Postoperative Heme Oxygenase Induction and Carbon Monoxide Production as a Novel Method to Assess Hepatic Regeneration and Predict Hepatic Related Morbidity After Partial Hepatectomy-1301GCC
Phase: NA
Conditions: Liver Cancer
Sponsor: University of Maryland, Baltimore

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Postoperative Heme Oxygenase Induction and Carbon 
Monoxide Production as a Novel Method to Assess Hepatic 
Regeneration and Predict Hepatic Related Morbidity After 
Partial Hepatectomy  
 
 
Protocol Number:  
 
HP-00054854  
NCT Number:  
 
NCT04195438 
Date:  
 
 
Version 1.0 5/7/2013 
 
 
 
 
Version 2.0 9/21/2023  
 
Principal Investigator:  
Cherif Boutros MD, MSc, FACS, FACG, FSSO 
 
 
 
  
Professor of Surgery  
University of Maryland School of Medicine  
  
 
Telephone:  
 
 
410-328-7320 
Fax: 
 
 
 
410-328-5919 
Email:   
 
 
cboutros@som.umaryland.edu  
 
 
 

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Protocol Summary 
Protocol Number: 
HP-00054854 
Protocol Title: 
Postoperative Heme Oxygenase Induction 
and Carbon Monoxide Production as a Novel 
Method to Assess Hepatic Regeneration and 
Predict Hepatic Related Morbidity After 
Partial Hepatectomy  
Study Rationale: 
The goal of this proposal is to elucidate the 
role of HO-1 induction in hepatic 
regeneration after partial hepatectomy (PH). 
There is a growing body of evidence that HO-
1 induction through CO production has an 
important role in cellular protection and 
regeneration. To test this concept, we will 
monitor endogenous CO production in 
patients who undergo PH and analyze the 
relationship between CO production and 
hepatic regeneration. Using this approach we 
will test the following two hypotheses: (1) 
HO-1 induction after PH is proportional to 
the extent of the surgical resection, and (2) 
failure to appropriately induce HO-1 is 
associated with impaired hepatic 
regeneration.  
Study Objectives:  
Specific Aim 1: To monitor the extent of HO-1 
induction (by measuring endogenous CO 
production) in relationship to the extent of 
hepatic resection 
1.1 Monitor CO in exhaled air of patients 
before liver resection, at 4 hours and 24 
hours after PH 
1.2 Monitor carboxyhemoglobin(COHb) in 
the arterial blood gas of patients before liver 
resection at 4 hours and 24 hours after PH 
1.3 Analyze the relationship between CO 
production and the extent of hepatic 
resection assessed by computed scans liver 
volumetric study at one and three months 
after PH, size and weight of the resected 
specimen and operative report. 
Specific Aim 2: To analyze the relationship 
between HO-1 induction and the quality of 
post-hepatectomy liver regeneration after PH 

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2.1 Analyze the relationship between CO 
production and the quantity and quality of 
hepatic regeneration assessed by computed 
scans liver volumetric study at one and three 
months after PH and liver function tests after 
PH. 
2.2 Determine the relation between 
postoperative liver related morbidity in 
patients undergoing PH and the extent of 
postoperative HO-1 induction 
Study Design: 
Study design: 
Aim 1.1: The monitoring of exhaled CO is 
based on previous report of assessing 
exhaled CO in septic patients. Exhaled CO will 
be determined with an infrared CO analyzer 
with a sensitivity of 0.1 ppm at 4 hours and 
24 hours after PH 
Aim 1.2: Arterial blood gas (ABG) will be 
collected from patients before liver 
resection, at 4 hours and 24 hours after PH. 
The ABG will be obtained through an arterial 
line usually placed as part of perioperative 
monitoring of the patient hemodynamic. This 
model was found accurate in reflecting HO-1 
induction in previous study in patient 
receiving liver transplantation. 
Aim 1.3: FLR will be calculated through liver 
volumetric study from a pre-resectional CT 
scan. The operative note will be checked for 
operative details regarding the number of 
resected hepatic segments and pathological 
examination of the specimen will allow 
accurate weight of the resected specimen. 
According to the resected amount of hepatic 
parenchyma, three groups of patients will be 
created (<25%, 25-50% and >50% PH) history 
of smoking and intraoperative blood 
transfusion will be reported for each patient. 
 
Procedures and Methods 
- Protocol: 
For collecting exhaled air specimen, each 
patient will be instructed to exhale 
completely, and then 

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take a deep breath, hold it for 20 seconds, 
and breathe out through D shaped flatpak 
mouth piece attached to CO analyzer 
 
- Equipment: 
A Micro+ CO monitor (Bedfont Scientific Ltd, 
Wales, England) will be used to analyze CO 
level in a single breath in exhaled air. The 
device will be used by applying Anti-bacterial 
filter and one-way valve for increased 
infection control. 
The device has adjustable breath-hold 
countdown timer for patients who are unable 
to hold their breath. Calibration will be 
performed every 6 months as per 
manufacture recommendation. The device is 
able to detect and analyze CO concentration 
within a range of: 0-250 ppm with a 
sensitivity of 0.1 ppm and accuracy of ±2% in 
< 20 seconds. 
Direct evaluation of the COHb level will be 
performed using an arterial blood gas 
analyzer (ABL 800 Flex, Radiometer, 
Copenhagen) and will be reported as % of the 
total Hb as well as calculated amount in 
mg/mL. 
 
- Assessment of postoperative liver function 
and regeneration: 
Liver volumetry will be measured using image 
J software downloaded from 
http://rsbweb.nih.gov/ij/. The accuracy of 
this method was reported in a prior study. 
Volumetric study will be performed based on 
CT scan studies obtained as standard of care 
preoperatively before each liver resection 
and at one and three months after each 
resection. 
Collected data will be reported as percentage 
compared to the preoperative hepatic 
volume. 
Specific Aim 2: To analyze the relationship 
between HO-1 induction and the quality of 
post-hepatectomy liver regeneration 

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2.1 Analyze the relationship between CO 
production and the quantity and quality of 
hepatic regeneration assessed by computed 
scans liver volumetric study at one and three 
months after PH and liver function tests. 
2.2 Determine the relation between hepatic 
related morbidity in patients undergoing 
partial hepatectomy and their ability to 
induce HO-1 
 
Study design: 
Aim 2.1: As CO is uniquely produced by HO 
reaction; its level was previously validated to 
check HO-1 induction. Checking this level and 
comparing outcome of the previously created 
three groups of patients (<25%, 25-50% and 
>50% PH) according to their ability to induce 
HO-1 after PH. Liver regeneration will be 
evaluated by radiological criteria at one and 
three months after PH as described above as 
well as laboratory criteria including liver 
function tests. 
Aim 2.2: HO-1 induction was found to offer 
beneficial protective effect in different 
clinical and surgical occasions. In this setting 
of PH, surgical complications in the 
perioperative (30 days) period graded by 
Clavien-Dindo Classification will be reported 
and analyzed regarding its relation with the 
ability to induce HO-1. 
 
Procedures and Methods: 
Liver function tests routinely checked on 
daily basis till normalization will be 
monitored. This includes serum protein, 
Albumin, liver enzymes (AST and ALT), 
alkaline phosphatase and bilirubin level. The 
duration needed to this normalization will be 
checked as well as the trend of each of these 
laboratory parameters. 
Hepatic morbidity will be evaluated using the 
criteria used by the international study group 
of liver surgery being normalization of the 
INR and bilirubin by POD#5 while the severity 

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of post hepatectomy liver failure is graded 
based on its impact on clinical management. 
Grade A post hepatectomy liver failure 
requires no change of the patient's clinical 
management. The clinical management of 
patients with grade B post hepatectomy liver 
failure deviates from the regular course but 
does not require invasive therapy. The need 
for invasive treatment defines grade C post 
hepatectomy liver failure. The Clavien-Dindo 
classification will be used to document 
surgical outcome of patients after PH. 
 
Analyses: 
All results will be collected in Excel 
Document. Analyses of groups based on the 
extent of liver resection and CO production. 
Results of different subgroups will be 
analyzed; comparison between groups will be 
performed by fisher test, paired and 
unpaired student t test, main Whitney test or 
ANOVA with repeated measurements when 
appropriate. Significance is defined as 
p<0.05. 
 
Potential problems and alternative strategy: 
It is possible that HO-1 induction varies 
according to preoperative and intraoperative 
factors beside the patient own ability to 
induce HO-1. The most important known 
other factors are smoking and intraoperative 
blood transfusion will be reported in the 
study, however, other factors may exist and 
influence the result of this study. 
Preoperative factors may include 
preoperative use of chemotherapy and the 
presence of underlying liver cirrhosis. 
Intraoperative factors may include operative 
time and surgical technique (open or 
minimally invasive). In case of significance 
variance between patient s in the same 
group ( same percentage of PH) Subgroup 
analysis including different variants  
(preoperative chemotherapy, perioperative 

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blood transfusion and open versus minimally 
invasive technique) will be used to assess the 
effect of these factors.  
Study Population: 
125 participants enrolled locally; 125 
enrolled worldwide 
Inclusion Criteria: 
1-Patients plans to have partial liver 
resection for primary liver pathology or 
metastatic disease 
2-Patients able to comprehend and willing to 
sign the written consent form 
Exclusion Criteria:  
1-Patients with age less than 18 years 
2-Patients identified as part of the vulnerable 
populations as defined in the current 
application, except women with child bearing 
potential with negative pregnancy test  
Study Treatment: 
For Aim 1, after the patient’s procedure, they 
will have to exhale into the device listed for 
collecting exhaled air specimen.  
For Aim 2, the patient’s blood samples that 
are routinely collected after a hepatectomy 
will be used for analyzing serum protein, 
Albumin, AST and ALT, alkaline phosphatase, 
and bilirubin level.  
The duration of an individual’s participation 
will be about 3 months +/- 2 weeks.  
Study Procedures and Assessments: 
Aim 1: 
Procedures and Methods 
- Protocol: 
For collecting exhaled air specimen, each 
patient will be instructed to exhale 
completely, and then 
take a deep breath, hold it for 20 seconds, 
and breathe out through D shaped flatpak 
mouth piece attached to CO analyzer 
 
- Equipment: 
A Micro+ CO monitor (Bedfont Scientific Ltd, 
Wales, England) will be used to analyze CO 
level in a single breath in exhaled air. The 
device will be used by applying Anti-bacterial 
filter and one-way valve for increased 
infection control. 
The device has adjustable breath-hold 

8 
 
countdown timer for patients who are unable 
to hold their 
breath. Calibration will be performed every 6 
months as per manufacture 
recommendation. The device is able to 
detect and analyze CO concentration within a 
range of: 0-250 ppm with a sensitivity of 0.1 
ppm and accuracy of ±2% in < 20 seconds. 
Direct evaluation of the COHb level will be 
performed using an arterial blood gas 
analyzer (ABL 800 Flex, Radiometer, 
Copenhagen) and will be reported as % of the 
total Hb as well as calculated amount in 
mg/mL. 
 
- Assessment of postoperative liver function 
and regeneration: 
Liver volumetry will be measured using image 
J software downloaded from 
http://rsbweb.nih.gov/ij/. The accuracy of 
this method was reported in a prior study. 
Volumetric study will be performed based on 
CT scan studies obtained as standard of care 
preoperatively before each liver resection 
and at one and three months after each 
resection. 
Collected data will be reported as percentage 
compared to the preoperative hepatic 
volume. 
 
Aim 2: 
Procedures and Methods: 
Liver function tests routinely checked on 
daily basis till normalization will be 
monitored. This includes serum protein, 
Albumin, liver enzymes (AST and ALT), 
alkaline phosphatase and bilirubin level. The 
duration needed to this normalization will be 
checked as well as the trend of each of these 
laboratory parameters. 
Hepatic morbidity will be evaluated using the 
criteria used by the international study group 
of liver surgery being normalization of the 
INR and bilirubin by POD#5 while the severity 

9 
 
of post hepatectomy liver failure is graded 
based on its impact on clinical management. 
Grade A post hepatectomy liver failure 
requires no change of the patient's clinical 
management. The clinical management of 
patients with grade B post hepatectomy liver 
failure deviates from the regular course but 
does not require invasive therapy. The need 
for invasive treatment defines grade C post 
hepatectomy liver failure. The Clavien-Dindo 
classification will be used to document 
surgical outcome of patients after PH. 
Study Endpoints: 
Aim 1: 
-CO output/production based on extent of 
liver resection, to be collected at 4 hrs and 24 
hrs after PH  
-CT scans looking at hepatic regeneration and 
one and three months after PH and liver 
function tests   
Statistical Analyses: 
Aim 1 Analyses: 
All results will be collected in Excel 
Document. Analyses of groups based on the 
extent of liver resection and CO production. 
Results of different subgroups will be 
analyzed; comparison between groups will be 
performed by fisher test, paired and 
unpaired student t test, main Whitney test or 
ANOVA with repeated measurements when 
appropriate. Significance is defined as 
p<0.05.  
 
Aim 2 Analyses: 
All results will be collected in Excel 
Document. Analyses of groups based on the 
extent of liver resection and CO production. 
Results of different subgroups will be 
analyzed; comparison between groups will be 
performed by fisher test, paired and 
unpaired student t test, main Whitney test or 
ANOVA with repeated measurements when 
appropriate. Significance is defined as 
p<0.05.  
Study Duration:  
15 years  
 

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1. INTRODUCTION  
Inducible Heme Oxygenase (HO - 1), an enzyme critical for heme catabolism, is also involved in 
cellular response to oxidative stress. We have previously shown that induction of HO-1 
expression in response to limb ischemia confers pulmonary cellular protection against oxidative 
stress suggesting a systemic factor; however, the mechanism of this protection is unclear. Our 
follow-up study showing that carbon monoxide (CO) the product of heme catabolism by HO-1, 
can mimic this protection suggests that CO itself may be the important active molecule. Recent 
animal work has independently shown that HO-1 induction may also influence cellular 
regeneration in the liver following partial hepatectomy. Independently, CO has emerged as 
growth-promoting signal molecule with anti-apoptotic effect that protects hepatocytes from 
hypoglycemia - induced cytotoxicity. Further, administration of CO significantly improved the 
survival of mice after initiation of fulminant hepatitis and enhances rapid and early hepatocytes 
proliferation after partial hepatectomy, when mice lacking functional HO-1 were unable to 
mount an appropriate regenerative response. Collectively, these data suggest that that the 
heme oxygenase - CO offers either direct or indirect protection against cellular injury and may 
promote regeneration in the liver. In this proposal, we will directly test the hypothesis that HO-
1 induction is critical to hepatic regeneration after partial hepatectomy. We will further test the 
relevance of this observation in human patients undergoing partial hepatectomy by measuring 
CO production and correlating this to parameters of liver regeneration. To achieve objective, 
we will pursue two specific aims: (1) we will monitor the extent of HO-1 induction (by 
measuring endogenous CO production) in relationship to the extent of hepatic resection (2) we 
will assess the production of CO in patients following liver resection and correlate these 
findings to the rate of liver regeneration and liver function. This research will establish a 
framework for understanding the role of HO-1 induction in liver regeneration and will provide a 
foundation for developing more effective approaches in enhancing hepatic regeneration and 
preventing liver failure after partial hepatectomy.  
2. STUDY OBJECTIVE   
The goal of this proposal is to elucidate the role of HO-1 induction in hepatic regeneration after 
partial hepatectomy (PH). There is a growing body of evidence that HO-1 induction through CO 
production has an important role in cellular protection and regeneration. To test this concept, 
we will monitor endogenous CO production in patients who undergo PH and analyze the 
relationship between CO production and hepatic regeneration. Using this approach, we will test 
the following two hypotheses: (1) HO-1 induction after PH is proportional to the extent of the 
surgical resection, and (2) failure to appropriately induce HO-1 is associated with impaired 
hepatic regeneration.   
3. INVESTIGATIONAL PLAN  
This is a University of Maryland Medical System multi-site study being done at University of 
Maryland Medical Center and UM Baltimore Washington Medical Center.  

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The study will monitor carbon monoxide production in patients undergoing liver resection and 
aims to identify the relationship between CO production and recovery after this resection.  
Carbon monoxide will be checked from arterial blood gas obtained routinely as a part of the 
postoperative care as well as from the exhaled air of the patient through a CO detector 
commercially available and used as smokerlyzer helping people to stop smoking. The results of 
the surgical resection will be collected from the patient routinely ordered tests after liver 
resection including pathology of the resected part of the liver, laboratory and radiology tests.  
The study duration for each individual patient is approximately 3 months +/- 2 weeks.  
4. STUDY TREATMENT  
Subjects who met all inclusion and exclusion criteria according to the data collected from 
medical records will have their carbon monoxide checked from arterial blood gas obtained as 
part of routine post-operative care, as well as from exhaled air measured through a CO 
detector. This study involves prospective data collection that does no interfere with treatment 
plan of participants.  
5. STUDY POPULATION  
The populations to be included are as follows:  
Gender: Female, Male 
Ages: 18-88 years (adult)  
Race/Ethnicity: All Races Included  
Languages: English  
No vulnerable populations are being targeted.  
5.1 Inclusion Criteria  
-Patient plans to have partial liver resection for primary liver pathology or metastatic disease  
-Patients is able to comprehend and willing to sign the written consent form  
5.2 Exclusion Criteria  
-Patients with age less than 18 years  
-Patients identified as part of the vulnerable populations as defined in the current application, 
except women with child bearing potential with negative pregnancy test  
6. STUDY VISITS 
 Patients planned to undergo partial hepatectomy for primary or metastatic hepatic pathology 
will be screened for eligibility to participate in this study. Objectives, methods of data 

12 
 
collection, and analysis will be discussed. After answering all patient questions and explaining 
the patient rights including refusal to participate, the patient will be offered to participate in 
the study. Patients who will accept to participate will be asked to sign the participation consent 
form. It is a prospective data collection without interference with the treatment plan.  
For Aim 1, CO will be monitored before a patient’s liver resection, and at 4- and 24-hours post-
resection. 
For Aim 2, CT scans of liver volume will be performed at one and three months post-
hepatectomy, as well as liver function tests PH.  
 
6.1 Withdrawal of Participants: 
The PI can remove a participant from the research study without the participants 
approval. Possible reasons include failure to follow instructions, or if the PI decides 
that the research study is no longer in the participant's best interest.  
During the study period, the patient can ask to withdraw from the study without 
that decision interfering with his/her treatment plan. 
For this purpose, a letter addressed to the PI or the provider in charge of follow up is 
needed. The Physician will inform the PI who will withdraw the patient information 
from the database. 
If the study is terminated for any other reason, the PI will be entitled to send a 
notification letter to all participants.  
The doctor/PI will speak to the participant about the withdrawal and the participant 
will have a chance to ask questions if this were to happen.  
 
7. STUDY ASSESSMENTS  
This study is a prospective data collection and thus poses no associated health risks. 
7.1 Measuring CO by exhaled air using a smokerlyzer device pre-liver resection, and at 4- 
and 24-hours post resection 
7.2 Measuring carboxyhemoglobin (COHb) in arterial blood gas pre-liver resection, and 
at 4 and 24 hours post resection 
7.3 CT scan at 1 and 3 months post liver resection 
  
8. STUDY ETHICAL CONSIDERATIONS  
The investigator agrees that the study will be conducted according to Good Clinical Practice 
principles of the ICH E6 (R1), ISO 14155 and the principles of the World Medical Association 
Declaration of Helsinki 1964 (including all amendments and Notes of Clarification). The 
Investigator will conduct all aspects of this study in accordance with all national, state, and local 
laws or regulations.  

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9. ADMINISTRATIVE CONSIDERATIONS  
All laboratory specimens, evaluation forms, reports, and other records will be identified in a 
manner designed to maintain subject confidentiality. All records will be kept in a secure storage 
area with limited access. Clinical information will not be released without the written 
permission of the subject (or the subject’s guardian), except as necessary for the FDA (or other 
international regulatory agency), or the IRB/IEC. 
The Investigator and all employees and coworkers involved with this study may not disclose or 
use for any purpose other than performance of the study, any data, record, or other 
unpublished confidential information disclosed to those individuals for the purpose of the 
study.   
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