| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Technical Briefs |
1 Department of Chemical Pathology, Women’s and Children’s Hospital, 72 King William Rd., North Adelaide, South Australia 5006, Australia
2 University of Adelaide, North Terrace, Adelaide, South Australia 5000, Australia
aauthor for correspondence: fax 61-8-81617100, e-mail david.johnson{at}adelaide.edu.au
A notable deficiency in the use of tandem mass spectrometry (MS/MS) for newborn screening is the inability to screen for urea cycle defects. The most common of these, with an incidence of 1 in 14 000 births (1), is the inherited X-linked disorder ornithine transcarbamylase deficiency (OTCD). A majority (60%) of hemizygous males risk death from hyperammonemic coma during the first week of life. The remainder, including 10% of heterozygous females, exhibit lethargy, vomiting episodes, and behavioral problems during childhood. The severity of the disorder and the potential for correction of OTCD by liver transplantation and gene therapy (2) provide adequate justification for newborn screening.
OTCD patients have low blood citrulline because of reduced conversion from carbamoyl phosphate. Citrulline is one of the amino acids routinely measured in MS/MS newborn-screening programs. Unfortunately, many protein-restricted newborns also have low blood citrulline (3). A more selective amino acid metabolite for OTCD is glutamine. The derivatization procedure used in many MS/MS screening programs (4), which uses butanol–hydrogen chloride, destroys glutamine. Approximately one-half of the glutamine is converted to glutamic acid dibutyl ester and is indistinguishable from that formed from endogenous glutamic acid in the blood. The surviving glutamine butyl ester is deaminated in acidic solution to a protonated form of pyroglutamic acid butyl ester in the electrospray source of the MS/MS. Again it is not possible to distinguish this pyroglutamic acid from what is already present in the blood. As a secondary consequence, the measurements of glutamic and pyroglutamic (and by analogy, aspartic) acids in blood spots after derivatization are grossly inaccurate. MS/MS newborn-screening programs that do not derivatize amino acids avoid solvolysis of glutamine and of pyroglutamic acid to glutamic acid. During electrospray ionization-MS/MS analysis, however, glutamine is again indistinguishable from pyroglutamic acid. Resolution is possible by separation with time-consuming liquid chromatography (5), which is unsuited to rapid screening programs.
Formamidene butyl esters of amino acids afford more stable ions than the corresponding butyl esters during electrospray ionization-MS/MS analysis (6). We have further optimized this derivatization method to quantify glutamine in dried blood spots from newborns and in plasma to monitor OTCD patients on treatment. Milder derivatization conditions minimized amide derivatization and solvolysis. The preparation of formamidene isobutyl esters increased MS/MS signal intensity. Additionally, glutamic and pyroglutamic acids were simultaneously quantified to determine the fate of glutamine in stored samples.
Amino acids were extracted with methanol from 3-mm dried blood spots or 2 µL of plasma adsorbed on a 3-mm filter-paper disk for 15 min. [2H5]Glutamine (2 nmol), [2H3]glutamic acid (1 nmol), and [2H3]pyroglutamic acid (1 nmol) were added as internal calibrators. The amino acids were treated with 240 µL of dimethylacetal dimethylformamide–acetonitrile–methanol (2:5:5 by volume) at room temperature for 5 min, excess reagents were evaporated, and the residue was treated with isobutanol–3 mol/L–hydrogen chloride (200 µL) at room temperature for 10 min and then was evaporated to dryness. Isobutanol affords a 50% increase in ion intensity during MS/MS analysis relative to n-butanol. The derivatives were dissolved in 2 mL of acetonitrile–water–formic acid (50:50:0.025 by volume).
An Applied Biosystems/MDS Sciex Model API3000 tandem mass spectrometer equipped with a TurboIonspray source (temperature, 100 °C) was used for analysis. An Agilent HP1100 LC pumped acetonitrile–water–formic acid (50:50:0.025 by volume) at a flow rate of 160 µL/min into the TurboIonspray via a Gilson 233 autosampler fitted with a 20-µL injection loop. Samples were injected from a 96-well tray at 2-min intervals. Multiple-reaction monitoring experiments with six ion pairs, representing a neutral loss of 73 atomic mass units (amu; for glutamine) and a neutral loss of 102 amu (for glutamic and pyroglutamic acids), and the corresponding set for the three labeled internal calibrators were used for data acquisition.
Calibration curves were constructed from the analyses of derivatized mixtures of the three amino acids. Statistical analysis revealed response linearity for glutamine (0–2.5 mmol/L; y = 0.999x - 0.09 µmol/L; R2 = 0.9999), glutamic acid (0–1.25 mmol/L; y = 0.999x + 0.26 µmol/L; R2 = 1), and pyroglutamic acid (0–1.25 mmol/L; y = 0.992x + 6.33 µmol/L; R2 = 0.9997). The imprecision in the measurement of an amino acid calibrator containing glutamine (1 mmol/L) was 2.3% (intraassay; n = 9) and 2.9% (interassay; n = 12). The imprecision in the measurement of glutamine in dried blood spots prepared from an adult blood sample (0.3 mmol/L) was 7.4% (intraassay; n = 9) and 12% (interassay; n = 10).
To determine whether a diagnosis of OTCD can be made in archived blood spots, we evaluated the stability of glutamine over time. The analyses of neonatal blood spots (five at each time point) stored at ambient temperature (15–25 °C) for up to 10 years are shown in Fig. 1
. We observed a rapid decrease in glutamine, a moderate decrease in glutamic acid, and a rapid increase in pyroglutamic acid. A duplicate dried blood spot from an OTCD patient stored at -20 °C in a sealed plastic bag for 6 months, however, retained 98% of its original glutamine concentration. We analyzed blood spots from seven OTCD patients, together with control blood spots of identical age and storage conditions, for glutamine (Table 1
). One patient was newly diagnosed, and the other six samples were from an archived collection stored at ambient temperature. Apart from the oldest sample, in which glutamine was unmeasurable, increased glutamine was observed for all of the OTCD patients except for one heterozygous female.
|
|
We also measured glutamine in freshly collected plasma samples (n = 36) referred to our laboratory for amino acid analysis and including some from OTCD patients on treatment. These samples were also analyzed for glutamine on a Waters 600E HPLC-based amino acid analyzer. Statistical analysis of the comparison (see Fig. 2 in the Data Supplement accompanying the online version of this Technical Brief at http://www.clinchem.org/content/vol49/issue4/) afforded excellent correlation (y = 1.02x - 4.39 µmol/L; R2 = 0.88).
Unfortunately, this method cannot be used as a substitute for the butylation method used in MS/MS newborn-screening programs, primarily because acylcarnitines are not fully derivatized at room temperature. It is doubtful that the incidence of OTCD justifies a separate newborn dried blood spot screen for glutamine with this method. One obvious role of the method is high-risk screening of all newborns with a familial history of OTCD. Another is as a second-tier screening method to reexamine dried blood spots with a low citrulline concentration. Our experience suggests that performing glutamine analysis on the lowest 2% of citrulline results should ensure the flagging of female OTCD heterozygotes. Since our MS/MS newborn screening program was introduced 4 years ago, dried blood spots from two diagnosed OTCD patients have been analyzed. A male had a citrulline concentration of 6 µmol/L (below the 0.1 percentile), and a heterozygous female had a citrulline concentration of 12 µmol/L (between the 1st and 2nd percentiles).
This study demonstrates the instability of glutamine and, to a lesser extent, glutamic acid in dried blood spots under typical storage conditions. Both are deaminated or dehydrated to pyroglutamic acid. Glutamine is not hydrolyzed to glutamic acid as is the case with plasma. After 1 year, pyroglutamic acid reaches a long-term stable concentration of 
500 µmol/L (see Fig. 1
) in dried blood spots from newborns. This is the case even in archived OTCD dried blood spots that initially contained much higher newborn glutamine concentrations.
When glutamine measurements are used as a second- tier screen, dried blood spots need to be analyzed within 2 weeks of collection to avoid more than a 5% decrease in concentration. This period can be extended if the dried blood spot samples are stored at a low temperature. Archived dried blood spots <10 years of age can be analyzed for glutamine if they are compared with age-matched and storage-condition-matched control blood spots.
This method for the quantification of glutamine in plasma supplements the measurements of plasma ammonia and urine orotic acid commonly used to monitor patients with urea cycle defects. It is a superior alternative to the use of an amino acid analyzer to monitor OTCD patients on treatment. Sample analysis time, without HPLC separation, is much shorter (2 vs 120 min), and the required sample volume is much smaller (2 vs 125 µL). Analysis can also be performed on dried blood spots from less invasive fingerprick samples that are sent from remote locations by post. Any other essential 
-amino acids can be quantified simultaneously, with detection limits comparable to those of glutamine, by adding the appropriate isotope-labeled internal calibrators. Amino acids containing amide and imine groups, such as citrulline, arginine, and asparagine, exhibited poor detection limits with the original formamidene derivatization method (6). The milder derivatization conditions and the use of isobutanol in this modified method improved MS/MS analysis of these amino acids dramatically.
Acknowledgments
We thank the Wellcome Trust for a grant to purchase the Model API3000 MS/MS instrument used in this study.
References
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  D. H. Chace, T. A. Kalas, and E. W. Naylor Use of Tandem Mass Spectrometry for Multianalyte Screening of Dried Blood Specimens from Newborns Clin. Chem., November 1, 2003; 49(11): 1797 - 1817. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
