Participants

We identified 414 individuals in the Wisconsin Alzheimer’s Disease Research Center (ADRC) clinical core (n = 277) and the Wisconsin Registry for Alzheimer’s Prevention (WRAP) study (n = 137) who had undergone lumbar puncture with CSF collection, as well as TMAO and biomarker quantification. The ADRC clinical core study consists of participants who fall along the clinical continuum of cognitive function, including AD dementia, MCI, and cognitively-unimpaired controls. The WRAP study is a large (> 1500 subjects), ongoing (> 15 years), prospective longitudinal investigation of the genetic, biological, and lifestyle factors that contribute to the development of AD dementia and cognitive decline [18]. Individuals in the WRAP study were recruited as cognitively-unimpaired, asymptomatic middle-aged adults and undergo biannual comprehensive medical and cognitive evaluation. Because both the WRAP study and the ADRC clinical core are enriched for risk of late-onset AD (~ 70% of WRAP subjects have a parental family history of AD, and ~ 50% of participants 45–65 years old in the ADRC study have a parental history of AD), the APOE ε4 genotype is more prevalent. General exclusion criteria for the ADRC and WRAP studies include any significant neurologic disease (other than AD dementia), history of alcohol/substance dependence, major psychiatric disorders (including untreated major depression), or other significant medical illness. APOE ε4 genotyping procedures have been described previously [19], and participants were categorized as noncarriers (zero ε4 alleles) or APOE ε4 carriers (one or two ε4 alleles). The University of Wisconsin Health Science Institutional Review Board approved all study procedures, and all experiments were performed in accordance with relevant guidelines and regulations. All participants provided written informed consent to be involved in this study.

Diagnostic classification

Participants underwent a comprehensive neuropsychological battery to determine their cognitive status. Participants with MCI and AD dementia were diagnosed using available clinical and cognitive information in accordance with the updated 2011 National Institute on Aging–Alzheimer’s Association workgroup diagnostic criteria [20, 21]. All participants in the ADRC clinical core are discussed at a consensus review committee consisting of physicians, neuropsychologists, and nurse practitioners. Biomarker data are not used in determining clinical diagnosis. Participants in the WRAP study are reviewed selectively when flagged after cognitive abnormalities are detected by algorithm on neuropsychological tests, at which point cases are discussed at a consensus review committee meeting [18]. Of the 414 identified participants, four individuals with a diagnosis of nonneurodegenerative cognitive impairment at the time of CSF collection were excluded from the present analyses, resulting in a total of 410 participants: n = 335 cognitively-unimpaired participants (Control group), n = 35 MCI (MCI group), and n = 40 AD dementia (AD group).

Lumbar puncture and CSF collection

Lumbar puncture and CSF collection procedures have been described previously [22]. Briefly, CSF was collected via lumbar puncture in the morning after a 12-h fast with a Sprotte 25 or 24-gauge spinal needle at the L3/4 or L4/5 interspace using gentle extraction into propylene syringes. CSF (~ 22 ml) was then combined, gently mixed, and centrifuged at 2000 × g for 10 min. Supernatants were frozen in 0.5 ml aliquots in polypropylene tubes and stored at − 80 °C.

CSF biomarker quantification

CSF AD biomarkers included the Aβ 42 /Aβ 40 ratio, phosphorylated tau (p-tau), and the p-tau/Aβ 42 ratio. CSF Aβ is an indicator of amyloid burden, with greater amyloid deposition in the brain being reflected by lower levels in the CSF. The Aβ 42 /Aβ 40 ratio (which normalizes CSF Aβ 42 for the total amount of Aβ peptides that are present in CSF) was used given that it shows better correspondence with brain amyloid deposition as well as superior diagnostic performance compared to CSF Aβ 42 alone [23]. p-tau is a marker of tau phosphorylation believed to be associated with neurofibrillary tangle pathology, with higher levels reflecting a more intense tau phosphorylation process; the ratio of p-tau/Aβ 42 incorporates both facets of pathology, with higher values indicating greater AD pathology [24]. For the Aβ 42 /Aβ 40 ratio, CSF Aβ 42 and CSF Aβ 40 were quantified separately by electrochemiluminescence (ECL) using an Aβ triplex assay (MSD Human Aβ peptide Ultra-Sensitive Kit; Meso Scale Discovery, Gaithersburg, MD, USA). For p-tau and the p-tau/Aβ 42 ratio, CSF p-tau and Aβ 42 were quantified using commercially available sandwich ELISAs (INNOTEST β-amyloid1–42 and Phospho-Tau[181 P], respectively; Fujirebio Europe, Ghent, Belgium).

CSF biomarkers of neuronal degeneration included total tau (t-tau), neurofilament light chain protein (NFL, a marker of axonal degeneration), and neurogranin (a marker of synaptic degeneration). CSF t-tau and NFL were quantified using commercially available sandwich ELISAs: t-tau, INNOTEST hTau Ag (Fujirebio Europe); and NFL, NF-Light ELISA kit (Uman Diagnostics AB, Umeå, Sweden). CSF neurogranin was quantified using a sandwich ELISA as described previously [25]. All CSF assays were performed in two batches (n = 192 samples in batch 1, n = 218 samples in batch 2), and all statistical analyses accounted for batch variation (see Statistical analysis).

CSF TMAO quantification

CSF TMAO was quantified via an untargeted plasma metabolomics analysis performed by Metabolon, Inc. (Durham, NC, USA) using ultrahigh performance liquid chromatography tandem mass spectrometry (UHPLC-MS) as described previously [26] (details presented in Additional file 1: Methods). All samples were sent to Metabolon in one shipment. Raw data were extracted, peak identified, and QC processed using Metabolon’s hardware and software. TMAO levels were expressed as scaled intensity units (SIU) using the QC-processed mass-to-charge ratio (m/z) area-under-the-curve values for TMAO and scaled to a median value of 1.

Statistical analysis