Recent findings have shown an inverse association between circulating C15:0/C17:0 fatty acids with disease risk, therefore, their origin needs to be determined to understanding their role in these pathologies. Through combinations of both animal and human intervention studies, we comprehensively investigated all possible contributions of these fatty acids from the gut-microbiota, the diet, and novel endogenous biosynthesis. Investigations included an intestinal germ-free study and a C15:0/C17:0 diet dose response study. Endogenous production was assessed through: a stearic acid infusion, phytol supplementation, and a Hacl1 −/− mouse model. Two human dietary intervention studies were used to translate the results. Finally, a study comparing baseline C15:0/C17:0 with the prognosis of glucose intolerance. We found that circulating C15:0/C17:0 levels were not influenced by the gut-microbiota. The dose response study showed C15:0 had a linear response, however C17:0 was not directly correlated. The phytol supplementation only decreased C17:0. Stearic acid infusion only increased C17:0. Hacl1 −/− only decreased C17:0. The glucose intolerance study showed only C17:0

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Mice lacking PMP34, a peroxisomal membrane transporter encoded by Slc25a17, did not manifest any obvious phenotype on a Swiss Webster genetic background, even with various treatments designed to unmask impaired peroxisomal functioning. Peroxisomal αand β-oxidation rates in PMP34 deficient fibroblasts or liver slices were not or only modestly affected and in bile, no abnormal bile acid intermediates were detected. Peroxisomal content of cofactors like CoA, ATP, NAD + , thiamine-pyrophosphate and pyridoxal-phosphate, based on direct or indirect data, appeared normal as were tissue plasmalogen and very long chain fatty acid levels. However, upon dietary phytol administration, the knockout mice displayed hepatomegaly, liver inflammation, and an induction of peroxisomal enzymes. This phenotype was partially mediated by PPARα. Hepatic triacylglycerols and cholesterylesters were elevated and both phytanic acid and pristanic acid accumulated in the liver lipids, in females to higher extent than in males. In addition, pristanic acid degradation products were detected, as wells as the CoAesters of all these branched fatty acids. Hence, PMP34 is important for the degradation of phytanic/pristanic acid and/or export of their metabolites. Whether this is caused by a shortage of peroxisomal CoA affecting the intraperoxisomal formation of pristanoyl-CoA (and perhaps of phytanoyl-CoA), or the SCPx-catalyzed thiolytic cleavage during pristanic acid β-oxidation, could not be proven in this model, but the phytol-derived acyl-CoA profile is compatible with the latter possibility. On the other hand, the normal functioning of other peroxisomal pathways, and especially bile acid formation, seems to exclude severe transport problems or a shortage of CoA, and other cofactors like FAD, NAD(P) + , TPP. Based on our findings, PMP34 deficiency in humans is unlikely to be a life threatening condition but could cause elevated phytanic/pristanic acid levels in older adults.

Circulating heptadecanoic acid (C17:0) is reported to be a pathology risk/prognosis biomarker and a dietary biomarker. This pathology relationship has been shown to be reliably predictive even when independent of dietary contributions, suggesting that the endogenous biosynthesis of C17:0 is related to the pathological aetiology. Little is known about C17:0 biosynthesis, which tissues contribute to the circulating levels, and how C17:0 is related to pathology. Hacl1+/− mice were mated to obtain Hacl1−/− and Hacl1+/+ control mice. At 14 weeks, they were anesthetized for tissue collection and fatty acid analysis. Compared to Hacl1+/+, C15:0 was not significantly affected in any Hacl1−/− tissues. However, the Hacl1−/− plasma and liver C17:0 levels were significantly lower: ~26% and ~22%, respectively. No significant differences were seen in the different adipose tissues. To conclude, Hacl1 plays a significant role in the liver and plasma levels of C17:0, providing evidence it can be endogenously biosynthesized via alpha-oxidation. The strong inverse association of C17:0 with pathology raises the question whether there is a direct link between α-oxidation and these diseases. Currently, there is no clear evidence, warranting further research into the role of α-oxidation in relation to metabolic diseases.

Long-chain aliphatic aldehydes are produced during various metabolic processes, such as microsomal oxidation of long-chain alcohols, lysosomal degradation of prenylated proteins, peroxisomal ␣ -oxidation of 3-methylbranched fatty acids and 2-hydroxy long-chain fatty acids, microsomal breakdown of phosphorylated sphingoid bases, attack of plasmalogens by myeloperoxidase (MPO)-derived hypochlorous acid, and microsomal degradation of (lyso)plasmalogens (see Fig. 1A ). The involved enzymes are fatty aldehyde dehydrogenase (ALDH3A2) ( 1, 2 ), prenylcysteine oxidase 1 (PCYOX1) ( 3, 4 ), 2-hydroxyacylCoA lyase (HACL1) ( 5, 6 ), sphingosine-1-phosphate lyase (SGPL1) ( 7-9 ), MPO ( 10 ), and lysoplasmalogenase ( 11,12 ). In particular, two of these enzymes, HACL1 and SGPL1, have intensively been studied in our laboratory. Their activity measurements are complicated by their low specifi c activity [ ‫ف‬ 115 ( 5 ) and ‫ف‬ 15 mU/g rat liver ( 13 ), respectively], low K m (10-15 M), and the considerable interference of enzymes acting on their substrates (acylCoA hydrolases for HACL1; phosphatases for SGPL1) or on their cofactors (phosphatases acting on TPP, cofactor for HACL1, and on pyridoxal-phosphate, cofactor for SGPL1). In addition, the generated aldehyde is chemically not stable and subject to further metabolism, and compounds normally used to trap the aldehyde or block its metabolism interfere with the cofactor (pyridoxalphosphate). Moreover, the presence of plasmalogens in mammalian tissues, which give rise to aldehydes under acidic conditions ( 14 ), can cause a high background when using nonradioactive substrates, especially in nervous tissue.To measure HACL1 and SGPL1 activities in tissue or cell lysates, various protocols were developed by our group.Abstract Long-chain aldehydes are commonly produced in various processes, such as peroxisomal ␣ -oxidation of longchain 3-methyl-branched and 2-hydroxy fatty acids and microsomal breakdown of phosphorylated sphingoid bases. The enzymes involved in the aldehyde-generating steps of these processes are 2-hydroxyacyl-CoA lyase (HACL1) and sphingosine-1-phosphate lyase (SGPL1), respectively. In the present work, nonradioactive assays for these enzymes were developed employing the Hantzsch reaction. Tridecanal (C13-al) and heptadecanal (C17-al) were selected as model compounds and cyclohexane-1,3-dione as 1,3-diketone, and the fl uorescent derivatives were analyzed by reversed phase (RP)-HPLC. Assay mixture composition, as well as pH and heating, were optimized for C13-al and C17-al. Under optimized conditions, these aldehydes could be quantifi ed in picomolar range and different long-chain aldehyde derivatives were well resolved with a linear gradient elution by RP-HPLC. Aldehydes generated by recombinant enzymes could easily be detected via this method. Moreover, the assay allowed to document activity or defi ciency in tissue homogenates and fi broblast lysates without an extraction step. In conclusion, a simple, quick, and cheap assay for the study of HACL1 and SGPL1 ac...