Patients and sample collection
Untreated HIV versus non-HIV-infected controls, population A
For the comparison of metabolic profiles of untreated HIV-1-infected patients to a non-HIV infected control population, 18 HIV-1-infected, cART naïve patients were selected from the outpatient clinic of the Erasmus Medical Center in Rotterdam, The Netherlands, archived sample bank. The plasma was stored at −80 °C. Inclusion criteria were age over 18 years and no previous treatment for HIV. Exclusion criteria were severe comorbidity (e.g. diabetes mellitus, cardiovascular disease, opportunistic infection), coinfection with hepatitis B or C, use of alcohol > 2 IU/day and use of co-medication. All patients had given written informed consent for inclusion in the database of the Dutch HIV monitoring foundation (Stichting HIV Monitoring; SHM) also known as the AIDS Therapy Evaluation in the Netherlands (ATHENA) cohort, and collection of demographic, laboratory and clinical data from the medical records and storage and future use for scientific research of biological material. Their plasma samples were compared to a non-HIV infected control population from volunteers (n = 23), without comorbidity or co-medication, which had all given their written informed consent.
Untreated HIV versus HIV-suppressed (12 months of cART), population B
For the comparison of metabolic profiles of untreated HIV-1-infected patients to the HIV-suppressed situation, plasma samples of 28 HIV-1-infected patients from the outpatient clinic of the Maastricht University Medical Center in Maastricht, The Netherlands were selected in the archived sample bank. The plasma was stored at −80 °C. All patients had given written informed consent for inclusion in the database of the Dutch HIV monitoring foundation and collection of demographic, laboratory and clinical data from the medical records and storage and future use for scientific research of biological material. From each included patient, a plasma sample was selected from before the start of cART and at 12 months after the start of cART. All patients were older than 18 years at the start of cART and had not received any previous ART therapy. All patients started with a cART regimen containing Abacavir. The patients had no diabetes mellitus and no diagnosed autoimmune diseases. We retrieved information regarding dyslipidaemia from the earlier laboratory results prior to the 12 months samples after the start of cART. Because the patients were generally not in care prior to the HIV diagnosis, no values were available from the blood sample prior to the start of cART. The study was performed according to the Helsinki Declaration and approved by the Ethical Committee of the Maastricht University Medical Center.
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Targeted LC-MS metabolomics
Targeted metabolomics analyses were performed using standard operating procedures derived from previously published methods49,50,51,52. Detailed procedures and target lists are provided in the Additional file 1 - Methods with a brief overview of the four platforms used given in Table 3. After LC-MS analyses, peak integration was done using the instrumental software, and the relative ratios between metabolites and their corresponding internal standards were determined.
Metabolomics quality controls
Quality control (QC) samples consisted of equal aliquots of a QC pool made by combining equal volumes (±25 µL) of all study samples. A set of QC samples was then included during the analyses of the experimental groups on the individual metabolomic platforms and evenly distributed across the randomized samples prior to LC-MS analyses. In addition, independent duplicate samples (10–15%) were randomly selected. Using the QC samples and duplicate samples, a double-QC approach was applied to include metabolites that were reliably measured by the individual metabolomics platforms by reporting and using only those metabolites for which both duplicate samples and QC samples showed an RSD < 30%.
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