Dennis J. Dietzen

Cholesterol oxidation products are sensitive and specific blood-based biomarkers for Niemann-Pick C1 disease

Oxysterols are biomarkers for diagnosis and drug treatment in Niemann-Pick C1 disease. Turning the Tables on Cholesterol A big push in disease research is to identify biochemical markers (biomarkers) in the blood that are early indicators of a disease that is already silently under way. By detecting the disease in its earliest stages, drugs and other therapeutic interventions have the best chance of halting or reversing the course of the disease before major tissue damage has been done. In a new study, Porter and colleagues set out to identify blood biomarkers for Niemann-Pick C1, a childhood neurological disease that is usually fatal. Niemann-Pick C1 disease is caused by mutations in the NPC1 or NPC2 proteins that result in mishandling of cholesterol and lipids in the endolysosomal system of cells. This leads to aberrant deposition of free cholesterol in the central nervous system, the death of neurons, and increasing motor and intellectual impairment, usually resulting in death during adolescence. The early symptoms of the disease are often difficult to distinguish from other childhood diseases, and thus, intervention in the form of a drug such as miglustat often comes too late. This prompted Porter and coworkers to search for possible molecules in the blood that could be used for early diagnosis of the disease and also to monitor the effectiveness of new drugs. On the basis of reports that aberrantly deposited free cholesterol is associated with increased oxidative stress, these investigators reasoned that cholesterol oxidation products (oxysterols) might be the long-sought biomarkers for Niemann-Pick C1 disease. Working in mice lacking the Npc1 gene, the researchers quickly identified two oxysterols that were markedly elevated in the plasma and tissues of the sick mice but not their healthy counterparts. Furthermore, the concentrations of these two oxysterols increased as the disease progressed. Moving into cats carrying an NPC1 mutation, which exhibit similar disease symptoms and progression as human patients, Porter and coworkers were able to decrease elevated concentrations of the two oxysterols and ameliorate disease symptoms by treating the animals with the experimental drug cyclodextrin. But could oxysterols be used as biomarkers in the human disease? The investigators demonstrated that the blood concentrations of two related oxysterol molecules were almost 10 times higher in Niemann-Pick C1 patients than in age-matched healthy controls or those with other diseases such as atherosclerosis or diabetes. Together, these compelling results suggest that the two oxysterol molecules are accurate diagnostic markers of early clinical disease and can be used not only to monitor disease progression but also to demonstrate drug efficacy. Free cholesterol may be at the root of Niemann-Pick C1 disease, but now, there is a way to turn the tables on cholesterol by using its oxidation products to diagnose and treat the disease in its earliest stages. Niemann-Pick type C1 (NPC1) disease is a rare progressive neurodegenerative disorder characterized by accumulation of cholesterol in the endolysosomes. Previous studies implicating oxidative stress in NPC1 disease pathogenesis raised the possibility that nonenzymatic formation of cholesterol oxidation products could serve as disease biomarkers. We measured these metabolites in the plasma and tissues of the Npc1−/− mouse model and found several cholesterol oxidation products that were elevated in Npc1−/− mice, were detectable before the onset of symptoms, and were associated with disease progression. Nonenzymatically formed cholesterol oxidation products were similarly increased in the plasma of all human NPC1 subjects studied and delineated an oxysterol profile specific for NPC1 disease. This oxysterol profile also correlated with the age of disease onset and disease severity. We further show that the plasma oxysterol markers decreased in response to an established therapeutic intervention in the NPC1 feline model. These cholesterol oxidation products are robust blood-based biochemical markers for NPC1 disease that may prove transformative for diagnosis and treatment of this disorder, and as outcome measures to monitor response to therapy.

Oligomerization of VIP21-caveolin in vitro is stabilized by long chain fatty acylation or cholesterol

VIP21‐caveolin is one of the components which form the cytoplasmic surface of caveolae. In vivo, this integral membrane protein is found in homo‐oligomers with molecular masses of approximately 200, 400 and 600 kDa. These oligomers are also formed by the addition of cytosol to the in vitro synthesized and membrane inserted VIP21‐caveolin. Here we show that long chain fatty acyl coenzyme A esters can completely substitute for cytosol in inducing 200 kDa and 400 kDa complexes, whereas 25‐hydroxy‐cholesterol can produce the 200 kDa oligomer. In order to understand whether acylation of VIP21‐caveolin itself is a prerequisite for oligomerization, we studied a mutant protein lacking all three cysteines. When analyzed by velocity sucrose gradient centrifugation in the presence of the non‐ionic detergent octylglucoside, both palmitoylated and non‐palmitoylated VIP21‐caveolin formed oligomers that were indistinguishable. However, only the oligomers of the non‐palmitoylated protein are disrupted when analyzed by SDS‐PAGE without boiling. These data suggest that the protein domains of VIP21‐caveolin are the primary determinants of oligomerization, but that palmitoylation of cysteine residues can increase the stability of the oligomers.

A sensitive and specific LC-MS/MS method for rapid diagnosis of Niemann-Pick C1 disease from human plasma

Niemann-Pick type C1 (NPC1) disease is a rare, progressively fatal neurodegenerative disease for which there are no FDA-approved therapies. A major barrier to developing new therapies for this disorder has been the lack of a sensitive and noninvasive diagnostic test. Recently, we demonstrated that two cholesterol oxidation products, specifically cholestane-3β,5α,6β-triol (3β,5α,6β-triol) and 7-ketocholesterol (7-KC), were markedly increased in the plasma of human NPC1 subjects, suggesting a role for these oxysterols in diagnosis of NPC1 disease and evaluation of therapeutics in clinical trials. In the present study, we describe the development of a sensitive and specific LC-MS/MS method for quantifying 3β,5α,6β-triol and 7-KC human plasma after derivatization with N,N-dimethylglycine. We show that dimethylglycine derivatization successfully enhanced the ionization and fragmentation of 3β,5α,6β-triol and 7-KC for mass spectrometric detection of the oxysterol species in human plasma. The oxysterol dimethylglycinates were resolved with high sensitivity and selectivity, and enabled accurate quantification of 3β,5α,6β-triol and 7-KC concentrations in human plasma. The LC-MS/MS assay was able to discriminate with high sensitivity and specificity between control and NPC1 subjects, and offers for the first time a noninvasive, rapid, and highly sensitive method for diagnosis of NPC1 disease.

Microvesicle‐associated tissue factor and Trousseau's syndrome

Summary.  Background: Trousseaus syndrome is a prothrombotic state associated with malignancy that is poorly understood pathophysiologically. Methods and Results: Here we report studies on the blood of a 55‐year‐old man with giant‐cell lung carcinoma who developed a severe form of Trousseaus syndrome. His clinical course was dominated by an extremely hypercoagulable state. Despite receiving potent antithrombotic therapy, he suffered eleven major arterial and venous thrombotic events over a 5 month period. We examined the patients blood for tissue factor (TF), the major initiator of coagulation, and found its concentration in his plasma to be forty‐one‐fold higher than the mean concentration derived from testing of 16 normal individuals. Conclusion: Almost all of the TF in the patients plasma was associated with cell‐derived microvesicles, likely shed by the cancer cells.

National Academy of Clinical Biochemistry laboratory medicine practice guidelines: follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry; executive summary.

BACKGROUND Almost all newborns in the US are screened at birth for multiple inborn errors of metabolism using tandem mass spectrometry. Screening tests are designed to be sufficiently sensitive so that cases are not missed. The NACB recognized a need for standard guidelines for laboratory confirmation of a positive newborn screen such that all babies would benefit from equal and optimal follow-up by confirmatory testing. METHODS A committee was formed to review available data pertaining to confirmatory testing. The committee evaluated previously published guidelines, published methodological and clinical studies, clinical case reports, and expert opinion to support optimal confirmatory testing. Grading was based on guidelines adopted from criteria derived from the US Preventive Services Task Force and on the strength of recommendations and the quality of the evidence. Three primary methods of analyte measurement were evaluated for confirmatory testing including measurement of amino acids, organic acids, and carnitine esters. The committee graded the evidence for diagnostic utility of each test for the screened conditions. RESULTS Ample data and experience were available to make strong recommendations for the practice of analyzing amino acids, organic acids, and acylcarnitines. Likewise, strong recommendations were made for the follow-up test menu for many disorders, particularly those with highest prevalence. Fewer data exist to determine the impact of newborn screening on patient outcomes in all but a few disorders. The guidelines also provide an assessment of developing technology that will fuel a refinement of current practice and ultimate expansion of the diseases detectable by tandem mass spectrometry. CONCLUSIONS Guidelines are provided for optimal follow-up testing for positive newborn screens using tandem mass spectrometry. The committee regards these tests as reliable and currently optimal for follow-up testing. .

Rapid comprehensive amino acid analysis by liquid chromatography/tandem mass spectrometry: comparison to cation exchange with post‐column ninhydrin detection

Ion-exchange chromatography with ninhydrin detection remains the gold standard for detecting inborn errors of amino acid catabolism and transport. Disadvantages of such analysis include long chromatography times and interference from other ninhydrin-positive compounds. The aim of this project was to develop a more rapid and specific technique using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Optimal fragmentation patterns for 32 amino acids were determined on a triple quadrupole mass spectrometer following butylation. Chromatographic characteristics of each of the amino acids were determined using C8 reversed-phase chromatography with 20% acetonitrile/0.1% formic acid as isocratic mobile phase. Quantitation using eleven deuterated internal standards was compared to cation exchange and ninhydrin detection on a Beckman 7300 system. Following methanol extraction and butylation, determination of 32 amino acids required 20 min. The dynamic range of each amino acid was generally 1-1000 micromol/L. Imprecision ranged from 7 to 23% (CV) over 6 months and recovery ranged from 88-125%. Deming regression with the Beckman 7300 yielded slopes from 0.4-1.2, intercepts from -21 to 65 micromol/L, correlation coefficients from 0.84-0.99 and Syx from 2-125 micromol/L. Isobaric amino acids were separated by chromatography (e.g. leucine, isoleucine) or by unique fragmentation (e.g., alanine, beta-alanine). LC/MS/MS is comparable to traditional LC-ninhydrin detection. Mass spectral detection shortens analysis times and reduces potential for interference in detecting inborn metabolic errors.

A GPI-anchored co-receptor for tissue factor pathway inhibitor controls its intracellular trafficking and cell surface expression.

Summary.  Background: Tissue factor pathway inhibitor (TFPI) lacks a membrane attachment signal but it remains associated with the endothelial surface via its association with an, as yet, unidentified glycosyl phosphatidylinositol (GPI)‐anchored co‐receptor. Objectives/methods: Cellular trafficking of TFPI within aerolysin‐resistant ECV304 and EA.hy926 cells, which do not express GPI‐anchored proteins on their surface, was compared with their wild‐type counterparts. Results and conclusions: Although aerolysin‐resistant cells produce normal amounts of TFPI mRNA, TFPI is not expressed on the cell surface and total cellular TFPI is greatly decreased compared with wild‐type cells. Additionally, normal, not increased, amounts of TFPI are secreted into conditioned media indicating that TFPI is degraded within the aerolysin‐resistant cells. Confocal microscopy and studies using metabolic inhibitors demonstrate that aerolysin‐resistant cells produce TFPI and transport it into the Golgi with subsequent degradation in lysosomes. The experimental results provide no evidence that cell surface TFPI originates from secreted TFPI that binds back to a GPI‐anchored protein. Instead, the data suggest that TFPI tightly, but reversibly, binds to a GPI anchored co‐receptor in the ER/Golgi. The co‐receptor then acts as a molecular chaperone for TFPI by trafficking it to the cell surface of wild‐type cells or to lysosomes of aerolysin‐resistant cells. TFPI that escapes co‐receptor binding is secreted through the same pathway in both wild‐type and aerolysin‐resistant cells. The data provide a framework for understanding how TFPI is expressed on endothelium.

Airway Hyperresponsiveness in Children With Sickle Cell Anemia

BACKGROUND The high prevalence of airway hyperresponsiveness (AHR) among children with sickle cell anemia (SCA) remains unexplained. METHODS To determine the relationship between AHR, features of asthma, and clinical characteristics of SCA, we conducted a multicenter, prospective cohort study of children with SCA. Dose response slope (DRS) was calculated to describe methacholine responsiveness, because 30% of participants did not achieve a 20% decrease in FEV1 after inhalation of the highest methacholine concentration, 25 mg/mL. Multiple linear regression analysis was done to identify independent predictors of DRS. RESULTS Methacholine challenge was performed in 99 children with SCA aged 5.6 to 19.9 years (median, 12.8 years). Fifty-four (55%) children had a provocative concentration of methacholine producing a 20% decrease in FEV1<4 mg/mL. In a multivariate analysis, independent associations were found between increased methacholine responsiveness and age (P<.001), IgE (P=.009), and lactate dehydrogenase (LDH) levels (P=.005). There was no association between methacholine responsiveness and a parent report of a doctor diagnosis of asthma (P=.986). Other characteristics of asthma were not associated with methacholine responsiveness, including positive skin tests to aeroallergens, exhaled nitric oxide, peripheral blood eosinophil count, and pulmonary function measures indicating airflow obstruction. CONCLUSIONS In children with SCA, AHR to methacholine is prevalent. Younger age, serum IgE concentration, and LDH level, a marker of hemolysis, are associated with AHR. With the exception of serum IgE, no signs or symptoms of an allergic diathesis are associated with AHR. Although the relationship between methacholine responsiveness and LDH suggests that factors related to SCA may contribute to AHR, these results will need to be validated in future studies.

First‐trimester prediction of preeclampsia using metabolomic biomarkers: a discovery phase study

We tested the hypothesis that first‐trimester metabolic biomarkers offered a unique profile in women with preeclampsia (PE) in the second half of pregnancy, compared with controls.

Ketogenesis prevents diet-induced fatty liver injury and hyperglycemia

Nonalcoholic fatty liver disease (NAFLD) spectrum disorders affect approximately 1 billion individuals worldwide. However, the drivers of progressive steatohepatitis remain incompletely defined. Ketogenesis can dispose of much of the fat that enters the liver, and dysfunction in this pathway could promote the development of NAFLD. Here, we evaluated mice lacking mitochondrial 3-hydroxymethylglutaryl CoA synthase (HMGCS2) to determine the role of ketogenesis in preventing diet-induced steatohepatitis. Antisense oligonucleotide-induced loss of HMGCS2 in chow-fed adult mice caused mild hyperglycemia, increased hepatic gluconeogenesis from pyruvate, and augmented production of hundreds of hepatic metabolites, a suite of which indicated activation of the de novo lipogenesis pathway. High-fat diet feeding of mice with insufficient ketogenesis resulted in extensive hepatocyte injury and inflammation, decreased glycemia, deranged hepatic TCA cycle intermediate concentrations, and impaired hepatic gluconeogenesis due to sequestration of free coenzyme A (CoASH). Supplementation of the CoASH precursors pantothenic acid and cysteine normalized TCA intermediates and gluconeogenesis in the livers of ketogenesis-insufficient animals. Together, these findings indicate that ketogenesis is a critical regulator of hepatic acyl-CoA metabolism, glucose metabolism, and TCA cycle function in the absorptive state and suggest that ketogenesis may modulate fatty liver disease.