A simple method for sphingolipid analysis of tissues embedded in optimal cutting temperature compound.

MS-assisted lipidomic tissue analysis is a valuable tool to assess sphingolipid metabolism dysfunction in disease. These analyses can reveal potential pharmacological targets or direct mechanistic studies to better understand the molecular underpinnings and influence of sphingolipid metabolism alterations on disease etiology. But procuring sufficient human tissues for adequately powered studies can be challenging. Therefore, biorepositories, which hold large collections of cryopreserved human tissues, are an ideal retrospective source of specimens. However, this resource has been vastly underutilized by lipid biologists, as the components of OCT compound used in cryopreservation are incompatible with MS analyses. Here, we report results indicating that OCT compound also interferes with protein quantification assays, and that the presence of OCT compound impacts the quantification of extracted sphingolipids by LC-ESI-MS/MS. We developed and validated a simple and inexpensive method that removes OCT compound from OCT compound-embedded tissues. Our results indicate that removal of OCT compound from cryopreserved tissues does not significantly affect the accuracy of sphingolipid measurements with LC-ESI-MS/MS. We used the validated method to analyze sphingolipid alterations in tumors compared with normal adjacent uninvolved lung tissues from individuals with lung cancer and to determine the long-term stability of sphingolipids in OCT compound-cryopreserved normal lung tissues. We show that lung cancer tumors have significantly altered sphingolipid profiles and that sphingolipids are stable for up to 16 years in OCT compound-cryopreserved normal lung tissues. This validated sphingolipidomic OCT compound-removal protocol should be a valuable addition to the lipid biologist's toolbox.

Lipidomic Profiling Reveals Biological Differences between Tumors of Self-Identified African Americans and Non-Hispanic Whites with Cancer.

In the US, the incidence and mortality of many cancers are disproportionately higher in African Americans (AA). Yet, AA remain poorly represented in molecular studies investigating the roles that biological factors might play in the development, progression, and outcomes of many cancers. Given that sphingolipids, key components of mammalian cellular membranes, have well-established roles in the etiology of cancer progression, malignancy, and responses to therapy, we conducted a robust mass spectrometry analysis of sphingolipids in normal adjacent uninvolved tissues and tumors of self-identified AA and non-Hispanic White (NHW) males with cancers of the lung, colon, liver, and head and neck and of self-identified AA and NHW females with endometrial cancer. In these cancers, AA have worse outcomes than NHW. The goal of our study was to identify biological candidates to be evaluated in future preclinical studies targeting race-specific alterations in the cancers of AA. We have identified that various sphingolipids are altered in race-specific patterns, but more importantly, the ratios of 24- to 16-carbon fatty acyl chain-length ceramides and glucosylceramides are higher in the tumors of AA. As there is evidence that ceramides with 24-carbon fatty acid chain length promote cellular survival and proliferation, whereas 16-carbon chain length promote apoptosis, these results provide important support for future studies tailored to evaluate the potential roles these differences may play in the outcomes of AA with cancer.

Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis.

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether sphingolipids are altered in a disease and investigate whether sphingolipids can be targeted clinically. However, properly powered prospective studies that acquire tissues directly from the surgical suite can be time consuming, and technically, logistically, and administratively challenging. In contrast, retrospective studies can take advantage of cryopreserved human specimens already available, usually in large numbers, at tissue biorepositories. Other advantages of procuring tissues from biorepositories include access to information associated with the tissue specimens including histology, pathology, and in some instances clinicopathological variables, all of which can be used to examine correlations with lipidomics data. However, technical limitations related to the incompatibility of optimal cutting temperature compound (OCT) used in the cryopreservation and mass spectrometry is a technical barrier for the analysis of lipids. However, we have previously shown that OCT can be easily removed from human biorepository specimens through cycles of washes and centrifugation without altering their sphingolipid content. We have also previously established that sphingolipids in human tissues cryopreserved in OCT are stable for up to 16 years. In this report, we outline the steps and workflow to analyze sphingolipids in human tissue specimens that are embedded in OCT, including washing tissues, weighing tissues for data normalization, the extraction of lipids, preparation of samples for analysis by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), mass spectrometry data integration, data normalization, and data analysis.

Improving the success rate of gluteal intramuscular injections.

OBJECTIVES: This study aimed to improve the success rate of gluteal intramuscular (IM) injection. METHODS: The outcomes of 328 intended gluteal IM injections in 115 patients receiving depot octreotide were evaluated using computed tomography performed in routine clinical practice. Patient-, nursing-, and technique-dependent factors were correlated with successful delivery of medication. Techniques associated with successful injection were taught to center nurses. RESULTS: At baseline, 52% of injections were successfully delivered (66% men, 36% women; P = 0.001). Factors associated with successful delivery included nurses' frequency of injections (P = 0.008), landmarks use to select injection site (P < 0.001), quick needle insertion (P < 0.001), and use of nonsyringe hand to compress injection site (P < 0.001). Patient-related factors included male sex (P < 0.001), lower body mass index (P < 0.001), and lower skin-to-muscle depth at injection site (P < 0.001). Techniques associated with successful injections were then taught to center nurses. After instruction, the success rate increased from 52% to 75% (P = 0.001). Importantly, improvements were observed in both men (66%-75%; P = 0.43) and women (38%-75%; P < 0.001). Successful injection was associated with better control of flushing among those with carcinoid syndrome (P = 0.005). CONCLUSIONS: Intended gluteal IM injections often are given into the subcutaneous space. Education in techniques associated with successful injections improves IM delivery rates.