Platelet Serotonin Concentration and Clinical Status in Alcohol Withdrawal Syndrome , Preliminary Results

INTRODUCTION Alcohol withdrawal syndrome is an important problem for management of alcoholism. It is known that alcohol alters the function of neurotransmitters such as serotonin, but our review found no studies associating serotonin concentration changes with patient clinical status during detoxifi cation. The authors hypothesized that total platelet serotonin concentration should signifi cantly increase during alcohol detoxifi cation. OBJECTIVE Assess possible association between total (endogenous and captured) platelet serotonin concentration and clinical status of patients with alcohol withdrawal syndrome, at beginning and end of detoxifi cation with clomethiazole. METHODS Thirty-one alcohol-dependent patients, diagnosed with alcohol withdrawal syndrome according to DSM-IV and classifi ed in three clinical groups (18–20, 21–22 and 23–26 points) per the Clinical Institute Withdrawal Assessment for Alcohol Scale, were included in a prospective case series from May 2009 through May 2011 at the Hermanos Ameijeiras Clinical-Surgical Teaching Hospital in Cuba. Patients were predominantly male (87%), ranging in age from 22 to 57 years; 40% were white, 40% mestizo and 20% black. All had been hospitalized for detoxifi cation in the hospital’s psychiatry service. Blood samples taken were mixed with 1% EDTA and centrifuged for isolating platelets. Serotonin concentrations (endogenous and captured) were measured within 2 hours of blood collection and mean values at treatment days 1 and 12 compared by t test (p <0.05). RESULTS Total serotonin concentration mean values (μg of serotonin per mg of total platelet protein) were statistically different between days 1 and 12 of treatment (1.329±0.916 μg/mg vs. 2.573±1.224 μg/ mg; p <0.001). There was a direct association between total serotonin concentration and patient clinical classifi cation both initially and at day 12. At day 1, the 29 patients in the group with 18–20 points had mean serotonin of 1.358±0.0.94 μg/mg; one patient with 21 points had serotonin of 1.25 μg/mg; and one patient with 24 points had serotonin of 0.740 μg/mg. At day 12, 26 patients had 0–1 points, with mean serotonin 2.688±1.244 μg/mg; and 5 patients had 2–8 points, with mean serotonin 1.244±0.596 μg/mg. No patient had >8 points at day 12. CONCLUSIONS Serotonin is a potential biomarker for initial clinical classifi cation and outcome monitoring and could be useful to psychiatrists working with patients in this area of medical practice and research. Further studies including more patients and variables are necessary to support these preliminary results.


INTRODUCTION
Alcoholism has a great importance in psychiatry due to its increasing social impact.A primary chronic disease that is progressive and often fatal its manifestations are infl uenced by genetic, psychosocial, and environmental factors.It is characterized by impaired control over drinking, preoccupation with alcohol and an uncontrollable urge to drink despite adverse consequences.[1][2][3] Reliable quantitative measures of alcohol intake and outcomes of alcoholic patients are needed to reduce the subjectivity of nonquantitative methods of classifying clinical status and monitoring response to treatment.[4] Serotonin's role in alcoholism and alcoholism withdrawal syndrome (AWS) may indicate potential usefulness as a biomarker of patient classifi cation and progress.
Functions of some neurotransmitters such as serotonin (also known as 5-hydroxitriptamine or 5-HT), gamma-aminobutyric acid (GABA) and dopamine can be drastically modifi ed under the effect of alcohol.The role of serotonin in alcoholism treatment has been studied for decades.[5,6] Alcoholics, who consume relatively larger quantities of alcohol, usually have lower brain serotonin than nonalcoholics.Chronic administration of ethanol reduces GABAergic neurotransmission by decreasing allosteric modulation of GABA receptors.[7,8] It has been hypothesized that genetically determined low serotonin levels may be related to a predisposition to alcoholism.[9] On the other hand, low serotonin has also been implicated in depression, obsessive-compulsive disorders, anxiety, impulsiveness, aggression and suicide, all of which may coexist with alcoholism.[10] Serotonin may also contribute to alcohol intoxication and abnormalities in the brain's serotonin system, where it appears to play an important role in brain processes underlying alcohol abuse and dependence.[11] Moreover, acute and long term alcohol ingestion also affects serotonin receptors that convert the chemical signal produced by serotonin into functional changes in signal-receiving cells.[12] Decreased serotonin in cerebrospinal fl uid, serum, plasma, urine and platelets has also been observed in alcohol withdrawal syndrome (AWS), [5] a phenomenon of major clinical importance.[13,14] Physical and psychological symptoms of AWS range from mild to severe and include nervousness, shakiness, anxiety, irritability or easy excitability, sudden emotional changes, headache, fatigue, nightmares, fuzzy thinking, fever, convulsion, blackouts, delirium tremens, involuntary eyelid movements, hand tremor, nausea, clammy or discolored skin, dilated pupils and rapid heart rate.[15,16] Several drugs can be used to treat and prevent AWS symptoms; clomethiazole (also called chlormethiazole), originally developed in the 1930s, is still one of the most widely used.It is structurally related to thiamine (vitamin B1) and has sedative, hypnotic, muscle relaxant and anticonvulsant effects.It is also used for management of agitation, restlessness, short-term insomnia, and Parkinson disease in the elderly.[17,18] Clomethiazole acts as a positive allosteric modulator at the barbiturate/picrotoxin site of the GABA-A receptor.GABA is the major inhibitory neurotransmitter in the brain, and has anxiolytic, anticonvulsant, sedative and hypnotic effects.

ABSTRACT
INTRODUCTION Alcohol withdrawal syndrome is an important problem for management of alcoholism.It is known that alcohol alters the function of neurotransmitters such as serotonin, but our review found no studies associating serotonin concentration changes with patient clinical status during detoxifi cation.The authors hypothesized that total platelet serotonin concentration should signifi cantly increase during alcohol detoxifi cation.OBJECTIVE Assess possible association between total (endogenous and captured) platelet serotonin concentration and clinical status of patients with alcohol withdrawal syndrome, at beginning and end of detoxifi cation with clomethiazole.
METHODS Thirty-one alcohol-dependent patients, diagnosed with alcohol withdrawal syndrome according to DSM-IV and classifi ed in three clinical groups (18-20, 21-22 and 23-26 points) per the Clinical Institute Withdrawal Assessment for Alcohol Scale, were included in a prospective case series from May 2009 through May 2011 at the Hermanos Ameijeiras Clinical-Surgical Teaching Hospital in Cuba.Patients were predominantly male (87%), ranging in age from 22 to 57 years; 40% were white, 40% mestizo and 20% black.All had been hospitalized for detoxifi cation in the hospital's psychiatry service.Blood samples taken were mixed with 1% EDTA and centrifuged for isolating platelets.Serotonin concentrations (endogenous and captured) were measured within 2 hours of blood collection and mean values at treatment days 1 and 12 compared by t test (p <0.05).

Original Research
Clomethiazole's hypothermic and neuroprotective effects appear to be related to another mechanism; it regulates serotonergic transmission through its modulatory action on GABA.[19,20] Chemical transmission regulates approximately one third of brain impulses, including in the adrenergic and serotonergic systems involved in the neurobiological basis of anxiety disorders.In fact, it has been proposed that the anxiolytic action of benzodiazepines may result from inhibition of serotonin release in limbic structures.[21] However, in our review of research over the past 30 years, we encountered no studies of AWS patients associating changes in serotonin concentration with clinical classifi cation and outcomes; hence our interest in pursuing this line of research.
Platelets have been proposed as a peripheral source of serotonin because they share similarities with synaptosome in mechanisms of serotonin capture, storage and release; and also because of the presence of serotonin 2A receptors in the cell membrane.The platelet serotonergic system has four main components: (i) the capture mechanism, (ii) intracellular storage organelles, (iii) serotonergic receptors in cytoplasmic membranes, and (iv) a mitochondrial enzyme for its metabolism.All these elements have physiological and pathophysiological similarities with the neuronal serotonergic system.In addition, serotonin capture by platelets occurs both actively and by passive diffusion.The active mechanism is mediated by a transporter protein similar to that of the neuronal active mechanism.There is also evidence that the serotonin transporter protein is encoded by the same gene in platelets and in the brain; and that the platelet carrier has the same functional and pharmacological characteristics as the neuronal transporter.[22][23][24][25][26][27][28] All of this makes platelets an appropriate model for studying neuronal serotonin.
Our research objective was to assess the possible association between serotonin concentration and both initial clinical classifi cation and eventual outcome in patients treated for AWS with clomethiazole, by measuring total (endogenous and captured) platelet serotonin concentration.

METHODS
The Hermanos Ameijeiras Clinical-Surgical Teaching Hospital is a tertiary care hospital for adults located in Havana, Cuba; it has 625 beds, 15 medical and 12 surgical units, and 3 intensive care units.

Study type and population
This preliminary prospective case series was carried out from May 2009 through May 2011.Alcohol-dependent patients included in the study were hospitalized in the psychiatry service (alcoholism consultation), with a history of heavy and prolonged alcohol consumption and recent interruption or reduction of alcohol.Thirty-one were admitted for a 15-day detoxifi cation program, and the study was conducted during the fi rst 12 days.Patients ranged in age from 22 to 57 years; 27 were male (87%) and 4 female (13%); 40% were white, 40% mestizo and 20% black.
Diagnosis Alcoholism was diagnosed per DSM-IV, [29] using the Schedules for Clinical Assessment in Neuropsychiatry.[30] The Clinical Institute Withdrawal Assessment for Alcohol Scale (CIWA-Ar) was administered by one of the authors (AJC) to diagnose AWS.[31] The interview covers signs and symptoms such as sweating, tachycardia, hand tremor, insomnia, nausea and/or vomiting, hallucinations, psychomotor agitation, anxiety and generalized tonic-clonic seizures.The CIWA-Ar range of possible scores is 0-30, with higher scores indicating greater severity of withdrawal symptoms.

Patient inclusion criteria
Age ranging 20 to 60 years old, signs and symptoms of AWS with CIWA-Ar score ≥18 points.[31] Patient exclusion criteria Use of other drugs that produce symptoms of anxiety during the month prior to the study; medical conditions (especially renal) that contraindicate treatment with clomethiazole; history of psychiatric illness (major depression, schizophrenia, epilepsy, bipolar disorder); moderate or profound mental retardation; and pregnancy or lactation.

Study variables
These included platelet endogenous, captured and total serotonin concentration in micrograms per milligram total platelet protein; and AWS status per CIWA-Ar.

Laboratory procedures
Early-morning blood samples were taken from fasting patients on days 1 (prior to fi rst dose of chlomethiazole) and 12 of the treatment period; 10 mL of venous blood was drawn and transferred to a plastic tube containing 1 mL of 1% EDTA, then centrifuged at 800 g for 30 min to obtain platelet-rich plasma (PRP).PRP is stable at 4°C for several hours, but all tests were conducted within 2 hours of venipuncture.

Platelet protein
The Lowry method was used to estimate total platelet protein.[32] Endogenous serotonin One milliliter of PRP was centrifuged at 800 g for 20 min; then plasma was discarded and the pellet washed twice with physiological saline (0.9% NaCl).Platelets were resuspended in distilled water with 0.5 mL of perchloric acid (0.4 mol/L) and shaken to promote platelet lysis.This material was then centrifuged again at 1500 g for 10 min, and 2 mL of supernatant used to determine serotonin concentration with a reverse-phase high performance liquid chromatography (RP-HPLC) C-18 column (Merck, Germany) with electrochemical detection (Knauer, Germany).Sample volume was 25 μL and the column was operated at 1 mL/min; 0.1 mol/L phosphate buffer was used as mobile phase; MW 387 (SIGMA-Aldrich, USA) was used as serotonin reference material.
Captured serotonin Serotonin capture by platelets was determined by calculating increase in intraplatelet serotonin content after incubation in a serotonin-rich medium.One milliliter of PRP was incubated for 10 min at 37°C, and then again for 5 min with the addition of 1 mg of serotonin.Incubation was stopped by adding 3 mL of cold 1% EDTA diluted in physiological saline.The tubes were centrifuged at 800 g for 15 min to obtain the platelet pellet and then 2 mL of supernatant were used to determine serotonin concentration, also using an RP-HPLC C18 column with electrochemical detection.The serotonin reference material used was the same for all measurements.
Total serotonin Total serotonin concentration estimation was done by summing platelet endogenous and captured serotonin concentrations.

Ethics
The Hermanos Ameijeiras Clinical-Surgical Teaching Hospital research ethics committee approved the study design.Written informed consent to participate was obtained from patients, or from next of kin or legal guardians.

RESULTS
Table 1 shows mean and individual serotonin concentrations for patients with AWS on days 1 and 12 of detoxifi cation.Total serotonin concentration is illustrated in Figure 1.Higher serotonin values were associated with lower scores, that is, better clinical status (Figures 1 and 2).At the beginning, total serotonin concentration values were as follows: for the group with 18-20 points (29 patients),  1).
Finally, Table 1 and Figure 2 show values of patient clinical status assessment per CIWA-Ar.Mean scores decreased after treatment, from 18.74±1.33points at day 1 to 0.77±2.29 points at day 12.This decrease coincided with a statistically signifi cant increase in mean endogenous, captured and total serotonin concentrations from day

DISCUSSION
Knowing that alcohol alters serotonin structure and function, [5] we hypothesized that serotonin concentration might be a potential biological marker for initial classifi cation of clinical status and for assessing progress in AWS patients treated with clomethiazole.In fact, we did fi nd a direct association between clinical status and total serotonin concentration in most patients studied.
Other researchers have also reported changes in serotonin concentration in patients during the withdrawal period, but these studies found similar serotonin concentrations in control groups and, in contrast to our fi ndings, serotonin levels declined and remained lower than controls' even after 14 days of abstinence.[7][8][9] However, those patients were receiving benzodiazepines, which may suppress serotonin activity.[10] In addition, the day of admission may not have been the actual beginning of withdrawal for all patients.Finally, all subjects were Caucasian men, limiting generalizability.None of our patients received benzodiazepines and admission day corresponded with the beginning of the withdrawal period, confi rmed by daily application of the patient evaluation scale.We intend to publish a further study using benzodiazepines.
It should be noted that there is no evidence suggesting a direct effect of clomethiazole on serotonin-its effi cacy in AWS is related to the GABA-A mechanism previously described-so it cannot be assumed that changes in serotonin concentration observed in this study are due to the drug.

Original Research
Interestingly, in the four patients whose serotonin concentrations decreased over the study period, this was due to a fall in captured serotonin concentration.Among these, patient 14's endogenous serotonin also failed to increase.Further study is needed to understand the paradox of decreased captured serotonin in the presence of palpable clinical improvement.All four of these patients were within the clinical subgroup of 0-1 points on day 12, that is, with virtually no clinical signs of AWS; interestingly, they reported depressive episodes during the detoxifi cation period, which could explain the lack of serotonin increase.Other authors have demonstrated that decreased brain serotonin is the core pathogenic factor in depression (a heterogeneous disease with diverse pathogenesis), pursuant to clinical observations that drugs enhancing extracellular or captured levels of serotonin have antidepressant effects in many patients.[23] Better AWS clinical status (assessed by two independent psychiatrists) was positively associated with total serotonin concentration on the fi rst day of detoxifi cation.From this preliminary evidence, we conclude that total serotonin concentration might be used as a biomarker to validate clinical classifi cation at initiation of AWS treatment.Furthermore, the positive association between total serotonin concentration and clinical improvement following detoxifi cation suggests that it could also be used to monitor clinical status during and after detoxifi cation.
Nevertheless, the apparent contradictory results for patient 11 cannot be ignored: this patient showed a signifi cant increase in total serotonin concentration at the end of detoxifi cation but not total recovery from AWS symptoms.In this case, endogenous serotonin concentration was unmodifi ed.Although conclusions cannot be drawn from results of a single patient, this might suggest a stronger specifi c role for endogenous serotonin.
As expected, all patients experienced AWS symptoms upon alcohol cessation and improved by study end.Another result, that of a patient who improved substantially but still had several symptoms at the end of the study, warrants further study.Differing alcohol use patterns, genetically determined differences in the nervous system, sex, age, or possible depression during detoxifi cation could also contribute to this variability in treatment response.
Trevisan reported that about 10% of AWS patients have serious symptoms (including delirium tremens, which has a mortality rate of 5-25%).[33] Ruusa concluded that testosterone levels infl uence AWS symptoms, observing that patients with low testosterone levels develop more neurotic-asthenic symptoms during alcohol withdrawal; that high levels of sex hormones are related to a history of seizures; and that younger alcoholics score higher on the paranoid-aggressive subscale of the comprehensive psychopathological rating scale.[34] This study must be considered preliminary because of its small sample size and lack of a control group; in addition, a number of potentially important variables could not be assessed.Accumulating a sample size adequate to take into consideration varying symptomatology, alcohol use history, genetic predisposition, sex, diet, medication use and clinical status at baseline is a serious challenge for this sort of research, and probably can only be done in a multicenter study.

CONCLUSIONS
This study provides preliminary evidence of a positive association between increased total platelet serotonin and clinical status in AWS patients treated with clomethiazole, making serotonin a candidate biomarker for monitoring and studying AWS.If validated, it could also be used in studies of other psychiatric disorders, such as depression, psychosis and anxiety.Nevertheless, larger studies able to address more variables are needed to confi rm these results.