Biomedical subjects
Publications and source records attributed to A Chung.
The aminobutyl derivative of phenobarbital, 5-phenyl-5-(4-aminobutyl)barbituric acid hydrochloride, was synthesized through two synthetic pathways for the preparation of immunogen in production of phenobarbital specific antibody. The produced antiserum had high titer, specificity, affinity and sensitivity (0.5ng/ml), when examined by radioimmunoassay.
The fat of less than Glu1-3H-labelled bradykinin-potentiating peptide 9a [BPP9a; less than Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro, an inhibitor of angiotensin-converting enzyme (peptidyl dipeptidase)] was studied in the rabbit. After intravenous injection, BPP9a was rapidly removed from blood and much of the associated radioactivity was excreted in urine. Approx. 8% of the radioactivity in urine collected 2h after drug administration occurred in the form of BPP9a itself, the remainder occurring in three lower homologues: less than Glu-Trp (60%), less Glu-Trp-Pro-Arg-Pro-Gln (20%) and less than Glu-Trp-Pro-Arg-Pro-Gln-Ile (12%). Hydrolysis was not accounted for by enzymes in blood or urine. Apparently hydrolysis occurred within the kidney, as less than Gl-Trp was obtained in 60% yield in urine of isolated rat kidney perfused with [less than Glu1-3H]BPP9a.
We have developed a sensitive, highly selective assay for human urinary kallikrein (HUK) that uses Pro-Phe-Arg-[3H]benzyl-amide as substrate. The substrate was prepared from Pro-Phe-Arg-3-iodo-benzylamide by dehalogenation in 3H2 gas. HUK is measured by its ability to release [3H]benzylamine. The pH optimum is 9.5. Urokinase, plasmin and thrombin do not interfere. The assay can measure as little as 5 ng of HUK in a 15 min incubation at 37 degrees C. Typically, we use 50 microliter of dialyzed urine for HUK assays. Reactions are terminated by adding 0.1 M NaOH, and reaction product is separated from substrate by partitioning with an equal volume of toluene. A sample of the toluene phase is submitted for liquid scintillation counting. As judged by separations obtained on molecular sieve chromatography (Sephacryl), only one urinary enzyme possesses the ability to hydrolyze our substrate. The enzyme MW 45,000, is inhibited by Trasylol but not by soya bean trypsin inhibitor (SBTI). It is reactive with and is inhibited by antibodies prepared against pure HUK.
As part of a program to prepare bradykinin (H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH) labelled at high specific radioactivities, we have synthesized three analogs for dehalogenation in tritium gas: [4-Br-Phe5]-bradykinin (BK), [4-Br-Phe8]-BK and [4-Br-Phe5,8]-BK. The analogs were synthesized by the Merrifield solid-phase method and were purified by molecular sieve and partition chromatography. The analogs themselves possess biological activity (as assayed for effects on mean arterial blood pressure and isolated rat uterus). [4-Br-Phe8]-BK was 1.5 to 3 times as active as bradykinin. [4-Br-Phe5,8]-BK was approx. 22% as active as BK and [4-Br-Phe5]-BK was approx. 18% as active. [4-Br-Phe5]-BK was submitted to catalytic dehalogenation with 10% Pd/C and 5% Rh/CaCO3 in H2O and DMF (1:1) plus 10 Ci of 3H2. [4-3H-Phe5]-BK was obtained at 6.7 Ci/mmole in an overall yield of 15%. [4-3H-Phe8]-BK was prepared similarly to yield an intrinsically-labelled peptide with a specific radioactivity of 21 Ci/mmole.
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Urines and sera (human, guinea pig and rat) contain low molecular weight inhibitors of angiotensin converting enzyme (ACE). The urines contain ACE, but the enzyme is scarcely measurable without prior ultrafiltration or dialysis. The activity increases strikingly through three ultrafiltration steps using a membrane with a 10,000 MW retention limit. As implied, the ultrafiltrates contain inhibitory activity and can prevent the hydrolysis of [3H]benzoyl-Gly-His-Leu by ACE from any source, including lung and serum. Human urinary ultrafiltrate contains at least three inhibitors separable on Bio-Gel P-2. The inhibitors are acidic and can be partially purified on Bio-Rex 70 developed with an acetic acid gradient. The smallest of the inhibitors can be purified to apparent homogeneity by partition chromatography (sephadex G-25; butanol, acetic acid, H2O; 4:1:5). The excretion of inhibitory activity varies in response to dietary salt: Activity is low when rats are maintained on a high NaCl diet and is high (3 x's control) on a low NaCl diet. Thus, the activity of ACE may be modulated in vivo by naturally-occurring enzyme inhibitors. Whether some hypertensive patients are deficient in ACE inhibitory activity remains to be determined.
Prior animal models of cholesterol gallstone formation have been criticized for their dissimilarity to the conditions of humans with gallstones. We present a new hamster model of cholesterol cholelithiasis that more closely approximates the human situation. Sixty female Golden Syrian hamsters (average weight 83.2 +/- 3.4 g) were allocated to six groups of 10 animals each. Groups were fed standard diet (containing 0.8 gm cholesterol/g of food) or increased cholesterol diet (containing 2.4 mg cholesterol/g of food), with or without ethinyl estradiol, 15 micrograms/kg/d. Two groups receiving both increased cholesterol and ethinyl estradiol also received either chenodeoxycholic acid or ursodeoxycholic acid, 20 mg/kg/d. The animsl were sacrificed at 12 wk. Cholesterol gallstones (78.3 +/- 5.0% cholesterol by weight) formed in 30% of the animals fed ethinyl estradiol, 50% of those fed increased cholesterol, and 90% of those fed the combination of both. Bile was saturated in all three groups, with the saturation index of the combination group (2.08 +/- 0.17) being the highest. In both groups receiving bile acid therapy, no gallstones were found, and the bile remained unsaturated. For the bile acid-fed groups, both hepatic HMG-CoAR and hepatic cholesterol 7 alpha-hydroxylase activities were reduced (P less than 0.01) when compared to the group fed standard diet and to the grou fed the combination. Thus, a new animal model of cholesterol gallstone formation has been developed in which chenodeoxycholic acid and ursodeoxycholic acid therapy prevented gallstone formation through mechanisms similar to those reported in cholesterol gallstone patients.
To develop means of measuring angiotensin converting enzyme of endothelial cells in culture, we have synthesized benzoyl-Phe-Ala-Pro-OH (I), benzoyl-Pro-Phe-Arg-OH (II) and benzoyl-Gly-His-Leu-OH (III), each bearing a 3H-atom on the para-position of its benzoyl moiety. All three of the acylated tripeptides are substrates for the enzyme. Substrate I exhibits the lowest Km (12.5 micrometer) and yields the most sensitive assay: the enzyme of 10(6) cells can be measured in a 30 min incubation at 37 degrees C. Radiolabelled reaction product is separated from substrate by extraction of acidified reaction mixture with an organic solvent, and the rate of formation of product can be quantified by liquid scintillation counting of the organic phase. Substrate III can also be used to measure angiotensin converting enzyme of cells but requires longer incubations (180--240 min) and high salt concentrations (0.75 M Na2SO4). Substrate II is not specific: it is hydrolyzed by more than one enzyme of endothelial cells.
Three colonic and three ileal loops were prepared in six rabbits pretreated with propranolol (PR) 4 mg./kg. I.V. and in five untreated rabbits. In random order, 1 ml. of either deoxycholic acid (DCA) 6 mM., prostaglandin E1 (PGE1) 20 microgram./ml., or saline was placed in each colonic loop and 1 ml. of either cholera enterotoxin (CE) 10 microgram./ml., PGE1 20 microgram./ml., or saline was placed in each ileal loop. In untreated animals, DCA and PGE1 in the colon and CE and PGE1 in the ileum stimulated (P less than 0.01) adenylate cyclase (AC) and net secretion. In the colon, PR abolished DCA-stimulation of AC and net secretion and decreased PGE1-stimulated AC (P less than 0.01) and net secretion. In conclusion, at the doses and times studied, colonic-AC and net secretion stimulated by PGE1 or DCA was distinguished from small bowel-AC and net secretion stimulated by PGE1 or CE.
Gallstone dissolution and biliary lipids were determined and compared in patients receiving either chenodeoxycholic acid (CDC), or CDC and phenobarbital (PB) for 11/2 to 2 years. Among patients with radiolucent gallstones, dissolution occurred in 53% of those receiving CDC alone and in only 25% of those receiving both CDC and PB. No dissolution occurred in 13 other patients with calcified gallstones. Patients with dissolution had a significantly greater molar percentage of CDC and a significantly lower saturation index in bile than those without dissolution. Diarrhea and transiently abnormal liver function tests were the most frequently observed side-effects but only diarrhea necessitated a reduction of the CDC dose. Gallstones recurred following dissolution in one of six patients followed for six months after discontinuation of CDC. In conclusion, PB did not enhance CDC-induced desaturation of bile or gallstone dissolution.
The influence of ethinyl estradiol (EE) on the effects of dietary cholesterol on the biliary saturation index and on the rate-limiting hepatic enzymes of cholesterol synthesis, hydroxymethylglutaryl-CoA-reductase, and bile acid synthesis, 7 alpha-hydroxylase, were determined. Four groups of 12 male hamsters were treated for 1 month with EE, 15 micrograms per kg per day, or placebo vehicle administered intraperitoneally and fed either a standard diet, 0.8 mg of cholesterol per g of food, or high cholesterol diet, 2.4 mg of cholesterol per g. The high cholesterol diet increased the saturation index to 1.00 +/- 0.03 (P less than 0.01) from 0.65 +/- 0.02 in untreated hamsters on the standard diet. EE treatment on the high cholesterol diet further increased (P less than 0.01) the saturation index to 1.15 +/- 0.02. The high cholesterol diet decreased (P less than 0.01) hydroxymethylglutaryl-CoA-reductase activity from 308 +/- 16 pmoles per mg per min in untreated hamsters on the standard diet. The addition of EE treatment had no effect on hydroxymethylglutaryl-CoA-reductase activity. The high cholesterol diet increased (P less than 0.01) 7 alpha-hydroxylase activity from 23 +/- 1.0 pmoles per mg per min in untreated hamsters on the standard diet. The addition of EE decreased (P less than 0.01) 7 alpha-hydroxylase activity from that in untreated hamsters on the standard diet. The conclusions are as follows: (1) EE prevented dietary cholesterol-induced stimulation of cholesterol 7 alpha-hydroxylase activity; (2) EE enhanced the ability of dietary cholesterol to induce saturated bile; and (3) gallstone formation in estrogen-treated women may result from impaired metabolism of dietary cholesterol.
Angiotensin-converting enzyme can be measured by the rate of release of 3H-labelled hippurate from p-[3H]benzoylglycylglycylglycine. The product is separable from the substrate by extraction of acidified reaction mixtures with ethyl acetate. Assay results for human serum angiotensin-converting enzyme can be obtained within 1.5 h of receipt of serum samples. Within the limits tested, the assay appears to be specific. However, interference by hitherto unrecognized enzymes of abnormal sera must be ruled out.
Experiments with lymphocyte and fibroblast chalones indicate that they are complexed strongly with RNA in tissue extracts; have a molecular weight of less than 10,000 and more than 1,000 daltons, and probably about 5,000 daltons; are strongly cationically charged; and contain mannose and perhaps other carbohydrates.
Four cases of squamous carcinoma arising in benign cystic teratoma of the ovary are presented, with a detailed correlation of clinical, operative and pathological findings with prognosis. Confinement of tumor to the ovary, with an intact ovarian capsule, is the most important prognostic factor since patients wih extracapsular extension succumb to their disease within 2 years. The literature dealing with this subject is reviewed.
Biliary cholesterol saturation has been correlated with disease variables that might effect bile acid loss in ileitis patients with (N = 9) or without (N = 8) intestinal resection having a defined prevalence of gallstones. In addition, cholesterol saturation was determined in ulcerative colitis patients (N = 7) and gallstone patients (N = 18) as well as in 5 normal controls. Biliary cholesterol saturation in ileitis patients both with and without resection was similar to that in gallstone patients yet the prevalence of gallstones was only 12%. Cholesterol saturation did not correlate with ileal resection nor the extent, duration, or activity of ileitis. Biliary cholesterol saturation was not different in ulcerative colitis patients from that in normal subjects. It is concluded that cholesterol saturation of bile alone does not account for the high prevalence of cholesterol gallstones that has been reported in ileitis patients.
Chenodeoxycholic acid, by reducing the concentration of biliary cholesterol relative to that of bile acid and phospholipid, dissolves cholesterol gallstones. This bile acid, however, has potential dose-related hepatotoxicity and causes dose-related diarrhea. Therefore, the feasibility of low-dose and intermittent therapy was assessed by studying the induction and persistence of chenodeoxycholic acid-induced biliary lipid changes. Biliary lipid composition with each of 3 doses of chenodeoxycholic acid was determined in bile samples obtained by cholecystokinin-stimulated duodenal drainage before, after one week and one month of treatment, and up to 9 weeks after discontinuation of treatment. The lowest dose that significantly reduced the relative concentration of biliary cholesterol was 250 mg/day. A significant reduction occurred one week after initiation of treatment and was maintained for 9 weeks following discontinuation of treatment. Thus, clinical trials on low-dose and intermittent chenodeoxycholic acid therapy for gallstone prophylaxis or dissolution are warranted.
Stimulation of net secretion by deoxycholic acid (DCA) in the colon and by cholera enterotoxin (CE) in the jejunum is mediated by cAMP. Propranolol (Pr) inhibits adenylate cyclase (AC) activity and net secretion induced by bile acid in the colon. The aim of this study was to assess the organ specificity of DCA and CE as well as the selectivity of Pr inhibition. Three colonic and three jejunal loops were prepared in each of 8 rabbits treated intravenously with Pr, 4 mg per kg, 1/2 hr before loop construction and in each of 10 untreated control rabbits. One milliliter of DCA, 6 mM, CE, 10 mug per ml, or heat-inactivated CE or 0.9% NaCl, as basal controls were injected in random order into each of the loops. The volume of luminal fluid and mucosal AC were measured in each intestinal loop 5 hr later. DCA in the colon stimulated AC 2-fold (P less than 0.01) and luminal fluid 15-fold (P less than 0.01). CE in the jejunum stimulated AC 2.3-fold (P less than 0.01) and luminal fluid 9-fold (P less that 0.01). No significant effects on volume or AC occurred in response to CE in the colon or to DCA in the jejunum. Pr pretreatment completely prevented the stimulation of AC and luminal fluid by DCA in the colon but did not affect the action of CE in the jejunum of the same animals. Thus, DCA and CE are organ-specific stimulants of cAMP systems, and Pr is a selective inhibitor of certain inducers of cAMP and net secretion.
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