Supplementary Digital Content 2
Extended Metabolomics Methodology
Metabolomic analysis of plasma and urine. All steps were carried out at room temperature,
unless otherwise stated. From AbsoluteIDQTM kit vials, lyophilized standards (200 L
methanol:water solution, 9:1, v/v) and QC plasma samples (in 200 L water) were prepared.
10 L plasma samples and internal standards were then added to the 96-well plate. Samples
were dried under nitrogen for 30 min, then 20 L of phenylisothiocyanate (PITC) reagent
(150 L PITC to 2850 L of ethanol/water/pyridine; 1:1:1, v/v/v) was added to each well.
The plate was left covered for 20 min then dried under nitrogen (45 min). 300 L of 5 mM
ammonium acetate (in methanol) extraction solvent was added to each well, the plate shaken
(Ika KS 260 Basic) for 30 min, then centrifuged (2 min, 500 g). Samples were diluted with
LC-MS running solvent (prepared in methanol from kit reagents). The sealed plate was
shaken (2 min) then placed in the autosampler (10 C).
For urine samples, calibration curves were prepared using EZ:Faast TM amino acid kitsupplied standard solutions; for amino acids whose concentrations were higher than those of
the highest solution from the kit, or not originally included with the kit (L-kynurenine,
xanthuric acid, kynurenic acid, 3-hydroxyanthanilic acid, cysteine, glycine, histidine, serine,
lysine, glutamine, alanine; Sigma, St. Louis, MO), new standard solutions of higher
concentration were prepared in water (see Supplementary Material I). Urine (100 μL) and the
internal standard (norvaline at 200 nmol·mL-1, 100 μL) were combined into glass vials and
mixed by vortex. For solid phase extraction, samples were extracted into sorbent-tipped
syringes (1.5 mL); washing solution (200 µL) was added to the vial and drawn through the
tip. Tips were detached and placed in the corresponding vial, then 200 µL of eluting medium
was added to each vial. The same sorbent tips were re-attached to new syringes (0.6 mL) and
wetted by drawing eluting medium; eluting medium was ejected back into the vial.
Chloroform (50 µL) was added and mixed by vortex, then iso-octane (100 µL) was added and
vortexed. The upper organic layer (50–100 µL) was transferred into a new vial by Pasteur
pipette. The solvent was evaporated to almost complete dryness under nitrogen (3 min) and
amino acid derivatives re-suspended in 100 µL re-dissolution solvent (iso-octane 80%,
chloroform 20%).
Urinary metabolomics calibration curves. Calibration curve standard solutions were made for
glutamine (500 nmol.mL-1), glycine (1500 nmol.mL-1), serine (500 nmol.mL-1), histidine
(1500 nmol.mL-1), alanine (500 nmol.mL-1), and lysine (500 nmol.mL-1). Urinary
concentrations of metabolites of tryptophan (many not included within the kit) were generally
very low. Therefore another calibration stock solution, in water (mix 1) including these
amino acids was prepared with kynurenine (50 nmol.mL-1), xanthuric acid (50 nmol.mL-1), 3hydroxyanthranilic acid (50 nmol.mL-1), and L-kynurenine (50 nmol.mL-1). All solutions
were stored at 4 C. After the preparation of calibration stocks, calibration solutions were
prepared by filling cartridges as described below (1-5), and then following the same
preparation procedure as described previously within Materials and Methods.
Reagents used were; Reagent 1 (internal standard solution): norvaline (internal standard
(Istd)) 0.2 mM, in N-propanol 10%. Reagent 2 (washing solution): N-propanol. Reagent 3A
(eluting medium component I): sodium hydroxide. Reagent 3B (eluting medium component
II): N-propanol. Reagent 4 (organic solution I): chloroform. Reagent 5 (organic solution II):
iso-octane. Reagent 6 (re-dissolution solvent): iso-octane 80 %, chloroform 20 %. Amino
acid standard mixtures were; SD1: α-aminoadipic acid, α-aminobutyric acid, allo-isoleucine,
alanine, aspartic acid, β-aminoisobutyric acid, cystine, glutamic acid, glycine, histidine, 4hydroxyproline, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline,
sarcosine, serine, threonine, tyrosine and valine. SD2: asparagine, glutamine and tryptophan.
SD3 (complementary urine amino acids): α-aminopimelic acid, cystathionine, glycine-proline
(dipeptide), hydroxylysine (2 isomers), proline-hydroxyproline (dipeptide) and thiaproline.
1. SD1 (10 µL) + SD2 (10 µL) + SD3 (10 µL) + reagent 1 (norvaline, 100 µL); equivalent to
20 nmol.mL-1 of each amino acid and 200 nmol.mL-1 of norvaline. Mix 1 (10 µL, each
metabolite at 5 nmol.mL-1) and cysteine (10 µL, at 20 nmol.mL-1) were added.
2. SD1 (25 µL) + SD2 (25 µL) + SD3 (25 µL) + reagent 1 (norvaline, 100 µL). Equivalent to
50 nmol.mL-1 of each amino acid and 200 nmol.mL-1 of norvaline. Mix 1 (25 µL, each
metabolite at 12.5 nmol.mL-1) and cysteine (25 µL, at 50 nmol.mL-1) were added.
3. SD1 (50 µL) + SD2 (50 µL) + SD3 (50 µL) + reagent 1 (norvaline, 100 µL). Equivalent to
100 nmol.mL-1 of each amino acid and 200 nmol.mL-1 of norvaline. Mix 1 (50 µL, each
metabolite at 25 nmol.mL-1) and cysteine (50 µL, at 100 nmol.mL-1) were added.
4. SD1 (150 µL) + SD2 (150 µL) + SD3 (150 µL) + reagent 1 (norvaline, 100 µL).
Equivalent to 300 nmol.mL-1 of each amino acid and 200 nmol.mL-1 of norvaline. Mix 1 (75
µL, each metabolite at 37.5 nmol.mL-1) and cysteine (150 µL, at 300 nmol.mL-1) were added.
5. Glutamine (100 µL) + glycine (100 µL) + serine (100 µL) + histidine (100 µL) + alanine
(100 µL) + lysine (100 µL) + reagent 1 (norvaline, 100 µL). Equivalent to 500 nmol .mL-1 of
each amino acid (except glycine and histidine which are 1500 nmol.mL-1) and 200 nmol.mL-1
norvaline. Mix 1 (100 µL); each amino acid at 50 nmol.mL-1.
Selected ion monitoring. The list below shows the amino acids analyzed and the ions used for
quantification (underlined); other ions listed were also recorded and used as qualifiers if
required. Quantification (in nmol.mL-1) was performed using the peak area ratio of each ion
obtained in SIM mode, and the norvaline peak area (internal standard). A calibration curve
was prepared and injected for each sequence.
Amino acid
Ions
Alanine (Ala)
130, 88
Sarcosine (Sar)
130, 217
Glycine (Gly)
116, 217
-aminobutyric acid (Aba)
144, 102
Valine (Val)
158, 116
-Aminoisobutyric acid (-AiB)
116, 172
Norvaline (Istd) (Norv)
158, 72
Leucine (Leu)
172, 86
allo-Isoleucine (aILe)
172, 130
Isoleucine (ILe)
172, 86
Threonine (Thr)
101, 160
Serine (Ser)
146, 203
Proline (Pro)
156, 243
Asparagine (Asn)
69, 155
Aspartic acid (Asp)
216, 130
Methionine (Met)
203, 277
4-Hydroxyproline (4Hyp)
172, 86
Glutamic acid (Glu)
230, 170
Phenylalanine (Phe)
206, 190
Cysteine (Cys)
248, 162
-Aminoadipic acid (AAA)
244, 98
Glutamine (Gln)
84, 187
Ornithine (Orn)
156, 70
Lysine (Lys)
170, 128
Histidine (His)
282, 168
Tyrosine (Tyr)
206, 107
Tryptophan (Trp)
130
Cystine (C-C)
248, 216
Xanthuric acid (Xa)
247
Thioproline (Tpr)
174, 147
3-Hydroxyproline (Hyp)
172, 259
-Aminopimelic acid (Apa)
198, 258
3-hydroxyanthanilic acid (Haa)
161
Glycyl-proline (Grp)
70, 300
Kynurenic acid (Ka)
145
Hydroxylysine (Hly)
129, 169
L-Kynurenine (L-k)
276
Proline-hydroxyproline (Php)
156, 186
Cystathionine (Cth)
203, 272