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ORIGINAL ARTICLES |
From the Departments of Psychiatry and Behavioral Sciences (R.B.W., J.C.B., G.L.C., K.M.G., M.J.H., I.C.S., E.C.S.), Anesthesiology (K.G., M.S.-S.), Pharmacology and Cancer Biology (C.M.K., S.M.S.), Pathology (J.G.L.), and Genetics (D.A.M., I.K.S.), Duke University Medical Center, Durham, North Carolina.
Address reprint requests to: Dr. Redford B. Williams, Box 3926, Duke University Medical Center, Durham, NC 27710. Email: redfordw{at}acpub.duke.edu
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ABSTRACT |
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METHODS: Lumbar puncture was performed on 54 healthy volunteers to obtain cerebrospinal fluid (CSF) for determination of 5-hydroxyindoleacetic acid (5HIAA) levels. Genotypes were determined with respect to a functional polymorphism of the serotonin transporter gene promoter region (5HTTLPR). Subjects then underwent mental stress testing.
RESULTS: Persons with one or two long (l) 5HTTLPR alleles had CSF levels of the major serotonin metabolite, 5HIAA, that were 50% higher than those of persons with the s/s 5HTTLPR genotype. Persons with one or two l alleles or higher CSF 5HIAA levels also exhibited greater blood pressure and heart rate responses to a mental stress protocol.
CONCLUSIONS: These findings suggest the 5HTTLPR polymorphism affects CNS serotonin function, and they are consistent with the general hypothesis that CNS serotonin function is involved in the regulation of potentially health-damaging biobehavioral characteristics. In particular, the l allele could contribute, through its association with increased cardiovascular reactivity to stress, to increased risk of cardiovascular disease.
Key Words: serotonin • brain • cardiovascular reactivity • stress • genes (serotonin transporter promoter polymorphism).
Abbreviations: ANOVA = analysis of variance; CNS = central nervous system; CSF = cerebrospinal fluid; CV = cardiovascular; HR = heart rate; MAP = mean arterial pressure; SES = socioeconomic; 5HIAA = 5-hydroxyindoleacetic acid; 5HTTLPR = polymorphism of the promoter region of the serotonin transporter gene.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Support for this hypothesis comes from a recent study in which low SES subjects showed blunted prolactin responses to fenfluramine challenge, indicative of reduced CNS serotonin function (12). To test this hypothesis further, we have been evaluating the relationship among psychosocial/biobehavioral risk characteristics and two indices of CNS serotonin function in human subjects. First, CSF 5HIAA levels provide an indirect measure of serotonin turnover, especially reflecting activity in the frontal cortex (13, 14). Low CNS serotonin function, as indexed by CSF 5HIAA, is traitlike and correlates with impulsive and aggressive behaviors, as well as tendencies toward alcohol abuse, in nonhuman primates (15) and humans (16, 17). Second, the serotonin transporter plays a critical role in regulating the magnitude and duration of both CNS and peripheral actions of serotonin. A recently described polymorphism of the promoter region of the serotonin transporter gene, 5HTTLPR, is associated with differential transcriptional efficiencies: Both basal and stimulated activity of the long (l) allele is approximately twice that of the short (s) variant (18). In a sample composed predominantly of white males, persons with the l/l 5HTTLPR genotype scored lower on several facets of the personality dimension of neuroticism (including anxiety, angry hostility, depression, and impulsiveness) than persons with one or two s alleles (18). Among African Americans and women, however, the association between 5HTTLPR genotype and negative affectivity may be opposite, with persons having the l allele exhibiting higher levels of negative affects (19).
Thus, there is evidence that both these indices of CNS serotonin function are associated with some of the health-damaging psychological and behavioral characteristics that tend to cluster in the same individuals and groups. We now report that persons with one or two copies of the l allele have higher CSF 5HIAA levels and that both the l allele and higher CSF 5HIAA levels are associated with greater CV responses to stress.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Subjects are recruited through ads in the public media, flyers distributed in supermarkets and other public locations, and outreach screening events at civic organization meetings and other public events (eg, street fairs). The ultimate stratified sample of 160 to 200 subjects will contain equal numbers of subjects with high and low SES (based on income and education distributions), men and women, and black and white subjects. The findings reported here are based on data obtained from the first 54 subjects studied to date (age range = 18–49 years, 56% white, 67% male).
Procedures
After undergoing screening to exclude those with medical or psychiatric disorders or current medication use and giving informed consent, subjects report to the General Clinical Research Center during the early afternoon. After completing admission procedures, they undergo lumbar puncture, performed by a board-certified anesthesiologist (K.G. or M.S.-S.). Initially, as in published studies (16), we obtained 10 to 12 cc of CSF, which was mixed (to abolish the expected gradient across successive samples during the collection) and then separated into 2-cc aliquots and frozen for later assay of monoamine metabolites using high-performance liquid chromatography. The not-unexpected 10% to 15% incidence of posttap headaches among the first female subjects led us to determine whether there is a gradient of 5HIAA levels in the next two subjects. There was no gradient, with 5HIAA concentrations in cc 11 to 12 being virtually identical to those in cc 1 to 2. A previous study (20) found that without strict bedrest before the lumbar puncture, body height was unrelated to CSF 5HIAA levels. Because our subjects had been ambulatory before lumbar puncture, it seems that CSF in the lumbar column had been mixed by their movements, thereby abolishing any gradient due to height. This has enabled us to use a smaller needle and obtain only 3 to 4 cc of CSF, thereby greatly reducing the incidence of posttap headaches.
Between 11 AM and noon on the first test day, following the sham depletion or infusion, all subjects underwent a 45-minute mental stress protocol, during which CV function was monitored. This began with a 5-minute rest period, followed by 5 minutes of reading from a neutral text, followed by a 5-minute anger recall task, a second 5-minute neutral reading task, and a 5-minute sadness recall task. Additional 5-minute rest periods followed each 5-minute stress period.
Measures
Blood pressure and heart rate were determined at 1-minute intervals using a Critikon automatic vital signs monitor. To determine 5HTTLPR genotypes, genomic DNA was extracted by standard procedure (Puregene D-50K isolation kit, Gentra, Minneapolis, MN) from fresh or frozen samples of peripheral blood collected from the subjects. Polymerase chain reaction amplification to generate a 484- or 528-base pair fragment corresponding to the short (s) and long (l) 5HTTLPR alleles, respectively, was performed as described elsewhere (18) with the following modifications: 100 ng of genomic DNA was used in each reaction mixture, deoxyguanosine triphosphate was substituted for 7-deaza-2'-deoxyguanosine triphosphate, and the final volume of each reaction mixture was 25 µl. The fragments were resolved by electrophoresis through 3% agarose gels. 5HIAA, the primary serotonin metabolite, was measured by high-pressure liquid chromatography with electrochemical detection. The method used is a trace-enrichment method that utilized sequential C-18 columns for sample cleanup and analytical separation (15). Samples are diluted in 0.2 N perchloric acid (PCA) containing 0.5 mM ethylenediaminetetraacetic acid and 0.5 mM sodium metabisulfite and injected directly onto the chromatograph. The sample is enriched on a C-18 precolumn using an aqueous mobile phase composed of 0.05 M citrate, 0.05 M dibasic sodium phosphate, and 0.5 mM ethylenediaminetetraacetic acid at pH 3.5. Then the sample is eluted onto a Waters Spherisorb 3 µM ODS2 C-18 column with a mobile phase containing 4% to 8% acetonitrile in addition to the components of the enrichment mobile phase. Samples are detected by electrochemical detection, with a detector potential set at +0.55 mV vs. Ag/AgCl reference electrode. Data are collected with a computer-based data collection system and quantitated with the use of internal standard and external standard curves. Sensitivity of the assay is 0.5 ng per sample.
Statistical Analysis
One-way ANOVA was used to compare CSF 5HIAA levels in 5HTTLPR genotype groups, and two-way, repeated-measures ANOVA was used to evaluate changes in CV measures between rest and stress periods as a function of CSF 5HIAA (high vs. low, based on median split) or 5HTTLPR (l/l, l/s, and s/s genotypes) groups. Sample sizes vary across analyses because of loss of data in subjects who experienced posttap headache or were unable to complete ingestion of the amino acid capsules required for sham tryptophan depletion.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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We next tested the relationship of the three genotypic groupings of the 5HTTLPR alleles (l/l, l/s, and s/s) to CSF 5HIAA levels in a one-way ANOVA controlling only for the significant main effect of gender (p<0.02). This test revealed significant (p = .02) differences among the three genotypes, with the three genotypes accounting for 13.3% of the variance (corresponding to an r of 0.365) in CSF 5HIAA levels. Similar results were obtained in analyses controlling for age, race, SES, or height. Inspection of the three means using pairwise tests adjusted for multiple testing (Bonferonni adjustment) indicated that each genotype containing the l allele differed significantly from the s/s genotype but not from each other (p = .68). By partitioning the sum of squares for the genotypic effect, we tested a directed comparison of the combination of both l-containing genotypes vs. the s/s genotype, resulting in a highly significant test (p = .006). As shown in Figure 1, CSF 5HIAA levels in persons with one or two copies of the l allele were about 50% higher than those in their s/s counterparts.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Although our data do not allow us to specify the mechanism whereby 5HTTLPR genotypes regulate CNS serotonin function as indexed by CSF 5HIAA, informed speculation is possible. First, the increased number of serotonin transporter sites on presynaptic nerve endings likely to be present (18) in persons with the l allele can be expected to result in more rapid reuptake of previously released serotonin. Under one scenario, this more rapid clearance of serotonin would lead to reduced extracellular serotonin being available for degradation to 5HIAA. This alone would lead, however, to lower CSF 5HIAA levels, a result opposite to what we observed in the present study. There is an alternative scenario that is consistent with our finding of higher CSF 5HIAA in persons with one or two copies of the l allele. Under this scenario, the more rapid clearance of serotonin in persons with the l allele would result in reduced stimulation of presynaptic 5-HT1A somatodendritic autoreceptors that are known to exert negative feedback on neuronal firing rates (24). With less inhibition of neuronal firing rates, there would be increased release of new serotonin into the synaptic cleft. And although the increased number of transporter sites in persons with the l allele would still clear this serotonin more rapidly, the net result could be more serotonin available for degradation, which would be consistent with the higher CSF 5HIAA levels we found in persons with l/l or l/s genotypes.
Results from a clinical study in depressed patients that examined the association between 5HTTLPR genotypes and response to the selective serotonin reuptake inhibitor fluvoxamine alone or in combination with the 5HT1A receptor antagonist pindolol (25) are consistent with the second mechanism just described. In that study, patients with the l/l or l/s genotype showed a more rapid decrease in depressive symptoms to fluvoxamine alone than patients with the s/s genotype. Among patients treated with a combination of fluvoxamine and pindolol, however, depression decreased in those with the s/s genotype just as rapidly as it did in their l/l or l/s counterparts. Our finding of a similar relationship of the l/l and l/s genotypes to increased CSF 5HIAA could reflect the neurochemical basis (ie, more rapid clearance of previously released serotonin resulting in less stimulation of inhibitory 5HT1A somatodendritic autoreceptors with a consequent increase in serotonergic neurotransmission) for the more rapid clinical response of patients with one or two l alleles. It will be possible to confirm this interpretation by showing that pindolol treatment increases CSF 5HIAA levels in persons with the s/s genotype to those of persons with the l/l or l/s genotype.
Our findings that both l/l or l/s genotypes and high CSF 5HIAA are correlated with each other and that both are associated with increased CV responses to mental challenge are consistent with the general hypothesis that variations in CNS serotonin function can play a role in the pathogenesis of CV disease, possibly through accelerated rates of endothelial injury associated with CV hyperreactivity (26). Further supporting a contribution of the l allele to the pathogenesis of CV disease, studies in Japanese populations, in whom the l allele is far less prevalent than in whites (21% vs. 57%), have found the l allele to be more prevalent in patients with coronary heart disease than healthy control subjects (27).
Results also indicated that of the two indicators of CNS serotonin function evaluated in the present study, only 5HTTLPR genotype is independently associated with blood pressure reactivity. Because CSF 5HIAA levels primarily reflect serotonin release in the frontal cortex (13, 14), this finding suggests that 5HTTLPR genotype affects CV reactivity through effects on serotonergic neurotransmission in other brain regions, such as the hypothalamus, where serotonergic stimulation results in decreased sympathetic nervous system outflow (28), where rates of serotonin release are far less likely to be reflected in lumbar CSF 5HIAA levels.
The greater CV responses exhibited by persons with high CSF 5HIAA levels were surprising to us in light of our expectation that decreased CNS serotonin function (ie, low 5HIAA) would be associated with increased biological reactivity. Because our results are quite robust statistically and internally consistent, it is unlikely that they are due to chance. Differential effects of 5HTTLPR genotypes on serotonin function in differing environmental settings could help explain this result. For example, in monkeys separated from the mother for the first 6 months of life, those with the l/l genotype have higher CSF 5HIAA than mother-reared monkeys, in which 5HTTLPR genotypes have no impact on 5HIAA levels, suggesting that 5HTTLPR genotype affects CNS serotonin function only in the setting of early environmental adversity (in this case, maternal deprivation). The maternally deprived monkeys are nevertheless characterized by more maladaptive behaviors than mother-reared monkeys (15, 23), suggesting that in the setting of environmental adversity the l/l genotype may be not only associated with increased CFS 5HIAA but also with biobehavioral characteristics that may damage health.
There is a marked global variation in the frequency of the l allele, ranging from 70%+ in Africa (and African Americans) to 50% to 60% in Europe, to <30% in China and Japan (29, 19). Although Gelernter et al. (29) note that this variation could be due to "random genetic drift" of a "selectively neutral or nearly neutral polymorphism," they also recognize that if the 5HTTLPR polymorphism is "ultimately proven to have a significant association with behavior or some other serotonin-related phenotype," then the global variation could be related to selection factors that may have operated differently at various places and times. The increased CV reactivity to stress we find associated with the l allele, regardless of race, gender, or age, could represent such a serotonin-related phenotype that has contributed, through selection advantage and disadvantage, to the persistence of the l allele in Africa and its decreased frequency in Europe and East Asia.
The findings of the present study indicate that 5HTTLPR genotypes regulate CSF 5HIAA levels and that 5HTTLPR genotype influences CV responses to stress, apparently independently of CSF 5HIAA level. The increased CV responses in persons with one or two l alleles and higher CSF 5HIAA could contribute to the pathogenesis of CV disease. Therefore, indices of CNS serotonin function could prove useful in identifying persons at increased risk of developing CV disease under conditions of chronic stress.
These implications suggest a number of questions that can be addressed in future research. For example, does the increased frequency of the l allele and associated CV hyperresponsivity to stress contribute to the increased rates of hypertension observed among African Americans? Are African Americans with the s/s genotype less likely to develop hypertension? Are persons newly exposed to a chronic life situation (eg, becoming a caregiver for a spouse with Alzheimer’s disease) more likely to develop CV disease if they have one or more l alleles? Are persons with the s/s allelotype relatively resistant to developing CV disease under these circumstances? It will also be important to investigate the combined effects of multiple polymorphisms on biological factors related to disease. For example, are the biological effects (and possible disease consequences) we find associated with the l allele of the 5HTTLPR even more pronounced in persons who also have the less functional allele of a recently described polymorphism (30) of the monoamine oxidase A gene promoter? Answers to these questions will have a profound influence on our understanding of the neurobiological pathways responsible for linkages between psychosocial factors and increased risk for development of major physical illnesses. This understanding could speed the development of more effective approaches to prevention and treatment.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Received for publication July 15, 2000.
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