Development of a Multiplexed Sphingolipids Method for Diagnosis of Inborn Errors of Ceramide Metabolism.

BACKGROUND: Sphingolipids play a crucial role in cellular functions and are essential components of cell membranes, signaling molecules, and lipid metabolism. In particular, ceramide is a key intermediate in sphingolipid metabolism and defects in ceramide metabolism can lead to various inborn errors of metabolism, making ceramides important targets for clinical screening and diagnosis. Detecting altered concentration patterns of sphingolipids is desirable for distinguishing related inborn errors of metabolism for diagnosis and treatment monitoring. METHODS: We developed a liquid chromatography-tandem mass spectrometry method with a pathway-oriented approach to focus on sphingolipids involved in ceramide metabolism. A total of 47 sphingolipids bearing different head groups and side chains were targeted. Precision/reproducibility, linearity, and spike recovery extraction efficiency tests were performed on plasma and serum samples from confirmed cases of sphingolipidosis. RESULTS: Linearity of the method showed the coefficient of determination (r2) for all standards to be >0.99 with a slope of 1.00 ± 0.01. Intra- and interday reproducibility of standards spiked into plasma and serum revealed a coefficient of variation <20%. Spike and recovery assessment showed recovery values of 80%-120% for all standards. Altered levels of sphingolipids from patients with hereditary sensory and autonomic neuropathy caused by pathogenic variants in SPTLC2 and hypomyelinating leukodystrophy related to variants in DEGS1 were detected, in agreement with trends reported in earlier studies confirming the utility of this pathway-centric method. CONCLUSIONS: This method can serve as a useful tool to simultaneously monitor sphingolipids, enabling screening and diagnosis of inborn errors of ceramide metabolism.

A reliable clinical test for detection of membranous nephropathy antigens using laser microdissection and mass spectrometry.

Membranous nephropathy (MN) results from accumulation of antigen-antibody immune complexes along the subepithelial region of the glomerular basement membranes. Over the last years, 13 target antigens have been discovered and include PLA2R, THSD7A, EXT1 and EXT2, NELL1, SEMA3B, NCAM1, CNTN1, HTRA1, FAT1, PCDH7, NTNG1, PCSK6 and NDNF, accounting for 80-90% of MN antigens. MN associated with many of these antigens have distinctive clinicopathologic findings. It is important to accurately identify the antigen in MN. Immunohistochemical (IHC) and/or immunofluorescence (IF) methods are currently used to detect PLA2R, THSD7A, NELL1, SEMA3B and EXT1/EXT2. However, for the remaining antigens, IHC/IF methods do not exist and are not practical for detection. Here, we developed laser microdissection-based mass spectrometry methodology (LMD/MS) as a one-stop clinical test for the detection of MN antigens using paraffin-embedded kidney biopsy tissue. The LMD/MS test was validated in two steps. LMD/MS was used to detect the antigen in 75 cases of MN with known antigens and correctly identified the antigen in all these cases. Next, LMD/MS was used to identify the antigen in 61 MN cases where the antigen was unknown and identified one of the known antigens in 40 of 61 cases including many of the less common antigens. This lower-than-expected detection rate is explained by intentional enrichment of the cohort with PLA2R-negative MN. Overall, PLA2R was identified in 16.4%, one of the other antigens detected in 49.1%, and in the remaining 34.5% of cases, none of the above antigens was detected. Thus, LMD/MS is an extremely useful and reliable method for the detection of known MN antigens and possibly indicating an unknown MN antigen for eventual discovery.

A new test method for biochemical analysis of plasmalogens in dried blood spots and erythrocytes from patients with peroxisomal disorders.

Measurement of plasmalogens is useful for the biochemical diagnosis of rhizomelic chondrodysplasia punctata (RCDP) and is also informative for Zellweger spectrum disorders (ZSD). We have developed a test method for the simultaneous quantitation of C16:0, C18:0, and C018:1 plasmalogen (PG) species and their corresponding fatty acids (FAs) in dried blood spots (DBS) and erythrocytes (RBC) by using capillary gas chromatography-mass spectrometry. Normal reference ranges for measured markers and 10 calculated ratios were established by the analysis of 720 and 473 unaffected DBS and RBC samples, respectively. Determination of preliminary disease ranges was made by using 45 samples from 43 unique patients: RCDP type 1 (DBS: 1 mild, 17 severe; RBC: 1 mild, 6 severe), RCDP type 2 (DBS: 2 mild, 1 severe; RBC: 2 severe), RCDP type 3 (DBS: 1 severe), RCDP type 4 (RBC: 2 severe), and ZSD (DBS: 3 severe; RBC: 2 mild, 7 severe). Postanalytical interpretive tools in Collaborative Laboratory Integrated Reports (CLIR) were used to generate an integrated score and a likelihood of disease. In conjunction with a review of clinical phenotype, phytanic acid, and very long-chain FA test results, the CLIR analysis allowed for differentiation between RCDP and ZSD. Data will continue to be gathered to improve CLIR analysis as more samples from affected patients with variable disease severity are analyzed. The addition of DBS analysis of PGs may allow for at-home specimen collection and second-tier testing for newborn screening programs.

Reliable, Rapid, and Robust Speciation of Arsenic in Urine by IC-ICP-MS.

BACKGROUND: Arsenic is a naturally occurring element with varying species and levels of toxicity. Inorganic arsenic (e.g., arsenite (AsIII) and arsenate (AsV)) are toxic, while its metabolites (e.g., monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA)) are less toxic). Symptoms of exposure can include headaches, confusion, diarrhea, and drowsiness. As these symptoms overlap with many other conditions, arsenic exposure can often be overlooked as a cause. Arsenic toxicity may be treated with chelation and/or electrolyte replacement therapy. However, treatment is not without risks and is unnecessary for exposure to organic (nontoxic) forms of arsenic. This makes screening and differentiation of arsenic important for clinical testing. METHOD: An IC-ICP-MS method was developed using a Dionex 5000 with ion exchange chromatography for separation and iCAP Q for detection. Nontoxic species are arsenobetaine and arsenocholine, and toxic species are AsIII, DMA, MMA, and AsV. RESULTS: Precision, linearity, and specificity studies produced acceptable results. For accuracy, proficiency testing and method comparison samples were analyzed and produced acceptable results. Carryover studies demonstrated single species carryover from the diluter at levels of 500 µg/L, which can be avoided by analysis rules in the standard operating procedure. Limit of detection studies yielded a lower limit of quantitation of 1 µg/L per species. CONCLUSIONS: Here, we present a rapid and reliable method for quantifying and differentiating toxic and nontoxic forms of arsenic to allow for swift and appropriate management of patients with exposure.