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NEWBORN SCREENING FOR HEPATORENAL TYROSINEMIA BY TANDEM MASS SPECTROMETRY:
ANALYSIS OF SUCCINYLACETONE EXTRACTED FROM DRIED BLOOD SPOTS
Allard P, Grenier A, Korson MS, Zytkovicz TH.
- Division of Genetics, Tufts-New England Medical Center, and Tufts University School of Medicine, Boston, MA 02111, USA
- Department of Genetic Medicine, Laval University Medical Centre, Sainte-Foy, Québec, Canada G1V 4G2
- New England Newborn Screening Program, University of Massachusetts Medical School, Jamaica Plain, MA 02130, USA
Hepatorenal tyrosinemia (HP) (known as tyrosinemia type I), the most severe form of tyrosinemia, is a rare autosomal recessive metabolic disorder characterized by life-threatening progressive liver and kidney dysfunction. Affected infants may die within the first month. Different screening methods to detect tyrosine and fumarylacetoacetase (FAH) have been applied but are neither specific nor sufficiently sensitive to screen for HT in newborns. Screening of SA via direct quantification seems more promising in a routine newborn-screening program. Hence, we intend to develop a method for quantifying SA in dried blood spot (DBS) using MS/MS that provides fast analysis.
This method is based on SA extraction from residual DBS with acetonitrile and water containing hydrazine hydrate, formic acid and unlabelled 5-7-dioxooctanoic as an internal standard. Using hydrazine-containing solution permits recovery of SA from residual DBS. Hydrazine is believed to cleave covalently linked SA-proteins adducts and to simultaneously form a hydrazone derivative, which is then analyzed by tandem mass spectrometry (MS/MS).
The suggested chemical reaction of the formation of a hydrazone from SA is described as follows:
First, SA is extracted from residual DBS with 100 μl of a solution of acetonitrile and water solution (80:20 by volume), containing, per litre, 1mL of formic acid, 15mmol of hydrazine hydrate and 100 nmol of 5,7-dioxooctanoic acid. Precautions must be taken as hydrazine is a 2nd class carcinogen. Microtitration plates are agitated gently and incubated at 370C covered with aluminium foil to prevent evaporation. The extract is then transferred to a 2nd plate for MS/MS analysis.
For calibration and quality-control samples, EDTA-blood is fortified with SA to concentration levels of 0, 2, 5, 10, 20, 50 and 100 μmol/L.
To compare the stability of SA in DBS under different temperature, DBS containing SA-fortified blood to concentrations of 2,10 or 50 μmol/L are kept at 370C, 200C, 40C or -200C. SA concentrations are weekly determined for a 4-week period.
For double-checking reliability of results using MS/MS, an established enzyme assay indirectly quantifying SA by inhibition of δ-aminolevulinic acid dehydratase is used using DBS fortified with 0-75 μmol/L SA.
Quantitative results don’t differ in residual DBS compared with unextracted DBS, but residual DBS shows lower background signal, giving higher sensitivity, thus using residual DBS is recommended.
The calibration curve is linear from 1 to 50 μmol/L and the lowest limit of quantification is 0.19 μmol/L.
Stability of SA in DBS at 4 different temperatures is consistent over a 4 week-period. However, SA concentration from samples kept at 370C, 200C, 40C are, on average, 20% higher than those kept at -200C.
There is good agreement between MS/MS analysis and indirectly quantifying SA by inhibition of δ-aminolevulinic acid dehydratase with a correlation coefficient of 0.998.
Although GC/MS and MS/MS methods have been published for direct SA quantification in blood and urine, these methods require significant purification and solvent extraction, making them slow and unsuitable for a newborn screening program.
Measurement of δ-aminolevulinic acid dehydratase activity, which is inhibited by SA, can give false-positive results due to its susceptibility from factors such as EDTA, high temperature and hereditary deficiency of this enzyme.
Many newborn screening programme working with MS/MS analysis requires extracted blood spots with absolute methanol to measure amino acids and acylcarnitines, and the remaining discarded residual DBS still contains SA. Making use of residual DBS saves sample materials and reduces sample preparation time. Using of MS/MS instruments requires little additional manual work and short analytical times. The required reagents are inexpensive. In conclusion, this method is believed to be the primary newborn screening method for HT.
Word Count: 592 words
Article from which summary is derived:
Pierre Allard, A. G. (2004). Newborn screening for hepatorenal tyrosinemia by tandem mass spectrometry: analysis of succinylacetone extracted from dried blood spots. Clinical Biochemistry , 1010-1015.
Location of Image:
Johannes Sande, N. J. (2011). Newborn Screening for Hepatorenal Tyrosinemia: Tandem Mass Spectrometric Quantification of Succinylacetone. Retrieved Dec Tuesday, 2011, from Clinical Chemistry: http://www.clinchem.org/content/52/3/482/F1.medium.gif