Prevalence of high serotonin uptake in lymphocytes of abstinent alcoholics.

An impairment in serotonergic neurotransmission may be associated with alcoholism. We recently identified a high-affinity serotonin transporter (5-HTT) in human peripheral blood lymphocytes (PBLs). Moreover, molecular analysis of RNA samples of human lymphocytes using reverse transcription, coupled with polymerase chain reaction, enabled us to confirm the expression of a 5-HTT identical to the one reported in neuronal tissues, as evidenced by hybridization and sequence analysis. In this investigation, we measured the serotonin (5-HT) uptake in PBLs of recovering alcoholics (N = 10) with long-term abstinence (2-10 years) and non-alcoholic controls (N = 10). 5-HT uptake was measured by incubating 1 x 10(7) cells of PBLs with [3H]5-HT (3-1000 nM; sp. act. 23 Ci/mmol) for 10 min at 37 degrees. The results of this preliminary study revealed that abstinent alcoholics had significantly (P < 0.01) increased uptake of 5-HT (43.6 +/- 5.70 pmol/10(7) cells) as compared with controls (23.33 +/- 2.50 pmol/10(7) cells). An enhanced uptake of 5-HT in PBLs of abstinent alcoholics agrees with previously reported observations of increased 5-HT uptake in brain and platelets of former alcoholics and their descendants. This suggested that a serotonergic mechanism may be linked to the heredity of alcoholism.

A cocaine-sensitive active dopamine transport in human lymphocytes.

Human lymphocytes possess a cocaine-sensitive high-affinity transport system for [3H]dopamine. [3H]Dopamine uptake was saturated with increasing dopamine concentrations and followed Michaelis-Menten kinetics. The uptake was temperature, sodium, and chloride dependent and was affected by the co-addition of ouabain, phloridzin, potassium cyanide, gramicidin, and other metabolic inhibitors. The uptake of dopamine was blocked significantly in a concentration-dependent manner by cocaine and its congeners. Furthermore, preliminary evidence is presented linking the possible relationship between decreased lymphocyte [3H]dopamine uptake and chronic cocaine abuse in humans.

Platelet monoamine oxidase activity in alcoholics, alcoholics with drug dependence, and cocaine addicts.

The main objective of this investigation was to study the influence of drug dependence on platelet monoamine oxidase (MAO) activity in the presence and absence of alcoholism. One hundred and thirteen admissions to alcohol and drug treatment facilities participated in the study. Twenty-six met the criteria for alcoholism (group I), seventy-eight subjects were alcohol-/cocaine- and cannabis-dependent (group II), and the remaining nine were patients with DSM-III-R diagnosis of cocaine addiction (group III). MAO activity was assayed radiochemically with [14C]tyramine as a substrate (221 microM). The results of this study showed that platelet MAO activity [nmol of product formed x (mg protein)-1 x hr-1] (mean +/- SE) was significantly (p < 0.01) lower in all of these subjects (group I, 5.50 +/- 0.80; group II, 3.90 +/- 0.50; group III, 4.3 +/- 1.60) as compared with controls (14.85 +/- 1.13). Measurements of platelet MAO activity may provide us with a reliable biochemical marker for alcoholism and perhaps addiction to other substances of abuse (i.e., cocaine).

The effect of cocaine abuse on plasma levels of sulfated dopamine and salsolinol in alcoholics.

This study investigated the effect of cocaine abuse on peripheral dopamine and its tetrahydroisoquinoline metabolite salsolinol in chronic alcoholics. Specifically, the concentration of dopamine sulfate and salsolinol sulfate was measured in plasma samples obtained from the blood of a group of alcoholics (n = 40) and alcoholics with cocaine dependence (n = 55). The concentrations of sulfoconjugated dopamine and salsolinol were measured by a radioenzymatic technique. The results of this study showed that chronic alcoholics (627 +/- 195 pg/ml) and alcoholics with cocaine addiction (409 +/- 76 pg/ml) had significantly (p < 0.05) elevated levels of salsolinol sulfate (mean +/- SEM) in their plasma as compared to controls (99.5 +/- 7.5 pg/ml). However, alcoholics with cocaine dependence produced significantly (p < 0.01) higher concentration of dopamine sulfate in their plasma (7520 +/- 1299 pg/ml) as compared to chronic alcoholics (3896 +/- 438 pg/ml) and controls (2124 +/- 104 pg/ml). Differences in plasma dopamine sulfate among alcoholics with cocaine dependence vs. alcoholics without cocaine dependence may be interpreted as a reflection of increased extracellular dopamine metabolism associated with chronic cocaine exposure.

Expression of a high-affinity serotonin transporter in human lymphocytes.

This study was undertaken to assess the capability of lymphocytes to actively transport serotonin (5-HT). The data we obtained showed that lymphocytes isolated from the blood of normal human subjects contained a high-affinity uptake system for [3H]5-HT. Kinetic analysis of the uptake data as computed by regression analysis from Lineweaver--Burk plots, yielded a Km of 180 +/- 20 nM and Vmax of 94 +/- pmole/10(7) cells. The uptake of [3H]5-HT was temperature, sodium and chloride dependent and was potently inhibited by the antidepressants clomipramine, imipramine, fluoxetine and fluvoxamine, which are specific for the 5-HT transporter. Compounds that are more selective for norepinephrine and dopamine transporters such as mazindol, desipramine, and GBR 19209 had a lower inhibitory effect on the uptake of [3H]5-HT in human lymphocytes. The expression of a 5-HT transporter in human lymphocytes that resembles 5-HT uptake by platelets and brain synaptosomes may provide insights into the potential role of 5-HT in immune function and its relationship to the neurobiology of affective and addictive disorders.

Active [3H]-dopamine uptake by human lymphocytes: correlates with serotonin transporter activity.

The main objective of the present investigation was to determine whether the uptake of [3H]-dopamine in human lymphocytes is mediated through a serotonin transporter. This was examined by studying the effects of various monoamine uptake inhibitors on the uptake of [3H]-dopamine in human lymphocytes. Among the compounds tested, indatraline, imipramine and fluoxetine, selective inhibitors of neuronal serotonin transporter, were the most potent inhibitors of [3H]-dopamine uptake in lymphocytes. The 50% inhibiting concentration (IC50) for these inhibitors was in the range of 3.5-17 nmol/l. Bupropion, GBR 12909, nomifensine and xylamine, selective inhibitors of dopamine and norepinephrine transporters, had low affinity for the dopamine uptake system in human lymphocytes with IC50 values ranging between 1,000 and 40,000 nmol/l. These findings provide supportive evidence for the participation of a serotonin transporter in the uptake of [3H]-dopamine in human lymphocytes. The existence of a high affinity transport system for dopamine and serotonin in human lymphocytes may serve as a readily accessible model to detect changes in the neuronal uptake of dopamine and serotonin in addictive and psychiatric disorders.

Elevated concentrations of dopamine sulfate in plasma of cocaine abusers.

This study investigated the effect of cocaine abuse on peripheral catecholamines. Specifically, we measured the concentration of free dopamine, dopamine sulfate, free norepinephrine, norepinephrine sulfate, free epinephrine and epinephrine sulfate in plasma samples obtained from the blood of a group of patients with cocaine addiction (N = 15). The concentrations of free and sulfoconjugated catecholamines in plasma were measured by a radioenzymatic technique. The results of this study revealed significant (P < 0.0001) elevation in plasma dopamine sulfate (8926 +/- 1204 pg/mL) of cocaine addicts upon admission to an in-patient treatment facility when compared with the level of this dopamine metabolite in plasma of control subjects (2356 +/- 121 pg/mL). Furthermore, there was a significant (P < 0.0001) relationship between elevation in plasma dopamine sulfate levels and severity of cocaine use among these patients, and in the majority of cases the plasma levels of dopamine sulfate declined appreciably in time with abstinence from cocaine. In contrast, no appreciable difference was observed in the concentrations of either free or sulfate-conjugated norepinephrine and epinephrine in plasma of cocaine addicts as compared with controls. Differences in plasma dopamine sulfate among these patients versus controls may be interpreted as a reflection of activation of extracellular dopamine metabolism associated with chronic cocaine exposure in humans.

Assessment of splanchnic blood flow in alcohol and drug abuse using radionuclide angiography.

Computerized radionuclide angiography (RA) is a noninvasive, quantitative, reproducible, and cost-effective method for measuring the portal venous fraction of total hepatic blood flow (represented by the Hepatic Perfusion Index, HPI), and also can be utilized to detect hemodynamic abnormalities in the spleen. A group of 105 men (aged 20-56) were evaluated at the time of admission to the Substance Abuse Program at the Department of Veterans Affairs Medical Center. These patients were classified into three groups: (a) alcohol dependence or abuse (Group A, n = 54); (b) polysubstance abuse without alcohol (Group B, n = 9); and (c) polysubstance abuse with alcohol (Group C, n = 42). Of the respective groups, 69%, 100%, and 79% had abnormal splanchnic flow (liver and/or spleen), whereas only 43%, 78%, and 48% had abnormal liver function tests. This method may be a sensitive, noninvasive detector of early pathophysiological changes in the splanchnic organs of alcohol and drug abusers.

Effect of astemizole on antigen-mediated histamine release from the blood of patients with allergic rhinitis.

The main objective of this study was to test the effectiveness of astemizole in vitro in blocking the release of histamine from blood of patients with allergic rhinitis. The results of this investigation indicated that astemizole inhibited allergen-mediated histamine release from blood basophils of patients with this allergic disorder. The inhibition by astemizole (33-156 mumol) was immediate, requiring no pre-incubation of the cells, and was dose-dependent, with maximal inhibition of about 91%. The relatively high potency of astemizole in inhibiting the immunologic release of histamine may provide an additional measure in the treatment of allergic rhinitis with this H1-receptor antagonist.

Interrelationship between activation of dopaminergic pathways and cerebrospinal fluid concentration of dopamine tetrahydroisoquinoline metabolite salsolinol in humans: preliminary findings.

The main objective of this study was to determine whether the activation of dopaminergic pathways, through adrenal-caudate transplantation, stimulated the production of dopamine and salsolinol in cerebrospinal fluid (CSF) of patients with Parkinson's disease. Dopamine sulfate and salsolinol sulfate in CSF specimens were measured by radioenzymatic technique. The results of this study demonstrated that the replacement of degenerative nigrostriatal neurons with new dopamine-producing cells by adrenal brain transplants in patients with Parkinson's disease resulted in significant increase (p less than 0.05) in CSF levels of free dopamine, dopamine sulfate, free salsolinol, and salsolinol sulfate as compared with preoperative levels. Moreover, the oral administration of L-dopa to these transplanted patients caused substantial (p less than 0.001) elevation in CSF levels of free dopamine (before L-dopa, 146 +/- 57 pg/ml; after L-dopa, 575 +/- 207 pg/ml), dopamine sulfate (before L-dopa, 1966 +/- 945 pg/ml; after L-dopa, 41679 +/- 29326 pg/ml), free salsolinol (before L-dopa, 43 +/- 29 pg/ml; after L-dopa, 186 +/- 90 pg/ml), and salsolinol sulfate (before L-dopa, 405 +/- 477 pg/ml; after L-dopa, 2908 +/- 2572 pg/ml), respectively.

Binding of [3H]-dopamine to human lymphocytes: possible relationship to neurotransmitter uptake sites.

Freshly isolated human lymphocytes from 11 healthy subjects had specific binding sites for dopamine which were dependent on time, temperature and sodium, and appeared to follow Michaelis-Menten kinetics. The apparent affinity constant (KD) of human lymphocytes for dopamine and the maximal number of binding sites (Bmax) were 109 +/- 21 nM and 2.66 +/- 1.75 pmol/10(7) cells, respectively. Dopamine binding was markedly affected by cocaine (IC50 = 150 nM) and other inhibitors of biogenic amine uptake. The relatively high potency of cocaine in competing for dopamine binding suggested that human lymphocytes may serve as a readily accessible model to detect changes in the neuronal uptake of dopamine and perhaps other monoamine neurotransmitters.

Abnormal vitamin B6 status in childhood leukemia.

Vitamin B6 is involved in many biological processes of potential relevance to carcinogenesis and tumor growth, including DNA synthesis and maintenance of immunocompetence, yet very little information exists on B6 nutritional status in childhood leukemia. Using a radioenzymatic assay, the authors measured plasma pyridoxal 5'-phosphate (PLP), the biologically active form of B6, in 11 newly diagnosed untreated children with leukemia and 11 age-matched controls. The children with leukemia had significantly lower PLP levels than the controls. In 26 additional leukemia patients and 26 additional controls, a high-performance liquid chromatography assay also demonstrated lower plasma PLP levels in childhood leukemia compared with controls. These differences were significant for both acute lymphoblastic leukemia (ALL) and for acute nonlymphoblastic leukemia (ANLL). The PLP values did not correlate with indices of leukemia cell burden, but did correlate with reported B6 intake, suggesting that illness-related diet changes are at least partially responsible for the low PLP levels. Before any chemotherapy, overall nutritional status was suboptimal in 53% of ALL cases and 57% of ANLL cases. Newly diagnosed children with leukemia have suboptimal overall nutrition as well as suboptimal vitamin B6 status.

A simplified radioenzymatic method for the study of whole blood histamine release in patients receiving radiographic contrast media.

With the availability of a newly developed, histamine-dependent allergy blood test, the authors studied the effect of ionic and nonionic radiocontrast media (RCM) on the in vitro release of histamine from whole blood obtained from 61 patients undergoing diagnostic arteriography. The ionic RCM, sodium meglumine diatrizoate (60 to 378 mmol/L), induced the release of a significantly (P less than .005) higher amount of histamine from blood compared with the nonionic agents, iopamidol and ioversol. Diatrizoate triggered significantly (P less than .05) more histamine release from the blood of eight patients with a history of adverse reactions to RCM than it did in 53 nonreactors; however, there was no appreciable difference in histamine release between reactive and nonreactive groups when stimulated with iopamidol and ioversol.

Effect of adrenal-caudate transplantation on CSF levels of salsolinol sulfate in patients with Parkinson's disease.

The main objective of this study was to determine whether the activation of dopaminergic pathways through adrenal-caudate transplantation stimulates the production of the dopamine cyclic metabolite salsolinol in CSF of patients with Parkinson's disease. Salsolinol sulfate in CSF samples was assayed by radioenzymatic technique. The outcome of this study revealed that the replacement of degenerative nigrostriatal neurons with new dopamine-producing cells by adrenal brain transplants resulted in significant increase in CSF concentration of salsolinol sulfate as compared to preoperative levels.

Effects of protamine on histamine release from human lung.

Animal mast cell models demonstrate direct histamine release by protamine. Investigators have proposed that protamine also releases histamine in man. We studied the effects of protamine alone and heparin-protamine mixtures on minced lung tissue for evidence of histamine release. We were unable to demonstrate the release of histamine despite positive anti-IgE controls. Nonimmunologic histamine release from human lung appears unlikely as a mechanism for protamine reactions in man.

Evaluation of patients at risk for protamine reactions.

Patients with neutral protamine Hagedorn and protamine-zinc insulin-dependent diabetes, a history of fish allergy, or prior vasectomy have been reported to be at an increased risk for protamine reactions after cardiopulmonary bypass because of prior sensitization. We prospectively evaluated cardiac surgical patients with prior vasectomies and fish allergies and retrospectively evaluated a cohort of 3245 consecutive cardiac surgical patients requiring cardiopulmonary bypass over a 2-year period for protamine-containing insulin use and clinical evidence of adverse reactions after protamine administration for heparin reversal after cardiopulmonary bypass. Clinical reactions to protamine did not occur in six patients with fish allergies or 16 patients with prior vasectomies. There was one reaction (0.6%) in 160 patients with neutral protamine Hagedorn insulin-dependent diabetes. The incidence of clinical reactions in the other patients was 2/3085 (0.06%). The incidence of clinical reactions in the patients with neutral protamine Hagedorn insulin-dependent diabetes is not significantly different from that in other patients. We conclude that prior neutral protamine Hagedorn insulin use, a history of fish allergy, or prior vasectomy does not represent a contraindication to protamine administration after cardiopulmonary bypass.

Elevation of plasma salsolinol sulfate in chronic alcoholics as compared to nonalcoholics.

This report describes a radioenzymatic assay for the measurement of salsolinol and dopamine sulfate levels in plasma. It is based on a sulfatase-catalyzed hydrolysis of the sulfoconjugates followed by catechol-O-methyltransferase and [methyl-3H]-S-adenosylmethionine-catalyzed O-methylation of the resulting free salsolinol and dopamine. Rapid thin-layer chromatographic separation of the formed labeled metabolites attributed to the specificity of the differential enzymatic assay of salsolinol and dopamine. This assay was used to study plasma salsolinol and dopamine levels in a group of adult males (n = 36) serving as controls and a group of hospitalized chronic alcoholics (n = 18). The results (mean and range) of this preliminary study show that alcoholics had significantly (p less than 0.0001) elevated plasma concentration of salsolinol sulfate (497; 50-1331 pg/ml) as compared to controls (93; 0-232 pg/ml). This was accompanied by significant (p less than 0.0003) elevation in plasma levels of dopamine sulfate. Elevation of plasma salsolinol sulfate reported here may be interpreted as a reflection of abnormalities in oxidative metabolism of dopamine, metabolically derived acetaldehyde, and/or biological carbonyls in chronic alcoholics.

Treatment of tyramine-induced brain edema with anion transport inhibitor L-644,711.

Tyramine induces coma in phenelzine-treated dogs. Development of coma in these animals is associated with brain edema, abnormal brain scans of Tc-99m-diethylene-triamine-penta-acetic acid (Tc-99m-DTPA), and elevated levels of CSF catecholamines. We found that the intravenous administration of 6-7 mg/kg of a single dose of L-644,711 given fifteen minutes after the oral administration of tyramine to phenelzine-pretreated animals followed by an infusion of normal saline containing 6-7 mg/kg of the drug given over a period of 2 hr caused reversal of brain injury. This was accompanied by full recovery within a period of 24 hr of all the animals tested. A follow-up study revealed that 24 hr after treatment with L-644,711 CSF levels of catecholamines and brain images of Tc-99m-DTPA were indistinguishable from normal controls. Animals that received no drug died from unresolved coma within 4 to 24 hr. Animals that had recovered due to therapy with L-644,711 were given 10-14 days rest followed by a repetition of the phenelzine and tyramine treatment but denied L-644,711 therapy. These animals also died of unresolved coma within 24 hr. This preliminary study suggest that the use of L-644,711 may constitute an important advance in treatment of brain edema of a wide range of neurological disorders.

Pyridoxal 5'-phosphate levels in children with sickle cell disease.

Pyridoxal 5'-phosphate (PLP), the major coenzyme form of vitamin B6, is known to have antisickling properties in vitro. Recently, low plasma PLP levels were reported in a group of adults with sickle cell anemia. We measured the plasma PLP levels in a group of 55 asymptomatic nontransfused children with sickle cell diseases (SCD) to determine the prevalence of low plasma PLP levels in this population. Comparative studies were made with the measurement of PLP in three other groups serving as controls: Group A (black children, n = 36); Group B (white children, n = 37); and Group C (black adults, n = 13). PLP was measured directly in plasma by a radioenzymatic technique. The results of these comparisons showed that there was no statistically significant difference in plasma PLP of black children with SCD (10.7 +/- 10.0 ng/ml) as compared with black control children (group A, 9.0 +/- 12.3 ng/ml). The low plasma levels PLP in these two groups were significantly lower than that of the plasma PLP of white control children (group B, 15.85 +/- 15.92 ng/ml). This data suggest that a high prevalence of low PLP levels exists in black children seen at Grady Memorial Hospital, both with and without SCD.

Prevalence of low monoamine oxidase function in alcoholism.

Several studies have found a trend for low platelet monoamine oxidase activity (MAO) in alcoholism but with a great deal of overlap in MAO activity of alcoholics versus controls. The main objective of this study was to carry out a detailed assessment of MAO function that included the measurement of key kinetic parameters (i.e., Km, Vmax) in three groups of male subjects: (a) 51 hospitalized chronic alcoholics, (b) 16 recovering alcoholics with 2-10 years of abstinence, and (c) 21 controls. MAO activity was assayed radiochemically with [14C]tyramine as substrate (43-729 microM). The present study demonstrated that alcoholics had low platelet MAO activity (p less than 0.05). Kinetic analysis revealed a substantial reduction (p less than 0.01) in enzyme Vmax values of chronic and recovering alcoholics. Greater than 95% of the alcoholics had Vmax values lower than the smallest value of control subjects. Moreover, 100% of the alcoholics in both groups exhibited exceedingly low Vmax values that were below the 25th percentile of controls. In summary, results of MAO Vmax determinations provided us with a better separation of the alcoholics from controls. Measurements of platelet MAO function that include enzyme Vmax may provide us a reliable biochemical marker for alcoholism.