The effects of smoking on the mood, cardiovascular and adrenergic reactivity of heavy and light smokers in a non-stressful environment.

Following a period of overnight deprivation, 58 smokers participated in a 90-min laboratory assessment in which they viewed a non-stressful movie and smoked two 0.5-mg nicotine-containing cigarettes. The first cigarette was given to all subjects following 25 min of adaptation and baseline. The next cigarette was provided at their request, which occurred 9-12 min later. "Heavy" and "light" smokers were grouped according to their average morning cotinine values, which fell above or below 250 ng/ml, respectively. The results showed that, relative to their baseline, heavy and light smokers experienced about the same level of post-smoking change in blood nicotine, heart rate and blood pressure. However, heavy smokers showed a significantly greater delta from baseline in post-smoking measures of epinephrine, norepinephrine, tension reduction and increase in vigor enhancement. A strong and consistent correlation was observed between post-smoking increases in epinephrine, tension reduction and increased vigor.

Antibodies and radioimmunoassays for phosphoserine, phosphothreonine and phosphotyrosine. Serologic specificities and levels of the phosphoamino acids in cytoplasmic fractions of rat tissues.

For antibody production, the O-phosphorylated derivative of tyrosine, threonine, or serine was covalently linked to succinylated bovine albumin via the carbodiimide reaction. Each conjugate was then complexed with methylated bovine albumin for immunization of rabbits. To determine binding, the corresponding O-phosphorylated [3H]amino acids were chemically synthesized. In addition, these 3H-phosphorylated derivatives were acylated (with succinic or acetic anhydride) to obtain ligands whose structures resemble those present in the immunogen. The acylated ligands bound to their respective antibodies more effectively: in some cases binding was about three orders of magnitude greater than their non-acylated counterparts. Radioimmunoassays were therefore developed using the N-succinyl-[3H]phosphoamino acids. When the unlabeled N-succinyl-phosphorylated amino acids were used as inhibitors in the homologous immune systems, 50% displacement of the labeled ligand was found with 0.06, 0.27 or 0.8 pmol of the tyrosine, threonine, or serine derivative, respectively. The antibodies were highly specific for the homologous hapten; the requirement for the phosphate group on the acylated amino acid was essentially absolute. Antibody content (expressed as mg/ml serum) and apparent binding constants for the N-succinyl derivatives in individual bleedings of immune sera were 1.9 and 1 X 10(10) M-1 for phosphotyrosine, 0.825 and 6 X 10(8) M-1 for phosphothreonine, and 0.150 and 2 X 10(8) M-1 for phosphoserine. The radioimmunoassays were used to quantitate the phosphoamino acids in cytoplasmic fractions of rat tissue extracts. The production of antibodies to phosphorylated O-tyrosine has been reported previously, but to our knowledge, this represents the first report of antibodies specific for O-phosphorylated serine and threonine residues.

Nicotine regulation among heavy and light smokers in a non-stressful environment.

The purpose of this study was to assess nicotine regulation among "heavy" and "light" smokers. Previous studies supporting the nicotine regulation model of smoking behavior have suggested that smokers compensate for a reduction in the amount of nicotine available in their cigarette by altering smoking frequency, puff volume, or other aspects of smoking topography. However, little is known about a smoker's decision to smoke a specific cigarette, and the concurrent changes in their blood nicotine. Manipulation of nicotine levels in the blood could play a critical role in smoking maintenance, by regulating the extent and quality of the CNS effects of smoking. In this study, 24 heavy and light smokers (cotinine above or below 260 ng/ml) smoked high- (1.0 mg) or low- (0.5 mg) dose nicotine cigarettes while watching non-stressful movies. Blood nicotine was assessed before and after smoking a preload and free operant cigarette. The results showed that blood nicotine levels after smoking the free operant cigarette were significantly more consistent (lower standard error) for the heavy smokers, following a low dose, as opposed to a high-dose preload. Light smokers showed a non-significant trend towards being more consistent when the high-dose nicotine preload was used. This suggests that heavy smokers may have maximized their dose of nicotine whenever available nicotine was in relatively short supply (low dose condition). However, light smokers may have minimized their exposure when available nicotine was relatively more plentiful (high dose condition).

Relative sensitivity and specificity of salivary and serum cotinine in identifying tobacco-smoking status of self-reported nonsmokers and smokers of tobacco and/or marijuana.

Serum and salivary cotinine levels were measured in 327 smoking and nonsmoking participants in a study of the health effects of marijuana with and without tobacco. These individuals had no reason to misrepresent their current tobacco-smoking status. The sensitivity, specificity, and predictive values positive and negative of the cotinine levels in distinguishing self-reported current tobacco smokers from nonsmokers was high (88-100%) and essentially the same for both fluids. Agreement between self-report and cotinine levels was not influenced by the presence or absence of marijuana smoking. A good correlation was found between serum and salivary cotinine levels in self-reported tobacco smokers (r = 0.84, p less than 0.001). Mean average levels were 279 +/- 144 ( +/- standard deviation) ng/ml for serum and 360 +/- 195 ng/ml for saliva. In a separate group of seven tobacco smokers, cotinine levels in saliva were found to be essentially independent of salivary flow rate. An analogous relationship has been observed by others for various compounds that are filtered to saliva from the blood. This may explain the close relationship observed between serum and salivary cotinine levels, and the observation made by others that the half-life of salivary cotinine is similar to that of serum cotinine.

A radioimmunoassay for the teleocidins using 26 (2'-aminoethylthio)-tetrahydroteleocidin A-2 as hapten.

A teleocidin A-2 derivative, 26 (2'-aminoethylthio)-tetrahydroteleocidin A-2, induced ornithine decarboxylase in mouse skin and inhibited the specific binding of [3H]12-O-tetradecanoyl-phorbol-13-acetate to a mouse skin particulate fraction with a potency that was weaker than that of teleocidin A-2 and stronger than that of (-)-indolactam-V. To obtain antibodies, 26 (2'-aminoethylthio)-tetrahydroteleocidin A-2 was covalently linked to bovine albumin with a carbodiimide and the resulting conjugate used for immunization of rabbits. Antibodies directed toward teleocidin were produced as measured by neutralization of teleocidin's capacity to stimulate arachidonic acid metabolism in rat liver cells (the C-9 cell line). An 125I-labeled ligand was prepared by reaction of the same derivative with radiolabeled Bolton-Hunter reagent. The antibodies bound this radiolabeled hapten, and the binding increased progressively with repeated immunizations. After absorption of the rabbit IgG with a goat anti-rabbit IgG, binding was reduced greater than 95%. The binding of the 125I-labeled ligand to the antibodies of one rabbit had an apparent average association constant of 2.84 x 10(9) M-1 at 0 degrees C. The serologic specificity of the antiserum was characterized by measuring the inhibition of binding by several teleocidins of varying structure as well as by other tumor promoters and toxins. The rank order of inhibitory activity expressed as concentration required for 50% inhibition (IC50) was for 26 (2'-aminoethylthio)-tetrahydroteleocidin A-2'(0.56 pmol) greater than teleocidin A-1 (6.5 pmol) greater than or equal to teleocidin A-2 (7.3 pmol) greater than (-)-indolactam-V (3.7 nmol) greater than teleocidin B-4 (13 nmol). Maitotoxin, aplysiatoxin, palytoxin, mezerein, okadaic acid and 12-O-tetradecanoylphorbol-13-acetate did not inhibit at the levels tested.

Production of antibodies and development of a radioimmunoassay for okadaic acid.

An okadaic acid immunogen, prepared by conjugation of okadaic acid to bovine albumin with carbodiimide, was used to immunize two rabbits. The rabbits responded by producing antibodies that neutralized okadaic acid's stimulation of arachidonic acid metabolism and this neutralization increased during the course of immunization. The immune sera bound 3H-okadaic acid and this binding also increased with repeated immunization. After absorption of the rabbit IgG with a goat anti-rabbit IgG, binding was reduced greater than 99%. The binding of okadaic acid to the antibodies in one antiserum was inhibited by as little as 0.2 pmoles of unlabelled okadaic acid. The apparent association constant for binding with this antiserum was 4.17 x 10(9) M-1 (35 degrees C). Maitotoxin, teleocidin, 12-O-tetradecanoylphorbol-13-acetate, aplysiatoxin, palytoxin and brevetoxin B when tested at 29, 228, 168, 169, 3.7 and 112 pmole levels, respectively, did not inhibit binding. The serologic and biological activities of okadaic acid after incubation for 60 min in 0.01 N HCl at 35 degrees C or at 100 degrees C at pH 7.2 were unaffected.

A radioimmunoassay for palytoxin.

Palytoxin, labelled with 125I-Bolton-Hunter reagent on its terminal amino group, bound specifically to rabbit anti-palytoxin. The extent of binding increased progressively with repeated immunizations. After absorption of the rabbit IgGs with a goat anti-rabbit IgG, binding was reduced greater than 95%. For 50% inhibition of binding in the 125I-palytoxin-antipalytoxin reaction 0.27 pmoles of unlabelled palytoxin was required. Maitotoxin, teleocidin, okadaic acid, debromoaplysiatoxin and 12-O-tetradecanoylphorbol-13-acetate, when tested at 10-100-fold higher concentrations than palytoxin did not affect binding. Palytoxin's serologic activity was stable after 60 min exposure to 100 degrees C and after 60 min exposure to 0.1 N HCl at 50 degrees C, but its capacity to stimulate the arachidonic acid metabolism of rat liver cells was reduced after the 60 min exposure to 0.1 N HCl treatments at 35 degrees C or 0.01 N HCl at 50 degrees C. The average binding constant (K0) as determined by separation of antibody-bound palytoxin from free palytoxin by the double antibody technique was 4.9 x 10(9) M-1 at 0 degrees C. This apparent average association constant increased with increasing temperature suggesting that palytoxin's epitope, most likely hydrophilic, is bound to H2O and the H2O is displaced before binding to the antibody's paratope.

Production of antibodies to palytoxin: neutralization of several biological properties of palytoxin.

Palytoxin stimulated arachidonic acid metabolism (in bovine aorta endothelial and smooth muscle cells, rat keratinocytes, porcine aorta endothelial cells and rat liver cells), hemolyzed rat erythrocytes and was lethal to mice when administered intraperitoneally. Serum from rabbits immunized with a conjugate in which palytoxin was covalently bound to bovine albumin through its free amino group neutralized these biologic activities of palytoxin. Ninety-nine per cent of the neutralizing activity of the immunized rabbit serum was removed after precipitation of the rabbit IgG with a goat anti-rabbit IgG.

Stereospecific monoclonal antibodies to nicotine and cotinine and their use in enzyme-linked immunosorbent assays.

Stereospecific monoclonal antibodies (McAb) have been prepared against the tobacco alkaloid (S)-(-)-nicotine and its major metabolite (S)-(-)-cotinine. Nine anti-nicotine and 4 anti-cotinine hybridomas, selected by a screening procedure that utilized immunoprecipitation of the 3H-labeled natural isomers of nicotine or continine, were grown in the ascites fluid of pristane-primed syngeneic BALB/c mice. Antibodies in concentrations up to 7.5 mg/ml ascites and with binding affinities that generally exceeded 10(8) M-1 were obtained. Enzyme-linked immunosorbent assays (ELISAs) were developed in which nicotine or cotinine derivatives bound covalently to poly-L-lysine were coated onto wells of polyvinyl chloride microtiter plates. Coated wells were incubated sequentially with McAb in the presence or absence of inhibitor, rabbit anti-mouse immunoglobulin, then horseradish peroxidase-labeled protein A (HRP-SpA) before addition of substrate. The antibodies are highly specific and show minimal cross-reactivity with several nicotine metabolites and other structurally related compounds. In the respective assays, only 0.25 ng (S)-(-)-nicotine and 0.12 ng (S)-(-)-cotinine are required to give 50% inhibition of antibody binding, and as little as 0.05 ng nicotine and 0.02 ng cotinine give 15% inhibition. These assays are 5-10 times more sensitive than analogous ELISAs developed with rabbit antisera and HRP-SpA or conventional radioimmunoassays (RIAs) that utilize the rabbit antisera and 3H-labeled ligands. There was good correlation between the levels of nicotine (r = 0.967) and cotinine (r = 0.981) found in saliva samples from smokers and non-smokers assayed by McAb-based ELISAs and conventional RIAs.

Radioimmunoassay for normeperidine: studies on the N-dealkylation of meperidine and anileridine.

A sensitive and specific radioimmunoassay for normeperidine has been developed that can detect as little as 100 pg of this metabolite. In competitive binding experiments with [125I]O-tyramyl-normeperidinic acid and an antiserum produced in rabbits immunized with a bovine serum albumin-normeperidinic acid conjugate, meperidine is only 0.01% as effective an inhibitor as normeperidine. Therefore, normeperidine can be determined in physiological fluids and in tissue extracts when relatively large amounts of meperidine or other N-phenylpiperidine esters are present. High pressure liquid chromatography can be used to confirm the results obtained by radioimmunoassay during the in vitro N-dealkylations of meperidine and anileridine to normeperidine. The normeperidine isolated by high pressure liquid chromatography accounts for all of the inhibitory reactivity determined in the enzymatic digests by the radioimmunoassay. Kinetic parameters (Km and Vmax) can be determined for the N-dealkylation of meperidine and anileridine. The formation of normeperidine with time can be followed in rabbits injected with meperidine or anileridine. Plasma levels may also be determined when meperidine is administered as an obsteric analgesic.

Radioimmunoassay for anileridine, meperidine and other N-substituted phenylpiperidine carboxylic acid esters.

Antibodies that bind an 125I-tyramyl derivative of N-succinylanileridine have been produced in animals immunized with N-succinylanileridine-hemocyanin conjugate. Several congeners and metabolites have been tested as competitors of this antigen-antibody reaction. The concentrations (in picomoles) required for 50% inhibition have been found to be: anileridine (0.2), meperidine (3.5), piminodine (3.8), diphenoxylate (20.5), normeperidine (20.0), meperidine acid (45,000) and anileridine acid (3,400). Although ester hydrolysis results in changes in inhibiting capacities on the order of 10(4), major structural changes in the substituent on the nitrogen of the piperidine ring are not readily recognized by the antibody. This radioimmunoassay can be used to study a variety of N-substituted phenylpiperidine carboxylic acid esters by relating the results to the standard curve obtained for the drug under investigation. For all practical purposes, alphaprodine, morphine and methadone do not interfere with the assay.

Specificity of the antibody receptor site to D-lysergamide: model of a physiological receptor for lysergic acid diethylamide.

Antibodies to D-lysergic acid have been produced in rabbits and guinea pigs and a radioimmunoassay for the hapten was developed. The specificity of this lysergamide-antilysergamide reaction was determined by competitive binding with unlabeled lysergic acid diethylamide (LSD), psychotomimetic drugs, neurotransmitters, and other compounds with diverse structures. LSD and several related ergot alkaloids were potent competitors, three to seven times more potent than lysergic acid itself. The N,N-dimethyl derivatives of several compounds, including tryptamine, 5-hydroxytryptamine, 4-hydroxytryptamine, 5-methoxytryptamine, tyramine, and mescaline, were only about ten times less effective than lysergic acid, even though these compounds lack some of the ring systems of lysergic acid. The pattern of inhibition by related compounds with various substituents suggests that the antibody receptor site recognizes structural features resembling the LSD molecule. In particular, the aromatic nucleus and the dimethylated ethylamine side chain in phenylethylamine and tryptamine derivatives may assume in solution a conformation resembling ring A and the methylated nitrogen in ring C of LSD. Among the tryptamine derivatives, a large percentage of the most potent competitors are also psychotomimetic compounds.