Metabolomic Profiling of Serum Retinol in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study

The role of retinol in the prevention of multifactorial chronic diseases remains uncertain, and there is sparse evidence regarding biological actions and pathways implicated in its effects on various outcomes. The aim is to investigate whether serum retinol in an un-supplemented state is associated with low molecular weight circulating metabolites. We performed a metabolomic analysis of 1,282 male smoker participants based on pre-supplementation fasting serum in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study. We examined the association between 947 metabolites measured by ultra-high performance LC-MS/GC-MS and retinol concentration (from HPLC) using linear regression that estimated the difference in metabolite concentrations per unit difference in retinol concentration as standardized β-coefficients and standard errors (SE). We identified 63 metabolites associated with serum retinol below the Bonferroni-corrected P-value (p < 5.3 × 10–5). The strongest signals were for N-acetyltryptophan (β = 0.27; SE = 0.032; p = 9.8 × 10−17), myo-inositol (β = 0.23; SE = 0.032; p = 9.8 × 10−13), and 1-palmitoylglycerophosphoethanolamine (β = 0.22; SE = 0.032; p = 3.2 × 10−12). Several chemical class pathways were strongly associated with retinol, including amino acids (p = 1.6 × 10−10), lipids (p = 3.3 × 10–7), and cofactor/vitamin metabolites (3.3 × 10−7). The strongest sub-pathway association was for inositol metabolism (p = 2.0 × 10–14). Serum retinol concentration is associated with circulating metabolites in various metabolic pathways, particularly lipids, amino acids, and cofactors/vitamins. These interrelationships may have relevance to the biological actions of retinol, including its role in carcinogenesis.


Discussion
This large-scale agnostic investigation of nearly 1,300 men identified 63 metabolites in several chemical classes associated with serum retinol concentrations after stringent correction for 947 retinol-metabolite comparisons. Amino acids and lipids were most highly represented among the top metabolites, with N-acetyltrytophan and myo-inositol (followed by the lipid 1-palmitoylglycerophosphoethanolamine) ranked first within these two chemical classes, respectively. In addition, N6-carbamoylthreonyladenosine, erythronate, erythritol, adsgegdfxaegggvr, and succinylcarnitine were represented as the top metabolites within the nucleotide, carbohydrate, xenobiotic, peptide, and energy metabolite classes. N-acetyltryptophan prevents oxidative degradation of proteins 33 , and the tryptophan/kynurenine pathway influences the regulation of inflammation, oxidative stress and immune activation 34,35 . The relevance of several other N-acetyl amino acids among the top metabolites (e.g., N-acetyl-3-methylhistidine, -valine, −1-methylhistidine, -lysine, and -threonine) is unclear, but may indicate perturbations in acetylation activity and influence on cell growth through histone-chromatin function and gene regulation [36][37][38] .
Retinol plays an important role in lipid metabolism through activation of RAR and PPARβ/δ signaling and gene expression enhancing lipid accumulation through control of adipocyte differentiation, fatty acid oxidation, and lipolysis [39][40][41][42][43] . Myo-inositol ranked first among lipid metabolites associated with retinol, which along with its phosphorylated derivatives including inositol 1-phosphate (I1P), may be related to mediation of retinoic acid signaling and cellular functions including adhesion, growth, vesicular trafficking, and cell survival 44,45 , and is involved in regulation of enzyme activity and hormone secretion 46 . Through its role in phosphatidylinositol-4-phosphate (IP 4 ) and phosphatidylinositol-4,5-bisphosphate (IP 4, 5 ) biosynthesis, myo-inositol and I1P are plasma membrane mediators of protein phosphorylation and second messenger signaling 47 .
This cross-sectional analysis was conducted in order to both contribute to elucidation of the molecular basis of retinol's diverse biological activities and provide mechanistic clues related to how higher vitamin A status might adversely impact prostate (and other) cancer risk. In this regard, the present findings regarding inositol and lysolipid metabolites are of interest in that they (e.g., I1P) were associated with aggressive prostate cancer in our recent serum metabolomic analysis 66 . In addition, findings from our group based on size and extent of primary prostate cancer cases demonstrated a positive association with histidine metabolites including N-acetyl-3-methylhistidine, and 1-and 3-methylhistidine in T2 prostate cancers 67 . Some of the sex steroids related to serum retinol here were associated with non-aggressive prostate cancer the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial 68 .
Fibrinopeptide A is the only metabolite we found to be inversely associated with serum retinol in this study. Fibrinopeptide A is produced from fibrinogen by thrombin during blood coagulation, and it is elevated in several malignancies, with persistent coagulation activation, thrombosis and disseminated intravascular coagulation being common complications [69][70][71][72] . Given that some cancers have been related to vitamin A status, the inverse fibrinopeptide A-retinol association observed here should be examined in other studies.
To our knowledge, this is the first metabolomic profiling analysis of vitamin A status. Strengths of the analysis include its relatively large sample size, assaying of serum collected after an overnight fast, and high laboratory quality control. Some limitations should also be acknowledged, including that our study was cross-sectional, which limits our ability to make causal inferences about the association between metabolites and retinol levels. In addition, the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) cohort constitutes a relatively homogeneous population of Finnish male smokers of European ancestry, which may limit the generalizability of our findings to other populations. Residual confounding by unidentified metabolic or other factors such as chronic health conditions is possible, although we extensively adjusted our models for several potential confounders including diabetes, body mass index (BMI), serum cholesterol, smoking intensity, and alcohol consumption, and the findings were unchanged after adjusting for serum creatinine, an important indicator of kidney function.
Our study identified a large number of metabolites in various metabolic pathways associated with serum retinol concentration, some of which directly relate to prostate cancer risk associations and other known biological functions. Lipid and amino acid biochemical classes are heavily represented, which may provide some molecular insights into the role of retinol in human health and disease. In particular, lipid bioactivity related to inositol, lysolipids and sterol/steroid compounds, may potentially provide biological clues relevant to the positive association between circulating retinol and the development of prostate cancer. Further studies in diverse populations are warranted to re-examine these findings.

Materials and methods
Study population. The ATBC Study has been described in detail elsewhere 73 . In brief, it is a 2 × 2 factorial randomized controlled trial in which 29,133 male Finish smokers aged 50-69 were assigned to receive either alpha-tocopherol (dl-α-tocopheryl-acetate, 50 mg/day), beta-carotene (20 mg/day), both vitamins, or placebo. Participants received the study capsules for a median of 6.1 years (range 5-8 years) until the trial ended on 30 April 1993. Information on general risk factors, smoking, and medical history was obtained through a questionnaire at study entry along with a validated food-frequency questionnaire. Serum was collected at baseline (i.e., pre-supplementation and years prior to cancer diagnoses) after overnight fast was protected from light and stored at −70 °C until assayed. Incident cancer diagnosed during 20 years of follow-up were identified from the Finnish Cancer Registry. All participants provided written informed consent. The trial was approved by institutional  Table 3. Gene set analysis for chemical class of metabolites for serum retinol review boards at the U.S. National Cancer Institute and the Finnish National Public Health Institute. All methods were performed in accordance with the relevant guidelines and regulations. The present analysis is based on participants included in seven on-going or completed analyses (case-control and other, subsequently referred to as "metabolomic sets") nested within the ATBC Study 66,74,75 . These included cases of prostate, lung, esophagus, and stomach cancer, along with their controls, as well as controls from a pancreatic cancer study. After excluding duplicate participants, 1,282 were included in the present analysis. Laboratory analysis. Baseline serum retinol concentration was measured using an isocratic high-performance liquid chromatography platform 76 . Assays were carried out from 1986-88 in a dedicated laboratory at the National Public Health Institute in Helsinki, Finland, which was certified by a National Institute of Standards and Technology quality-control testing program. Serum samples were subsequently analyzed at Metabolon, Inc. (Durham, N.C.) using ultrahigh performance liquid chromatography (LC)/mass spectroscopy (MS) and gas chromatography (GC)/mass spectrometry (MS) as described before 66,74,75 . The methods of sample accessioning, sample preparation, quality control, data extraction and compound identification have also been described in detail 77 . Briefly, each of the samples were analyzed using three different analytical conditions: ultrahigh performance LC-MS/MS ( + electrospray ionization (ESI)), ultrahigh performance LC-MS/MS (-ESI), and GC-MS. To remove proteins, dissociate small molecules bound to protein or trapped in the precipitated protein matrix, and to recover chemically diverse metabolites, each of the samples were extracted using an aqueous methanol extraction procedure. The methanol contained four recovery standards (DL-2-fluorophenylglycine, tridecanoic acid, d6-cholesterol and 4-chlorophenylalanine) to allow confirmation of extraction efficiency. For each sample, four aliquots were obtained from the extract and dried. Two aliquots of each sample were then reconstituted in 50 µl of 6.5 mM ammonium bicarbonate in water (pH 8) for the negative ion analysis, and the other two aliquots of each were reconstituted using 50 µl 0.1% formic acid in water (pH ~3.5) for the positive ion analysis. The resulting extracts were divided into fractions for analysis by ultrahigh performance LC/MS/MS (positive mode), ultrahigh performance LC/MS/MS (negative mode), and GC/MS. Samples were placed briefly on a TurboVap ® (Zymark) to remove the organic solvent. Each sample was then frozen and dried under vacuum. The samples were then prepared for the appropriate instrument, either ultrahigh performance LC/MS/ MS or GC/MS. The raw data was further extracted, peak-identified and quality control (QC) processed using Metabolon's hardware and software. Internal controls included extraction process (5 recovery standards), injection (up to 11 standards), and alignment standards for quality assurance/quality control procedures to control for experimental variability. Compounds were identified by comparison to library entries of purified standards or recurrent unknown entities. Metabolon maintains a library based on authenticated standards that contains the retention time/index (RI), mass to charge ratio (m/z), and chromatographic data (including MS/MS spectral data) on all molecules present in the library. Furthermore, biochemical identifications are based on three  Table 4. Gene set analysis for chemical sub-class of metabolites for serum retinol at 8.5 × 10 −4 level of statistical significance.
criteria: retention index within a narrow RI window of the proposed identification, accurate mass match to the library + /− 0.005 amu, and the MS/MS forward and reverse scores between the experimental data and authentic standards. The MS/MS scores are based on a comparison of the ions present in the experimental spectrum to the ions present in the library spectrum. More than 2400 commercially available purified standard compounds have been acquired and used in both the LC and GC platforms for determination of their analytical characteristics 78 . Batch variability was standardized by dividing the signal strength by the batch median value for each metabolite and subject. Metabolite signal strength were log-transformed and centered according to normalization. Within each metabolomic set, metabolite values below the limit of detection were imputed to have the minimum of all non-missing values.
After excluding metabolites that had fewer than 10 non-missing values across all metabolomic sets, or single-value metabolites in individual metabolomic set, 947 identified compounds remained eligible for analysis. Based upon a standard chemical classification scheme, 920 of the metabolites were categorized in eight mutually exclusive chemical classes: amino acids and amino acid derivatives (referred to as "amino acids"), carbohydrates, cofactors and vitamins, energy metabolites, lipids, nucleotides, peptides, and xenobiotics. Blinded quality control samples (9%) from a pooled sample were included in each batch to assess the technical reliability of data, and coefficient of variation (CV) was calculated. The median and interquartile range of the CV% across the metabolites was 9% (4%-20%). These CVs are similar to those previously observed for blood samples analyzed by the same laboratory 77,78 , and previous studies have reported a high reliability for the metabolomics platform used in the present study 79 . Statistical analysis. We investigated the association between serum levels of retinol and each metabolite, using linear regression adjusted for age at randomization (continuous), BMI (continuous), case status (binary, case or control status in each metabolomic set), metabolomic sets (categorical), serum cholesterol (continuous), number of cigarettes per days (continuous), and baseline alcohol consumption (continuous). We further performed additional analysis to adjust for creatinine in the model. For each model, we computed the standardized beta-coefficient which represents the change in the level of each metabolite per 1-SD increase in the levels of retinol.
For all analyses, we used log-transformed metabolite concentrations. To control for multiple comparisons (n = 947 retinol-metabolite associations), we applied the Bonferroni correction 80 using p = 0.05/947 = 5.3 × 10 −5 as the adjusted significance threshold. We further performed sensitivity analyses additionally adjusted for storage time (from blood collection to metabolomics measurement).
Within each metabolomic set, we applied Gene-Set Analysis (GSA), which is a pathway analysis method, to examine whether pre-defined metabolic pathways including super-and sub-pathways were related to retinol within individual metabolomic sets 81 . For Z values (i.e. Z 1 to Z s ) tested from S metabolites in a pre-defined pathway, GSA examines the "maxmean" statistic max that is the average of all Z values (both positive and negative) and calculates the p values by 10,000 permutations. With the seven individual metabolomic sets, we used Fisher's method, namely sum of logs method, to combine p value for each pre-defined pathway.
Analyses were performed in SAS 9.4, and R 3.2.3. All statistical tests and reported p values are two-sided.