A series of self-measurements by the patient is a reliable alternative to ambulatory blood pressure measurement

BACKGROUND: Several studies have reported overdiagnosis and overtreatment of hypertensive patients, especially in borderline hypertensives. AIM: To find a blood pressure measurement procedure that reduces the risk of misclassification to an acceptable level. METHOD: Comparative, prospective study over seven months of primary care patients with elevated initial blood pressures. Blood pressure measurements made by general practitioners (GPs), practice nurses, and patients were compared with ambulatory blood pressure measurements. RESULTS: Ninety-nine patients completed the study. Mean differences (systolic blood pressure) between different measurement procedures and ambulatory measurement ranged from +10 mmHg (doctor) to -1 mmHg (patient), and (diastolic) from +4 mmHg (doctor) to -2 mmHg (patient). Standard deviations of mean differences ranged from 12 mmHg (doctor/systolic) to 10 mmHg (patient/systolic), and from 8 mmHg (doctor/diastolic) to 7 mmHg (patient/diastolic). CONCLUSION: Self-measurements by the patient appear to be a reliable alternative to ambulatory blood pressure measurement. In diagnosing and managing mild hypertension, we recommend the use of a valid self-measuring device.     

Molecular models of two competitive inhibitors, IL-2delta2 and IL-2delta3, generated by alternative splicing of human interleukin-2

Molecular models of IL-2delta2 and IL-2delta3, two alternative splice variants of human IL-2 without exon 2 and 3, respectively, are described. These alternative splice variants attract particular interest as potential competitive inhibitors of the cytokine. Tertiary structure of IL-2 consists of four-helix bundle including helices A, B, C and D and a beta-pleated sheet. Exon 2 encodes the A-B loop (Asn30-Lys49 residues) linking helices A and B running in one direction. Rotation of the helix A around putative centre during the construction of IL-2delta2 model have not produced any significant changes in the hydrophobic core of IL-2 molecule. However, a large hole was formed on the surface of IL-2delta2 molecule instead of A-B loop in IL-2 fold. A high affinity IL-2 receptor is formed by combination of alpha, beta, and gamma(c) chains. Comparison of the model of the receptor bound IL-2 with the model of IL-2delta2 has shown that their beta-chain binding sites have minimum differences as distinct from alpha and gamma(c) chain-binding sites. Exon 3 encodes Ala50-Lys97 fragment which forms helices B and C with their short connecting loop. Model IL-2delta3 consists of helices A and D and long linking loop. This loop was composed of A-B and C-D loops which run in opposite directions in IL-2 structure and contain beta-strands making a beta-pleated sheet. Conformation of the linking loop relatively to helices A and D was stabilized by creation of a disulphide bond between cysteines 105 and 125. In addition, the hydrophobic residues of beta-sheet interact with the hydrophobic surface of A-D helical complex and close the latter from contacts with solution. Comparison of the model of IL-2 bound to receptor with IL-2delta3 model has shown that absence of helices B and C in IL-2delta3 model results in insignificant conformational changes only in residues interacting with gamma(c) chain of the receptor. The beta/gamma(c) heterodimer is an intermediate affinity receptor of IL-2. Most likely, both IL-2delta2 and IL-2delta3 are naturally occurring IL-2 antagonists since they keep the ability of binding with an intermediate affinity receptor of this cytokine and fail to engage the alpha chain of its high affinity receptor.     

Molecular mechanics studies on the conformations of 2',3'-dideoxy-2',3'-didehydroguanine nucleoside, D4G

Conformational energy calculations have been presented on guanine nucleoside in which the furanose ring is replaced by 2',3'-dideoxy-2',3'-didehydrofuran using molecular mechanics and conformational analysis. Conformational energies have been evaluated using the MM2 and AMBER94 force field parameters at two different dielectric constants. The results are presented in terms of isoenergy contours in the conformational space of the glycosidic (chi) and C4'-C5' (gamma) bonds torsions. In general, the chi-gamma interrelationships differ from the corresponding plots for unmodified nucleosides and nucleotides, reported previously. Consistency of the calculated preferred conformations with the x-ray data is sensitive to the force field employed.     

2-aminotetralin-derived substituted benzamides with mixed dopamine D2, D3, and serotonin 5-HT1A receptor binding properties: a novel class of potential atypical antipsychotic agents

A new chemical class of potential atypical antipsychotic agents, based on the pharmacological concept of mixed dopamine D2 receptor antagonism and serotonin 5-HT1A receptor agonism, was designed by combining the structural features of the 2-(N,N-di-n-propylamino)tetralins (DPATs) and the 2-pyrrolidinylmethyl-derived substituted benzamides in a structural hybrid. Thus, a series of 35 differently substituted 2-aminotetralin-derived substituted benzamides was synthesized and the compounds were evaluated for their ability to compete for [3H]-raclopride binding to cloned human dopamine D2A and D3 receptors, and for [3H]-8-OH-DPAT binding to rat serotonin 5-HT1A receptors in vitro. The lead compound of the series, 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (12a), displayed high affinities for the dopamine D2A receptor (Ki = 3.2 nM), the dopamine D3 receptor (Ki = 0.58 nM) as well as the serotonin 5-HT1A receptor (Ki = 0.82 nM). The structure-affinity relationships of the series suggest that the 2-aminotetralin moieties of the compounds occupy the same binding sites as the DPATs in all three receptor subtypes. The benzamidoethyl side chain enhances the affinities of the compounds for all three receptor subtypes, presumably by occupying an accessory binding site. For the dopamine D2 and D3 receptors, this accessory binding site may be identical to the binding site of the 2-pyrrolidinylmethyl-derived substituted benzamides.     

Molecular basis for the stabilization and inhibition of 2, 3-dihydroxybiphenyl 1,2-dioxygenase by t-butanol

The steady-state cleavage of catechols by 2,3-dihydroxybiphenyl 1, 2-dioxygenase (DHBD), the extradiol dioxygenase of the biphenyl biodegradation pathway, was investigated using a highly active, anaerobically purified preparation of enzyme. The kinetic data obtained using 2,3-dihydroxybiphenyl (DHB) fit a compulsory order ternary complex mechanism in which substrate inhibition occurs. The Km for dioxygen was 1280 +/- 70 microM, which is at least 2 orders of magnitude higher than that reported for catechol 2,3-dioxygenases. Km and Kd for DHB were 22 +/- 2 and 8 +/- 1 microM, respectively. DHBD was subject to reversible substrate inhibition and mechanism-based inactivation. In air-saturated buffer, the partition ratios of catecholic substrates substituted at C-3 were inversely related to their apparent specificity constants. Small organic molecules that stabilized DHBD most effectively also inhibited the cleavage reaction most strongly. The steady-state kinetic data and crystallographic results suggest that the stabilization and inhibition are due to specific interactions between the organic molecule and the active site of the enzyme. t-Butanol stabilized the enzyme and inhibited the cleavage of DHB in a mixed fashion, consistent with the distinct binding sites occupied by t-butanol in the crystal structures of the substrate-free form of the enzyme and the enzyme-DHB complex. In contrast, crystal structures of complexes with catechol and 3-methylcatechol revealed relationships between the binding of these smaller substrates and t-butanol that are consistent with the observed competitive inhibition.     

Molecular composition of the 16S toxin produced by a Clostridium botulinum type D strain, 1873

The 16S toxin was purified from a Clostridium botulinum type D strain 1873 (D-1873). Furthermore, the entire nucleotide sequences of the genes coding for the 16S toxin were determined. It became clear that the purified D-1873 16S toxin consists of neurotoxin, nontoxic nonhemagglutinin (NTNH), and hemagglutinin (HA), and that HA consists of four subcomponents, HA1, HA2, HA3a, and HA3b, the same as type D strain CB16 (D-CB16) 16S toxin. The nucleotide sequences of the nontoxic components of these two strains were also found to be identical except for several bases. However, the culture supernatant and the purified 16S toxin of D-1873 showed little HA activity, unlike D-CB16, though the fractions successively eluted after the D-1873 16S toxin peak from an SP-Toyopearl 650S column showed a low level of HA activity. The main difference between D-1873 and D-CB16 HA molecules was the mobility of the HA1 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Therefore it was presumed that the loss of HA activity of D-1873 16S toxin might be caused by the differences of processing HA after the translation.     

Enzyme kinetic modelling as a tool to analyse the behaviour of cytochrome P450 catalysed reactions: application to amitriptyline N-demethylation

1. To determine kinetic parameters (Vmax, K(m)) for cytochrome P450 (CYP) mediated metabolic pathways, nonlinear least squares regression is commonly used to fit a model equation (e.g., Michaelis Menten [MM]) to sets of data points (reaction velocity vs substrate concentration). This method can also be utilized to determine the parameters for more complex mechanisms involving allosteric or multi-enzyme systems. Akaike's Information Criterion (AIC), or an estimation of improvement of fit as successive parameters are introduced in the model (F-test), can be used to determine whether application of more complex models is helpful. To evaluate these approaches, we have examined the complex enzyme kinetics of amitriptyline (AMI) N-demethylation in vitro by human liver microsomes. 2. For a 15-point nortriptyline (NT) formation rate vs substrate (AMI) concentration curve, a two enzyme model, consisting of one enzyme with MM kinetics (Vmax = 1.2 nmol min-1 mg-1, K(m) = 24 microM) together with a sigmoidal component (described by an equation equivalent to the Hill equation for cooperative substrate binding; Vmax = 2.1 nmol min-1 mg-1, K' = 70 microM; Hill exponent n = 2.34), was favoured according to AIC and the F-test. 3. Data generated by incubating AMI under the same conditions but in the presence of 10 microM ketoconazole (KET), a CYP3A3/4 inhibitor, were consistent with a single enzyme model with substrate inhibition (Vmax = 0.74 nmol min-1 mg-1, K(m) = 186 microM, K1 = 0.0028 microM-1). 4. Sulphaphenazole (SPA), a CYP2C9 inhibitor, decreased the rate of NT formation in a concentration dependent manner, whereas a polyclonal rat liver CYP2C11 antibody, inhibitory for S-mephenytoin 4'-hydroxylation in humans, had no important effect on this reaction. 5. Incubation of AMI with 50 microM SPA resulted in a curve consistent with a two enzyme model, one with MM kinetics (Vmax = 0.72 nmol min-1 mg-1, K(m) = 54 microM) the other with 'Hill-kinetics' (Vmax = 2.1 nmol min-1 mg-1, K' = 195 microM; n = 2.38). 6. A fourth data-set was generated by incubating AMI with 10 microM KET and 50 microM SPA. The proposed model of best fit describes two activities, one obeying MM-kinetics (Vmax = 0.048 nmol min-1 mg-1, K(m) = 7 microM) and the other obeying MM kinetics but with substrate inhibition (Vmax = 0.8 nmol min-1 mg-1, K(m) = 443 microM, K1 = 0.0041 microM-1). 7. The combination of kinetic modelling tools and biological data has permitted the discrimination of at least three CYP enzymes involved in AMI N-demethylation. Two are identified as CYP3A3/4 and CYP2C9, although further work in several more livers is required to confirm the participation of the latter.     

G proteins in adipocytes and preadipocytes: characterization, subcellular distribution, and potential roles for Gi2 and/or Gi3 in the control of cell proliferation

Guanosine triphosphate (GTP)-binding protein subunits were studied by immunoblot analysis in particulate fractions from mature adipocytes, confluent preadipocytes, and in vitro-differentiated preadipocytes. Mature adipocytes express Gi alpha 1, Gi alpha 2, Gi alpha 3, Go alpha, Gq/11 alpha, G13 alpha and the long and short isoforms of Gs alpha, but no Gz alpha or G12 alpha. Confluent and differentiated preadipocytes differ in having a higher content of Gi alpha 3 and G13 alpha and expressing G12 alpha. In contrast, they lack Gi alpha 1, Go alpha, and the short from of Gs alpha. The G-protein alpha subunits Gi alpha 2, Gs alpha (long isoform), and Gq/11 alpha, and G-protein beta subunits were unchanged throughout the differentiation process. By immunoblot and indirect immunofluorescence studies on confluent preadipocytes, we showed that Gi alpha 2 is present in the endoplasmic reticulum and marginally in plasma membranes and nuclei. In contrast, antibodies to Gi alpha 3 stained the Golgi apparatus. The role of G proteins on preadipocyte proliferation was studied using Bordetella pertussis toxin. Exposure of growing cells to this toxin in the presence of fetal calf serum (FCS) decreased [3H]thymidine incorporation by 40% and induced a 40% increase in doubling time. This resulted in a 30% decrease in cell number per well after 48 h. These effects of B. pertussis toxin did not appear to be related to an increase in cyclic adenosine monophosphate (cAMP) concentration, because forskolin had the opposite effect on cell proliferation. Finally, B. pertussis toxin prevented serum-induced Raf1 association to the plasma membrane, possibly by disrupting FCS-induced G beta gamma effects on the Ras/Raf1 pathway. Since Go alpha and Gi alpha 1 subunits were absent in preadipocytes, we conclude that Gi2 and/or Gi3 proteins transduce some mitogenic signals of FCS through release of G beta gamma subunits. The subcellular distribution of Gi alpha 2 and Gi alpha 3 suggests that part of their functions result from interactions with components other than the plasma membrane.     

Pharmacokinetics and distribution over the blood-brain barrier of zalcitabine (2',3'-dideoxycytidine) and BEA005 (2', 3'-dideoxy-3'-hydroxymethylcytidine) in rats, studied by microdialysis

Microdialysis was applied to sample the unbound drug concentration in the extracellular fluid in brain and muscle of rats given zalcitabine (2',3'-dideoxycytidine; n = 4) or BEA005 (2', 3'-dideoxy-3'-hydroxymethylcytidine; n = 4) (50 mg/kg of body weight given subcutaneously). Zalcitabine and BEA005 were analyzed by high-pressure liquid chromatography with UV detection. The maximum concentration of zalcitabine in the dialysate (Cmax) was 31.4 +/- 5. 1 microM (mean +/- standard error of the mean) for the brain and 238. 3 +/- 48.1 microM for muscle. The time to Cmax was found to be from 30 to 45 min for the brain and from 15 to 30 min for muscle. Zalcitabine was eliminated from the brain and muscle with half-lives 1.28 +/- 0.64 and 0.85 +/- 0.13 h, respectively. The ratio of the area under the concentration-time curve (AUC) (from 0 to 180 min) for the brain and the AUC for muscle (AUC ratio) was 0.191 +/- 0.037. The concentrations of BEA005 attained in the brain and muscle were lower than those of zalcitabine, with Cmaxs of 5.7 +/- 1.4 microM in the brain and 61.3 +/- 12.0 microM in the muscle. The peak concentration in the brain was attained 50 to 70 min after injection, and that in muscle was achieved 30 to 50 min after injection. The half-lives of BEA005 in the brain and muscle were 5.51 +/- 1.45 and 0.64 +/- 0.06 h, respectively. The AUC ratio (from 0 to 180 min) between brain and muscle was 0.162 +/- 0.026. The log octanol/water partition coefficients were found to be -1.19 +/- 0.04 and -1.47 +/- 0.01 for zalcitabine and BEA005, respectively. The degrees of plasma protein binding of zalcitabine (11% +/- 4%) and BEA005 (18% +/- 2%) were measured by microdialysis in vitro. The differences between zalcitabine and BEA005 with respect to the AUC ratio (P = 0.481), half-life in muscle (P = 0.279), and level of protein binding (P = 0.174) were not statistically significant. The differences were statistically significant in the case of the half-life in the brain (P = 0.032), clearance (P = 0.046), volume of distribution (P = 0.027) in muscle, and octanol/water partition coefficient (P = 0.019).     

Comparison of 2D and 3D algorithms for adding a margin to the gross tumor volume in the conformal radiotherapy planning of prostate cancer

OBJECTIVE: To determine whether electron beam computed tomography (CT) adds to conventional risk factor assessment in the prediction of angiographic coronary artery disease. BACKGROUND: Electron beam CT scanning can be used to predict the severity of coronary atherosclerosis, but whether it does so independently of conventional risk factors is unclear. METHODS: Electron beam CT scans were performed and conventional risk factors were measured in 290 men and women undergoing coronary arteriography for clinical indications. The association of the electron beam CT-derived coronary artery calcium score and conventional risk factors with the presence and severity of angiographically defined coronary atherosclerosis was analyzed by logistic regression and receiver-operator characteristics analysis. RESULTS: Age, the ratio of total cholesterol to high-density lipoprotein (HDL) cholesterol and the coronary calcium score were significantly and independently associated with the presence of any coronary disease and obstructive coronary disease. In association with any coronary disease, odds ratios for age, the ratio of total cholesterol to HDL cholesterol and calcium score, highest quartile vs. lowest quartile, were 6.01 (95% confidence interval 2.87 to 12.56), 3.14 (1.56 to 6.31) and 94.08 (21.06 to 420.12), respectively. For obstructive coronary disease, highest quartile vs. lowest quartile, the respective odds ratios for age, the ratio of total cholesterol to HDL and calcium score were 3.86 (1.86 to 8.00), 4.11 (1.98 to 8.52) and 34.12 (12.67 to 91.86). Male gender was also significantly associated with any coronary disease (odds ratio 2.19, p=0.04) and obstructive coronary disease (odds ratio 2.07, p=0.04). Cigarette smoking was significantly associated with any coronary disease (odds ratio=2.74, p=0.004), and diabetes was significantly associated with obstructive disease (odds ratio 3.16, p=0.01). After adjustment for the coronary calcium score and other risk factors, it was determined that triglycerides, family history and hypertension were not significantly associated with any disease state. A coronary calcium score >80 (Agatston method) was associated with an increased likelihood of any coronary disease regardless of the number of risk factors, and a coronary calcium score > or = 170 was associated with an increased likelihood of obstructive coronary disease regardless of the number of risk factors (p < 0.001). CONCLUSIONS: Electron beam CT scanning offers improved discrimination over conventional risk factors in the identification of persons with any angiographic coronary disease or angiographic obstructive coronary disease.     

Multiple human cytochromes contribute to biotransformation of dextromethorphan in-vitro: role of CYP2C9, CYP2C19, CYP2D6, and CYP3A

Cytochromes mediating the biotransformation of dextromethorphan to dextrorphan and 3-methoxymorphinan, its principal metabolites in man, have been studied by use of liver microsomes and microsomes containing individual cytochromes expressed by cDNA-transfected human lymphoblastoid cells. In-vitro formation of dextrorphan from dextromethorphan by liver microsomes was mediated principally by a high-affinity enzyme (Km (substrate concentration producing maximum reaction velocity) 3-13 microM). Formation of dextrorphan from 25 microM dextromethorphan was strongly inhibited by quinidine (IC50 (concentration resulting in 50% inhibition) = 0.37 microM); inhibition by sulphaphenazole was approximately 18% and omeprazole and ketoconazole had minimal effect. Dextrorphan was formed from dextromethorphan by microsomes from cDNA-transfected lymphoblastoid cells expressing CYP2C9, -2C19, and -2D6 but not by those expressing CYP1A2, -2E1 or -3A4. Despite the low in-vivo abundance of CYP2D6, this cytochrome was identified as the dominant enzyme mediating dextrorphan formation at substrate concentrations below 10 microM. Formation of 3-methoxy-morphinan from dextromethorphan in liver microsomes proceeded with a mean Km of 259 microM. For formation of 3-methoxymorphinan from 25 microM dextromethorphan the IC50 for ketoconazole was 1.15 microM; sulphaphenazole, omeprazole and quinidine had little effect. 3-Methoxymorphinan was formed by microsomes from cDNA-transfected lymphoblastoid cells expressing CYP2C9, -2C19, -2D6, and -3A4, but not by those expressing CYP1A2 or -2E1. CYP2C19 had the highest affinity (Km = 49 microM) whereas CYP3A4 had the lowest (Km = 1155 microM). Relative abundances of the four cytochromes were determined in liver microsomes by use of the relative activity factor approach. After adjustment for relative abundance, CYP3A4 was identified as the dominant enzyme mediating 3-methoxymorphinan formation from dextromethorphan, although CYP2C9 and -2C19 were estimated to contribute to 3-methoxymorphinan formation, particularly at low substrate concentrations. Although formation of dextrorphan from dextromethorphan appears to be sufficiently specific to be used as an in-vitro or in-vivo index reaction for profiling of CYP2D6 activity, the findings raise questions about the specificity of 3-methoxymorphinan formation as an index of CYP3A activity.     

Internal cleavage of the inhibitory 7B2 carboxyl-terminal peptide by PC2: a potential mechanism for its inactivation

The neuroendocrine protein 7B2 contains two domains, a 21-kDa protein required for prohormone convertase 2 (PC2) maturation and a carboxyl-terminal (CT) peptide that inhibits PC2 at nanomolar concentrations. To determine how the inhibition of PC2 is terminated, we studied the metabolic fate of the 7B2 CT peptide in RinPE-7B2, AtT-20/PC2-7B2, and alphaTC1-6 cells. Extracts obtained from cells labeled for 6 h with [3H]valine were subjected to immunoprecipitation using an antibody raised against the extreme carboxyl terminus of r7B2, and immunoprecipitated peptides were separated by gel filtration. All three cell lines yielded two distinct peaks at about 3.5 kDa and 1.5 kDa, corresponding to the CT peptide and a smaller fragment consistent with cleavage at an interior Lys-Lys site. These results were corroborated using a newly developed RIA against the carboxyl terminus of the CT peptide which showed that the intact CT peptide represented only about half of the stored CT peptide immunoreactivity, with the remainder present as the 1.5-kDa peptide. Both peptides could be released upon phorbol 12-myristate 13-acetate stimulation. We investigated the possibility that PC2 itself could be responsible for this cleavage by performing in vitro experiments. When 125I-labeled CT peptide was incubated with purified recombinant PC2, a smaller peptide was generated. Analysis of CT peptide derivatives for their inhibitory potency revealed that CT peptide 1-18 (containing Lys-Lys at the carboxyl terminus) represented a potent inhibitor, but that peptide 1-16 was inactive. Inclusion of carboxypeptidase E (CPE) in the reaction greatly diminished the inhibitory potency of the CT peptide against PC2, in line with the notion that the CT peptide cleavage product is not inhibitory after the removal of terminal lysines by CPE. In summary, our data support the idea that PC2 cleaves the 7B2 CT peptide at its internal Lys-Lys site within secretory granules; deactivation of the cleavage product is then accomplished by CPE, thus providing an efficient mechanism for intracellular inactivation of the CT peptide.     

Continuous monitoring of brain tissue PO2: a new tool to minimize the risk of ischemia caused by hyperventilation therapy

Secondary ischemic events worsen the outcome of patients with severe head injury. Such a secondary ischemic event may be caused by a forced hyperventilation. A consequence of the induced vasoconstriction is the risk of ischemia with an adverse effect on outcome. As a reliable and on-line technique, brain tissue pO2 (p(ti)O2) is used for monitoring regional microcirculation, to detect critical hypoperfusion. On 22 patients with a severe head injury 70 hyperventilation tests were performed from day 0-9 after trauma, calculating TCD-CO2-reactivity (% change of mean flow velocity per mm Hg paCO2 change). Additionally brain p(ti)O2-CO2-reactivity (% change of brain p(ti)O2 per mm Hg paCO2 change) was calculated and introduced. Group A +2 (p(ti)O2 < or = 15 mm Hg, TCD-CO2-reactivity > or = 2.5%, p(ti)O2-CO2-reactivity > 0%) and group B +2 (p(ti)O2 > 15 mm Hg, TCD-CO2-reactivity > or = 2.5%. p(ti)O2-CO2-reactivity > 0%) was formed. P(ti)O2 values in group A+2 decreased to an ischemic level or ischemia aggravated during hyperventilation. In group B+2 no ischemic events occurred. TCD-CO2-reactivity, p(ti)O2-CO2-reactivity and decrease of paCO2 were not significantly different in both groups. 6 out of 22 patients showed, from day 0-9, at least once a risk of (aggravating) ischemia by hyperventilation therapy.     

1- and 3-hydroxylations, in addition to 4-hydroxylation, of debrisoquine are catalyzed by cytochrome P450 2D6 in humans

Twenty-one healthy Swedish Caucasian volunteers, representing different groups with 0-13 functional cytochrome P450 (CYP) 2D6 genes, were given a single oral dose of 20 mg of debrisoquine. The hypothesis of further oxidation of the main metabolite, (S)-4-hydroxydebrisoquine, in subjects with multiple CYP2D6 genes was tested by screening the 0-8-hr urine samples for dihydroxylated metabolites of debrisoquine with protonated molecular ions at m/z 208, using LC/MS. Three peaks were detected in a subject with 13 functional CYP2D6 genes. One compound was identified as dihydroxylated debrisoquine (presumably with hydroxylation at position 4 plus one of the positions in the aromatic ring). This metabolite had not been previously demonstrated in humans and was detected only in this subject. The other two compounds, which were measurable in various amounts in all subjects investigated, were identified as 2-(guanidinomethyl)phenylacetic acid and 2-(guanidinoethyl)benzoic acid. They had been previously detected in the urine of humans, dogs, and rats. They were distinguished by acid-catalyzed deuterium exchange of the hydrogens at the alpha-position, with respect to the carboxylic acid group, of the former but not the latter acid. The acids are formed by 3- and 1-hydroxylation of debrisoquine, respectively, followed by ring opening to aldehydes, which are further oxidized to acids. Strong Spearman rank correlations between debrisoquine products of 1- or 3-hydroxydebrisoquine and debrisoquine/4-hydroxydebrisoquine ratios (rS = 0.97 and rS = 0.96, respectively), using the intensity of the peaks of the reconstructed ion-current chromatograms, clearly showed that both hydroxylation steps are catalyzed by CYP2D6. Because reference compounds for the two acids were not available, the absolute quantities could not be determined.