The composition of human preimplantation embryo culture media and their stability during storage and culture.

STUDY QUESTION: What is the composition and stability during storage and culture of fifteen commercially available human preimplantation embryo culture media? SUMMARY ANSWER: No two culture media had the same composition, and both storage and culture had an effect on the concentrations of multiple components. WHAT IS KNOWN ALREADY: The choice of embryo culture medium not only affects the success rate of an IVF treatment, but also affects the health of the future child. Exact formulations of embryo culture media are often not disclosed by manufacturers. It is unknown whether the composition of these media changes during storage or culture in the IVF laboratory. Without details on the exact concentrations, it is not possible to determine which components might be responsible for the differences in IVF success rates and health of the resulting children. STUDY DESIGN, SIZE, DURATION: Between October 2014 and October 2015, all complete human preimplantation embryo culture media, i.e. ready to use for IVF, that were commercially available at that time, were included (n = 15). Osmolality and the concentration of thirty seven components including basic elements, metabolites, immunoglobulins, albumin, proteins and 21 amino acids were tested immediately upon arrival into the IVF laboratory, after three days of culture without embryos (sham culture) starting from the day of arrival, just before the expiry date, and after three days of sham culture just before the expiry date. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ions, glucose, immunoglobulins, albumin and the total amount of proteins were quantified using a combination of ion selective electrodes and photometric analysis modules, and lactate, pyruvate and 21 amino acids were analysed by ultra performance liquid chromatography mass spectrometry. Osmolality was analysed by an advanced micro-osmometer. Statistical analysis was done using multivariate general linear models. MAIN RESULTS AND THE ROLE OF CHANCE: The composition varied between media, no two media had the same concentration of components. Storage led to significant changes in 17 of the 37 analyzed components (magnesium, chloride, phosphate, albumin, total amount of proteins, tyrosine, tryptophan, alanine, methionine, glycine, leucine, glutamine, asparagine, arginine, serine, proline, and threonine). Storage affected the osmolality in 3 of the 15 media, but for all media combined this effect was not significant (p = 0.08). Sham culture of the analyzed media had a significant effect on the concentrations of 13 of the 37 analyzed components (calcium, phosphate, albumin, total amount of proteins, tyrosine, alanine, methionine, glycine, leucine, asparagine, arginine, proline, and histidine). Sham culture significantly affected the osmolality of the analysed culture media. Two media contained 50% D-lactate, which a toxic dead-end metabolite. In a secondary analysis we detected human liver enzymes in more than half of the complete culture media. LIMITATIONS, REASONS FOR CAUTION: The analyzed culture media could contain components that are not among the 37 components that were analyzed in this study. The clinical relevance of the varying concentrations is yet to be determined. WIDER IMPLICATIONS OF THE FINDINGS: The presence of D-lactate could be avoided and the finding of human liver enzymes was surprising. The wide variation between culture media shows that the optimal composition is still unknown. This warrants further research as the importance of embryo culture media on the efficacy and safety in IVF is evident. Companies are urged to fully disclose the composition of their culture media, and provide clinical evidence supporting the composition or future changes thereof. STUDY FUNDING/COMPETING INTEREST(S): None.

Rapid screening for lipid storage disorders using biochemical markers. Expert center data and review of the literature.

BACKGROUND: In patients suspected of a lipid storage disorder (sphingolipidoses, lipidoses), confirmation of the diagnosis relies predominantly on the measurement of specific enzymatic activities and genetic studies. New UPLC-MS/MS methods have been developed to measure lysosphingolipids and oxysterols, which, combined with chitotriosidase activity may represent a rapid first tier screening for lipid storage disorders. MATERIAL AND METHODS: A lysosphingolipid panel consisting of lysoglobotriaosylceramide (LysoGb3), lysohexosylceramide (LysoHexCer: both lysoglucosylceramide and lysogalactosylceramide), lysosphingomyelin (LysoSM) and its carboxylated analogue lysosphingomyelin-509 (LysoSM-509) was measured in control subjects and plasma samples of predominantly untreated patients affected with lipid storage disorders (n=74). In addition, the oxysterols cholestane-3ß,5α,6ß-triol and 7-ketocholesterol were measured in a subset of these patients (n=36) as well as chitotriosidase activity (n=43). A systematic review of the literature was performed to assess the usefulness of these biochemical markers. RESULTS: Specific elevations of metabolites, i.e. without overlap between controls and other lipid storage disorders, were found for several lysosomal storage diseases: increased LysoSM levels in acid sphingomyelinase deficiency (Niemann-Pick disease type A/B), LysoGb3 levels in males with classical phenotype Fabry disease and LysoHexCer (i.e. lysoglucosylceramide/lysogalactosylceramide) in Gaucher and Krabbe diseases. While elevated levels of LysoSM-509 and cholestane-3ß,5α,6ß-triol did not discriminate between Niemann Pick disease type C and acid sphingomyelinase deficiency, LysoSM-509/LysoSM ratio was specifically elevated in Niemann-Pick disease type C. In Gaucher disease type I, mild increases in several lysosphingolipids were found including LysoGb3 with levels in the range of non-classical Fabry males and females. Chitotriosidase showed specific elevations in symptomatic Gaucher disease, and was mildly elevated in all other lipid storage disorders. Review of the literature identified 44 publications. Most findings were in line with our cohort. Several moderate elevations of biochemical markers were found across a wide range of other, mainly inherited metabolic, diseases. CONCLUSION: Measurement in plasma of LysoSLs and oxysterols by UPLC-MS/MS in combination with activity of chitotriosidase provides a useful first tier screening of patients suspected of lipid storage disease. The LysoSM-509/LysoSM ratio is a promising parameter in Niemann-Pick disease type C. Further studies in larger groups of untreated patients and controls are needed to improve the specificity of the findings.

Deoxysphingolipid precursors indicate abnormal sphingolipid metabolism in individuals with primary and secondary disturbances of serine availability.

Patients with primary serine biosynthetic defects manifest with intellectual disability, microcephaly, ichthyosis, seizures and peripheral neuropathy. The underlying pathogenesis of peripheral neuropathy in these patients has not been elucidated, but could be related to a decrease in the availability of certain classical sphingolipids, or to an increase in atypical sphingolipids. Here, we show that patients with primary serine deficiency have a statistically significant elevation in specific atypical sphingolipids, namely deoxydihydroceramides of 18-22 carbons in acyl length. We also show that patients with aberrant plasma serine and alanine levels secondary to mitochondrial disorders also display peripheral neuropathy along with similar elevations of atypical sphingolipids. We hypothesize that the etiology of peripheral neuropathy in patients with primary mitochondrial disorders is related to this elevation of deoxysphingolipids, in turn caused by increased availability of alanine and decreased availability of serine. These findings could have important therapeutic implications for the management of these patients.

Development and validation of a severity scoring system for Zellweger spectrum disorders.

The lack of a validated severity scoring system for individuals with Zellweger spectrum disorders (ZSD) hampers optimal patient care and reliable research. Here, we describe the development of such severity score and its validation in a large, well-characterized cohort of ZSD individuals. We developed a severity scoring system based on the 14 organs that typically can be affected in ZSD. A standardized and validated method was used to classify additional care needs in individuals with neurodevelopmental disabilities (Capacity Profile [CAP]). Thirty ZSD patients of varying ages were scored by the severity score and the CAP. The median score was 9 (range 6-19) with a median scoring age of 16.0 years (range 2-36 years). The ZSD severity score was significantly correlated with all 5 domains of the CAP, most significantly with the sensory domain (r = 0.8971, P = <.0001). No correlation was found between age and severity score. Multiple peroxisomal biochemical parameters were significantly correlated with the severity score. The presently reported severity score for ZSD is a suitable tool to assess phenotypic severity in a ZSD patient at any age. This severity score can be used for objective phenotype descriptions, genotype-phenotype correlation studies, the identification of prognostic features in ZSD patients and for classification and stratification of patients in clinical trials.

When silence is noise: infantile-onset Barth syndrome caused by a synonymous substitution affecting TAZ gene transcription.

Barth syndrome (BTHS) is an X-linked inborn error of metabolism which affects males. The main manifestations are cardiomyopathy, myopathy, hypotonia, growth delay, intermittent neutropenia and 3-methylglutaconic aciduria. Diagnosis is confirmed by mutational analysis of the TAZ gene and biochemical dosage of the monolysocardiolipin/tetralinoleoyl cardiolipin (MLCL:L4-CL) ratio. We report a 6-year-old boy who presented with severe hypoglycemia, lactic acidosis and severe dilated cardiomyopathy soon after birth. The MLCL:L4-CL ratio confirmed BTHS (3.90 on patient's fibroblast, normal: 0-0.3). Subsequent sequencing of the TAZ gene revealed only the new synonymous variant NM_000116.3 (TAZ):c.348C>T p.(Gly116Gly), which did not appear to affect the protein sequence. In silico prediction analysis suggested the new c.348C>T nucleotide change could alter the TAZ mRNA splicing processing. We analyzed TAZ mRNAs in the patient's fibroblasts and found an abnormal skipping of 24 bases (NM_000116.3:c.346_371), with the consequent ablation of 8 amino acid residues in the tafazzin protein (NP_000107.1:p.Lys117_Gly124del). Molecular analysis of at risk female family members identified the patient's sister and mother as heterozygous carriers. Apparently harmless synonymous variants in the TAZ gene can damage gene expression. Such findings widen our knowledge of molecular heterogeneity in BTHS.

Mitochondrial neurogastrointestinal encephalomyopathy: Clinical and biochemical impact of allogeneic stem cell transplantation in a Greek patient with one novel TYMP mutation.

We describe the case of a Greek female patient with the Classic form of the ultra- rare and fatal autosomal recessive disorder Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) and the impact of allogeneic hematopoietic stem cell transplantation on the biochemical and clinical aspects of the disease. The patient presented at the age of 15 years with severe gastrointestinal symptoms, cachexia, peripheral neuropathy and diffuse leukoencephalopathy. The diagnosis of MNGIE disease was established by the increased levels of thymidine and deoxyuridine in plasma and the complete deficiency of thymidine phosphorylase activity. The novel c.[978dup] (p.Ala327Argfs*?) variant and the previously described variant c.[417 + 1G > A] were identified in TYMP. The donor for the allogeneic hematopoietic stem cell transplantation was her fully compatible sister, a carrier of the disease. The patient had a completely uneventful post- transplant period and satisfactory PB chimerism levels. A marked and rapid decrease in thymidine and deoxyuridine plasma levels and an increase of the thymidine phosphorylase activity to the levels measured in her donor sister was observed and is still present sixteen months post-transplant. Disease symptoms stabilized and some improvement was also observed both in her neurological and gastrointestinal symptoms. Follow up studies will be essential for determining the long term impact of allogeneic hematopoietic stem cell transplantation in our patient.

Bizarre behavior and decreased level of consciousness in an adult patient.

This case report presents an adult patient with decreased levels of consciousness and bizarre behavior. A silent delirium was first suspected however, symptoms did not improve and further examination revealed elevated ammonia levels. A hepatic cause and portosystemic shunting were excluded and eventually a diagnosis of ornithine transcarbamylase deficiency was made. After treatment with high carbohydrate intake, a low protein diet and supplementation with arginine and sodium benzoate, the patient recovered.

Right ventricular pressure overload alters cardiac lipid composition.

INTRODUCTION: Right ventricular (RV) failure due to pressure load is an important determinant of clinical outcome in pulmonary hypertension, congenital heart disease and left ventricular failure. The last decades it has become clear that metabolic dysregulation is associated with the development of RV-failure. However, underlying mechanisms remain to be unraveled. Recently, disruption of intracardiac lipid content has been suggested as potential inducer of RV failure. In the present study, we used a rat model of RV-dysfunction and aimed to obtain insight in temporal changes in RV-function, -remodelling and -metabolism and relate this to RV lipid content. METHODS AND RESULTS: Male Wistar WU rats were subjected to pulmonary artery banding (n = 25) or sham surgery (n = 14) and cellular, hemodynamic and metabolic assessments took place after 2, 5 and 12 weeks. In this model RV dysfunction and remodelling occurred, including early upregulation of oxidative stress markers. After 12 weeks of pressure load, lipidomics revealed significant decreases of myocardial diglycerides and cardiolipins, driven by (poly-)unsaturated forms. The decrease of cardiolipins was driven by its most abundant form, tetralinoleoylcardiolipin. Mitochondrial capacity for fatty acid oxidation preserved, while the capacity for glucose oxidation increased. CONCLUSION: RV dysfunction due to pressure load, is associated with decreased intracardiac unsaturated lipids, especially tetralinoleoylcardiolipin. This was accompanied with preserved mitochondrial capacity regarding fatty acids oxidation, with increased capacity for glucose oxidation, and early activation of oxidative stress. We suggest that early interventions should be directed towards preservation of lipid availability as possible mean in order to prevent RV failure.

Single vagus nerve stimulation reduces early postprandial C-peptide levels but not other hormones or postprandial metabolism.

A recent study in rheumatoid arthritis (RA) patients using electrical vagus nerve stimulation (VNS) to activate the inflammatory reflex has shown promising effects on disease activity. Innervation by the autonomic nerve system might be involved in the regulation of many endocrine and metabolic processes and could therefore theoretically lead to unwanted side effects. Possible effects of VNS on secretion of hormones are currently unknown. Therefore, we evaluated the effects of a single VNS on plasma levels of pituitary hormones and parameters of postprandial metabolism. Six female patients with RA were studied twice in balanced assignment (crossover design) to either VNS or no stimulation. The patients selected for this substudy had been on VNS therapy daily for at least 3 months and at maximum of 24 months. We compared 10-, 20-, and 30-min poststimulus levels to baseline levels, and a 4-h mixed meal test was performed 30 min after VNS. We also determined energy expenditure (EE) by indirect calorimetry before and after VNS. VNS did not affect pituitary hormones (growth hormone, thyroid stimulating hormone, adrenocorticotropic hormone, prolactin, follicle-stimulating hormone, and luteinizing hormone), postprandial metabolism, or EE. Of note, VNS reduced early postprandial insulin secretion, but not AUC of postprandial plasma insulin levels. Cortisol and catecholamine levels in serum did not change significantly. Short stimulation of vagal activity by VNS reduces early postprandial insulin secretion, but not other hormone levels and postprandial response. This suggests VNS as a safe treatment for RA patients.

3-Methoxytyramine: An independent prognostic biomarker that associates with high-risk disease and poor clinical outcome in neuroblastoma patients.

INTRODUCTION: Prognosis of neuroblastoma patients is very diverse, indicating the need for more accurate prognostic parameters. The excretion of catecholamine metabolites by most neuroblastomas is used for diagnostic purposes, but their correlation with prognosis has hardly been investigated. Therefore, we performed an in-depth analysis of a panel of elevated urinary catecholamine metabolites at diagnosis and their correlation with prognosis. PATIENTS AND METHODS: Retrospective study of eight urinary catecholamine metabolites in a test (n = 96) and validation (n = 205) cohort of patients with neuroblastoma (all stages) at diagnosis. RESULTS: Multivariate analyses, including risk factors such as stage and MYCN amplification, revealed that 3-methoxytyramine (3MT) was an independent risk factor for event-free survival (EFS) and overall survival (OS). Furthermore, only 3MT appeared to be an independent risk factor for both EFS and OS in high-risk patients, which was independent of modern high-risk therapy and immunotherapy. Among high-risk patients, those with elevated 3MT and older than 18 months had an extremely poor prognosis compared to patients with non-elevated 3MT and younger than 18 months (5-year EFS of 14.3% ± 4% and 66.7% ± 18%, respectively, p = 0.001; 5-year OS of 21.8% ± 5% and 87.5% ± 12%, respectively, p < 0.001). CONCLUSIONS: Elevated 3MT at diagnosis was associated with high-risk disease and poor prognosis. For high-risk patients, elevated 3MT at diagnosis was the only significant risk factor for EFS and OS. 3MT was also able to identify subgroups of high-risk patients with favourable and extremely poor prognosis.

Biological profiling of prospective antidepressant response in major depressive disorder: Associations with (neuro)inflammation, fatty acid metabolism, and amygdala-reactivity.

BACKGROUND: A better understanding of factors underlying antidepressant non-response may improve the prediction of which patients will respond to what treatment. Major depressive disorder (MDD) is associated with alterations in fatty acid metabolism, (neuro)inflammation and amygdala-reactivity. However, their mutual relations, and the extent to which they are associated with prospective antidepressant-response, remain unknown. PURPOSE: To test (I) alterations in (neuro)inflammation and its associations with fatty acid metabolism and amygdala-reactivity in MDD-patients compared to controls, and (II) whether these alterations are associated with prospective paroxetine response. METHODS: We compared 70 unmedicated MDD-patients with 51 matched healthy controls at baseline, regarding erythrocyte membrane omega-6 arachidonic acid (AA), inflammation [serum (high-sensitivity) C-reactive protein (CRP)], and in a subgroup amygdala-reactivity to emotional faces using functional magnetic resonance imaging (fMRI) (N=42). Subsequently, we treated patients with 12 weeks paroxetine, and repeated baseline measures after 6 and 12 weeks to compare non-responders, early-responders (response at 6 weeks), and late-responders (response at 12 weeks). RESULTS: Compared to controls, MDD-patients showed higher CRP (p=0.016) and AA (p=0.019) after adjustment for confounders at baseline. AA and CRP were mutually correlated (p=0.043). In addition, patients showed a more negative relation between AA and left amygdala-reactivity (p=0.014). Moreover, AA and CRP were associated with antidepressant-response: early responders showed lower AA (p=0.018) and higher CRP-concentrations (p=0.008) than non-responders throughout the study. CONCLUSION: Higher observed CRP and AA, their mutual association, and relation with amygdala-reactivity, are corroborative with a role for (neuro)inflammation in MDD. In addition, observed associations of these factors with prospective antidepressant-response suggest a potential role as biomarkers. Future studies in independent samples are needed to replicate and test the clinical applicability of these biological predictors for treatment response to result in a precision/personalized medicine approach for treatment.

L-carnitine is synthesized in the human fetal-placental unit: potential roles in placental and fetal metabolism.

Carnitine plays an indispensable role in fatty acid oxidation. Previous studies revealed that fetal carnitine is derived from the mother via transplacental transfer. Recent studies demonstrated the presence and importance of an active fatty acid oxidation system in the human placenta and in the human fetus. In view of these findings we decided to study carnitine metabolism in the fetal-placental unit by measuring carnitine metabolites, intermediary metabolites of carnitine biosynthesis, as well as the activity of carnitine biosynthesis enzymes in human term placenta, cord blood and selected embryonic and fetal tissues (5-20 weeks of development). Placenta contained low but detectable activity of gamma-butyrobetaine dioxygenase. This enzyme, which was considered to be expressed only in kidney, liver and brain, catalyzes the last step in the carnitine biosynthesis pathway. In addition, our results show that human fetal kidney, liver and spinal cord already have the capacity to synthesize carnitine. The ability of the placenta and fetus to synthesize carnitine suggests that in circumstances when maternal carnitine supply is limited, carnitine biosynthesis by the fetal-placental unit may supply sufficient carnitine for placental and fetal metabolism.

Cardiolipin or MTCH2 can serve as tBID receptors during apoptosis.

During apoptosis, proapoptotic BAX and BAK trigger mitochondrial outer membrane (MOM) permeabilization by a mechanism that is not yet fully understood. BH3-only proteins such as tBID, together with lipids of the MOM, are thought to play a key role in BAX and BAK activation. In particular, cardiolipin (CL) has been shown to stimulate tBID-induced BAX activation in vitro. However, it is still unclear whether this process also relies on CL in the cell, or whether it is more dependent on MTCH2, a proposed receptor for tBID present in the MOM. To address this issue, we deleted both alleles of cardiolipin synthase in human HCT116 cells by homologous recombination, which resulted in a complete absence of CL. The CL-deficient cells were fully viable in glucose but displayed impaired oxidative phosphorylation and an inability to grow in galactose. Using these cells, we found that CL was not required for either tBID-induced BAX activation, or for apoptosis in response to treatment with TRAIL. Downregulation of MTCH2 in HCT116 cells also failed to prevent recruitment of tBID to mitochondria in apoptotic conditions. However, when both CL and MTCH2 were depleted, a significant reduction in tBID recruitment was observed, suggesting that in HCT116 cells, CL and MTCH2 can have redundant functions in this process.

Betulinic acid induces a novel cell death pathway that depends on cardiolipin modification.

Cancer is associated with strong changes in lipid metabolism. For instance, normal cells take up fatty acids (FAs) from the circulation, while tumour cells generate their own and become dependent on de novo FA synthesis, which could provide a vulnerability to target tumour cells. Betulinic acid (BetA) is a natural compound that selectively kills tumour cells through an ill-defined mechanism that is independent of BAX and BAK, but depends on mitochondrial permeability transition-pore opening. Here we unravel this pathway and show that BetA inhibits the activity of steroyl-CoA-desaturase (SCD-1). This enzyme is overexpressed in tumour cells and critically important for cells that utilize de novo FA synthesis as it converts newly synthesized saturated FAs to unsaturated FAs. Intriguingly, we find that inhibition of SCD-1 by BetA or, alternatively, with a specific SCD-1 inhibitor directly and rapidly impacts on the saturation level of cardiolipin (CL), a mitochondrial lipid that has important structural and metabolic functions and at the same time regulates mitochondria-dependent cell death. As a result of the enhanced CL saturation mitochondria of cancer cells, but not normal cells that do not depend on de novo FA synthesis, undergo ultrastructural changes, release cytochrome c and quickly induce cell death. Importantly, addition of unsaturated FAs circumvented the need for SCD-1 activity and thereby prevented BetA-induced CL saturation and subsequent cytotoxicity, supporting the importance of this novel pathway in the cytotoxicity induced by BetA.

Carnitine biosynthesis. Purification of gamma-butyrobetaine hydroxylase from rat liver.

gamma-Butyrobetaine hydroxylase catalyse the last step in carnitine biosynthesis, the formation of L-carnitine from gamma-butyrobetaine, a reaction dependent on Fe2+, alpha-ketoglutarate, ascorbate and oxygen. Initial attempts to purify the protein from rat liver showed that gamma-butyrobetaine hydroxylase is unstable. We, therefore, determined the influence of various compounds on the stability of gamma-butyrobetaine hydroxylase at different storage temperatures. The enzyme activity was best conserved by storing the protein at 4 degrees C in the presence of 200 g/l glycerol and 10 mM DTT. We subsequently purified the enzyme from rat liver to apparent homogeneity by liquid chromatography.

Considering Fabry, but Diagnosing MPS I: Difficulties in the Diagnostic Process.

INTRODUCTION: Recent studies have indicated that a proportion of patients with renal failure, left ventricular hypertrophy, or cryptogenic stroke have sequence variants in their aGal A gene (Fabry disease), which has resulted in an increase in diagnostic activities for this disorder. The diagnostic process for lysosomal storage disorders may result in findings of unknown clinical significance. Here we report such an unexpected outcome. CASE: A 32-year-old male presented at the emergency department because of a transient ischemic attack. Extensive investigations revealed no cause and an initial diagnosis of cryptogenic stroke was made. Subsequently, aGal A activity was measured in a bloodspot and was shown to be normal, but the activity of alpha-L-iduronidase (IDUA), used as reference enzyme, was unexpectedly low: 0.5 umol/L (ref = 1.7-14.3). A diagnosis of IDUA deficiency, mucopolysaccharidosis type 1S or Scheie disease was considered. IDUA gene analysis revealed two homozygous sequence alterations: a silent sequence change (979C > T) in exon 7 (N297N) and an unknown missense mutation 875A > T (R263W). Physical examination was completely normal, without clinical signs of mucopolysaccharidosis type I (MPS I). Leukocyte IDUA activity was also low: 2.1 nmol/mg prot/h (ref = 14-40 nmol prot/h), but higher than the patient range of <0.1 nmol/mg prot/h. Urinary glycosaminoglycan levels were normal both quantitatively and qualitatively. It was concluded that there was low IDUA activity without clinical symptoms and the diagnosis of mucopolysaccharidosis I was discarded. CONCLUSION: The diagnostic process for lysosomal storage disorders may result in biochemical abnormalities of unknown clinical significance. Early evaluation by a specialist in inborn errors of metabolism may help to avoid anxiety in patients and unnecessary additional analyses.

BID is cleaved by caspase-8 within a native complex on the mitochondrial membrane.

Caspase-8 stably inserts into the mitochondrial outer membrane during extrinsic apoptosis. Inhibition of caspase-8 enrichment on the mitochondria impairs caspase-8 activation and prevents apoptosis. However, the function of active caspase-8 on the mitochondrial membrane remains unknown. In this study, we have identified a native complex containing caspase-8 and BID on the mitochondrial membrane, and showed that death receptor activation by Fas or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced the cleavage of BID (tBID formation) within this complex. tBID then shifted to separate mitochondria-associated complexes that contained other BCL-2 family members, such as BAK and BCL-X(L). We report that cells stabilize active caspase-8 on the mitochondria in order to specifically target mitochondria-associated BID, and that BID cleavage on the mitochondria is essential for caspase-8-induced cytochrome c release. Our findings indicate that during extrinsic apoptosis, caspase-8 can specifically target BID where it is mostly needed, on the surface of mitochondria.