High Power Liquid Chromatography Research Articles

Endothelin-1 stimulates its own synthesis in human endothelial cells

We studied whether endothelin-1 (ET-1) would affect its own synthesis. Human umbilical cord vein endothelial cells in methionine-poor culture medium containing [ 35 S] methionine were treated with synthetic ET-1 or ET-3. Immunoprecipitation of 35 S -labeled ET-1 was performed with rabbit ET-1 antiserum. ET-1 caused an 40 ± 4 % (mean ± SEM) increase of immunoprecipitable 35 S -labeled ET-1 as confirmed by its elution point in reversed phase high power liquid chromatography (HPLC). ET-3 caused a 23 ± 2 % increase in ET-1 concentration. Amplification of cDNA by PCR showed both ET-1 and ET B receptor mRNAs in human cord vein endothelial cells. We conclude that ET-1 increases its own synthesis in endothelial cells. This suggests a positive autocrine feed-back action of ET-1 on its own synthesis, an effect which is probably mediated by non-specific ET B receptors.

Tissue distribution and half-life of 125I-endothelin in the rat: Importance of pulmonary clearance

Plasma half-life and organ uptake of endothelin 1 (ET-1) was studied in anaesthetized Wistar rats administering a bolus of 125I-labeled ET-1 into jugular vein, with and without a preceding dose of 1–80 ng of unlabeled ET-1, via the same route. Plasma half-life of intact 125I-ET-1 was 40 sec and that of total radioactivity 68 sec. Immunoreactivity of circulating 125I-ET was rapidly reduced, by 23% at 30 sec and by 43% at 360 sec, respectively. High power liquid chromatography of plasma extracts revealed a corresponding decrease of intact 125I-ET-1 peak with time. There was a predominant uptake of 125I-ET-1 in the lungs, 82%, and lesser by kidneys, 10%, heart 3.6%, liver, 2.7%, and spleen, 1.5%, respectively. Pretreatment with unlabeled ET-1 caused a dose-dependent shift of uptake from lungs to kidneys. We conclude that circulating ET-1 is rapidly cleared, primarily by the lungs, which may play an important role in protecting the systemic circulation from the vasoconstricting actions of ET-1.

抗原誘発後の鼻粘膜ＳＲＳ‐Ａ含有量の経時的変化

We evaluated the time course of production of SRS-A in the nasal mucosa after antigen challenge in the guinea pigs with nasal allergy and the effect of Ketotifen sprayed topically over the nasal mucosa on SRS-A production. Fifty-six Hartley strain guinea pigs were sensitized passively by intravenous injection of 0.4 ml of sera containing antiovaTbumin IgE and γ1 antibody (7 days' PCA titer, ×1664; 48 hours' RCA titer, ×5632). After 7 days, all animals received nasal challenge with 50 μl×2 of 1 % ovalbumin. Thirty-two animals were subdivided into 8 groups that were killed before the challenge and 15 min, 30 min, 1 hr, 3 hr and 12 hr after antigen challenge. The nasal mucosa was removed and the amount of LTC4, LTD4 and LTE4 in the nasal mucosa was measured using high-powered liquid chromatography combined with radioimmunoassay. In 12 animals, nasal spray of 0.1 mg Ketotifen dissolved in normal saline was given once a day for 6 days until the day of antigen challenge and another 12 animals were given nasal spray of normal saline as a control. Thirty minutes after nasal antigen challenge, the animals were killed and the nasal mucosa was removed to measure the amount of SRS-A. LTC4, LTD4 and LTE4 in the nasal mucosa increased significantly between 15 and 60 min after antigen challenge and then decreased markedly. Ketotifen sprayed topically over the nasal mucosa significantly inhibited production of LTC4, LTD4 and LTE4 by as much as 50%.

Concentration gradient of CSF monoamine metabolites in children and adolescents

Whether the lumbar cerebrospinal fluid (CSF) concentration gradient of monoamine metabolites found in adults is influenced by age or pubertal status was studied in 26 children ranging from 6.5 to 17.3 years of age. Homovanillic acid (HVA), 5-hydroxyin-doleacetic acid (5-HIAA), and 3-methoxy-4-hydroxyphenylglycol (MHPG) were assayed by high-power liquid chromatography (HPLC) with electrochemical detection. Eight patients were prepubertal (Tanner stage I). The slopes in units of picomoles/milliliter/milliliter for regression lines for CSF monoamine metabolite concentrations versus milliliter of CSF collected were 5.07 ± 0.65, 10.13 ± 2.0, and 0.67 ± 0.22 for 5-HIAA, HVA, and MHPG, respectively, for the group as a whole. Significant correlations with age, height, weight, or Tanner stage were not found for the HVA or MHPG concentration gradients. Tanner stage and 5-HIAA slope were significantly correlated. Three of eight prepubertal patients had nonsignificant 5-HIAA gradients. CSF studies in pediatrie populations must control for aliquot collected, as the size of the gradient could produce differences sufficient to mimic a “positive” clinical study if the aliquots collected are not the same.

Bioassay for A-56268 (TE-031) and identification of its major metabolite, 14-hydroxy-6-O-methyl erythromycin.

A-56268 is a new macrolide which is generally two-fold more potent than erythromycin. A new bioassay is described in which plasma samples are extracted with acetonitrile prior to bioassay. The concentration range for the assay is between 0.05-4.0 micrograms/ml, and the concentrations measured are within 6% of those measured by high-power liquid chromatography. An active metabolite which is as active as erythromycin was identified in the plasma. The plasma half-life and area under the plasma curve values of A-56268, as determined by bioassay, were significantly greater than those of erythromycin.

The release and percutaneous permeation of anthralin products, using clinically involved and uninvolved psoriatic skin

The release and permeation of 1% Westragel and 1% Westrastick and three commercial 1% anthralin products were investigated in vitro with the use of a Franz diffusion cell unit. An inert Teflon membrane with a mesh opening of 74 mu was used for measuring the rate of release. Involved psoriatic and uninvolved human skin collected from the same subjects were used for the permeation study. The permeation of danthron and dianthrone, the major degradation products of anthralin, was also studied with the use of microemulsion gels of 1% danthron and 1% dianthrone, which were prepared in the same way as 1% Westragel. The penetrating anthralin, danthron, and dianthrone were stabilized by a modified receptor fluid, and sample solutions were analyzed by a high-power liquid chromatography method. Involved psoriatic skin was found to be much more permeable to anthralin than was uninvolved psoriatic skin. The slow permeation rate of anthralin, danthron, and dianthrone through normal skin and uninvolved skin indicates that the stratum corneum is the rate-limiting barrier. In involved psoriatic skin, the release rate of anthralin from the topical product becomes the rate-determining step. Large individual variations were found in the permeation of anthralin through involved psoriatic skin, suggesting that the permeation rate of anthralin also may depend on the disease state of psoriatic patients. The anthralin molecule, possessing both hydrophilic and lipophilic centers, diffused significantly faster than did danthron and dianthrone. Westragel, 1%, showed the highest diffusion rate as well as the highest driving force when compared to other commercial 1% anthralin products. This suggests that 1% Westragel may be an optimal design, especially for short-contact anthralin therapy.

Plasma and urinary kinetics of furosemide in newborn infants

Plasma and urinary furosemide kinetics were assayed by high-power liquid chromatography in six newborn infants receiving furosemide (1 mg/kg body weight IV) for the treatment of fluid overload. Mean +/- SD for plasma half-life, apparent volume of distribution, and plasma clearance were, respectively, 9.5 +/- 4.4 hours, 173 +/- 28 ml/kg, and 15.3 +/- 8.4 ml/hr/kg. There was close correspondence between plasma and urinary half-lives and between plasma clearance and renal clearance. In the first 24 hours, mean estimated urinary recovery of unchanged furosemide was 90% of the injected dose (range 61% to 106%). The results suggest that in the newborn infant furosemide is virtually all excreted unchanged in the urine and that the absence of significant nonrenal elimination, together with the immaturity of neonatal renal function, accounts for its prolonged half-life in newborn infants.

Single-dose ceftriaxone pharmacokinetics in pediatric patients with central nervous system infections

Ceftriaxone has greater in vitro and in vivo efficacy against many common bacteria than other third-generation cephalosporins. Single-dose ceftriaxone pharmacokinetics were studied in 17 patients, aged 0.6 to 52 months, with infections of the central nervous system. Patients received a randomized dose of 50 or 75 mg/kg ceftriaxone intravenously over 5 minutes on the second to fifth day of illness. Serial blood samples were collected over 24 hours in all patients, and cerebrospinal fluid (CSF) was obtained 1 to 4.5 hours after injection. Ceftriaxone mean peak plasma concentrations, determined by high-power liquid chromatography, were 267 and 184 microgram/ml for the 75 and 50 mg/kg dosage groups, respectively. The harmonic mean elimination half-life was 4.2 hours, and the mean percent drug penetrance into CSF was 4.8 +/- 3.5%. Of CSF studies evaluated, the glucose concentration was correlated most closely (inversely) with CSF penetration of ceftriaxone. Individual CSF concentrations of ceftriaxone exceeded the minimal inhibitory concentrations of the respective bacteria causing infection by 480 to 5,600 times. Ceftriaxone may be useful in the treatment of serious pediatric infections, including meningitis.

Metabolism of thienamycin and related carbapenem antibiotics by the renal dipeptidase, dehydropeptidase.

Thienamycin (THM), the N-formimidoyl thienamycin derivative MK0787, and related carbapenem antibiotics were metabolized extensively in mice, rats, rabbits, dogs, rhesus monkeys, and chimpanzees. Urinary recovery of THM ranged from a low of 5% in dogs to 58% in rhesus monkeys. Renal clearance rates in dogs and chimpanzees were unusually low, less than glomerular filtration rates. The reduction in clearance of THM and MK0787 from plasma of rats and rabbits after ligation of renal arteries indicate that the kidneys are responsible for 35 and 92%, respectively, of metabolic drug clearance. Degradation was detected only in kidney homogenates. The enzyme activity was membrane bound and sensitive to inhibitors of Zn-metalloenzymes such as EDTA. A renal dipeptidase, dehydropeptidase-I (DHP-I), EC 3.4.13.11, was found to be responsible for the metabolism of the THM-class antibiotics, which exhibit a structural homology to dehydropeptides. A parallel increase in specific activity against THM and the substrate of DHP-I, glycyldehydrophenylalanine, was observed during solubilization and purification of the enzyme from porcine and human renal cortex. DHP-I was found to catalyze the hydrolysis of the beta-lactam ring in THM and MK0787. The products of the enzyme reaction were identical by high-powered liquid chromatography to their respective metabolites found in the urine. Nonbasic N-acylated THM and natural N-acylated carbapenems (epithienamycins and olivanic acids) were degraded 4- to 50-fold faster than THM when exposed to the enzymatic hydrolysis of DHP-I. Good correlations were obtained between the increased susceptibility of the carbapenem antibiotics to DHP-I as measured in the in vitro enzyme assay and the generally lower recoveries of active antibiotic in the urine of test animals. Despite this unusual degree of metabolism localized in the kidney, the plasma half-life of MK0787 and its efficacy against experimental systemic infections in animals remain satisfactory.

N-Acetyl-procainamide kinetics in the elderly.

Plasma and saliva N-acetyl-procainamide (NAPA) concentrations were measured by high-power liquid chromatography (HPLC) after intravenous infusion of 750 mg to 14 elderly patients (x age = 69 yr). The plasma NAPA disappearance curve can best be described by a two-compartment body model. Mean total body clearance was 10.6 1/hr, Vdss 125.8 1, and terminal half-life (t 1/2) 8.8 hr. A nonrenal clearance of 2.72 1/hr was calculated, that is, 19% of the expected total body clearance with normal kidney function. Saliva concentrations show huge inter- and intraindividual variability and are probably not usable for NAPA monitoring in older patients.