A Simplified Sample Preparation Method for the Assessment of Plasma Ascorbic Acid in Clinical Settings.

BACKGROUND: Vitamin C deficiency is difficult to diagnose on the basis of clinical presentation alone and requires plasma levels for confirmation. Reference laboratories typically specify shipment of plasma on dry ice. This requirement may complicate clinic work flow and delay vitamin C measurement. Additionally, patients with vitamin C deficiency may experience unnecessary testing and increased health-care costs, as other diagnoses are often considered first. We examined an alternative, more practical shipping method. METHODS: Plasma was collected from 17 healthy volunteers by use of heparin tubes with gel separators, and all tubes were centrifuged immediately to separate the plasma layer from the cells. Baseline vitamin C was measured in plasma obtained immediately after specimen collection. Remaining sample tubes were held in Styrofoam containers with cold packs for 30 h or 48 h, followed by vitamin C measurement. Additional samples were exposed to conditions that simulated harsher shipping conditions. RESULTS: Mean plasma vitamin C was 69.6 µmol/L (SD = 21.5 µmol/L). Vitamin C losses were 5.4% at 30 h (SD = 5.55%, P < 0.05) and 7.6% at 48 h (SD = 5.56%, P < 0.05), which is slightly more than freeze-and-thaw treatment (average loss of 1.4%, SD = 6.9%, NS). The vitamin C method had an intraday variation of 1.88%. Vigorous shaking of 2 samples for 24 h resulted in a -1.9% change in 1 sample, and a +4.1% change in another sample. Exposure of the shipping container to elevated temperature (35 °C for 30 h) did not change the internal temperature of the container. CONCLUSIONS: The shipping procedure uses routine sample handling, standard vacutainers, and can be replicated by health-care centers seeking to evaluate patient vitamin C status.

Influence of a GSK3ß phosphorylation site within the proximal C-terminus of Neurofilament-H on neurofilament dynamics.

Phosphorylation of the C-terminal tail of the heavy neurofilament subunit (NF-H) impacts neurofilament (NF) axonal transport and residence within axons by fostering NF-NF associations that compete with transport. We tested the role of phosphorylation of a GSK-3ß consensus site (S493) located in the proximal portion of the NF-H tail in NF dynamics by transfection of NB2a/d1 cells with NF-H, where S493 was mutated to aspartic acid (S493D) or to alanine (S493A) to mimic constitutive phosphorylation and non-phosphorylation. S493D underwent increased transport into axonal neurites, while S493A displayed increased perikaryal NF aggregates that were decorated by anti-kinesin. Increased levels of S493A co-precipitated with anti-kinesin indicating that reduced transport of S493A was not due to reduced kinesin association but due to premature NF-NF interactions within perikarya. S493D displayed increased phospho-immunoreactivity within axonal neurites at downstream C-terminal sites attributable to mitogen-activated protein kinase and cyclin-dependent kinase 5. However, S493D was more prone to proteolysis following kinase inhibition, suggesting that S493 phosphorylation is an early event that alters sidearm configuration in a manner that promotes appropriate NF distribution. We propose a novel model for sidearm configuration.

Omega-3 Hastens and Omega-6 Delays the Progression of Neuropathology in a Murine Model of Familial ALS.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive disease of motor neurons that has no cure or effective treatment. Any approach that could sustain minor motor function during terminal stages would improve quality of life. OBJECTIVE: We examined the impact of omega-3 (Ω-3) and Ω-6, on motor neuron function in mice expressing mutant human superoxide dismutase-1 (SOD-1), which dominantly confers familial ALS and induces a similar sequence of motor neuron decline and eventual death when expressed in mice. METHOD: Mice received standard diets supplemented with equivalent amounts of Ω-3 and Ω-6 or a 10x increase in Ω-6 with no change in Ω-3 commencing at 4 weeks of age. Motor function and biochemical/histological parameters were assayed by standard methodologies. RESULTS: Supplementation with equivalent Ω-3 and Ω-6 hastened motor neuron pathology and death, while 10x Ω-6 with no change in Ω-3 significantly delayed motor neuron pathology, including preservation of minor motor neuron function during the terminal stage. CONCLUSION: In the absence of a cure or treatment, affected individuals may resort to popular nutritional supplements such as Ω-3 as a form of "self-medication". However, our findings and those of other laboratories indicate that such an approach could be harmful. Our findings suggest that a critical balance of Ω-6 and Ω-3 may temporarily preserve motor neuron function during the terminal stages of ALS, which could provide a substantial improvement in quality of life for affected individuals and their caregivers.

Plasma oxalate levels in prevalent hemodialysis patients and potential implications for ascorbic acid supplementation.

OBJECTIVES: Ascorbic acid (AA) supplementation may increase hemoglobin levels and decrease erythropoiesis-stimulating agent dose requirement in patients with end stage renal disease (ESRD). While plasma AA levels >100µM may be supratherapeutic, levels of at least 30µM may be needed to improve wound healing and levels may need to reach 70µM to optimize erythropoiesis. Of concern, oxalate (Ox), an AA metabolite, can accumulate in ESRD. Historically, if plasma Ox levels remain ≥30µM, oxalosis was of concern. Contemporary hemodialysis (HD) efficiencies may decrease the risk of oxalosis by maintaining pre-HD Ox levels <30µM. This study focuses on the plasma Ox levels in HD patients. DESIGN AND METHODS: A prospective, observational study of 197 HD patients with pre-HD AA levels and pre-HD and post-HD Ox levels. RESULTS: Mean plasma Ox levels decreased 71% during the intradialytic period (22.3±11.1µM to 6.4±3.2µM, P<0.001). In regression analysis, pre-HD plasma AA levels ≤100µM were not associated with a pre-HD plasma Ox level≥30µM, even if ferritin levels were increased. Pre-HD plasma Ox levels ≥20 or ≥30µM were not associated with lower cumulative 4-year survival. CONCLUSIONS: Pre-HD plasma AA levels up to 100µM in HD patients do not appear to be associated with an increased risk of developing secondary oxalosis, as the corresponding pre-HD plasma Ox level appears to be maintained at tolerable levels.

Plasma ascorbic acid concentrations in prevalent patients with end-stage renal disease on hemodialysis.

OBJECTIVE: To determine the prevalence of vitamin C (ascorbic acid [AA]) deficiency in patients with end-stage renal disease, the effect of supplemental AA on plasma AA concentrations, and the extrinsic and intrinsic factors that affect plasma AA concentrations in this patient population. DESIGN: In study 1, we compared the effect of hemodialysis (HD) on plasma AA concentrations between patients with low and high pre-HD AA concentrations. In study 2, we analyzed kinetic and nonkinetic factors for their association with increased plasma AA concentrations in patients on maintenance HD. Study 1 was performed in a single outpatient HD clinic in Cherry Hill, New Jersey. Study 2 was performed in 4 outpatient HD clinics in Southern New Jersey. SUBJECTS AND INTERVENTION: In study 1, we collected plasma samples from 8 adult patients on maintenance HD at various time points around their HD treatment and assayed them for AA concentration. In study 2, we enrolled 203 adult patients and measured pre-HD plasma AA concentrations. We ascertained supplemental AA use and assessed dietary AA intake. MAIN OUTCOME MEASURE: In study 1, plasma AA concentrations were compared during the intradialytic and interdialytic period. In study 2, pre-HD plasma AA concentrations were correlated with supplement use and demographic factors. RESULTS: Study 1 showed that over the course of a single HD treatment, the plasma AA concentration decreased by a mean (±standard deviation) of 60% (±6.6). In study 2, the median pre-HD plasma AA concentration was 15.7 µM (interquartile range, 8.7-66.8) in patients who did not take a supplement and 50.6 µM (interquartile range, 25.1-88.8) in patients who did take a supplement (P < .001). Supplement use, increasing age, and diabetes mellitus were associated with a pre-HD plasma AA concentration ≥30 µM. CONCLUSION: HD depletes plasma AA concentrations, and AA supplementation allows patients to achieve higher plasma AA concentrations.