Study of Perioperative Plasma Levels of Endothelial Glycocalyx Markers in Patients Undergoing Colon Surgery.
Conditions: Endothelial Dysfunction
Sponsor: Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunitat Valenciana

	  
	  
	  
RESEARCH	  PROTOCOL	  
	  
	  
	  
STUDY OF PERIOPERATIVE PLASMA LEVELS 
OF ENDOTHELIAL GLYCOCALIX MARKERS IN 
PATIENTS UNDERGOING COLON SURGERY. 
 
 
 
	  
Principal	   Investigator:	   NURIA	   GARCIA	   DEL	   OLMO.	   NIF:	   26748077C	   Telephone:	  
619660434.	  Email:	  nuriagarciadelolmo@gmail.com	  
	  
Data:	  1/2/2022	  
	  
	  
	  
	  
	  
	  
	  
	  
	  
	  
	  
	  
	  
	  
	  
	  
	  
	  
	  
	  

SUMMARY:	   The	   glycocalyx	   is	   a	   layer	   of	   macromolecules	   that	   covers	   the	   luminal	  
surface	   of	   the	   endothelium,	   playing	   an	   important	   role	   in	   vascular	   homeostasis.	  
Several	  studies	  have	  shown	  that	  in	  conditions	  of	  sepsis	  there	  is	  a	  degradation	  of	  the	  
glycocalyx	  mediated	  by	  inflammatory	  mechanisms,	  releasing	  its	  components	  into	  the	  
bloodstream.	  These	  glycocalyx	  fragments	  can	  be	  measured	  in	  blood	  plasma,	  showing	  
a	  correlation	  between	  these	  markers	  and	  organ	  dysfunction,	  severity	  and	  mortality	  
in	   sepsis	   situations.	   However,	   despite	   the	   great	   interest	   in	   glycocalyx	   biomarkers,	  
their	  values	  in	  patients	  undergoing	  colorectal	  surgery	  are	  not	  well	  known.	  Therefore,	  
verifying	   whether	   plasma	   levels	   of	   glycocalyx	   damage	   markers	   increase	   in	   the	  
perioperative	   period	   of	   colorectal	   surgery	   is	   of	   great	   interest	   at	   the	  
pathophysiological	   and	   clinical	   level,	   since	   the	   correlation	   of	   these	   markers	   with	  
postoperative	  clinical	  evolution	  is	  unknown.	  In	  this	  study	  we	  want	  to	  determine	  the	  
plasma	   levels	   of	   Heparan-­‐sulphate	   and	   Syndecan-­‐1,	   two	   of	   the	   most	   important	  
constituents	  of	  the	  glycocalyx,	  and	  perform	  several	  measurements	  to	  form	  a	  "time	  
course"	   of	   the	   plasma	   levels	   of	   these	   markers,	   in	   order	   to	   compare	   the	   different	  
subgroups	   of	   patients	   undergoing	   colorectal	   surgery	   based	   on	   their	   clinical	   and	  
surgical	  characteristics.	  
	  
	  
BACKGROUND:	  	  
The	  endothelial	  glycocalyx	  (EG)	  is	  a	  complex	  layer	  of	  macromolecules	  that	  lines	  the	  
luminal	  surface	  of	  the	  vascular	  endothelium.	  This	  concept	  was	  proposed	  more	  than	  
70	  years	  ago	  and	  its	  composition	  is	  well	  studied	  as	  detailed	  in	  two	  reviews	  (1,	  2).	  
However,	   its	   role	   in	   the	   mechanisms	   of	   endothelial	   protection	   and	   injury,	   and	   its	  
subsequent	  clinical	  implications,	  have	  recently	  become	  evident.	  The	  GE	  consists	  of	  a	  
variety	   of	   molecules,	   including	   glycoproteins	   and	   proteoglycans,	   that	   provide	   the	  
basis	   for	   plasma-­‐endothelial	   cell	   interaction.	   The	   structure	   of	   GE,	   although	   well	  
characterized	   in	   vitro,	   is	   poorly	   defined	   in	   vivo	   because	   its	   composition	   changes	  
dynamically	   by	   self-­‐assembly	   and	   shear-­‐dependent	   enzymatic	   degradation	   or	  
detachment	   of	   its	   elements.	   Its	   major	   components	   are	   hyaluronic	   acid,	  
proteoglycans	   (PG)	   such	   as	   syndecan-­‐1,	   glycoproteins,	   plasma	   proteins	   and	  
glycosaminoglycans	  (GAG),	  whose	  main	  component	  is	  Heparan	  Sulfate.	  The	  GE	  as	  a	  
whole	  forms	  an	  endothelial	  surface	  layer	  that	  acts	  as	  a	  barrier	  to	  circulating	  cells	  and	  
large	  molecules.	  Considerable	  prognostic-­‐therapeutic	  promise	  lies	  in	  the	  emergence	  
of	   the	   EG	   as	   a	   key	   mediator	   of	   endothelial	   dysfunction	   in	   pathogenic	   states,	  
particularly	   with	   respect	   to	   vascular	   permeability	   and	   edema	   formation.	   Several	  
studies	  have	  demonstrated	  the	  role	  of	  GE	  in	  plasma/interstitial	  fluid	  balance	  (3,4,5),	  
mechano-­‐transduction	   that	   couples	   intravascular	   pressure,	   and	   endothelial	   cell	  
responses,	  i.e.,	  biochemical	  signals,	  (6)	  to	  the	  inflammatory	  response	  cascade	  (7,8,9).	  
EG	   damage	   affects	   a	   number	   of	   important	   endothelial	   cell	   functions	   leading	   to	  
impaired	  mechano-­‐transduction	  with	  changes	  in	  fluid	  passage	  (6,10),	  activation	  of	  

coagulation	   pathways	   (11),	   leukocyte	   adhesion	   (7,11	   )	   and	   platelets	   (12)	   to	   the	  
surface	   of	   endothelial	   cells,	   and	   leakage	   of	   fluid	   and	   plasma	   proteins	   into	   the	  
interstitium	  (13,14),	  resulting	  in	  tissue	  edema	  (15).	  The	  specific	  pathophysiological	  
triggers	   that	   lead	   to	   EG	   damage	   are	   still	   actively	   investigated	   and	   remain	   poorly	  
understood.	  Nevertheless,	  EG	  protection	  appears	  to	  be	  a	  promising	  target	  in	  many	  
clinical	   scenarios,	   especially	   since	   its	   degradation	   is	   closely	   associated	   with	   the	  
pathophysiology	   of	   inflammation,	   capillary	   leak,	   and	   edema	   formation	   in	   various	  
injuries	  and	  disease	  states;	  including	  ischemia/reperfusion,	  hypoxia,	  inflammation,	  
trauma,	  hypervolemia,	  atherosclerosis,	  diabetes,	  and	  hypertension	  (16).	  In	  patients	  
undergoing	  major	  abdominal	  surgery	  (including	  digestive,	  urological,	  gynecological	  
or	  other	  surgery)	  there	  is	  very	  little	  information	  on	  the	  potential	  endothelial	  damage	  
secondary	  to	  surgery	  (19).	  There	  are	  some	  reviews	  that	  have	  studied	  the	  damage	  of	  
the	  SG	  in	  septic	  patients	  of	  medical	  and	  surgical	  origin	  (17,	  18).	  However,	  in	  relation	  
to	  digestive	  abdominal	  surgery,	  and	  specifically	  in	  colorectal	  surgery,	  only	  one	  study	  
has	   been	   carried	   out,	   with	   only	   18	   patients	   undergoing	   colectomy	   (without	  
mentioning	   the	   pathologies)	   and	   they	   are	   included	   with	   other	   patients	   of	   major	  
abdominal	  surgery	  of	  the	  pancreas,	  liver,	  gynecology	  and	  urology	  (19).	  
	  
	  
EXPECTED	  RESULTS:	  	  
In	  this	  study,	  it	  is	  expected	  that	  in	  the	  patients	  who	  underwent	  elective	  surgery,	  the	  
markers	   of	   glycocalyx	   damage	   will	   increase	   their	   values	   in	   the	   postoperative	  
laboratory	  tests	  compared	  to	  the	  values	  of	  the	  baseline	  (control)	  laboratory	  tests.	  It	  
is	  equally	  expected	  that	  in	  patients	  undergoing	  urgent	  surgery,	  generally	  in	  clinical	  
states	   of	   occlusion,	   perforation	   or	   sepsis,	   with	   activation	   of	   the	   inflammatory	  
response,	   dehydration	   with	   decreased	   oncotic	   pressure	   and	   generation	   of	   tissue	  
edema	  and	  third	  space,	  the	  markers	  of	  damage	  of	  glycocalyx	  are	  already	  elevated	  in	  
the	  baseline	  analysis,	  with	  a	  maintenance	  or	  greater	  elevation	  of	  its	  values	  in	  the	  
postoperative	  period.	  Likewise,	  a	  correlation	  would	  be	  expected	  between	  glycocalyx	  
damage	  markers	  and	  other	  markers	  involved	  in	  the	  inflammatory	  response,	  such	  as	  
leukocytes,	  C-­‐reactive	  protein,	  procalcitonin,	  and	  lactate;	  as	  well	  as	  with	  associated	  
patient	   comorbidities	   that	   lead	   to	   endothelial	   degeneration,	   such	   as	   high	   blood	  
pressure,	  obesity,	  diabetes,	  atherosclerosis,	  etc.	  If	  the	  results	  of	  this	  study	  were	  as	  
expected,	   glycocalyx	   damage	   markers	   (Heparan	   sulfate	   and	   Syndecan-­‐1)	   could	   be	  
used	   together	   with	   other	   inflammatory	   markers	   (CRP,	   procalcitonin,	   etc.)	   and	   be	  
very	  useful	  in	  postoperative	  follow-­‐up.	  of	  surgical	  patients,	  being	  able	  to	  anticipate	  
possible	  postoperative	  complications.	  However,	  if	  elevations	  in	  glycocalyx	  markers	  
were	   not	   observed	   in	   the	   perioperative	   period,	   it	   could	   be	   due	   to	   a	   lack	   of	   the	  
number	  of	  Time-­‐course	  determinations;	  that	  is	  to	  say,	  that	  an	  elevation	  of	  markers	  
did	  occur,	  but	  we	  would	  not	  have	  detected	  it	  in	  the	  measurement	  times;	  or	  a	  larger	  

study	  population	  might	  also	  be	  needed.	  In	  either	  case,	  the	  study	  could	  be	  completed	  
in	  subsequent	  research	  projects.	  
	  
	  
OBJECTIVES	  	  
Primary	   Objectives:	   To	   estimate	   the	   plasma	   levels	   of	   the	   2	   most	   representative	  
markers	  of	  damage	  to	  the	  endothelial	  glycocalyx	  (Heparan-­‐sulfate	  and	  Syndecan-­‐1)	  
in	   patients	   undergoing	   colorectal	   surgery	   during	   the	   perioperative	   period	   (“time-­‐
course”).	  	  
	  
Secondary	  Objectives:	  	  
-­‐ 
To	  analyze	  a	  possible	  correlation	  of	  the	  glycocalix	  damage	  markers	  with	  other	  
analytical	   parameters	   (PCR,	   procalcitonin,	   lactate,	   complete	   blood	   count,	  
coagulation	  and	  basic	  biochemistry).	  	  
-­‐ 
To	   analyze	   the	   possible	   relationship	   of	   glycocalyx	   damage	   markers	   with	  
different	   parameters	   depending	   on	   the	   patient	   (age,	   sex	   and	   associated	  
comorbidities)	  and	  the	  surgical	  intervention	  (surgical	  time,	  approach,	  type	  of	  
surgery...).	  	  
-­‐ 
To	   analyze	   other	   clinical	   parameters	   that	   may	   indicate	   a	   possible	  
postoperative	   complication	   and	   their	   relationship	   with	   markers	   of	  
endothelial	  glycocalyx	  damage	  will	  also	  be	  studied.	  
	  
	  
METHODOLOGY	   DESIGN:	   The	   present	   work	   is	   about	   a	   clinical,	   prospective,	  
observational,	  and	  longitudinal	  study	  of	  single	  center	  groups	  (Hospital	  Lluís	  Alcanyís,	  
Xàtiva).	  Always	  after	  the	  approval	  of	  the	  Research	  Ethics	  Committee	  of	  said	  Hospital.	  	  
	  
STUDY	   POPULATION:	   The	   study	   population	   (value	   of	   n)	   has	   been	   calculated	   on	   a	  
population	  of	  100	  patients	  who	  will	  undergo	  surgery	  approximately	  one	  year.	  With	  a	  
confidence	  level	  of	  95%	  and	  a	  margin	  of	  error	  of	  5%,	  a	  sample	  size	  of	  80	  patients	  has	  
been	  calculated.	  These	  80	  patients	  will	  be	  of	  legal	  age,	  men	  and	  women,	  undergoing	  
elective	   or	   emergency	   colorectal	   surgery	   (right	   hemicolectomy,	   extended	   right	  
hemicolectomy,	   left	   hemicolectomy,	   sigmoidectomy,	   anterior	   resection	   of	   the	  
rectum	  or	  abdominoperineal	  amputation).	  	  
	  
CHOICE	  OF	  PATIENTS	  AND	  INFORMED	  CONSENT:	  The	  selection	  of	  patients	  and	  the	  
drafting	   of	   the	   informed	   consent	   documents	   will	   be	   carried	   out	   in	   a	   reasoned	  
manner	  as	  established	  in	  article	  32	  of	  the	  Declaration	  of	  Helsinki	  and	  in	  article	  58.2	  
of	   the	   Research	   Law.	   Biomedical.	   It	   should	   be	   considered	   taking	   into	   account	   the	  
Declaration	  of	  Helsinki	  of	  the	  World	  Medical	  Association	  in	  its	  latest	  revision.	  And	  
according	  to	  its	  latest	  version	  of	  the	  Helsinki	  Declaration	  of	  Fortaleza	  (Brazil)	  October	  

2013.	  Patients	  will	  be	  included	  in	  the	  study	  upon	  preoperative	  hospital	  admission,	  
after	   verifying	   compliance	   with	   the	   inclusion	   criteria.	   To	   participate,	   it	   will	   be	  
necessary	  to	  sign	  the	  informed	  consent.	  
	  
	  
INCLUSION	  CRITERIA:	  	  
To	  be	  included	  in	  the	  study,	  the	  following	  criteria	  must	  be	  met:	  	  
•	  Signature	  of	  the	  informed	  consent	  document.	  	  
•	  Age	  equal	  to	  or	  greater	  than	  18	  years.	  	  
•	  ASA	  surgical	  risk	  ≤	  III.	  	  
• 	  Being	  operated	  on	  for	  colorectal	  surgery	  	  
	  
EXCLUSION	  CRITERIA:	  	  
The	  	  presence	  of	  any	  of	  the	  following	  criteria	  will	  be	  excluding:	  	  
• Younger	  than	  18	  years	  old	  
• Pregnant	  women	  
	  
WITHDRAWAL	  FROM	  THE	  STUDY:	  	  
Patients	  will	  be	  withdrawn	  from	  the	  study	  when	  any	  of	  the	  following	  events	  occur:	  	  
• Non-­‐compliance	  inclusion	  criteria	  	  
• Due	  to	  voluntary	  abandonment	  of	  the	  patient	  
	  
	  
METHOD:	  	  
Blood	  samples	  will	  be	  drawn	  from	  an	  intravenous	  line,	  in	  BD	  Vacutainer®	  LH	  PST™	  II	  
tubes	   with	   lithium	   heparin	   at	   different	   times:	   1st)	   basal	   time	   (preoperative),	   at	  
hospital	  admission;	  2nd)	  2	  hours	  after	  the	  end	  of	  the	  surgery,	  3rd)	  6	  hours	  after	  the	  
end	  of	  the	  surgery,	  4th)	  24	  hours	  after	  the	  end	  of	  the	  surgery	  and	  5th)	  48	  hours	  after	  
the	   surgery.	   The	   blood	   samples	   will	   be	   centrifuged	   at	   3000	   rpm/12	   min	   and	   the	  
supernatants	   will	   be	   collected	   and	   stored	   at	   -­‐80ºC	   until	   the	   determination	   of	   the	  
markers.	   Plasma	   levels	   of	   Syndecan-­‐1	   and	   Heparan	   Sulfate	   will	   be	   determined	   by	  
ELISA	  
(Enzyme-­‐Linked	  
Immunosorbent	  
Assay),	  
PCR	  
(C-­‐reactive	  
protein),	  
procalcitonin,	   lactate,	   blood	   count,	   renal	   function,	   basic	   coagulation	   and	   general	  
biochemistry	  techniques.	  The	  blood	  samples	  will	  be	  drawn	  by	  the	  nursing	  team	  of	  
the	  general	  surgery	  hospitalization	  ward,	  and	  will	  be	  transported	  to	  the	  laboratory	  
by	   the	   auxiliary	   staff	   of	   said	   hospitalization	   ward.	   The	   laboratory	   staff	   will	   be	   in	  
charge	   of	   centrifuging	   the	   samples	   and	   storing	   the	   supernatants	   in	   the	   pertinent	  
conditions	  mentioned	  above.	  Later,	  when	  all	  the	  samples	  have	  been	  collected,	  the	  
determination	  of	  the	  markers	  will	  be	  carried	  out,	  and	  the	  result	  will	  be	  displayed	  
through	   the	   Orion	   clinic	   computer	   program	   or	   in	   paper	   format,	   which	   will	   be	  
delivered	   to	   me	   personally.	   In	   addition,	   all	   the	   usual	   anesthetic,	   surgical	   and	  

postoperative	   care	   protocols	   of	   the	   Institution	   (General	   and	   Digestive	   Surgery	  
Department.	  Lluís	  Alcanyís	  Hospital)	  will	  be	  maintained	  for	  these	  patients.	  
	  
	  
STATISTICAL	   ANALYSIS	   PLAN	   (SAP):	   Continuous	   data	   are	   expressed	   as	   mean	   ±	  
standard	   deviation.	   The	   normality	   of	   the	   data	   obtained	   was	   determined	   with	   the	  
Kolmogorov-­‐Smirnov	   and	   Shapiro-­‐Wilk	   tests.	   Differences	   between	   baseline	   values	  
and	   different	   moments	   after	   the	   "time	   course"	   of	   plasma	   levels	   of	   glycocalyx	  
damage	   markers	   and	   other	   parameters	   will	   be	   analyzed	   by	   one-­‐way	   analysis	   of	  
variance	  (ANOVA).	  Data	  sets	  in	  which	  F	  is	  significant	  will	  be	  examined	  for	  multiple	  
comparisons	  by	  Tukey's	  test.	  The	  Pearson	  correlation	  coefficient	  (r)	  will	  be	  calculated	  
between	  the	  values	  of	  the	  EG	  damage	  markers	  and	  other	  parameters.	  The	  possible	  
differences	  between	  the	  different	  population	  subgroups	  will	  be	  examined	  with	  the	  
Student's	   t-­‐test.	   P	   <	   0.05	   will	   be	   considered	   statistically	   significant.	   All	   statistical	  
calculations	  will	  be	  performed	  using	  the	  SPSS	  program.	  	  
	  
MEDIA:	  We	  have	   sufficient	  infrastructure	  and	   biochemical	  laboratory	  reagents,	  as	  
well	  as	  an	  ELISA	  reader.	  We	  also	  have	  all	  the	  consumable	  material	  necessary	  for	  the	  
determination	   of	   glycocalyx	   damage	   markers,	   as	   well	   as	   all	   other	   biochemical	  
determinations.	  
	  
	  
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