CASE
A 5-day-old male infant with an increased dried blood spot
propionylcarnitine (C3-carnitine) value of 7.93 [micro]mol/L (cutoff
< 6.79 [micro]mol/L) was identified by the New Jersey state newborn
screening program. C3-carnitine is used as a screening tool for
methylmalonic and propionic acidemias, potentially fatal but treatable
inborn errors of metabolism. The initial screen values provided a
calculated C3:C2 carnitine ratio of 0.23 (cutoff < 0.32, mean 0.074)
and a C3:C16 ratio of 2.51 (cutoff < 4.16, mean 0.96). The child was
an inpatient at an outlying neonatal intensive care unit. He was born at
35 weeks estimated gestational age, required continuous positive airway
pressure for a short time after birth, and transitioned quickly to room
air. He was taking regular feedings with a cow's milk
protein--based formula.
On day of life 6, the patient developed a mild acidosis (pH 7.24 on
arterial blood gas testing). Because methylmalonic and propionic
acidemia could not be excluded while confirmatory test results were
pending, feedings were discontinued, intravenous hydration with
glucose-containing fluids was initiated, and the infant was transferred
to our institution. On arrival the child appeared well, was alert, and
had normal growth parameters and no tachypnea. He had good tone and
normal reflexes, and laboratory studies showed no acidosis. We allowed
normal feedings and proceeded with the diagnostic evaluation.
DISCUSSION
Although C3-carnitine appears in the blood, the active metabolite
within the mitochondrion is propionyl-CoA. Propionyl-CoA is an
intermediate in the degradation of several amino acids. It can also
appear as an intermediate of odd-chain fatty acid metabolism and
exogenously as a derivative of propionate that is generated by
gastrointestinal flora. Normally propionyl-CoA is metabolized to
methylmalonyl-CoA by the action of propionyl-CoA carboxylase (PCC), but
if the metabolite is in excess the propionyl species is released from
the mitochondrion after conversion by carnitine palmitoyl transferase II
to the corresponding acylcarnitine (Fig. 1).
The differential diagnosis for increased C3-carnitine in a newborn
includes inborn errors of metabolism, vitamin B12-deficiency, and
false-positive results (1). The associated inborn errors of metabolism
include PCC defects that cause propionic acidemia. Children with this
condition potentially have the greatest elevations in C3-carnitine
because of an immediate backup of metabolic flux resulting in increased
concentrations of propionyl-CoA. Defects in processing of the cofactor
for PCC, biotin, could in theory lead to C3-carnitine elevation, but
isolated elevations of C3carnitine in patients with biotinidase
deficiency or holocarboxylase synthetase deficiency have not been
reported.
The largest group of defects associated with C3-carnitine elevation
involves the downstream enzyme methylmalonyl-CoA mutase
(MMM).MMMconverts methylmalonyl-CoA to succinyl-CoA, an intermediate in
the Krebs cycle. This enzyme is one of 2 in the body that uses vitamin
B12 as a cofactor. C3-carnitine is the newborn screen metabolite used
for detection of MMMdeficiency (known as methylmalonic acidemia) because
C4-dicarboxylcarnitine elevations (MMA-carnitine or succinyl-carnitine)
are not consistently or sufficiently increased to enable differentiation
of patients from those who are unaffected; the backup to propionyl-CoA
and C3-carnitine is more readily detectable. A broad variety of defects
in vitamin B12 processing, known as cobalaminopathies, can lead to
disorders with a biochemical overlap with methylmalonic acidemia.
Maternal vitamin B12 deficiency, and vertical transmission of this
deficiency, is a known cause of C3-carnitine elevation (2, 3). This
defect is not isolated to the newborn period; breast-fed infants of
vegan mothers with B12 deficiency have been reported with neurological
impairment and methylmalonate excretion (4). Commercially available
formulas and term breast-milk from mothers with normal B12 metabolism
have adequate concentrations of B12 to avoid such complications.
[FIGURE 1 OMITTED]
The use of diagnostic laboratory evaluations can help to
differentiate the causes of C3-carnitine elevations (Table 1). The
acylcarnitine profile of our patient on day of life 5 did not detect
C3-acylcarnitine, nor did a repeat acylcarnitine analysis on day of life
6. There was no increased methylmalonate, the diagnostic species of
methylmalonic acidemia, in urine organic acids measured by GC-MS.
Homocysteine, often increased in B12 deficiency and some defects of
cobalamin metabolism, was not detected. Methylcitrate and 3-hydroxy
propionate, additional markers of propionic acidemia, were absent from
urine organic acids, effectively ruling out defects in PCC. A serum B12
concentration obtained on day of life 5 was in the lower end of the
normal range (3300 pg/L, normal reference interval 2930-12 080 pg/L).
On further review of the history, we learned that the mother was
diagnosed with anemia during the pregnancy. On closer questioning, she
disclosed that 3 years before the pregnancy she had undergone gastric
bypass for the purpose of weight loss. She could not recall having
received supplemental vitamin B12 following the procedure.
DIAGNOSIS
The diagnosis was vitamin B12 deficiency due to maternal vitamin
B12 deficiency after gastric bypass.
RESOLUTION OF CASE
Vertical transmission of vitamin [B.sub.12] deficiency caused a
metabolic disturbance in the child on the second day of life, at the
time of newborn screening. The B12 deficiency in our patient, however,
had been corrected by the time of transfer to our institution. After 5
days of enteral feeding, provision of dietary vitamin [B.sub.12] in
infant formula had corrected both the intramitochondrial defect (as
determined by C3-carnitine concentrations) and plasma concentrations of
B12. The cause of the patient's transient acidosis on day of life 6
was unclear, but metabolic studies confirmed that it was unrelated to
defects in branched-chain amino acid metabolism. The mother was told to
inform her primary care physician that she had B12 deficiency and would
require lifelong supplementation.
Newborn screening is designed to detect inborn errors of metabolism
in a time course that improves prospects for treatment and survival.
Newborn screening was initially developed for detection of
phenylketonuria, but has expanded in most states to include other
conditions such as fatty acid oxidation defects, amino acidopathies, and
organic acidopathies. Increased C3carnitine presents a diagnostic
challenge because of the wide range of possible causes, including
false-positive results, vitamin deficiency, and life-threatening
disorders such as methylmalonic or propionic acidopathy. Severe disease
was unlikely in this case, because the typical infant with methylmalonic
or propionic acidopathy presents with dehydration, moderate
hepatomegaly, increasedammonia, ketoacidosis, poor feeding, drowsiness,
axial hypotonia, and limb hypertonia.
Detection of maternal pathology (in this case B12 deficiency)
through the newborn screen is not unique to this condition. Elevations
in C5-OH acylcarnitine are indicative of a variety of pathologies,
including 3-methylcrotonyl-CoA carboxylase deficiency, and have
frequently resulted in a diagnosis of this deficiency in the mother
rather than in the newborn (5). The mechanism in these patients is
probably distinct, with 3-methylcrotonyl-CoA carboxylase elevations due
to a direct maternal transfer of the acylcarnitine species. Defects in
the maternal carnitine transporter have also been detected by low
concentrations of carnitine on a newborn screen (6).
The mechanism of B12 deficiency in the patient's mother is
also not uncommon. The number of gastric bypasses performed on women of
reproductive age has increased as roux-en-Y gastric bypasses in the
general population have increased from 16 000 per year in the early
1990s to 103 000 in 2003 (7). Patients who have had gastric bypass are
at significant risk for B12 deficiency because of the loss of
intrinsic-factor secretion that is required for absorption. Patients
should receive 1 mg intramuscular B12 every 3months or 500[micro]g
intranasal B12 weekly following gastric bypass (8).
This case illustrates an unusual mechanism leading to the elevation
of a diagnostic metabolite. The patient's benign presentation in
combination with family history and laboratory studies revealed the
cause before any form of treatment was instituted in the infant. In this
case, the mother elected to feed her child commercial infant formula,
which corrected the vitamin deficiency. Had the infant been breastfed,
the deficiency would have continued and acidosis resulting from impaired
MMM function could have resulted. As a result of the newborn screening
program, the maternal deficiency in B12 was detected and treated before
the emergence of neurological symptoms, although she had already
developed anemia. This unusual route of diagnosis, although it may
provoke anxiety in the clinician and family, can be considered an
unexpected benefit of the newborn screening program. History,
examination, and metabolic laboratory studies are sufficient to
expeditiously separate cases of methylmalonic and propionic acidemia
from false-positive or nutritional causes of C3-carnitine elevation on
newborn screening.
POINTS TO REMEMBER
* The diagnosis of a metabolic disease cannot be made exclusively
on the basis of newborn screening. An abnormal result must be confirmed
by additional testing pursued in consideration of the clinical
presentation and family history.
* Elevations in C3-acylcarnitines are used in the diagnosis of
methylmalonic aciduria, although this metabolite is several steps
removed from the metabolic defect.
* Newborn screen findings can reveal maternal defects in the case
of several inborn errors of metabolism, including 3-methylcrotonyl-CoA
carboxylase deficiency and carnitine transport defect, and also can
reveal nutritional deficits in the mother.
* Newborn screening is designed to accept an increased
false-positive rate to have an excellent sensitivity and negative
predictive value.
Author Contributions: Each author confirmed they have contributed
to the intellectual content of this paper and have met the following 3
requirements: (a) significant contributions to the conception and
design, acquisition of data, or analysis and interpretation of data; (b)
drafting or revising the article for intellectual content; and (c) final
approval of the published article.
Authors' Disclosures of Potential Conflicts of Interest: Upon
manuscript submission, all authors completed the Disclosures of
Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: M. J. Bennett, Board of Directors, AACC,
and member of the editorial board of Clinical Chemistry.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: K. A. Chapman is the recipient of grant
2T32GM008638, NIH, National Institute of General Medical Sciences; and
N. Sondheimer is the recipient of grant 2K12HD043245, NIH, National
Institute of Child Health and Human Development. Expert Testimony: None
declared.
Role of Sponsor: The funding organizations played no role in the
design of study, choice of enrolled patients, review and interpretation
of data, or preparation or approval of manuscript.
References
(1.) ACMG Newborn Screening Workgroup. Newborn screening ACT sheets
and confirmatory algorithms. 2006 (Accessed March 2008).
(2.) Bjorke Monsen AL, Ueland PM, Vollset SE, Guttormsen AB,
Markestad T, Solheim E, et al. Determinants of cobalamin status in
newborns. Pediatrics 2001;108:624 -30.
(3.) Campbell CD, Ganesh J, Ficicioglu C. Two newborns with
nutritional vitamin B12 deficiency: Challenges in newborn screening for
vitamin B12 deficiency. Haematologica 2005;90(12 Suppl):ECR45.
(4.) Kuhne T, Bubl R, Baumgartner R. Maternal vegan diet causing a
serious infantile neurological disorder due to vitamin B12 deficiency.
Eur J Pediatr 1991;150:205-8.
(5.) Koeberl DD, Millington DS, Smith WE, Weavil SD, Muenzer J,
McCandless SE, et al. Evaluation of 3-methylcrotonyl-CoA carboxylase
deficiency detected by tandem mass spectrometry newborn screening. J
Inherit Metab Dis 2003;26:25-35.
(6.) Schimmenti LA, Crombez EA, Schwahn BC, Heese BA, Wood TC,
Schroer RJ, et al. Expanded newborn screening identifies maternal
primary carnitine deficiency. Mol Genet Metab 2007;90:441-5.
(7.) Steinbrook R. Surgery for severe obesity. N Engl J Med
2004;350:1075-9.
(8.) Clements RH, Katasani VG, Palepu R, Leeth RR, Leath TD, Roy
BP, et al. Incidence of vitamin deficiency after laparoscopic roux-en-Y
gastric bypass in a university hospital setting. Am Surg
2006;72:1196,202; discussion 1203-4.
Commentary
Charles P. Venditti
Supplemental newborn screening using acylcarnitine ester profiling
has greatly expanded the scope of detection of newborns with a wide
range of fatty acid oxidation disorders and organic acidemias. Each
metabolite, or group of metabolites, measured by tandem mass
spectrometry in the newborn blood spot carries the potential to identify
infants at risk for metabolic disease. Among these markers, a positive
test for increased C3 species--propionylcarnitine--causes a
characteristic and immediate reaction of initiating an emergency
metabolic evaluation for the at-risk infant, with good reason. Increased
propionylcarnitine in the blood is a biochemical hallmark of isolated
methylmalonic acidemia, propionic academia, and disorders of
intracellular cobalamin processing, all potentially lethal disorders of
intermediary metabolism. However, experienced clinicians and newborn
screening laboratories alike recognize that increased propionylcarnitine
is not a perfect disease marker and in some large series, an infant with
increased C3 species will more likely be categorized as a false positive
vs affected with ametabolic disorder (1), an outcome far more desirable
than the diagnosis of propionic or methylmalonic acidemia.
Although hereditary metabolic disorders comprise the most common
true-positive subset within the group of babies with increased
propionylcarnitine (1), another category includes infants born to
mothers with diminished maternal vitamin B12 stores. Maternal B12
deficiency is recognized to produce a spectrum of symptoms in the
infant--ranging from frank encephalopathy with severe metabolic
derangements (2) to increased propionylcarnitine (1) with mild
methylmalonic aciduria (3). The mother in the current case had undergone
gastric bypass without subsequent vitamin B12 supplementation, and
although her biochemical and hematological parameters were not
documented in this report, she presumably was vitamin B12 deficient as
was the proband when initially screened. Fortunately, other than mild
prematurity, the baby was well and without biochemical abnormalities
such as methylmalonic aciduria. In the end, a positive newborn screen
enabled recognition of untreated maternal disease and demonstrates the
unexpected societal benefit that can be derived from expanded newborn
screening. One now wonders whether other infants in the false-positive
category for increased C3 species might be instructing us to pay more
attention to whether maternal pathology is present.
Author Contributions: Each author confirmed they have contributed
to the intellectual content of this paper and have met the following 3
requirements: (a) significant contributions to the conception and
design, acquisition of data, or analysis and interpretation of data; (b)
drafting or revising the article for intellectual content; and (c) final
approval of the published article.
Authors' Disclosures of Potential Conflicts of Interest: No
authors declared any potential conflicts of interest.
Role of Sponsor: The funding organizations played no role in the
design of study, choice of enrolled patients, review and interpretation
of data, or preparation or approval of manuscript.
References
(1.) Chace DH, DiPerna JC, Kalas TA, Johnson RW, Naylor EW. Rapid
diagnosis of methylmalonic and propionic acidemias: quantitative tandem
mass spectrometric analysis of propionylcarnitine in filter-paper blood
specimens obtained from newborns. Clin Chem 2001;47:2040-4.
(2.) Higginbottom MC, Sweetman L, Nyhan WL. A syndrome of
methylmalonic aciduria, homocystinuria, megaloblastic anemia and
neurologic abnormalities in a vitamin B12-deficient breast-fed infant of
a strict vegetarian. N Engl J Med 1978;299:317-23.
(3.) Marble M, Copeland S, Khanfar N, Rosenblatt DS. Neonatal
vitamin B12 deficiency secondary to maternal subclinical pernicious
anemia: identification by expanded newborn screening. J Pediatr
2008;152:731-3.
Genetic Disease Research Branch, National Human Genome Research
Institute, National Institutes of Health, Bethesda, MD
Address correspondence to the author at: Genetic Disease Research
Branch, National Human Genome Research Institute, National Institutes of
Health, Bethesda, MD 20892. e-mail venditti@mail.nih.gov. Received
August 22, 2008; accepted August 26, 2008. Previously published online
at DOI: 10.1373/clinchem.2008.113449
Commentary
Dietrich Matern
Newborn screening is used to detect disease in the newborn. Rarely
do we consider that screening may uncover disease in the mother. Only in
newborn screening for infectious diseases such as HIV is the
identification of disease in the mother also considered a goal (1).
Furthermore, it is also generally assumed that abnormal results are
indicative of an underlying genetic disease and not dietary or
iatrogenic factors. As this case nicely demonstrates, newborn screening
can have implications and benefits not only for the baby but also for
the mother. In addition, newborn screening can also benefit others when
follow-up of positive test results includes further testing of
asymptomatic family members, such as in medium chain acyl-CoA
dehydrogenase deficiency (2).
This case also exemplifies an issue in newborn screening that needs
to be considered. Many screening programs require a second dried blood
spot sample to repeat the newborn screen when the first test is
abnormal. If the second sample yields normal results, then the baby is
deemed healthy and the first result is overruled as a false positive. By
now, it should be well known that this approach is inappropriate for
several fatty acid oxidation disorders, in particular very long-chain
acyl-CoA dehydrogenase deficiency (3, 4). Had the same approach been
applied to this case, the maternal condition would again have escaped
detection because the results of follow-up acylcarnitine profiles in the
baby were already normal.
With the expansion of newborn screening and the associated increase
in complexity, the American College of Medical Genetics developed
follow-up guidelines (freely available at www.ACMG.net) to help the
practitioner efficiently and comprehensively follow up on abnormal
newborn screening results. These practice guidelines are based on the
analytes that can be abnormal in newborn screening and not just the
conditions officially screened for in a particular screening program.
These guidelines also include suggestions to evaluate family members
when indicated.
Author Contributions: Each author confirmed they have contributed
to the intellectual content of this paper and have met the following 3
requirements: (a) significant contributions to the conception and
design, acquisition of data, or analysis and interpretation of data; (b)
drafting or revising the article for intellectual content; and (c) final
approval of the published article.
Authors' Disclosures of Potential Conflicts of Interest: No
authors declared any potential conflicts of interest.
Role of Sponsor: The funding organizations played no role in the
design of study, choice of enrolled patients, review and interpretation
of data, or preparation or approval of manuscript.
References
(1.) Cameron T. Mandatory HIV testing of newborns in New York
State: what are the implications? J Health Soc Policy 2002;14:59 -78.
(2.) Matern D, Rinaldo P. Medium-chain acyl-coenzyme A
dehydrogenase deficiency.
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=gene&part= mcad
(Accessed October 2008).
(3.) Browning MF, Larson C, Strauss A, Marsden DL. Normal
acylcarnitine levels during confirmation of abnormal newborn screening
in long-chain fatty acid oxidation defects. J Inherit Metab Dis
2005;28:545-50.
(4.) Boneh A, Andresen BS, Gregersen N, Ibrahim M, Tzanakos N,
Peters H, et al. VLCAD deficiency: pitfalls in newborn screening and
confirmation of diagnosis by mutation analysis. Mol Genet Metab
2006;88:166-70.
Biochemical Genetics Laboratory, Mayo Clinic College of Medicine,
Rochester, MN
Address correspondence to the author at: Biochemical Genetics
Laboratory, Mayo Clinic College of Medicine, 200 First Street SW,
Rochester, MN 55905. E-mail Matern@mayo.edu.
Received July 14, 2008; accepted August 18, 2008.
Previously published online at DOI: 10.1373/clinchem.2008.113456
Kimberly A Chapman, (1) Michael J. Bennett, (2) and Neal Sondheimer
(3) *
Departments of (1) Genetics, (2) Pathology and Laboratory Medicine,
and (3) Child Development, Rehabilitation, and Biochemical Genetics, The
Children's Hospital of Philadelphia, Philadelphia, PA
* Address correspondence to this author at: The Children's
Hospital of Philadelphia, 34th Street and Civic Center Blvd,
Philadelphia, PA, 19104. Fax 215-5904297; e-mail
Sondheimer@email.chop.edu.
Received March 21, 2008; accepted July 10, 2008.
Previously published online at DOI: 10.1373/clinchem.2008.107581
Table 1. Diagnostic workup and expected values
for newborns with elevated C3-carnitine (1).
Acylcarnitine
True diagnosis B12 profile
Propionic acidemia Normal [up arrow] C3
Methylmalonic acidemia Normal [up arrow] C3
Cobalaminopathy Normal [up arrow] C3
B12 deficiency Low [up arrow] C3
Corrected B12 Normal Normal
deficiency
True diagnosis Urine organic acids
Propionic acidemia [up arrow] Methylcitrate, 3-OH
propionate, propionyl glycine
Methylmalonic acidemia [up arrow][up arrow] Methylmalonate
Cobalaminopathy [up arrow] Methylmalonate
B12 deficiency [up arrow] Methylmalonate
Corrected B12 Normal
deficiency
True diagnosis Plasma amino acids Other
Propionic acidemia [up arrow] Glycine, [up arrow][up arrow]
Normal homocysteine Ammonia, acidosis
Methylmalonic acidemia Normal homocysteine Severe acidosis,
[up arrow] Ammonia
Cobalaminopathy Elevated homocysteine None
B12 deficiency Elevated homocysteine None
Corrected B12 Normal None
deficiency