and cholesterol are reesterifi ed and packaged with apoB48, apoA1, apoA4, the C apos, phospholipids, and unesterifi ed cholesterol into chylomicrons. The chylomicrons enter the bloodstream where, in adipose and muscle tissue, the core TG is hydrolyzed by LPL. The known activators of LPL include apoC2, apoA4, apoA5, and lipase maturation factor 1, whereas apoC3 and angiopoietin-like proteins 3 and 4 inhibit LPL ( 3, 4 ). Remnant chylomicron particles are formed by the removal of the TG core. These remnants are then taken up into the liver. The endogenous pathway of TG metabolism starts with TG synthesized by the liver. These TGs are then secreted into the circulation within the core of VLDL particles. At the cellular level, the synthesis and secretion of VLDL particles resembles that of chylomicrons, except that a different B apo (B100 instead of B48) is required, with apoE and C proteins possibly acting as modifi ers. Once in the plasma, VLDL TG is hydrolyzed by LPL, generating smaller and denser VLDL and IDL particles, with the latter either taken up by the liver or further remodeled to become LDL ( 3 ).Clearly, apos such as B48, B100, A4, A5, E, C2, and C3 play important roles in TG metabolism. Accurate determination of the concentrations and the turnover kinetics of these proteins in healthy and dyslipidemic individuals could shed new light on the regulation of plasma TG levels and, therefore, facilitate the development of better treatments. Because these proteins simultaneously interact with both the exogenous and endogenous pathways of TG metabolism, an ideal method would be one that allowed investigators to examine their interplay. Although good Abstract LC/MS quantifi cation of multiple plasma proteins that differ by several orders of magnitude in concentration from a single sample is challenging. We present a strategy that allows the simultaneous determination of the concentration and turnover kinetics of higher and lower abundant proteins from a single digestion mixture. Our attention was directed at a cluster of proteins that interact to affect the absorption and interorgan lipid traffi cking. We demonstrate that apos involved in TG metabolism such as apoC2, C3, E, and A4 (micromolar concentration), and apoB48 and apoA5 (single-digit nanomolar concentration) can be quantifi ed from a single digestion mixture. A high degree of correlation between LC/MS and immunobased measurements for apoC2, C3, E, and B48 was observed. Moreover, apoA5 fractional synthesis rate was measured in humans for the fi rst time. Finally, the method can be directly applied to studies involving nonhuman primates because peptide sequences used in the method are conserved between humans and nonhuman primates. There is growing evidence to support an association between elevated TG levels and CVD ( 1 ). However, despite decades of research, the precise role of TG in CVD is still a subject of intense debate ( 2 ). Abbreviations: CV, coeffi cient of variation; FSR, fractional synthesis rate; ISTD, internal standard; LLOQ, lower limit o...
Numerous studies have implicated dyslipidemia as a key factor in mediating insulin resistance. Ceramides have received special attention since their levels are inversely associated with normal insulin signaling and positively associated with factors that are involved in cardiometabolic disease. Despite the growing literature surrounding ceramide biology, there are limited data regarding the activity of ceramide synthesis and turnover in vivo. Herein, we demonstrate the ability to measure ceramide kinetics by coupling the administration of [H]water with LC-MS/MS analyses. As a "proof-of-concept" we determined the effect of a diet-induced alteration on ceramide flux; studies also examined the effect of myriocin (a known inhibitor of serine palmitoyltransferase, the first step in sphingosine biosynthesis). Our data suggest that one can estimate ceramide synthesis and draw conclusions regarding the source of fatty acids; we discuss caveats in regards to method development in this area.
The integrated semi-automated bench-top system provided the ability to overcome many of the drawbacks of existing in vitro models. It can be used for various simple or complicated pharmacokinetic/pharmacodynamic studies efficiently and conveniently.
ObjectiveCeramides are putative mediators of insulin resistance and lipotoxicity; an up‐regulation of ceramide biosynthesis has been well described in rodent models of metabolic disease. A precise, robust and specific LC/MS method was developed to quantify ceramide profiles and support determinations of ceramide flux. The LC/MS method was used to determine the effects of diet‐induced obesity (DIO) in C57B1/6J mice ±treatment with myriocin (1mg/kg, over 7 days), an inhibitor of ceramide synthesis.MethodPlasma and tissue samples were collected from mice (lean, high‐fat fed ± myriocin) at various times following administration of stable isotope tracers. The ceramides were extracted with organic solvents and analyzed using electrospray ionization tandem mass spectrometry; concentrations were determined by normalization to an internal standard (d31‐c16:0 ceramide).ResultsIn contrast to lean mice, DIO‐mice demonstrated an increased concentration and synthesis of circulating ceramides. Myriocin treatment inhibited ceramide synthesis and led to a normalization of circulating levels.ConclusionLC/MS analyses can enable characterization of circulating concentrations of ceramide subspecies and enable stable isotope determinations of in vivo kinetics.Support or Funding InformationAll authors are employed by Merck.
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