Lawrence Fisher

The Association of Vitamin D Status With Pediatric Critical Illness

OBJECTIVES: Vitamin D is a pleiotropic hormone important for the proper functioning of multiple organ systems. It has been hypothesized that vitamin D deficiency could contribute to or worsen outcomes in critical illness. The study objective was to determine the prevalence of vitamin D deficiency, risk factors for its presence, and potential association with clinically relevant outcomes in critically ill children. METHODS: A prospective cohort study, conducted from 2005 to 2008 in 6 tertiary-care PICUs in Canada. Data and biological samples from 326 critically ill children up to 17 years of age were available for analysis. Total serum 25 hydroxyvitamin D or 25(OH)D was measured by using liquid chromatography-mass spectrometry. RESULTS: The prevalence of 25(OH)D <50 nmol/L was 69% (95% confidence interval, 64–74), and 23% (95% confidence interval, 19–28) for 25(OH)D between 50 to 75 nmol/L. Lower levels were associated with hypocalcemia, catecholamine utilization, and significant fluid bolus administration. Vitamin D deficiency was independently associated with a longer PICU length of stay (+1.92 days, P = .03) and increasing severity of illness as determined by the Pediatric Risk of Mortality score with every additional point increasing the likelihood of being vitamin D deficient by 8% (P = .005). CONCLUSIONS: This study provides evidence that vitamin D deficiency is both common among critically ill children and associated with greater severity of critical illness. Further research will determine whether targeted vitamin D supplementation or rapid restoration will improve outcome.

A digital microfluidic method for dried blood spot analysis

Blood samples stored as dried blood spots (DBSs) are emerging as a useful sampling and storage vehicle for a wide range of applications. Unfortunately, the surging popularity of DBS samples has not yet been accompanied by an improvement in automated techniques for extraction and analysis. As a first step towards overcoming this challenge, we have developed a prototype microfluidic system for quantification of amino acids in dried blood spots, in which analytes are extracted, mixed with internal standards, derivatized, and reconstituted for analysis by (off-line and in-line) tandem mass spectrometry. The new method is fast, robust, precise, and most importantly, compatible with automation. We propose that the new method can potentially contribute to a new generation of analytical techniques for quantifying analytes in DBS samples for a wide range of applications.

Measurement of homocysteine and related metabolites in human plasma and urine by liquid chromatography electrospray tandem mass spectrometry

The sulfur amino acids, methionine and cysteine play crucial roles in cells as a substrate for protein synthesis, as a methyl donor, and for the synthesis of sulfur-containing compounds, including the key intracellular tripeptide, glutathione. Homocysteine is an intermediary metabolite formed during the metabolism of methionine to cysteine. Dysregulation of homocysteine metabolism is implicated in adverse clinical outcomes such as increased risk of cardiovascular disease, stroke, Alzheimers disease dementia and osteoporosis. While hyperhomocysteinemia is commonly observed in those conditions, the impact on other related metabolites is condition-specific. Therefore, there exists a need to establish precise and sensitive analytical techniques that allow for the simultaneous measurement of homocysteine and related metabolites in biological samples. The current review outlines the development and use of liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS) to simultaneously measure metabolites involved in sulfur amino acid metabolism. Additionally, extensions of the technique in relation to the measurement of sulfur amino acid and one-carbon kinetics in vivo are discussed. The LC-MS/MS technique has the capacity for unambiguous analyte identification and confirmation, due to its high specificity and sensitivity. It has the greatest potential of being accepted and utilized as a dedicated homocysteine and its related metabolite Standard reference method (SRM).

Impact of anesthesia and surgery for congenital heart disease on the vitamin d status of infants and children: a prospective longitudinal study.

Background:Vitamin D is recognized as a pleiotropic hormone important for the functioning of organ systems, including those central to critical illness pathophysiology. Recent studies have reported associations between vitamin D status and outcome among critically ill adults and children. Preoperative vitamin D status, impact of operative techniques, and relationship between immediate postoperative vitamin D levels and clinical course have not been described in the pediatric congenital heart disease (CHD) population. The objective of this study was to describe the impact of CHD surgery on vitamin D status and relationship between postoperative levels and clinical course. Methods:A prospective cohort study was conducted from 2009 to 2011 at a single tertiary care pediatric hospital. A total of 58 children with CHD were enrolled and blood collected preoperatively, intraoperatively, and postoperatively. Serum 25-hydroxyvitamin D (25OHD) was measured using liquid chromatography–mass spectrometry. Results:The mean preoperative 25OHD was 58.0 nM (SD, 22.4), with 42% being deficient (<50 nM). Postoperatively, we identified a 40% decline in 25OHD to 34.2 nM (SD, 14.5) with 86% being deficient. Intraoperative measurements determined that initiation of cardiopulmonary bypass coincided with abrupt decline. CHD patients requiring catecholamines had lower postoperative 25OHD (38.2 vs. 26.5 nM, P = 0.007), findings confirmed through multivariate logistic regression. Lower postoperative 25OHD was associated with increased fluid requirements and intubation duration. Conclusions:Most CHD patients are vitamin-D deficient postoperatively due to low preoperative levels and a significant intraoperative decline. Interventional studies will be required to determine whether prevention of postoperative vitamin D deficiency improves outcome.

The first three years of screening for medium chain acyl-CoA dehydrogenase deficiency (MCADD) by newborn screening ontario

BackgroundMedium chain acyl-CoA dehydrogenase deficiency (MCADD) is a disorder of mitochondrial fatty acid oxidation and is one of the most common inborn errors of metabolism. Identification of MCADD via newborn screening permits the introduction of interventions that can significantly reduce associated morbidity and mortality. This study reports on the first three years of newborn screening for MCADD in Ontario, Canada.MethodsNewborn Screening Ontario began screening for MCADD in April 2006, by quantification of acylcarnitines (primarily octanoylcarnitine, C8) in dried blood spots using tandem mass spectrometry. Babies with positive screening results were referred to physicians at one of five regional Newborn Screening Treatment Centres, who were responsible for diagnostic evaluation and follow-up care.ResultsFrom April 2006 through March 2009, approximately 439 000 infants were screened for MCADD in Ontario. Seventy-four infants screened positive, with a median C8 level of 0.68 uM (range 0.33-30.41 uM). Thirty-one of the screen positive infants have been confirmed to have MCADD, while 36 have been confirmed to be unaffected. Screening C8 levels were higher among infants with MCADD (median 8.93 uM) compared to those with false positive results (median 0.47 uM). Molecular testing was available for 29 confirmed cases of MCADD, 15 of whom were homozygous for the common c.985A > G mutation. Infants homozygous for the common mutation tended to have higher C8 levels (median 12.13 uM) relative to compound heterozygotes for c.985A > G and a second detectable mutation (median 2.01 uM). Eight confirmed mutation carriers were identified among infants in the false positive group. The positive predictive value of a screen positive for MCADD was 46%. The estimated birth prevalence of MCADD in Ontario is approximately 1 in 14 000.ConclusionsThe birth prevalence of MCADD and positive predictive value of the screening test were similar to those identified by other newborn screening programs internationally. We observed some evidence of correlation between genotype and biochemical phenotype (C8 levels), and between C8 screening levels and eventual diagnosis. Current research priorities include further examining the relationships among genotype, biochemical phenotype, and clinical phenotype, with the ultimate goal of improving clinical risk prediction in order to provide tailored disease management advice and genetic counselling to families.

A novel method for quantitation of acylglycines in human dried blood spots by UPLC-tandem mass spectrometry

BACKGROUND Several acylcarnitines used as primary markers on dried blood filter papers (DBS) for newborn screening lack specificity and contribute to a higher false positive rate. The analysis of urine acylglycines is useful in the diagnosis of inborn errors of metabolism (IEM) including medium chain acyl-CoA dehydrogenase deficiency (MCADD), isovaleric acidemia, and beta-ketothiolase deficiency (BKTD). Currently, no method for analyzing acylglycines from DBS has been published. METHODS Acylglycines were extracted from two 3.2 mm DBS punches and butylated using Butanol-HCl. Ultra Performance Liquid Chromatography (UPLC-MS/MS) with run time of 10 min permits resolution and quantitation of 15 acylglycines; including several isobaric. Method development was completed. Reference intervals (n = 573) were established for four birth weight groups. Furthermore, samples from patients with a confirmed IEM (n = 11), and false positive screens (n = 78) were analyzed to validate the interpretation obtained from the newly established reference intervals. RESULTS Calibration curves were linear from 0.005 to 25.0 μM. Ion suppression was evaluated as minimal (2 to 10%). Samples from known patients were used to validate the reference intervals. For C5OH-related disorders, tiglylglycine (TG), TG/acetylglycine (AG) ratio, 3methylcrotonylglycine (3MCG) and 3MCG/AG ratio increased specificity. Propionylglycine (PG) and PG/acetylglycine ratio were two discriminatory markers in the investigation of C3-related disorders. Hexanoylglycine (HG), octanoylglycine (OG), suberylglycine (SG), and the ratios HG/AG, OC/AG and SG/AG were excellent markers of MCADD deficiency. CONCLUSION This method shows potential application as a second tier screen in order to reduce the false positive rate for a number of IEM targeted by newborn screening.

Quantification of DNA in Neonatal Dried Blood Spots by Adenine Tandem Mass Spectrometry

Newborn screening programs have expanded to include molecular-based assays as first-tier tests and the success of these assays depends on the quality and yield of DNA extracted from neonatal dried blood spots (DBS). To meet high throughput and rapid turnaround time requirements, newborn screening laboratories adopted rapid DNA extraction methods that produce crude extracts. Quantification of DNA in neonatal DBS is not routinely performed due to technical challenges; however, this may enhance the performance of assays that are sensitive to amounts of input DNA. In this study, we developed a novel high throughput method to quantify total DNA in DBS. It is based on specific acid-catalyzed depurination of DNA followed by mass spectrometric quantification of adenine. The amount of adenine was used to calculate DNA quantity per 3.2 mm DBS. Reference intervals were established using archived, neonatal DBS (n = 501) and a median of 130.6 ng of DNA per DBS was obtained, which is in agreement with literature values. The intra- and interday variations were <15%. The limits of detection and quantification were 12.5 and 37.8 nmol/L adenine, respectively. We demonstrated that DNA from neonatal DBS can be successfully quantified in high throughput settings using instruments currently deployed in NBS laboratories.