Excretion of β-Carbolines Harman and Norharman in 24-Hour Urine of Chronic Alcoholics During Withdrawal and Controlled Abstinence

Animal experiments suggest that endogenous substances that could result from the interaction between neurotransmitters (dopamine and indoleamines) and ethanol and its metabolite acetaldehyde might be involved in the pathogenesis and maintenance of alcohol dependence. Therefore, aromatic β-carbolines (norharman and harman) were investigated repeatedly in 24-hr urine of 13 male severe alcoholics without any psychiatric comorbidity during a controlled inpatient abstention program of up to 8 weeks. Harman excretion was ˜2-fold above levels in control subjects, with a steady decline after 3 weeks of abstinence and lower levels in patients with a longer duration of alcohol dependence. Severity of withdrawal symptoms and actual feelings of anxiety/depression were negatively associated with urinary harman excretion. Positive associations could be established with daily ethanol consumption the month before admission and the score on the scale "reward dependence" according to Cloninger's Tridimensional Personality Questionnaire. Moreover, patients without alcohol-dependent first-degree relatives and higher "reward dependence" exhibited an increased excretion of harman. Therefore, harman levels might characterize a distinct subgroup of alcoholic patients, who in part resemble the so-called type I alcoholics of Cloninger. However, this awaits further study in a larger number of individuals. In contrast, norharman excretion was elevated up to 6-fold, compared with nonalcoholics over 6 to 8 weeks of controlled abstention. No correlations to demographic or clinical variables could be observed. Therefore, increased norharman levels might be proposed as a "residual marker" or a trait variable. Whether the observed changes are specific markers of at least certain aspects of alcoholism or dependence remain to be elucidated.     

β-Carbolines in Chronic Alcoholics Undergoing Elective Tumor Resection

The prevalence of chronic alcoholism in patients with carcinomas of the upper digestive tract exceeds 60%. The patient's history and laboratory markers, preoperatively, are often not sensitive or specific enough to detect alcohol-dependent patients, preoperatively, who are at risk of developing alcohol withdrawal syndrome (AWS) during their postoperative intensive care unit (ICU) stay. Previously, it was found that plasma norharman was elevated in chronic alcoholics, suggesting marker characteristics for chronic ethanol misuse and possibly alcohol dependence. We investigated whether β-carbolines (i.e., harman and norharman) were different between chronic alcoholics and nonalcoholics with carcinoma, and how the levels change in alcohol-dependent patients during their hospital stay. Ninety-seven patients with oral, pharyngeal, laryngeal, or esophageal carcinomas were evaluated regarding their drinking habits. Sixty patients were transferred to the ICU following tumor resection. Chronic alcoholics met the DSM-III-R and ICD-10 criteria for alcohol dependence or chronic alcohol abuse/harmful use. The daily ethanol intake in chronic alcoholics was ≥60 g. Blood samples were collected on admission to the hospital, preoperatively, on admission to the ICU and on days 2, 4, and 7 in the ICU. Harman and norharman were determined by HPLC. Elevated norharman was found in chronic alcoholics on admission to the hospital, whereas harman did not differ between groups. On admission, the area under the receiver operating characteristics curve was significantly larger for carbohydrate-deficient transferrin and preoperatively for norharman. The preoperative norharman levels were significantly correlated with the period of mechanical ventilation and the length of ICU stay. Postoperatively, norharman decreased in all patients, except a group of 11 alcohol-dependent patients who developed AWS during their ICU stay. The finding that elevated norharman levels were found in chronic alcoholics on admission to the hospital and preoperatively supports the view of a specific marker for alcoholism. Preoperative norharman was superior to carbohydrate-deficient transferrin and was associated with a prolonged ICU stay and a prolonged period of mechanical ventilation. Further studies are required to determine whether norhaman aids in the preoperative diagnosis of chronic alcohol misuse with respect to the prevention of postoperative complications.     

Increases and Time-Course Variations in β-Hexosaminidase Isoenzyme B and Carbohydrate-Deficient Transferrin in Serum from Alcoholics Are Similar

β -Hexosaminidase B-isoforms ( β -hexosaminidase B, P, and intermediate forms; abbreviated herein as "Hex B") and serum carbohydrate-deficient transferrin (CDT) are two markers of alcohol abuse. In the present study, we have compared "Hex B" with CDT as markers of alcohol abuse in a group of alcoholics hospitalized for detoxification after a period of heavy alcohol abuse. We have also followed the disappearance rate of these two markers from circulation. "Hex B" was elevated in 38 of 42 patients hospitalized for detoxication, whereas CDT was elevated in 35 of 42 patients. A highly significant correlation was noted between "Hex B" and CDT in these patients ( p = 0.52, p < 0.001). Neither "Hex B" nor CDT correlated with γ-glutamyltransferase or AST. The disappearance rates from serum of "Hex B" and CDT were determined in 21 hospitalized patients followed for up to 15 days. "Hex B" and CDT showed similar time-course variation and half-lives, 6.5 ± 3.7 (mean × sd ) and 8.6 ± 4.1 days, respectively. The possible reasons for a relation between these two markers are discussed, and it is concluded that more experience of both "Hex B" and CDT in unselected populations is needed to establish the diagnostic potential of these tests as markers of alcohol abuse.     

Elevated Plasma Homocysteine in Alcoholics

A significantly higher concentration of plasma homocysteine compared with controls was noted in a group of alcoholics ( n = 42) hospitalized for detoxication. Normal concentrations of plasma homocysteine were reached within 1 or 2 weeks after admission to the hospital. In another group of abstinent alcoholics ( n = 16) plasma homocysteine did not deviate from that of controls. Since hyperhomocysteinemia has been associated with premature vascular disease, we speculate that the increased plasma homocysteine in alcoholics might cause the increased incidence of stroke found in these patients.     

Hormonal (ACTH, Cortisol, β-Endorphin, and Met-Enkephalin) and Cardiovascular Responses to Hyperthermic Stress in Chronic Alcoholics

Chronic alcohol drinking causes profound alterations in hypothalamic-pituitary function. In the present study, endocrine [corticotropin (ACTH), β-endorphin, cortisol, and met-enkephalin] and cardiovascular (blood pressure) changes in response to hyperthermic stress (sauna at 90°C for 30 min) were evaluated in 25 normal men (25 to 50 years old) and in 48 male alcoholic subjects (34 to 56 years old) after 5 weeks of abstinence. Significantly lower increments in systolic blood pressure were observed in alcoholics than in control subjects. Furthermore, alcoholics showed lower ACTH, β-endorphin, and cortisol increments in response to sauna than normal controls. In contrast, sauna-induced hyperthermia did not change significantly the circulating met-enkephalin levels in either normal controls or chronic alcoholics. These data suggest that an impairment in the adaptive response to stress affects alcoholic men even after a few weeks of abstinence from alcohol.     

Serum β-Hexosaminidase Isoenzyme: A Sensitive Marker for Alcohol Abuse

Serum β-hexosaminidase (Hex) isoenzymes analyzed by enzyme immunoassay were investigated in a group of alcoholics (n = 38) hospitalized for detoxication, in another group of alcoholics (n = 22) abstinent between 6 days and 10 years and in a reference group (n = 20). Hex 'B' isoenzyme was elevated in all 38 patients hospitalized for detoxication but only 35 of these had total Hex values above the upper limit of the reference group. The Hex A isoenzyme, γglutamyltransferase, and aspartate aminotransferase showed considerable overlap between these patients and the reference group. In the group of 22 abstinent patients only one had an increased level of Hex A, Hex 'B,' and total Hex, whereas 10 had γ-glutamyltransferase values above the upper limit of the reference group. It is concluded that Hex 'B' is a useful marker for alcohol abuse.     

Homozygous β-thalassaemia resulting in the β-thalassaemia carrier state phenotype

Summary. This paper describes the phenotypic manifestations of a very mild β-thalassaemia mutation detected in several members of two families of Italian descent. The molecular defect, defined by denaturing gradient gel electrophoresis analysis and direct sequencing. consists of a C G substitution at position 844 of IVSII of the β-globin gene within the consensus sequence of IVSII acceptor splice site. Heterozygotes for this mutation show a haematological phenotype ranging in severity from silent β-thalassaemia to that of a mild β-thalassaemia carrier silent β-thalassaemia to that of a mild β-thalassaemia carrier state, whereas homozygotes have the typical manifestations commonly resulting from heterozygosity for a β-thalassaemia mutation. Compound heterozygotes for the IVSII nt844 (C G) mutation and a severe β-thalassaemia mutation have the phenotype of thalassaemia intermedia.
This paper indicates that the presence of borderline red blood cell indices or HbA₂ values should make one suspect the presence of a very mild or silent β-thalassaemia.     

Stimulation of thrombocytopoiesis decreases platelet β2 but not β1 or β3 integrins

The expression of CD29, CD61, CD18 and CD11a on platelets was examined by flow cytometry in mice treated with leukaemia inhibitory factor (LIF) or megakaryocyte growth and development factor (PEG-rHuMGDF or mpl-ligand). Treatment for 7–14 d with PEG-rHuMGDF or LIF increased the number of platelets in peripheral blood from 0.9 up to <2.0×10⁶/μl. These treatments decreased the expression of CD11a and CD18, whereas that of CD29 or CD61 was not markedly changed. Study after various doses or times of PEG-rHuMGDF administration indicated that a decrease of CD18 expression occurred when platelet counts started to rise. Platelet RNA content was increased in mice treated with PEG-rHuMGDF but double staining indicated that expression of CD18 was not correlated with RNA content. To evaluate integrin expression as a function of time in circulation, platelets were biotinylated in vivo . In normal or PEG-rHuMGDF-treated mice, the expression of CD29 or CD61 did not change, whereas that of CD18 decreased significantly as a function of time in circulation. These findings indicate, firstly, that stimulation of thrombocytopoiesis leads to the release of platelets with a low content of β2 integrin and, secondly, that this integrin is also selectively lost while in the circulation.     

Effect of Short Photoperiod on Hypothalamic Methionine-Enkephalin and LHRH Content and Serum β-Endorphin-Like Immunoreactivity (β-end LI) Levels in Golden Hamsters

Adult female golden hamsters were used to study the effect of short photoperiod on the endogenous opioid system and the effect of pinealectomy on the serum beta-endorphin-like immunoreactivity (beta-end LI) levels. Hamsters were housed under either long photoperiod (14L:10D) or short photoperiod (2L:22D) and the regularity of the estrous cycles was determined by daily vaginal exfoliative cytology. Hamsters under short photoperiod became acyclic after about 7 wk. At the end of 8 wk, all the hamsters were decapitated and medial basal hypothalamic (MBH) content of LHRH and methionine-enkephalin (met-enkephalin) were measured by specific radioimmunoassays (RIA). Both LHRH and met-enkephalin levels of the MBH were significantly elevated in the short-photoperiod hamsters as compared to the normally cycling control animals under long photoperiod. In a second experiment, the effect of pinealectomy (PNX) on the serum levels of beta-end LI in the short-photoperiod hamsters was determined. The serum beta-end LI levels were increased approximately threefold in the noncyclic hamsters housed under 8 wk of short-photoperiod conditions. Pinealectomized hamsters kept under 8 wk of short-photoperiod exhibited lower serum beta-end LI levels similar to those of normally cycling hamsters kept under long photoperiod. These results indicate a possible functional relationship between increased pineal activity (as a result of short photoperiod) and increased MBH met-enkephalin, LHRH, and serum beta-end LI levels.     

Hydroxyurea therapy in β-thalassaemia intermedia: improvement in haematological parameters due to enhanced β-globin synthesis

Summary. The β-thalassaemias represent a heterogenous group of diseases resulting from decreased erythroid β-globin mRNA expression and imbalanced a/β-globin chain synthesis which are manifest clinically by ineffective erythropoiesis and excessive haemolysis. Increasing levels of haemoglobin F (HbF) by pharmacological agents has been proposed to ameliorate the severity of the disease by improving the balance in globin chain synthesis. Hydroxyurea (HU), as an effective agent with low toxicity for activating 7-globin gene, has been shown to enhance HbF synthesis in experimental animals and in patients with sickle cell anaemia. However, previous trials of HU in β-thalassaemia patients are ambiguous, with a small number having increased HbF synthesis. In a recent study of HU effects in Chinese j3 -thalassaemia patients we unexpectedly found that two unrelated patients with β-thalassaemia intermedia demonstrated an improvement in the effectiveness of erythropoiesis reflected by an increase in haemoglobin concentration (from 4-1 to 6-3 g/dl, patient 1; from 6-5 to 97 g/dl, patient 2) and in red cell volume (from 68 to 104 fl, patient 1; from 68 to 85 fl, patient 2) after a period of excess of 300 d of low-dosage HU treatment. These effects, however, appear to be due to increased,3-globin biosynthesis, because the percentage of HbF decreased in each patient as total Hb increased. This was reflected by changes in the β/a ratio (from 0'301 to 0-581, patient 1; from 0'348 to 0-487, patient 2) with minimal changes in 7-globin biosynthesis. We conclude that in addition to its known effects in stimulating 7-globin production, hydroxyurea may have a more general role in augmenting globin synthesis, including β-globin in some thalassaemia intermedia patients who maintain the capacity to express normal β-globin chains.     

Harman and Norharman in Alcoholism: Correlations with Psychopathology and Long-Term Changes

In the search for mechanisms specific for alcoholism, it has become evident that β-carbolines (BCs; e.g., harman and norharman) are compounds that may act on brain reward systems, thereby mediating an increase in voluntary ethanol (ETOH) drinking in animals. This study was undertaken to analyze relationships between these compounds and clinical variables (e.g., family history, personality data, and affect) in alcoholics and to trace the time course of blood concentrations in subjects abstaining from alcohol for at least 6 months. Nonalcoholics were investigated during sober and ETOH-loading conditions (1 g ETOH/kg body weight). Levels of harman were elevated in the chronically intoxicated alcoholics and correlated with the scores on the self-rating depression (SDS) and the self-rating anxiety (SAS) scales. The group of alcoholics with at least one alcoholic parent had higher levels than the group without such a history. Levels remained elevated for 6 months. Norharman levels were only slightly elevated on the day of admission. They were correlated to high harm avoidance and SDS scores. A family history of alcoholism and the severity of alcoholism as assessed by the number of ICD-10 criteria fulfilled were correlated with norharman levels. Long-term observation revealed elevated levels of norharman after 3 months of abstinence, but not after 6 months. The association of harman levels with anxiety and depression demonstrated in the present study suggests that alcoholics with high harman levels use alcoholic beverages as self-medication in an attempt to overcome possible anxiogenic/depressiogenic actions of harman. Norharman levels are less strongly associated with these mood states, but significantly correlated to harm avoidance tendencies. It has been suggested that the activity of the indolergic neurons is relatively high in individuals with a high harm avoidance score. Biosynthesis of norharman might be stimulated under these conditions (tryptamine serves as precursor).     

A novel β-thalassaemia mutation in the 5'untranslated region of the β-globin gene

Summary. A thymidine deletion at position +10 of the 5'untranslated region of the β-globin gene was detected in a β-thalassaemia intermedia patient carrying a β; 39 stop codon mutation on the other chromosome; this new mutation, +10(-T), was detected by automated fluorescent DNA sequencing and verified by dot-blot allele-specific hybridizations.
The +10(-T) mutation is a 'silent carrier', is associated with a reduced amount of steady-state β-globin mRNA, and establishes a connection between the 5'untranslated region of the β-globin gene and the regulation of its expression.     

α-θ Brainwave Training and β-Endorphin Levels in Alcoholics

An alpha-theta brainwave biofeedfack training program was applied as a novel treatment technique for chronic alcoholics. Following a temperature biofeedback pretraining phase, experimental subjects completed 15 30-min sessions of alpha-theta biofeedback training. Compared to a nonalcoholic control group and a traditionally treated alcoholic control group, alcoholics receiving brainwave training (BWT) showed significant increases in percentages of EEG record in alpha and theta rhythms, and increased alpha rhythm amplitudes. Alcoholics receiving BWT showed a gradual increase in alpha and theta brain rhythms across the 15 experimental sessions. These experimentally treated alcoholics showed sharp reductions in self-assessed depression (Beck's Depression Inventory) compared to the control groups. Alcoholics receiving standard medical treatment (abstinence, group psychotherapy, antidepressants) showed a significant elevation in serum beta-endorphin levels at the conclusion of the experiment. This neuropeptide is an index of stress and a stimulant of caloric (e.g., ethanol) intake. Application of brainwave treatment, a relaxation therapy, appears to counteract the increase in circulating beta-endorphin levels seen in the control group of alcoholics. 13-month follow-up data indicate sustained prevention of relapse in alcoholics that completed alpha-theta brainwave training.     

A novel amber mutation in a β°-thalassaemia gene (β37TGG→TAG), with direct detection by mapping the restriction fragments in amplified genomic DNA

Summary. A novel amber mutation, a G to A substitution at the second position of codon 3 7 in the β-globin gene that changes the tryptophan coding triplet (TGG) to a termination codon (TAG), was found in a Chinese β-thalassaemia carrier. The mutant gene creates an additional Dde I recognition site and eliminates the Ava II site, so this point mutation can be directly identified by restriction enzyme analysis.     

Serum β-hexosaminidase and α-mannosidase Activities as Markers of Alcohol Abuse

In 25-alcohol-dependent patients in whom detoxication treatment has been introduced serum total beta-hexosaminidase, thermostable beta-hexosaminidase, alpha-mannosidase and gamma glutamylotransferase (GGT) and serum high density lipoprotein (HDL) cholesterol were determined during alcohol withdrawal. The assays were also performed in a group of dependent individuals after a 1 month or longer period of abstinence. Marked increase in beta-hexosaminidase activity was observed in intoxicated patients. The activity decreased rapidly after the cessation of drinking, resembling the decrease in HDL cholesterol level in its dynamics. The alpha-mannosidase activity rise was less pronounced and its normalization was slow, similar to the GGT activity normalization rate. The rise of beta-hexosaminidase activity was mostly due to the thermostable component of the enzyme. Total beta-hexosaminidase or thermostable beta-hexosaminidase activity determinations appear to be simple and reliable markers of alcohol abuse.     

Severe inclusion body β-thalassaemia with haemolysis in a patient double heterozygous for β°-thalassaemia and quadruplicated α-globin gene arrangement of the anti-4.2 type

We describe a new case of an association of alpha-globin gene quadruplication of the anti-4.2 type with beta(0)-thalassaemia. The patient, a young woman of mixed Brazilian-Portuguese origin, suffered from chronic haemolytic anaemia with splenomegaly. Bone marrow supravital staining with brilliant cresyl blue and electron microscopy studies showed large inclusion bodies in about 3% of erythroblasts. Upon immunofluorescent staining these inclusions reacted with a monoclonal antibody to alpha- but not to beta-globin. Analysis of alpha-globin cluster by Southern blotting showed the presence of pathologic fragments specific for the anti-4.2 alpha-globin gene quadruplication. Alpha/beta mRNA ratio was higher than in cases combining alpha-globin triplication and beta(0)-thalassaemia or in cases of beta(0)-thalassaemia heterozygous state alone (18, 14.7 and 10.1 respectively). Our data confirmed the hypothesis that the clinically detectable haemolysis in this beta(0)-thalassaemic patient was due to an unusually high amount of precipitated alpha-globin in erythroid precursors. This considerable excess of alpha-globin chains was due partly to the beta-globin deficit caused by the presence of the beta(0)-thalassaemic gene, but also to the presence of 6 active alpha-globin genes resulting from alpha-globin gene quadruplication in one chromosome.     

Diagnosis of thalassaemia by non-isotope detection of α/β and ζ/α mRNA ratios

Summary. The α/β and ζ/α messenger RNA (mRNA) ratios in the thalassaemia syndromes were investigated by polymerase chain reaction (PCR) with silver staining of the PCR products. In this study we used the PCR to amplify cDNA copies of circulating erythroid cell mRNA in order to measure the relative amounts of α-, β- and ζ-globin contained within. Quantitation was performed by scanning the silver stain of specific globin cDNA bands. We found that there were significant differences of α/β-mRNA and ζ/α-mRNA in patients with Hb H disease and α-thalassaemia-1 compared to normal subjects. There was a marked increase in the α/β-mRNA ratio but not in the ζ/α-mRNA ratio in patients with β-thalassaemia. In two β-thalassaemia cases abnormal increases of ζ-globin bands were noted and they were confirmed through DNA analysis to be combined with α-thalassaemia-1. This method provides a simple, rapid and non-radioactive approach to detect thalassaemia syndromes, and can help to screen cases of β-thalassaemia with α-thalassaemia-1.