topical_update_v14i1Biochemical genetics in the expanded newborn
screening era
Volume
14, Issue 1 January 2019
(download full article in pdf)
Editorial note
In this topical update, Dr. Calvin Chong reviews and updates
on the technological development of biochemical genetics for the three
major classes of metabolic disorders, namely aminoacidopathies, organic
acidurias and fatty acid oxidation defects. These conditions have
increasing local awareness, in particular with the introduction of
universal expanded newborn screening. With a rising clinical demand for
confirmatory tests in biochemical genetics, the ways to achieve better
analytical quality and capacity were discussed. We welcome any feedback
or suggestions. Please direct them to Dr. Sammy Chen (email: chenpls@ha.org.hk)
of Education Committee, the Hong Kong College of Pathologists. Opinions
expressed are those of the authors or named individuals, and are not
necessarily those of the Hong Kong College of Pathologists.
Dr. Calvin Yeow-Kuan Chong
Department of Pathology, Princess Margaret Hospital
Introduction
Biochemical genetics refers to the diagnosis of genetic
disorders with biochemical markers. Sir Archibald Garrod first
described the biochemical features of alkaptonuria in 19021,
and is often named as the founding father of biochemical genetics.
Throughout the past century, the practice of biochemical genetics has
evolved from spot chemical tests towards the use of chromatography and
mass spectrometry2,3. The recent
implementation of the pilot study of expanded newborn screening means,
on one hand, disorders are diagnosed earlier, and patients with such
conditions would fare better, and on the other hand, this represents an
increase in the use of confirmatory tests in biochemical genetics which
is most acutely felt at the major chemical pathology laboratories.
The screening panel of the government-initiated pilot study
included 8 aminoacidopathies, 7 organic acidurias, 6 fatty acid
oxidation defects, and 3 other disorders4.
Apart from the three disorders which required separate measurements,
all three other major classes of disorders (viz. the aminoacidopathies,
organic acidurias, and fatty acid oxidation defects) are screened by
the use of tandem mass spectrometric measurement of succinylacetone,
amino acids and acylcarnitines, all done within a period of around two
minutes.
Table 1 lists the confirmatory markers and tests for the
screened conditions 5,6: as can be seen, plasma
amino acids, urine organic acids, and plasma acylcarnitines represents
the main bulk work as confirmatory tests for these conditions. The
present article aims therefore to explore the contemporary techniques
available for these three major confirmatory tests in biochemical
genetics and attempts to identify the approaches a laboratory may seek
in order to cater for the increased demands.
Table 1. Confirmatory markers and tests for conditions covered in the government-initiated pilot study of expanded newborn screening. Specialized tests not discussed in the present article are italicized.
Amino acids
Quantitative analysis of amino acids in plasma is the first-line
confirmatory test for most aminoacidopathies. Amino acids are
characterized by the presence of primary amine and carboxylic acid
groups in one molecule, though some imino acids, namely proline and
hydroxyproline, containing an imino (a functional group containing
carbon-nitrogen double bond) as well as a carboxylic acid groups are
also considered under the umbrella term amino acids in medical parlance
and this use of terminology is adopted in the present article.
The analytical difficulties for amino acids are obvious when one
examine their chemical structures: they are amphoteric, very small
(glycine has a molecular mass of 75.067 g/mol), and most of them do not
possess conjugated double bonds which gives absorbance in the
ultraviolet spectra
7. The first and second
characteristics make
separation difficult when reverse phase chromatography is used, whereas
the last two give rise to problems in mass spectrometric detection and
ultraviolet detection respectively. There are three major methods
commonly used in clinical laboratories: amino acid analysers, high
performance liquid chromatography (HPLC) with pre-column
derivatization, and liquid chromatography-tandem mass spectrometry
(LC-MS/MS). Irrespective of the methods employed, protein precipitation
is necessary before analysis.
Amino acid
analysers
Amino acid analysers (AAAs) are standalone machines that employ
cation-exchange chromatography using a lithium buffer system, with
post-column derivatization with ninhydrin (at some 120-135 degree
Celsius) and monitoring at two wavelengths
8. It
is considered the
gold standard method for amino acid analysis. This method overcomes the
difficulty in separation of amino acids by utilizing ion-exchange
chromatography which relies on the charge of a molecule at a particular
pH rather than the hydrophobicity and steric interactions of a molecule
and that of detection by formation of purple complexes which absorb
strongly at 570 nm for primary amino acids, and yellow complexes which
absorb strongly at 440 nm for proline and hydroxyproline
9,10.
When
compared with other methods, AAAs provides higher degree of automation
and require less expertise from the laboratory; the major issues with
AAAs are the lengthy analytical run (120 minutes is common), the cost
of the analytical instruments and the proprietary nature of the
reagents. Analytical interference in AAAs are rare but do occur with
dipeptides, which occur in prolidase deficiency
8,11,
and
aspartylglucosamine, which occur in aspartylglucosaminuria
8,
can be
spotted by the 570/440 absorbance ratio.
HPLC and
LC-MS/MS methods
HPLC coupled with pre-column derivatization is used in the author’s
laboratory for PAA determination. The method relied on the pre-column
(immediately before injection) derivatization of amino acids by
orthophthalaldehyde (OPA) and 3-mercaptopropionic acid (MPA), followed
by reversed phase chromatography and ultraviolet detection
12-14.
In
the presence of a thiol reagent, OPA forms a fluorescent derivative
with primary amino acids with peak absorbance/excitation at 340 nm and
emission wavelength at 450 nm
7,8. The major
drawback of this method
is the inability to detect proline (and hydroxyproline) as OPA does not
react with imino acids.
A newer derivatization reagent, AccQ-Tag
(6-aminoquinolyl-N-hydroxysuccinimidyl carbamate), which converts both
primary and secondary amino acids into stable fluorescent derivatives,
has been used with ultra-high performance liquid chromatography with
ultraviolet detection
15. This derivatization
reagent has the
advantages of covering both primary and secondary amino acids and as
the derivatization products are stable, advanced autosamplers which can
pipet reagent from one vial to another is not required
16.
Complete
chemistry kit-sets that use this derivatization reagent is commercially
available
17.
Liquid chromatography coupled with tandem mass spectrometry is also
used locally for PAA determination. As these methods are often based on
reversed phase chromatography, despite the use of mass spectrometric
detector, derivatization is still often employed
7.
A method based on
the proprietary derivatization reagent AccQ-Tag has been recently
described in the literature with a run-time of 6 minutes
18.
Isotopic
internal standards are required for LC-MS/MS-based methods and it is
important to note that the impracticality of having isotopic internal
standards for each and every analyte would mean that the robustness of
the assay is considerably weaker than optical-based methods
19.
Compared with methods based on optical detection, LC-MS/MS methods had
a shorter analytical runtime and better specificity. This is
counterweighed by the higher capital and maintenance cost of acquiring
an LC-MS/MS (and not to mention the cost of having a backup LC-MS/MS
system), as well as the technical expertise necessary to operate and
troubleshoot an LC-MS/MS.
Choice of method
and the future
The question is different depending on the volume of testing as well as
equipment availability. For laboratories that has existing HPLC-DAD
equipment employing OPA derivatization there may be little incentive in
adopting a new procedure: there is probably little competition for HPLC
analyser time, and the inability to detect proline may not be a major
issue as hyperprolinaemia type I is benign whereas hyperprolinaemia
type II is classically diagnosed by the presence of
N-(Pyrrole-2-carboxyl)-glycine in urine organic acids
20.
On the other hand, for a laboratory seeking to provide amino acids
analysis for the first time, the use of stable derivatization reagent
such as AccQ-Tag mentioned above has the advantage of not requiring
higher-end liquid chromatographs and the availability of commercial
kits means that development time is reduced. For a laboratory
anticipating a high workload, a dedicated liquid chromatograph with UV
detection appears to be the simplest solution as it is robust and
inexpensive. On the other hand, for laboratories with lower service
demand, mass spectrometric detection may in fact be more feasible as
the notion of spare LC-MS/MS capacity means that the major downside of
LC-MS/MS detection, viz capital cost and technical expertise, are sunk
cost to the laboratory.
Organic acids
Organic acidurias are diagnosed most commonly by urine organic acid
analysis with gas chromatography-mass spectrometry (GC-MS)
21,
and
this technique is used by many local hospitals. Though the term organic
acids refers to organic compounds with a carboxylic acid group, a
broader spectrum of compounds, such as uracil and xanthine, are
detected in practice. Urine organic analysis is usually qualitative
though quantitative analyses of some compounds (e.g. orotic acid,
methylmalonic acid) are often offered.
GC-MS
based urine organic acid analysis
For GC-MS analysis of urine organic acids, a
creatinine-corrected amount of urine is subject to liquid-liquid
extraction with ethyl acetate after acidification using hydrochloric
acid, the organic extract is then dried and derivatized by
N,O-bis-tri(methylsilyl)trifluoroacetamide (BSTFA) with 1%
trimethylchlorosilane (TMCS)22. The resultant
product is injected to
the GC-MS operating in scan mode. For reliable detection of ketones,
oximation with hydroxylamine hydrochloride can be performed prior to
acidic extraction23.
An alternative way of performing urine organic acid analysis
is to bypass the extraction step. As no acidic extraction step is done,
a limited panel of amino acids, purine, pyrimidines, and mono- and
di-saccharides can be detected in the same analytical run. This method
has been in-use in the author’s laboratory in the past 2
decades24,25, and allows the detection of a much
wider range of
metabolites in urine. With no extraction, the chromatograms are
extremely complex due to co-elution of analytes and therefore this
approach requires expertise in post-analytical data processing to be
viable as a routine method.
The interpretation of GC-MS data for urine organic acid
analysis is commonly based on examination of the chromatogram (Figure
1), followed by a combination of peak integration in the total ion
chromatogram followed by library searching and examination of extracted
ion chromatogram at particular retention times for analytes which gives
lower responses (Figure 2). The raw analyte response is then compared
to locally-established age-specific reference intervals which allow
clinical interpretation23.
Figure 1. Total ion chromatogram of a urine sample from a patient with malonic acidemia circulated in the ERNDIM qualitative organic acid program in 2016. The two abnormal peaks are indicated by asterisks: the first peak represents malonic acid (di-TMS derivative) and the second peak represents methylmalonic acid (di-TMS derivative).
Figure 2. Extracted ion chromatogram showing the quantifier ions (m/z 375, for aconitic acid in red; and m/z 254, for orotic acid, in black) and qualifier ions (m/z 285, for aconitic acid in green; and m/z 357, for orotic acid, in blue). They are co-eluting in many GC-MS based urine organic acid assays.
LC-MS
based urine organic acid analysis
In the recent years, liquid chromatography-mass
spectrometry-based methods have been published for analysis of urine
organic acids. The benefit of liquid chromatography-mass spectrometry
is clear: there is no need for the cumbersome derivatization step, and
heat-labile analytes can be detected26,27. The
major drawbacks of
LC-MS based methods are the poorer separation of analytes and higher
susceptibility towards ion suppression, as electrospray ionization is
much less robust against matrix effects compared to electron ionization
as used in GC-MS based methods28. The difficulty
of developing an
in-house LC-MS based method for urine organic acids lies in the
procurement of a practically endless list of organic acid standards to
establish the retention time and multiple reaction monitoring ratios.
At the time of writing, there is at least one commercial kit that has
been made available (Zivak Organic Acids LC-MS/MS analysis kit), but
this commercial kit did not utilize isotopic internal standards even
for critical analytes (such as hexanoylglycine and orotic acid) and one
may wish to validate extensively the robustness of such assays against
matrix effects.
Choice
of method and the future
Out of the three assays discussed in the present article,
urine organic acid analysis represents the most difficult of the three
to establish in a laboratory. There is first the difficulty in
establishing age-specific reference intervals, and then difficulty in
acquiring a large number of chemical standards. A good starting point
would be obtaining the bi-level quality controls for urine organic acid
and special assays for urine from the ERNDIM
network which consist of a
number of critical analytes. As for choice of internal
standards, while traditional choices such as tropic acid and
pentadecanoic acid are often employed, deuterated internal standards
covering critical analytes are much more available nowadays (e.g.
methylmalonic acid-d3 and orotic acid-1,3-15N2) and their addition may
improve quantitation of these critical analytes, the former being
commonly quantified and the latter being prone to analytical errors29.
For laboratories with existing GC-MS based urine organic acid
assay, the question is probably whether to adopt LC-MS based solution.
The belief that organic aciduria always results in sky-high level of
abnormal metabolites in urine is flawed: the low-excretor phenotype of
glutaric aciduria type I can serve as the classical example30.
The difficulty may be mitigated somewhat if acylcarnitines and acylglycines
are measured by the LC-MS based assays as glutarylcarnitine has been
shown to be informative even for low-excretor GA I patients31.
Overall, it remains to be seen whether LC-MS based organic acid
analysis could stand alone replacing, rather than complementing, GC-MS
based assays.
Free carnitine and acylcarnitines
Quantitative analysis of free carnitine and acylcarnitines in plasma
represents the first-line confirmatory test for fatty acid oxidation
disorders. Analysis of free carnitine with calculation of fraction of
excretion can be helpful in the diagnosis of carnitine uptake defect,
as is measurement of urine glutarylcarnitine for the diagnosis of
glutaric aciduria type I as discussed above
31.
Carnitine is a quaternary ammonium compound with a carboxylic acid group, as well as a
hydroxyl group with which acyl groups are attached to form
acylcarnitines; as such, it forms zwitterions under physiological pH.
Derivatization to esters by incubation with an alcohol (typically
butanol) has been employed
32 though locally
underivatized analysis at
acidic pH is commonly used
33.
For underivatized analysis, plasma is first diluted with a mixture of
isotopic internal standards, and acidified acetonitrile is slowly added
to precipitate plasma proteins. The sample is vortexed, centrifuged,
followed by evaporation of the supernatant to dryness and reconstituted
for analysis by positive electrospray ionization-tandem mass
spectrometry with or without liquid chromatography separation. Data can
be collected in multiple reaction monitoring (MRM) mode, or in
precursor ion scan with accumulation of ions, commonly known as
multichannel acquisition (MCA) mode
34 (Figure
3). The American
College of Medical Genetics Guideline, published in 2008, suggested the
use of precursor ion scan as it permitted the evaluation of the whole
acylcarnitine profile, as well as the detection of drug artefacts,
interfering compounds and assessment of derivatization
35.
Figure 3. Cumulative precursor scan mass spectrum for a blood-spot sample distributed in the ERNDIM Qualitative Acylcarnitine Program in 2014. In this specimen, elevated signals at m/z ratio 260 (C6-carnitine), 288 (C8-carnitine), and 314 (C10:1-carnitine) can be seen. The pattern would be compatible with MCAD deficiency. (Mass spectra courtesy of Mr CK Lai, Chemical Pathology Laboratory, PMH)
Derivatization
and chromatographic separation
In the analysis of carnitine and acylcarnitines, butyl-ester
derivatization enhances the formation of positively-charged ion by
reacting with carboxylic groups, and causes mass separation of
dicarboxylic-carnitines and hydroxy-acylcarnitines (e.g. C4DC-carnitine
and C5OH-carnitine), which are isobaric when underivatized36.
Derivatization is typically performed at highly acidic conditions (e.g.
3N hydrochloric acid at 65°C for 15 minutes)35.
On the other hand,
chromatographic separation allows the separate determination of
individual isomeric constituents (e.g. C4DC-carnitines include
succinylcarnitine and methylmalonylcarnitine; and C5OH-carnitines
include 3-hydroxyisovalerylcarnitine and
2-methyl-3-hydroxybutyrylcarnitine) (Figure 4). With meticulous
chromatographic separation, most biologically relevant isomeric species
could be separately quantified37.
Choice
of method and the future
The question for the laboratory is, first, whether to employ
the derivatization procedure. The advantage of derivatization is the
mass separation of hydroxylacylcarnitines and carnitine derivatives of
dicarboxylic acids; the problems associated with derivatization are the
partial hydrolysis of acylcarnitines because of the high temperature
and strongly acidic condition employed33. The
other considerations
are whether to employ chromatography and if so, how extensive should it
be: it would then be a delicate balance between through-put, diagnostic
specificity, and analyser-time that is available. With the improvement
of separation capability of liquid chromatographs and sensitivity of
modern mass spectrometers, it is suggested that a short UHPLC program
combined with both scheduled MRM and precursor ion scan function would
be a good compromise.
Figure 4. SRM chromatogram of m/z 262>85 showing chromatographic separation of different species of isobaric (C4DC/C5OH) acylcarnitines (viz. succinylcarnitine at 4.68 minutes, two diastereomeric peaks of methylmalonylcarnitine at 5.2 and 5.35 minutes, and 3-hydroxyisovalerylcarnitine at 5.82 minutes) could be individually identified and quantified with liquid chromatography-tandem mass spectrometry without derivatization. (a) sample with normal amounts of succinylcarnitine; (b) sample with abnormal amounts of methylmalonylcarnitines (Chromatograms courtesy of Mr CK Lai, Chemical Pathology Laboratory, PMH)
Conclusions
The three assays discussed above represent the bulk of workload for
most metabolic laboratories. The planned implementation of universal
expanded newborn screening in Hong Kong means that the demand would
increase, and the phenotype will be less defined, as tests are
requested for patients who may not yet present with features of an
inborn error and importantly, patients with only borderline elevation
of analytes.
From a Bayesian point of view, this change in pre-test probability
would mean that, if the analytical quality and interpretative capacity
remains the same (which affect the likelihood ratio of positive
results), the post-test probability would suffer from a negative
impact. The quest for the metabolic bench of any major pathology
laboratory is then to improve both throughput and quality of analysis
at the same time, an impossible task, as the Duke of Norfolk wrote in
1538, “a man can not have his cake and eat his cake”
38.
The
technology improvements as reviewed in the present article may aid in
the analytical quality but the quest for improved interpretative
capacity remains on the training and education of our present and
coming generations of pathologists.
Reference
-
Garrod, AE. The incidence of alkaptonuria: a study in
chemical individuality. The Lancet. 1902 Dec 13;160(4137):1616–20.
-
Millington DS, Kodo N, Norwood DL, Roe CR. Tandem mass
spectrometry: a new method for acylcarnitine profiling with potential
for neonatal screening for inborn errors of metabolism. Journal of
inherited metabolic disease. 1990;13(3):321–4.
-
Schulze A, Lindner M, Kohlmüller D, Olgemöller K,
Mayatepek E, Hoffmann GF. Expanded newborn screening for inborn errors
of metabolism by electrospray ionization-tandem mass spectrometry:
results, outcome, and implications. Pediatrics. 2003;111(6):1399–406.
-
Mak, CM. Newborn Screening: Past, Present and the Future.
Topical Update, The Hong Kong College of Pathologists. 2016;11(2):1–10.
-
Ozben T. Expanded newborn screening and confirmatory
follow-up testing for inborn errors of metabolism detected by tandem
mass spectrometry. Clinical Chemistry and Laboratory Medicine.
2012;51(1):157–176.
-
Saudubray J-M, Berghe G van den, Walter JH. Inborn
Metabolic Diseases. Springer-Verlag Berlin Heidelberg; 2012.
-
Kaspar H, Dettmer K, Gronwald W, Oefner PJ. Advances in
amino acid analysis. Anal Bioanal Chem. 2009 Jan;393(2):445–52.
-
Duran M. Amino Acids. In: Blau N, Duran M, Gibson KM,
editors. Laboratory Guide to the Methods in Biochemical Genetics
[Internet]. Berlin, Heidelberg: Springer Berlin Heidelberg; 2008 [cited
2018 Nov 24]. p. 53–89. Available here.
-
Friedman M, Williams LD. Stoichiometry of formation of
Ruhemann’s purple in the ninhydrin reaction. Bioorganic Chemistry.
1974;3(3):267–80.
-
Cunico RL, Schlabach T. Comparison of ninhydrin and
o-phthalaldehyde post-column detection techniques for high-performance
liquid chromatography of free amino acids. Journal of Chromatography A.
1983;266:461–70.
-
Ferreira CR, Cusmano-Ozog K. Spurious Elevation of
Multiple Urine Amino Acids by Ion-Exchange Chromatography in Patients
with Prolidase Deficiency. JIMD Rep. 2016 Apr 12;31:45–9.
-
Fürst P, Pollack L, Graser TA, Godel H, Stehle P.
Appraisal of four pre-column derivatization methods for the
high-performance liquid chromatographic determination of free amino
acids in biological materials. Journal of Chromatography A.
1990;499:557–69.
-
Schuster R. Determination of amino acids in biological,
pharmaceutical, plant and food samples by automated precolumn
derivatization and high-performance liquid chromatography. Journal of
Chromatography B: Biomedical Sciences and Applications. 1988;431:271–84.
-
Terrlink T, Van Leeuwen PA, Houdijk A. Plasma amino acids
determined by liquid chromatography within 17 minutes. Clinical
chemistry. 1994;40(2):245–9.
-
Narayan SB, Ditewig-Meyers G, Graham KS, Scott R, Bennett
MJ. Measurement of plasma amino acids by Ultraperformance® Liquid
Chromatography. Clinical Chemistry and Laboratory Medicine.
2011;49(7):1177–1185.
-
van Wandelen C, Cohen SA. Using quaternary
high-performance liquid chromatography eluent systems for separating
6-aminoquinolyl-N-hydroxysuccinimidyl carbamate-derivatized amino acid
mixtures. Journal of Chromatography A. 1997;763(1–2):11–22.
-
Armenta JM, Cortes DF, Pisciotta JM, Shuman JL, Blakeslee
K, Rasoloson D, et al. A sensitive and rapid method for amino acid
quantitation in malaria biological samples using AccQ•Tag
UPLC-ESI-MS/MS with multiple reaction monitoring. Anal Chem. 2010 Jan
15;82(2):548–58.
-
Gray N, Zia R, King A, Patel VC, Wendon J, McPhail MJW, et
al. High-Speed Quantitative UPLC-MS Analysis of Multiple Amines in
Human Plasma and Serum via Precolumn Derivatization with
6-Aminoquinolyl-N-hydroxysuccinimidyl Carbamate: Application to
Acetaminophen-Induced Liver Failure. Anal Chem. 2017 Feb
21;89(4):2478–87.
-
Sharer JD, Biase ID, Matern D, Young S, Bennett MJ, Tolun
AA. Laboratory analysis of amino acids, 2018 revision: a technical
standard of the American College of Medical Genetics and Genomics
(ACMG). Genetics in Medicine. 2018 Oct 19;1.
-
Walker V, Mills GA. N-(pyrrole-2-carboxyl) glycine a
diagnostic marker of hyperprolinaemia type II: mass spectra of
trimethylsilyl derivatives. Clin Chim Acta. 2009 Jul;405(1–2):153–4.
-
Christou C, Gika HG, Raikos N, Theodoridis G. GC-MS
analysis of organic acids in human urine in clinical settings: a study
of derivatization and other analytical parameters. Journal of
Chromatography B. 2014;964:195–201.
-
Rinaldo P. Organic Acids. In: Blau N, Duran M, Gibson KM,
editors. Laboratory Guide to the Methods in Biochemical Genetics
[Internet]. Berlin, Heidelberg: Springer Berlin Heidelberg; 2008 [cited
2018 Nov 30]. p. 137–69. Available here.
-
Gallagher RC, Pollard L, Scott AI, Huguenin S, Goodman S,
Sun Q, et al. Laboratory analysis of organic acids, 2018 update: a
technical standard of the American College of Medical Genetics and
Genomics (ACMG). Genet Med. 2018;20(7):683–91.
-
Lee H-CH, Lai C-K, Yau K-CE, Siu T-S, Mak CM, Yuen Y-P, et
al. Non-invasive urinary screening for aromatic l-amino acid
decarboxylase deficiency in high-prevalence areas: A pilot study.
Clinica Chimica Acta. 2012 Jan 18;413(1):126–30.
-
Au KM, Lai CK, Yuen YP. Diagnosis of dihydropyrimidine
dehydrogenase deficiency in a neonate with thymine-uraciluria. Hong
Kong Medical Journal. 2003;9(2):130–3.
-
Körver-Keularts IMLW, Wang P, Waterval HWAH, Kluijtmans
LAJ, Wevers RA, Langhans C-D, et al. Fast and accurate quantitative
organic acid analysis with LC-QTOF/MS facilitates screening of patients
for inborn errors of metabolism. J Inherit Metab Dis. 2018 May
1;41(3):415–24.
-
Yassine MM, Dabek-Zlotorzynska E. Application of
ultrahigh-performance liquid chromatography–quadrupole time-of-flight
mass spectrometry for the characterization of organic aerosol:
Searching for naphthenic acids. Journal of Chromatography A. 2017 Aug
25;1512:22–33.
-
Taylor PJ. Matrix effects: the Achilles heel of
quantitative high-performance liquid chromatography–electrospray–tandem
mass spectrometry. Clinical biochemistry. 2005;38(4):328–34.
-
Scott C, Langhans C-D, Roux C. Qualitative Organic Acid.
In: ERNDIM Workshop. Manchester, UK; 2017.
-
Kölker S, Christensen E, Leonard JV, Greenberg CR, Boneh
A, Burlina AB, et al. Diagnosis and management of glutaric aciduria
type I–revised recommendations. Journal of inherited metabolic disease.
2011;34(3):677.
-
Tortorelli S, Hahn SH, Cowan TM, Brewster TG, Rinaldo P,
Matern D. The urinary excretion of glutarylcarnitine is an informative
tool in the biochemical diagnosis of glutaric acidemia type I.
Molecular Genetics and Metabolism. 2005 Feb 1;84(2):137–43.
-
Dhillon KS, Bhandal AS, Aznar CP, Lorey FW, Neogi P.
Improved tandem mass spectrometry (MS/MS) derivatized method for the
detection of tyrosinemia type I, amino acids and acylcarnitine
disorders using a single extraction process. Clinica Chimica Acta.
2011;412(11–12):873–9.
-
Ho CS, Cheng BSS, Lam CWK. Rapid Liquid
Chromatography–Electrospray Tandem Mass Spectrometry Method for Serum
Free and Total Carnitine. Clinical Chemistry. 2003 Jul 1;49(7):1189–91.
-
Matern D. Acylcarnitines, Including In Vitro Loading
Tests. In: Blau N, Duran M, Gibson KM, editors. Laboratory Guide to the
Methods in Biochemical Genetics [Internet]. Berlin, Heidelberg:
Springer Berlin Heidelberg; 2008 [cited 2018 Nov 30]. p. 171–206.
Available here.
-
Rinaldo P, Cowan TM, Matern D. Acylcarnitine profile
analysis. Genetics in Medicine. 2008 Feb;10(2):151–6.
-
Vreken P, Van Lint AEM, Bootsma AH, Overmars H, Wanders
RJA, Van Gennip AH. Quantitative plasma acylcarnitine analysis using
electrospray tandem mass spectrometry for the diagnosis of organic
acidaemias and fatty acid oxidation defects. Journal of inherited
metabolic disease. 1999;22(3):302–6.
-
Giesbertz P, Ecker J, Haag A, Spanier B, Daniel H. An
LC-MS/MS method to quantify acylcarnitine species including isomeric
and odd-numbered forms in plasma and tissues. Journal of lipid
research. 2015;jlr. D061721.
-
Letters and Papers, Foreign and Domestic, Henry VIII,
Volume 13 Part 1, January-July 1538 [Internet]. 1892. Available here.
Abbreviations:Plasma
amino acids (PAA):
Arginine (Arg),
Citrulline (Cit), Homocystine (Hcy), Isoleucine (Ileu), Leucine (Leu),
Methionine (Met), Ornithine (Orn), Phenylalanine (Phe), Threonine
(Thr), Tyrosine (Tyr), Valine (Val).
Urine
organic acids (UOA):
ethylmalonic acid (EMA),
glutaric acid (GA), 2-hydroxyglutaric acid (2-OHGA), 3-hydroxyglutaric
acid (3-OHGA), 3-methylglutaric acid (3-MGA), 3-methylglutaconic acid
(3-MGCA), hexanoylglycine (HG), 3-hydroxyisovaleric acid (3-OHIVA),
Isovalerylglycine (IVG), 3-methylcrotonylglycine (3-MCG), Methylcitric
acid (MCA), Methylmalonic acid (MMA), 3-hydroxypropionic acid (3-OHPA),
propionylglycine (PG), phenylpropionylglycine (PPG), Tiglylglycine (TG).
Plasma
acylcarnitines (PAC):
Free carnitine (C0).
Others:
6-pyruvoyl-tetrahydropterin
synthase (PTPS), 3-hydroxy-3-methylglutaryl CoA (HMG-CoA),
Carnitine-acylcarnitine translocase (CACT), Carnitine
palmitoyltransferase II (CPT-II), Medium-chain acyl-CoA dehydrogenase
(MCAD), Very long-chain acyl-coA dehydrogenase (VLCAD). 
