Protocol for : l_mtbllipi1_ex05_1_0617s
Introduction
High throughput metabolite profiling was performed on plasma specimens collected from individuals in
the Framingham Offspring cohort attending Examination 5. This dataset includes measurements made
on blood specimens (pre-OGTT) that were collected from a subset of the Offspring Cohort participants
who attended Examination 5 (and were selected for inclusion in nested case-control studies of incident
diabetes and cardiovascular disease). The concentration of plasma metabolites was assessed using a
liquid chromatography / mass spectrometry (LC/MS) platform. In the dataset, each value represents the
plasma concentration of an analyte (in this case, a lipid analyte) that was assayed. The first row includes
column headings that serve as names for each variable.
Notes Regarding Sampling Variables
Lipid profiling was performed on plasma collected from individuals who were selected for the purpose
of conducting an initial series of case-control and case-cohort studies of incident diabetes,
cardiovascular disease, and kidney disease. This sampling strategy has been described, in part, in Nat
Med 2011;17:448-453 and J Clin Invest 2011;121:1402-1411. Additional details are provided below:
Variable Name
DM_pair
Label / Description
Pair ID# assigned for diabetes case-control study,
where cases were matched to controls based on
propensity score (to within <0.10 score)
DM_case
Diabetes case status (cases developed CVD within
12 years of follow up)
CVD_pair
Pair ID# assigned for CVD case-control study,
where cases were matched to controls based on
Framingham Risk Score (to within 1 point)
CVD_case
CVD case status (cases developed CVD within 12
years of follow up)
Random
CKD_case
Randomly selected as additional control for the
incident diabetes study
Chronic kidney disease case status (cases
developed CKD within 8 years of follow up, as part
of case-cohort study)
Values
Ordinal: range, 1-189
(missing=not paired)
Binary: 0=control, 1=case
(missing=neither case, nor
matched control)
Ordinal: range, 1-181
(missing=not paired)
Binary: 0=control, 1=case
(missing=neither case, nor
matched control)
Binary: 0=no, 1=yes
Binary; 0=no, 1=yes
Notes Regarding Analyte Variables
Units: There are no standard units for these data, as all values are normalized to an internal standard
and then to pooled plasma.
Range of values: There is also no established range of normal values for these measurements.
Missing values: For some analytes, there are missing values because the platform was unable to
reliably resolve quantity of these analytes for these particular individuals. In these cases, the values
should be coded as missing. There should be no zero values.
Distribution of values: For these types of analytes, distribution of values is often relatively skewed.
Skewed values can be log transformed prior to analyses (there are no zero values).
Naming: For these types of analytes (lipids), variables are named according to a specific conventional
nomenclature. In this convention, the first letter “C” stands for carbon and the number that follows
denotes the total carbon number across the lipid acyl chains (i.e. fatty acids). The first underscore
symbol (“_”) stands in place of what is conventionally a colon symbol (“:”) , which is followed by the
total number of double bonds across the lipid acyl chains (i.e. fatty acids). The second underscore
symbol (“_”) is followed by one of the following abbreviations, which denotes the type of lipid molecule:
CE, cholesterol ester
DAG, diacylglycerol
LPC, lysophosphatidylcholine
LPE, lysophosphatidylethanolamine
MS, mass spectrometry
PC, phosphatidylcholine
SM, sphingomyelin
TAG, triacylglycerol
Therefore, as an example of how this nomenclature works, the variable in the dataset called
“C14_0_LPC” represents the analyte that is conventionally named “C14:0 LPC”. This analyte is a lipid
molecule of the lysophosphatidylcholine type and has 14 carbon atoms and zero double bonds.
Lipid metabolite profiling
Lipid metabolite profiling was performed using a 4000 QTRAP triple quadrupole mass
spectrometer (Applied Biosystems/Sciex; Foster City, CA), coupled to a 1200 Series pump
(Agilent Technologies; Santa Clara, CA) and an HTS PAL autosampler (Leap Technologies;
Carrboro, NC). MultiQuant software (Version 1.1; Applied Biosystems/Sciex; Foster City, CA)
was used for automated peak integration and peaks were also manually reviewed for quality of
integration. Ammonium acetate, acetic acid, and LC-MS grade solvents were purchased from
Sigma-Aldrich (St. Louis, MO). 10μL of plasma were extracted with 190μL of isopropanol
containing an internal standard, 1-dodecanoyl-2-tridecanoyl-sn-glycero-3-phosphocholine
(Avanti Polar Lipids; Alabaster, AL). Following centrifugation, supernatants were injected
directly, followed by reverse phase chromatography using a 150 x 3.0 mm Prosphere HP C4
column (Grace; Columbia, MD); mobile phase A: 95:5:0.1 v/v/v 10 mM ammonium
acetate/methanol/acetic acid; mobile phase B: 99.9:0.1 v/v methanol/acetic acid. The column
was eluted isocratically with 80% mobile phase A for 2 minutes followed by a linear gradient to
20% mobile phase A over 1 minute, a linear gradient to 0% mobile phase A over 12 minutes,
then 10 minutes at 0% mobile phase A. Electrospray ionization and Q1 scans in the positive ion
mode were used for MS analyses. Ion spray voltage was 5.0 kV, and source temperature was
400ºC.