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Vol. 73, No. 2 February 2014 pp. 115Y122
ORIGINAL ARTICLE
Serotonin Metabolites in the Cerebrospinal Fluid in Sudden Infant Death Syndrome Ingvar J. Rognum, MD, Hoa Tran, PhD, Elisabeth A. Haas, Keith Hyland, PhD, David S. Paterson, PhD, Robin L. Haynes, PhD, Kevin G. Broadbelt, PhD, Brian J. Harty, MA, Othon Mena, MD, Henry F. Krous, MD, and Hannah C. Kinney, MD
Abstract Forensic biomarkers are needed in sudden infant death syndrome (SIDS) to help identify this group among other sudden unexpected deaths in infancy. Previously, we reported multiple serotonergic (5-HT) abnormalities in nuclei of the medulla oblongata that help mediate protective responses to homeostatic stressors. As a first step toward their assessment as forensic biomarkers of medullary pathology, here we test the hypothesis that 5-HTYrelated measures are abnormal in the cerebrospinal fluid (CSF) of SIDS infants compared with those of autopsy controls. Levels of CSF 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA), the degradative products of 5-HT and dopamine, respectively, were measured by highperformance liquid chromatography in 52 SIDS and 29 non-SIDS autopsy cases. Tryptophan (Trp) and tyrosine (Tyr), the substrates of 5-HT and dopamine, respectively, were also measured. There were no significant differences in 5-HIAA, Trp, HVA, or Tyr levels between the SIDS and non-SIDS groups. These data preclude the use of 5-HIAA, HVA, Trp, or Tyr measurements as CSF autopsy biomarkers of 5-HT medullary pathology in infants who have died suddenly and unexpectedly. They do, however, provide important information about monoaminergic measurements in human CSF at autopsy and their developmental profile in infancy that is applicable to multiple pediatric disorders beyond SIDS. Key Words: 5-Hydroxyindoleacetic acid, Brainstem, Dopamine, High-performance liquid chromatography, Postmortem interval, Tyrosine, Tryptophan. From the Department of Pathology, Boston Children’s Hospital and Harvard Medical School, Boston, Massachusetts (IJR, HT, DSP, RLH, KGB, HCK); Faculty of Medicine, University of Oslo, Oslo, Norway (IJR); Department of Pathology, Rady Children’s Hospital, San Diego, California (EAH, HFK); Medical Neurogenetics, Atlanta, Georgia (KH); New England Research Institutes, Watertown, Massachusetts (BJH); and San Diego County Medical Examiner’s Office, San Diego, California (OM). Send correspondence and reprint requests to: Ingvar J. Rognum, MD, Department of Pathology, Boston Children’s Hospital, Enders Bldg 1109, 61 Binney St, Boston, MA 02115; E-mail: [email protected] This study was funded by the Translational Research Program, Boston Children’s Hospital, Boston, MA; Norwegian ExtraFoundation for Health and Rehabilitation (Ingvar Rognum); Norwegian SIDS and Stillbirth Society (Ingvar Rognum); First Candle/SIDS Alliance; CJ Foundation for SIDS; Jacob Neil Boger Foundation for SIDS; Marley Jaye Cerella Foundation for SIDS; Eunice Kennedy Shriver National Institute of Child Health and Development (P01-042774441) (Hannah Kinney); and Intellectual and Developmental Disabilities Research Center, Boston Children’s Hospital, Boston, MA (P30-HD18655).
INTRODUCTION Sudden infant death syndrome (SIDS) is defined as the sudden death of an infant younger than 12 months that is related to a sleep period and remains unexplained after a complete autopsy, death scene investigation, and review of the clinical history (1). It is the leading cause of postneonatal infant mortality in westernized countries today (2). During the last 2 decades, our group has provided mounting evidence for serotonergic (5-HT) pathology in the homeostatic network of the medulla oblongata in a majority of SIDS infants (3Y5). In 4 independent studies, we have reported altered 5-HT receptor binding (3, 5, 6) in autonomic- and respiratory-related nuclei of the medulla that are composed of 5-HT neurons and/or receive 5-HT projections, that is, the so-called medullary 5-HT homeostatic network (7), in SIDS cases. Moreover, other investigators have confirmed the finding of medullary abnormalities in 5-HT receptors in SIDS infants by immunocytochemical methods (8, 9). Furthermore, we demonstrated a significant reduction in SIDS of 5-HT itself and tryptophan (Trp) hydroxylase 2 (TPH2), the major biosynthetic enzyme for 5-HT synthesis, in two 5-HT source nuclei with altered 5-HT1A binding in SIDS, that is, the raphe obscurus and paragigantocellularis lateralis (3). These results point to a deficiency of 5-HT in the pathogenesis of the medullary 5-HT pathology in affected SIDS cases. The medullary 5-HT network plays a key role in mediating protective responses to homeostatic stressors such as hypercarbia, hypoxia, and asphyxia (7, 10, 11). Thus, defects in this system may impair the SIDS infant’s responses to such stressors, for example, asphyxia in the prone (face down) sleep position, thereby leading to sudden death in a critical developmental period (12, 13). Although 5-HT abnormalities are not the only reported neurochemical abnormalities in SIDS brainstems, they are among the most robust and reproducible (11), and these findings have suggested our formulation that at least a subset of SIDS is part of a spectrum of ‘‘developmental serotonopathies’’ that include known inborn errors of 5-HT metabolism, autism, and prenatal exposure to selective serotonergic reuptake inhibitors (10). The objective of this study was to determine if levels of 5-HTYrelated parameters are decreased in the cerebrospinal fluid (CSF) of SIDS infants as a potential reflection of reported 5-HT abnormalities in the medulla in SIDS (3, 5). Currently, the determination of the medullary 5-HT abnormalities in the
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medulla in SIDS infants is highly specialized, labor intensive, and expensive, for example, tissue receptor autoradiography in multiple frozen sections of the medulla and biochemical assays of micropunched samples of frozen medulla. Thus, markers of these abnormalities in a more readily studied source at autopsy, such as CSF, with a cost-effective and standard technique, such as high-performance liquid chromatography (HPLC), would be of considerable value in the forensic identification of infants who die suddenly with a 5-HT abnormality. We further reasoned that new information accrued in this study about the methodology of 5-HTYrelated measurements in human CSF and their developmental profile would be directly relevant to multiple neurologic disorders involving 5-HT beyond the SIDS problem (10). The major 5-HTYrelated parameters that can be readily measured in human CSF are 5-hydroxyindoleacetic acid (5-HIAA) and Trp, the essential amino acids involved in 5-HT synthesis, both using clinically available HPLC techniques. In this study, we tested the hypothesis that 5-HIAA and/or Trp levels in the CSF are decreased in SIDS infants compared with those in controls dying of known causes. We reasoned that the decreased tissue levels of 5-HT in the medullary (raphe and extraraphe) nuclei we previously reported in SIDS infants result in a secondary decrease of tissue 5-HIAA (3) and, thus, its decreased release into the CSF with decreased levels in that compartment. Because we did not previously find any differences in dopamine metabolite levels or turnover directly in the medullary 5-HT system in SIDS cases compared with controls (3), we hypothesized here that there are no differences in dopamine metabolites in the CSF in SIDS versus non-SIDS groups. In addition, we tested the hypothesis that 5-HIAA and Trp levels in the CSF in SIDS infants are significantly different from those of infants dying with unequivocal asphyxia to determine if the putative 5-HTYrelated alterations in SIDS are potentially secondary to terminal asphyxia. To further explore the potential effects of hypoxia/ anoxia on 5-HIAA and Trp levels, we also included a group of SIDS infants who had a cardiopulmonary arrest and anoxic encephalopathy and were ventilated mechanically for 6 to 31 hours before death. Information about the potential effects of terminal hypoxia-ischemia on monoaminergic levels in postmortem CSF is of importance in its own right in assessing such levels in human autopsy populations across the age spectrum. We further tested the hypotheses that there are no significant differences between 5-HIAA and/or Trp levels in the CSF between infants who are adjudicated as SIDS and those adjudicated as ‘‘undetermined’’ (defined in Materials and Methods), and that both groups have levels significantly decreased from those of infants dying of known causes of death. Our hypothesis is based on the premises that 1) this undetermined group of sudden and unexplained deaths share common underlying abnormalities with those of the SIDS group, 2) the distinction between the 2 groups is largely a semantic one, and 3) the 2 groups share a common biomarker such as altered 5-HIAA and/or Trp levels in the CSF. Finally, we were interested in determining if any SIDS case met the diagnostic screening criteria for a known inborn error of 5-HT metabolism, thereby potentially unmasking a subgroup of infants
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with genetic bioaminergic disorders that present as sudden death in seemingly well infants (14).
MATERIALS AND METHODS Clinical Database All CSF specimens were obtained between 2004 and 2011 from the San Diego County Medical Examiner’s Office, San Diego, CA. Samples were accrued in accordance to California law Chapter 955, Statutes of 1989 (SB1069). This law permits the use of autopsy tissues from infants with sudden death for research without parental consent. The SIDS cases were defined as above (1). ‘‘Acute’’ controls (ACs) were infants younger than 12 months who died unexpectedly (in some instances with a minor or acute illness within 48 hours of death) and in whom an autopsy and death scene investigation established a known cause of death (15). ‘‘Chronic’’ controls (CCs) were infants younger than 12 months who had clinical chronic illnesses but who nevertheless died suddenly and unexpectedly; a complete autopsy confirmed the presence of a chronic illness (15). The acute asphyxia (AA) group was defined as infants younger than 12 months in whom the death scene investigation determined unequivocally that there was an acute lethal asphyxia event (e.g. overlaying, crib accident) and the autopsy itself did not demonstrate a chronic or lethal intrinsic disease process. The resuscitated SIDS (RSIDS) group was defined as infants younger than 12 months who were discovered unresponsive, underwent successful cardiopulmonary resuscitation, and were subsequently ventilated; these infants were withdrawn from the ventilator because of severe postarrest anoxic encephalopathy and were classified as SIDS at autopsy. The undetermined (UND) group was defined as infants younger than 12 months in whom the findings at autopsy or death scene may have contributed to death, but their causative role in death was uncertain (e.g. bed sharing). Clinicopathologic variables were recorded on review of autopsy and death scene investigation reports, including gestational age, race, sex, and circumstances of death (e.g. position found on discovery). The diagnosis in each case and control was made without knowledge of the CSF data. The Committee on Clinical Investigation at Boston Children’s Hospital, Boston, MA, approved this study.
Neurotransmitter-Related Measurements in CSF The site of CSF collection was either posterior lumbar subarachnoid space or cisterna magna. On collection, all CSF samples were stored at j80-C until analysis. Obviously bloody specimens were excluded from biochemical analysis because of red blood cell contamination. Samples were thawed on ice and centrifuged at 3,500 g at 4-C for 10 minutes. Reverse-phase HPLC with electrochemical detection was performed on CSF supernatants for 5-HIAA and homovanillic acid (HVA) analyses at Medical Neurogenetics, Atlanta, GA, with internal standards, according to previously published protocols (16, 17). Measurements of the CSF levels of Trp and tyrosine (Tyr) were performed in the same set of SIDS cases and non-SIDS controls at Medical Neurogenetics by reverse-phase HPLC with fluorescence detection (18). All Ó 2014 American Association of Neuropathologists, Inc.
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bioaminergic CSF measurements were made without knowledge of diagnosis, age, or other clinical factors.
Statistical Analysis Descriptive statistics were performed with regard to demographic information and study measures for each of the study groups (SIDS and non-SIDS). A one-way analysis of variance was used for the comparison of age and postmortem interval (PMI) between the different groups, and the Fisher exact test was used for the comparison of the percentage of sex and prematurity between groups. Models of analysis of covariance (ANCOVA) were used to compare the levels of 5-HIAA, HVA, Trp, and Tyr between the SIDS group and various non-SIDS groups, separately and combined, controlling for postconceptional age (PCA) and PMI. A TukeyKramer adjustment was used to account for the multiple comparisons being made within each model.
RESULTS Clinical Database The total data set consisted of 86 cases, with the following classification: SIDS, n = 52; AC, n = 9; CC, n = 2; AA, n = 8; RSIDS, n = 5; and UND, n = 10 (Table 1). There was no significant difference in PCA, gestational age, postnatal age, percentage of preterm birth or sex, or PMI among the study groups (Table 1). The median PMI for each of the groups was less than 24 hours (data not shown), with a range of 5.5 to 37 hours (only 1 case longer than 30 hours). There was no significant effect of increasing PMI on CSF levels of 5-HIAA (Fig. 1), in contrast to adverse effects on Trp and Tyr levels within certain groups (see below). The causes of death in the non-SIDS groups and the circumstances of death in the AA group are provided in Table 2. All 86 cases were included in the analysis of 5-HIAA, HVA, Trp, and Tyr (Tables 3Y6, respectively). There was no effect of increasing age for any of the 4 measures (5-HIAA, HVA, Trp, Tyr) in any group or all groups combined (Fig. 2) (Trp and Tyr not shown).
Analysis of CSF Serotonin Metabolites in SIDS
Measurements of 5-HIAA in the CSF of SIDS Versus Non-SIDS Groups There were no significant differences in mean 5-HIAA levels between the SIDS group and the AC, CC, AA, RSIDS, and UND groups (p 9 0.05) for all 5 comparisons (Table 3). In a separate ANCOVA model, there was no significant difference in 5-HIAA levels between the SIDS group, non-SIDS group (defined as combined AC, CC, and AA groups), RSIDS group, and UND group, adjusting for PCA and PMI (data not shown). Although there was a minimal reduction (9%) in 5-HIAA levels on average in the SIDS group versus nonSIDS groups, it was not statistically significant (p = 0.824).
Measurements of Trp in the CSF of SIDS Versus Non-SIDS Groups There was a significant positive correlation in the nonSIDS group between Trp and PMI (correlation coefficient [r] = 0.48, p = 0.037), as previously suggested in human CSF autopsy analysis (19), and there were nonsignificant positive correlations in the SIDS (r = 0.14, p = 0.313), RSIDS (r = 0.03, p = 0.965), and UND (r = 0.53, p = 0.113) groups. Using an ANCOVA model to compare the groups while adjusting for PCA and PMI, we found no significant differences in mean Trp levels between the SIDS cases and the AC, CC, AA, RSIDS, and UND groups (p 9 0.05 for all 5 comparisons) (Table 4).
Measurements of HVA in the CSF of SIDS Versus Non-SIDS Groups In fitting an ANCOVA model for the HVA data, a significant PCA-diagnosis interaction (p G 0.0001) was observed. The difference between HVA and diagnoses, adjusting for PMI, varied with PCA, with an increase in HVA levels with increasing age (data not shown). In exploring this interaction further, we found that it was caused by 2 outliers: 1 case from the RSIDS group (HVA, 15,420 nmol/L; PCA, 67 weeks) and 1 case from the UND group (HVA, 15,520 nmol/L; PCA, 82 weeks). Exclusion of these 2 outliers eliminated the significant PCA-diagnosis interaction (Fig. 2). There were significant
TABLE 1. Demographics of the Entire Study Cohort Male n SIDS 52 AC 9 CC 2 AA 8 RSIDS 5 UND 10 Non-SIDS controls (AC + CC + AA) 19 p value comparing demographic by diagnosis
Premature (GA G37 weeks)
Mean (SD)
n (%)
n (%)
PCA, Mean (SD), weeks
GA, weeks
PNA, weeks
PMI, hours
25 (48) 7 (78) 2 (100) 3 (38) 2 (40) 7 (70) 12 (63) 0.27
6 (12) 2 (22) 1 (50) 2 (25) 1 (20) 2 (20) 5 (26) 0.37
52.7 (8.6) 50.6 (12.2) 40.8 (3.2) 54.8 (11.7) 57.5 (8.0) 53.0 (14.0) 51.3 (11.8) 0.45
39.0 (3.1) 38.7 (1.9) 38.5 (3.5) 38.3 (3.2) 38.2 (2.7) 37.8 (4.1) 38.5 (2.5) 0.91
13.9 (8.1) 11.9 (10.9) 2.3 (0.4) 16.5 (11.4) 19.3 (8.0) 15.1 (12.5) 12.8 (11.1) 0.31
22.2 (5.4) 21.1 (8.1) 23.5 (4.9) 16.5 (8.8) 16.7 (2.2) 20.1 (8.3) 19.4 (8.2) 0.14
A one-way analysis of variance was used for the comparison of age and PMI between the different groups; Fisher exact test was used for the comparison of the percentage of sex and prematurity between groups. AA, acute asphyxia; AC, acute controls; CC, chronic controls; GA, gestational age; n, sample size; PCA, postconceptional age; PMI, postmortem interval; PNA, postnatal age; RSIDS, resuscitated SIDS; UND, undetermined cases.
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FIGURE 1. There is no significant effect of increasing postmortem interval (PMI) on CSF levels of either 5-hydroxyindoleacetic acid (5-HIAA) levels (left) or homovanillic acid (HVA) (right) in the SIDS or non-SIDS groups (the latter composed of acute controls, chronic controls, and acute asphyxia cases combined). Solid circles, SIDS; open circles, non-SIDS.
differences in HVA levels among the pairwise comparisons between the SIDS and AC groups and between the AA and AC groups (Table 5). On excluding the single outlier (HVA, 11,070 nmol/l) in the AC group from the analyses, the significant differences disappeared. There was no significant difference in HVA levels between the SIDS and AA groups (p = 0.989) (Table 5). A separate ANCOVA model was performed to compare HVA levels between the SIDS, non-SIDS
(AC, CC, and AA combined), RSIDS, and UND groups, adjusting for PCA and PMI.
Measurements of Tyr in the CSF of SIDS Versus Non-SIDS Groups There was a significant positive correlation of Tyr with PMI in the non-SIDS group (r = 0.50, p = 0.034), as previously suggested in human CSF autopsy analysis (19). There
TABLE 2. Demographic Information and Cause of Death in Acute Controls, Chronic Controls, and Acute Asphyxial Group Acute Controls
Sex
GA, weeks
PNA, weeks
PCA, weeks
M F M M M M M M M
40 40 35 36 38 40 40 39 36
21.0 3.5 2.0 0.3 15.5 11.0 24.5 1.0 2.5
61.0 43.5 37.0 36.3 53.5 51.0 64.5 40.0 38.5
Hyperthermia (excessive bedding) Histiocytoid cardiomyopathy Bronchopulmonary dysplasia secondary to lung disease of prematurity Fatty acid oxidation disorder Acute aspiration pneumonia Acute bacterial meningitis Drowning Acute gastrointestinal illness of unknown etiology with dehydration Congenital heart defect (ventricular septal defect and bicuspid aortic valve with stenosis) with minimal acute symptoms preceding death
Chronic controls 1 2
M F
41 40
2.0 28.5
43.0 68.5
Disseminated herpes simplex type II viral infection Hemolytic anemia
Acute asphyxia 1 2 3 4 5 6 7 8
M F F F M F M F
40 40 36 40 39 31 40 40
9 13 32 23 6 12 33 4
49 53 68 63 45 43 73 44
1 2 3 4 5 6 7 8 9
Cause of Death
Findings on Death Scene Investigation Found face down in mattress with lower body on ‘‘boppy’’ pillow (feet in air) Found with head inside pillow case between two pillows Found with body wedged between bed springs and air mattress Found with head covered by adult pillow Found between arm and torso of intoxicated mother in adult bed Found face down in unsafe sleeping environment Found with head wrapped in blanket Found with face and head wedged between cosleeper and sofa back
F, female; GA, gestational age; M, male; PCA, postconceptional age; PNA, postnatal age.
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Analysis of CSF Serotonin Metabolites in SIDS
FIGURE 2. The levels of 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) across all groups are relatively constant over infancy, the age range studied. Solid circles, SIDS; open circles, non-SIDS group; gray circles, resuscitated SIDS (RSIDS).
were nonsignificant positive correlations in the SIDS (r = 0.16, p = 0.250), RSIDS (r = 0.38, p = 0.522), and the UND (r = 0.48, p = 0.161) groups. Using an ANCOVA model to compare the groups while adjusting for PCA and PMI, we found no significant differences in mean Tyr levels between the SIDS cases and the AC, CC, AA, RSIDS, and UND groups (p 9 0.05 for all 5 comparisons) (Table 6).
DISCUSSION
To determine if the entire SIDS cohort contained cases with low 5-HIAA and HVA levels in the range of those found in known inborn errors of monoamine metabolism, we compared the 5-HIAA and HVA levels published in inborn errors (20, 21) as a reference. There were no SIDS cases that demonstrated combined low levels of 5-HIAA and HVA within this disease reference range (Fig. 3).
Measurement of 5-HIAA and Trp in CSF provides an important tool in clinical practice for detecting and monitoring disorders of brain 5-HT levels and metabolism (16Y18). The relevance of these tools to SIDS, as well as to multiple other neurologic disorders known to involve 5-HT in children (10, 14, 18, 21Y25) and adults (26Y31), is of interest. In this study, we did not find a significant difference in the two standard measures of 5-HT metabolism in human CSF, that is, 5-HIAA and Trp, between SIDS cases and infants dying of a range of acute and chronic disorders. Thus, our data suggest that 5-HIAA or Trp is not a reasonable postmortem biomarker for SIDS, and that the need to correlate CSF levels with medullary tissue 5-HT markers in the same SIDS cases is negated. We also did not find a difference in dopaminergic markers between the SIDS and non-SIDS cases as anticipated because we did not find differences in dopaminergic parameters in
TABLE 3. 5-HIAA Comparisons by Diagnosis, Adjusting for PCA and PMI
TABLE 4. Trp Comparisons by Diagnosis, Adjusting for PCA and PMI
Analysis for Potential Known Inborn Errors of 5-HT Metabolism
Trp Levels
5-HIAA Adjusted Mean (SE), nmol/L SIDS, n = 52 AC, n = 9 CC, n = 2 AA, n = 8 RSIDS, n = 5 UND, n = 10
1,191.8 (67.6) 1,399.9 (161.0) 1,546.8 (347.8) 1,126.7 (175.1) 1,150.3 (220.5) 904.7 (152.6)
Pairwise Comparisons Between Groups of Interest (p) SIDS vs AC (0.839) SIDS vs CC (0.915) SIDS vs AA (0.999) SIDS vs RSIDS (1.000) SIDS vs UND (0.526) AC vs CC (0.999) AC vs AA (0.861)
A Tukey-Kramer adjustment was used to account for the multiple comparisons. The p values are in parentheses. There was no difference in 5-HIAA levels between the SIDS and non-SIDS (AC, CC, and AA groups combined) (p = 0.824). 5-HIAA, 5-hydroxyindoleacetic acid; AA, acute asphyxia; AC, acute controls; CC, chronic controls; PCA, postconceptional age; PMI, postmortem interval; RSIDS, resuscitated SIDS; UND, undetermined cases.
Adjusted Mean (SE), Kmol/L SIDS, n = 52 AC, n = 9 CC, n = 2 AA, n = 8 RSIDS, n = 5 UND, n = 10
52.1 (2.8) 55.0 (6.6) 50.1 (14.2) 51.2 (7.2) 45.8 (9.0) 47.0 (6.2)
Pairwise Comparisons Between Groups of Interest (p) SIDS vs AC (0.999) SIDS vs CC (1.000) SIDS vs AA (1.000) SIDS vs RSIDS (0.985) SIDS vs UND (0.975) AC vs CC (1.000) AC vs AA (0.999)
A Tukey-Kramer adjustment was used to account for the multiple comparisons. The p values are in parentheses. There was no difference in Trp levels between SIDS and non-SIDS (AC, CC, and AA groups combined) (p = 0.999). AA, acute asphyxia; AC, acute controls; CC, chronic controls; PCA, postconceptional age; PMI, postmortem interval; RSIDS, resuscitated SIDS; Trp, tryptophan; UND, undetermined cases.
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TABLE 5. HVA Comparisons by Diagnosis Adjusting for Postconceptional Age and Postmortem Interval HVA Levels Adjusted Mean (SE), nmol/L SIDS, n = 52 AC, n = 9 CC, n = 2 AA, n = 8 RSIDS, n = 5 UND, n = 10
1,557.7 (177.4) 3,305.2 (423.0) 2,321.9 (915.6) 1,245.9 (462.4) 2,755.1 (645.3) 914.2 (424.0)
Pairwise Comparisons Between Groups of Interest (p) SIDS vs AC (0.004) SIDS vs CC (0.963) SIDS vs AA (0.989) SIDS vs RSIDS (0.485) SIDS vs UND (0.729) AC vs CC (0.923) AC vs AA (0.019)
A Tukey-Kramer adjustment was used to account for the multiple comparisons. The p values are in parentheses. There was no difference in HVA levels between the SIDS and non-SIDS (AC, CC, and AA groups combined) (p = 0.130). AA, acute asphyxia; AC, acute controls; CC, chronic controls; HVA, homovanillic acid; PCA, postconceptional age; PMI, postmortem interval; RSIDS, resuscitated SIDS; UND, undetermined cases.
medullary tissues between SIDS and control (3). We also did not find significant differences in 5-HIAA, Trp, HVA, or Tyr measures between the UND and the SIDS group, indicating that these metabolites in the CSF do not provide a distinguishing biomarker. Of note, there were no changes in the levels of any bioaminergic CSF parameter analyzed in the SIDS cases or controls within the first year of life, suggesting that this time frame is not a critical developmental period for these CSF parameters. Irrespective of the SIDS problem, this study also provides information directly relevant to assessment of monoaminergic metabolites in postmortem human CSF, including potential effects of terminal hypoxia-ischemia on them, as highlighted below.
Serotonergic Parameters in the CSF of SIDS Versus Non-SIDS Groups Synthesis of 5-HT in the brain occurs in raphe and extraraphe neurons of the entire brainstem through the conversion of the precursor molecule Trp to 5-hydroxytryptophan by TPH2 and then to 5-HT via the enzymatic action of aromatic
FIGURE 3. There are no SIDS cases in the entire data set (n = 52) that demonstrate combined low levels of 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) within the reference levels of known inborn errors of serotonin (5-HT) metabolism, indicated in the shaded rectangle in the lower left portion of the graph (21). LYamino acid decarboxylase, which has 5-hydroxytryptophan and L-DOPA as substrates (22). Tryptophan is also metabolized
in the kynurenine pathway and used for protein synthesis (31). Serotonin is degraded to 5-HIAA by monoamine oxidase (32). Terminals from the raphe 5-HT source neurons in the brainstem form supra- and subependymal plexuses around the lining of the fourth ventricle and release extrasynaptic 5-HT, including directly into the CSF (Fig. 4) (33, 34). In addition, 5-HIAA in the brain extracellular space diffuses into the CSF, such that this compartment serves as a so-called 5-HIAA sink (35). Serotonin itself cannot reliably be measured in the CSF because its levels are constitutively negligible (i.e. G10 pg/mL) (36). Nevertheless, the CSF concentration of 5-HIAA correlates with 5-HT levels and turnover in brain parenchyma (37, 38). Tryptophan crosses the blood-brain barrier (in competition with other large neutral amino acids) for incorporation into brain 5-HT (39, 40). It is not entirely surprising that we did not find reduced 5-HIAA levels in the CSF in SIDS cases because we did not previously observe significantly
TABLE 6. Tyr Comparisons by Diagnosis Adjusting for PCA and PMI Tyr Levels Adjusted Mean (SE), Kmol/L SIDS, n = 52 AC, n = 9 CC, n = 2 AA, n = 8 RSIDS, n = 5 UND, n = 10
224.0 (11.2) 254.2 (26.8) 248.0 (81.7) 208.6 (29.1) 211.3 (36.6) 198.7 (25.4)
Pairwise Comparisons Between Groups of Interest (p) SIDS vs AC (0.902) SIDS vs CC (1.000) SIDS vs AA (0.997) SIDS vs RSIDS (1.000) SIDS vs UND (0.943) AC vs CC (1.000) AC vs AA (0.859)
A Tukey-Kramer adjustment was used to account for the multiple comparisons. The p values are in parentheses. There was no difference in Tyr levels between SIDS and non-SIDS (AC, CC, and AA groups combined) (p = 0.966). AA, acute asphyxia; AC, acute controls; CC, chronic controls; PCA, postconceptional age; PMI, postmortem interval; RSIDS, resuscitated SIDS; Tyr, tyrosine; UND, undetermined cases.
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FIGURE 4. Diagram of the cerebrospinal fluid (CSF) ‘‘sink’’ of tissue 5-hydroxyindoleacetic acid (5-HIAA). The 5-HIAA in the CSF diffuses freely from the brainstem parenchyma or, alternatively, represents degradation of serotonin (5-HT) released directly into the CSF at the ependymal plexuses of the 5-HT terminals of 5-HT source neurons in the raphe and extraraphe regions of the entire brainstem. Ó 2014 American Association of Neuropathologists, Inc.
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reduced 5-HIAA or 5-HT/5-HIAA levels in the medullary tissue in SIDS infants. Our inability to detect a significant reduction in Trp in the CSF in SIDS cases suggests that the reduction in medullary 5-HT may be caused by TPH2 deficiency and not to the dietary availability of Trp or the capability to transport Trp across the blood-brain barrier. Two previous studies reported significantly increased levels of 5-HIAA, HVA, Trp, and Tyr in the CSF of SIDS infants compared with those of controls (41, 42). Our lack of confirmation of these studies may reflect a discrepancy in the control populations, that is, the previous studies mainly involved CSF measurements in living control infants for comparison, whereas our study only incorporated autopsy controls. In a previous report, the metabolite levels were similar between 10 autopsy controls and 28 living controls, and the SIDS values were elevated relative to these two control populations (42). We found, however, that the autopsy controls in our study were more than 3-fold higher than published values of living controls (20), suggesting that autopsy and living control values cannot be combined. These data need to be considered in selecting controls for CSF assessments in other pediatric (10, 14, 18, 21Y25) and adult (26Y31) disorders affecting central monoaminergic metabolism.
Potential Relationship of CSF Bioaminergic Biomarkers to Asphyxia The potential effect of asphyxia/hypoxia on CSF levels of 5-HIAA and HVA is of concern in SIDS research because of experimental reports of abnormal brainstem measures of 5-HT (and dopaminergic) parameters secondary to hypoxiaischemia (3, 23) and elevated CSF levels in adults who have undergone successful cardiopulmonary resuscitation after cardiac arrest (30). Thus, interpretation of potentially altered levels in SIDS infants must account for (possible) asphyxia engendered in an unsafe sleeping environment (7, 10, 11). However, we found no significant differences in 5-HIAA or Trp (or Tyr) levels in the CSF between the SIDS and AA groups. There were also no differences in these metabolite concentrations between the SIDS and RSIDS groups, the latter group with potential secondary chronic (6Y31 hours) hypoxic-ischemic injury complicating the putative intrinsic defect.
Analysis of Potential Inborn Errors of 5-HT Metabolism Measures of 5-HIAA and HVA are used to clinically screen for inborn errors of 5-HT metabolism, including a genetic deficiency of LYamino acid decarboxylase (14). In this study, however, we found no SIDS outliers with significant decreases in combined 5-HIAA and HVA levels versus controls. Thus, these data suggest that infants with known inborn errors of 5-HT metabolism are not likely embedded within the SIDS population. Infants with known inborn bioaminergic errors are recognized by a clinical phenotype of mild to severe developmental delay, dystonia, and seizures (14, 24); they may be associated with autonomic dysfunction, such as temperature instability and hypotension, with rare reports of cardiorespiratory arrest (14). Nevertheless, sudden death in a clinically healthy infant, as in SIDS, has not been described.
Analysis of CSF Serotonin Metabolites in SIDS
Potential Limitations of the Study The potential compromise of the validity of postmortem measures of 5-HT parameters by lengthy PMIs (the definition of ‘‘lengthy’’ may be within minutes or even seconds after death) is of concern. Importantly, our data indicate no change in 5-HIAA and HVA levels with an increasing PMI between 5.5 and 37 hours. This observation is supported by the report of stable levels up to a PMI of 30 hours (37). Still, our finding that the 5-HIAA and HVA levels in the entire autopsy data set (SIDS and non-SIDS controls) were more than 3-fold higher than that of published values for living infants and children (irrespective of neurologic disease) (20) suggests that rapid changes in these parameters occur immediately after death. Thus, comparison of an autopsy study group must be made to an appropriate autopsy control group, not living control standards. A second potential limitation of this study is that CSF was obtained at autopsy by two approaches, that is, lumbar puncture and cisternal tap, which were not always specifically documented. Because an uneven concentration gradient in CSF metabolites has been reported in older children and adults, including at autopsy (37, 43), values may vary by rostral or caudal site. Nevertheless, this putative gradient may not be present in infants; a 5-HIAA and HVA gradient was not observed in 6 living infants who underwent neurosurgery for hydrocephalus (25).
CONCLUSIONS We report no alterations in 5-HTYrelated parameters in the CSF of SIDS infants compared with those of infants dying of various disorders, including asphyxia. The finding of unaltered levels of 5-HIAA in SIDS is consistent with our previous report of likewise unaltered levels of this metabolite in medullary tissues of SIDS infants with altered 5-HT levels (3). Thus, this study does not negate the 5-HT brainstem hypothesis in SIDS. This study focuses the ongoing search for 5-HT biomarkers of 5-HT medullary pathology in SIDS infants in other readily accessible tissue compartments, notably in serum and platelets, or with different 5-HTYrelated markers (e.g. TPH2, 5-HT1A receptor protein by proteomics) in future studies. It also provides important information about the methodology of monoaminergic measurements in human CSF at autopsy, including considerations related to PMI and terminal hypoxia-ischemia at autopsy, as well as their developmental profile in infancy that is applicable to multiple disorders of the pediatric and adult brain beyond SIDS.
ACKNOWLEDGMENTS The authors are grateful for the dedicated assistance in this study of the medical examiners of the San Diego County Medical Examiner’s Office, San Diego, CA. The authors also appreciate the helpful input of Dr Siri Hauge Opdal and Professor A˚shild Vege, University of Oslo, Oslo, Norway, in the course of the study. The authors thank Drs Eugene E. Nattie and Richard D. Goldstein for critical reading of the manuscript in preparation.
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ORIGINAL ARTICLE
Serotonin Metabolites in the Cerebrospinal Fluid in Sudden Infant Death Syndrome Ingvar J. Rognum, MD, Hoa Tran, PhD, Elisabeth A. Haas, Keith Hyland, PhD, David S. Paterson, PhD, Robin L. Haynes, PhD, Kevin G. Broadbelt, PhD, Brian J. Harty, MA, Othon Mena, MD, Henry F. Krous, MD, and Hannah C. Kinney, MD
Abstract Forensic biomarkers are needed in sudden infant death syndrome (SIDS) to help identify this group among other sudden unexpected deaths in infancy. Previously, we reported multiple serotonergic (5-HT) abnormalities in nuclei of the medulla oblongata that help mediate protective responses to homeostatic stressors. As a first step toward their assessment as forensic biomarkers of medullary pathology, here we test the hypothesis that 5-HTYrelated measures are abnormal in the cerebrospinal fluid (CSF) of SIDS infants compared with those of autopsy controls. Levels of CSF 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA), the degradative products of 5-HT and dopamine, respectively, were measured by highperformance liquid chromatography in 52 SIDS and 29 non-SIDS autopsy cases. Tryptophan (Trp) and tyrosine (Tyr), the substrates of 5-HT and dopamine, respectively, were also measured. There were no significant differences in 5-HIAA, Trp, HVA, or Tyr levels between the SIDS and non-SIDS groups. These data preclude the use of 5-HIAA, HVA, Trp, or Tyr measurements as CSF autopsy biomarkers of 5-HT medullary pathology in infants who have died suddenly and unexpectedly. They do, however, provide important information about monoaminergic measurements in human CSF at autopsy and their developmental profile in infancy that is applicable to multiple pediatric disorders beyond SIDS. Key Words: 5-Hydroxyindoleacetic acid, Brainstem, Dopamine, High-performance liquid chromatography, Postmortem interval, Tyrosine, Tryptophan. From the Department of Pathology, Boston Children’s Hospital and Harvard Medical School, Boston, Massachusetts (IJR, HT, DSP, RLH, KGB, HCK); Faculty of Medicine, University of Oslo, Oslo, Norway (IJR); Department of Pathology, Rady Children’s Hospital, San Diego, California (EAH, HFK); Medical Neurogenetics, Atlanta, Georgia (KH); New England Research Institutes, Watertown, Massachusetts (BJH); and San Diego County Medical Examiner’s Office, San Diego, California (OM). Send correspondence and reprint requests to: Ingvar J. Rognum, MD, Department of Pathology, Boston Children’s Hospital, Enders Bldg 1109, 61 Binney St, Boston, MA 02115; E-mail: [email protected] This study was funded by the Translational Research Program, Boston Children’s Hospital, Boston, MA; Norwegian ExtraFoundation for Health and Rehabilitation (Ingvar Rognum); Norwegian SIDS and Stillbirth Society (Ingvar Rognum); First Candle/SIDS Alliance; CJ Foundation for SIDS; Jacob Neil Boger Foundation for SIDS; Marley Jaye Cerella Foundation for SIDS; Eunice Kennedy Shriver National Institute of Child Health and Development (P01-042774441) (Hannah Kinney); and Intellectual and Developmental Disabilities Research Center, Boston Children’s Hospital, Boston, MA (P30-HD18655).
INTRODUCTION Sudden infant death syndrome (SIDS) is defined as the sudden death of an infant younger than 12 months that is related to a sleep period and remains unexplained after a complete autopsy, death scene investigation, and review of the clinical history (1). It is the leading cause of postneonatal infant mortality in westernized countries today (2). During the last 2 decades, our group has provided mounting evidence for serotonergic (5-HT) pathology in the homeostatic network of the medulla oblongata in a majority of SIDS infants (3Y5). In 4 independent studies, we have reported altered 5-HT receptor binding (3, 5, 6) in autonomic- and respiratory-related nuclei of the medulla that are composed of 5-HT neurons and/or receive 5-HT projections, that is, the so-called medullary 5-HT homeostatic network (7), in SIDS cases. Moreover, other investigators have confirmed the finding of medullary abnormalities in 5-HT receptors in SIDS infants by immunocytochemical methods (8, 9). Furthermore, we demonstrated a significant reduction in SIDS of 5-HT itself and tryptophan (Trp) hydroxylase 2 (TPH2), the major biosynthetic enzyme for 5-HT synthesis, in two 5-HT source nuclei with altered 5-HT1A binding in SIDS, that is, the raphe obscurus and paragigantocellularis lateralis (3). These results point to a deficiency of 5-HT in the pathogenesis of the medullary 5-HT pathology in affected SIDS cases. The medullary 5-HT network plays a key role in mediating protective responses to homeostatic stressors such as hypercarbia, hypoxia, and asphyxia (7, 10, 11). Thus, defects in this system may impair the SIDS infant’s responses to such stressors, for example, asphyxia in the prone (face down) sleep position, thereby leading to sudden death in a critical developmental period (12, 13). Although 5-HT abnormalities are not the only reported neurochemical abnormalities in SIDS brainstems, they are among the most robust and reproducible (11), and these findings have suggested our formulation that at least a subset of SIDS is part of a spectrum of ‘‘developmental serotonopathies’’ that include known inborn errors of 5-HT metabolism, autism, and prenatal exposure to selective serotonergic reuptake inhibitors (10). The objective of this study was to determine if levels of 5-HTYrelated parameters are decreased in the cerebrospinal fluid (CSF) of SIDS infants as a potential reflection of reported 5-HT abnormalities in the medulla in SIDS (3, 5). Currently, the determination of the medullary 5-HT abnormalities in the
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medulla in SIDS infants is highly specialized, labor intensive, and expensive, for example, tissue receptor autoradiography in multiple frozen sections of the medulla and biochemical assays of micropunched samples of frozen medulla. Thus, markers of these abnormalities in a more readily studied source at autopsy, such as CSF, with a cost-effective and standard technique, such as high-performance liquid chromatography (HPLC), would be of considerable value in the forensic identification of infants who die suddenly with a 5-HT abnormality. We further reasoned that new information accrued in this study about the methodology of 5-HTYrelated measurements in human CSF and their developmental profile would be directly relevant to multiple neurologic disorders involving 5-HT beyond the SIDS problem (10). The major 5-HTYrelated parameters that can be readily measured in human CSF are 5-hydroxyindoleacetic acid (5-HIAA) and Trp, the essential amino acids involved in 5-HT synthesis, both using clinically available HPLC techniques. In this study, we tested the hypothesis that 5-HIAA and/or Trp levels in the CSF are decreased in SIDS infants compared with those in controls dying of known causes. We reasoned that the decreased tissue levels of 5-HT in the medullary (raphe and extraraphe) nuclei we previously reported in SIDS infants result in a secondary decrease of tissue 5-HIAA (3) and, thus, its decreased release into the CSF with decreased levels in that compartment. Because we did not previously find any differences in dopamine metabolite levels or turnover directly in the medullary 5-HT system in SIDS cases compared with controls (3), we hypothesized here that there are no differences in dopamine metabolites in the CSF in SIDS versus non-SIDS groups. In addition, we tested the hypothesis that 5-HIAA and Trp levels in the CSF in SIDS infants are significantly different from those of infants dying with unequivocal asphyxia to determine if the putative 5-HTYrelated alterations in SIDS are potentially secondary to terminal asphyxia. To further explore the potential effects of hypoxia/ anoxia on 5-HIAA and Trp levels, we also included a group of SIDS infants who had a cardiopulmonary arrest and anoxic encephalopathy and were ventilated mechanically for 6 to 31 hours before death. Information about the potential effects of terminal hypoxia-ischemia on monoaminergic levels in postmortem CSF is of importance in its own right in assessing such levels in human autopsy populations across the age spectrum. We further tested the hypotheses that there are no significant differences between 5-HIAA and/or Trp levels in the CSF between infants who are adjudicated as SIDS and those adjudicated as ‘‘undetermined’’ (defined in Materials and Methods), and that both groups have levels significantly decreased from those of infants dying of known causes of death. Our hypothesis is based on the premises that 1) this undetermined group of sudden and unexplained deaths share common underlying abnormalities with those of the SIDS group, 2) the distinction between the 2 groups is largely a semantic one, and 3) the 2 groups share a common biomarker such as altered 5-HIAA and/or Trp levels in the CSF. Finally, we were interested in determining if any SIDS case met the diagnostic screening criteria for a known inborn error of 5-HT metabolism, thereby potentially unmasking a subgroup of infants
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with genetic bioaminergic disorders that present as sudden death in seemingly well infants (14).
MATERIALS AND METHODS Clinical Database All CSF specimens were obtained between 2004 and 2011 from the San Diego County Medical Examiner’s Office, San Diego, CA. Samples were accrued in accordance to California law Chapter 955, Statutes of 1989 (SB1069). This law permits the use of autopsy tissues from infants with sudden death for research without parental consent. The SIDS cases were defined as above (1). ‘‘Acute’’ controls (ACs) were infants younger than 12 months who died unexpectedly (in some instances with a minor or acute illness within 48 hours of death) and in whom an autopsy and death scene investigation established a known cause of death (15). ‘‘Chronic’’ controls (CCs) were infants younger than 12 months who had clinical chronic illnesses but who nevertheless died suddenly and unexpectedly; a complete autopsy confirmed the presence of a chronic illness (15). The acute asphyxia (AA) group was defined as infants younger than 12 months in whom the death scene investigation determined unequivocally that there was an acute lethal asphyxia event (e.g. overlaying, crib accident) and the autopsy itself did not demonstrate a chronic or lethal intrinsic disease process. The resuscitated SIDS (RSIDS) group was defined as infants younger than 12 months who were discovered unresponsive, underwent successful cardiopulmonary resuscitation, and were subsequently ventilated; these infants were withdrawn from the ventilator because of severe postarrest anoxic encephalopathy and were classified as SIDS at autopsy. The undetermined (UND) group was defined as infants younger than 12 months in whom the findings at autopsy or death scene may have contributed to death, but their causative role in death was uncertain (e.g. bed sharing). Clinicopathologic variables were recorded on review of autopsy and death scene investigation reports, including gestational age, race, sex, and circumstances of death (e.g. position found on discovery). The diagnosis in each case and control was made without knowledge of the CSF data. The Committee on Clinical Investigation at Boston Children’s Hospital, Boston, MA, approved this study.
Neurotransmitter-Related Measurements in CSF The site of CSF collection was either posterior lumbar subarachnoid space or cisterna magna. On collection, all CSF samples were stored at j80-C until analysis. Obviously bloody specimens were excluded from biochemical analysis because of red blood cell contamination. Samples were thawed on ice and centrifuged at 3,500 g at 4-C for 10 minutes. Reverse-phase HPLC with electrochemical detection was performed on CSF supernatants for 5-HIAA and homovanillic acid (HVA) analyses at Medical Neurogenetics, Atlanta, GA, with internal standards, according to previously published protocols (16, 17). Measurements of the CSF levels of Trp and tyrosine (Tyr) were performed in the same set of SIDS cases and non-SIDS controls at Medical Neurogenetics by reverse-phase HPLC with fluorescence detection (18). All Ó 2014 American Association of Neuropathologists, Inc.
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bioaminergic CSF measurements were made without knowledge of diagnosis, age, or other clinical factors.
Statistical Analysis Descriptive statistics were performed with regard to demographic information and study measures for each of the study groups (SIDS and non-SIDS). A one-way analysis of variance was used for the comparison of age and postmortem interval (PMI) between the different groups, and the Fisher exact test was used for the comparison of the percentage of sex and prematurity between groups. Models of analysis of covariance (ANCOVA) were used to compare the levels of 5-HIAA, HVA, Trp, and Tyr between the SIDS group and various non-SIDS groups, separately and combined, controlling for postconceptional age (PCA) and PMI. A TukeyKramer adjustment was used to account for the multiple comparisons being made within each model.
RESULTS Clinical Database The total data set consisted of 86 cases, with the following classification: SIDS, n = 52; AC, n = 9; CC, n = 2; AA, n = 8; RSIDS, n = 5; and UND, n = 10 (Table 1). There was no significant difference in PCA, gestational age, postnatal age, percentage of preterm birth or sex, or PMI among the study groups (Table 1). The median PMI for each of the groups was less than 24 hours (data not shown), with a range of 5.5 to 37 hours (only 1 case longer than 30 hours). There was no significant effect of increasing PMI on CSF levels of 5-HIAA (Fig. 1), in contrast to adverse effects on Trp and Tyr levels within certain groups (see below). The causes of death in the non-SIDS groups and the circumstances of death in the AA group are provided in Table 2. All 86 cases were included in the analysis of 5-HIAA, HVA, Trp, and Tyr (Tables 3Y6, respectively). There was no effect of increasing age for any of the 4 measures (5-HIAA, HVA, Trp, Tyr) in any group or all groups combined (Fig. 2) (Trp and Tyr not shown).
Analysis of CSF Serotonin Metabolites in SIDS
Measurements of 5-HIAA in the CSF of SIDS Versus Non-SIDS Groups There were no significant differences in mean 5-HIAA levels between the SIDS group and the AC, CC, AA, RSIDS, and UND groups (p 9 0.05) for all 5 comparisons (Table 3). In a separate ANCOVA model, there was no significant difference in 5-HIAA levels between the SIDS group, non-SIDS group (defined as combined AC, CC, and AA groups), RSIDS group, and UND group, adjusting for PCA and PMI (data not shown). Although there was a minimal reduction (9%) in 5-HIAA levels on average in the SIDS group versus nonSIDS groups, it was not statistically significant (p = 0.824).
Measurements of Trp in the CSF of SIDS Versus Non-SIDS Groups There was a significant positive correlation in the nonSIDS group between Trp and PMI (correlation coefficient [r] = 0.48, p = 0.037), as previously suggested in human CSF autopsy analysis (19), and there were nonsignificant positive correlations in the SIDS (r = 0.14, p = 0.313), RSIDS (r = 0.03, p = 0.965), and UND (r = 0.53, p = 0.113) groups. Using an ANCOVA model to compare the groups while adjusting for PCA and PMI, we found no significant differences in mean Trp levels between the SIDS cases and the AC, CC, AA, RSIDS, and UND groups (p 9 0.05 for all 5 comparisons) (Table 4).
Measurements of HVA in the CSF of SIDS Versus Non-SIDS Groups In fitting an ANCOVA model for the HVA data, a significant PCA-diagnosis interaction (p G 0.0001) was observed. The difference between HVA and diagnoses, adjusting for PMI, varied with PCA, with an increase in HVA levels with increasing age (data not shown). In exploring this interaction further, we found that it was caused by 2 outliers: 1 case from the RSIDS group (HVA, 15,420 nmol/L; PCA, 67 weeks) and 1 case from the UND group (HVA, 15,520 nmol/L; PCA, 82 weeks). Exclusion of these 2 outliers eliminated the significant PCA-diagnosis interaction (Fig. 2). There were significant
TABLE 1. Demographics of the Entire Study Cohort Male n SIDS 52 AC 9 CC 2 AA 8 RSIDS 5 UND 10 Non-SIDS controls (AC + CC + AA) 19 p value comparing demographic by diagnosis
Premature (GA G37 weeks)
Mean (SD)
n (%)
n (%)
PCA, Mean (SD), weeks
GA, weeks
PNA, weeks
PMI, hours
25 (48) 7 (78) 2 (100) 3 (38) 2 (40) 7 (70) 12 (63) 0.27
6 (12) 2 (22) 1 (50) 2 (25) 1 (20) 2 (20) 5 (26) 0.37
52.7 (8.6) 50.6 (12.2) 40.8 (3.2) 54.8 (11.7) 57.5 (8.0) 53.0 (14.0) 51.3 (11.8) 0.45
39.0 (3.1) 38.7 (1.9) 38.5 (3.5) 38.3 (3.2) 38.2 (2.7) 37.8 (4.1) 38.5 (2.5) 0.91
13.9 (8.1) 11.9 (10.9) 2.3 (0.4) 16.5 (11.4) 19.3 (8.0) 15.1 (12.5) 12.8 (11.1) 0.31
22.2 (5.4) 21.1 (8.1) 23.5 (4.9) 16.5 (8.8) 16.7 (2.2) 20.1 (8.3) 19.4 (8.2) 0.14
A one-way analysis of variance was used for the comparison of age and PMI between the different groups; Fisher exact test was used for the comparison of the percentage of sex and prematurity between groups. AA, acute asphyxia; AC, acute controls; CC, chronic controls; GA, gestational age; n, sample size; PCA, postconceptional age; PMI, postmortem interval; PNA, postnatal age; RSIDS, resuscitated SIDS; UND, undetermined cases.
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FIGURE 1. There is no significant effect of increasing postmortem interval (PMI) on CSF levels of either 5-hydroxyindoleacetic acid (5-HIAA) levels (left) or homovanillic acid (HVA) (right) in the SIDS or non-SIDS groups (the latter composed of acute controls, chronic controls, and acute asphyxia cases combined). Solid circles, SIDS; open circles, non-SIDS.
differences in HVA levels among the pairwise comparisons between the SIDS and AC groups and between the AA and AC groups (Table 5). On excluding the single outlier (HVA, 11,070 nmol/l) in the AC group from the analyses, the significant differences disappeared. There was no significant difference in HVA levels between the SIDS and AA groups (p = 0.989) (Table 5). A separate ANCOVA model was performed to compare HVA levels between the SIDS, non-SIDS
(AC, CC, and AA combined), RSIDS, and UND groups, adjusting for PCA and PMI.
Measurements of Tyr in the CSF of SIDS Versus Non-SIDS Groups There was a significant positive correlation of Tyr with PMI in the non-SIDS group (r = 0.50, p = 0.034), as previously suggested in human CSF autopsy analysis (19). There
TABLE 2. Demographic Information and Cause of Death in Acute Controls, Chronic Controls, and Acute Asphyxial Group Acute Controls
Sex
GA, weeks
PNA, weeks
PCA, weeks
M F M M M M M M M
40 40 35 36 38 40 40 39 36
21.0 3.5 2.0 0.3 15.5 11.0 24.5 1.0 2.5
61.0 43.5 37.0 36.3 53.5 51.0 64.5 40.0 38.5
Hyperthermia (excessive bedding) Histiocytoid cardiomyopathy Bronchopulmonary dysplasia secondary to lung disease of prematurity Fatty acid oxidation disorder Acute aspiration pneumonia Acute bacterial meningitis Drowning Acute gastrointestinal illness of unknown etiology with dehydration Congenital heart defect (ventricular septal defect and bicuspid aortic valve with stenosis) with minimal acute symptoms preceding death
Chronic controls 1 2
M F
41 40
2.0 28.5
43.0 68.5
Disseminated herpes simplex type II viral infection Hemolytic anemia
Acute asphyxia 1 2 3 4 5 6 7 8
M F F F M F M F
40 40 36 40 39 31 40 40
9 13 32 23 6 12 33 4
49 53 68 63 45 43 73 44
1 2 3 4 5 6 7 8 9
Cause of Death
Findings on Death Scene Investigation Found face down in mattress with lower body on ‘‘boppy’’ pillow (feet in air) Found with head inside pillow case between two pillows Found with body wedged between bed springs and air mattress Found with head covered by adult pillow Found between arm and torso of intoxicated mother in adult bed Found face down in unsafe sleeping environment Found with head wrapped in blanket Found with face and head wedged between cosleeper and sofa back
F, female; GA, gestational age; M, male; PCA, postconceptional age; PNA, postnatal age.
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Analysis of CSF Serotonin Metabolites in SIDS
FIGURE 2. The levels of 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) across all groups are relatively constant over infancy, the age range studied. Solid circles, SIDS; open circles, non-SIDS group; gray circles, resuscitated SIDS (RSIDS).
were nonsignificant positive correlations in the SIDS (r = 0.16, p = 0.250), RSIDS (r = 0.38, p = 0.522), and the UND (r = 0.48, p = 0.161) groups. Using an ANCOVA model to compare the groups while adjusting for PCA and PMI, we found no significant differences in mean Tyr levels between the SIDS cases and the AC, CC, AA, RSIDS, and UND groups (p 9 0.05 for all 5 comparisons) (Table 6).
DISCUSSION
To determine if the entire SIDS cohort contained cases with low 5-HIAA and HVA levels in the range of those found in known inborn errors of monoamine metabolism, we compared the 5-HIAA and HVA levels published in inborn errors (20, 21) as a reference. There were no SIDS cases that demonstrated combined low levels of 5-HIAA and HVA within this disease reference range (Fig. 3).
Measurement of 5-HIAA and Trp in CSF provides an important tool in clinical practice for detecting and monitoring disorders of brain 5-HT levels and metabolism (16Y18). The relevance of these tools to SIDS, as well as to multiple other neurologic disorders known to involve 5-HT in children (10, 14, 18, 21Y25) and adults (26Y31), is of interest. In this study, we did not find a significant difference in the two standard measures of 5-HT metabolism in human CSF, that is, 5-HIAA and Trp, between SIDS cases and infants dying of a range of acute and chronic disorders. Thus, our data suggest that 5-HIAA or Trp is not a reasonable postmortem biomarker for SIDS, and that the need to correlate CSF levels with medullary tissue 5-HT markers in the same SIDS cases is negated. We also did not find a difference in dopaminergic markers between the SIDS and non-SIDS cases as anticipated because we did not find differences in dopaminergic parameters in
TABLE 3. 5-HIAA Comparisons by Diagnosis, Adjusting for PCA and PMI
TABLE 4. Trp Comparisons by Diagnosis, Adjusting for PCA and PMI
Analysis for Potential Known Inborn Errors of 5-HT Metabolism
Trp Levels
5-HIAA Adjusted Mean (SE), nmol/L SIDS, n = 52 AC, n = 9 CC, n = 2 AA, n = 8 RSIDS, n = 5 UND, n = 10
1,191.8 (67.6) 1,399.9 (161.0) 1,546.8 (347.8) 1,126.7 (175.1) 1,150.3 (220.5) 904.7 (152.6)
Pairwise Comparisons Between Groups of Interest (p) SIDS vs AC (0.839) SIDS vs CC (0.915) SIDS vs AA (0.999) SIDS vs RSIDS (1.000) SIDS vs UND (0.526) AC vs CC (0.999) AC vs AA (0.861)
A Tukey-Kramer adjustment was used to account for the multiple comparisons. The p values are in parentheses. There was no difference in 5-HIAA levels between the SIDS and non-SIDS (AC, CC, and AA groups combined) (p = 0.824). 5-HIAA, 5-hydroxyindoleacetic acid; AA, acute asphyxia; AC, acute controls; CC, chronic controls; PCA, postconceptional age; PMI, postmortem interval; RSIDS, resuscitated SIDS; UND, undetermined cases.
Adjusted Mean (SE), Kmol/L SIDS, n = 52 AC, n = 9 CC, n = 2 AA, n = 8 RSIDS, n = 5 UND, n = 10
52.1 (2.8) 55.0 (6.6) 50.1 (14.2) 51.2 (7.2) 45.8 (9.0) 47.0 (6.2)
Pairwise Comparisons Between Groups of Interest (p) SIDS vs AC (0.999) SIDS vs CC (1.000) SIDS vs AA (1.000) SIDS vs RSIDS (0.985) SIDS vs UND (0.975) AC vs CC (1.000) AC vs AA (0.999)
A Tukey-Kramer adjustment was used to account for the multiple comparisons. The p values are in parentheses. There was no difference in Trp levels between SIDS and non-SIDS (AC, CC, and AA groups combined) (p = 0.999). AA, acute asphyxia; AC, acute controls; CC, chronic controls; PCA, postconceptional age; PMI, postmortem interval; RSIDS, resuscitated SIDS; Trp, tryptophan; UND, undetermined cases.
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TABLE 5. HVA Comparisons by Diagnosis Adjusting for Postconceptional Age and Postmortem Interval HVA Levels Adjusted Mean (SE), nmol/L SIDS, n = 52 AC, n = 9 CC, n = 2 AA, n = 8 RSIDS, n = 5 UND, n = 10
1,557.7 (177.4) 3,305.2 (423.0) 2,321.9 (915.6) 1,245.9 (462.4) 2,755.1 (645.3) 914.2 (424.0)
Pairwise Comparisons Between Groups of Interest (p) SIDS vs AC (0.004) SIDS vs CC (0.963) SIDS vs AA (0.989) SIDS vs RSIDS (0.485) SIDS vs UND (0.729) AC vs CC (0.923) AC vs AA (0.019)
A Tukey-Kramer adjustment was used to account for the multiple comparisons. The p values are in parentheses. There was no difference in HVA levels between the SIDS and non-SIDS (AC, CC, and AA groups combined) (p = 0.130). AA, acute asphyxia; AC, acute controls; CC, chronic controls; HVA, homovanillic acid; PCA, postconceptional age; PMI, postmortem interval; RSIDS, resuscitated SIDS; UND, undetermined cases.
medullary tissues between SIDS and control (3). We also did not find significant differences in 5-HIAA, Trp, HVA, or Tyr measures between the UND and the SIDS group, indicating that these metabolites in the CSF do not provide a distinguishing biomarker. Of note, there were no changes in the levels of any bioaminergic CSF parameter analyzed in the SIDS cases or controls within the first year of life, suggesting that this time frame is not a critical developmental period for these CSF parameters. Irrespective of the SIDS problem, this study also provides information directly relevant to assessment of monoaminergic metabolites in postmortem human CSF, including potential effects of terminal hypoxia-ischemia on them, as highlighted below.
Serotonergic Parameters in the CSF of SIDS Versus Non-SIDS Groups Synthesis of 5-HT in the brain occurs in raphe and extraraphe neurons of the entire brainstem through the conversion of the precursor molecule Trp to 5-hydroxytryptophan by TPH2 and then to 5-HT via the enzymatic action of aromatic
FIGURE 3. There are no SIDS cases in the entire data set (n = 52) that demonstrate combined low levels of 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) within the reference levels of known inborn errors of serotonin (5-HT) metabolism, indicated in the shaded rectangle in the lower left portion of the graph (21). LYamino acid decarboxylase, which has 5-hydroxytryptophan and L-DOPA as substrates (22). Tryptophan is also metabolized
in the kynurenine pathway and used for protein synthesis (31). Serotonin is degraded to 5-HIAA by monoamine oxidase (32). Terminals from the raphe 5-HT source neurons in the brainstem form supra- and subependymal plexuses around the lining of the fourth ventricle and release extrasynaptic 5-HT, including directly into the CSF (Fig. 4) (33, 34). In addition, 5-HIAA in the brain extracellular space diffuses into the CSF, such that this compartment serves as a so-called 5-HIAA sink (35). Serotonin itself cannot reliably be measured in the CSF because its levels are constitutively negligible (i.e. G10 pg/mL) (36). Nevertheless, the CSF concentration of 5-HIAA correlates with 5-HT levels and turnover in brain parenchyma (37, 38). Tryptophan crosses the blood-brain barrier (in competition with other large neutral amino acids) for incorporation into brain 5-HT (39, 40). It is not entirely surprising that we did not find reduced 5-HIAA levels in the CSF in SIDS cases because we did not previously observe significantly
TABLE 6. Tyr Comparisons by Diagnosis Adjusting for PCA and PMI Tyr Levels Adjusted Mean (SE), Kmol/L SIDS, n = 52 AC, n = 9 CC, n = 2 AA, n = 8 RSIDS, n = 5 UND, n = 10
224.0 (11.2) 254.2 (26.8) 248.0 (81.7) 208.6 (29.1) 211.3 (36.6) 198.7 (25.4)
Pairwise Comparisons Between Groups of Interest (p) SIDS vs AC (0.902) SIDS vs CC (1.000) SIDS vs AA (0.997) SIDS vs RSIDS (1.000) SIDS vs UND (0.943) AC vs CC (1.000) AC vs AA (0.859)
A Tukey-Kramer adjustment was used to account for the multiple comparisons. The p values are in parentheses. There was no difference in Tyr levels between SIDS and non-SIDS (AC, CC, and AA groups combined) (p = 0.966). AA, acute asphyxia; AC, acute controls; CC, chronic controls; PCA, postconceptional age; PMI, postmortem interval; RSIDS, resuscitated SIDS; Tyr, tyrosine; UND, undetermined cases.
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FIGURE 4. Diagram of the cerebrospinal fluid (CSF) ‘‘sink’’ of tissue 5-hydroxyindoleacetic acid (5-HIAA). The 5-HIAA in the CSF diffuses freely from the brainstem parenchyma or, alternatively, represents degradation of serotonin (5-HT) released directly into the CSF at the ependymal plexuses of the 5-HT terminals of 5-HT source neurons in the raphe and extraraphe regions of the entire brainstem. Ó 2014 American Association of Neuropathologists, Inc.
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J Neuropathol Exp Neurol Volume 73, Number 2, February 2014
reduced 5-HIAA or 5-HT/5-HIAA levels in the medullary tissue in SIDS infants. Our inability to detect a significant reduction in Trp in the CSF in SIDS cases suggests that the reduction in medullary 5-HT may be caused by TPH2 deficiency and not to the dietary availability of Trp or the capability to transport Trp across the blood-brain barrier. Two previous studies reported significantly increased levels of 5-HIAA, HVA, Trp, and Tyr in the CSF of SIDS infants compared with those of controls (41, 42). Our lack of confirmation of these studies may reflect a discrepancy in the control populations, that is, the previous studies mainly involved CSF measurements in living control infants for comparison, whereas our study only incorporated autopsy controls. In a previous report, the metabolite levels were similar between 10 autopsy controls and 28 living controls, and the SIDS values were elevated relative to these two control populations (42). We found, however, that the autopsy controls in our study were more than 3-fold higher than published values of living controls (20), suggesting that autopsy and living control values cannot be combined. These data need to be considered in selecting controls for CSF assessments in other pediatric (10, 14, 18, 21Y25) and adult (26Y31) disorders affecting central monoaminergic metabolism.
Potential Relationship of CSF Bioaminergic Biomarkers to Asphyxia The potential effect of asphyxia/hypoxia on CSF levels of 5-HIAA and HVA is of concern in SIDS research because of experimental reports of abnormal brainstem measures of 5-HT (and dopaminergic) parameters secondary to hypoxiaischemia (3, 23) and elevated CSF levels in adults who have undergone successful cardiopulmonary resuscitation after cardiac arrest (30). Thus, interpretation of potentially altered levels in SIDS infants must account for (possible) asphyxia engendered in an unsafe sleeping environment (7, 10, 11). However, we found no significant differences in 5-HIAA or Trp (or Tyr) levels in the CSF between the SIDS and AA groups. There were also no differences in these metabolite concentrations between the SIDS and RSIDS groups, the latter group with potential secondary chronic (6Y31 hours) hypoxic-ischemic injury complicating the putative intrinsic defect.
Analysis of Potential Inborn Errors of 5-HT Metabolism Measures of 5-HIAA and HVA are used to clinically screen for inborn errors of 5-HT metabolism, including a genetic deficiency of LYamino acid decarboxylase (14). In this study, however, we found no SIDS outliers with significant decreases in combined 5-HIAA and HVA levels versus controls. Thus, these data suggest that infants with known inborn errors of 5-HT metabolism are not likely embedded within the SIDS population. Infants with known inborn bioaminergic errors are recognized by a clinical phenotype of mild to severe developmental delay, dystonia, and seizures (14, 24); they may be associated with autonomic dysfunction, such as temperature instability and hypotension, with rare reports of cardiorespiratory arrest (14). Nevertheless, sudden death in a clinically healthy infant, as in SIDS, has not been described.
Analysis of CSF Serotonin Metabolites in SIDS
Potential Limitations of the Study The potential compromise of the validity of postmortem measures of 5-HT parameters by lengthy PMIs (the definition of ‘‘lengthy’’ may be within minutes or even seconds after death) is of concern. Importantly, our data indicate no change in 5-HIAA and HVA levels with an increasing PMI between 5.5 and 37 hours. This observation is supported by the report of stable levels up to a PMI of 30 hours (37). Still, our finding that the 5-HIAA and HVA levels in the entire autopsy data set (SIDS and non-SIDS controls) were more than 3-fold higher than that of published values for living infants and children (irrespective of neurologic disease) (20) suggests that rapid changes in these parameters occur immediately after death. Thus, comparison of an autopsy study group must be made to an appropriate autopsy control group, not living control standards. A second potential limitation of this study is that CSF was obtained at autopsy by two approaches, that is, lumbar puncture and cisternal tap, which were not always specifically documented. Because an uneven concentration gradient in CSF metabolites has been reported in older children and adults, including at autopsy (37, 43), values may vary by rostral or caudal site. Nevertheless, this putative gradient may not be present in infants; a 5-HIAA and HVA gradient was not observed in 6 living infants who underwent neurosurgery for hydrocephalus (25).
CONCLUSIONS We report no alterations in 5-HTYrelated parameters in the CSF of SIDS infants compared with those of infants dying of various disorders, including asphyxia. The finding of unaltered levels of 5-HIAA in SIDS is consistent with our previous report of likewise unaltered levels of this metabolite in medullary tissues of SIDS infants with altered 5-HT levels (3). Thus, this study does not negate the 5-HT brainstem hypothesis in SIDS. This study focuses the ongoing search for 5-HT biomarkers of 5-HT medullary pathology in SIDS infants in other readily accessible tissue compartments, notably in serum and platelets, or with different 5-HTYrelated markers (e.g. TPH2, 5-HT1A receptor protein by proteomics) in future studies. It also provides important information about the methodology of monoaminergic measurements in human CSF at autopsy, including considerations related to PMI and terminal hypoxia-ischemia at autopsy, as well as their developmental profile in infancy that is applicable to multiple disorders of the pediatric and adult brain beyond SIDS.
ACKNOWLEDGMENTS The authors are grateful for the dedicated assistance in this study of the medical examiners of the San Diego County Medical Examiner’s Office, San Diego, CA. The authors also appreciate the helpful input of Dr Siri Hauge Opdal and Professor A˚shild Vege, University of Oslo, Oslo, Norway, in the course of the study. The authors thank Drs Eugene E. Nattie and Richard D. Goldstein for critical reading of the manuscript in preparation.
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