The Fatty Acid Desaturation Index in Human Plasma: Comparison of Different Analytical Methodologies for the Evaluation of Dietary Effects

Abstract (252 words) Background: Stearoyl-CoA Desaturase (SCD1) plays a role in the development of obesity and related conditions, such as insulin resistance, and potentially also in neurological and heart diseases. The activity of SCD1 can be monitored using the desaturation index (DI), the ratio of product (16:1n-7 and 18:1n-9) to precursor (16:0 and 18:0) fatty acids. Here we analyzed the DI in the plasma total triglyceride fraction and very low density lipoproteins (VLDL) in volunteers on a high carbohydrate diet using a dual ultra-high pressure liquid chromatography – mass spectrometry (UHPLC-MS) strategy. Methods: One assay was based on a simple extraction followed by neutral loss triglyceride – fatty acid analysis, the other on saponification of triglycerides followed by fatty acid analysis (specific for the position of the double bond within monosaturated fatty acids (MUFA). Both assays were applied to the isolated VLDL or to the plasma total triglyceride fraction. Assays were compared using plasma from a study in which 8 lean and obese healthy individuals received a high carbohydrate diet for 3 days. Results: All assays showed acceptable accuracies (75-125) and precisions (<20%) for the analysis of fatty acids in VLDL and plasma. Analysis of human plasma samples revealed increased DI (up to 136% of control, p< 0.05 after a high carbohydrate diet of 3 days. Conclusion: Only the specific fatty acid UHPLC-MS analysis of human plasma VLDL samples reflects the complete biological pathway and showed that a significant increase in DI and associated SCD1 activity –in healthy individuals after just 3 days of high carbohydrate diet

Low-Salt Diet and Cyclosporine Nephrotoxicity: Changes in Kidney Cell Metabolism

Cyclosporine (CsA) is a highly effective immunosuppressant used in patients after transplantation; however, its use is limited by nephrotoxicity. Salt depletion is known to enhance CsA-induced nephrotoxicity in the rat, but the underlying molecular mechanisms are not completely understood. The goal of our study was to identify the molecular effects of salt depletion alone and in combination with CsA on the kidney using a proteo-metabolomic strategy. Rats (<i>n</i> = 6) were assigned to four study groups: (1) normal controls, (2) low-salt fed controls, (3) 10 mg/kg/d CsA for 28 days on a normal diet, (4) 10 mg/kg/d CsA for 28 days on low-salt diet. Low-salt diet redirected kidney energy metabolism toward mitochondria as indicated by a higher energy charge than in normal-fed controls. Low-salt diet alone reduced phospho-AKT and phospho-STAT3 levels and changed the expression of ion transporters PDZK1 and CLIC1. CsA induced macro- and microvesicular tubular epithelial vacuolization and reduced energy charge, changes that were more significant in low-salt fed animals, probably because of their more pronounced dependence on mitochondria. Here, CsA increased phospho-JAK2 and phospho-STAT3 levels and reduced the phospho-IKKγ and p65 proteins, thus activating NF-κB signaling. Decreased expression of lactate transport regulator CD147 and phospho-AKT was also observed after CsA exposure in low-salt rats, indicating a decrease in glycolysis. In summary, our study suggests a key role for PDZK1, CD147, JAK/STAT, and AKT signaling in CsA-induced nephrotoxicity and proposes mechanistic explanations on why rats fed a low-salt diet have higher sensitivity to CsA