The effect of pyridoxamine supplementation on vascular function and insulin sensitivity; a double-blind randomized placebo controlled trial in abdominally obese subjects.

A growing body of evidence demonstrates that increased adipose mass, especially
visceral adipose tissue, contributes directly towards an increase in systemic
inflammation, vascular dysfunction and the burden of CVD, insulin resistance
and type 2 diabetes. The induced expression and secretion of adipokines most
likely plays a causal role in this process.
Advanced glycation/lipoxidation endproducts (AGEs/ALEs) are a heterogeneous
family of unavoidable by-products, which are formed by reactive metabolic
intermediates derived from glucose and lipid oxidation. In addition to the
overwhelming amount of data demonstrating the role of AGEs/ALEs in the
development of vascular disease, AGEs/ALEs are also implicated in the
development of insulin resistance. We recently investigated the accumulation of
AGEs/ALEs in the expanding adipose tissue and found that the activation of the
AGE-RAGE axis contributes to the dysregulation of adipokines and insulin
resistance. In animal models we recently demonstrated that pyridoxamine, a
natural vitamin B6 vitamer and inhibitor of AGE/ALE generation, attenuates
dysregulation of adipokines, vascular dysfunction, and the development of
insulin resistance . We hypothesize that accumulation of AGEs and ALEs could
influence obesity-associated vascular dysfunction, insulin resistance, and an
impaired adipokine profile, and want to investigate the physiological influence
of a dietary intervention with pyridoxamine.


*** Addendum: Substudy of the Pyridoxamine trial ***
Objective: The objective is to study the compliance and metabolisation of
pyridoxamine in plasma and urine with UPLC-MS/MS. To measure the uptake and
bioavailability in plasma following 3 x daily intake of pyridoxamine (protocol
1). To measure the plasma/urine metabolites and pharmacokinetics (protocol 2).
Participants will be recruited locally via posters and our website/facebook
page. We can also contact previously-screened individuals who where too healthy
to participate before.
Study design: We will conduct a small study with the dietary supplement
pyridoxamine dihydrochloride. Five subjects will undergo the following
protocols.
1) Intake of 3 times 66.66 mg pyridoxamine separated over 3 standardized meals
during the day (i.e. identical as the pyridoxamine trial; 66.66 mg/capsule). In
total 20 plasma samples of 4 mL will be collected over 24 hours and a 24h urine
collection will be performed.
2) Intake of 200 mg of pyridoxamine at once in the morning (this is for the
calculation of the pharmacokinetics, to show the metabolisation with a single
admission dosage). In total 20 samples of 4 mL will be collected over 24 hours
and a 24h urine collection will be performed.
3) As control for dietary effects or possible Vitamin B6 content, 2 subjects
will eat the same as abovementioned protocols (1 & 2) but without pyridoxamine
supplementation.
Protocol 1 and 2 will be performed during one single day; the day will be
completely standardised with regard to food intake and physical activity. All
participants will receive the exact same meals and drinks, at the same time
points. A financial compensation of 75 euros will be offered for each visit
(two or three visits in total).
Study population: The study population will consist of five healthy subjects
older than 18, with no disease or medication use. Volunteers (men and women)
will be locally recruited and telephonically screened for possible exclusion
criteria (medication use & current diseases).
Intervention: All 5 subjects will take the same pyridoxamine supplement and
during the day blood will be collected. With a single venous cannula, a maximum
collection of 100mL (80mL specifically) of blood will be collected during one
day. We also collect 24h urine samples.
To reduce variation, this protocol will be performed twice by the same subjects
(protocol 1 and 2). Two volunteers will run through protocol 3 as a control for
dietary intake. *

The objective is to study the possible effects of AGE/ALE reduction on vascular
function as primary endpoint, insulin sensitivity, and adipokine levels in
abdominally obese subjects. We will investigate the following parameters:
1) Micro- and macrovascular function by means of skeletal muscle
contrast-enhanced ultrasound (CEUS) measurements, skin capillary
videomicroscopy, skin laser doppler flowmetry (LDF), flow mediated dilation
(FMD), and arterial stiffness (pulse wave velocity and local stiffness).
2) Whole body insulin sensitivity using a hyperinsulinemic, euglycemic clamp
and glucose metabolism using an OGTT
3) AGE measurements in blood plasma and skin (skin autofluorescence (SAF)).
4) Adipokine, endothelial function marker, and inflammation marker measurements
in plasma.

We will conduct a short-term (8 weeks), double blind, randomized,
placebo-controlled trial with the dietary supplement pyridoxamine
dihydrochloride, a metabolic variant of pyridoxal phosphate and a member of the
vitamin B6 group. The study has a parallel design and includes three study
groups of each 40 participants, bringing the number of participants at a total
of 120. The groups consist of a placebo treatment, a 25 mg/day pyridoxamine
dosage, and a 200 mg/day pyridoxamine dosage. After baseline measurements all
subjects will be randomized in one of these three groups.

All subjects will undergo an intervention consisting of three capsules per day,
during 8 continuous weeks. One group will receive a placebo treatment, another
a dosage of 25 mg pyridoxamine per day, and the last will receive a dosage of
200 mg pyridoxamine per day.

Participants will visit the study center for a screening visit and two main
study days, one before and one after the intervention, for measurements of
vascular function and a hyperinsulinemic euglycemic clamp test. Additionally, a
24h urine collection and 24h blood pressure measurement will be returned in a
short visit, which we aim to combine with an OGTT (during which a food
frequency questionnaire (FFQ) will be filled out).
At the study days, measurements of vascular function will be performed in a
fasted state, before and during a hyperinsulinemic euglycemic clamp test for
determination of insulin sensitivity. Participants will be requested to abstain
from alcohol 24 hours prior to the measurements and not to perform strenuous
exercise 48 hours before the study days.
The total amount of blood drawn during a study day is approximately 180 ml (9
ml during the screening visit). This amount does not carry any risk, however
subjects are not allowed to donate blood 8 weeks prior to investigation.
Micro- and macrovascular measurements carry virtually no risk. The contrast
agent administered during the contrast-enhanced ultrasound of the skeletal
muscle has proven to be a safe imaging modality in previous investigations and
post-marketing studies. An infrequent side effect of the hyperinsulinemic,
euglycemic clamp test is hypoglycemia.
The two main study days will occupy ~7 hours. The screening visit only takes 1
hour. Although the number of measurements performed during a study day is
considerable, the burden for the participants is limited since these
measurements are all performed in the supine position and are merely
non-invasive, with the exception of contrast-enhanced ultrasound.
Pyridoxamine: In multiple Phase 1 and 2 clinical studies with pyridoxamine,
adverse event rates were similar to those seen in subjects receiving placebo.
The only adverse events that showed an increase over placebo were observed in
the highest dose of 600 mg daily, these were small increases in diarrhea and
constipation and this mild increase was not noticeable in the 300mg group. No
studies have yet described any directly related side affects of pyridoxamine
supplementation, while in many studies higher dosages than the ones
supplemented in this study (up to 200 mg daily) were used. Furthermore, the
participating subjects in our study are abdominally obese individuals without
emerged cardiac, pulmonary, or metabolic disease. Placebo treatment holds no
risks.
Patients from the control group (placebo) will not have any direct health
benefit from participation. As for the other groups, the potential benefit is
directly related to the beneficiary effects of pyridoxamine. Pyridoxamine is a
natural form of vitamin B6 and it is proven that this molecule is able to
effectively inhibit the formation of AGEs and ALEs.