Persistent behavior at high rates maintained by intravenous self-administration of nicotine.

Squirrel monkeys pressed a level at high rates under a second-order schedule of reinforcement in which level pressing produced a brief visual stimulus that was occasionally contiguous with an intravenous injection of nicotine. The rate of lever pressing could be markedly reduced either by substituting saline for nicotine injections or by blocking the effects of nicotine with mecamylamine. The rate of level pressing could be reduced by eliminating the brief visual stimulus. These results show that nicotine can function as an effective reinforcer under a second-order schedule of drug self-administration and that an environmental stimulus associated with nicotine intake can contribute to the maintenance of persistent drug-seeking behavior.

Coupled optical rate determinations of amino acid oxidase activity.

Methods are described in which liberation of ammonia from amino acid substrates by the D- and L-amino acid oxidases may be coupled with the NADH-dependent reductive amination of 2-oxoglutarate catalysed by exogenous glutamate dehydrogenase (L-glutamate: NAD oxidoreductase (deaminating), EC 1.4.1.2). The inhibition of D-amino acid oxidase (D-amino acid:O2 oxidoreductase (deaminating), EC 1.4.3.3) by ADP needed to activate and stabilise glutamate dehydrogenase was relieved by FAD, and the substrate was D-alanine at approximately 6-fold Km concentration. Neither FAD or FMN were required in the L-amino acid oxidase (L-amino acid:O2 oxidoreductase (deaminating), EC 1.4.3.2) assay; this utilised L-leucine as substrate in a concentration approximately 7-fold the Km value. The methods were reasonably sensitive and precise, and a linear relationship between activity and enzyme concentration prevailed up to an absorbance change of 0.050 per min. They have the advantage of being amenable to automation and to employment of fluorescence techniques should greater sensitivity be required.

Effect of chronic heparin administration on serum lipolytic activity and some aspects of lipid metabolism.

Chronic heparin administration to rats for periods up to 8 days by i.p. implantation of mini pumps, increased serum total lipolytic activity in a dose-dependent manner up to infusion rates of 10 U/h per 100 g body weight. This augmentation was predominantly due to lipoprotein lipase (LPL). Synchronously, heart muscle demonstrated a dose-dependent reduction in LPL activity and adipose tissue showed a biphasic response, LPL activity decreasing at low doses and rising towards control levels at higher doses. Lipolytic activities of skeletal muscle and liver were unaffected. Increased serum LPL could not be attributed to altered enzyme clearance from the circulation in chronically heparinised rats, but was accompanied by a reduced response to i.v. high-dose heparin indicating reduction in the pool of endothelial-bound enzyme. Fasting serum concentrations of triacylglycerol and glycerol were unaffected in chronically heparinised animals although accelerated clearance of exogenous 14C-labelled VLDL was demonstrated, together with enhanced uptake of the isotope by liver and heart. Since de novo synthesis of fatty acids and triacylglycerol from 3H2O was not increased by heparin, we suggest that serum triacylglycerol concentrations were maintained by enhanced re-esterification of preformed fatty acids taken up by the liver. Hepatic cholesterol synthesis from 3H2O was augmented by heparin; this observation is consistent with reported increases in serum total and HDL-cholesterol mediated by chronic heparin administration in man and dog.

Histoplasmosis in patients with the acquired immune deficiency syndrome.

Five patients with disseminated histoplasmosis are reviewed. Four of five had the acquired immune deficiency syndrome (AIDS) and one was receiving steroid therapy. All were immigrants to the United States from Puerto Rico, the Dominican Republic, or South America, and none had a history of travel to regions of the United States where Histoplasma is endemic. Histoplasma complement fixation titers to mycelial antigen were not demonstrable in three of three patients in whom they were measured. Of the four patients with AIDS, Histoplasma capsulatum was isolated from bone marrow aspirates in two patients and from lymph node and liver biopsy specimens in one patient each. One of the bone marrow specimens showed organisms on Gomori-methenamine silver stain. In the other three cases, results of staining were falsely negative and diagnosis awaited culture results weeks later. Amphotericin B therapy resulted in rapid clinical improvement in the three patients that were treated. Intravenous therapy was followed by treatment with oral ketoconazole. Follow-up has not been long enough to determine the ultimate efficacy of ketoconazole. Disseminated histoplasmosis should be considered in all patients from the Caribbean or South America with AIDS or who are receiving immunosuppressive therapy.

Modulation of lipoprotein production in Hep G2 cells by fenofibrate and clofibrate.

Fenofibrate and other fibrate derivatives are commonly used to treat hyperlipidemia. It is not yet clear how they exert their modulatory effects on plasma lipoproteins. To investigate whether these drugs act on the liver to primarily inhibit very low density lipoprotein production, we utilized the highly differentiated human hepatoma cell line, Hep G2. At concentrations greater than 15 micrograms/mL, fenofibrate caused a 30% decrease in secreted apolipoprotein B (apo B) after 4 days of treatment. Pulse-chase studies demonstrated that this was not due to inhibition of apo B synthesis. Triglyceride synthesis by fenofibrate-treated Hep G2 cells was decreased by 30%, and the amount secreted into the medium was reduced by 50%. At a low concentration of drug (5 micrograms/mL), triglyceride secretion was reduced markedly while apo B secretion remained unchanged. Thus, apo B secretion is less sensitive to fenofibrate than the synthesis and secretion of triglyceride, and may be secondary to changes in the latter. Fenofibrate has also been shown to raise plasma high density lipoprotein concentrations. We found that low concentrations of fenofibrate caused a 20-101% increase in secreted apolipoprotein AI (apo AI), and pulse-chase immunoprecipitation studies showed that this was due to an increase in apo AI synthesis. Fenofibrate was compared to clofibrate to investigate whether their relative effects on lipoprotein production in Hep G2 cells were comparable to their relative effects on plasma lipoproteins. Both fibrates decreased the secretion of apo B to the same extent, but only fenofibrate increased apo AI secretion. Fenofibrate was more effective than clofibrate in inhibiting the secretion of lipids by these cells. Thus, the known effects of fenofibrate on plasma lipoproteins can be attributed to its direct modulation of lipoprotein synthesis in the liver cell. Hep G2 cells may thus be useful in testing the relative efficacy of fibric acid derivatives in vitro.

Serum enzyme tests in diseases of the liver and biliary tree.

Eight serum enzyme tests were performed over a three-year period in 1,147 cases of patients with suspected hepatobiliary disease, of whom 580 had identifiable primary disease of the liver or biliary system. Individually, aminotransferase assays did not provide good discrimination among the various categories of hepatobiliary disease, but when expressed as a ratio a useful degree of discrimination was obtained. Isocitrate dehydrogenase, guanase and glutamate dehydrogenase alone were poor discriminants of the various disease categories studied; combination of the latter enzyme with the aminotransferases in various ratios did not achieve worthwhile improvement. Adenosine deaminase was normal in most patients with extrahepatic obstruction and abnormal in most patients with parenchymal hepatic disease, and is potentially a useful test additional to the aminotransferases in routine diagnosis. 5'-Nucleotidase was more sensitive and specific than alkaline phosphatase in diagnosing hepatobiliary disorders. Abnormalities of all these enzymes were encountered in patients who did not have hepatobiliary disease, most frequently among subjects with cancer, diabetes mellitus, and diseases of the respiratory and cardiovascular systems.

Biochemical features of intrahepatic cholestasis.

PIP: A pattern of results is reported which was found to be common among patients who had intrahepatic cholestasis (IHC) which was rarely found in patients with other hepatic conditions. The pattern was recognized from over 1000 cases suspected of hepatobiliary disease. 29 were diagnosed with IHC, and excluding 4, 25 revealed the following etiological pattern: chlorpromazine (12 patients); pregnancy and oral contraceptive use (8); and other (5). As opposed to patients with acute and chronic hepatic disease, IHC sufferers had relatively normal values for immunoglobulins and antibody titers. A disproportionate elevation of serum bilirubin vis-a-vis serum enzymatic activities separated potential IHC cases into intra- and extrahepatic cholestasis. The following factorial evaluations were useful in distinguishing hepatic disease states: 1) when the sum of the activities of serum alkaline phosphatase, 5'-nucleotidase, aspartate and alanine amiotransferases, and isocitrate dehydrogenase was divided by the serum bilirubin concentration, there was good resolution of the distinction between patients with IHC and those with primary biliary cirrhosis, early and late viral hepatitis, cholelithiasis, and pancreatic and bile duct cancers. 2) Resolution was also achieved when the numerator included alkaline phosphatase, 5'-nucleotidase, and aspartate aminotransferase, but not when alkaline phosphatase alone, or alkaline phosphatase combined with 5'-nucleotidase, was used. The essential lesion in IHC is an excretory defect.^ieng

Activation of serum 5' nucleotidase by magnesium ions and its diagnostic applications.

Magnesium ions increase the hydrolysis of adenosine 5' monophosphate (5' AMP) by human serum at pH 7.9 but not at pH 9.3. The additional hydrolysis at pH 7.9 is predominantly due to increased activity of the specific phosphatase 5' nucleotidase (5Nase). This increase is proportional to enzyme concentration and has been employed as a measure of 5Nase activity in a sensitive micro-estimation. The normal range for serum 5Nase activity by this technique was 0 to 20 mIU/ml. In a series of over 200 patients, raised values were found frequently in hepatobiliary disease and infrequently in bone disease. Assay of 5Nase activity gave a more reliable indication of the source of raised serum alkaline phosphatase than isoenzyme electrophoresis in agar gel. The correlation between activities of the two enzymes was low in bone disease generally, and fairly good in hepatobiliary disease. The closest correlation was found in patients with parenchymal liver disease.

Measurement of mercury in human urine.

Four methods of determining the concentration of mercury in human urine have been studied. A simple method suitable for general laboratory use is recommended and the requirements for accurate results are defined. The method employs mild oxidation with permanganate and HS(2)O(4) followed by dithizone extraction and measurement of absorbance at 485 nm and 620 nm.No mercury was detected in any of 74 urines from unexposed laboratory controls and hospital patients. A random urine sample seems adequate for the investigation of clinical or industrial mercury poisoning. Two individuals, free of symptoms, but subjected to moderate exposure, excreted 3.0-9.7 mug of mercury per 100 ml of urine. After the administration of an organic mercurial to two volunteers, urinary excretion was rapid and virtually complete within 48 hours.

An assessment of serum acid and alkaline phosphatase determinations in prostatic cancer with a clinical validation of an acid phosphatase assay utilizing adenosine 3'-monophosphate as substrate.

Serum acid phosphatase (AcPase) was measured by a colorimetric method utilizing adenosine 3' -monophosphate as substrate in 389 patients. In about half the cases blood was taken shortly after a rectal examination. The upper reference limit (mean + 2SD) for 116 cases with miscellaneous illness after eliminating outliers was 4.1 International Units per litre (U/I) at 37 degrees C, and no correlation existed between AcPase activity and age in these subjects (r = 0.040). Eight of 18 patients with untreated carcinoma confined within the prostate gland had AcPase activities below 4.1 U/l, and all of 27 cases with extension to pelvic soft tissues or to bone exceeded this value. AcPase activities above 4.1 U/l were found in 6% of cases with benign hypertrophy of the prostate, in 5% of cases with non-prostatic cancer, and in none of 22 cases with other urological illness. Raised serum alkaline phosphatase (APase) activity was found in 60% of patients with untreated prostatic cancer and in only 6% of patients free of prostatic cancer, in most of whom there was a clinical explanation for the elevation. The correlation between the two phosphatase activities was not significant (r = 0.294). While APase activity does not reflect the stage of the disease as closely as AcPase activity, and is not so frequently elevated, it provided useful confirmation of the diagnosis in five patients of the present series whose AcPase levels were normal or only minimally elevated.

Colorimetric determination of serum acid phosphatase activity using adenosine 3'-monophosphate as substrate.

The hydrolysis of adenosine 3'-monophosphate by serum acid phosphatase has been coupled to the liberation of ammonia from the adenosine generated through the action of exogenous adenosine deaminase. The ammonia is measured at the end of the incubation by a modification of the phenol-hypochlorite reaction of Berthelot. Optimum conditions for the enzyme reaction have been defined. Inhibition of the Berthelot reaction by the serum used in the assay is small, and may be compensated by a correction factor. Although the value for the control is high in relation to the test over the normal range, this is largely outweighed by the good sensitivity and precision of the method. The substrate is not significantly hydrolysed by erythrocyte acid phosphatase within the limits encountered in haemolysed sera. Experience of the method in routine hospital diagnosis compared favorably with that of a standard method employing disodium phenyl phosphate as substrate. It is suggested that activities greater than 3.1 IU/l should be further investigated and those greater than 3.7 IU/l should be regarded as definitely raised. The stability of human serum AcPase when promptly separated and held at 4 degrees C or - 20 degrees C was confirmed. At room temperature, acidification to pH 6.0 greatly improved stability.

An optimized semi-automatic rate method for serum glutathione reductase activity and its application to patients with malignant disease.

An improved and optimized method for serum glutathione reductase is described. The reference range for normal subjects is 47-79 IU/1. The method is more sensitive than conventional enzyme tests in the detection of malignant disease. It was not raised more frequently in patients with clinical evidence of metastases than in those clinically free of such metastases, and it did not seem to correlate with prognosis among those patients who failed to survive six months from the time the analysis was first conducted.

Serological and histological diagnosis of primary biliary cirrhosis.

A simple immunofluorescence test for antibody to a mitochondrial antigen present in many tissues is a reliable method of distinguishing most cases of primary biliary cirrhosis from jaundice due to extrahepatic biliary tract obstruction. Of 30 cases diagnosed as primary biliary cirrhosis, 26 had antimitochondrial antibody whereas none of 77 cases with jaundice due to extrahepatic bile duct obstruction showed this serological abnormality. The antibody was also found in the serum of three of 42 patients who had other forms of cirrhosis and in two of 266 patients with no evidence of liver disease.Clinical, biochemical, and serological findings favour the view that primary biliary cirrhosis is a real entity which, in our present state of knowledge, cannot be defined clearly by any single method of investigation. In particular, the liver may show a variety of histological appearances which, interpreted without regard to the other features of the case, may lead to errors in diagnosis.

The effects of sertraline on nicotine self-administration and food-maintained responding in squirrel monkeys.

Recent reports suggested the involvement of serotonergic mechanisms in nicotine self-administration. The present study assessed the effects of sertraline, a selective serotonergic uptake inhibitor, on the reinforcing effects of i.v. nicotine (30 microgram/kg per injection) in squirrel monkeys responding under a fixed-ratio schedule. Nicotine (10-100 micrograms/kg per injection) produced a significant inverted U-shaped distribution on FR rate. Vehicle or sertraline (3, 6, 12, 24 mg/kg, p.o.) produced no changes in the response rates maintained by 30 micrograms/kg per injection i.v. nicotine, but sertraline produced non-significant increases response rates maintained by 10 micrograms/kg per injection nicotine and vehicle. In a separate group of monkeys, sertraline given in combination with i.m. doses of nicotine produced a significant dose-dependent decrease in responding maintained by food-pellet delivery. Thus, sertraline produced differential effects on response rates that may be related to (1) route of nicotine administration and (2) whether the behavior was maintained by nicotine or food. In addition, the results of the self-administration study suggest that sertraline would not disrupt well-maintained responding for nicotine.

The enzymology of intestinal disease.

The role of enzyme estimations is reviewed. Serum levels of most enzymes do not alter significantly in intestinal diseases because dying mucosal cells slough off into the lumen. Similarly, biopsy material may provide misleading results because of lack of homogeneity between diseased and normal segments of bowel, whether in inflammatory or neoplastic conditions. Lactase deficiency is the most common intestinal enzyme deficiency. The once popular lactose tolerance test is lately giving way to the breath hydrogen test, which generally reflects unabsorbed carbohydrate reaching the colon. This test and its clinical usefulness are reviewed in some detail, and the use of lactulose as an indicator of intestinal transit is also discussed.

Demographic and analytic factors affecting the normal range of serum enzyme activities.

1. The effect of demographic variables such as age, sex, body weight, social class and blood pressure upon the activity of serum enzymes in healthy humans is reviewed. A system developed by the author and his colleagues, which automatically adjusts the results of enzyme activity measurements to allow for demographic influences, is described. This allows derivation of a "demographically corrected" normal range; for most enzymes this range is much narrower than that provided by the conventional mean +/- 2 SD approach. 2. The influence of precision and analytical factors upon the normal range for serum enzymes is discussed. Data from a British national quality-control survey reveal a depressing picture of interlaboratory precision for commonly determined enzyme estimations. Examples are given (serum aspartate aminotransferase and guanase) which illustrate the influence that precision of a method may have on the normal range for that enzyme.

Functional, chemical, and clinical aspects of proteolytic enzymes in the alimentary canal.

Knowledge of the structure and catalytic function of the pancreatic proteases has increased rapidly in recent years. These advances have been less spectacular in the case of pepsin, and information concerning mucosal enzymes of the small intestine and colon has not even reached the point where one can be certain of their number, far less their nature. The role of these enzymes in disease processes is probably not significant so far as the alimentary tract is concerned, and it is not even certain that the pancreatic proteases influence the course of acute pancreatitis -- they are of more interest to the clinician as diagnostic aids. Since undesirable consequences result from their deficient output, future research should be directed towards improving replacement therapy.

Advances in the application of biochemical tests to diseases of the liver and biliary tract: their role in diagnosis, prognosis, and the elucidation of pathogenetic mechanisms.

Despite the biochemical complexity of the liver, few laboratory tests provide discriminatory diagnostic information in patients with hepatobiliary disease. Recent efforts have concentrated upon tests which assess the function of the liver, the severity of the disease state, and underlying pathological processes. Bile Acids: The emergence of facile technology and widespread application has brought the realization that these assays are not as sensitive in detecting liver disease as previously believed, although the cholate/chenate ratio may be useful in distinguishing cholestasis from chronic liver disease. The presence of unusual bile acids in serum or urine may be helpful in some cases. Drug Metabolism: A number of tests provide good evidence about liver function, hepatic blood flow and portal shunting, but the aminopyrine breath tests is the most useful, giving prognostic information in acetaminophen overdose and alcoholic liver disease. The antipyrine half-life identifies surgical cases at risk from poor hepatic function. Proteins and Immunochemical Tests: Interest has developed in plasma proteins such as prealbumin and retinol-binding protein to monitor hepatic protein synthetic function. Secretory IgA is more elevated in biliary tract disease, unlike the native protein which is increased principally in cirrhosis. Type III procollagen can be measured in serum, and correlates with the activity of collagen synthesis and the degree of fibrosis in biopsy samples. Reye's Syndrome: Biochemical tests play an essential role in diagnosis of this recently discovered disease. These will be presented and discussed.

Lipolytic enzymes as markers of induction and differentiation.

Factors leading to microsomal enzyme induction are associated with hypertriglyceridemia in man. Phenobarbital (PB) increases hepatic synthesis of triglyceride but lowers its serum concentration in rats due to increased postheparin plasma activities of lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL); these changes are accompanied by increased activity of these lipolytic enzymes in adipose tissue and liver. The present work explores the cellular mechanisms whereby PB increases the tissue content of these enzymes, using primary cultures of rat liver hepatocytes and a continuous cell line of mouse fibroblasts (preadipocytes) that undergo differentiation into mature fat cells. Secretion and synthesis of HTGL in primary rat hepatocytes increased 50% with insulin; when PB was added with insulin, activity was enhanced an additional 50%. By contrast, insulin inhibited HTGL secretion from the well differentiated rat hepatoma cell line, FU-5-5, C8, and this inhibition was partly overcome by PB. These results suggest that different control mechanisms govern the synthesis and secretion of HTGL in normal rat liver cells and hepatoma. In cultured pre-adipocytes (3T3-F442A) insulin promoted differentiation when added to confluent cultures. PB (0.5 mM) resulted in marked enhancement of conversion of adipocytes characterized by a two- to threefold increase in extracellular LPL and a 10-fold increase in intracellular enzyme. These results suggest that PB promotes conversion of uncommitted cells into pre-adipocytes at an early stage in the differentiation of adipose tissue.

Biochemical tests in the diagnosis of chronic pancreatitis and in the evaluation of pancreatic insufficiency.

Chronic pancreatitis (adults) and cystic fibrosis (children) are the most common diseases leading to exocrine pancreatic insufficiency that, when reduced to < 5% of normal function, is characterised by steatorrhoea. The pathogenesis of the former condition is outlined, and recent concepts are emphasized. Biochemical tests to detect pancreatic insufficiency and to identify pancreatic disease as the cause of steatorrhoea include: serum enzyme tests (lipase, amylase, trypsin); stool chymotrypsin; isotopic tests based upon the assimilation of [14C] lipids and starch or excretion of the isotope as breath CO2, as well as the dual-labelled Schilling test; oral function tests utilising substrates hydrolysed by pancreatic enzymes such as benzoyl tyrosyl-p-aminobenzoic acid and fluorescein dilaurate; and duodenal intubation studies following meal-induced or hormonal stimulation of the pancreas. The rationale for these tests and the cumulative clinical experience of their utility are reviewed. A recommended diagnostic strategy is briefly presented. The role of various biochemical procedures to evaluate the efficacy of pancreatic enzyme replacement therapy is also described.