Mitochondria receive cholesterol from late endosomes and lysosomes (LE/LYSs) or from the plasma membrane for production of oxysterols and steroid hormones. This process depends on the endo-lysosomal sterol transfer protein Niemann Pick C2 (NPC2). Using the intrinsically fluorescent cholesterol analog, cholestatrienol, we directly observe sterol transport to mitochondria in fibroblasts upon treating NPC2 deficient human fibroblasts with NPC2 protein. Soft X-ray tomography reveals the ultrastructure of mitochondria and discloses close contact to endosome-like organelles. Using fluorescence microscopy, we localize endo-lysosomes containing NPC2 relative to mitochondria based on the Euclidian distance transform and use statistical inference to show that about 30% of such LE/LYSs are in contact to mitochondria in human fibroblasts. Using Markov Chain Monte Carlo image simulations, we show that interaction between both organelle types, a defining feature of membrane contact sites (MCSs) can give rise to the observed spatial organelle distribution. We devise a protocol to determine the surface fraction of endo-lysosomes in contact with mitochondria and show that this fraction does not depend on functional NPC1 or NPC2 proteins. Finally, we localize MCSs between LE/LYSs containing NPC2 and mitochondria in time-lapse image sequences and show that they either form transiently or remain stable for tens of seconds. Lasting MCSs between endo-lysosomes containing NPC2 and mitochondria move by slow anomalous sub-diffusion, providing location and time for sterol transport between both organelles. Our quantitative imaging strategy will be of high value for characterizing the dynamics and function of MCSs between various organelles in living cells.

Niemann-Pick disease type C2 is a lipid storage disorder in which mutations in the NPC2 protein cause accumulation of lipoprotein-derived cholesterol in late endosomes and lysosomes (LE/LYSs). Whether cholesterol delivered by other means to NPC2 deficient cells also accumulates in LE/LYSs is currently unknown. We show that the close cholesterol analog dehydroergosterol (DHE), when delivered to the plasma membrane (PM) accumulates in LE/LYSs of human fibroblasts lacking functional NPC2. We measured two different time scales of sterol diffusion; while DHE rich LE/LYSs moved by slow anomalous diffusion in disease cells (D ∼ 4.6∙10^-4 μm²/sec; α∼0.76), a small pool of sterol could exchange rapidly with D ∼ 3 μm²/s between LE/LYSs, as shown by fluorescence recovery after photobleaching (FRAP). By quantitative lipid mass spectrometry we found that esterification of ¹³C-labeled cholesterol but not of DHE is reduced 10-fold in disease fibroblasts compared to control cells. Internalized NPC2 rescued the sterol storage phenotype and strongly expanded the dynamic sterol pool seen in FRAP experiments. Together, our study shows that cholesterol esterification and trafficking of sterols between the PM and LE/LYSs depends on a functional NPC2 protein. NPC2 likely acts inside LE/LYSs from where it increases non-vesicular sterol exchange with other organelles.

Vitamin B12 (B12) is present in foods of animal origin, and vegans are encouraged to take supplements with synthetic B12 in order to ensure a sufficient uptake. Recent rat studies suggest that natural (hydroxo-B12, HO-B12) and synthetic (cyano-B12, CN-B12) B12 behave differently in the body. Here, we test if a daily vitamin pill matches dietary B12 in ability to restore a low B12 status in rats. B12-depleted male Wistar rats (n = 60) were divided into five groups (n = 12 in each) and subjected to two weeks intervention with various schemes of B12 supplementation. Two “dietary” groups received a low-B12 chow that was fortified with either HO-B12 or CN-B12 providing a continuous supply. Two “pill” groups received a single daily dose of CN-B12, where the vitamin content either matched or exceeded by factor four the provisions for the “dietary” groups. A control group received the low-B12 chow without B12 fortification. B12 was measured in plasma and tissues. Dietary B12 provides 35% more B12 to the tissues than an equivalent single daily dose (p < 0.0001). Natural B12 delivers 25% more B12 to the liver than synthetic B12 (p = 0.0007). A fourfold increase in B12, supplemented as a single daily dose, does not provide any extra B12 to the tissues (p = 0.45). We conclude that dietary B12 is better at rescuing a low B12 status than a daily vitamin pill.

Lactovegetarians (n = 35) with low vitamin B12 (B12) status were intervened for eight weeks capsules containing cyano-B12 (CN-B12), (2 × 2.8 µg/day), or equivalent doses of endogenous B12 (mainly hydroxo-B12 (HO-B12)) in whey powder. Blood samples were examined at baseline, every second week during the intervention, and two weeks post-intervention. The groups did not differ at baseline in [global median (min/max)] plasma B12 [112(61/185)] pmol/L, holotranscobalamin [20(4/99)] pmol/L, folate [13(11/16)], the metabolites total homocysteine [18(9/52)] µmol/L and methylmalonic acid [0.90(0.28/2.5)] µmol/L, and the combined indicator of B12 status (4cB12) [−1.7(−3.0/−0.33)]. Both supplements caused significant effects, though none of the biomarkers returned to normal values. Total plasma B12 showed a higher increase in the capsule group compared to the whey powder group (p = 0.02). However, the increase of plasma holotranscobalamin (p = 0.06) and the lowering of the metabolites (p > 0.07) were alike in both groups. Thereby, the high total plasma B12 in the capsule group was not mirrored in enhanced B12 metabolism, possibly because the B12 surplus was mainly accumulated on an “inert” carrier haptocorrin, considered to be of marginal importance for tissue delivery of B12. In conclusion, we demonstrate that administration of whey powder (HO-B12) or capsules (CN-B12) equivalent to 5.6 µg of B12 daily for eight weeks similarly improves B12 status but does not normalize it. We document that the results for plasma B12 should be interpreted with caution following administration of CN-B12, since the change is disproportionately high compared to the responses of complementary biomarkers.

Introduction. The aim of this study was to establish a radio synthesis of ^99mTc-HYNIC-lactadherin for in vivo studies and to perform biodistribution analysis studies in mice, comparing ^99mTc-HYNIC-lactadherin to ^99mTc-HYNIC-annexin V. Methods. The radiochemical purity of ^99mTc-HYNIC-lactadherin was optimized by varying the amount of SnCl₂ in the synthesis. Furthermore, the need for bovine serum albumin (BSA) as a stabilizing agent was evaluated by following the stability by radiochemical purity measurement with and without the addition of BSA. A total of 24 mice were assigned to groups of 15 and nine mice, respectively. The animals were sacrificed at different time points; 10 min, 60 min, and 180 min. Results. The synthesis of ^99mTc-HYNIC-lactadherin for in vivo studies has been optimized to give a stable product without addition of BSA and with a radiochemical purity of more than 95%. Approximately 60% of the injected dose of ^99mTc-HYNIC-lactadherin was found in the liver and 4–5% could be assigned to kidneys. In contrast, ^99mTc-HYNIC-annexin V distributes with around 13% and 45% of the injected dose in liver and kidneys, respectively. Over the experimental period (10–180 min) only small distributional changes were observed for both probes. Conclusion. In conclusion, the biodistribution of ^99mTc-HYNIC-lactadherin, a potential new tracer for in vivo quantification of apoptosis, was evaluated. The small renal uptake of ^99mTc-HYNIC-lactadherin makes it possible to image apoptosis in the kidneys, but the high liver clearance may be a disadvantage during myocardial perfusion.