Published on Web 04/21/2010
Using Isotopic Tools to Dissect and Quantitate Parallel Metabolic Pathways
Sushabhan Sadhukhan,†,§ Yong Han,†,§ Guo-Fang Zhang,‡ Henri Brunengraber,‡ and
Gregory P Tochtrop*,†
Department of Chemistry and Department of Nutrition, Case Western ReserVe UniVersity, 10900 Euclid AVenue,
CleVeland, Ohio 44106
Received January 15, 2010; E-mail: tochtrop@case.edu
The existence of parallel metabolic pathways is typically
indicative of fundamental pathways that are critical for normal
physiology. For example, conversion of glucose to pyruvate is
dogmatic across biology, but the eventual fate of pyruvate can
proceed down different parallel pathways dictated by physiologic
conditions. In aerobic organisms under normal conditions, pyruvate
enters the citric acid cycle to be eventually catabolized to carbon
dioxide. However, if that organism is transferred to anaerobic condi-
tions, a parallel metabolic step is utilized whereby pyruvate is converted
to lactate.1 This example is by no means unique, with other prominent
examples including bile acid biosynthesis occurring via either neutral
(classic) or acidic (alternative) pathways2 and ꢀ-oxidation occurring
in either the peroxisome or mitochondria. These examples further
illustrate that this parallelism may occur in the same compartment or
be separately compartmentalized within the cell.3
acid catabolism can proceed via two parallel pathways (shown in
Figure 1 and supplementary Figure S1) that involve either a
phosphorylation and isomerization of the C-4 hydroxyl (denoted
Pathway A) or a ꢀ-oxidation/R-oxidation sequence (Pathway B).6
This represented the first report on the catabolism of this dogmatic
class of biological molecules.
4-Hydroxyacids are omnipresent in ViVo, derived from either
exogenous or endogenous sources. Exogenously these molecules
are typically introduced through drugs of abuse including γ-hy-
droxybutyrate (GHB, the date rape drug)4 or its emerging alterna-
tive, γ-hydroxypentanoate.5 Endogenously, 4-hydroxyacids are
derived from the oxidation and saturation of the ubiquitous lipid
peroxidation product, 4-hydroxy-2-(E)-nonenal (4-HNE).6,7 Since
its discovery in 1964,8 4-HNE has been generally accepted as a
modulator of numerous cellular systems and implicated in the
pathogenesis of a number of degenerative diseases including
Alzheimer’s disease, atherosclerosis, cataracts, and cancer.9,10 The
generally accepted pathogenesis of 4-HNE is linked to the γ-hy-
droxy-R,ꢀ-unsaturated aldehyde acting as a strong electrophile,
which can form adducts with a variety of cellular nucleophiles via
Michael addition or Schiff base formation. This interesting patho-
genesis is linked to the general abundance of this molecule. Under
physiological conditions typical concentrations of 4-HNE range
from 0.1 to 0.3 µM in all tissues, but under severe oxidative stress
this concentration can rise substantially in a localized manner to
between 10 µM and 5 mM.11-13 Consequently, understanding the
mechanisms for elimination of 4-HNE is critical, as it may give
key insights into the pathogenesis of the molecule.
Despite its physiologic importance, little has been known about
the catabolic fate of 4-HNE. Previous reports consisted of a pathway
for elimination of 4-HNE via glutathionylation by glutathione
S-transferase (GST) and subsequent excretion through the kidneys,14
as well as a report of hepatocyte metabolism through the alcohol
dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH),15
though little was known about the latter process.
Recently, we reported the catabolic fates of 4-hydroxyacids in
perfused rat livers using a combination of metabolomics and mass
isotopomer analysis. A key finding of this work is that 4-hydroxy-
Figure 1. Parallel pathways of the catabolism of 4-hydroxy nonanoic acid.
Essential to this finding was the use of isotopically labeled
4-hydroxyacids to define the catabolic pathway for 4-HNE (via
4-hydroxyacids). Here we report the synthesis and evaluation of a
series of tools that were critical for this work and demonstrate how
these tools can be used to quantify the differential catabolic flux
of 4-hydroxacids down either Pathway A or Pathway B.
The logic behind our labeling strategy can be seen in Figure 1.
The ultimate fate of 4-hydroxyacids lies in an initial catabolic step
that can proceed either via a phosporylation and subsequent
isomerization to a 3-hydroxyacid (Pathway A) or via an initial round
of ꢀ-oxidation followed by the loss of formate via R-oxidation
(Pathway B).6 To define the two pathways, we devised a labeling
strategy that could incorporate either a single 13C label at position
C-3 or a doubly labeled molecule with 13C at positions C-3 and
C-4. The label at C-3 was used primarily to confirm that the
R-oxidation step in Pathway B leads to [13C]formate. The C-3,4
molecule allowed us to confirm both pathways’ existence and, as
we report here, has allowed us to quantify the relative flux down
each pathway.
Our synthetic approach is illustrated in Figure 2 (details can be
found in Supporting Information) and is based on a convergent
route that minimizes the number of reactions needed to be
optimized. For the C-3 labeled molecule we began with n-hexyl
bromide and introduced the carbon via a Grignard reaction with
† Department of Chemistry.
‡ Department of Nutrition.
§ These authors contributed equally.
9
10.1021/ja100399m 2010 American Chemical Society
J. AM. CHEM. SOC. 2010, 132, 6309–6311 6309