Unsaturated fats with trans double bonds are commonly referred to as "trans fats." There has been much discussion about the effects of dietary trans fats on health. In their investigations of the effects of trans fatty acid metabolism on health, Yu and colleagues (2004) showed that a model trans fatty acid was processed differently from its cis isomer. They used three related 18 -carbon fatty acids to explore the difference in $\beta$ oxidation between cis and trans isomers of the same-size fatty acid. The researchers incubated the coenzyme A derivative of each acid with rat liver mitochondria for 5 minutes, then separated the remaining CoA derivatives in each mixture by HPLC (high-performance liquid chromatography). The results are shown below, with separate panels for the three experiments.
In the figure, IS indicates an internal standard (pentadecanoyl-CoA) added to the mixture, after the reaction, as a molecular marker. The researchers abbreviated the CoA derivatives as follows: stearoyl-CoA, $C_{18}-$ CoA; cis$-\Delta^{5}$ -tetradecenoyl-CoA, $\mathrm{c} \Delta^{5} \mathrm{C}_{14}$ -CoA; oleoyl-CoA, $\mathrm{c} \Delta^{9} \mathrm{C}_{18}-\mathrm{CoA} ;$ trans- $\Delta^{5}$ -tetradecenoyl-CoA, $\mathrm{t} \Delta^{5} \mathrm{C}_{14}-\mathrm{CoA} ;$ and elaidoyl-CoA, $\mathrm{t} \Delta^{9} \mathrm{C}_{18}-\mathrm{CoA}$
(a) Why did Yu and colleagues need to use CoA derivatives rather than the free fatty acids in these experiments?
(b) Why were no lower molecular weight CoA derivatives found in the reaction with stearoyl-CoA?
(c) How many rounds of $\beta$ oxidation would be required to convert the oleoyl-CoA and the elaidoyl-CoA to $c$ is- $\Delta^{5}$ -tetradecenoyl-CoA and trans$=\Delta^{5}$ -tetradecenoyl-CoA, respectively?
$\mathrm{Yu}$ and coworkers measured the kinetic parameters of two forms of the enzyme acyl CoA dehydrogenase: long-chain acyl-CoA dehydrogenase (LCAD) and very-long-chain acyl-CoA dehydrogenase (VLCAD). They used the CoA derivatives of three fatty acids:
tetradecanoyl-CoA $\left(\mathrm{C}_{14}-\mathrm{CoA}\right),$ cis- $\Delta^{5}$ -tetradecenoyl-CoA $\left(\mathrm{c} \Delta^{5} \mathrm{C}_{14}-\mathrm{CoA}\right),$ and trans- $\Delta^{5}$ tetradecenoyl-CoA $\left(\mathrm{t} \Delta^{5} \mathrm{C}_{14}-\mathrm{CoA}\right)$. The results are shown below. (See Chapter 6 for definitions of the kinetic parameters.)
(d) For $\mathrm{LCAD}$, the $K_{\mathrm{m}}$ differs dramatically for the cis and trans substrates. Provide a
plausible explanation for this observation in terms of the structures of the substrate molecules. (Hint: You may want to refer to Fig. $10-1$.)
(e) The kinetic parameters of the two enzymes are relevant to the differential processing of these fatty acids only if the LCAD or VLCAD reaction (or both) is the rate-limiting step in the pathway. What evidence is there to support this assumption?
(f) How do these different kinetic parameters explain the different levels of the CoA derivatives found after incubation of rat liver mitochondria with stearoyl-CoA, oleoyl-CoA, and elaidoyl-CoA (shown in the three-panel figure)?
Yu and coworkers measured the substrate specificity of rat liver mitochondrial thioesterase, which hydrolyzes acyl-CoA to CoA and free fatty acid. This enzyme was approximately twice as active with $\mathrm{C}_{14}$ -CoA thioesters as with $\mathrm{C}_{18}$ -CoA thioesters.
(g) Other research has suggested that free fatty acids can pass through membranes. In their experiments, $Y u$ and colleagues found trans- $\Delta^{5}$ -tetradecenoic acid outside (i.e., in the medium surrounding) mitochondria that had been incubated with elaidoyl-CoA. Describe the pathway that led to this extramitochondrial trans$-\Delta^{5}$ -tetradecenoic acid. Be sure to indicate where in the cell the various transformations take place, as well as the enzymes that catalyze the transformations.
(h) It is often said in the popular press that "trans fats are not broken down by your cells and instead accumulate in your body." In what sense is this statement correct and in what sense is it an oversimplification?