Human short chain fatty acid metabolism and microbiota

Recent evidence suggests that gut microbiota play a major role in the
pathophysiology of obesity and its related disorders. Data points towards the
existence of quantitative and qualitative differences in gut microbiota between
lean and obese and between diabetic and non-diabetic individuals. These
distinct differences in gut microbiota have been suggested to influence the
energy extraction of ingested foods, intestinal permeability and transit time,
mucosal immunity and systemic inflammation and as such, may play an important
role in the development of obesity and its related disorders. In line with
this, germ-free mice are protected from obesity when fed a high-fat Western
diet. Apparently, germ-free mice are devoid of short chain fatty acids (SCFA),
indicating the importance of gut microbiota providing products that affect host
metabolism.
However, a detailed understanding of the SCFA metabolism and the interorgan
exchange of SCFA in vivo in humans is lacking, and the relation gut
microbiota-host metabolism remains largely unclear.

Knowledge about the physiological metabolism of SCFAs and the interorgan
exchange of SCFAs across different abdominal organs in humans is lacking to
some extent.
To this end, the following objective will be addressed in the current study:

-Determine in vivo in humans the role of different abdominal organs in the
production or extraction of SCFAs

To assess SCFAs handling of individual organs, we will once sample 10 ml blood
from the above indicated blood vessels during surgery and then we will make use
of a recently validated, highly accurate HPLC-MS method to determine
concentrations of the SCFAs acetic acid, propionic acid and butyric acid in
human plasma samples34. Additionally, 1 liver and 1 duodenal biopsy will be
taken allowing detailed study of SCFA metabolism in the gut-liver axis in vivo
in humans.

* We hypothesize that intestinal butyric acid and proprionate release by the
gut is equaled by hepatic uptake of these SCFAs
o We hypothesize that the net organ fluxes of butyric acid and
proprionate will indicate release of these SCFAs by the gut
o We hypothesize that the net organ fluxes of butyric acid and
proprionate will indicate uptake of these SCFAs by the liver

* We hypothesize that the net organ fluxes of the SCFAs across the colon will
be higher compared to the net organ fluxes of these SCFAs across the small
intestine pointing out the colon as the dominant part of the gut in producing
these SCFAs
* We hypothesize that the net organ flux of acetic acid across the liver will
be close to zero indicating no significant uptake/release of acetic acid by the
liver
* We hypothesize that the concentration of acetic acid in the hepatic vein will
be lower compared to the concentration of acetic acid in the arterial radialis
indicating peripheral absorption

This is a cross-sectional descriptive study.
For this study, patients undergoing pp Whipple will be recruited at the
surgical outpatient clinic of Maastricht University Medical Center (MUMC+).
First, patients will consult their surgeon for the so called *preoperative
screening*. This screening is intended to determine whether and what kind of
operation is suitable for the patient involved. In case the surgeon assumes
that a patient may be suitable for participation in this study, he will briefly
cite the study. Once the patient has indicated to be willing to receive more
information about this study, the researcher will be informed by the surgeon.
Subsequently, the researcher will check in a consult with the patient
immediately following the preoperative screening, if he/she is indeed suitable
for participation in this study on the basis of the inclusion and exclusion
criteria as described below. During this consult, patients are given the
opportunity to ask questions. Afterwards, the patients go home with the written
information as provided by the researcher. Should they encounter questions,
they always can contact the researcher to ask questions. In addition, it is
possible to contact an independent medical doctor. Finally, the day of
admission, generally one day before surgery, there is again time to ask
questions. If the patient decides to participate in the study, informed consent
will be obtained. Subjects are participating entirely voluntarily in this study
and can stop their participation at any time for any reason if they wish to do
so without any consequences.
From the patients participating in the study blood samples will be collected.
Blood will be sampled from the portal vein, hepatic vein, superior mesenteric
vein, inferior mesenteric vein, splenic vein, renal vein and the radial artery
according to Figure 1. Arteriovenous differences (ΔAV) and net organ fluxes
(flow x ΔAV) are a quantitative measure of the role of the liver, PDV, the
splanchnic area, small intestine, colon, spleen and the kidneys in producing or
extracting SCFAs.

All patients undergoing pp Whipple have an arterial catheter during surgery
from which we can sample blood. The mentioned veins will be punctured directly
using 25G needles as described previously in several MEC protocols and in a
publication of Van de Poll et al (MEC 02-045, MEC 03-032, MEC 06-2067 and
[35]). Only once 10 ml of arterial blood and 10 ml intra-abdominal blood from
the different vessels separately will be sampled resulting in a total amount of
maximal 70 ml. All blood samples will be collected in both pre-chilled EDTA and
heparinized vacuum tubes and thereafter centrifuged at 3500 rpm at 4°C for 10
minutes. Plasma will be stored in Eppendorf cups at -80°C until further
analysis. Blood flow will be measured using intra-operative Duplex
ultrasonography and the concentration of the SCFAs acetic acid, propionic acid
and butyric acid in human plasma samples will be measured using HPLC-MS as
previously described34. Additionally, 1 liver and 1 duodenal biopsy will be
taken allowing detailed study of SCFA metabolism in the gut-liver axis in vivo
in humans. The methods applied, i.e. intra-abdominal blood sampling and
collecting liver biopsies, have been previously used without any problems for
the surgical procedures or the patients. Besides, there are no additional
risks to the collection of duodenal biopsies since these are part of the
duodenum which is resected anyway.


The corresponding AV differences will be calculated as follows:
ΔAV PDV = [PV] - [A]
ΔAV Splanchnic area = [HV] - [A]
ΔAV IMV = [IMV] - [A]
ΔAV SMV = [SMV] - [A]
ΔAV SV = [SV] - [A]
ΔAV RV = [RV] - [A]

Fluxes will be calculated as follows:
F PDV = portal plasma flow*([PV]-[A])
F Splanchnic area = splanchnic plasma flow*([HV] - [A])
F Liver = F splanchnic - F PDV
F Colon = inferior mesenteric plasma flow*([IMV] - [A])
F Small intestine = superior mesenteric plasma flow*([SMV] - [A])
F Spleen = splenic plasma flow*([SV] - [A])
F Kidneys = renal plasma flow*([SV] - [A])

ΔAV = Arteriovenous differences
PDV = Portal drained viscera
A = Blood sample from arterial line
PV = Blood sample from portal vein
HV = Blood sample from hepatic vein
IMV = Blood sample from inferior mesenteric vein
SMV = Blood sample from superior mesenteric vein
SV = Blood sample from splenic vein
RV = Blood sample from renal vein

Of note, positive fluxes indicate release, whilst negative fluxes indicate
uptake.

The methods applied, i.e. intra-abdominal blood sampling and collecting liver
biopsies, have been used previously without any problems for the surgical
procedures or the patients (MEC 02-045, MEC 03-032, MEC 06-2067) as published
by Van de Poll et al. Besides, there are no additional risks to the collection
of duodenal biopsies since these are part of the duodenum which is resected
anyway.


Although the results of this project have no direct positive effects for the
patients involved, they do contribute to the understanding of the SCFA
metabolism, and thereby form a basis in the process of unraveling the relation
between gut microbiota, SCFAs and human energy and substrate metabolism.
Future nutritional modulation of gut microbiota may beneficially affect gut and
host health.