Deoxysphingoid bases as plasma markers in diabetes mellitus

Abstract

Background: Sphingoid bases are formed from the precursors L-serine and palmitoyl-CoA-a reaction which is catalyzed by the serine-palmitoyltransferase (SPT). SPT metabolizes, besides palmitoyl-CoA also other acyl-CoAs but shows also variability towards the use of other amino acid substrates. The enzyme is also able to metabolize alanine, which results in the formation of an atypical deoxy-sphingoid base (DSB). This promiscuous activity is greatly increased in the case of the sensory neuropathy HSAN1, and pathologically elevated DSB levels have been identified as the cause of this disease. Clinically, HSAN1 shows a pronounced similarity to the diabetic sensory neuropathy (DSN), which is the most common chronic complication of diabetes mellitus. Since serine and alanine metabolism is functionally linked to carbohydrate metabolism by their precursors 3-phosphoglycerate and pyruvate, we were interested to see whether the levels of certain sphingoid base metabolites are altered in patients with diabetes.

Results: In a case-control study we compared plasma sphingoid base levels between healthy and diabetic individuals. DSB levels were higher in the diabetic group whereas C16 and C18 sphingoid bases were not significantly different. Plasma serine, but not alanine levels were lower in the diabetic group. A subsequent lipoprotein fractionation showed that the DSBs are primarily present in the LDL and VLDL fraction.

Conclusion: Our results suggest that DSBs are a novel category of plasma biomarkers in diabetes which reflect functional impairments of carbohydrate metabolism. Furthermore, elevated DSB levels as we see them in diabetic patients might also contribute to the progression of the diabetic sensory neuropathy, the most frequent complication of diabetes.

Figure 1

De-novo sphingolipid synthesis pathway. De-novo ceramide synthesis involves several steps. Serine Palmitoyltransferase (SPT) catalyzes the initial conjugation of palmitoyl-CoA with L-serine to form 3-keto-sphinganine which is subsequently reduced to sphinganine (SA). SA is acetylated by ceramide synthase (CerS) and desaturated by ceramide desaturase (DES) to form ceramide. The degradation pathway starts with the deacetylation of ceramide by ceramidase. The sphingosine (SO) formed is then phosphorylated by SO-Kinase and finally degraded to hexadecenal and phosphoethanolamine by the action of the sphingosine-1-phospate lyase (SO1P-lyase).

Figure 2

A) Boxplot representation of C₁₆, C₁₈ and DSB levels in the control (C) and diabetic (D) cohort. Whereas C₁₆SA, C₁₆SO, C₁₈SA and C₁₈SO were not different between the two groups were doxSA and doxSO levels higher in the diabetic group. (Box represents the upper and lower quartile, whiskers show the 5% and 95% percentile, the horizontal line represents the median) C) Receiver operator curve (ROC) for doxSO D) Area under the curve (AUC) and p-values for all analyzed variables were calculated from a ROC analysis. Most significant parameters are marked (p values were calculated with the null hypothesis (H0) as AUC = 0.5). E) Distribution of the DSB and C16 sphingoid bases in the distinct lipoprotein fractions. Human plasma was separated by a four step density gradient ultracentrifugation. The individual fractions were assayed for cholesterol (not shown), triglycerides (TG; dashed line), doxSA (triangle) doxSO (triangle) and C₁₆SO. Both doxSA and doxSO are only found in the VLDL and LDL but not in the HDL lipoprotein fraction. C₁₆-SO, in contrast, was present in LDL and HDL but not in VLDL. The sum of all fractions was defined as 100% and values are given as a percentage of the total.