Sample collection materials and methods 35

Each  of  the  samples  listed  were  collected  by  the  following  methods.   Time%of% sample% %(hours)% 0% 6% 12% 24% 48% 72% 96% 120% Gastric% Aspirate! X! %! %! %! %! %! %! %! Blood% Samples! X! X! X! X! X! X! X! X! ET%% Aspirates! X! X! X! X! X! X! X! X! Urine% Samples! X! X! X! X! X! X! X! X!

2.2.1  Blood  samples  

Blood   samples   (250µl)   were   collected   using   ethylenediaminetetraacetic   acid  

(EDTA)  as  anti-­‐coagulant  from  an  indwelling  arterial  line  at  baseline  then  6,  12,  24,  

48,  72,  96  and  120  hours  after  the  start  of  the  [methyl-­‐D9]choline  chloride  infusion  

for   measurement   of   labelled   phospholipid   which   will   provide   a   measure   of   liver   lipoprotein   phospholipid   as   a   control   for   the   ET   aspirate   incorporation   analysis   and  to  characterise  liver  PC  metabolism.    

2.2.2  Endotracheal  aspirate  specimens  

Specimens  of  lung  fluid  were  collected  by  gentle  suctioning  after  instillation  of  up   to  0.5  to  1ml  of  sterile  saline  down  the  ET  tube.  As  with  blood  samples  this  was   performed  at  baseline  then  6  hours  after  the  start  of  administration  of  the  [methyl-­‐ D9]choline   chloride,   at   12   hours   and   then   at   daily   intervals   from   24   hours   after  

infusion  for  5  days  or  until  ventilation  is  discontinued,  whichever  was  the  shorter   period.  This  technique  is  a  component  of  routine  endotracheal  toilet  in  intubated   neonates.    

2.2.3  Urine  specimens  

Urine  samples  were  collected  at  similar  intervals  as  blood  and  ET  aspirate  samples.     They   were   analysed   for   choline   metabolites   to   provide   an   assessment   of   the   enrichment  of  stable  isotope  label  in  the  choline  pool.    

2.2.4  Stabilisation  solutions  

Once   collected   the   samples   were   stabilised   by   the   addition   of   a   pre-­‐prepared   aliquot  of  stabilisation  solution,  designed  for  each  sample  type.  

i)   Gastric   aspirate,   urine   and   endotracheal   aspirate   stabilisation   solution   (GUESS)  

For   the   gastric   aspirate,   urine   and   endotracheal   aspirate   samples   an   eppendorf   containing   510µl   of   stabilisation   solution   was   added.   This   contained   10µl   butylhydroxytoluene   (BHT)   20g/l   stock   solution   as   an   antioxidant   and   500µl   RNAlater®  _{to  prevent  degradation  of  bacterial  or  viral  DNA/RNA.  This  stabilisation}  

solution,   for   use   with   gastric   aspirate,   urine   and   ETA   (GUESS),   was   refrigerated   prior  to  use.  

ii)  Blood  only  stabilisation  solution  (BOSS)  

For  blood  samples,  measuring  250µl  in  EDTA  bottles,  a  blood-­‐specific  stabilisation   solution   of   10µl   BHT   20g/l   stock   solution   and   200µl   0.9%   NaCl   was   added   immediately   following   collection.   This   blood-­‐only   stabilisation   solution   (BOSS)   was  refrigerated  prior  to  use.  

In  all  cases  the  samples  were  labelled  with  study  identification  numbers,  sample   type,   date   and   time   of   collection   and,   after   addition   of   stabilisation   solution,   refrigerated  immediately  using  the  designated  refrigerator  on  the  NICU.  

2.2.5  Sample  collection  

Samples  were  collected  daily  and  transported  on  ice  to  the  preparation  laboratory   in  the  Wellcome  Trust  Clinical  Research  Facility  (WTCRF)  in  Southampton  General   Hospital.  

2.2.6  Sample  processing  

The   samples   were   then   processed   according   to   the   TSuNaMI   Sample   Processing   Protocol  (Appendix  III)  before  freezing  in  aliquots  down  to  -­‐80˚C.  

The  method  of  processing  was  adapted  slightly  for  use  by  the  research  nurses  in   Portsmouth   where   ETA   samples   were   not   spun   prior   to   aliquoting   due   to   restricted  laboratory  space  on  the  NICU.  Prior  to  being  moved,  on  dry  ice,  to  the   WTCRF,  Portsmouth  samples  were  frozen  at  -­‐20˚C.  

i)  Blood  sample  processing  

Blood  samples  of  250µl  were  collected  into  standard  paediatric  EDTA  blood  bottles   (750µl)   before   addition   of   the   specific   blood   stabilisation   solution,   as   detailed   above.  Samples  were  refrigerated  at  4˚C  until  processing,  mainly  <24  hours  later.   250µl  samples  of  blood  were  centrifuged  at  3000rpm  for  15  minutes  at  4˚C.  The   upper   plasma   layer   was   carefully   aspirated   and   separated   into   eppendorfs   as   100µl  aliquots.  The  remaining  red  blood  cell  pellets  were  retained  and  frozen.  The   aliquots   of   plasma   were   labelled   and   frozen   at   -­‐80˚C   until   lipid   extraction   was   performed.    

ii)  ETA  sample  processing  

ETA   samples   were   processed   by   addition   of   200µl   of   0.9%   NaCl   by   Hamilton   syringe.   This   fluid   could   be   used   to   flush   any   suction   tubing   or   ET   catheters   provided   with   the   sample   to   ensure   as   much   residue   as   possible   was   removed.   Samples  were  separated  into  eppendorfs  in  300µl  aliquots  before  freezing  at  -­‐80˚C.  

iii)  Urine  sample  processing  

Urine  was  collected  from  cotton  wool  pads  placed  towards  the  front  of  the  nappies   of   participating   infants.   After   stabilisation   solution   was   added   the   samples   were   separated  into  2x  0.5ml  aliquots  and  1ml  aliquots  thereafter.  Again  samples  were   frozen  at  -­‐80˚C.  

iv)  Gastric  aspirate  sample  processing  

Gastric   aspirates   were   not   separated   into   aliquots   and,   after   addition   of   stabilisation  solution,  were  stored  whole  at  -­‐80˚C.  

2.2.7  Internal  standards  

Prior  to  analysis  of  lipids  extracted  from  samples  a  reliable  internal  standard  must   be   prepared   with   which   to   compare.   Three   internal   standards   were   prepared   in   chloroform  with  different  concentrations  used  in  plasma  and  ETA  samples.      

For  plasma  samples:  

a) 10nmol  DMPC  (0.1ml  of  100nmol/ml  solution)   b) 2nmol  DMPG  (0.1ml  of  20nmol/ml  solution)   c) 1nmol  LHPC  (0.1ml  of  10nmol/ml  solution)   For  endotracheal  aspirate  samples:  

a) 1nmol  DMPC  (0.01ml  of  100nmol/ml  solution)   b) 0.2nmol  DMPG  (0.01ml  of  20nmol/ml  solution)   c) 0.1nmol  LHPC  (0.01ml  of  10nmol/ml  solution)   DMPC  is  dimyristoylglycerophosphocholine,    

DMPG  is  dimyristoylglycerophosphoglycerol,   LHPC  is  1-­‐heptadecanoylglycerophosphocholine.  

2.2.8  Lipid  extraction  

Lipids  were  extracted  and  purified  using  a  technique  modified  from  Bligh  and  Dyer   (Bligh  and  Dyer,  1959)  as  described  below.  

Samples   (100µl   plasma   or   300µl   ETA)   had   volumes   adjusted   to   800µl   with   0.9%NaCl.  Then  2ml  of  methanol  were  added  followed  by  100µl  of  each  internal  

standard   (above)   and   700µl   of   chloroform.   The   samples   were   centrifuged   at   3000rpm,  4-­‐10˚C  for  10  minutes.  The  supernatant  was  carefully  drawn  off  to  a  new   drying  tube  and  the  remaining  protein  pellet  sealed  and  frozen  for  future  protein   analysis.  To  the  supernatant  1ml  distilled  H2O  and  1ml  chloroform  were  added  and  

centrifugation  repeated.  The  lower  (chloroform)  layer  was  then  carefully  aspirated,   placed  in  a  new  drying  tube  and  dried  in  a  concentrator  at  37˚C  under  continuous   low  flow  of  N2  gas.  Once  dry  a  further  500µl  chloroform  was  added,  samples  were  

transferred  to  brown-­‐glass  mass  spectrometry  bottles  and  the  drying  process  was   repeated.  Once  drying  was  completed  the  bottles  were  capped,  labelled  and  stored   at   -­‐20˚C   prior   to   analysis   by   electrospray   ionisation   tandem   mass   spectrometry   (ESI  MS/MS).