPE) was analyzed by LC-MS/MS with an electrospray ionization (ESI) interface. The mobile phase consisted of acetonitrile:water:acetic acid (35:65:0.1, v/v/v) in an isocratic elution mode pumped at 1.0ml/min. The analytical column (250mmx4.6mm i.d.) was packed with Kromasil C(18) material (5.0mum). The standard curve was linear from 16.5 to 5000ng/ml. The assay was specific, accurate (R.S.D.<2.6%), precise and reproducible (within- and between-day precisions R.S.D. <7.0% and <9.0%, respectively). Adrafinil in rat serum was stable over three freeze-thaw cycles at ambient temperature for 6h. The method had a lower limit of quantitation of 16.5ng/ml, which offered high sensitivity for the determination of adrafinil in serum. The method was successfully applied to pharmacokinetic studies of adrafinil after an oral administration to rats.     

New method for peptide desorption from abundant blood proteins for plasma/serum peptidome analyses by mass spectrometry

This report describes a new method for desorption of low-molecular weight (LMW) peptides from abundant blood proteins for use in subsequent mass spectrometry analyses. Heating of diluted blood serum to 98°C for 15min resulted in dissociation of LMW peptides from the most abundant blood proteins. Application of blood plasma/serum fractionation using magnetic beads with a functionalized surface followed by heating of the resultant fractions significantly increases the number of LMW peptides detected by MALDI-TOF MS, enhances the general reproducibility of mass spectrometry profiles and considerably increases the number of identified blood serum peptides by LC-MS/MS using an Agilent 6520 Accurate-Mass Q-TOF.     

Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. III. Identification of cysteine residues whose modification with N-ethylmaleimide leads to loss of the Ca2+-transporting activity

The reactive sulfhydryl group (SHD) (Kawakita et al. (1980) J. Biochem. 87, 609-617) which is essential for the decomposition of the E-P intermediate of Ca2+-transporting ATPase of the rabbit skeletal muscle sarcoplasmic reticulum has been identified. One sample of sarcoplasmic reticulum membranes was reacted for 3 min with 0.4 mM N-[3H]ethylmaleimide at pH 7.0 at 30 degrees C to a labeling density of 1 mol/mol ATPase without loss of the Ca2+-transporting activity. Another sample of the membranes was treated similarly with non-radioactive N-ethylmaleimide and then labeled with 0.4 mM N-ethyl[14C]maleimide for 17 min. An extensive loss of the Ca2+-transporting activity occurred during the period of this radio-labeling, thus substantiating the 14C-labeling of SHD. The labeled membranes were digested by thermolysin, and the labeled peptides were fractionated by gel filtration and reversed-phase HPLC. Two major radioactive peptides were present in both 3H- and 14C-labeled thermolytic digests, and each of the major components of 14C-labeled peptides had a counterpart in the major components of 3H-labeled peptides which behaved identically on HPLC. The major 14C-labeled peptides were purified and found to be identical with the two SHN peptides, TL-I and TL-II (Saito-Nakatsuka et al. (1987) J. Biochem. 101, 365-376), and 0.5 mol/mol ATPase each of Cys344 and Cys364 was assigned as SHD. It seems that the Ca2+-transport system retains its activity while either of the two Cys residues is unoccupied, but loses it when both of them are modified with N-ethylmaleimide.     

IR spectroscopy of isotope-labeled helical peptides: probing the effect of N-acetylation on helix stability

The effect of N-acetylation on the conformation of alanine-rich helical peptides is examined using isotope-edited Fourier transform infrared (FTIR) spectroscopy. A series of peptides with sequence AA(AAKAA)(3)AAY has been prepared; each peptide incorporates four (13)C-labeled alanines. These peptides have two amide I' bands in their FTIR spectra: one corresponding to the (12)C amino acids, and one assigned to the (13)C amino acids. The intensity and frequency of the (13)C amide I' band varies systematically with the position of the labels in the sequence and the presence or absence of an N-acetyl capping group. The intensity of the (13)C amide I' band correlates with helix stability at the labeled residues as predicted by thermodynamic models of the helix-coil transition. These results suggest that FTIR spectroscopy combined with specific isotope labeling can be used to dissect the conformation of helical peptides at the residue level.     

Determination of baicalin in rat cerebrospinal fluid and blood using microdialysis coupled with ultra-performance liquid chromatography-tandem mass spectrometry

An in vivo microdialysis sampling method coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was employed for continuous simultaneous monitoring of unbound baicalin in rat blood and brain. Microdialysis probes were inserted into the jugular vein and brain cerebrospinal fluid (CSF) of Sprague-Dawley rats then, following administration of baicalin at doses of 24mg/kg via the candal vein, samples were collected every 20min and injected directly into the UPLC-MS/MS system. In vitro recoveries of the probes were 19.26% and 18.38%, while in vivo recoveries of the probes were 15.0% and 17.52% for blood and brain, respectively. This improved method offers a rapid quantitative procedure for the determination of baicalin with a retention time of only 1.6min. The lower limit of quantification (LLOQ) and the lower limit of detection (LLOD) based on a signal-to-noise ratio of 5 were 2.37 and 0.1ng/ml for anticoagulant citrate dextrose (ACD) solution, and 1.185 and 0.3ng/ml for artificial cerebrospinal fluid (aCSF), respectively. The pharmacokinetics results indicated that baicalin could pass through the blood-brain barrier (BBB) and was detectable in brain dialysate. These in vivo microdialysis-based measurements provide a technique for simple sampling and rapid sensitive analysis of unbound baicalin in rat blood and CSF and for further application in pharmacokinetic studies.     

Gastric emptying, absorption, and carbohydrate oxidation during prolonged exercise.

This study was designed to examine aspects of digestive function that may limit assimilation of water and oxidation of orally ingested carbohydrate (CHO) during exercise. Eight males completed a crossover study in which each cycled on four occasions for 80 min at 70% maximal O2 consumption. Beverage was consumed at 0, 20, 40, and 60 min. Beverages were water, 4.5% glucose (4.5G), 17% glucose (17G), and 17% maltodextrin (17MD). CHO beverages contained 20 meq/l NaCl and were 13C enriched to measure exogenous CHO oxidation. Gastric (beverage) volume was measured at 80 min. Water uptake was estimated by including 2H2O in the beverage and measuring 2H accumulation in blood. Jejunal perfusion tests were conducted at rest with the same subjects and beverages. In 60 min, 1,294 +/- 31 (SE) ml were ingested; at 80 min, volumes emptied with H2O (1,257 +/- 32 ml) and 4.5G (1,223 +/- 32 ml) were greater than with 17G (781 +/- 56 ml) and 17MD (864 +/- 71 ml; P less than 0.05). Total CHO oxidized was similar with all beverages, but there was a greater increase in exogenous CHO oxidation over time with 17G and 17MD than with 4.5G; 54, 19, and 18% of the CHO ingested with 4.5G, 17G, and 17MD, respectively, was oxidized. This represents 57, 32, and 27%, respectively, of the CHO emptied from the stomach. 2H accumulation in the blood was more rapid with H2O and 4.5G than with 17G or 17MD. Net jejunal water absorption was greater from 4.5G than from water. Net water absorption was also observed from 17MD, whereas net secretion was observed with 17G.(ABSTRACT TRUNCATED AT 250 WORDS)     

Systemic delivery of enkephalin peptide through eyes

Leu-enkephalin is one of the important peptides which could become useful drugs in the clinics because of its analgesic action and its availability in mass quantity through biotechnology production. It is found in this study that enkephalin can be effectively absorbed systemically through eyes without using a surfactant as absorption enhancer. Enkephalin at 0.125% (31.25 micrograms/25 microliter) reached a plateau of blood concentration at 11.5 ng/ml in 3-4 hrs and stayed high for 8-9 hrs. In contrast, the blood concentration of enkephalin declined rapidly after i.v. administration with a T1/2 of less than 30 min and reached the lowest point at 22 ng/ml in 5 hrs. With higher concentrations at 1% (.25 mg/25 microliter) and 5% (1.25 mg/25 microliter) similar absorption kinetics was observed except that they reached higher plateau of blood concentration at 72 ng/ml and 233 ng/ml, respectively.     

Muscle pump function during locomotion: mechanical coupling of stride frequency and muscle blood flow

We imposed opposing oscillations in treadmill speed and grade on nine rats to test for direct mechanical coupling between stride frequency and hindlimb blood flow. Resting hindlimb blood flow was 15.5 +/- 1.7 ml/min. For 90 s at 7.5 m/min, rats alternated walking at -10 degrees for 10 s and +10 degrees for 10 s. This elicited oscillations in hindlimb blood flow having an amplitude of 4.1 +/- 0.5 ml/min (18% of mean flow) with a delay presumably due to metabolic vasodilation. Similar oscillations in speed (5.5-9.5 m/min) elicited oscillations in hindlimb blood flow (amplitude 3.4 +/- 0.5 ml/min, 15% of mean flow) with less of a delay, possibly due to changes in vasodilation and muscle pump function. We then simultaneously imposed these speed and grade oscillations out of phase (slow uphill, fast downhill). The rationale was that the oscillations in vasodilation evoked by the opposing oscillations in speed and grade would cancel each other, thereby testing the degree to which stride frequency affects hindlimb blood flow directly (i.e., muscle pumping). Opposing oscillations in speed and grade evoked oscillations in hindlimb blood flow having an amplitude of 3.3 +/- 0.6 ml/min (16% of mean flow) with no delay and directly in phase with the changes in speed and stride frequency. The finding that hindlimb blood flow changes directly with speed (when vasodilation caused by changes in speed and grade oppose each other) indicates that there is a direct coupling of stride frequency and hindlimb blood flow (i.e., muscle pumping).     

ACE activity during the hypotension produced by standardized aqueous extract of Cecropia glaziovii Sneth: A comparative study to captopril effects in rats

To evaluate the effect of the standardized aqueous extract (AE) of Cecropia glaziovii Sneth on the plasma angiotensin I converting enzyme (ACE-EC 3.4.15.1) activity, rats were treated with a single dose of AE (1 g/kg, p.o.) or repeatedly (0.5 g/kg/bid, p.o.) for 60 days. Captopril (50 mg/kg, p.o.) was used as positive control on the same animals. The effects on the blood pressure were recorded directly from the femoral artery (single dose), or indirectly by the tail cuff method (repeated doses) in conscious rats. The plasma ACE activity was determined spectrofluorimetrically using Hypuril-Hystidine-Leucine as substrate. The arterial blood pressure, heart rate and plasma ACE activity were not significantly modified within 24 h after a single dose administration of AE. Comparatively, blood pressure in captopril treated rats was reduced by 7-16% and heart rate was increased by 10-20% from 30 min to 24 h after drug administration. ACE activity after captopril presented a dual response: an immediate inhibition peaking at 30 min and a slow reversal to 32% up-regulation after 24 h. To correlate the drug effects upon repeated administration of either compound, normotensive rats were separated in three groups: animals with high ACE (48.8+/-2.6 nmol/min/ml), intermediate ACE (39.4+/-1.4 nmol/min/ml) and low ACE (23.5+/-0.6 nmol/min/ml) activity, significantly different among them. Repeated treatment with AE reduced the mean systolic blood pressure (121.7+/-0.5 mm Hg) by 20 mm Hg after 14 days. The hypotension was reversed upon washout 60 days afterwards. Likely, repeated captopril administration decreased blood pressure by 20 mm Hg throughout treatment in all groups. After 30 days treatment with AE (0.5 g/kg/bid, p.o.) the plasma ACE activity was unchanged in any experimental group. After captopril (50 mg/kg/bid, p.o.) administration the plasma ACE activity was inhibited by 50% within 1 h treatment but it was up-regulated by 120% after 12 h in all groups. It is concluded that the hypotension produced by prolonged treatment with AE of C. glaziovii is unrelated to ACE inhibition.