Thorsten Hornemann

Comparable increase of B-type natriuretic peptide and amino-terminal pro-B-type natriuretic peptide levels in patients with severe sepsis, septic shock, and acute heart failure.

Objective:B-type natriuretic peptide (BNP) and N-terminal pro-BNP measurements are used for the diagnosis of congestive heart failure (HF). However, the diagnostic value of these tests is unknown under septic conditions. We compared patients with severe sepsis or septic shock and patients with acute HF to unravel the influence of the underlying diagnosis on BNP and N-terminal pro-BNP levels. Design:Prospective, clinical study. Setting:Academic medical intensive care unit (ICU). Patients:A total of 249 consecutive patients were screened for the diagnosis of sepsis or HF. Sepsis was defined according to published guidelines. HF was diagnosed in the presence of an underlying heart disease and congestive HF, pulmonary edema, or cardiogenic shock. Interventions:BNP and N-terminal pro-BNP were measured from blood samples that were drawn daily for routine analysis. Measurements and Main Results:We identified 24 patients with severe sepsis or septic shock and 51 patients with acute HF. At admission, the median (range) BNP and N-terminal pro-BNP levels were 572 (13–1,300) and 6,526 (198–70,000) ng/L in patients with sepsis and 581 (6–1,300) and 4,300 (126–70,000) ng/L in patients with HF. The natriuretic peptide levels increased during the ICU stay, but the differences between the groups were not significant. Nine patients with sepsis and eight patients with HF were monitored with a pulmonary artery catheter. Mean (sd) pulmonary artery occlusion pressure were 16 (4.2) and 22 (5.3) mm Hg (p = .02), and cardiac indexes were 4.6 (2.8) and 2.2 (0.6) L/min/m2 (p = .03) in patients with sepsis and HF, respectively. Despite these clear hemodynamic differences BNP and N-terminal pro-BNP levels were not statistically different between the two groups. Conclusion:In patients with severe sepsis or septic shock, BNP and N-terminal pro-BNP values are highly elevated and, despite significant hemodynamic differences, comparable with those found in acute HF patients. It remains to be determined how elevations of natriuretic peptide levels are linked to inflammation and sepsis-associated myocardial dysfunction.

Hereditary Sensory Neuropathy Type 1 Is Caused by the Accumulation of Two Neurotoxic Sphingolipids

HSAN1 is an inherited neuropathy found to be associated with several missense mutations in the SPTLC1 subunit of serine palmitoyltransferase (SPT). SPT catalyzes the condensation of serine and palmitoyl-CoA, the initial step in the de novo synthesis of sphingolipids. Here we show that the HSAN1 mutations induce a shift in the substrate specificity of SPT, which leads to the formation of the two atypical deoxy-sphingoid bases (DSBs) 1-deoxy-sphinganine and 1-deoxymethyl-sphinganine. Both metabolites lack the C1 hydroxyl group of sphinganine and can therefore neither be converted to complex sphingolipids nor degraded. Consequently, they accumulate in the cell, as demonstrated in HEK293 cells overexpressing mutant SPTLC1 and lymphoblasts of HSAN1 patients. Elevated DSB levels were also found in the plasma of HSAN1 patients and confirmed in three groups of HSAN1 patients with different SPTLC1mutations. The DSBs show pronounced neurotoxic effects on neurite formation in cultured sensory neurons. The neurotoxicity co-occurs with a disturbed neurofilament structure in neurites when cultured in the presence of DSBs. Based on these observations, we conclude that HSAN1 is caused by a gain of function mutation, which results in the formation of two atypical and neurotoxic sphingolipid metabolites.

Cloning and Initial Characterization of a New Subunit for Mammalian Serine-palmitoyltransferase

Serine-palmitoyltransferase (SPT) catalyzes the rate-limiting step of the de novo synthesis of sphingolipids. SPT is considered to be a heterodimer composed of two subunits, SPTLC1 and SPTLC2. Here we report the identification of a novel, third, SPT subunit (SPTLC3) that shows 68% homology to the SPTLC2 subunit. Quantitative real-time PCR revealed that SPTLC3 expression is highly variable between different human tissues and cell lines. The highest expression was observed in placenta tissue and human trophoblast cell lines. The overexpression of SPTLC3 in Hek293 cells, which otherwise have very little endogenous SPTLC3, led to a 2- to 3-fold increase in cellular SPT activity. Silencing of SPTLC3 expression in HepG2 cells or human trophoblast cells by transfecting SPTLC3-specific siRNA resulted in a significant reduction of cellular SPT activity. The expression of two SPT isoforms could be a cellular mechanism to adjust SPT activity to tissue-specific requirements of sphingolipid synthesis.

Mutations in the SPTLC2 Subunit of Serine Palmitoyltransferase Cause Hereditary Sensory and Autonomic Neuropathy Type I

Hereditary sensory and autonomic neuropathy type I (HSAN-I) is an axonal peripheral neuropathy associated with progressive distal sensory loss and severe ulcerations. Mutations in the first subunit of the enzyme serine palmitoyltransferase (SPT) have been associated with HSAN-I. The SPT enzyme catalyzes the first and rate-limiting step in the de novo sphingolipid synthesis pathway. However, different studies suggest the implication of other genes in the pathology of HSAN-I. Therefore, we screened the two other known subunits of SPT, SPTLC2 and SPTLC3, in a cohort of 78 HSAN patients. No mutations were found in SPTLC3, but we identified three heterozygous missense mutations in the SPTLC2 subunit of SPT in four families presenting with a typical HSAN-I phenotype. We demonstrate that these mutations result in a partial to complete loss of SPT activity in vitro and in vivo. Moreover, they cause the accumulation of the atypical and neurotoxic sphingoid metabolite 1-deoxy-sphinganine. Our findings extend the genetic heterogeneity in HSAN-I and enlarge the group of HSAN neuropathies associated with SPT defects. We further show that HSAN-I is consistently associated with an increased formation of the neurotoxic 1-deoxysphinganine, suggesting a common pathomechanism for HSAN-I.

Oral l-serine supplementation reduces production of neurotoxic deoxysphingolipids in mice and humans with hereditary sensory autonomic neuropathy type 1

Hereditary sensory and autonomic neuropathy type 1 (HSAN1) causes sensory loss that predominantly affects the lower limbs, often preceded by hyperpathia and spontaneous shooting or lancinating pain. It is caused by several missense mutations in the genes encoding 2 of the 3 subunits of the enzyme serine palmitoyltransferase (SPT). The mutant forms of the enzyme show a shift from their canonical substrate L-serine to the alternative substrate L-alanine. This shift leads to increased formation of neurotoxic deoxysphingolipids (dSLs). Our initial analysis showed that in HEK cells transfected with SPTLC1 mutants, dSL generation was modulated in vitro in the presence of various amino acids. We therefore examined whether in vivo specific amino acid substrate supplementation influenced dSL levels and disease severity in HSAN1. In mice bearing a transgene expressing the C133W SPTLC1 mutant linked to HSAN1, a 10% L-serine–enriched diet reduced dSL levels. L-serine supplementation also improved measures of motor and sensory performance as well as measures of male fertility. In contrast, a 10% L-alanine–enriched diet increased dSL levels and led to severe peripheral neuropathy. In a pilot study with 14 HSAN1 patients, L-serine supplementation similarly reduced dSL levels. These observations support the hypothesis that an altered substrate selectivity of the mutant SPT is key to the pathophysiology of HSAN1 and raise the prospect of l-serine supplementation as a first treatment option for this disorder.

The SPTLC3 Subunit of Serine Palmitoyltransferase Generates Short Chain Sphingoid Bases

The enzyme serine palmitoyltransferase (SPT) catalyzes the rate-limiting step in the de novo synthesis of sphingolipids. Previously the mammalian SPT was described as a heterodimer composed of two subunits, SPTLC1 and SPTLC2. Recently we identified a novel third SPT subunit (SPTLC3). SPTLC3 shows about 68% identity to SPTLC2 and also includes a pyridoxal phosphate consensus motif. Here we report that the overexpression of SPTLC3 in HEK293 cells leads to the formation of two new sphingoid base metabolites, namely C16-sphinganine and C16-sphingosine. SPTLC3-expressing cells have higher in vitro SPT activities with lauryl- and myristoyl-CoA than SPTLC2-expressing cells, and SPTLC3 mRNA expression levels correlate closely with the C16-sphinganine synthesis rates in various human and murine cell lines. Approximately 15% of the total sphingolipids in human plasma contain a C16 backbone and are found in the high density and low density but not the very low density lipoprotein fraction. In conclusion, we show that the SPTLC3 subunit generates C16-sphingoid bases and that sphingolipids with a C16 backbone constitute a significant proportion of human plasma sphingolipids.

Creatine kinase: an enzyme with a central role in cellular energy metabolism.

The enzyme creatine kinase (CK), catalyzing the reversible transfer of the N-phosphoryl group from phosphocreatine (PCr) to ADP to regenerate ATP, plays a key role in the energy homeostasis of cells with intermittently high, fluctuating energy requirements, e.g. in skeletal and cardiac muscle, neurons, photoreceptors, spermatozoa and electrocytes. Cytosolic CK isoenzyme(s) (MM-, MBand BB-CK) are always coexpressed in a tissue-specific fashion together with a mitochondrial isoform. Using biochemical fractionation and in situ localization, one was able to show that the CK isoenzymes, earlier considered to be strictly soluble, are in fact compartmentalized subcellularly and coupled functionally and/or structurally either to sites of energy production (glycolysis and mitochondria) or energy consumption (cellular ATPases, such as the actomyosin ATPase and SR-Ca 2+-ATPase), thus forming an intricate, highly regulated energy distribution network, the PCr-circuit or PCr-shuttle (Fig. 1, [1]). This non-equilibrium energy transport model has been challenged, based upon global 31p-NMR experiments, measuring CK-mediated flux in muscles at different work-loads [2,3]. The conclusions reached were that the CK system is in equilibrium with the substrates and behaves like a solution of well-mixed enzymes, that effects of compartmentation were negligible with respect to total cellular bioenergetics and that thermodynamic characteristics of the cytosol could be predicted as if the CK metabolites were freely mixing in solution.

Is the mammalian serine palmitoyltransferase a high-molecular-mass complex?

SPT (serine palmitoyltransferase) catalyses the rate-limiting step for the de novo synthesis of sphingolipids. Mammalian SPT is believed to be a heterodimer composed of two subunits, SPTLC1 and SPTLC2. We reported previously the identification of a new third SPT subunit, SPTLC3. In the present study, we have investigated the structure of the SPT complex in more detail. Pull-down assays with antibodies against SPTLC3 concomitantly co-precipitated SPTLC1 and SPTLC2 in human placenta extracts and SPTLC3 overexpressing human embryonic kidney-293 cells. By size exclusion chromatography, we determined the molecular mass of the functional SPT complex to be approx. 480 kDa. By Blue-native-PAGE experiments we demonstrated that all three SPT subunits (SPTLC1-3) are co-localized within a single SPT complex. On the basis of these results we conclude that the functional SPT is not a dimer, but a higher organized complex, composed of three distinct subunits (SPTLC1, SPTLC2 and SPTLC3) with a molecular mass of 480 kDa. The stoichiometry of SPTLC2 and SPTLC3 in this complex seems not to be fixed and is probably changed dynamically in dependence of the tissue specific SPTLC2 and SPTLC3 expression levels. Based on our own and earlier published data we propose a model of an octameric SPT structure. The observed dynamic composition of the SPT complex could provide a cellular mechanism to adjust SPT activity to tissue specific requirements in sphingolipid synthesis.

Premature Atrial Contractions in the General Population Frequency and Risk Factors

Background— Premature atrial contractions (PACs) are independent predictors of atrial fibrillation, stroke, and death. However, little is known about PAC frequency in the general population and its association with other cardiovascular risk factors. Methods and Results— We performed a cross-sectional analysis among participants of the population-based Swiss cohort Study on Air Pollution and Lung Diseases in Adults (SAPALDIA). 24-hour Holter electrocardiograms to assess PAC prevalence and frequency were performed in a random sample of 1742 participants aged ≥50 years. The median (interquartile range) number of PACs per hour was 0.8 (0.4–1.8), 1.1 (0.5–2.4), 1.4 (0.7–4.6), 2.3 (0.8–6.9), and 2.6 (1.2–6.5) among participants aged 50 to 55, 55 to 60, 60 to 65, 65 to 70, and ≥70 years, respectively (P<0.0001). Only 18 (1.0%) participants did not have at least 1 PAC during Holter monitoring. In multivariable negative binomial regression models, PAC frequency was significantly associated with age (risk ratio [RR] per SD 1.80; P<0.0001), height (RR per SD 1.52; P<0.0001), prevalent cardiovascular disease (RR 2.40; P<0.0001), log-transformed N-terminal pro B-type natriuretic peptides (RR per SD 1.27; P<0.0001), physical activity ≥2 hours per day (RR 0.69; P=0.002), and high-density lipoprotein cholesterol (RR per SD 0.80; P=0.0002). Hypertension and body mass index were not significantly related to PAC frequency. Conclusions— To our knowledge, this is the first study to assess risk factors for PAC frequency in the general population aged ≥50 years. PACs are common, and their frequency is independently associated with age, height, history of cardiovascular disease, natriuretic peptide levels, physical activity, and high-density lipoprotein cholesterol. The underlying mechanisms of these relationships need to be addressed in future studies.

Overexpression of the Wild-Type SPT1 Subunit Lowers Desoxysphingolipid Levels and Rescues the Phenotype of HSAN1

Mutations in the SPTLC1 subunit of serine palmitoyltransferase (SPT) cause an adult-onset, hereditary sensory, and autonomic neuropathy type I (HSAN1). We previously reported that mice bearing a transgene-expressing mutant SPTLC1 (tgSPTLC1C133W) show a reduction in SPT activity and hyperpathia at 10 months of age. Now analyzed at a later age, we find these mice develop sensory loss with a distal small fiber neuropathy and peripheral myelinopathy. This phenotype is largely reversed when these mice are crossed with transgenic mice overexpressing wild-type SPTLC1 showing that the mutant SPTLC1 protein is not inherently toxic. Simple loss of SPT activity also cannot account for the HSAN1 phenotype, since heterozygous SPTLC1 knock-out mice have reduced SPT activity but are otherwise normal. Rather, the presence of two newly identified, potentially deleterious deoxysphingoid bases in the tgSPTLC1C133W, but not in the wild-type, double-transgenic tgSPTLC1WT + C133W or SPTLC1+/− mice, suggests that the HSAN1 mutations alter amino acid selectivity of the SPT enzyme such that palmitate is condensed with alanine and glycine, in addition to serine. This observation is consistent with the hypothesis that HSAN1 is the result of a gain-of-function mutation in SPTLC1 that leads to accumulation of a toxic metabolite.